Recent Advances in Scientific Research


Scientific Study, 2018
146 Pages

Free online reading

Contents

Preface

Contributors

Chapters

1 Recent Advances in Biological Applications of Schiff Base Complexes
Dr. Malabika Borah

2 Liver - Its Threats and Herbal Cure Dr. Munmi Borkataky

3 Phytochemical Studies and Traditional Use Nature’s Most Valuable Plant of Bael (Aegle Marmelos) in Assam
Dr. Monmi Saikia, and Bidanshree Basumatary

4 Polyphenols: Its present and future perspective
Dr. Archana Borah

5 Recent Development of Ruthenium(II) Complexes with P,N-Donor Ligands
Dr. Devajani Boruah

6 Applicability of Genetic Parameters in Rice Improvement Programmes: Present Initiatives and Future Prospects
Dr. Pallabi Dutta

7 Antioxidants: The Modern Elixire
Dr. Manas Pratim Boruah

8 Phosphatase Activity: A study on indigenous Tea RhizospherePhosphate Solubilizing Microorganisms
Dr. Rakhi Phukan, R. Samanta, and B. K. Barthakur

Preface

In recent years there has been a significant development in science and technology. This book is a compilation of eight chapters relating to different areas of scientific research. The efforts of our extreme contributors of this book are highly commendable.

In this book we tried to cover the areas relating to the exploitation of natural products of North-East region of India and their application in food and drugs for the benefit of mankind. Application of genetic variability for the crop improvement programme through breeding. Some of the chapters highlight the use of microbial strains as an alternate source of biofertilizer. The rich and diverse coordination chemistry of ruthenium metal with hemilabile P,N- donor ligands has been overviewed which received much attention in the last few couple of years due to academic interest as well as industrial needs. Specially, in the field of catalysis for different chemical transformations that generate various bio-active natural products, agrochemicals, pharmaceuticals, polymers, etc. Moreover, the antioxidant and anticancer activities of Schiff base complexes have got recent attention as with the progress of their structure-activity relationship, Schiff base complexes have the potential to emerge as a good future therapeutic agent.

In the compilation of this book, we have received admirable cooperation from the contributors of each chapter. In fact, it is the contributors of chapters who construct the heart of this book. We feel the pleasure to express our gratitude to all of them. We express our thanks to our family members and well wishers for their inspiring attitude, and assistance in this effort.

We hope that this book will inspire our budding researchers to take up further in depth studies in the field of science.

Neither the editors nor the publishers are responsible for the data, results and comments reported by the respective authors on the book.

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Contributors

Dr. Malabika Borah, Assistant Professor, Department of Chemistry, B. N. College, Dhubri, Assam, India

Dr. Munmi Borkataky, Department of Plant Physiology and Breeding, Tea Research Association, Tocklai Tea Research Institute, Jorhat, Assam-785008, India,

Dr. Monmi Saikia, Department of Chemistry, Bodoland University, Kokrajhar, BTAD, Assam, India

Bidanshree Basumatary, Department of Chemistry, Bodoland University, Kokrajhar, BTAD, Assam, India

Dr. Archana Borah, Department of Life Sciences, Dibrugarh University, Assam, India

Dr. Devajani Boruah, Assistant Professor, Department of Chemistry, Silapathar Science College, Dhemaji, Assam, India

Dr. Pallabi Dutta, Assistant Professor, Department of Botany, Silapathar Science College, Dhemaji, Assam, India

Dr. Manas Pratim Boruah, Assistant Professor, Department of Chemistry, Dhemaji College, Dhemaji, Assam, India

Dr. Rakhi Phukan, Department of Mycology & Microbiology, Tocklai Experimental Station, Tea Research Association, Tocklai Tea Research Institute, Jorhat, Assam, India

Professor R. Samanta, Department of Life Sciences, Dibrugarh University, Assam, India

Professor B. K. Barthakur, Department of Life Sciences, Dibrugarh University, Assam, India

Chapter 1
Recent Advances in Biological Applications of Schiff Base Complexes

Dr. Malabika Borah

Introduction

Schiff bases are versatile compounds that are formed from the condensation of a primary amine with an aldehyde or a ketone under specific conditions. Structually, a Schiff base (also known as imine or azomethine) is a nitrogen analogue of an aldehyde or ketone in which the carbonyl group (CO) has been replaced by an imine or azo-methine group[1,2].

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Figure 1: Structure of azomethine group.

Schiff bases are a very interesting group of compounds for studying hydrogen bond properties. The important physical and biological properties of schiff bases are directly related to the presence of the intramolecular hydrogen bond and a proton transfer equilibrium. This group of compounds is characterized by great biological activity and they play an important role in biological system. Generally, Schiff bases have widely applied in food industry, dye industry, analytical chemistry, catalysis, fungicidal, agrochemical and biological activities[3]. It is also used in the development of modern co-ordination chemistry, inorganic biochemistry, catalysis and optical materials[4]. The versatility of Schiff base ligands and biological, analytical and industrial applications of their complexes make further investigations in this area highly desirable.

General synthesis

The acid/base catalysis or heating is employed for the synthesis of Schiff bases as their reactions are mostly reversible. The Schiff bases are formed by the reaction of amines with carbonyl compounds but it does not follow simple nucleophilic addition, but give an unstable addition compound called carbinolamine[5]. The compound thus obtained is unstable and loses water molecule. The removal of product or separation of water from the reaction mixture assists the formation of product[6]. The aqueous acids or bases may hydrolyze Schiff bases towards their respective aldehydes or ketones and amines as well. In this regard, high concentration of acid is not needed due to basic character of amines. This is the reason that mildly acidic pH are quite good for the formation of Schiff bases.

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Scheme 1: Formation of Schiff base ligand through carbinolamine intermediate.

Schiff base ligands are easily synthesized and form complex with almost all metal ions[1]. The chelating ability is enhanced when nitrogen atom is present in the vicinity of one or more donor groups. The azomethine group carrying ligands i.e., Schiff bases have achieved a considerable position and become ligands of interest in coordination chemistry due to the fact that formation of such compounds proceeds with greater ease. The condensation of a diamine derivative with salisaldehyde leads to the formation of a Schiff base which possesses such a structural set up that two nitrogen atoms and two oxygen atoms are available for chelation[7]. These ligands are known as Salen ligands and are analogous to the porphyrin in structural aspects but can be easily prepared[8]. Since the tetradentate ligands obtained by condensation of salicyldehyde and ethylenediamine were originally termed as salen ligands but another term “salen-type” is now employed to discuss the class of (O, N, N, O) tetradentate bis-Schiff ligands in literature[9] (Figure 2).

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Figure 2: Structures of two salen-type Schiff-base ligands.

Biological applications

Recently, a number of researchers are expressing keen interest and are working in the field of functionalizing metal complexes with biomolecules and nanomaterials for therapeutic applications. The nano-functionalized metal complexes of Schiff-base ligands have been acted towards targeted delivery and are used to demonstrate the broad range of opportunities and challenges of this promising approach[9-12].

As antibacterial agent

Metal complexes of Schiff base derived from 2-thiophene carboxaldehyde and 2-aminobenzoic acid (HL) and Fe(III) or Co(II) or Ni(II) or UO2(II) showed a good antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus pyogenes. Fe(III), Cu(II), Zn(II) and UO2(II) complexes caused inhibition for E. coli. The importance of this lies in the fact that these complexes could be applied fairly in the treatment of some common diseases caused by E. coli. However, Fe(III), Co(II), Cu(II), Zn(II) and UO2(II) complexes were specialized in inhibiting Gram-positive bacterial strains (S. pyogenes and P. aeruginosa). The importance of this unique property of the investigated Schiff base complexes lies in the fact that, it could be applied safely in the treatment of infections caused by any of these particular strain[13]. Tetra and hexa co ordinate metal chelate complexes with Schiff base ligands were found to possess remarkable bacterial properties, it is however interesting that the biological activity gets enhanced on undergoing complexation with the metal ion[13].

Neutral tetradentate complexes of transition metals with Schiff bases derived from 2-aminophenol/2-aminothiophenol and 1-phenyl-2,3-dimethyl-4(4-iminopentan-2-one)-pyrazol-5-one showed antimicrobial activity against Staphylococcus aureus, Bacillus Subtilis, Salmonella typhi, Aspergillus niger and Trichoderma Viride. Most of the complexes have higher activity than the free ligands[14]. Complex of transition metal with Schiff base derived from 2,3-dihydrazinoquinoxaline (DHQ) showed antimicrobial activity. Preliminary testing of the ligand and metal complexes for antimicrobial activity on the Gram positive S. aureus and Gram negative E. coli shows that the ligand is active only against S. saureus and the activity enhanced by complexation. The metal complexes exhibit more bacteriostatic activity against E. coli. The appearance of activity may be due to synergetic mechanism[15].

A tridentate Schiff base formed from the condensation of s-benzyldithiocarbamate with salicylaldehyde and transition metal complexes showed significant bioactivity against Pseudomonas aeruginosa (Gram-negative) and bacillus cereus (Gram positive) while the uranium analogue was effective against Bacillus cereus and showed very weak activity against Candida albicans fungi [ 16 ]. A series of novel Schiff base derivatives with different substituent were screened were antibacterial activity against S. aureus and these compounds were found to show a significant antibacterial activity [ 17 ].

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Figure 3: Mn(II)complex of 5-aminosalicylic acid.

Soliman and his co-workers have synthesized Mn(II) complex with 5-aminosalicylic acid and examined its antibacterial activity in E. coli, E. subtilis etc. The experimental results showed that the metal complex (Figure 3) exhibits higher activity than the free ligand 5-amino-salicylic acid[18]. Transition metal complexes of Mn(II) with 2-acetyl thiophene thiosemicarbazone having general composition [MnL2X2] (Figure 4) had been synthesized. The spectral studies of the ligand and complex confirms the bidentate nature of the ligand and forms hexacoordinated Mn(II) complexes with octahedral geometry. It was also established that it shows considerable antibacterial activity [ 19 ].

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Figure 4: Mn(II)complex of 2-acetylthiophene thiosemicarbazone.

As antifungal agent

Metal complexes Cu(II), Co(II), Ni(II) and Mn(II) are synthesized with Schiff bases derived from o -phthalaldehyde and amino acids viz., glycine L-alanine, L-phenylalanine, then tested against three fungi. It was clear that Cu(II) and Ni(II) complexes exhibit inhibition towards all the studied microorganisms. However, Co(II) and Mn(II) complexes exhibit less inhibition and VO(II) complexes have no activity towards the microorganisms[20]. From the antifungal screening data it is concluded that the activity of Co(II) complexes have shown better antifungal activity compared to the ligand and the corresponding metal salts[21].

The ligands hydrazine and carbothioamide[22] and their metal complexes show antifungal activities against A. alternata and H. graminicum. Ruthenium(II) complexes [ 23 ] with Schiff base salicylamine, thalium(I) complexs[24 ] with benzothiazothiazolines, copper(II) complexes [ 25 ] with benzoylpyridine Schiff base show antifungal activities. In addition, oxovanadium(IV)complexes [ 26 ] with triazole shows antifungal activity.

Complexes of Mn(II) had been synthesized with macrocyclic Schiff’s base ligand 2,3,9,10-tetraketo-1,4,8,11-tetraazacycoletradecane (Figure 5). The ligand was derived by the condensation of diethyloxalate and 1,3-diamino propane and characterized by various spectrochemical techniques[27]. On the basis of spectral studies an octahedral geometry had been assigned. The antifungal activity of the ligand and its metal complexes were evaluated against some fungi species i.e ., F. odum, A. niger and R. bataticola which gave significant results.

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Figure 5: Mn(II) complex with2,3,9,10-tetraketo-1,4,8,11-tetraazacycoletradecane.

As anticancer agent

In recent years, various Schiff base derivatives have been found to be associated with anticancer properties. Some Schiff bases [ 28] and their metal complexes containing Cu, Ni, Zn and Co were synthesized from salicylaldehyde, 2,4-dihydroxybenzaldehyde, glycine and L-alanine that possess antitumour activity and their order of reactivity with metal complexes was found to be Ni>Cu>Zn>Co. Amino Schiff bases [ 29 ] derived from aromatic and heterocyclic amine possess high activity against human tumour cell lines. Aryl–azo Schiff base[30 ] exhibit anticancer activity.

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Figure 6: Proposed structure of the Schiff base complexes exhibiting anticancer activity where X=Cl-,NO3-,NCS- ; M=Sm(III),Gd(III),Dy(III)[1].

As antioxidant

The search for metal-derived antioxidants has received much attention and effort in order to identify the compounds having high capacity in scavenging free radicals related to various disorders and diseases associated with oxidative damage, caused by reactive oxygen species (ROS)[1]. Presently, synthetic antioxidants are widely used because they are effective and cheaper than natural antioxidants. Currently a number of Schiff-base metal complexes have been investigated as effective scavengers of ROS, acting as antioxidants.

The antioxidant capacities of ferrocenyl Schiff bases including o -(1 - ferrocenylethylideneamino)phenol (OFP), m -(1ferrocenylethylideneamino)phenol (MFP) and p -(1-ferrocenylethylideneamino)phenol (PFP) were evaluated by Li and his co-workers in 2,2/-azo bis (2-amidinopropane hydrochloride) (AAPH), Cu2+/glutathione (GSH) and hydroxyl radical (OH-) induced oxidation of DNA. All the ferrocenyl Schiff bases employed herein behaved as prooxidants in Cu2+/GSH and OH- induced oxidation of DNA except that OFP exhibited weak antioxidant activity in OH- induced oxidation of DNA. PFP, OFP and MFP can terminate about 15.2, 11.3, and 9.4 radical-chain propagations in AAPH-induced oxidation of DNA. Especially, the introduction of ferrocenyl group to Schiff base increased the antioxidant effectiveness more remarkably than benzene related Schiff bases[31 ].

As antiviral agent

Schiff bases of gossypol [ 32 ] show high antiviral activity. Silver complexes [ 33 ] in oxidation state(I) showed inhibition against Cucumber mosaic virus; glycine salicylaldehyde Schiff base Ag(I) [ 33 ] gave effective result upto 74% towards C. mosaic virus. A new series of 3-(benzylideneamino)-2-phenylquinazoline-4(3H)-ones were prepared through Schiff base formation of 3-amino-2-phenyl quinazoline-4(3)-H-one with various substituted carbonyl compounds. Their chemical structures were elucidated by spectral studies. Cytotoxicity and antiviral activity were evaluated against herpes simplex virus-1 (KOS), herpes simplex virus-2 (G), vaccinia virus, vesicular stomatitis virus, herpes simplex virus-1 TK-KOS ACVr, para influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, feline corona virus (FIPV), feline herpes virus, respiratory syncytial virus, influenza A H1N1 subtype, influenza A H3N2 subtype, and influenza B virus. The synthesized compound showed better antiviral activity against the entire tested virus [ 34 ].

As anti-inflammatory agent

Schiff base derived from 4-aminoantipyrine (4-amino-1,5-dimethyl-2-phenylpyrazole-3-one) and benzaldehyde derivative was tested for its anti-inflammatory property. The results showed promising anti-inflammatory activity which could be beneficial for use in the treatment of inflammatory diseases. The results of this study may lead to the development of a new therapeutic agent useful in fighting diseases caused by oxidative stress and inflammation [ 35 ].

Conclusion

Schiff bases are considered as a very important class of organic compounds because of their ability to form complexes with transition metal ion and of their pharmacological properties. Transition metal complexes containing Schiff bases have been of much interest over the last few years largely because of its various application in biological, food industry, dye industry, analytical chemistry, catalysis, agrochemical processes and potential application in designing new therapeutic agents. But still there is a need to explore the biological properties of these synthesized transition metal complexes and to synthesize new complexes with more properties. With proper designing and structure activity relationship, Schiff base compounds with a good therapeutic activity can be synthesized. Transition metal complexes of Schiff base ligands appear to be a new future medicinal candidate.

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Chapter 2
Liver: Its Threats and Herbal Cure

Dr. Munmi Borkataky

Introduction

The human body is culmination of a complex metabolic processes governed by different but specific organs working in sync to sustain the human life. Liver occupies a central position in this complicated interlace of various biochemical and physiological processes. Liver is appropriately referred to as the ‘metabolic hub’1owing to its involvement in virtually all biochemical pathways relating to energy production, growth and homeostasis. On account of its participation in intense metabolic activities, liver is ceaselessly exposed to endogenous and exogenous toxins, xenobiotics and oxidative stress. Consequently, liver is the primary target of toxins introduced into the body either intentionally (e.g. Ethanol) or generated during the metabolism of drugs (e.g. Paracetamol). Since a healthy liver is essential for the overall well-being of an individual, liver injury often proves lethal. Liver disorders like- hepatitis, cirrhosis, fatty liver etc. are recognized as major clinical problems since many years. Although the predominance of different types of liver diseases is subject to variation based on region, dietary habits and life-style, the treatment options for the common liver diseases are limited and debatable.

Chronic liver diseases are prevalent worldwide and are resultant of long-term exposure to toxic chemicals, drugs, alcohol and viral infection2,3. Repetitive hepatic injury leads to chronic inflammation associated with dynamic healing response, ultimately leading to cirrhosis and other life threatening complications4. In the absence of an effective pharmacological therapy for such pathological processes, the liver diseases are claiming the lives of millions worldwide. The current pharmacopoeia has little to offer to the patients of hepatic disorders. The effectiveness of treatments such as interferons, colchicines, penicillamine and corticosteroids are inconsistent and often have side-effects associated with them5. As a result, the search of alternatives for the treatment of liver diseases has increased all over the world6. With various works highlighting the role of oxidative stress in liver cirrhosis, antioxidants have been proposed as a treatment for cirrhosis2. Several studies have demonstrated the protective effects of antioxidants against induced liver injury by reducing oxidative stress in cells7,8. A number of herbal products antioxidant activities, for example Silymarin, have effectively been used for the treatment of liver diseases9. The ancient science of medicine- Ayurveda and other traditional systems report a large number of plants that may prove effective to meet up to the challenge of treating hepatic diseases10. Approximately, 160 phytoconstituents from 101 plants have been claimed to possess liver protective activities. In India, 87 plants are used in 33 patented proprietary multi-ingredient formuations which have been shown to possess hepatoprotective activity11,12,13. Silybum marianum, Piccorrhiza kurroa, Schizandra chinensis and Phyllanthus niruri are some of the well-known examples of plants that have been used as anti-hepatotoxic drugs since ancient times14. A large number of other plants and formulations have been claimed to exhibit hepatoprotective effects. Therefore, it becomes important to evaluate and validate such claims so that a safe and reliable drug may be available for the treatment of liver disorders.

Functions of Liver

The liver performs many different functions and is known as ‘metabolic hub’ of the human body. Many of its functions are interrelated with one another and this fact becomes evident especially during liver disorders when many of its functions are disturbed simultaneously. The major functions of liver include- filtration and storage of blood, metabolism of carbohydrates, proteins, fats, hormones, and foreign chemicals, formation of bile, storage of vitamins and iron, and formation of coagulation factors15.

The Liver as a Blood Reservoir

Liver is an expandable organ and large quantities of blood can be stored in its blood vessels. Its normal blood volume is about 450 ml, which is almost 10 per cent of the body's total blood volume. Occasionally, the hepatic veins and sinuses may also store 0.5 to 1 liters of extra blood. This occurs especially in case of cardiac failure with peripheral congestion. Thus, liver is capable of acting as a valuable blood reservoir in times of excess blood volume and capable of supplying extra blood in times of diminished blood volume.

Metabolic functions of Liver

The liver is a large; chemically reactive pool of cells that have a high rate of metabolism and which synthesize multiple substances that are transported to different parts of the body where they perform myriad of other metabolic functions.

