Isolation and Characterisation of Protoberberines with Antioxidant, Antiproliferative and Protease Inhibitory Activity from Berberis Aristata

CONTENTS

Chapter 1 Introduction and review of literature
1.1 Introduction
1.2 Classification of antioxidants
1.3 Applications of antioxidants
1.4 Source of natural antioxidants
1.4.1 Berberis aristata
1.4.2 Carum carvi
1.4.3 Foeniculum vulgare
1.4.4 Glycyrrhiza glabra
1.4.5 Madhuca indica
1.4.6 Myristica fragrans
1.4.7 Nardostacy’s jatamansi
1.4.8 Swertia chirayita
1.4.9 Trachyspermum ammi
1.4.10 Zingiber officinale
1.5 Protoberberine

Chapter 2 Screening of medicinal plant extracts for antioxidant activity and phytochemicals
2.1 Introduction
2.2 Aim and Objectives
2.3 Materials and Methods
2.3.1 Preparation of plant extracts
2.3.2 DPPH radical scavenging assay
2.3.3 Superoxide radical scavenging assay
2.3.4 Hydroxyl radical scavenging assay
2.3.5 In-vitro inhibition of lipid peroxidation
2.3.6 Total antioxidant assay by FRAP method
2.3.7 Screening of medicinal plant extracts for phytochemicals
2.3.7.1 Tests for alkaloids
2.3.7.2 Tests for flavonoids and polyphenolics
2.3.7.3 Test for saponins
2.3.7.4 Test for tannins
2.4 Statistical analysis
2.5 Results and Discussion
2.6 Conclusion

Chapter 3 Activity guided isolation of antioxidants from B.aristata
3.1 Introduction
3.2 Aim and Objectives
3.3 Materials and Methods
3.3.1 Fractionation of ethanolic extract of B.aristata
3.3.2 Antioxidant activity
3.3.3 Phytochemical analysis
3.3.3.1 UV-Visible absorption spectral analysis
3.3.3.2 Qualitative analysis
3.3.3.3 Quantification of total alkaloids
3.3.3.4 Quantification of total phenolics
3.3.3.5 Quantification of total flavonoids
3.4 Statistical analysis
3.5 Results and Discussion
3.6 Conclusion

Chapter 4 Antioxidant potential of ethyl acetate fraction against oxidative stress in E.coli model, on erythrocytes and DNA
4.1 Introduction
4.2 Aim and Objectives
4.3 Materials and Methods
4.3.1 Effect of H2 O2 induced oxidative stress in E.coli model
4.3.2 Effect of AAPH radical induced oxidative stress on RBC
4.3.3 DNA fragmentation assay
4.4 Statistical analysis
4.5 Results and Discussion
4.6 Conclusion

Chapter 5 Purification and characterisation of antioxidant alkaloids from B.aristata
5.1 Introduction
5.2 Aim and Objectives
5.3 Materials and Methods
5.3.1 Purification using silica gel column chromatography
5.3.2 High performance liquid chromatography
5.3.3 Structure prediction
5.3.3.1 UV-Visible absorption spectral analysis
5.3.3.2 IR spectroscopy
5.3.3.3 Nuclear magnetic resonance spectroscopy
5.4 Results and Discussion
5.5 Conclusion

Chapter 6 In silico docking studies of berberrubine, jatrorrhizine and thalifendine with caspase 3, cathepsin B, MMP-9 and telomeric DNA
6.1 Introduction
6.2 Aim and Objectives
6.3 Materials and Methods
6.3.1 Retrieval of receptors from Protein DataBank (PDB)
6.3.2 Designing of ligands
6.3.3 Docking with hex v6.3
6.3.4 Docking with iGEMDOCK v2.1
6.4 Results and Discussion
6.5 Conclusion

Chapter 7 Antiproliferative and protease inhibitory activities of protoberberines
7.1 Introduction
7.2 Aim and Objectives
7.3 Materials and Methods
7.3.1 Antiradical activity of berberrubine, jatrorrhizine and thalifendine
7.3.2 Cell proliferation assay
7.3.3 In vitro inhibition of cathepsin B enzyme activity
7.3.4 In vitro inhibition of MMP-9 enzyme activity
7.4 Statistical analysis
7.5 Results and Discussion
7.6 Conclusion

Bibliography

DECLARATION

I hereby declare that this thesis comprises of my own work except where specifically stated to the contrary and it is not substantially the same, to the best of my knowledge as any thesis which has been submitted at any other university for the award of any degree or diploma.

Preface:

Antioxidants as vital substances protecting cells from oxidative damage of biological molecules like DNA, proteins, lipids. Antioxidants find applications in medicine as immunoregulators, antiageing agents and inducers of apoptosis and regulators of cell cycle in some cancer cell lines. Studies have suggested that medicinal plants are potential and inexpensive source of antioxidants. Medicinal plants are endowed with large number of bioactive compounds such as glycosides, flavonoids, tannins, lignans, terpinoids, phenols and alkaloids. The use of certain medicinal plants in folk medicine for treating various ailments especially radical related damage, inflammation and cancer is widely practiced.

Berberies aristata is belongs to family Berberidaceae grown in Himalayas, Nepal and Bhutan. It is commonly called as Daruhaldi or Chitra and is distributed in temperate and sub-tropical parts of Asia, Europe and America. Traditionally, B. aristata is used for the treatment of skin diseases, inflammation, diarrhoea and jaundice. The decoction prepared from the roots is used for cleaning infected wounds and ulcers and also reported to promote healing and cicatrisation.

The ethanolic and aqueous extracts of root reported to show significant anti-bacterial, anti-fungal and anti-depressant activities . The alcoholic root extract showed DNase, RNase, aldolase, alkaline phosphatase, acid phosphatase, amylase and protease inhibitory activity. Alcoholic root extract exhibited antidiabetic, anti-inflammatory and antiradical activities.

In the present study, some of the Indian medicinal plants popular in folk medicine are considered to screen for their antioxidant activities using DPPH, superoxide, hydroxyl and peroxide radical scavenging assays. Among them ethanolic roots extract B.aristata exhibit highest antioxidant ability and least IC50 value.

Hence, ethanolic extract of B.aristata was subjected to activity guided isolation of antioxidants using liquid-liquid fractionation and silica gel column chromatography. The structures of the isolated antioxidants were predicted as berberrubine, jatrorrhizine and thalifendine based on UV- visible, IR and NMR spectral data. All these compounds exhibited significantly high antioxidant activity in terms of DPPH radical scavenging potential. In silico docking of berberrubine, jatrorrhizine and thalifendine with caspase 3, MMP-9, cathepsin B and telomeric DNA reveals that all the three compounds formed considerably stable complex. Further, three compounds exhibited antioxidant, antiproliferative and antiproteases activities.

The thesis has been broadly divided into seven chapters. In chapter I, introduction, survey of literature with reference to antioxidants, therapeutic applications of medicinal plants.

Chapter 2 included screening of different solvent extracts of medicinal plants for their antioxidant potential against different radicals like DPPH, superoxide, hydroxyl and peroxide. Among them ethanolic roots extract B.aristata exhibit highest antioxidant ability and least IC50 value.

Chapter 3 comprises with the In addition, the IC50 for DPPH radical scavenging and inhibition of lipid peroxidation were significantly less compared to crude ethanol extract indicating quality of antioxidants in ethyl acetate fraction. Spectral and qualitative analysis revealed the presence of alkaloids, phenolics and flavonoids. Thus upon quantitative analysis, the results confirm the high levels of alkaloids compared to polyphenolics and flavonoids. Hence, ethyl acetate fraction of root extract of B.aristata is potential source of antioxidant alkaloids.

Chapter 4 deals with the protective role of ethyl acetate fraction of B.aristata against hydrogen peroxide induced stress in E.coli and APPH induce lysis in RBC models indicating the protective effect of B.aristata on lipid and protein peroxidation and H2O2 induced lymphocyte genomic DNA fragmentation respectively, as membrane lipids, proteins and nucleic acid are highly susceptible to free radicals associated damage.

In chapter 5, purification of antioxidant compounds from ethyl acetate fraction by silica gel column chromatography, purity by TLC and HPLC, structure prediction by UV- visible, IR and NMR spectroscopic methods was presented. Three different protoberberines were purified and their structure was predicted as berberrubine, jatrorrhizine and thalifendine. These three compounds exhibited significantly high antioxidant activity in terms of DPPH radical scavenging potential.

Chapter 6 deals with docking of protoberberines with ECM proteases, caspase-3 and telomeric DNA. In silico docking study of berberrubine, jatrorrhizine and thalifendine with caspase 3, MMP-9, cathepsin B and telomeric DNA reveals that all the three compounds formed considerably stable complex with caspase 3, cathepsin B and telomeric DNA, but berberrubine and thalifendine did not show significant results with MMP-9. Hence, considering these results further their antiproliferative and antiprotease activity was determined by in vitro methods using cancer cell lines and enzymatic inhibition assay.

Chapter 7 comprised with the antioxidant, antiproliferative and antiprotease effects of these protoberberines. Berberrubine, jatrorrhizine and thalifendine exhibited high antioxidant activity and this property may be due to the presence of free phenolics group in their structure as reported earlier. Further, antiproliferative activity of berberrubine, jatrorrhizine and thalifendine on ovarian, lymphoma and colon on cancer cell lines reveals that berberrubine exhibited high antiproliferative activity on the cell lines followed by thalifendine and jatrorrhizine. These compounds were as effective as doxorubicin, a well known anticancer drug. In addition, berberrubine and thalifendine was more effective in inhibiting cathepsin B activity, but jatrorrhizine was effective against MMP-9.

In end the principal findings of the present work have been summarized followed by references cited have been listed alphabetically.

ACKNOWLEDGEMENTS

Many people contributed to this thesis in innumerable ways. I have immense pleasure in expressing my sincere gratitude and profound sense of indebtness to my teacher and research supervisor Dr. Rama Rao Malla, Department of Biochemistry, GITAM University, Visakhapatnam, for suggesting the problem, able guidance and meticulously assessing the progress of the work at every stage. I am very appreciative of his generosity with his time, advice, to name a few of his contributions.

I express my thanks to Prof. Y.L.N. Murthy, Dr. C. Kamakshi and K. Suhasini, Department of Organic Chemistry, Andhra University, for spectral analysis.

I express my sincere gratitude to Dr. K.V.V.V Satyanaraya, Department of Chemistry, GITAM University, for interpretation of IR and NMR spectral data.

My Thanks to Dr. V. Rama Rao, Secretary and correspondent, M.V.R College of PG studies, Visakhapatnam, for moral support and providing lab facilities for successful completion of my project.

I express my thanks to Dr. P.V. Arjun Rao for his valuable suggestions during course of this project.

I am appreciative to G. Kishore, University of Hyderabad , for providing cell lines and carrying MTT assay. Sudheer Kumar Rai, Tezpur University for providing chemicals to maintain the continuity of my project.