Carbohydrate metabolism

The liver performs the following functions in carbohydrate metabolism:

1. Synthesis and storage of glycogen
2. Conversion of galactose and fructose to glucoseillustration not visible in this excerpt
3. Gluconeogenesis

The liver performs an important function in the maintenance of normal blood glucose levels. Liver converts excess glucose in the blood to glycogen and stores it. When the blood glucose concentration falls below normal, liver breaks down glycogen to glucose and returns it to the blood. This homeostatic function of liver is termed as the glucose buffer function.

Gluconeogenesis in the liver is also important in maintaining normal blood glucose levels. Gluconeogenesis occurs when the glucose concentration falls below normal. In such a case, large amounts of amino acids and glycerol from triglycerides are converted into glucose, thereby maintaining a relatively stable blood glucose concentration15.

Fat metabolism

Although most cells of the body metabolize fat, liver performs some of the vital functions in fat metabolism. Specific functions of liver in fat metabolism include:

1. Oxidation of fatty acids to supply energy for other body functions
2. Synthesis of cholesterol, phospholipids and lipoproteins
3. Synthesis of fat from proteins and carbohydrates

Liver splits neutral fats into glycerol and fatty acids; and then the fatty acids are metabolized by beta-oxidation into two-carbon acetyl radicals that form acetyl coenzyme A (acetyl-CoA). Acetyl-CoA then is oxidized in the citric acid cycle to liberate tremendous amounts of energy. Although beta-oxidation can take place in all cells of the body, it occurs especially rapidly in the hepatic cells. The liver itself cannot use all the acetyl-CoA that is formed; and converts it into acetoacetic acid which is highly soluble and is transported throughout the body to be absorbed by other tissues. These tissues reconvert the acetoacetic acid into acetyl-CoA and then oxidize it in the usual manner. Thus, the liver is responsible for a major part of the metabolism of fats.

About 80 per cent of the cholesterol synthesized in the liver is converted into bile salts and the other 20% is transported as lipoproteins by blood to the other parts of the body. Similarly, phospholipids are synthesized in the liver and are also transported in the lipoproteins. Both cholesterol and phospholipids are used by the cells to form membranes, intracellular structures, and multiple chemical substances that are important to cellular function15.

Protein metabolism

The liver performs the following important roles in protein metabolism:

1. Deamination of amino acids
2. Formation of urea for removal of ammonia from the body fluids
3. Formation of plasma proteins
4. Inter-conversion of the various amino acids and synthesis of other compounds from amino acids

Deamination of amino acids occurs in liver prior to their conversion into carbohydrates or fats or energy production. Formation of urea by the liver removes ammonia from the body fluids. Excessive ammonia in the blood results in extremely toxic conditions.

Almost all the plasma proteins are formed by the hepatic cells and accounts for about 90 per cent of all the plasma proteins. The liver can form plasma proteins at a maximum rate of 15 to 50 g/day. The synthesis of certain amino acids and certain important metabolic intermediates from amino acids constitutes one of the other important functions of liver.

Liver Diseases

Fatty liver disease (FLD) is a reversible condition developed due to the formation of large vacuoles of triglyceride fats in liver cells by the process of steatosis (i.e. abnormal retention of lipids within a cell). Fatty liver disease occurs primarily due to excessive alcohol intake and obesity and is associated with diseases that influence fat metabolism. Alcoholic FLD and non-alcoholic FLD are difficult to distinguish morphologically and both exhibit micro-vesicular and macro-vesicular fatty changes at different stages. Changes associated with this condition include intra-cytoplasmic accumulation of triglyceride (neutral fats) in the form of small fat vacuoles (liposomes) around the nucleus (micro­-vesicular fatty change) of the hepatocytes. At this initial stage, liver cells are filled with multiple fat droplets that do not displace the centrally located nucleus but during the later stages push the nucleus to the periphery of the cell. These vesicles are well delineated and optically "empty" because the fats dissolve during the processing of tissue. Large vacuoles may coalesce and produce fatty cysts which are irreversible lesions. Macro-vesicular steatosis is the most common form and is typically associated with alcohol, diabetes, obesity and corticosteroids16.

Viral hepatitis is an inflammation of the liver caused by one of the five hepatitis viruses: A, B, C, D and E. These are transmitted through via different routes- Hepatitis A and E through contaminated food and water; Hepatitis B – through unsafe blood and other bodily fluids; Hepatitis C – mostly through infectious blood; and Hepatitis D – serving as an additional infection in the presence of Hepatitis B. These viruses all cause acute hepatitis which is characterized by fatigue, loss of appetite, fever and jaundice17. Acute hepatitis seldom results in death and most patients recover fully. In addition, hepatitis B and C infections can become chronic, thus leading to cirrhosis and liver cancer. The hepatitis infections are present throughout the world. Although data on the prevalence of infection are still not available for many countries; however, an estimate states that half a billion people are chronically infected with hepatitis B or C virus. Such chronic infections are responsible for an estimated 57% of cases of liver cirrhosis and 78% of cases of primary liver cancer17.

Cirrhosis is the end-stage of most liver diseases. The term- Cirrhosis was originally used to refer to a particular pattern of scar tissue in the liver but has now come to be used to describe the consequences of this heavy scarring of the liver. These consequences include liver failure, as a result of replacement of functioning liver tissue by scar tissue, and an increase in the pressure in the veins leading into the liver (portal hypertension) as a result of destruction of blood vessels within the liver. Cirrhosis can be completely silent clinically, with no features on radiology or on blood tests. As a result, many patients with cirrhosis are not diagnosed until complications develop. The most common complications of cirrhosis are internal bleeding from distended veins in the esophagus or stomach (bleeding varices), or an accumulation of fluid in the abdomen (ascites), or behavioral changes (hepatic encephalopathy). All patients with cirrhosis are also at risk for the development of liver cancer (hepatocellular carcinoma). This occurs at a rate of between 1-8% per year, depending on the severity of the underlying liver disease. Liver cancer is also silent for much of its development. Thus, with both the predisposing cirrhosis and the developing cancer being silent, most patients will only come to medical attention with the development of symptoms of liver failure or with cancer symptoms, such as weight loss.

The incidence and prevalence of liver-related diseases described above has increased and also has the number of deaths. The current facilities available to manage end-stage liver diseases are barely enough, and are likely to become completely overwhelmed in near future. The only treatment for end-stage liver diseases is liver transplantation, and the magnitude of the situation can be gauged by the fact that only in 8% transplants annually are accomplished successfully18. Therefore, liver transplantation is not the ultimate solution to chronic liver diseases.

Currently, no effective treatment is available in medical sciences to counteract the deleterious hepatic disorders. There are no specific allopathic medicines for use as hepatoprotective agents, though different research works are going in this regard19. Herbal drugs are more widely used than allopathic drugs as hepatoprotective agents because they are inexpensive, have better cultural acceptability, better compatibility with the human body and minimal side effects. Nearly 150 phytoconstituents from 101 plants have been claimed to possess liver protecting activity19. At the same time, surprisingly, there are no readily available satisfactory plant drugs or formulations to treat severe liver diseases. Therefore, many folk remedies from plant origin are being tested for their potential hepatoprotective liver damage in experimental animal model. Silymarin, which is one of the plant derived medicines, has been approved for use in liver cirrhosis and alcoholic liver diseases. The efficacy of silymarin for recovering hepatic damage has been established in a number of studies20,21,22,23. Silymarin is a mixture of flavono-lignans from the fruits of Silybum marianum that has been known since ancient times and recommended in traditional European and Asian medicines primarily for the treatment of liver disorders24,25. Hepatoprotective activity of ethanol and aqueous extracts of Momordica dioica Roxb. leaves were evaluated against carbon tetrachloride (CCl4) induced hepatic damage in rats and similar results were reported26.

Research on Plants which can be used in liver disorders

One of the traditionally used plants of East, Central, and West Africa the baobab (Adansonia digitata Linn.) has been reported for its hepatoprotective activity against Carbon tetrachloride (CCl4) induced toxicity in rats. The pulp of the plant has been shown to exhibit significant hepatoprotective activity and the consumption of the fruit has been reported to play an important role in human resistance to liver damage27,28,29. Natanzi et al. (2009) reported the hepatoprotective activity of Nasturtium officinale (watercress) against acetaminophen-induced hepatic damage30. Similar study on Scoparia dulcis L. against carbon tetrachloride-induced acute liver injury in mice showed that the plant possesses potential hepatoprotective activity, which was attributed to its free radical scavenging potential due to the presence of terpenoids31. Hepatoprotective activity was also observed to be significantly higher in Eclipta alba (L.) Hassk . in CCl4 induced hepatic damage. The administration of plant extracts was observed to significantly restore the levels of hepatic marker enzymes, total proteins and antioxidant enzymes in the hepatotoxin treated groups which may be considered as an evidence of the hepatoprotective activity of the plants under study32. In vitro study by Gnanasekaran et al. (2012 a)and Gnanasekaran et al. (2012 b) showed that the whole plant extracts of Indigofera tinctoria and Sida cordata exerted positive hepatoprotective activity on the normal human liver33,34. Beta vulgaris Linn.root is consumed as a vegetable in many parts of the world. The leaves of the plant have been reported to exert hepatoprotective action against ethanol-mediated hepatotoxicity35. Zeashen et al. (2009), studied hepatoprotective and antioxidant activity of ethanolic extract of whole plant of Amaranthus spinosus L. against carbon tetrachloride (CCl4) induced hepatic damage in rats. The results of this study strongly indicate that whole plants of Amaranthus spinosus have potent hepatoprotective activity36. Srinivasa et al.(2010) has reported the methanolic extract of whole plant of Amaranthus spinosus L . for hepatoprotective activity against paracetamol induced liver damage in wistar rats. Presence of compounds such as amino acids, flavonoids and phenolic compounds in the methanolic extract was responsible for its chemoprotective and antioxidant activities against paracetamol induced liver damage in wistar rats37. Hepatoprotective activity of ethanol and aqueous extracts of Momordica dioica Roxb. Leaves were evaluated against carbon tetrachloride (CCl4) induced hepatic damage in rats and similar results were reported26. Souza et al. (2007) studied the aqueous and alcoholic extract of fruit pulp of Litchi chinensis for hepatoprotective activity using carbon tetrachloride induced hepatotoxicity and reported that both alcoholic and aqueous extracts exhibited significant hepatoprotective activity38. Vidhya Malar and MettildaBai (2009) studied the efficacy of the medicinal plant Phyllanthus emblica in preventing paracetamol induced hepatotoxicity in rats. Treatment with aqueous extract of fruits of P. emblica showed the appearance of normal hepatocytes, offset of necrosis and consequent appearance of leucocytes thus suggesting the hepatoprotective effect of this medicinal plant39. Another compound Glycyrrhizin significantly inhibits the CCl4- induced release of aspartate aminotransferase (AST) and Lactate dehydrogenase (LDH) by altering the membrane fluidity was isolated from important medicinal plant Glycyrrhiza glabra Linn40. Hepatoprotective activity of the n-heptane extract of Cassia fistula L . leaves was investigated in rats by inducing hepatotoxicity with carbon tetrachloride:liquid paraffin (1:1). The extract has been shown to possess significant protective effect by lowering the serum levels of transminases- serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase (SGOT and SGPT), bilirubin and alkaline phosphatase (ALP)41. The effects of Solanum nigrum extract was evaluated on thioacetamide (TAA)-induced liver fibrosis in mice and it was found that Oral administration of extract significantly reduces TAA-induced hepatic fibrosis in mice. In other study, the protective effects of aqueous extract of Solanum nigrum) against liver damage were evaluated in CCl4 - induced chronic hepatotoxicity in rats. The results showed that the treatment of the extract significantly42. Some other plants which are also proved to have significant hepatoprotective activity are Curcuma longa L., Daucus carota L., Moringa oleifera Lam., Bauhinia variegata L., Canna indica L., Ficus carica L, Asparagus racemosus Willd , Spondias pinnata (L.f) Kurz . , Ricinus Communis L ., Ocimum sanctum, Casuarina equisetifolia L. , Cajanus cajan L ., Glycosmis pentaphylla (Retz.)DC., Bixa orellana L ., Argemone Mexicana L ., Physalis minima L ., Caesalpinia bonduc (L.). Roxb . etc. 41,42,43,44.

Conclusion

The role of medicinal plants in curing various liver diseases caused by toxic metabolites is of very much essence in modern life. But solely finding new prototype of pure compounds as drugs may not be that effective and hence active extracts or fractions or combination of fractions may prove to be very effective for therapeutic use. Plant extract therefore possesses sufficient efficacy to curb many liver diseases caused by toxic chemicals, excess alcohol intake, viruses etc. Therefore a deliberate effort towards developing drugs using medicinal plants should be focused by standard protocol adopting proper pharmacological experiments and clinical trials. This approach could go a long way in finding new drugs for therapeutic use of indigenous medicinal plants for liver disorders.

References

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3. Vitaglione P,Morisco F, Caporaso N, Fogliano V (2004) Dietary antioxidant compounds and liver health.Crit.Rev.Food Sci.Nutr 44:575-586.

4. Lotersztajn S, Julien B, Teixeira-Clerc F, Grenard P, Mallat A (2005). Hepatic fibrosis: molecular mechanisms and drug targets . Annu.Rev.Pharmacol.Toxicol 45: 605-628.

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6. Girish C, Pradhan SC (2008) Drug development for liver diseases: focus on picroliv, ellagic acid and curcumin. Fundam Clin Pharmacol 22: 623-32.

7. Bansal AK, Bansal M, Soni G, Bhatnagar D (2005) Protective role of Vitamin E pre-treatment on nitrosodiethylamine induced oxidative stress in rat liver. Chem Biol interact 156: 101-111.

8. Cederbaum AI, Lu Y, Wu D (2009) Role of oxidative stress in alcohol-induced liver injury. Arch.Toxicol 83: 519–548.

9. Stickel F, Schuppan D (2007) Herbal medicine in the treatment of liver diseases. Dig Liver Dis 39: 293–304.

10. Chatterjee TK (2000) Medicinal Plants with Hepatoprotective Properties.Herbal Options.3rd edn.Books and Applied Allied (P) Ltd.; Calcutta: 135- 143.

11. Khin M, Nyunt N, and Maung T (1978) The protective effects of Eclipta alba on carbon tetrachloride Induced Acute Liver Damage. Toxicol.and Applied Pharmacol 45:723-728.

12. Handa SS, Sharma A, Chakraborti KK (1986) Natural products and plants as liver protecting drugs. Fitoterapia 57: 307-352.

13. Tabassum N, Agrawal, SS (2004) Hepatoprotective Activity of Eclipta alba Hassk.Against Paracetamol Induced Hepatocellular Damage in Mice. Experimental Medicine 11: 278-280.

14. Trivedi PC (2006) Medicinal plants: Ethnobotanical Approach.1st edn.Agroboios (India); Jodhpur 342002: 399.

15. Guyton C and Hall JE (2006) Textbook of Medical Physiology.11th edn.Elsevier Saunders, Philadellphia; Pennsylvania: 1116.http://www. studentconsult. com/content/default. cfm.

16. Sharma N, Rana AC, Kumar S, Singh R (2012) Different models of hepatotoxicity and related liver diseases: A review.InternationalResearch Journal of Pharmacy 3: 86–95.

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18. Tarba R (2013) An assessment of liver disease in Canada. Canadian Liver Foundation; Ontario: 70.

19. Rao BG, Rao YV, Rao TM. (2012) Hepatoprotective activity of Spillanthes acmella Extracts against CCl4-induced liver toxicity in rats.Asian Pacific Journal of Tropical Disease 1: S208-S211.

20. Saraswat B, Visen PKS, Patnaik GK, Dhawan BN (1993). Anticholestic effect of picroliv, active hepatoprotective principle of Picrorhiza kurrooa, against carbon tetrachloride induced cholestatis. Indian J Exp Biol 31:316-318.

21. Subramonium A, Pushpangadan P (1999) Development of phytomedicines for liver diseases. Indian J Pharmacol 31: 166- 175.

22. Sherlock S, Dooley J (2002). Diseases of liver and biliary system. 11th edn.Oxford: Blackwell Scientific Publications; 322-356.

23. Salam OMEA, Sleem AA, Omara EA, Hassan NS (2009). Hepatoprotective effects of misoprostol and silymarin on carbon tetrachloride-induced hepatic damage in rats. Fundamental & Clinical Pharmacology 23: 179-88.

24. Zlatkis A, Zak BB, Boyle GJB (1953) A colorimetrical method for determination of cholesterol. J Lab Clin Med 41:486-496.

25. Deshmukh RD, Pokharkar RD, Takate SB, Gite VN (2007) Amelioration of CCl4-induced Hepatosuppression by Nyctanthes arbortristis Linn.leaves in Wistar Albino Rats. Pharmacologyonline 1: 203-208.

26. Jain A, Soni M, Deb L, Jain A, Rout SP, Gupta VB, Krishna KL (2008) Antioxidant and hepatoprotective activity of ethanolic and aqueous extracts of Momordica dioica Roxb.leaves. J Ethnopharmacol 115: 61-66.

27. Beckier B (1983) The contribution of wild plants to human nutrition in the Ferlo (Northern Senegal). Agroforestry-Systems 1: 257-267.

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29. Al-Qarawi AA, Al-Damegh MA, El-Mougy SA (2003) Hepatoprotective Influence of Adansonia digitata Pulp. Journal of Herbs, Spices & Medicinal Plants 10:1-6.

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31. Praveen TK, Dharmaraj S, Bajaj J, Dhanabal SP, Manimaran S, Nanjan MJ, Razdan R (2010) Hepatoprotective activity of petroleum ether, diethyl ether, and methanol extract of Scoparia dulcis L.against CCl4-induced acute liver injury in mice. Indian J Pharmacol 41: 110-114.

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34. (b) Gnanasekaran D, Umamaheswara Reddy C, Jaiprakash B, Narayanan N, Hannah Elizabeth S, Ravi Kiran Y (2012) In vitro hepatoprotective activity of a selected siddha medicinal plant Sida cordata using chang liver cells. American Journal of PharmTech Research 2: 466-473.

35. Jain N K, Singhai AK (2012) Protective Role of Beta vulgaris L.leaves extract and Fractions on Ethanol-Mediated Hepatic Toxicity. Drug Research 69: 945-950.

36. Zeashen H, Amresh G, Singh S and Rao CV (2009) Hepatoprotective and antioxidant activity of Amaranthus spinosus against CCL4 induced toxicity. J Ethnopharmacol 125: 364-366.

37. Srinivasa B, Ashok kumar BA, Lakshman K, Jayaveera KN, Shekar DS, Nandeesh R, Velmurugan C (2010) Chemoprotective and antioxidant activities of methanolic activities of Amaranthus spinosus leaves on paracetamol induced liver damage in rats. Acta Medica Saliniana 39: 68-74.

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40. Damle M (2014) Glycyrrhiza glabra (Liquorice) - a potent medicinal herb. International Journal of Herbal Medicine 2: 132-136.

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43. Hari Kumar C, Ramesh A, Suresh kumar JN, Mohammad Ishaq B (2011) A Review on Hepatoprotective activity of Medicinal Plants. International Journal of Pharmaceutical sciences and Research 2: 501-515.

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Chapter 3
Phytochemical studies and traditional use nature’s most valuable plant of Bael (Aegle marmelos) in Assam

Dr. Monmi Saikia, and Bidanshree Basumatary

Introduction

From the early days mankind is using plant source to alleviate or cure illnesses. Plants constitute a source of novel chemical compounds which of potential use in medicine and other application. Alkaloids, steroids, tannins, glycosides, volatile oils, fixed oils, resins, phenols and flavanoids are present as active components in plant, which are deposited in their specific parts such as leaves, flowers, bark, seeds, fruits, roots, etc [1]. World is bestowed with a rich wealth of medicinal plants. Man cannot survive on this earth for long life without the plant kingdom because the plant products and their active constituents played an important role [2]. Plants have been utilized as a natural source of medicinal compounds since thousands of years and human is using numerous plants and plant derived products to cures and relief from various physical and mental illness [3]. Photochemicals (a Greek word: phyto means plants) are biologically active, naturally occurring chemical compounds found in plants, which provide health benefits for humans further than those attributed to macronutrients and micronutrients. They protect plants from disease and damage and contribute to the plant’s color, aroma and flavour. In general, phytochemicals are the plant chemicals that protect plant cells from environmental hazards such as pollution, stress drought, UV exposure and pathogenic attack; where they accumulate in different parts of the plants, such as in the roots, stems, leaves, flowers fruits or seeds [4].

Phytochemicals are not essential nutrients for the human body for sustaining life but have important properties to prevent or to fight some common diseases. Many of these benefits suggest a possible role for phytochemicals in the prevention and treatment of many diseases [4]. A. marmelos is a native plant of India, belongs to Rutaceae family and commonly known as wood apple. In India, A. marmelos is grown as a temple garden plant and the leaves are used to pray Lord Shiva. A. marmelos is an important medicinal plant with several ethnomedicinal applications in traditional and folk medicinal systems [3]. India is a treasure trove of aromatic and medicinal plants. In recent days medicinal plants play a major role as pillar of traditional healthcare systems of medicine in many developing countries like India. Since from the ancient times, different drugs have been formulated using the bioactive compounds present in these medicinal plants. More than 60% of the world’s population depends on phytomedicines derived from these medicinal plants for primary health care needs. The Phytoconstituents from these medicinal plants serve as lead compounds in the modern era in drug discovery and design.

Many of the researchers have validated the pharmacological importance of different parts of Aegle marmelos which includes antioxidant, free radical scavenging antibacterial, antiviral, anti-diarrheal, hepatoprotective, anti-diabetic, cardioprotective, gastroprotective, anti-ulcerative colitis and radioprotective effects. As at present scenario, only a few articles are available on the phytochemical and pharmacological values of fruit pulp of A. marmelos, here we overview the therapeutic use of the fruit as well as to summarize the different bioactive compounds present in the fruit pulp of the plant which contributes to its medicinal properties in curing different aliments [5].

Physical description of the plant

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Plant profile

Scientiic classification

Kingdom- Plantae

Order- Sapindales

Family- Rutaceae

Subfamily- Aurantioideae

Genus- Aegle

Species- Aegle marmelos

Botanical name- Aegle marmelos

Aegle marmelos is a slow-growing, medium sized tree, up to 12-15 meter tall with short trunk, thick, soft, flaking bark, and spreading, sometimes spiny branches in which the lower ones drooping. Young branches bear many stiff and straight spines. The deciduous and alternate leaves are composed of 3 to 5 oval, pointed and shallowly toothed leaflets, 4-10 cm long, 2-5 cm wide, the terminal one with a long petiole [7]. Bael tree is generally considered as sacred tree by the Hindus, as its leaves are offered to lord Shiva during worship.

Phytochemicals of the plant

From early days people have given importance on medicinal the plants and herbs in overcoming disease. Bael is reported to have number of coumarins, alkaloids, steroids, and essential oils as chemical constituents. Root and fruits of bael have coumarins such as scoparone, scopoletin, umbelliferone, marmesin and skimming. In addition to this fruits contain xanthotoxol, imperatorin and alloimperation and alkaloids like aegeline and marmelline. Polysaccharides like galactose, arabinose, uronic acid and L -rahaminose, which may obtain after hydrolysis may present in bael. Due to the presence of different types of cerotenoids in the Aegle marmelos as reported are responsible for the imparting yellow pale colour to fruit. Minor constituents are like ascorbic acid, sitosterol, crude fibres, tannins, α -amyrin, carotenoids and crude proteins are also resent. Apart from these chemical constituents more than 100 compounds have been isolated these are γ-sitosterol, aegelin, lupeol, rutin, marmesinin, β -sitosterol, flavone, glycoside, O-isopentenylhalfordiol, marmeline and phenylethyl cinnamamides, skimminine, aegelin, lupeol, cineole, citral, citronellal, cuminaldehyde, eugenol, marmesinin, marmelosine, luvagetin, aurepten, psoralen, marmelide, fagarine, marmin, and tennins have been proved to be biologically active against various major and minor disease [8].

Nutritional values of Aegle marmelos

The fruit of A. marmelos possess high nutritional value. The fruit is used to make juice, jam, syrups, jelly, toffee and other products. The pulp is reported to contain water, sugars, protein, fibre, fat, calcium, phosphorous, potassium, iron, minerals and vitamins (vitamin A, Vitamin B, vitamin C and Riboflavin) [3].

Pharmacological studies

In recent history this plants is reported for various medicinal properties [3].

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Antimicrobial Activity

A. marmelos has been traditionally used for the treatment of various infectious diseases and been reported to inhibit the broad range of pathogenic microorganisms where many in vitro studies proved the antimicrobial potential of A. marmelos extracts towards the pathogenic microorganisms including bacteria and fungi. The antimicrobial activity of the leaves of Aegle marmelos was reported by various authors. It was found that the petroleum ether extract of leaves was checked against multi resistant strains of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi, Proteus vulgaris, Pseudomonas aeruginosa and Klebsiella pneumoniae. It was observed that the antimicrobial activity against gram-negative strains was higher than that of gram positive strains [9]. The antifungal activity of the leaves of Aegle marmelos was reported against clinical isolates of dermatophytes and A. marmelos leaf extracts and fractions were found to have fungicidal activity against Trichophyton mentagrophytes, T. rubrum, Microsporum canis, M. gypseum, Epidermophyton floccosum [10]. Literature reviewed that the different solvent extracts of Aegle marmelos leaves have antibacterial activity. Different extracts showed antibacterial activity against Micrococcus glutamicus, Streptococcus faecalis, Staphylococcus aureus, S. pyogenes, Bacillus stearothermophilus, Micrococcus luteus, E. coli and Pseudomonas denitrificans; but the Petroleum ether extract did not resulted in any activity while ethanol and chloroform extract exhibits maximum activity [11].

Antidiarrheal Activity

Antidiarrheal activity is one of the important medicinal properties of A. marmelos and traditionally it is widely used to control chronic diarrhea and dysentery. The in vitro antidiarrheal activity of dried fruit pulps of A. marmelos was reported in different solvents; in which the ethanolic extract showed good activity against Shigella boydii, S. sonnei and S. flexneri, moderate against S. dysenteriae [12].

Antidiabetic Activity

Diabetes can be controlled by using A. marmelos in traditional medicinal system. Many in vivo scientific studies have been conducted in animal models to evaluate the ant-diabetic activity of different organic extracts and fresh juice of A. marmelos. All the extracts can reduce the blood sugar level in streptozotozin diabetic rabbits, however, among the various extracts, the methanol extracts of the leaf and callus resulted about the maximum anti-diabetic effect [13].

Cytoprotective Effect

The cytoprotective effect of the leaves of Aegle marmelos was reported in freshwater fish named Cyprinus carpio exposed to heavy metals, where C. carpio was exposed to heavy metals followed by the treatment with the dried powder of Aegle marmelos leaves [14].

Hepatoprotective Effect

The hepatoprotective effect of the leaves of A. marmelos and were reported in alcohol induced liver injury in Albino rats. The induced rats were fed with leaves of A. marmelos for 21 days, which indicates the excellent hepatoprotective effect of the leaves of A. Marmelos [15].

Insect controlling properties

Essential oil extracted from the leaves of A. marmelos was reported for showing insecticidal activity against four stored grain insect pests included Callosobruchus chinensis (L.), Rhyzopertha dominica (F.), Sitophilus oryzae (L.) and Tribolium castaneum. The oil treatment significantly reduced the grain damage as well as weight loss in fumigated grains samples [16].

Analgesic activity

Analgesic activity was reported in leaves of A. marmelos. Methanol extract of leaves of A. marmelos was screened for analgesic activity by Acetic acid-induced writhing test in Swiss mice. In tail flick test methanol extract showed significant analgesic activity in the [17].

Antiarthritis activity

Leaves of A. marmelos were reported to possess anti arthritis activity against collagen induced arthritis in Wistar rats. Methanol extract treatment of rats showed the reduction of paw swelling and arthritic index [18].

Anti-inflammatory activity

Unripe fruit pulp of A. marmelos was reported to possess anti-inflammatory activity.

Extract treatment of the inflammated rats significantly reduced the λ carrageenan induced inflammation [19].

Medicinal uses and traditional uses of Aegle Marmelos

Bael has been used as a folklore medicine since ancient time to cure various human diseases. These fruits reported to be valuable Ayurvedic medicine for chronic diarrhea, tonic for heart and brain, anti-viral activity, hypoglycemic activity, anti-bacterial activity, and against parasites as in. The ripe fruit is aromatic, cooling, alternative, laxative and nutritive. It is useful in habitual constipation, chronic dysentery and dyspepsis in Assam. It also relieves flatulent colic in patients suffering from a condition of chronic gastro intestinal catarrh. From early days ripe fruit mamalade is used as prevention during cholera epidimices in entire Assam. Powder of the dried fruit pulp is used as febrifuge, antiscorbutic, nauseant, stimulant and antipyretic as in. Unripe fruit powder is found to be effective against intestinal parasite Entamoeba histolytica and Ascaris lumberiodes [20].

A. marmelos is traditionally used to treat jaundice, constipation, chronic diarrhea, dysentery, stomachache, stomachic, fever, asthma, inflammations, febrile delirium, acute bronchitis, snakebite, abdominal discomfort, acidity, burning sensation, epilepsy, indigestion, leporsy, myalgia, smallpox, spermatorrhoea, leucoderma, eye disorders, ulcers, mental illnesses, nausea, sores, swelling, thirst, thyroid disorders, tumors, ulcers and upper respiratory tract infections [3].

The medicinal value of Bael fruit is enhanced due to presence of Tanin, the evaporating substance in its rind. The literature reported that rind contains 20% and the pulp has only 9% of Tanin. This substance helps to cure diabetes. Treatment of Asthma it was popularly used from ancient time by grinding 5 gms of Bael leaves with 1 spoon of honey, the mixture is taken orally in morning and evening for relief. Cure of anaemia the extract of pulp of Bael is used. One spoonful dry and grinded powder form is added to boiled cow milk. Some sugar candy is added and if this drink is taken twice a day in morning and evening for a long period, anaemia can be cured completely. In fractures extract of the pulp of raw bael in powder form with pure ghee, turmeric powder and luke warm water should be taken orally twice a day. It is also used in healing of wound. Swollen Joints can be cured by taking few bael pulp mixed with hot mustard oil to be applied on the affected area twice a day during morning and evening. High blood pressure can be controlled by taking bael leaves every morning. The juice of bael leaves added with honey can also be taken every morning. Jundice can be cured using juice of soft bael leaves and black pepper. It is also used in diarrhoea. Troubles during pregnancy can be cured by taking one spoon of raw fruit pulp twice a day, stops frequent vomiting nausea during pregnancy. Little sugar candy may be added to the pulp for taste. Weakness due to typhoid can be cured using bael leave juice with honey. Ripe fruit is taken with fresh cream (butter) and sugar candy powder for healthy mind and brain, which sharpens concentration and intelligence.

Conclusion

A. marmelos which is an important medicinal plant has an effective antioxidant potential also and thus is a good natural antioxidants source. This study highlighted the important medicinal use and chemical constituents present in bael as well as discussed different properties of the plant. Specific studies on detailed chemistry of antioxidant compounds as well as other active compounds present in A. marmelos remains an area with scope for research.

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[11] Rajasekaran, C.; Meignanam, E.; Premkumar, N.; Kalaivani, T.; Siva, R.; Vijayakumar, V.; Ramya, S.; Jayakumararaj, R. Ethnobotanical Leaflets, 2008, 12, 1124-1128.

[12] Joshi, P. V.; Patil, R. H.; Maheshwari, V. L. Natural Product Radiance, 2009, 8, 498-502.

[13] Arumugam, S.; Kavimani, S.; Kadalmani, B.; Ahmed, A. B. A. ; Akbarsha, M. A. ; Rao, M.V. ScienceAsia, 2008, 34, 317-321.

[14] Vinodhini, R.; Narayanan, M. International Journal of Integrative Biology, 2009, 7, 124-129.

[15] Singanan, V.; Singanan, M.; Begum, H. International Journal of Science & Technology, 2007, 2, 83-92.

[16] Kumar, R.; Kumar, A.; Prasa, C. S.; Dubey, N. K.; Samant, R. Internet Journal of Food Safety, 2008, 10, 39-49.

[17] Shankarananth, V.; Balakrishnan, N.; Suresh, D.; Sureshpandian, G.; Edwin, E.; Sheeja, E. Fitoterapia 2007, 78, 258-259.

[18] Trivedi, H. P.; Pathak, N. L.; Gavaniya, M. G. ; Patel, A. K.; Trivedi, H. D.; Panchal, N. M. International Journal of Pharmaceutical Research and Development, 2011, 3, 38-45.

[19] Rao, Cb.V.; Ojha, A.S.K. ; Mehrotra, S. ; Pushpangadan, P. Acta Pharmaceutica Turcica, 2003, 45, 85-91.

[20] Varughese, B.; Tripathi, J. Advances in Life Science and Technology, 2013, 8, 8-12.

Chapter 4
Polyphenols: Its present and future perspective

Dr. Archana Borah

Introduction

There is currently tremendous interest in the studies related to the free radical scavenging properties of plant polyphenols. This lead to the characterization and identification of major phenolic compounds as antioxidant from natural sources. Recent studies showed that there is an immense health benefits of polyphenols which has an inverse correlation with the intake of polyphenols and the occurrence of certain cardiovascular and cancer diseases. It is the established facts that consumption of certain types of food substances assists the body’s repair and interfere the complicated chemical reaction of life processes. The non nutritional exogenous phytochemicals like alkaloids, Flavonoids, terpenes and glycosides have several therapeutic potential. Certain foods are essential for maintaining the nutrition and health in humans. However, foods particularly those of plant origin contain a wide range of non-nutrient phytochemicals which have been found effective against several ailments (Anonymous, 1987). These non nutrients include phenols, Flavonoids, glycosides etc. When a food material serves as a source of nutrients as well as medicine they are termed as neutraceuticals. Thus neutraceuticals are nutritionally enhanced food with health benefits. They act either as enhancers of body’s immune system or play a key role in maintaining the body’s enzymatic defenses against free radicals.

Structure of Phenolic compounds

Phenolic compounds are the secondary metabolites of plants origin. They are generally derived from phenylalanine. The phenolic compounds are classified on the basis of number of carbon atom in the molecules (Harbone and Simmonds, 1964). The structure may vary from simple molecules (eg. Phenolic acids with a single ring structure), biphenyls and Flavonoids having 2-3 phenolic rings (Harbone, 1980; Vattem et al., 2005). Polyphenols is the another group which contain 12-16 phenolic groups and are found abundantly in fruits and vegetables. These polyphenols are classified as condensed proanthocyanidins, tannins which include galloyl and hydroxydiphenoyl (or ellagoyl) esters and their derivatives or phlorotannins (Harbone, 1980; Vattem et al., 2005). Flavonoids are one of the potential polyphenolic compounds. They occur as glycosides in plants while aglycones (lacking sugar moieties) occur less frequently. They contain several phenolic hydroxyl groups attached to ring structures designated as A, B and C (Fig 1).

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Fig 1. Flavonoid ring structure

The various classes of Flavonoids differ in the level of oxidation and patterns of substitution of the C ring. These structural variation within the rings subdivided the Flavonoids into several families (Rice-Evans et al., 1997). The families are: flavanols, flavons, flavan-3-ols, isoflavones.

a. Flavanols: Compounds belonging to this family has 3-hydroxy pyran-4-one ring. Conjugation occurs commonly at the 3- position of the ring, although 5, 7, 4’, 3’ and 5’ substitutions also occur (Herman, 1976). Examples are quercetin and kaempherol.
b. Flavones: Flavones are structurally similar to flavonols, however they lack 3-hydroxyl group. Luteolin, chrysin represent this family.
c. Flavan-3-ols: They lack 2,3-double bond and the 4-one structures. Catechin belongs to this family and is found highest concentration in tea and red wine (Arts et al., 2000).
d. Isovlavones: They are characterized by having the B ring attached at the C-3 of the phenylchromane structure. Daidzen and genistein represent this family.

Plant as a source of polyphenols

Phenolic phytochemicals constitute one of the most abundant groups of natural metabolites and form an important part of both human and animal diets (Vattem et al., 2005). There are more than 8000 identified polyphenols found in food such as fruits, vegetables, wine, tea, extra virgin olive oil, chocolate and other cocoa products (Han et al., 2007). They function in the body as free radical scavengers, complexes of pro-oxidant metals, reducing agents and quenchers of singlet-oxygen formation (Andlauer and Furst, 1998). There are numerous different types of phenolic phytochemicals which are characterized by an aromatic ring possessing one or more hydroxyl substituent. Harbone and Simmonds (1964) classified phenolic compounds into several groups depending on the number of carbons in the molecule. They vary in structure from simple phenolics having C6 carbon in their molecule to betacyanins which contain C18 carbon. Polyphenols are the compounds that have more than one phenolic hydroxyl group attached to one or more benzene rings. They are characteristics of plants and as a group they are usually found as esters or glycosides rather than as free compounds (Vermerris and Nicholson, 2006). Polyphenols are divided into several classes, according to the number of phenol rings that they contain and to the structural elements that bind these rings to one another (Grassi et al., 2010).

Polyphenolic compounds are found to be reported in both edible and inedible plants, which have multiple applications in food, cosmetic and pharmaceutical industries (Kahkonen et al., 1999). Surveswaran et al. (2007) screened natural phenolic antioxidants from 133 Indian medicinal plants. Du et al. (1975) reported the presence of anthocyanins and the 3-glucosides and 3,5-diglucosides of delphinidins, cyanindin and pelargonidins in pomegranate juice. Patuletin (quercetagetin 6-methyl ether), jaceidin and spinacetin (quercetagetin 6,3’-dimethyl ether) conjugates are the major Flavonoids reported in spinach leaves (Naczk and Shahidi, 2006).

Structural and antioxidant relationship of polyphenols

Phenolic compounds could easily donate hydroxyl hydrogen due to resonance stabilization (Fessenden and Fessenden, 1994). Boland and ten-Have (1947) postulated that phenolic antioxidants interfere with lipid oxidation by rapid donation of a hydrogen atom to lipid radicals. This redox property of phenolic compounds allows them to act as reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators (Balasundram et al., 2006).

In case of Flavonoids, the antioxidant activities depend on their molecular structure. Structure activity relationship of some phenolic compounds (eg. Flavonoids, phenolic acid, tannins) has been widely studied (Rice-Evans et al., 1996; Lien et al., 1999; Son and Lewis, 2002) and their hydroxylation pattern (related to the position and numbers of hydroxyl groups) which is responsible for their antioxidant activity (Pratt and Hudson, 1990). It was reported that O-dihydroxylation of the B-ring contributes to the antioxidant activity. The p-quinol structure of the B-ring was found to impart the greater activity than o-quinol. However, para and meta hydroxylation of the B-ring do not occur naturally (Pratt and Hudson, 1990). Other important features include a carbonyl group at position 4 and a free hydroxyl group at position 3 and 5 (Dziedzic and Hudson, 1983b). Uri (1961) observed the other important sites of hydroxylation.

Flavonols are known to chelate metal ions because of the presence of 3-hydroxy-4-keto group and or 5-hydroxy-4-keto group (when the A-ring is hydroxylated at the fifth position). An O-quinol group at the B-ring can also demonstrate metal chelating activity (Pratt and Hudson, 1990).