I would like to extend my deepest gratitude to my family, my parents Yogendra Prasad Singh, Raj Rani Devi, my brother Kameshwar Singh, my sister Reema Singh, and my supporting husband Alok Kumar Nanda. They always have provided unwavering love and encouragement. Thank you for believing in me.

ABBREVIATIONS USED

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LIST OF TABLES

Table 1: Common and scientific names of indigenous plants used for the treatment of various ailments like inflammation, hepatic disorder and cancer

Table 2: Effect of medicinal plant extracts on DPPH radical scavenging activity

Table 3: Effect of different concentrations of ethanolic extracts on DPPH radical scavenging activity

Table 4: Effect of different concentrations of ethanolic extracts on superoxide radical scavenging activity

Table 5: Effect of different concentrations of ethanolic extracts on hydroxyl radical scavenging activity

Table 6: Effect of different concentrations of ethanolic extracts on inhibition of lipid peroxidation

Table 7: Effect of different concentrations on ferric iron reducing ability (FRAP)

Table 8: Effect of different concentrations of ethanolic extracts B.aristata on various radicals

Table 9: Screening of ethanolic extracts of medicinal plants for phytochemicals

Table10: DPPH radical scavenging activity and inhibition of lipid peroxidation of fractions at 1000 µg/ml

Table 11: Comparison of antiradical and IC50 values of ethanolic extract and ethyl acetate fraction

Table 12: Phytochemical screening of ethyl acetate fraction of B.aristata using qualitative assays

Table 13: Quantification of total alkaloids, phenols and flavonoids

Table14: Column purification of ethyl acetate fraction.

Table 15: Binding energy values (kcal/mol) of protoberberines with receptors

Table 16: Comparison of IC50 values at different levels of extraction and purification

LIST OF FIGURES

Figure 1: IC 50 values of ethanolic extracts for DPPH radical scavenging

Figure 2: IC 50 values of ethanolic extracts for superoxide radical scavenging.

Figure 3: IC 50 values of ethanolic extracts for hydroxyl radical scavenging

Figure 4: IC 50 values of ethanolic extracts for inhibition of lipid peroxidation

Figure 5: Ferrous ammonium sulphate calibration curve for estimation of total antioxidant ability

Figure 6: Comparison of total antioxidant ability of ethanolic extracts for medicinal plants

Figure 7: Effect of different concentrations of ethyl acetate fraction on DPPH radical scavenging activity

Figure 8: Effect of different concentrations of ethyl acetate fraction on inhibition of lipid peroxidation

Figure 9: U.V-Visible maxima of ethyl acetate fraction.

Figure 10: Atropine standard curve for estimation of total alkaloid

Figure 11: Gallic acid standard curve for estimation of total phenolics

Figure 12: Quercetine standard curve for estimation of total flavonoids

Figure 13: Effect of different concentrations of hydrogen peroxide induced oxidative stress on viability of E.coli cells

Figure 14: Effect of different concentrations of ethyl acetate fraction against hydrogen peroxide induced oxidative stress in E.coli

Figure 15: Effect of different concentrations of ethyl acetate fraction on APPH induced red blood cell lysis

Figure 16: Effect of different concentrations of ethyl acetate fraction on hydroxyl radical-induced DNA fragmentation

Figure 17: HPLC chromatograms of isolated compounds BA1, BA2 and BA3

Figure 18: UV-Visible absorption spectra of BA1, BA2 and BA3

Figure 19: IR spectra of BA1

Figure 20: IR spectra of BA2

Figure 21: IR spectra of BA3

Figure 22: NMR spectra (C13) of BA1

Figure 23: NMR spectra (C13) of BA2

Figure 24: NMR spectra (H1) of BA3

Figure 25: Determination of Melting point of BA1, BA2 and BA3

Figure 26: Predicted structure of berberrubine (BA1)

Figure 27: Predicted structure of jatrorrhizine (BA2)

Figure 28: Predicted structure of thalifendine (BA3)

Figure 29: Docking pose of caspase 3 with doxorubicin

Figure 30: Docking pose of caspase 3 with berberine

Figure 31: Docking pose of caspase 3 with berberrubine

Figure 32: Docking pose of caspase 3 with jatrorrhizine

Figure 33: Docking pose of caspase 3 with thalifendine

Figure 34: Docking pose of MMP-9 with doxorubicin

Figure 35: Docking pose of MMP-9 with berberine

Figure 36: Docking pose of MMP-9 with berberrubine

Figure 37: Docking pose of MMP-9 with jatrorrhizine

Figure 38: Docking pose of MMP-9 with thalifendine

Figure 39: Docking pose of cathepsin B with doxorubicin

Figure 40: Docking pose of cathepsin B with berberine

Figure 41: Docking pose of cathepsin B with berberrubine

Figure 42: Docking pose of cathepsin B with jatrorrhizine

Figure 43: Docking pose of cathepsin B with thalifendine

Figure 44: Docking pose of telomeric DNA with doxorubicin

Figure 45: Docking pose of telomeric DNA with berberine

Figure 46: Docking pose of telomeric DNA with berberrubine

Figure 47: Docking pose of telomeric DNA with jatrorrhizine

Figure 48: Docking pose of telomeric DNA with thalifendine

Figure 49: Effect of different concentrations of berberrubine, jatrorrhizine and thalifendine on DPPH radical scavenging activity

Figure 50: Effect of different concentrations of berberrubine, jatrorrhizine and thalifendine on inhibition of lipid peroxidation

Figure 51: Effect of berberrubine, jatrorrhizine and thalifendine on hydroxyl radical induced DNA fragmentation

Figure 52: Effect of different concentrations of berberrubine, jatrorrhizine and thalifendine on proliferation of COLO205 cell line