Determination of Polyphenols

Polyphenols are determined by Folin assay method. Sum of the content of polyphenols are determined by Chromatography method (Han et al., 2007)). Due to their reducing properties, polyphenols can be determined by electrochemical detection. A colourimetric detector where the analyte flows through the porous electrodes is sensitive and can provide the valuable information about chemical structure, because electrochemical response is directly related to the structure of the compound (Abay et al., 2004; Milbury 2001). In recent years, LC-MS is commonly used for the identification of the phenolic compounds. MS detection provides information about the molecular mass and fragmentation pattern of the analyte. According to most studies, for both APCI and ESI, the negative mode provides the highest sensitivity. Negative ionization results in limited fragmentation, making it most suited to deduce the molecular mass of Flavonoids, especially in cases where the concentrations are low (Cuyckens and Claeys, 2004). The combined use of both ionization modes yield complementary information which may aid in the identification of unknowns (De Rijke et al., 2006).

Bioavailability of polyphenols

Different researchers gave different definition of bioavailability. The most commonly accepted definition of bioavailability is the proportion of the nutrient that is digested, absorbed and metabolized through normal pathways (Archivio et al., 2007). It was suggested that a compound may have strong antioxidative or other biological activities in vitro, it would have little biological activity in vivo if little or none of the compound gets to the target tissues. The most abundant polyphenols present in the diets may not necessarily be those that have the best bioavailablility profile. Therefore, it is not only important to know how much of nutrient is present in specific food or dietary supplement, but it is even more important to know how much of it is bioavailable (Archivio et al., 2010). One of the main objectives of bioavailbility studies is to determine, among the hundreds of dietary polyphenols, which are better absorbed and which lead to the formation of active metabolites (Manach et al., 2005). Different polyphenolic glycosides are present in different plants, which results in marked variation in bioavailability of polyphenols from different dietary sources (Hollman et al., 1999). Bioavailability studies of polyphenols from berries (Erlund et al., 2006) and tea (Rio et al., 2010) was done extensively.

Conclusion

Fruits, vegetables and beverages such as tea, red and white wine are rich sources of dietary polyphenols. The evidence from numerous studies has shown that they have therapeutic effects. Therefore, it is essential to identify the type of polyphenols and its nature of metabolism in vivo. There are tremendous prospects of developing preventive and therapeutic nutritional strategies by incorporating plants polyphenols in food. This will also give new insights to the food industries for developing new product by virtue of the presence of specific polyphenols.

References

1. Aaby K., Hvattum E., Skrede G. (2004). Analysis of Flavonoids and other phenolic compounds using high-performance liquid chromatography with coulometric array detection: relationship to antioxidant activity. J. Agric. Food Chem., 52, 4595-4603.

2. Andlauer W. and Furst P. (1998). Antioxidative power of phytochemicals with special reference to cereals. Cereal Foods world, 43, 356-359.

3. Anonymous. (1987). Medicinal plants of India, Vol I and II. Indian council of Medical Research, New Delhi, India.

4. Archivio M D, Filesi C, Benedetto R D, Gargiulo R, Giovannini C and Masella R (2007). Polyphenols dietary sources and bioavailability. Ann Ist Super Sanita. 43 (4). 348-361.

5. Archivio M D, Filesi C, Vari R, Scazzocchio B and Masella R (2010). Bioavailability of the polyphenols: Status and Controversies. Int. J. Mol. Sci. 11, 1321-1342.

6. Arts I. C., Hollman P. C., Fesken E. J., Bueno de Mesquita H. B. and Kromhout D. (2000). Catechin intake and associated dietary and lifestyle factors in a representative sample of Dutch men and women. European Journal of Clinical Nutrition, 55, 76-81.

7. Balasundram N., Sundram K. and Sammar S. (2006). Phenolics compounds in plants and agri-industrial by products: Antioxidant activity, occurrence and potential uses. Food Chemistry, 68, 191-203.

8. Boland J. L. and ten-Have P. (1947). Kinetics in the chemistry of rubber and related materials; the inhibitory effect of hydroquinone on the thermal oxidation of ethyl linoleate. Trans Faraday Soc., 43, 201-204.

9. Cuyckens F. and Claeys M. (2004). Mass spectrometry in the structural analysis of Flavonoids. Journal of mass spectrometry, 39, 1-15.

10. De R. E., Out P., Niessen W. M. A., Ariese F., Gooijer C. and Brinkman U. A. Th. (2006). Review-Analytical separation and detection methods for Flavonoids. Journal of chromatography A, 1112, 31-63.

11. Du C. T., Wang P. L., Francis F. J. (1975). Anthocyanins of pomegranate, Punica granatum. J Food Sci., 40, 417-418.

12. Dziedzic S. Z. and Hudson B. J. F. (1983b). Polyhydroxy chalcones and flavonones as antioxidants for edible oils. Food chem., 12, 205-212.

13. Erlund I, Freese R, Marniemi J, Hakala P and Alfthan G (2006). Bioavailability of quercetin from berries and the diet. Nutrition and cancer. 54(1), 13-17.

14. Fessenden R. J., Fessenden J. S. (1994). Organic chemistry. 5th ed., Brooks/cole publishing: Belmont, C A., 246-260.

15. Grassi D., Desideri G. and Ferri C. (2010). Flavonoids: Antioxidants against atherosclerosis. Nutrients, 2, 889-902.

16. Han X, Shen T and Lou H (2007). Dietary polyphenols and their biological significance. Int. J. Mol. Sci. 8(9). 950-988.

17. Harbone J. B. (1980). Plant phenolics. In: Encyclopedia of plant physiology (Bell E. A. and Charlwood B .V. eds., 329-395. Berlin Heidelberg.

18. Harbone J. B. and Simmonds N. W. (1964). Natural distribution of the phenolic aglycones. In: Biochemistry of phenolic compounds (Harbone, J B ed.), 77-128, London: Academic Press.

19. Harman D. (1956). Ageing: a theory based on free radical and radiation chemistry. Journal of Gerontology, 11 (3), 298-300.

20. Hollman P C, Bijsman M N, Van Gameren Y, Cnossen E P, de Varies J H (1999). The sugar moiety is a major determinant of the absorbtion of dietary flavonoid glycosides in man. Free Radic Res. 31, 569-573.

21. Kahkonen M. P., Hopio A. I., Rauha J. P., Pihlaja K. and Kujala T. S. (1999). Antioxidant activity of plant extracts containing phenolic compounds. Journal of agricultural and food chemistry, 47, 3954-3962.

22. Lien E. J., Ren S., Bui H. H., Wang R. (1999). Quantitative structure activity relationship analysis of phenolic antioxidants. Free Radical Biology and Medicine, 26, 285-294.

23. Manach C, Williamson G, Morand C, Scalbert A and Remesy C (2005). Bioavailability and bioefficacy of polyphenols in humans. 1. Review of 97 bioavailability studies. Am J Clin Nutr. 81, 230S-42S.

24. Milbury P. E. (2001). Analysis of complex mixture of Flavonoids and polyphenols by HPLC electrochemical detection methods. Methods Enzymol., 335, 15-26.

25. Naczk M, Shahidi F. (2006). Phenolics in cereals, fruits and vegetables: occurrence, extraction and analysis. J. Pharm. Biomed. Anal. 41. 1523-1542.

26. Pratt D. E. and Hudson B. J. F. (1990). Natural antioxidants not exploited commercially. In Food antioxidants. Hudson B J F Ed., Elsevier applied science, London. 171-192.

27. Rice-Evans C. A., Miller N. J. and Paganga G. (1997). Antioxidant properties of phenolic compounds. Trends in plant science, 2 (4), 152-159.

28. Rice-Evans C. A., Miller N. J., Paganga G. (1996). Structure-antioxidant activity relationships of Flavonoids and phenolic acids. Free Radical Biology and Medicine, 20 (7), 933-956.

29. Rio D D, Calani L, Scazzina F, Jechiu L, Cordero C and Brighenti F (2010). Bioavailability of catechins from ready to drink tea. Nutrition. 26, 528-533.

30. Son S. and Lewis B. A. (2002). Free radical scavenging and antioxidant activity of caffeic acid amide and ester analogues: structure-activity relationship. Journal of Agricultural and Food Chemistry, 50 (3), 468-472.

31. Surveswaran S., Cai Y-Z, Corke H. and Sun M. (2007). Systematic evaluation of natural phenolic antioxidants from 133 Indian medicinal plants. Food Chemistry, 102, 938-953.

32. Uri N. (1961). Mechanism of antioxidation . In Autoxidation and antioxidants. Lunbert W O. Ed., Interscience Publishers, New York. 133-169.

33. Vattem D. A., Ghaedian R. and Shetty K. (2005). Enhancing health benefits of berries through phenolic antioxidant enrichment: focus on cranberry. Asia Pac J Clin Nutr., 14 (2): 120-130.

34. Vermerris W. and Nicholson R. (2006). Phenolic compound biochemistry. Springer, Netherlands.

Chapter 5
Recent Development of Ruthenium(II) Complexes with P,N-Donor Ligands

Dr. Devajani Boruah

Introduction

In the last few decades the ruthenium chemistry with different types of multifunctionalized P,N-donor ligands have received considerable importance in both coordination chemistry and homogeneous catalysis[1,2,3]. Multifunctional ligands bearing both soft-hard assembly (e.g., soft ‘P’ and hard ‘N’) of donor atoms exhibits interesting structural diversity, stability, unusual reactivity and catalytic activity in homogeneous as well as heterogeneous catalysis. So, much efforts have been devoted in the last few years to develop and design such catalysts for homogeneous catalysis [4,5,6].The main advantage of such metal complexes as catalyst precursors is that by the process of simple ‘ ligand tailoring ’ their activity and selectivity can be affected significantly.

The combination of “soft” phosphorus and “hard” nitrogen donor atoms possess distinctly different π-acceptor strength. They stabilize the metal ions in different oxidation states and geometries. The relatively good σ-donor ability of the “hard” nitrogen makes them able to stabilize the “soft” metal ions in their higher oxidation state, while the π-acceptor character of the “soft” phosphorus can stabilize the metal ions in lower oxidation state. In the chelated complexes, the metal-phosphorus bond is comparatively strong enough due to the presence of dπ-pπ back bonding. The metal-nitrogen bond which is weak, may dissociate easily to create reversibly a “vacant coordination site” [4] for binding an incoming substrate. This occurs by displacement of the labile “hard” group of the ligand which although remain available for recoordination (Scheme 1) and confer additional stabilities to the metal complexes by chelate effect in absence of the substrate [7,8,9].

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Scheme 1. Ring “Opening and Closing” mechanism of hemilabile P,N donor ligand

Thus, this type of ligands exhibit interesting dynamic stereo-chemistry by this reversible “Opening and Closing” mechanism [10]. Because of this special feature they are also termed as “hemilabile or half-labile” ligand[11].The concept of “hemilabile” was first introduced by Rauchfuss about 33 years ago which in fact, originates from Pearson’s hard-soft acid-base (HSAB) principle [12,13].

Coordination modesof P,N-donor ligands

There are different types of P,N-donor ligands which favour several different coordination modes depending on their exact structure. Some examples are given below.

P^N type:

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P^NN type:

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P^NP type:

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Many complexes are reported with such ligands due to their different bonding characteristics (Scheme 2.).

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η1-(P) monodentate

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η1-(N) monodentate

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η2-(P,N) bridging chelate

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η2-(P,N) bidentate chelate

Scheme 2. Possible bonding modes of hemilabile P,N-ligands in complexes

In case of P,N-ligands bearing further functionality can bind to metals in tridentate fashion (Scheme 3.).

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η1-(P) monodentate

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η3-(P,N,N) bidentate bridging chelate

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meridian

Scheme 3. Possible bonding modes of tridentate hemilabile P,N,N- and N,P,N-ligands in complexes

Advantages of P,N-donor ligands over the homofuctional ligands

The P,N-donor hemilabile ligands exhibit distinct advantages over the homofuctional ligands like P-P, N-N, O-O, S-S etc. as comparatively, they have greater ability to make the metal center more electron rich by their direct M-N or M-P interactions. This makes the complexes more susceptible for oxidative addition reactions [14]. Because of the unique properties of such ligands, they got immense importance as catalyst for different homogeneous systems like hydrogenation [15], carbonylation [7], transfer hydrogenation [16], C-C cross-coupling reactions [17].

Ruthenium metal complexes containing different types of P,N-donor ligands

Ruthenium metal exhibits the widest scope of oxidation states among all other elements in the periodic table, viz., -2, 0, +2, +3, +4, +5, +6 and +8 and different coordination geometries with each electronic configurations. This makes the coordination chemistry of ruthenium very rich and diverse. Among the various types of Ru metal catalysts, the phosphorus and nitrogen ligated Ru complexes show greater potentiality for control of reactivity, selectivity in catalytic reaction [18].

In fact, there are several processes such as carbonylation of methanol and alkynes, olefin oligomerizations and polymerization [7], asymmetric hydrogenation of highly substituted alkenes, asymmetric hydroboration [15], transfer hydrogenation of acetophenone[19] in which complexes of P,N-ligands are the catalysts of choice. Ruthenium(II) derivatives containing tertiary phosphine ligands are found to be important precursors in catalytic processes, particularly hydrogenation [20]. Complexes with the formula [RuCl2Ln], [RuClHLn] (n = 3, 4), [RuCl2(CO)Ln] or [RuClH(CO)L3] are efficient catalysts in the hydrogenation of alkenes, alkynes, aromatic heterocycles or hydrocarbons, aldehydes and ketones [21,22].

The coordination chemistry of ruthenium has been drawing attention for its possible applications in the treatment of lots of diseases including cancer [23]. Furthermore, some aminophosphine complexes have the potential to exhibit cytotoxicity either by disrupting mitochrondrial function as bis-chelated (ring closed) lipophilic cations or by direct binding to DNA bases as ring-opened complexes [24,25]. Morries et. al. have reported Ru(II) complexes of diphenyl-phosphinoethylamine which undergoes facile chelate ring opening in DMSO and CH3CN solutions and might be promising for anticancer activity involving attack on DNA bases [5].For the facile redox interchange between the two oxidation states Ru(II) to Ru(III), it is an ideal metal for preparation of various complexes of coordination number five or six. It is comparatively inexpensive than the other Group 8 metals such as rhodium, and preparation aof a great variety of ruthenium complexes have been reported till date. In addition to this ruthenium complexes often exhibit isomerism in its coordination number six. A large variety of ruthenium complexes of phosphorus functionalized ligands have been prepared from RuCl3.xH2O, (x ≈ 3). Some very important selected starting ruthenium complexes such as [RuCl2(PPh3)3], [Ru(CO)2Cl2]n, [Ru(cod)Cl2]n, [RuCl2(dmso)4], [Ru(p -cymene)Cl2]2 etc. have been prepared from RuCl3.xH2O. The coordination variety of ruthenium ions is relatively complicated in comparison to the four-coordinated species like palladium, rhodium and iridium [26].

J.-Y. Shen et. al. have reported [27] the synthesis of dark red complex [RuCl2{η2-(P,N)PPh2CH2CH2NMe2}2] (1) by the treatment of RuCl3.3H2O with two equivalent of PPh2CH2CH2NMe2 in the presence of Zn with 80% yield and also by the reaction of RuCl2(PPh3)3 with two equivalent of PPh2CH2CH2NMe2 with 87% yield. The hemilabile behavior of the ligand in the complex was studied by the reaction of CO and HC≡CPh resulting complexes [RuCl2{η2-(P,N)PPh2CH2CH2NMe2}{η1-(P)PPh2CH2CH2NMe2}(CO)] (2) (67% isolated yield) and [RuCl2{η2-(P,N)PPh2CH2CH2NMe2}{η1-(P)PPh2CH2CH2NMe2}(HC≡CPh)] (3) (46% yield) as an air-stable yellow solid respectively (Scheme 4.).

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Scheme 4. Synthesis and hemilabile behavior of [RuCl2{η2-(P,N)PPh2CH2CH2NMe2}2] (1)

Removal of halide by NaBPh4 from the neutral complex [RuCl2{η2-(P,N)PPh2CH2CH2NMe2}2] results a five-coordinated cationic complexes [RuCl{η2-(P,N)PPh2CH2CH2NMe2}2]+[BPh4]– (4) (Scheme 5.). Reaction of [RuCl{η2-(P,N)PPh2CH2CH2NMe2}2]+ with CO, CH3CN, HC≡CR (R = Ph, SiMe3, n-Bu) afforded corresponding cationic complexes [RuCl{η2-(P,N)PPh2CH2CH2NMe2}2(CO)]+ (5), [RuCl{η2-(P,N)PPh2CH2CH2NMe2}2(CH3CN)]+ (6) and [RuCl{η2-(P,N)PPh2CH2CH2NMe2}2(=C=HR)]+ (R = Ph, SiMe3, n-Bu) (7-9), containing BPh4- as counter ion (Scheme 5.).

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Scheme 5. Synthesis and reaction of five-coordinated cationic complex [RuCl{η2-(P,N)PPh2CH2CH2NMe2}2]+ (4) with CO, CH3CN and HC≡CR (R = Ph, SiMe3 and n-Bu) (5-9)

Similar type of dichloro ruthenium complex of P,N-donor ligands Ph2PCH2CMe2NH2 are also synthesized by L. Dahlenburg et. al. [28] using starting compound RuCl3.3H2O to get [RuCl2{η2-(P,N)(Ph2PCH2CMe2NH2}2] (Scheme 6.).

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Scheme 6. Synthesis of [RuCl2{η2-(P,N) (Ph2PCH2CMe2NH2)2}]

The dimer [RuCl2(p -cymene)]2 is a versatile precursor complex known for Ru(II) chemistry. Synthesis of a great quantity of P,N-coordinated Ru(II) complexes of general formulae [RuCl2(P,N)2] have been prepared by reacting the ligand with substitutionally labile metal precursors like [RuCl2(p -cymene)]2 [29], [RuCl2(Me2SO)4] [5,30], [RuCl2(PPh3)3] [27] and [Ru(cod)Cl2] [5,30]. The advantage of using hydrated Ru(III) chloride as precursor is that it avoids additional reaction intermediates [31].

R. J. Lundgren and his group reported the synthesis of cationic complexes of general formula [{(p -cymene)RuCl}(PN)]+X- [X = Cl, BF4, SO3CF3 or B(C6F5)4] and its zwitterion and screened their catalytic activities for transfer hydrogenation of ketones. The zwitterions have been proved to be highly active precatalyst for reduction of acetophenone to 1-phenylethanol with 99% conversion (TOF = 1,80,000 h-1) after 5 min of reaction. The cationic complexes show modest activity for the same reaction [32]. For transfer hydrogenation of ketones catalyzed by ruthenium complexes supported by structurally different ancillary ligand containing Ru-NH linkage in general exhibit the highest level of efficacy and selectivity [33].

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Scheme 7. Structure of the ligand PN

I. Angurell et. al. recently reported the synthesis of the tridentate P,N,N-donor ligand 4-amino(N-methylendiphenylphosphino)pyridine (PPh2CH2NH-4-py) and its coordination behavior towards [RuCl2(p -cymene)]2. Upon treatment of one molar equivalent of [RuCl2(p -cymene)]2 with two molar equivalent of [PPh2CH2NH-4-py] in mild reaction condition afforded the new product [(p -cymene)RuCl2{η1-(P)PPh2CH2NH-4-py}]. The complex was characterized by single crystal X-ray diffraction. The ligand bonded to the Ru center through phosphorus atom and the existing other two N-donors are not involved in coordination [34] as shown in the structure given below (Scheme 8.).

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Scheme 8. Structure of [(p -cymene)RuCl2{η1-(P)PPh2CH2NH-4-py}]

A. Bacchi and his co-workers reported the synthesis of some half-sandwich neutral ruthenium(II) complexes of 2-(diphenyphosphanyl)aniline (PNH2) and 2-(diphenylphosphanyl)- N,N -dimethylaniline (PNMe2) of molecular composition [(p -cymene)RuCl2{η1-(P)PNR2}] (R = H, Me) [35] . The complexes had been synthesized by the treatment of [RuCl2(p -cymene)]2 with two fold excess of PNH2 and PNMe2 at r.t. Moreover, the complexes exhibit solvent-promoted isomerism to give the corresponding ionic complexes [(p -cymene)RuCl{η2-(P,N)PNH2}]Cl (R = H, Me), when it is treated with i PrOH (Scheme 9.). The complexes [(p -cymene)RuCl{η2-(P,N)PNH2}]Cl (R = H, Me) have been found to be effective precatalyst for homogeneous hydrogen transfer reaction of acetophenone [35].

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Scheme 9. Synthesis of Ru complexes containing PPh2(o -C6H4NR2) (R = H and Me) ligand

Synthesis of some ionic pseudooctahedral ruthenium(II) complexes of the type [(p -cymene)RuCl{η2-(P,N)PNH2}]Y, (Y = PF6, BF4, BPh4 and TfO) have also been reported [35].

Recently J. Schroer et. al. have reported few monomeric complexes [RuCl2{η2-(P,N)PPh2CH2(o -C6H4NH2)}(PPh3)(CH3CN)], [RuCl2{η2-(P,N)PPh2CH2(o -C6H4NH2)}2] and a chloro-bridged dimer [RuCl2{η2-(P,N)PPh2CH2(o -C6H4NH2)}(PPh3)]2 of Ru(II) with P,N-donor [36]. The complexes have been synthesized by the reaction of [RuCl2(PPh3)3] with 2-(Diphenylphosphinomethyl)aniline {PPh2CH2(o -C6H4NH2)} applying different reaction conditions ( Scheme 10). It has also been reported that the reaction for the formation of [RuCl2{η2-(P,N)PPh2CH2(o -C6H4NH2)}2] proceeds via [RuCl2{η2-(P,N)PPh2CH2(o -C6H4NH2)}(PPh3)]2, as shown below in scheme 10.

illustration not visible in this excerpt

Scheme 10. Synthesis of Ru(II) complexes containing PPh2CH2(o -C6H4NH2) ligand

Another mononuclear pale yellow chelated complex [RuCl2{η2-(P,N)PPh2CH2(o -C6H4NH2)}(dmso)2] have been prepared by using [RuCl2(dmso)4] (2 molar ratio amount) as starting material with one equivalent amount of the ligand under stirring at r.t. [36] (Scheme 11.).

illustration not visible in this excerpt

Scheme 11. Synthesis of [RuCl2{η2-(P,N)PPh2CH2(o -C6H4NH2)}(dmso)2]

C.-C. Lee and his group described the geometric isomers and substitution reactions of a series of ruthenium(II) complexes of o -(diphenylphosphino)aniline {PPh2(o -C6H4NH2)} ligand. Reactions of this ligand with different Ru(II) precursors, [RuCl2(CO)3(THF)], [RuCl2(dmso)4] and [RuCl2(PPh3)3] afforded the complexes [RuCl2{η2-(P,N)PPh2(o -C6H4NH2)}(CO)2], [RuCl2{η2-(P,N)PPh2(o -C6H4NH2)}(dmso)2] and [RuCl2{η2-(P,N)-PPh2(o -C6H4NH2)}(PPh3)], respectively. Moreover, when [RuCl2{η2-(P,N)PPh2(o -C6H4NH2)}(dmso)2] was treated with CH3CN a substitution reaction occurred to yield [RuCl2{η2-(P,N)PPh2(o -C6H4NH2)}(dmso)(CH3CN)] [26]. The five coordinated complex [RuCl2{η2-(P,N)PPh2(o -C6H4NH2)}(PPh3)] exist in two isomeric forms (Scheme 12.) as evident by the IR absorptions at 345, 294 and 261 cm-1 for υ(Ru-Cl).

illustration not visible in this excerptillustration not visible in this excerpt

Scheme 12. Isomeric forms of[RuCl2{η2-(P,N)PPh2(o -C6H4NH2)}(PPh3)]

All the species undergo ligand substitution reactions with acetonitrile, dmso and carbon monoxide.

Conclusion

To the rapid growth of chemical industries of different sectors like petrochemicals, petroleum, pharmaceuticals in production of more efficient drugs, fine chemicals and energy conservation etc., the contribution of catalysis is now unquestionable. Application of catalysis has made significant impact on the development of our society, improvement of our quality of everyday life and economy. For different chemical transformations, the coordination chemistry of ruthenium metal occupies a special position in catalysis. The catalytic application of platinum group metals complexes containing hemilabile ligand has indeed a remarkable effect on the rate of many reactions.

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Chapter 6
Applicability of Genetic Parameters in Rice Improvement Programmes: Present Initiatives and Future Prospects

Dr. Pallabi Dutta

Introduction

Rice (Oryza sativa L.) is a plant belonging to the family of grasses, Poaceae. It is staple food for over one third of the world’s people (Poehlman and Sleeper, 1995), especially in those areas of the world, where population densities are highest (Ironan, 1972). Rice provides 75% of the calories and 55% of the protein in the average daily diet of the people (Rashid et al., 2009). In India rice is the main food source for more than 60% of population and it contributes around 45% of cereal production in the country (Siddiq, 2002). More than 90% of the world’s rice is produced and consumed in Asia (Virmani, 1996). The demand for rice is expected to increase further keeping in view the expected increase in the population. It is foreseen that the world’s population may exceed 8 billion by 2025 (Duwayri et al., 1999) and half of this increase will in Asia where rice is the staple food (Gregory et al., 2000). Meeting the nutritional needs of such a large population will require increasing agricultural productivity through a combination of methods. Considering these facts, it is very important to keep pace between population growth rate and rice production growth rate. This increase in rice production must be achieved through utilization of less land, less water and little agro-chemical use as well as by growing high yielding varieties of rice so that the difference between potential and actual yield can be bridged up.

Land races play an important role in the local food security and sustainable development in agriculture (Tang et al., 2002). The green revolution has remarkably increased crop productivity over the past four decades. (Mann, 1997). However this agricultural transformation has also resulted in problems, including the loss of crop genetic diversity (Tilman, 1998). Due to agricultural intensification, on-farm losses of traditional crop varieties are increasing, as most farmers prefer high yielding modern varieties (Maikhuri et al., 2001). FAO (1997) estimated that about three-quarters of the original varieties of agricultural crops have been already lost from the farm fields between 1950 and 1995. In India, a good example is the fast erosion of aromatic rice cultivars and landraces (Singh and Singh 1998). Because, in the past, emphasis has been mainly on increasing yield, the release and spread of high-yielding varieties have caused the fast replacement of low-yielding locally grown scented rice cultivars. Although these traditional varieties or landraces appear to be inferior to modern types in terms of yield potential, they possess many vital qualities such as pest resistance, drought-resistance, high protein content, flavour etc. showing unique source of genetic variation. The major objective in rice breeding programme is to maintain these desirable traits with an increase in the yield potential of these land races.

Yield is a complex inherited character, influenced by its component traits, such as number of panicles per plant, number of spikelets per plant, 1000 seeds weight etc. Hence, direct selection for yield often misleads breeders. Character association of various component traits with yield and among themselves is, therefore, very important. The estimation of character association could identify the relative importance of independent characters.

Genetic improvement mainly depends on the amount of genetic variability present in the population which is a ubiquitous property of all species in nature. The importance of genetic diversity in the selection of suitable genotypes for hybridization has been stressed by several scientists in different crops (Ivy et al. 2007, Mondal et al. 2007 and Haydar et al. 2007). It results due to differences either in the genetic constitutions of the individuals of the population or due to the differences in the environment in which they grown. Estimation of genetic variability present in the germplasm of a crop is pre-requisite for making any effective breeding programme. These genetic variations might be either heritable or non-heritable. It could be of interest to know the magnitude of variation due to heritable component, which in turn would be a guide for selection for crop improvement programmes of the population. Thus genetic variability for agronomic traits is the key component of breeding programs for broadening the gene pool of rice and other crops. For any trait of interest, observed phenotypic differences among individuals may be due to differences in the genetic codes for that trait or may be the result of variation in environmental condition. Most agronomically significant characters are known to be affected by environmental factors. Selection based on the phenotype might not be fruitful in such traits. In breeding programs, it is often difficult to manipulate such traits, since several inter-componential characters indirectly control these traits (Hittalmani et al., 2003). The measurement of genetic variation and understanding of mode of inheritance of quantitative traits, therefore, are essential steps in any crop improvement programme. Heritability estimates provide authentic information about a particular genetic attribute which will be transmitted to the successive generations. A broad-sense heritability estimate provides information on the relative magnitude of genetic and environmental variation in the population (Dudley and Moll, 1969; Marwede et al., 2004; Rafi and Nath, 2004) and help breeders to determine the possible extent for improvement through selection. Again the heritable portion of the total variation might not be always due to additive gene action. Thus estimates of heritability alone give no clear indication of the associating genetic progress that would result from selecting the best plants. It is also essential to find out the relative magnitude of additive and non additive genetic variances with regard to the characters of concern. The heritability along with phenotypic variance and the selection intensity, however, promise the estimation of genetic advance or response to selection which is more useful in the selection of promising lines (Johnson et al., 1955; Iqbal et al., 2003; Rohman et al., 2003).

To formulate a sustainable breeding programme precise knowledge about genetic divergence for yield components is a crucial one as varietal improvement depends mainly on the selection of parents with high genetic divergence in hybridization which is supposed to increase the chance of obtaining maximum heterosis and give broad spectrum of variability in segregating generations. Information on the nature and degree of genetic divergence would help the plant breeder in choosing the right parents for breeding programme (Vivekananda and Subramanian, 1993). Now a days, the magnitude of genetic diversity among all the possible pairs of populations at genotypic level before effecting actual crosses in modelling the genotypes in a desired genetic architecture has become possible with the help of advanced biometrical methods such as multivariate analysis (Rao,1952) based on Mahalanobis’s (1936) D2 statistic.

A thorough understanding of the genetic diversity of yield and its attributes, extent of variation, genetic architecture of the plant and heritability of the characters would help in developing sound plant improvement programmes.

In this context, a survey of pertinent literatures that are related to the various aspects was made to get insight to the proposed investigation under the following sub-headings.

Morpho-physiological and nutritional aspects

Important yield and yield attributing traits

Different traits may be important to increase the rice grain production. The considered traits may include short plant height, strong culms, moderate tillering, short and erect leaves, large and compact panicles and early maturation (Paterson et al., 2005). Yield and nutrient uptake by rice is affected by moisture regimes, puddling and time of application of organic matter (Das and Mandal, 1986). Prasad et al., 2001 reported that Plant height has significant correlation with grain yield. Mohammad et al., 2002 observed significant variation among the studied cultivars for the trait plant height. Rajesh et al., 2010 reported a range of 78.80 cm to 217.80 cm and Chakraborty and Chakraborty, 2010 reported a range of 100 cm to 175 cm plant height for rice.

Tillering in rice is one of the most important agronomic characters for grain production (Smith and Dilday, 2003), because the tiller number per plant determines the panicle number, a key component of grain yield (Yan et al., 1998). Miller et al., (1991) reported that tillering is a major determinant for production in rice. According to Gallagher and Biscoe (1978), tillering ability affects total yield in rice. Sinha and Biswas (1987), believed that although tillering ability of crop was not significantly affected by the environment, crop suffers from tiller mortality is due to the cool temperature. Kusutani et al., (2000) and Dutta et al., (2002) suggested that, genotypes producing higher number of effective tillers per hill showed higher grain yield in rice. Jamal et al., (2009) revealed significant variation among the genotypes for this trait. Rajesh et al., (2010) reported a range of 7 to 25 tillers per plant and Adeyemi et al., (2011) reported a range of 3 to 23 tillers per plant in rice.

Rangel et al., (1991) reported 76.00 days to 229.00 days of 50% flowering in rice. According to Ashrafuzzaman et al., (2009), breeding efforts are underway to develop short lived varieties with high yield potential.

Increased leaf area gives opportunity to increase total chlorophyll content and ultimately increase yield. Wang et al., (2008) believed that increasing chlorophyll content is an effective way to increase biomass production and grain yield. Adeyemi et al., (2011) observed wide range of 2.86 cm2 to 106 cm2 for leaf area index when studying 25 upland cultivars.

Das et al., (1981) believed that flag leaf has impact on grain size and weight. Bashar et al., (1990) and Rao, (1992) reported that flag leaf plays an important role in panicle size. Bharali and Chandra (1994) observed higher direct effect of flag leaf area on grain yield. According to Jamal et al., (2009), flag leaf characters differ from cultivars to cultivars and are affected by the temperature, photoperiod and other traits like, plant height and plant population density. Chakraborty and Chakraborty, (2010) reported significant variation for flag leaf length with a range of 16 cm. to 45 cm. and flag leaf breadth with a range of 0.93 cm to 2.3 cm.

Raj and Tripathi, (2000) revealed that correlation between leaf area and yield suggested that chlorophyll and leaf area are important in determining the yield. According to Ramesh et al., (2002), Leaf chlorophyll content is the best indicator of photosynthetic activity in rice. Ashrafuzzaman et al., (2009) observed significant difference among the cultivars for this trait.

According to Gravois and Helms (1996) and Tan et al., (1999) prime among the various grain quality characters in deciding the overall grain quality in rice are grain length, breadth, shape, and its weight. Adeyemi et al., (2011) found significant variation for the trait grain length with a range 4.4 mm to 11.4 mm.

According to Murty and Govindaswami , (1967) enormous variation in size and the shape of grain exists among the rice varieties. According to Nanda et al., (1976) some high yielding varieties from India had 5.2 mm to 6.8 mm in length and 1.9 mm to 2.5 mm in breadth. According to Thongbam et al., (2010) rice kernel length roughly varies from 5.0 mm to 7.5 mm and breadth from 1.9 mm to 3.0 mm.

According to Redona and Mackill, (1998) rice grain features such as length, breadth, and shape have a direct effect on the marketability, and therefore on the commercial success of modern rice cultivars. According to Yoshida et al., (2002) 4 to 5 genes are effective on grain length and breadth in rice. Govindaraj et al., (2005) reported that grain breadth showed polygenic inheritance. Rathi et al., (2010) reported significant variation for this trait while studying 100 upland rice genotypes from Assam with a range of 2.00 mm to 3.5 mm.

Grain length breadth ratio determines grain type and the marketing value of a variety. According to Siddiqui et al., (2007) existence of great diversity in the seed morphology i.e. length, breadth and thickness indicates the presence of other related agronomic, physiological, cooking, nutritional traits or cultural aspects for their selection and adoption.

Harvest index reflects translocation and alternatively dry matter partitioning of a given genotype to the economic parts. According to Miah et al., (1996), high yield is determined by physiological process leading to a high net accumulation of photosynthates and their partitioning. Kusutani et al., (2000) highlighted the contribution of high harvest index to yields. Singh et al., (2010ii) observed significant difference between cultivars for this trait.

Rangel et al., (1991) reported significant differences among the cultivars regarding panicle length. Sharma (2002) worked with fine grain rice and reported that there had been significant variation in panicle length. However Shrirame and Muley (2003) observed that panicle length had no significant difference among the genotypes studied. Singh et al., (2010i) and Rajesh et al., (2010) also observed significant differences for this trait. Chakraborty and Chakraborty, (2010) observed a range of 18 cm to 30 cm and Rajesh et al., (2010) observed a range of 24.66 cm to 37.00 cm for this trait.

Mondal et al. (2005) studied 17 modern cultivars of transplant aman rice and reported that 1000-grains weight differed significantly among the cultivar studied. Mustafa and Elsheikh (2007), found minimum and maximum values for 1000 grains weight as 28.1gm and 41.9 gm respectively. Okelora et al., (2007) and Adeyemi et al., (2011) reported significant variation for this trait in rice while studying five upland rice cultivars and twenty-four genotypes of rice respectively.

Spikelet’s per panicle is an important character contributing to grain yield. According to Kusutani et al., (2000) and Dutta et al., (2002), genotypes producing higher number of grains per panicle showed higher grain yield in rice.

Rangel et al., (1991) observed 51.70% to 91.00% of viable seeds in rice.

Gawai et al., (2006) concluded that spikelet density had highly significant positive correlation with grain yield per plant. Ali et al., (2000) reported a range of 3.24 g/cm to 5.50 g/cm spikelet density in rice. Zhenmin et al., 1987 observed correlation of spikelet density with plant height, flag-leaf length, flag-leaf breadth, panicle length, grain number per panicle, sterility and yielding capacity of the individual plant.

Mirza et al, (1992) reported positive correlation among number of panicle per plant, panicle length, number of grains per panicle and 1000-grains weight and grain yield plant. According to Mahpattra, (1993) variation in the grains yield might be due to the environment or the correlation of grain yield per plant with various yield contributing characteristics like; fertility of soil, flag leaf area, grains per panicle, number of grains per panicle and grain weight. Varietal differences of grain yield were reported by Biswas et al., (1998) also. Jamal et al., (2009) observed a range of 82.2 g/plant to 124.9 g/plant grain yield studying five exotic and one local check rice genotypes.

Nutritional traits

Carbohydrate is the main component of rice grains. Pedersen and Eggum (1983) and Juliano et al., (1985) reported a range of 73-87% total carbohydrate content in brown rice. Devi et al., (2008) concluded that milled rice has contained about 78% carbohydrate. Devi et al., (2008) also reported 73.77% to 85.33% of carbohydrate content studying fifteen indigenous rice cultivars. Devi et al., (1012) observed a range of 70% to 89.25% of total carbohydrate content studying eighteen indigenous cultivars of North-Eastern hill regions of India. 79% carbohydrate content is recommended by USDA Nutritional database, U.S.

The starch content of glutinous rice varieties of Assam varied from 72.0 to 76.0 percent and in non-glutinous varieties it ranged from 70.5 to 84.5 percent (Kandali et al., 1995). Deka, 2003 observed that the starch content in ten boro rice varieties grown in Assam ranged from 66.46 to 77.40 percent. Chakraborty et al., (2009) reported that bold grained rice genotypes had starch content varied from 65.60 to 79.88 percent. Devi et al., (2008) and Rathi et al., (2010) reported significant variation among cultivars for total starch content while studying fifteen indigenous rice and 100 upland rice cultivars respectively.

Juliano, (1971) concluded that low amylose levels are associated with cohesive, tender, and glossy cooked rice. Conversely, high levels of amylose cause rice to absorb more water and consequently expand more during cooking, and the grains tend to cook dry, fluffy, and separate. The apparent amylose content is highly influenced by environmental conditions, which cause amylose content to vary up to 6 percent for a given cultivar (Juliano and Pascual, 1980). Juliano, (1979a), (1979b); and Webb (1985) described that amylose content is considered to be the single most important characteristics for predicting rice cooking and processing behaviours. According to Webb, (1991) it is commonly used as an objective index for cooked rice texture. Sinha et al., (2001) confirmed that amylose, in combination with the water-insoluble polymer ethyl-cellulose, which is necessary to control the swelling of amylose, has been exploited as a film coating in colonic drug delivery . According to Govindaswami, (1985) and Nakagahara et al., (1986) amylose content in rice varied from 0 to about 37%. Kandali et al., (1995) reported that the amylose content in glutinous rice of Assam varied from 0.95 to 23.2 percent and in case of non-glutinous rice; it varies from 16.41 to 21.14 percent while Bhattacharjee, (2006) observed 0.136 to 11.4 percent of amylose content in glutinous rice cultivars of Assam. Ahmed et al., (1998) reported that scented rice varieties of Assam had amylose content varying from 18.9 to 23.2 percent. Rathi et al., (2010) concluded that varieties with intermediate amylose content are generally most preferred because they look dry and fluffy retaining their soft texture even after cooling.