Figure 53: Effect of different concentrations of berberrubine, jatrorrhizine and thalifendine on proliferation of SupT1 cell line

Figure 54: Effect of different concentrations of berberrubine, jatrorrhizine and thalifendine on proliferation of A2780 cell line

Figure 55: Comparison of IC50 values of berberrubine, jatrorrhizine, thalifendine and doxorubicin

Figure 56:Inhibition of cathepsin B enzyme activity by berberrubine, jatrorrhizine and thalifendine

Figure 57:Inhibition of Matrix metalloproteinase -9 enzyme activity by berberrubine, jatrorrhizine and thalifendine

Chapter 1

Introduction and Review of Literature

1.1 Introduction

Antioxidants are vital substances required in small quantity to prevent oxidation of biological molecules like DNA, proteins, lipids and also to maintain normal homeostasis in cell (Parola, 2001). To protect cell from oxidative stress, a highly sophisticated and complex antioxidant system has been evolved, which includes endogenous and exogenous systems working interactively and synergistically. A variety of free radical scavenging agents exists and they may be derived from different dietary sources (Effat et al., 2008). Various studies have also suggested that medicinal plants are potential and inexpensive source of antioxidants as they are enriched with bioactive compounds (Joy et al., 1998).

The history of antioxidant discovery dates back to several decades, but most of them took place in the 19th and 20th centuries. The first documented evidence regarding existence of antioxidants came from the Berthollet’s theory of catalytic poisoning of oxidative reactors (Moureu and Dufraisse, 1926). However, first report on the use of antioxidants to prevent lipid peroxidation came from Deschamps study (Wanasundara and Shahidi, 2005). Moureu and Dufraisse (1928) have reported the role of synthetic chemical compounds in delaying the oxidative decomposition of dietary lipids. In the early 1960’s, the concept of antioxidant mechanisms evolved as a significant area of research in medicine. In the last two decades, search for novel antioxidants from natural sources has emanated and myriad number of antioxidants has been reported as therapeutic agents in medicine.

The main role of antioxidants is to delay or prevent the deleterious effects of reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated during metabolic reactions (Buyukokuroglu, 2001). Generally, ROS are eliminated by endogenous antioxidant defence system (Valko, 2006; Chatterjee et al., 2007) but defect in this system leads to threat for cellular components (Bayani et al., 2009). Oxidative stress is one of the major source of radicals and causing disorders like multiple sclerosis, Alzheimer’s, Parkinson’s (Smith et al., 2000; Bolton et al., 2000; Guidi et al., 2006) and cardiovascular diseases (Subhash, 2010). In addition, the role ROS and RNS have been extensively studied in cancer initiation and progression (Lara et al., 2010).

For a long time, synthetic antioxidants are in use, but due to their toxicity and mutagenicity, in the recent past their usage is limited (Nagulendran et al., 2007). In this scenario, there is scope for extensive research in the field of medicinal plants for exploring natural bioactive compounds as antioxidants (Saikat et al., 2010). Reports also demonstrated that natural antioxidants from medicinal plants are compatible with human body (Dash et al., 2007).

1.2 Classification of antioxidants

Mechanism of antioxidant action involves donation of its hydrogen to radicals to form underactive intermediate which are less reactive in propagation of reaction and leads to termination of free radicals (Buettner, 1993; Decker, 2002). Lately, Pham et al., (2007) proposed new mechanism of action of antioxidants. According to this concept antioxidants scavenge free radical either by donating proton or by electron transfer as shown below.

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Based on the mechanism of action, antioxidants can be classified as primary, secondary and sequestering/ chelating antioxidants. Primary antioxidants act as free radical scavengers and delay or inhibit initiation and propagation of free radicals formation cascade. Examples for primary antioxidants are mono and polyhydroxy phenols (Wanasundara and Shahidi, 2005). Secondary antioxidants slow down the oxidation reaction without converting free radicals to stable molecules. They act by chelating pro-oxidants, deactivating singlet oxygen by providing hydrogen to primary antioxidant. The secondary antioxidants citric acid, succinic acid and ethylenediaminetetreacetic acid chelates metal ions, ascorbic acid and erythrobic acid reduces singlet oxygen and carotenoids quench singlet oxygen (Mielnik et al., 2003). Sequestering antioxidants including transferrin, lactoferrin, ferritin and carnosin sequester / chelate metal ions and ceruloplasmin and albumin sequesters copper (Decker et al., 2001).

Based on nature, antioxidants are classified into natural and synthetic antioxidants (Shiv kumar, 2011). Natural antioxidants are low molecular weight molecules with different physical, chemical and biological properties. These include enzymatic antioxidants and non-enzymatic antioxidants. Enzymatic antioxidants such as superoxide dismutase (SOD), catalase and glutathione peroxidase inhibit generation of ROS by removing potential oxidants or by transforming ROS/RNS into relatively stable compounds. Non-enzymatic antioxidants delay or inhibit cellular damage mainly through their free radical scavenging property (Chan, 1998; Pryor, 2000; Temme et al., 2001; Padayatty, 2003). Tocopherol, carotenoids, quinones, bilirubin, polyphenols, ascorbic acid, uric acid and alkaloids are some of the non -enzymatic antioxidants.

1.3 Applications of antioxidants

Antioxidants have been bestowed with diverse functions like prevention of deterioration and maintenance of nutritive value of foods (Bahman et al., 2009). They also have great potential to act as immunoregulators (Wilankar et al., 2011). Moreover, antioxidants are also known to regulate the expression of hormones like T4, leptin and adipokine and fight against ageing (Al-suhaimi, 2011). Antioxidants were reported to induce apoptosis, cell cycle arrest and also inhibit angiogenesis, metastasis in some cancer cell lines (Lopez et al., 2011; Lara Gibellini et al., 2011).