Rao et al., 1961 believed that, in glutinous or waxy rice, the composition of starch could entirely be the amylopectin. The amylopectin content in some glutinous rice varieties of Assam varied from 83.05 to 99.30 percent, whereas in non glutinous rice it was in between 77.61 to 83.49 percent (Kandali et al., 1995; Bahar, 2000; Bhagabati, 2000). Ahmed et al., 1998 and Ahmed, 2003 observed 75.49 to 81.80 percent amylopectin content in some scented rice grown in Assam while Deka, 2003 reported 74.48 to 78.97 percent amylopectin content in ten boro rice varieties of Assam. Amylopectin isolated from Assam bora rice were characterized for use as plasma volume expander by Ahmed and Bhattacharya in 2010.

The nutritional quality of rice depends on the protein content and quality of protein depends on the composition of amino acids (FAO, 1970). According to Eggum, (1979) rice is the poor sources of protein among the cereals, but rice protein is considered as superior and unique because of its composition of essential amino acids. Coffman and Juliano, (1987) and Juliano and Villareal (1993) revealed that protein content and other constituents such as amylose, starch, crude fibre, ash and total fat can be present in different amounts in different rice varieties. Ahmed et al., (1998) reported 9.17 to 11.77% of protein in rice collected from Assam. However Saikia and Bains (1990) and Singh et al., (1998) reported low protein content (around 6% to 7% in both brown and milled rice of Assam). In another study by Govindaswami and Ghosh (1973), on protein content of indigenous and exotic varieties, observed a range of 5.5 to 14%. Devi et al., (2008) also reported as high as 12.07% protein studying local cultivars from Manipur.

1.1.3 Additional traits related to yield and abiotic stresses.

There are some additional traits such as germination percentage, flag leaf sheath etc, which have indirect effect on yield. Seed weight, whether heavy, medium or light, was negatively correlated with germination percentage (Teng et al., 1992). Cui et al., (2002) indicated that germination rate and early seedling growth were interrelated in rice.

Pang and Dong, (1995) concluded that chlorophyll a and b perform important function in the absorption and exploitation of light energy, thereby influencing photosynthetic efficiency. According to wang et al., (2008) grain yield can be increased by increasing these components. Ashrafuzzaman et al., (2009) revealed that Chlorophyll a and b play vital role in viable seeds and are most important elements of photosynthesis. They reported a range of 2.81 mg/g to 2.85 mg/g for chlorophyll a content and 1.13 mg/g to 1.83 mg/g for chlorophyll b content, studying six aromatic rice varieties.

Ashrafuzzaman, et al., (2009) believed that the overlapping character of leaf sheath on internodes might be the basis of resistance to lodging in high yielding varieties.

Grain length breadth ratio determines the grain type, which is a commercially important character. According to Unnevehr et al., (1992) and Juliano and Villareal, (1993) long and slender grain is generally preferred for indica rice by the majority of consumers in China, USA and most Asian countries. Borah, (2002) reported that some of the high yielding rice varieties of Assam were extra long with respect to seed length, which ranged from 7.8 to 9.9 mm. Medhi et al., 2004 reported twenty-eight long grained, ten medium grained and two short grained rice varieties among forty indigenous scented rice cultivars.

Sharma and Reddy, (1991) observed positive correlation between root xylem vessel numbers per plant and grain yield. Allah et al., (2010a) reported a range of 4.00 to 7.10 xylem vessel number studying five rice varieties in drought condition.

According to Muhammad Shahid et al., (2002) number of stomata has prominent impact on development of drought resistant rice varieties. They reported 36.57 to 63.70 numbers of stomata studying eight varieties of rice and their five crosses.

Character association

Sharma and Sharma, (2007) believed that grain yield is a complex trait of integration of a number of component traits. According to Kishor et al., (2008), development of high yielding varieties through breeding requires a thorough knowledge of the association of the yield components. The knowledge of association i.e., genotypic and phenotypic correlation between yield and its component characters is essential for yield improvement through selection programmes (Ismail et al., 2001; Kumar and Sukla, 2002). Yadav et al., (2010i); Rathi et al., (2010) and Chakraborty et al., (2010i) observed that genotypic correlation coefficients were of higher in magnitude than the corresponding phenotypic correlation coefficients. According to Dewey and Lu, (1959), very close values for genotypic and phenotypic correlations might be due to reduction in error (environmental) variance to minor proportions. Rao and Gomathinayagam (1997), Prasad et al., (2001), Surek and Beser (2003) and Yogamenakshi et al. (2004) reported strong inherent association between maximum numbers of pairs of characters.

Singh et al., (1979) observed that yield per plant was positively correlated with plant height and panicles per plant. Sarma and Dwivedi, (1980) reported positive correlation between grain yield per plant and panicle length. Pushpa et al., (1999); Meenakshi et al., (1999), Rao and Saxena (1999); Ray and Debi (1999); Janardhanam et al., (2001) and Mustafa and Elsheikh, (2007) emphasized the importance of grains per panicle in determining grain yield in rice. Biswas et al., (2000); Singh et al. (2002); Hossain and Haque (2003) and Chakraborty and Chakraborty (2010), reported positive significant association of panicle length with grain yield per plant. Sharma and Shar­ma (2007) observed positive correlation of grain yield with grains/panicle and 100 grain weights.

According to NeWall and Eberhart (1961) when two characters show negative phenotypic and genotypic correlation it would be difficult to exercise simultaneous selection for these characters in the development of a variety. Saini and Gag­neja (1975) reported negative genotypic correlation of yield/plant with panicle length. Ganesan et al., (1998) and Chakraborty et al., (2010i) reported significant positive correlation in both genotypic and phenotypic plane for harvest index with panicles per plant.

Drought and pre-harvest sprouting tolerance

Plant-water relationships are vital to understanding crop growth and development. Begg and Turner (1976) point out that all plants undergoing transpiration can experience some degree of daily water deficit at any given time. Stress is viewed as an excess deficit that develops more slowly over a period of days and that significantly reduces plant growth and productivity.

Water loss from plant tissues under drought conditions results in growth inhibition and in a number of other metabolic and physiological changes. These include decreased photosynthesis (Hsiao, 1973), solute accumulation (Skriver et al., 1973, Bray, 1991), stomatal closure (MacRobbie., 1991), changes in leaf water potential (Premachandra et al., 1995) and abscisic acid (ABA) accumulation (Mansfield and McAinsh, 1995).

Boyer (1982) pointed out that plant growth and productivity are negatively affected by water stress and other environmental stress. Genetic improvement of water stress tolerance is important to agricultural plants.

According to Inthpan and Fukai (1988) rice is known to be more susceptible to shortage of irrigation water than most of other crops because rice is a semi aquatic plant species and is commonly grown in lowland paddies where there is standing water during all stages of growth.

Drought is a major abiotic stress, affecting 20% of the total rice growing area of Asia (Pandey and Bhandari, 2008). It is one of the main environmental problems of rice production in Assam. Great changes in the growth and production of rice plant have takes place due to this stress condition and the only answer to this problem is the development of new variety and new technology suitable for drought stress.

Seshu and Akbar (1995) reported that drought stress causes yield reduction and somewhat total crop failure in rain fed rice area of Asia, Africa and Latin America. Achieving drought resistance in rice will be necessary for meeting the growing water shortage of the world, and it requires a deep understanding of the mechanisms that could facilitate drought resistance (Serraj et al., 2011).

Bhattacharjee et al., (1971) stated that seedling survival rate is more in drought tolerant variety than drought susceptible variety.

McMichael (1980) shows evidence for intraspecific as well as interspecific differences in the ability of plants to cope with severe plant water stress and to maintain plant turgor and photosynthetic capacity. Chang et al., (1979) leave no doubt that such variation exists in both dry land and wetland rice cultivars. They also recognize the need to characterize or diagnose the location-specific parameters of drought. Thangaraj and Subramaniam (1990) also reported the varietal variation in drought recovery and leaf rolling in upland rice.

Considerable work has recently been undertaken to understand the genetic basis of putative drought-adaptive traits in rice (Price and Courtois, 1999; Courtois et al., 2000; Price et al., 2002a, b; Babu et al., 2003; Robin et al., 2003), but it has been difficult to identify genetic segments with clear and repeatable effects on yield under stress.

According to Russell, 1959, root development of a plant has long been recognized as an important factor in determining its adaptability to water stress conditions. When water deficit occurs, the most effective resistance mechanism available to the rice plant is a deep root system consisting of mostly thick roots that enables the plant to avoid the adverse effects of internal water deficit (Chang et al., 1972). According to Kramer, (1969); Newman, (1972); Hurd, (1976); Blum, (1982) and Passioura, (1982), root size, structure, morphology, depth, length, density and branching or distribution in soil horizons are important in maintaining high leaf water potential against evapo-transpiration demand under water deficit. The possession of a deep and thick root system which allows access to water deep in the soil profile is crucially considered important in determining drought tolerance in upland rice and substantial genetic variation exists for this (O'Toole, 1982; Yoshida and Hasegawa, 1982; Ekanayake et al., 1985; Fukai and Cooper, 1995). Gomathinayagam and Soundrapandian (1987), also reported that drought resistant rice variety have certain root characteristics such as longer, widely spreading more number of roots ranging from 276-359 and greater root/shoot ratio. On the other hand Rao, et al., (1994) reported that deep rooting system does not have any additional advantage for recovery from drought.

According to Loresto et al., 1983, root thickness and xylem vessel number can be used as selection indices in breeding for drought resistance in rice. He also pointed out that root thickness is stable and is not greatly affected by the environment. According to Yambo et al., (1992) also thick roots might have greater capacity for water uptake from deeper soil layers. Thick roots are also hypothesized to confer drought tolerance because root branching is related to root thickness (Fitter, 1991).

Minabe (1951), IRRI (1975) and Parao et al., (1976) reported a high positive association between root thickness and drought resistance. Chang et al., (1972) also obtained a significant association between root length and field resistance to drought. On the other hand Minabe (1951) and Chang et al. (1972) reported negative relationship between root number and drought resistance.

Mambani and Lal, (1983) and Lilley and Fukai, (1994) reported that there is a positive association between root length and grain yield. In contrast, Ingram et al. (1994), found no significant association between these two traits.

Haque et al., in 1989 reported a positive association between drought resistance and root length, root thickness, number of xylem vessels and xylem vessel area and a negative association between root number and drought resistance. They also concluded that thick root and many xylem vessels can be useful criteria in selecting for drought resistance. Allah et al., (2010b) reported that root xylem vessel number per plant had significant correlations at genotypic level with all other traits except grain yield.

Liu, (1982) pointed out a relationship in number of stomata, number of veins per unit area and leaf thickness with the moisture availability. According to Bormotov and Smirnova, (1981) stomata size is a highly heritable character. Tanzrella in 1984 concluded that stomatal frequency can be altered by breeding to improve water use. Muhammad Shahid et al., (2002) pointed out that number of stomata merits special attention in projects focused on development of drought resistant rice varieties and concluded that effort may be made to incorporate this character in the upcoming commercial rice varieties. They also concluded that denser flag leaf venation helps in moisture economy through judicious distribution.

According to Muhammad Shahid et al., (2002) flag leaf with short length is one of the key weapons against drought.

According to Allah et al., 2010a, a highly tillered plants tends to have a short root system and hence a negative relationship with drought resistance. Low tillering capacity appears to be one desirable characteristic when rice plant has to depend on soil moisture retained in the deep soil layers during drought stress. They also pointed out that the number of xylem vessels were closely related to the capacity of the plant to transport water and nutrients absorbed from the soil.

Variability, heritability and genetic advance

The progress in breeding for yield and its contributing characters depends on many factors such as environment in which they grown, nature of genetic variability and genes controlling the characters (Singh et al., 2000). Genetic variability, interrelationship and cause effect analysis are pre-requisites for improvement of any crop for selection of superior genotypes and improvement of any trait (Krishnaveni et al., 2006). It is very difficult to judge whether observed variability is highly heritable or not. Moreover, knowledge of heritability is essential for selection based improvement as it indicates the extent of transmissibility of a character into future generations (Ullah et al., 2011).

Variability, heritability and genetic advance of important yield and yield attributing traits

Huang et al., (1996) believed that plant height in rice is controlled by both qualitative and quantitative genes. Ashrafuzzaman et al., (2009) also considered that plant height is mostly governed by genetic makeup of the cultivar, but the environmental factors also influence it. Okelola et al., (2007) and Prajapati et al., (2011) reported minimum differences between GCV and PCV values for the trait plant height. Pal and Sabesan (2010); Ahmadikhah (2010); Lal and Chauhan (2011) and Prajapati et al., (2011), reported high GCV and PCV, high heritability and high genetic advance as percentage of mean for this trait. On the other hand Okelola et al., (2007), Ahmed et al., (2010a) and Ullah et al., (2011), reported moderate GCV and PCV, high heritability and high genetic advance as percentage of mean for this trait and Chakraborty and Chakraborty, (2010) revealed moderate GCV and PCV, high heritability and moderate genetic advance as percentage of mean for this trait.

Sinha, et al., (2004) and Padmaja et al., (2008) reported high GCV-PCV, high heritability and high genetic advance as percentage of mean the trait tillers per plant. Anbanandan et al., (2009) and Prajapati et al., (2011), reported high heritability and high genetic advance as percentage of mean for this trait. Singh et al., (2010ii) found moderate coefficients of variation for this trait. Bisne et al., (2009) reported moderate heritability and Ahmad et al., (2010) reported moderate GCV and PCV, high heritability and high genetic advance for this trait. High genetic advance for tillers per plant was also recorded by Kuldeep et al., (2004) and Karthikeyan et al., (2010).

Sawant et al. (1994); Padmaja et al., (2008) and Prajapati et al., (2011) reported high phenotypic and genotypic variances by the trait days to 50% flowering. GCV and PCV were high and PCV was higher than corresponding GCV for this trait but is noteworthy that the Shahidullah et al., (2009a), reported minimum differences between GCV and PCV values. Moderate GCV and PCV, high heritability and moderate genetic advance for days to 50% flowering were found by Ahmed et al., (2010a) and Prajapati et al., (2011) and moderate GCV and PCV, high heritability and high genetic advance were reported by Vange, (2009). Bisne et al., (2009) also reported moderate genetic advance for this trait.

According to Ahmed et al., (2010i), high phenotypic and genotypic coefficients of variability, heritability, genetic advance and genetic advance as percentage of mean were exhibited by leaf area index.

According to Singh (1980) heritability estimates were moderate to high for flag leaf length. Muhammad Shahid et al., (2002) also get similar results. Ahmed et al., (2010a) reported high heritability for flag leaf area. Chakraborty and Chakraborty, (2010) reported moderate genotypic and phenotypic coefficients of variation, moderate heritability and moderate genetic advance as percentage of mean for this trait whereas Yadav, et al., (2010ii), observed high GCV and PCV, high heritability and high genetic advance as percentage of mean for flag leaf length. Lal and Chauhan (2011) also reported moderate phenotypic and genotypic coefficients of variability for flag leaf length.

Muhammad Shahid et al., (2002) revealed low genotypic and phenotypic coefficient of variation along with high heritability for the trait flag leaf breadth. Chakraborty and Chakraborty (2010); Yadav et al., (2010ii) and Prajapati et al., (2011), observed moderate phenotypic and genotypic coefficients of variation, high heritability and high genetic advance as percentage of mean for this trait.

Sedeek et al., (2009) and Laxuman et al., (2010) reported moderate GCV and PCV, high heritability and high genetic advance as percentage of mean for total chlorophyll content in rice. Ubarhande et al., (2009) found very high heritability. Wani et al., (2011) observed moderate GCV and PCV, high heritability and moderate genetic advance as percentage of mean for 50 elite wheat genotypes for this trait. On the other hand Ullah et al., (2011) recorded low GCV and PCV, high heritability and low genetic advance as percentage of mean for 10 biroin rice.

Bisne et al., (2009), revealed low genotypic and phenotypic coefficients of variation, high heritability and moderate genetic advance as percentage of mean for grain length whereas Rathi et al., 2010 reported moderate GCV and PCV, high heritability and high genetic advance as percentage of mean for the trait grain breadth.

Munhot et al., (2000) and Lal and Chauhan (2011) reported high genotypic and phenotypic coefficients of variation and high genetic advance for L/B ratio. Sharma and Sharma (2007) reported high GCV and PCV, high heritability and high genetic advance as percentage of mean for grain length breadth ratio in forty-four extra early and early maturing rice genotypes. Bharadwaj et al., (2007) also reported high heritability coupled with high genetic advance as percentage of mean in two filial generations of three new plant type based crosses for rice in two environments of normal and high dose of nitrogen. Vanaja and Babu, (2006) and Devi et al., (2012) reported moderate GCV and PCV, high heritability and high genetic advance as percentage of mean for grain length breadth ratio.

Habib et al., (2005) and Ahmed et al., (2010i) reported moderate genotypic and phenotypic coefficients of variation, high heritability and high genetic advance as percentage of mean for the trait harvest index. Whereas for this trait Karim et al., (2007) and Bisne et al., (2009) Prajapati et al., (2011) found high GCV and PCV, high heritability and high genetic advance.

Vange et al., (2009) reported moderate GCV and PCV, high heritability and high genetic advance as percentage of mean for panicle length. Ahmadikhah (2010) also observed high heritability for this trait. Prajapati et al., (2011) also observed moderate GCV and PCV, high heritability and moderate genetic advance as percentage of mean for this trait. Habib et al., (2005) reported low GCV and PCV and high heritability, Bisne et al., (2009) observed high heritability and moderate genetic advance, Shahidullah et al., (2009a) observed low GCV and PCV, Chakraborty and Chakraborty, (2010) reported low heritability and low genetic advance as percentage of mean and Singh et al., (2011) reported low GCV PCV and low genetic advance as percentage of mean for this trait.

Akanda et al., (1997), Choudhury and Das (1997), Karim et al., (2007) and Ullah et al., (2011) had reported high GCV-PCV, high heritability and high genetic advance as percentage of mean for 1000 seeds weight. Habib et al., (2005) reported moderate GCV and PCV, high heritability and high genetic advance as percentage of mean for this trait. Bisne et al., (2009) reported high GCV and PCV, Anbanandan et al., (2009) observed high heritability and high genetic advance and Kumar et al., (2009) revealed high heritability for this trait. On the other hand Lal and Chauhan (2011) reported moderate GCV and PCV and high genetic advance as percentage of mean for this trait.

Mustafa and Elsheikh, (2007) reported high genotypic and phenotypic coefficient of variation for the trait spikelet per panicle. Prajapati et al., (2011); Ullah et al., (2011); Singh et al., (2011) and Lal and Chauhan (2011) reported high GCV and PCV and high genetic advance for this trait.

Karim et al., (2007) reported high GCV and PCV, moderate heritability and high genetic advance as percentage of mean for the trait percentage of viable seeds.

Ali et al., (2000) observed Moderate variability, high heritability and high genetic advance as percentage of mean and Ahmed et al., (2010i) reported low GCV and PCV, moderate heritability and low genetic advance as percentage of mean for spikelet density.

Lal and Chauhan, (2011) reported moderate genotypic and phenotypic coefficients of variation for spikelets per plant.

Karim et al., (2007) and Singh et al., (2011) observed high genotypic and phenotypic coefficients of variability for panicles per plant. Prajapati et al., (2011) reported high heritability and high genetic advance as percentage of mean for this trait. Karim et al., (2007) and Lal and Chauhan, (2011) also reported high genetic advance as percentage of mean for this trait. Habib et al., (2005) reported moderate GCV and PCV, moderate heritability and moderate genetic advance as percentage of mean and Ullah et al., (2011) observed moderate GCV and PCV, high heritability and moderate genetic advance as percentage of mean for the trait panicles per plant.

Habib et al., (2005); Anbanandan et al., (2008); Vange (2009) and Ahmadikhah et al., (2010) reported highest GCV and PCV by grain yield among studied traits. Karim et al., 2007; Vange, 2009 and Ahmadikhah et al., 2010 emphasised on the role of environment in the expression of the trait grain yield. Das et al., (1992), Kumar et al., (1998), Habib et al., (2005), Anbanandan et al., (2008), Bisne et al., (2009) and Ullah et al., (2011) reported high heritability and high genetic advance as percentage of mean for grain yield. Mustafa and Elsheikh, (2007) and Prajapati et al., (2011) observed high GCV and PCV, high heritability and moderate genetic advance as percentage of mean for this trait. Thongbam et al., (2010) reported low GCV and PCV, moderate heritability and low genetic advance while studying thirteen indigenous medicinally used cultivars of Manipur while Devi et al., (2012) observed low GCV-PCV, high heritability, moderate genetic advance as percentage of mean studying eighteen indigenous rice cultivars of Tripura.

Variability, heritability and genetic advance of nutritional traits

Thongbam et al., (2010) reported low GCV and PCV, moderate heritability and low genetic advance while studying 13 indigenous medicinally used cultivars of Manipur while Devi et al., (2012) observed low GCV and PCV, high heritability, moderate genetic advance as percentage of mean studying eighteen indigenous rice cultivars of Tripura for the trait total carbohydrate content.

Chakraborty et al., (2009) and Chakraborty et al., (2010ii) reported high heritability with low genetic advance for total starch content.

According to Sinha et al., (2001), amylose, in combination with the water-insoluble polymer ethyl-cellulose, which is necessary to control the swelling of amylose, has been exploited as a film coating in colonic drug delivery. Subbaiah et al., (2011) and Devi et al., (2012) reported high GCV and PCV, high heritability and high genetic advance as percentage of mean for amylose content. Vanaja and Babu, (2006) and Devi et al., (2010) reported moderate GCV and PCV, high heritability and high genetic advance and Chakraborty et al., (2010ii) reported high heritability with moderate genetic advance for this trait.

Chakraborty et al., (2010ii) reported high heritability with low genetic advance for the trait amylopectin content.

Chakraborty et al., (2010ii) observed high heritability and moderate genetic advance and Thongbam et al., (2010) reported moderate GCV and PCV, moderate heritability and moderate genetic advance as percentage of mean. Samak et al., (2011) reported low GCV, moderate PCV, moderate heritability and moderate genetic advance as percentage of mean for protein content and Devi et al., 2012 reported moderate GCV and PCV, high heritability, moderate genetic advance as percentage of mean for the trait.

Variability, heritability and genetic advance of additional characters related to yield and abiotic stresses

Okelola et al., (2007) reported high heritability and moderate genetic advance as percentage of mean for germination percentage in rice.

Wani et al., 2011 observed moderate GCV and PCV for chlorophyll a and chlorophyll b content when studying 50 lines of wheat. He also reported high heritability accompanied with high genetic advance as percentage of mean for these traits.

Tiwari et al., (2011) reported moderate GCV and PCV, high heritability and high genetic advance as percentage of mean for percentage of pollen viability. Manju and Shreelathakumary, (2002) reported high GCV and PCV, high heritability and high genetic advance as percentage of mean for pollen viability percentage in hot chilli.

Pal and Sabesan, (2010) reported moderate GCV and PCV, moderate heritability and moderate genetic advance as percentage of mean in rice for the trait length of flag leaf sheath. Nirmalakumari et al., (2010) reported high variability and heritability with high genetic advance as percentage of mean in little millet germplasms for this trait.

Roy et al., (2009) observed moderate GCV, high PCV, moderate heritability and moderate genetic advance and Mohankumar et al., (2011) reported moderate GCV, high PCV, high heritability and moderate genetic advance as percentage of mean for length of root in rice. Sumathi et al., 2010 reported low GCV and PCV, high heritability and low genetic advance as percentage of mean for length of root while studying 47 pearl millet genotypes.

Muhammad Shahid et al., (2002) reported moderate to high heritability for number of stomata studying five F2 population and their eight parents of rice.

Genetic divergence

According to Tang et al., (2002) land races play an important role in the local food security and sustainable development in agriculture. In both cross and self pollinated crops, genetic diversity is one of the most important tools to quantify genetic variability (Griffing and Lindstrom, 1954 and Murty and Arunachalam, 1966). Information on the nature and degree of genetic divergence would help the plant breeder in choosing the right parents for breeding programme (Vivekananda and Subramanian, 1993). Tomooka (1991) reported that evaluation of genetic diversity within available germplasm is important to know the source of gene for a particular trait. Genetic divergence analysis quantifies the genetical distance among the selected genotypes and reflects the relative contribution of specific traits towards the total divergence (Iftekhruddaula et al., 2002). According to Banumathy et al., 2010 also genetic divergence is an efficient tool for the selection of parents used in hybridization programme. Several workers have emphasized the importance of genetic divergence for selection of desirable parents (Arunachalam, 1981; Pradhan and Roy 1990; Roy and Panwar, 1993). To help the breeders in the process of identify the parents that nick better, several methods of divergence analysis based on quantitative traits have been proposed to suit various objectives, of which Mahalanobis’s generalized distance occupy a unique place and an efficient method to estimate the extent of diversity among genotypes, which quantify the differences among several quantitative traits. According to Rao, (1952) Multivariate analysis based on Mahalanobis-D2 statistics and canonical variant analysis has been considered as an important tool in quantifying the genetic divergence in different crops. Joshi and Dhawan (1966) inferred that Mahalanobis’s D2 statistics was a powerful tool for choosing parents for hybridization aiming at hybrid improvement. The use of Mahalanobis D2 statistic for estimating genetic divergence has also been emphasized by Shukla et al., (2006) and Sarawgi and Binse (2007).

Early workers regarded geographical isolation as a reasonable index of genetic diversity (Vavilov, 1926; Joshi and Dhawan, 1966). The varieties, which come from different localities are usually presumed to diverse and are utilized in hybridization programme. However, several workers in different crop species have emphasized that there is no parallelism in geographical distribution and genetic diversity (Murthy and Anand, 1966 in linseed; Bhatt, 1970 in greegram; Maurya and Singh, 1977 and De et al., 1992 in rice) advocating that varieties with the same geographical origin could have undergone changes under selection pressure. Thus, for estimation of variation within the germplasm, divergence study in the form of classification into different homogenous groups is an important practice. Some of the earlier reports on genetic divergence study in rice have been reviewed below:

Rangel et al., (1991) evaluated seventy two local rice landraces adopted to lowland conditions considering ten traits of agronomic importance and partitioned these genotypes into four groups applying Mahalanobis generalised distance.

Sarawgi et al., (1998) assessed genetic divergence of one-hundred thirty-two rice genotypes considering eighteen grain quality traits and grouped the genotypes into 10 clusters. Considering cluster mean and cluster distances, they concluded that cultivars viz. Bakal-B, Jondhera dhan, Gonda jhul, Proova, IR-36, Kranchi, X-12, Moti bakiya, Kranti and Assam chudi were the most promising varieties and may be utilized in future plant breeding programme.

Cheema et al., (2004) studied the genetic variation among seventeen mutants and their respective parents and classified the genotypes into eleven groups.

Misra et al., (2004) divided one hundred sixteen germplasms of semi-deep water ecology into nine clusters considering eleven traits.

Bose and Pradhan (2005) assessed the nature and magnitude of genetic divergence among thirty-five deep water rice genotypes from India using Mahalanobis D2 statistics. The genotypes were grouped into ten clusters showing fair degree of relationship between geographic distribution and genetic divergence. Traits such as plant yield, days to 50% flowering, and plant height were the major contributors to genetic divergence.

Naik et al., (2006) conducted genetic divergence study to estimate the nature and magnitude of diversity in fifty aromatic rice accessions and partitioned them into seven clusters. The conclusion drawn by the cluster analysis is that, in that studied population, high variability was observed between the genotypes of different clusters for different characters.

Singh et al., (2006) assessed genetic divergence of fifty-two traditional lowland rice (Oryza sativa L.) genotypes from five states of North Eastern Region of India using Mahalanobis D 2 statistic and were grouped into six clusters. Plant height and leaf angle and leaf area contributed maximum (32.43%) to the formation of clusters. They concluded that geographical origin was not found to be a good parameter of genetic divergence.

Bisht et al., (2007) evaluated genetic diversity of thirteen landraces of paddy using multivariate analysis of Mahalanobis D2 statistics and grouped them into seven distinct clusters.

Chandra et al., (2007) studied fifty-seven upland rice genotypes including thirty-two local rice germplasm for the nature and magnitude of genetic divergence among them based on fourteen agro-morphological traits and grouped the genotypes into five clusters. They observed that clustering patterns of the genotypes were quite at random indicating that the geographical origin and genetic diversity were not related and traits, viz. 1000-grains weight, grain yield and biological yield were the main contributor to divergence.

Kumar (2008) reported that plant height, panicle length and grain yield contributed significantly for the genetic diversity and no parallelism between genetic diversity and geographical distribution studying seventy-one red rice genotypes by Mahalanobis D2 statistic. On the basis of genetic distance, they grouped genotypes into ten clusters and concluded that genotypes (Dodiga, Sharavathi, Akkalu, Bettasali, IRLON/90/39 and KHRS-17) from these clusters may be used as potential donors for future hybridization programme to develop higher grain yield with red kernel type.

Akter et al., (2009) evaluated genetically diverged forty-four restorers of rice hybrids using Mahalanobis’s statistic (D2) and principal component analysis. The population was divided into five physiological groups. The study also revealed wider genetic diversity among the genotypes of different groups and no parallelism between the geographical diversity and genetic diversity. Plant height was found to be the maximum contributors towards the total divergence.

Sabesan et al., (2009) evaluated genetic diversity among twenty six genotypes of rice using Mahalanobis D2 statistic based on twelve morphological and quality characters and were grouped into thirteen clusters. They concluded that geographical origin was not a good parameter of genetic divergence and number of grains per panicle (42.71%) followed by days to first flower (25.62%) contributed maximum to total divergence.

The genetic divergence of forty genotypes were assessed by Shahidullah et al., (2009b) using univariate and multivariate analyses for grain quality and nutrition aspects and partitioned them into six clusters. They concluded that the most contributing traits were kernel weight, kernel length and L/B ratio.

Ahmed et al., (2010i) assessed sixteen genotypes of rice (Oryza sativa L.) and revealed that genotypes differed significantly among themselves for all the characters viz., morpho-physiological, biochemical, yield and yield attributing traits. The population was divided into six clusters.

Ahmed et al., (2010ii) experimented thirty-six accessions of traditional Boro rice germplasms of three different groups (20 accessions as Kaliboro, 12 as Jagliboro and 4 as Tepiboro) using D2 statistic and partitioned into six clusters.

Banumathy et al., (2010) divided fifty three rice genotypes into 11 clusters and an examination of characters chosen revealed that important contribution of grain yield (50.87%), days to 50 per cent flowering (15.02%), total grains per panicle (10.52%) and plant height (10.23%) to the divergence.

Hosan et al., (2010) assessed twenty rice landraces, partitioned them into five clusters based on twelve characters and observed no parallel relationship between genetic and geographical divergence. They estimated higher inter-cluster distances than intra-cluster distances reflecting wider genetic diversity among the genotypes of different groups and traits viz. number of filled grains number /panicle, number of panicles/plant, biomass index and grain yield as main contributor towards total divergence.

Rajesh et al., (2010) assessed genetic diversity in twenty-nine land races of rice using Mahalanobis’s D2 statistics considering eight quantitative characters and partitioned them into five clusters. They concluded that characters viz. days to first flowering and single plant yield contributed maximum towards genetic divergence and geographical diversity and genetic diversity were not related.

Saravanan and Sabesan, (2010) subjected forty six genotypes of rice to multivariate analysis and grouped them into six clusters. They also revealed that characters viz., total number of grains per panicle, number of filled grain per panicles and plant height contributed maximum towards total genetic divergence.

Mathure et al., (2011) studied eighty-eight aromatic cultivars collected from Maharashtra state for determinants of kernel quality and concluded that to increase the yield, improvement in length of panicle and increasing number of productive tillers in medium or mild scented cultivars would be the best strategy.

Conclusions

The success of any plant breeding programme aimed at the evolution of high yielding, better quality and disease resistant varieties depends upon the selection of suitable genotypes to be utilized in breeding programme. The development of superior rice population is mainly depends on the intelligent use of available genetic variability of that particular population. Grain yield is a complex character, which depends on its main components viz; number of panicles per plant, panicle length, number of grains per panicle, 1000 grains weight etc. In both favourable and unfavourable environments, grain yield is the primary trait targeted for improvement of rice productivity from its present level. These grain yield components are further dependent for their expression on several morphological and developmental traits, which are interrelated with each other. Hence, the parents selected for the breeding programmes aimed at increased seed yield should possess wide range of genetic variation for the above mentioned morphological and developmental characters. Besides, it could be of interest to know the magnitude of variation due to heritable component, which in turn would be a guide for selection for the improvement of a population. In crop improvement programme, genetic variability for agronomic traits as well as quality traits in almost all the crops is important, since this component is transmitted to the next.Thus the knowledge of nature and extent of genetic variation and diversity available in the germplasm or breeding material helps the breeder for planning sound breeding programmes.

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Chapter 7
Antioxidants: The Modern Elixire

Dr. Manas Pratim Boruah

Antioxidants for health

Oxidation is essential in living organisms for the production of energy to fuel biological processes. Free radicals and other reactive oxygen species (ROS) such as hydroxyl radical (OH•), superoxide radical anion (O2•–), hydrogen peroxide (H2O2) etc comprise an entire class of highly reactive species derived from the metabolism of oxygen and is often generated as by-products of biological reactions or from exogenous factors [1-3]. Some of these ROS’s play positive roles in cell/physiology; however they may also cause great damage to cell membranes and DNA, including oxidation that causes membrane lipid peroxidation, decreased membrane fluidity, and other DNA mutations, leading to cancer, degenerative and other diseases [4]. Thus, there is an increased evidence for the participation of these free radicals in the aetiology of diseases like cancer, diabetes, cardiovascular diseases, autoimmune disorders, neurodegenerative disorders, ageing, etc. Almost all organisms are well protected against free radical damage by enzymes such as superoxide dismutase (SOD) and catalase, or low molecular weight compounds such as ascorbic acid, tocopherols and glutathione [5]. Catalase and hydroperoxidase enzymes convert hydrogen peroxides to non-radical forms and function as natural antioxidants in human body [6]. Thus, there are enzymatic and non enzymatic compounds to counteract the damaging effects of ROS, which are called antioxidants.

Antioxidants are agents which scavenge the free radicals and prevent the damage caused by them. The immune system is vulnerable to oxidative stress. Oxidative stress results when reactive oxygen species are not adequately removed. This can happen if antioxidants are depleted and/or if the formation of reactive oxygen species is increased beyond the ability of the defences to cope with them [7]. During certain diseased states as well as during ageing, there is a need to boost the antioxidant abilities, thereby potentiating the immune mechanism. The antioxidants preserve an adequate function of immune cells against homeostatic disturbances. Antioxidant supplementation constitutes important defence against variety of diseases and environmental stresses. Hence, antioxidant compounds in food play an important role as a health-protecting factor.

In human body, two categories of antioxidants are present. They are classified depending upon whether they are soluble in water (hydrophilic) or in lipids (hydrophobic). Generally water soluble antioxidants react with oxidants in the cell cytosol and blood plasma e.g., - Vitamin C, Glutathione, Lipoic acid and Uric acid while lipid soluble antioxidants protect cell membranes from lipid peroxidation e.g., - Carotenes, Vitamin E, Ubiquinol etc. [7].

Many synthetic antioxidants are reported to possess carcinogenic activities and also cause liver damage [8], so natural antioxidants are getting importance as they are safer and also possess antiviral, anti-inflammatory, anti-cancer, anti-mutagenic, anti-tumour, antimicrobial and hepatoprotective properties [9-11]. Natural antioxidants have already been isolated from varieties of plant materials such as leafy vegetables, fruits, seeds, cereals and algae [8]. Fruits and vegetables contain different antioxidant compounds such as Vitamin C, Vitamin E and Carotenoids, whose activities have been recently established. Therefore, the search for antioxidants from natural sources has received much attention, and efforts have been made to identify new natural resources for active antioxidant compounds. In addition, these naturally occurring antioxidants can be formulated to give nutraceuticals, which can help to prevent oxidative damage from occurring in the body.

Plants as source of Antioxidants

Plants are a rich source of antioxidants. Among the plant based antioxidants, ascorbic acid, tocopherols and carotenoids have been known for years. But in recent years, plant polyphenols have attracted attention of researchers. Polyphenols are a large group of natural compounds which possess wide spectrum of biological activity. Polyphenols play key role in antioxidant activity of plant extracts [12]. Phenolic compounds are commonly found in both edible and non-edible plants. Crude extracts of fruits, herbs, vegetables, cereals and other plant materials rich in phenolics are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food [13]. The importance of the antioxidant constituents of plant materials in the maintenance of health and protection from coronary heart disease and cancer is also receiving interest among scientists, food manufacturers and consumers as the trend of the future is moving toward functional food with specific health effects. Flavonoids and other phenolics have been suggested to play a preventive role in the development of cancer and heart disease.

Potential sources of antioxidant compounds have been searched in several types of plant materials such as vegetables, fruits, leaves, oilseeds, cereal crops, barks and roots spices and herbs, and crude plant drugs. Flavonoids and other plant phenolics, such as phenolic acids, stilbenes, tannins, lignans and lignin, are especially common in leaves, flowering tissues, and woody parts such as stems and barks [14]. They are important in the plant for normal growth development and defense against infection and injury. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. In addition, they have a metal chelation potential [15]. Some examples of plant polyphenolic structures are given in table 1.1.

Table - 1.1: Examples of some basic plant phenolic structures

illustration not visible in this excerpt

Antioxidant activity of medicinal plants: prophylactic action

In many countries such as India and China, where thousands of tribal communities still use folklore medicinal plants till today to cure sicknesses. The great interest in the use and importance of Indian medicinal plants by the World Health Organisation in many developing countries has led to intensified efforts on the documentation of ethnomedicinal data of medicinal plants [16-18]. Traditional medicinal practice has been known for centuries in many parts of the world for the treatment of various human ailments. According to World Health Organization (WHO), more than 80% of the world's population relies on plant-based traditional medicine for their primary healthcare needs [19]. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases [20]. The search for eternal health and longevity and for remedies to relieve pain and to heal diseases drove early man to explore the natural resources surroundings him particularly the plants. Nature has bestowed upon us a very rich botanical wealth and a large number of diverse types of plants grow wild in different parts of our country. In Asia, the use of different parts of several medicinal plants to cure specific illness has been in use from ancient times [21]. Use of herbal medicines in Asia represents a long history of human interactions with the environment.

The increasing interest on traditional ethno medicine may lead to discovery of novel therapeutic agents. Traditional healing systems around the world that utilize herbal remedies are an important source for the discovery of new antibiotics [22]; some traditional remedies have already produced compounds that are effective against antibiotic-resistant strains of bacteria [23]. The results of this indicate the need for further research into traditional health systems [24]. It also facilitates pharmacological studies leading to synthesis of a more potent drug with reduced toxicity [25, 26]. There are numerous reports on the use of plants in traditional healing by either tribal people or indigenous community [27-31].

In the last two decades, there has been an increasing interest in the role of antioxidant in human health [32]. Consequently, many medicinal plants have been investigated for evaluation of their antioxidant activities [33]. A medicinal plant contains many phytochemicals besides the actual active principle against certain disease. If these phytochemicals are found to have antioxidant activities, then the plant is not only important in view of its therapeutic action against the disease, but it may also have prophylactic action against that particular disease or some other related disease. Hence, due to the antioxidant action, the medicinal value of the plant is augmented.