Antioxidants also find a valuable position in modern system of medicine especially in the treatment of cystic fibrosis, acquired immunodeficiency and skin diseases (Hamid et al., 2010; Ratz et al., 2012). Lee et al., (2011) reported the importance of antioxidant therapy in premature infancy. Ozten et al., (2011) emphasised the efficacious use of lycopene in treatment of prostate cancer. Withanone, a natural alkaloid shown to have protective role against methoxyacetic acid induced cytotoxicity in normal human fibroblasts (Priyandoko et al., 2011). Zhao et al., (2012) reported the importance of theanine, an antioxidant in the treatment of neurodegenerative diseases.

Some of the natural antioxidants also used in the food industry for the prevention of peel browning during post harvesting process of certain foods (Xuqiao et al., 2004). Natural antioxidants from the plant rosemary are used for maintaining the quality of paprika (Jorge et al., 1997). Another antioxidant, lignin used in cosmetic industry and topical formulation (Vinardell 2008). Antioxidants like oleoresin, ascorbic acid and coniferyl alcohol are used as a preservative and to increase the shelf life of frozen fish, canned mushrooms, meat, juice and general foods (Mielnik et al., 2003; Pinedo et al., 2007; Pereira et al., 2012).

1.4 Sources of natural antioxidants

Based on the biological diversity, three major sources of natural antioxidants have been identified including micro-organisms, animal products and medicinal plants. Microorganisms are most diversified living organisms on the earth and are good source of antioxidants like phenolics, flavonoids, pestacins and graphislactone A (Newman and Cragg, 2007; Liu et al., 2010; Mariana et al., 2011). Animal products can also serve as source of natural antioxidants including protein hydrolysates, chitosan, vitamin E and carotenoids (Decker et al., 2000; Surai, 2003; Elzbieta et al., 2008). Medicinal plants contain an innumerable number of bioactive constituents with antioxidant activity including glycosides, flavonoids, tannins, lignans, phenols and alkaloids (Latha and Daniel, 2001). The source of antioxidant may be a whole plant or a part of plant such as leaves, stem, bark, root, flower and seeds or excretory products like gum, resin and latex (Joy, 1998).

Antioxidant activities of several medicinal plants have been reported several centuries ago (Ottolenghi et al., 1959; Osawa et al., 1981; Rafatullah et al., 1993; Mackeen et al., 1997). Sabu et al., (2002) demonstrated the antioxidant activity in Terminalia chebula, Terminalia belerica and Emblica officinalis. Later Reddy et al., (2005) reported the antidiabetic and antioxidant activity in seeds of Hydnocarpus wightiana. Aggarwal et al., (2007) reported the antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal and anticancer activities of Curcuma longa. Recently, phenolic acid content, antioxidant and antimicrobial activity of Lingusticum mutellina was reported (Sieniawska et al., 2012). In the present study, some of the Indian medicinal plants popular in folk medicine are considered to screen for their antioxidant activities (Table 1).

Table 1: Common and scientific names of indigenous plants used for the treatment of various ailments like inflammation, hepatic disorder and cancer

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1.4.1 Berberis aristata

Berberis is a genus which belongs to family Berberidaceae with nearly 450-500 species of shrubs or small trees. Berberis aristata is a spinous shrub grown in Himalayas, Nepal and Bhutan. It is commonly called as Daruhaldi or Chitra and is distributed in temperate and sub-tropical parts of Asia, Europe and America (Chopra et al., 1956). These species are well known for their curative properties in folk medicine from several centuries (Sharma et al., 2011).

It is an erect spiny shrub grows upto 2 to 3m in height with hard and yellow coloured wood. The bark appears yellowish brown from outside and dark yellow from inside. The leaves are in tufts, lanceolated, toothed and sessile. The flowers are stalked, yellow coloured, complete and inflorescence is a simple corymbose racemose. The fruits are globose to ovoid with violet colour and contain 2 to 5 seeds (Parmar et al., 1982; Rashmi et al., 2008).

Traditionally, B. aristata is used for the treatment of skin diseases, inflammation, diarrhoea and jaundice (Rashmi et al., 2008). Crude extract from leaves is demonstrated to have hepatoprotective activity (Gilani et al., 1995) and crude extract from fruits have hypocholesterolemic activity (Janbaz et al., 2000). Sharma et al., (2011) reviewed that B.aristata stem have anticancerous activity on HT29 cell lines.

The decoction prepared from the roots is used for cleaning infected wounds, ulcers and also reported to promote healing and cicatrisation (Khory and Kartak, 1985; Kirtikar and Basu, 1995). The ethanolic and aqueous extracts of root showed significant anti-bacterial and anti-fungal activity (Sharma et al., 2008). Kulkarni et al., (2008) reported anti-depressant activity of berberine, an alkaloid from B.aristata. The alcoholic root extract showed inhibitory activity against DNase, RNase, aldolase, alkaline phosphatase, acid phosphatase, amylase and protease (Sohni et al., 1995). Anti-platelet activating factor (PAF) activity of alcoholic root extract was reported by Tripathi et al., (1996). The ethanolic root extract of B.aristata was reported to exhibit antidiabetic effect in alloxan-induced diabetic rat models (Semwal et al., 2009). Shahid et al., (2009) reported that the aqueous and alcoholic root extracts of B.aristata showed significant anti-inflammatory activity. The alcoholic root extract of B.aristata was reported to exhibit strong antiradical activity (Singh et al., 2009). Adhikay, (2010) reported the nephroprotective activity of stem bark decoction of B.aristata. Yogesh et al., (2011) reported anti-osteoporosis activity of aqueous-methanolic root extract in ovariectomized rats. Mittal et al., (2012) reported the antidiabetic, antihyperlipidemic and cytoprotective activity of ethanolic root extract of B.aristata.

Rasanjana or Rasaut or Rasavanti is a thick aqueous extract mainly prepared from root bark of B.aristata. It is generally prepared by boiling the chopped roots in water for 5-6 h until its consistency becomes syrupy (Rajasekaran et al., 2009). The rasaut is a bitter tonic and reported to be used as cholagogue, stomachic, astringent, laxative, diaphoretic, antipyretic, and antiseptic, also used in treatment of conjunctivitis, ulcers and haemorrhoids (Rajasekaran et al., 2009).

B. aristata is reported as rich source of protoberberines like berberine, berbamine, palmatine, oxyacanthine, aromoline, oxyberberine, karachine, taxiamine, pseudopalmatinechloride, pseudoberberinechloride, pakistanine, tetrahydropalmatine (Chatterjee, 1951; Bhakuni et al., 1968; Blasko, 1982; Blasko et al., 1982; Lect et al.,1983).

1.4.2 Carum carvi

This belongs to the family Apiaceae, commonly called as caraway. It is an annual herb, grown in India, Iran and Egypt. The leaves are pinnately dissected, flowers are white, dense and umbel. The fruits are schizocarp, yellow brown in colour and aromatic (Zargari et al., 1996). The fruit used in as carminative, mild stomachic and in the treatment of dyspepsia, hysteria, microbial infection and gingivitis (Thangam et al., 2003). Methanolic extract was reported to show antibacterial activity against multidrug resistant Salmonella typhi (Rani et al., 2004). Antifertility activity of C.carvi on female mice was reported by Priya et al., (2012).

P-cymene, a hydrophobic compound with antibacterial activity was reported in the essential oils of C.carvi (Burt et al., 2004). Germacrene D trans-dihydrocarvone, dillapiole, germacrene, β-selinene, nothoapiole, Carvone, limonene and monounsaturated fatty acids (MUFA) were reported in essential oils of seeds (Bochra et al., 2011).

1.4.3 Foeniculum vulgare

Foeniculum vulgare (fennel) belongs to the family Apiaceae, is a widespread annual plant with aromatic odour. Its fruits are used as a culinary spice in many countries (Traboulsi et al., 2005). It is used for the treatment of bronchitis (Boskabady et al., 2004) and gastric mucosal lesions (Birdane et al., 2007). Methanolic extract is used in the treatment of dementia and Alzheimer’s disease (Joshi, 2006). Fennel seeds have been used in the treatment of various gastric and respiratory disorders (Raffo et al., 2011).

The seeds, leaves and fruits are reported to exhibit antioxidant activity (Surveswaran et al., 2007; Sandhu et al., 2005; Marino et al., 2007). Anticancerous activity of fennel was reported by Celik and Islik, (2008). Recently, antioxidant and antimicrobial activity of F.vulgare was reported by Martin et al., (2012).

Presence of flavonoids such as quercetin, arabinoside, 3-caffeoylquinic acid, 4-caffeoylquinic acid, 1.5-Odicaffeoylquinicacid, rosmarinic acid, eriodictyol-7-Orutinoside, quercetin-3-O-galactoside, kaempferol-3-Orutinoside and kaempferol-3-O-glucoside were reported in fennel with antioxidant ability (Harborne et al., 1971, 1972, 1984). The fruits reported to contain characteristic fatty acid, petroselinic acid (Charvet et al., 1991; Reiter et al., 1998). Prominent components like trans-anethole, trans-anisole, estragole, fenchone and 1- octen-3-ol were identified in the essential oil (Mimica-Dukie et al., 2003; Diaz-Maroto et al., 2005). Parejo et al., (2004) reported the presence of hydroxycinnamic acid and its derivatives, such as flavonoids glycosides and flavonoids aglycones. Recently, chemical constituents including fatty acids, phenylpropanoids, monoterpenoids, sesquiterpenes, coumarins, triterpenoids, tannins, flavonoids, cardiac glycosides and saponins were also reported in fennel (Weiping He, et al., 2011).

1.4.4 Glycyrrhiza glabra

Glycyrrhiza glabra, also known as licorice or sweetwood, grown in Mediterranean countries and China. It is a shrub belonging to the family Leguminosae with oval leaflets, white to purplish flower in clusters and flat pods (Olukoga et al., 1998). The licorice reported to possess steroid like anti-inflammatory activity and platelets aggregation (Okimasu et al., 1983; Ohuchi, 1982). Moreover, licorice extracts have been used to treat chronic hepatitis and also to inhibit the growth of Cytomegalovirus (CMV), and Herpes simplex viruses (Pompei, 1979; Partridge, 1984; Numazaki, 1994). Studies indicate that Glycyrrhizin reduces ischemia reperfusion induced lipid peroxidation in animal models (Nagai, 1991). Deglycyrrhizinated licorice (DGL) is an herbal supplement which has been used in treatment of ulcers, skin eruption healing, psoriasis and herpetic lesions (Goso et al., 1996). Glycyrrhiza obtained from the root and is often used as flavouring and sweetening agent and also as an expectorant (Obolentseva et al., 1999). Licorice was reported to inhibit abnormal cell proliferation in breast, liver and skin cancers (Tamir, 2000; Shiota et al., 1999). In 2001, Tamir et al., demostrated that isoflavones glabridin and hispaglabridins A and B from G.glabra exhibited estrogen like and antioxidant activities. In addition, glycyrrhizin and glabridin reported to inhibit the formation of ROS at the site of inflammation (Wang and Nixon, 2001). Earlier studies have demonstrated that isoflavones from licorice inhibit iron induced mitochondrial lipid peroxidation in rat liver cells (Haraguchi and Yoshida et al. 2000). Licorice constituents also showed hepatoprotective activity by lowering liver enzyme levels and improving tissue pathology in hepatitis patients (Van Rossum, 2001). Ojha et al., (2011) demonstrated the cardioprotective role G. glabra in myocardial infarction. Qamar et al., (2012) reported that G.glabra showed reduction of caspase activity in Benzo (a) pyrene induced lung injury. Recently, Lateef et al., (2012) reported the antiurease activity of ethyl acetate fraction.