Antimicrobial properties of the plants

The use of plant extracts and phytochemicals, with established antimicrobial properties, could be of great significance in preventive and/or therapeutic approaches. Numbers of investigations have identified compounds within herbal plants that are effective antibiotics [34]. Numerous studies have shown that medicinal plants are sources of diverse nutrient and non-nutrient molecules, many of which can protect the human body against both cellular oxidation reactions and pathogens. Several studies indicated that medicinal plants contain substances like phenols, quinonoids, flavonoids, coumarins, terpenoids, alkaloids, etc. These compounds are potentially significant in therapeutic applications against human and animal pathogens, including bacteria, fungi and viruses. Naturally derived compounds and other natural products may have applications in controlling bacteria in foods and other products. These compounds have been safe, have been shown to have varying degree of antimicrobial activity, and could provide another hurdle to growth of pathogens and spoilage bacteria, thereby improving the shelf-life of natural products. Numerous studies have reported that medicinal plants produce a large number of secondary metabolites with antimicrobial effects on pathogens [35, 36]. Medicinal plant extracts, therefore, for the control of the growth of pathogens and spoilage bacteria are emerging as alternatives to conventional natural preservatives as they are generally safe to humans, and environmentally friendly [37]. However, natural antimicrobial activity of medicinal plants and their essential oils is often variable. Most research on medicinal plants as natural antimicrobial has been conducted in vitro in microbiological media. The increasing prevalence of multi-drug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raised the specter of ‘untreatable’ bacterial infections and adds urgency to the search for new infection-fighting strategies [38-41]. Contrary to synthetic drugs, antimicrobials of plant origin usually are not associated with many side effects and have an enormous anti-infective potential in numerous infectious diseases. Some of the known antimicrobial phytochemicals are listed in table 1.2.

Table-1.2: Examples of phytochemicals having antimicrobial activities

illustration not visible in this excerpt

Hence the use of plant extracts and phytochemicals, both with known antimicrobial properties, can be of great significance in therapeutic treatments. In other words, medicinal plants are finding their way into pharmaceuticals, nutraceuticals, cosmetics and food supplements. Thus medicinal plants are one of the best resources for the invention and development of novel bioactive substances.

The use of antibiotics has revolutionized the treatment of various bacterial infections. In recent years, multiple drug resistance in human pathogenic micro-organisms has been developed due to indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of such diseases. Over the last three centuries, the intensive efforts have been made to discover clinically useful antimicrobial drugs [42-44]. Plant based antimicrobial drugs are of importance as these are found to be without side effects.

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43. Manas Pratim Boruah* and Jyotirekha G . Handique , Free Radicals and Their Biological Damage: Role of Antioxidants, Lambert Academic Publishing, Germany, 2013. ISBN: 978-3-659-47230-5

44. Jyotirekha G. Handique*, Manas Pratim Boruah and Dipika Kalita, Antioxidant Activities and Total Phenolic and Flavonoid Contents in certain Indigenous Medicinal Vegetables of North East India, Natural Product Communications , Vol. 7(8), 1021-1023, 2012.

Chapter 8
Phosphatase Activity: A study on indigenous Tea RhizospherePhosphate Solubilizing Microorganisms

Dr. Rakhi Phukan, R. Samanta, and B. K. Barthakur

Introduction

Research on phosphate solubilizing microorganisms’ dates back to 1960s. Recently research is ongoing towards enhancing the availability of native soil P by different crops.Microorganisms with phosphate solubilizing potential increase the availability of soluble phosphate and enhance theplant growth by improving biological nitrogen fixation (Ponmurugan andGopi, 2006). Bacteria were more effective in alkaline soils and fungi in acid soils ( Satter& Gaur 1985). When fungi dissolve P from rock phosphate, this P can again get converted to less soluble forms on ceasing the fungal growth. Phosphorus may exist in 2 forms: soluble and insoluble forms. Phosphorus ininsoluble forms, such as tricalcium phosphate [Ca3(PO4)2], iron phosphate (FePO4) and aluminiumphosphate (AlPO4), was unavailable to living organisms including microorganisms and plants. Therefore phosphate solubilizing organisms play a great role in soil.Phosphorus solubilizers play role in phosphorus nutrition by enhancing itsavailability to plants through release from inorganic and organic soil P pools by solubilization and mineralization.Principal mechanism in soil for mineral phosphate solubilization is lowering of soil pH by microbial production oforganic acids and mineralization of organic P by acid phosphatases (Ahmad et .al 2009). Enzyme activities have been proposed as a tool tomonitor changes in soil ecology resulting from the interactionsbetween inoculants and indigenous microbialpopulations of soil (Doyle and Stotzky, 1993).Phosphatase enzymemediates the release of inorganicphosphorus from organically bound phosphorusand returned it to soil (Jha et al.,1992). Hence, study of Phosphatase enzymes that releases phosphorus from organic compounds by dephosphorylation ofphospho-ester or phosphoanhydride bonds in organic matter (Rodriguez et .al 2006) is an important tool in initiating biofertilizer for specific crop.Acid and alkalinephosphatases catalyze such a reaction at acid and alkaline conditions respectively.

Occurrence of Phosphate solubilizing microorganisms in tea soil have been reported earlier (Patgiri&Bezbaruah1990; Selvaraj et al., 2006). Gnanachitra and Govindarajan (2002) screened Azospirillum cultures isolated from acid soils of tea grown areas in South India for P-solubilizing ability and found four out of 27 isolates to solubilize 2.00 to 3.25 per cent tri calcium (TCP) phosphate against Bacillusmegaterium which solubilized 5.65 per cent TCP. In this chapter detail methodology of phosphatase activity of rhizosphere phosphate solubilizing microorganism has been described. This type of study provides an effective alternate source of phosphate biofertilizer from microbial source for the tea industry.

Materials and Method

I. Isolation of phosphate solubilizing microbes:

Soil samples were collected randomly from 20 different commercial tea estates of Assam for isolation of phosphate solubilising. The soil samples were air dried and used for isolation following Dilution Plate Technique (Waksman, 1922) in Pikovaskaya’s mediawhich contains (per liter): 0.5 g yeast extract, 10 g Glucose, 5 g Ca3(PO4)2 0.5 g (NH4)2SO4, 0.2 g KCl, 0.1 g MgSO4.7H2O, 0.0001 g MnSO4.H2O, 0.0001 g FeSO4.7H2O and 15 g agar . For isolation, soil samples were serially diluted to 10-5 and incubated at 28 oC for seven days. At the end of incubation, PSM colonies were visuallyidentified by the formation of clear zone around the colony.

II. Screening of PSM :

To detect the phosphate solubilising microorganismsisolated strains were streaked onto Pikovskaya’s agar medium,. After 3 days of incubation at 28 oC ± 2, strains that produced clear zone around the colonies were considered as positive.

III. Measurement of Phosphatase activity:

The strains were cultured in Nutrient broth as well as Pikovaskaya’sbroth and used to study the phosphatase activity. Alkaline [Orthophosphoric Monoester Phosphohydrolase (EC 3.1.3.1)] and Acid [Orthophosphoric Monoester Phosphohydrolase (EC 3.1.3.2)] phosphatase activitywas estimated for 24 ,48, 72 and 96 hours of incubation to record the difference at different growth rate of the cultures. The total cell counts in inoculums were examined by the standard plate count method. The initial cell density of strain MM PSM 10 was 38 x 104 colony forming unit (CFU)/ ml for 24 hour incubated culture at room temperature while for MM PSM DR/BS the initial cell density was 11.5 x 106 CFU/ ml .

i) Alkaline [Orthophosphoric Monoester Phosphohydrolase (EC 3.1.3.1)]: The experiment was conducted following two protocols to estimate the enzyme activity. Firstly the whole Culture cells were centrifuged after imposing the treatment directly to the inoculums. In this experiment after incubating the culture of 24, 48, 72 and 96 hour for 60 minute , the reaction was stopped .Secondly, the culture cells were centrifuged at beginning , after this taking the supernatantand the cell suspensionseparately treatment was imposed andincubate it for 60 minute. Cultures were centrifuge at 10,000 rpm for 30 mins, temperature 25 0C at acceleration 9 in Hermle Z 36 HK Refrigerated Centrifuge in all the experiments. For alkaline phosphatase activity 1 ml of 5 M p -nitrophenol phosphate in 0.1 M Tris–HCL buffer (PH 9.5 ) was incubated with 1 ml properly diluted enzyme for 60 minutes at 30 ± 2 0C. The reaction is stopped with the addition of 3 ml of 0.1 N NaOH. The control is prepared by terminating the reaction at zero time. The absorbance of the solution was taken at UV-Visible spectrophotometer (Pharmacia ,Ultrospec mode) at 420 nm. Amount of p-nitro phenol (liberated) was calculated using standard curve of p-nitro phenol.

ii) Acid [Orthophosphoric Monoester Phosphohydrolase (EC 3.1.3.2)]: The assay of acid phosphatase will remain the same as alkaline phosphatase except the substrate p-nitro phenol phosphate will be dissolve in 0.05 N/ Citrate buffer (PH 4.5). (Ph.D thesis Choudhury.M.2002).1 ml of each culture cells, supernatant and cell suspension was added as per the experiment protocol. Reaction was incubated at 37 0C for 60 min and terminated by adding 3 ml of 0.1 N NaOH. Distilled water was used in the reaction instead of the culture cells, supernatants and cell suspension to prepare blanks. Hydrolysis of p -nitrophenyl phosphate was estimated by measuring the concentration of p -nitrophenol with a spectrophotometer at wavelength 420 nm. The concentration of p -nitrophenol was determined by comparison with a standard curve. One unit of enzyme activity is expressed as m moles p -nitrophenol liberated per min per mg protein.(Stumpf et.al 1946; Tso, S. C. and Chen, Y. R.

iii) Also from the experiment, Cell pellets were washed, resuspended in 10 ml sterile water and taken for protein estimation by Lowey et .al (1951) method at 660 nm.

Results

Table 1. Protein content of the screened phosphate solubilizing microbes during different growth phase.

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Values are mean of 3 replicas ± SD

Table2.Alkaline and acid phosphatase activity of MM PSM 10 at different growth period (Nutrient Broth)

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Table 3. Alkaline and acid phosphatase activity of MM PSM DR/BS at different growth period(Nutrient Broth)

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ND = Not Detected under present experimental conditions. Culture cells: cells with medium; Supernatant : Extracellular enzyme; Cell suspension: cell pellets suspended in water / cell bound enzyme.#SD = Standard Deviation

Table 4. Alkaline and acid phosphatase activity of MM PSM 10 at different growth period (Pikovskaya’s media)

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Table 5. Alkaline and acid phosphatase activity of MM PSM DR/BS at different growth period (Pikovskaya’s media)

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ND = Not Detected under present experimental conditions. Culture cells: cells with medium; Supernatant : Extracellular enzyme; Cell suspension: cell pellets suspended in water / cell bound enzyme.#SD = Standard Deviation

Discussion

The growth of the phosphate solubilizing microbes were determined in two different medium to observe the amount of phosphatase activity .Table 1 showed the protein content of the screened phosphate solubilizing microbes in Nutrient broth and Pikovskaya’s broth. The protein content of MM PSM 10 ranged from 66.3 ± 0.15 to 95.68 in Nutrient broth and 41.68 ±1.33 to 128.61 ± 4.95 in Pikovskaya’s broth. While in case of MM PSM DR/BS the protein content ranged from 164.8 ± 1.04 to 197.6 ± 1.05 in nutrient broth and 47.30 ± 7.37 to 196.37 ±8.53 µg/ml cultures in Pikovskaya’s Broth (Table1).

The present investigation reveals the production of both acid and alkaline phosphatase activity in the selected strains. The culture cells without disruption also depict both acid and alkaline phosphatase enzyme activities. Phosphatase activities were measured in cell free supernatants (extracellular enzyme) as well as in cell suspension (cell bound enzyme) in both the strains. MM PSM 10 strain which is a fungi ( Table 2) reveals a increasing trend of alkaline phosphatase activity during its incubation period from initial 24 hours culture ( 2.655 ± 0.043 ) to 96 hours culture ( 5.238 ± 0.055 ) in Nutrient broth. In MM PSM 10 highest enzyme activity was recorded at 96 hours grown culture (5.238 ± 0.055). The cell suspension record presence of more enzyme activity (2.765 ±0.001) than supernatant (0.773 ± 0.001).

While in case of Acid phosphatase 72 hour grown culture record more enzyme activity (3.207 ± 0.001) with a increasing trend from 24 to 72 hour but at 96 hour its activity drops to 0.007 ± 0.002. The acid enzyme activity was not recorded in cell suspension in 96hours in MM PSM 10.

The strain MMPSM DR/BS (Table 3) which is a bacteria shows highest alkaline phosphatase activity at 48 hour grown culture (1.249 ± 0.046) .The phosphatase activity shows no similar trend in case of MMPSM DR/BS both in supernatant and cell suspension. Acid phosphatase activity was not recorded at 24, 48 hour grown culture but at 72 hour it shows 0.631 ± 0.032 . This indicates that the culture is more active after 3 days of growth and produce specific enzyme. Both the strains show specific activity at Nutrient broth culture which means both strains adapt to different nutrient parameters.

Another experiment was conducted on Pikovskaya’s broth taking TCP (tri-calcium phosphate) as sole source of insoluble phospahte. The isolate MM PSM 10 recorded both alkaline and acid phosphatase activity in Pikovskaya’s broth at different hours of incubation (Table4).The alkaline phosphatase enzyme liberated increased with incubation time from 24 hours to 96 hours. The maximum alkaline enzyme liberated were 0.023±0.002, 0.037±0.006, 0.048±0.004 and 0.094±0.012 units/ml in the culture cells from 24 to 96 hours, respectively. The isolate MM PSM 10 liberated maximum acidic phosphatase enzyme at 72 hours grown culture cells (0.183 ± 0.004 units /ml).

Table 5 showed the activity of MM PSM DR/BS in Pikovskaya’s broth. The isolate MM PSM DR/BS also liberated both alkaline and acidic phosphatase enzyme. The maximum alkaline enzyme was liberated at 72 hours (0.066±0.004 units/ml).While in case of acid phosphatase enzyme only at 96 hours of incubation enzyme was liberated (0.023±0.002 units/ml).

Research shows that fungi have been reported to be more effective than bacteria as solubilizer of rock phosphate(Satter& Gaur 1985), while many bacteria are capable of dissolving tri calcium phosphate (Bardiya and Gaur 1972) . Ferric phosphate was best solubilized by Aspergillus niger (Varsha et.al 1994 ). Hence, from the above investigation and from the past records we can conclude that both the strains MM PSM 10 (Aspergillusniger) and MM PSM DR/BS (Bacteria) proves to be an alternative for increased yield by solubilizing the fixed or applied P in the soil due to its presence of acid and alkaline phosphatase activity. The use of PSM will improve the phosphate nutrient of both the tea plant and the soil.

Acknowledgement

Authors are thankful to the Director TRA, Tocklai for providing laboratory facilities and the support extended to carry out the investigation. This is a part of research during my thesis.

References

1. Ahmad, A. K, Ghulam .J, Mohammad S. A., Syed M. S.N, Mohammad .R. (2009): Phosphorus Solubilizing Bacteria: Occurrence, Mechanisms and their Rolein Crop Production. J. AGRIC. BIOL. SCI. 1(1):48-58.

2. Bardiya, M.C and Gaur, A.C ( 1972): Rock phosphatase dissolution by bacteria . Indian .J.Microbial. 12:269-271.

3. Choudhury.M.(2002) :Biochemical Characteristics of shade tree Rhizobium in tea gardens of Upper Assam with special reference to the effect of pesticides.:Ph.D thesis. Dibrugarh University.

4. Doyle, D.J., Stotzky, G. (1993). Methods for the detection of changes in the microbial ecology of soil caused by the introduction of micro-organisms. Microbial Rel. 2, 63–72.

5. Gnanachitra, M. and Govindarajan, K. (2002) Solubilization of insoluble phosphates by acid tolerant Azospirillum isolates. In: Natl. Symp. on Mineral Phosphate Solubilization, Eds. A. R. Alagawadi, P. U. Krishnaraj, M. S. Kuruvinashetti, C.K. Dodagoudar and K. S. Maheshkumar, Univ. Agric. Sci., Dharwad, pp 96.

6. Jha ,D.K., Sharma, G.D., Mishra, R.R.(1992) Soil microbial population numbers and enzyme activities in relation to altitude and forest degradation. Soil Biol. Biochem. 24, 761– 767.

7. Lowery,O.H,Rosebrough, N,J., Farr, A.L and Randall ,R.J.(1951): Protein measurement with Folin phenol agent . J.Biol.Chem. 193:265-275.

8. Patgiri, I. and Bezbaruah, B. (1990). Strains contributing to phosphorus mobilization in acid soils. Ind., J. Agri. Sci. 60: pp197-200.

9. Pikovskaya, R. I.(1948): Mobilization of phosphorus in soils in connection with vital activityof some microbial species, Microbiologia, 17, 362-370.

10. Ponmurugan, P. and C. Gopi. (2006). Distribution pattern and screening of phosphate solubilizing bacteria isolated from different food and forage crops. J. Agron. 5: pp 600-604.

11. Rodriguez, H., Fraga1, R., Gonzalez1, T. and Bashan, Y. (2006). Genetics of phosphate solubilization and its potential applications for improving plant growth-promoting bacteria. Plant and Soil, 287(1/2), 15-21.

12. Sattar, M.A and Gaur, A.C (1985): Characterization of phosphate dissolving microorganisms isolated from some Bangladesh soil samples. Bangladesh J.Microbial. 2:22-28

13. SelvarajVenkatesan and VeluKalaipandianSenthurpandian (2006): Comparison of enzyme activity with depth under tea plantation and forested sites in South India .In GeodermaVolm 137, Issues 1-2 pp 212-216.

14. Stumpf, P.K., Green, D.E and Smith,Jr. F.W (1946): Ultrasonic disintegration as a method of extracting bacterial enzymes .J.Bacteriol., 51,487-493.

15. Tso, S. C. and Chen, Y. R. (1997) : Isolation and characterization of a group III isozyme of acid phosphatase from rice plants, Botanical Bulletin of Academia Sinica, 38 , 245-250.

16. Varsha –Narian,Jugnu,Thakkar and Patel,H.H.1994.Isolation and screening of phosphate solubilizing fungi. Indian.J.Microbial. 34(2):113-118.

17. Waksman, A. (1922). Microbiological analysis of soil as an index of soil fertility II. Methods of the study of numbers of microorganisms in the soil. Soil Sci., 14, 283-298.

About the editors

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Devajani Boruah (b. 1980) was born and brought up at Dhakuakhana, a subdivision of Lakhimpur district of Assam. She obtained M.Phil and Ph.D from Dibrugarh University, Assam. She is working as an Assistant Professor in the Department of Chemistry, Silapathar Science College, Silapathar, Dhemaji, Assam. She published a good number of research papers in nationally and internationally reputed research journals. Besides, Dr. Boruah is the author of the book- Interaction of Amino Acids with Transition Metal Ions. Her area of interest are inorganic synthesis and catalysis.

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Archana Borah (17th January’ 1982). She has completed her B.Sc with major in Zoology from Dibrugarh University. She then completed her M.Sc in Life Sciences with specialization in Biochemistry from Dibrugarh University. She later persued her Ph.D in plant biochemistry from Dibrugarh University. During that period she published several research papers in both national and international Journal. She has also contributed two chapters in Book.

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Title
Recent Advances in Scientific Research
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2018
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V435253
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Devajani Boruah (General editor)Archana Borah (Editor), 2018, Recent Advances in Scientific Research, Munich, GRIN Verlag, https://www.grin.com/document/435253

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