Licorice contains flavonoids like liquiritin, isoliquiritin, which impart yellow colour to the plant (Yamamura et al., 1992). In addition, numerous active compounds were isolated from this plant including triterpenes, saponins, flavonoids, polysaccharides, pectins, simple sugars, amino acids, mineral salts and various other substances (Obolentseva et al., 1999).

1.4.5 Madhuca indica

Madhuca indica, synonym of Madhuca longifolia belongs to the family Sapotaceae, is an important economic tree growing throughout India. The flowers, seeds and seed oil of Madhuca have great medicinal value. Bark is used as a remedial agent for itching, swellings, fracture and snake bites (Khaleque et al., 1969). Traditionally, M. longifolia bark has been used against rheumatism, ulcers, bleeding and tonsillitis (Khare, 2000). Pawar, (2004) reported the inhibitory effect of madhucoside A and B on superoxide in phagocytes. Mahua oil is used to cure skin diseases and also for the treatment rheumatism, headache, piles and haemorrhoids (Eichler, 2005). In animal model, ethanolic leaf extract of M. longifolia showed a significant reduction in wound area as well as epithelisation (Smitha, et al., 2010). Samaresh et al., (2010) reported the hepatoprotective activity of ethanolic extract of M. longifolia against CCl4 induced liver damage in rats. Ethanolic bark extract of M.indica exhibited antihyperglycemic activity against streptozotocin (STZ) induced diabetes in rats (Anu chaudhary et al., 2011).

Sandip et al., (2011) reported that methanolic extract showed antiepileptic activity against pentylenetetrazole (PTZ) induced convulsion in mice. Dinesh, (2001) reported chemical constituents like stigmasterol, quercetine, dihydro quercetine, triterpenoids, and myricetine in the leaves of M. longifolia.

1.4.6 Myristica fragrans

Myristica fragrans (nutmeg) belongs to the family of Myristiceae, is an evergreen tree, native of Asia and Australia (Gils and Cox, 1994). It is used in the treatment of tuberculosis, cold, respiratory ailments, skin diseases (Zaitschek, 1964). Nutmeg is also reported to be useful in increasing the blood circulation (Lindley, 1981).It is an important spice and reported to act as a nerve stimulant (Ainslie, 1979), stomachic, carminative (Khory and Katrak, 1985), aphrodisiac, hypolipidemic, anti-thrombotic, anti-fugal, anti-dysenteric and anti-inflammatory properties (Tajuddin, 2005).

M. fragrans has been shown to contain antibacterial and anti-hyperlipidaemia activities (Dorman, 2000), antioxidant properties (Murcia, et al., 2004; Olaleye et al., 2006). The petroleum ether extracts of M. fragrans fruits possess anti-diarrheal property, where as n-hexane extract has been reported to have memory enhancing effect in mice (Parle, 2004). Lee et al., (2005) reported the anticancer property of myristicin, a constituent of M.fragran against neuroblastoma SK-N-SH cell lines. Lee et al., (2011) reported anti-inflammatory activity of myristicin an important bioactive compound of M.fragran related to the inhibition of nitric oxide, cytokines and growth factors in dsRNA stimulated macrophages.

A very few reports appeared in literature on phytochemical constituents of M.fragran. Isogai et al.,(1973) & Janssen and Laeckman (1990) identified volatile oil, a fixed oil, proteins, fats, starch and mucilage.

1.4.7 Nardostacys jatamansi

Nardostacys jatamansi commonly called as Jatamansi or Indian spikenard belongs to family Valerianaceae. It is a perennial herb with sessile and ovate leaves, dark pink coloured flowers. Rhizomes are short, dark grey coloured with typical smell. The rhizomes are used as bitter tonic, stimulant, and antispasmodic, also used in treatment of epilepsy, hysteria, cornea, palpitation and convulsions (Bagchi, et al., 1991). The decoction of roots has been used in treatment of insomnia, mental and circulatory disorders (Uniyal et al., 1969). The alcoholic extract was reported to exhibit hepatoprotective effect against thiacetamide induced liver damage and iron induced lipid peroxidation in rats (Ali et al., 2000). Subashini et al., (2006) reported that ethanolic extract of N. jatamansi exhibited radical scavenging activity against doxorubicin (DOX) induced myocardial damage in rats. Rasheed et al., (2010) reported that the aqueous root extract of N. jatamansi showed antioxidant ability and reduced haloperidol catalepsy induced in rats. Recently, Bae et al., (2011) reported that the aqueous extract of N. jatamansi inhibits lipopolysaccharides induced endotoxin shock in mice.

Arora et al., (1965) and Bagchi et al., (1991) reported that the presence of alkaloids, lignin, neolignan and sesquiterpenes in N.jatamansi which impart medicinal values. Amritpal et al., (2009) reported that the volatile jatamansic oil and other bioactive compounds from different fractions of roots and rhizomes. Terpenoids like nardal, jatamansic acid and nardin were reported in rhizome by Gottumukkala et al., (2011).

1.4.8 Swertia chirayita

Swertia chirayita is native to India and belongs to the family Gentianaceae. It is an annual herb grows upto 1.5m with opposite leaves, pale green colour flowers and ovoid fruits. Reports suggested that whole plant has medicinal importance like anti-helmintic, hypoglycaemic, anti-pyretic, and also used in gastric disorder and anaemia (Karan, 1996 and 1999). Sampath et al., (2010) reported the medicinal importance of S.chirayata in diabetes, fever, skin diseases, and bronchial asthma and in treatment of malaria. Hepatoprotective activity of S.chirayata was reported by Nagalekshmi et al., (2011) against paracetamol induced hepatic damage.

Enicoflavine (Dalal, 1956), episwertenol, sweroside seco-iridoid glycoside, swerta-7, 9(11)-dien-3- b -ol and swertane (Chakravarty, 1992), sweroside-2-O-3², 5²-trihydroxy biphenyl-2² carboxylic acid ester and seco-iridoid glycoside were reported (Chaudhuri, 1996). Other phytochemical constituents reported are swerchirin, swertiamarin and 1, 3, 5, 8-tetrahydroxyxanthone xanthone (Verma, et al., 2001).

1.4.9 Trachyspermum ammi

Trachyspermum ammi commonly known as omum belongs to the family Umbellifereae. It is a small, erect, annual, herbaceous plant with branched leafy stem, feather like leaves (2.5 cm long) and ray flower heads bearing 6 -16 flowers. The fruits are minute, greyish-brown coloured and omum shaped (Gurinder and Daljit, 2010).

It is used in household remedies for stomach disorders, its fruits are used for relieving colic pains, asthma, anti-aggregatory effects (Srivastava, 1988). Due to its characteristic aromatic smell and pungent taste, it is used as flavouring agent in medicine and for manufacturing perfumes (Pruthi, 1992). According to Evans, (1996); Blumental, (2000); Amin, (2005) this plant has numerous pharmacological properties and therapeutic uses. The fruits have been reported to show anti-helminthic (Lateef et al., 2006), anti-hyperlipidaemic (Javed et al., 2006), nematicidal activities (Park et al., 2007), anti-filarial (Mathew et al., 2008), insecticidal (Chaubey, 2008), kidney stone inhibitory properties (Kaur et al., 2009). Singh et al., (2010) reported the chemopreventive role of seeds against DMBA induced carcinogenesis.

Phytochemical analysis of seeds showed the presence of tannins, glycosides, saponins, flavones, calcium, phosphorous, iron and nicotinic acid (Pruthi, 1992). T. ammi seeds also contain essential oil with phenols, thymol, carvacrol, thymine, beta-pinene and limonenes (Raghavan, 2006).

1.4.10 Zingiber officinale

Zingiber officinale, commonly known as ginger, belongs to the family Zingiberaceae. It is a rhizomatous perennial herb with sheathed leaves which are alternate, linear and lanceolate. It has been used as a flavouring agent, anti-emetic, digestive aid and also as an ingredient in the preparation of Maha-oushadha . It is conventionally used for the treatment of several of gastrointestinal disorders including diarrhoea (Nadkarni et al., 1976). It is commonly used spice with several medicinal properties and also used in household remedies for treating cough, cold, nausea, respiratory disorders, cardiovascular diseases and rheumatic disorders (Polasa and Nirmala, 2003). It has immunomodulatory properties and known to inhibit various inflammation regulating mediators such as prostaglandins and proinflammatory cytokines (Sharma, et al., 1994; Grzanna 2005). It has been considered as a safe herbal medicine because of no side effects (Ali and Blunden, 2008). Nazan et al., (2006) reported that the ethanolic extract of Z. officinale enhances the antioxidant defence against isoproterenol induced oxidative myocardial injury.

Poonam et al., (2010) gave an insight regarding the anti-diarrhoeal activity of Z. officinale. The phytochemical analysis showed the presence of flavonoids and tannins. Riazur Rehman et al., (2011) reported the presence of zingiberol, zingiberene, sesquiterpenes and bisapolene.

1.5 Protoberberines

Protoberberine is a group of isoquinoline alkaloids found different parts of Berberis aquifolium, Berberis vulgaris, Berberis aristata, Hydrastis Canadensis, Phellodendron amurense, Coptis chinensis and Tinospora cordifolia (Zhang et al., 2010).

A large number of tetrahydroprotoberberine with tranquilizing effect on dopamine receptors have been reported in chloral hydrate-anesthetized and gallamine-paralyzed rats (Huang et al., 1992). Alkaloid like tetrandrine has been proved to exhibit antitumor properties in multidrug resistant cancers (Fu et al., 2002). Ivanovska and Philipov, (1996) reported the anti-inflammatory activity of berberine and oxyacanthine. Protoberberines inhibited the DNA synthesis in microorganisms, viruses and herbivores (Schmeller et al., 1997). Kim et al., (1998) reported the inhibitory activity of protoberberine on Topoisomerase I and II. Meyerson, (2000) reported telomerase inhibitory activity of berberine in leukemia cell lines.

Thus based on the literature regarding the use of the above mentioned medicinal plants in folk medicine for treating various ailments especially radical related damage, inflammation and cancer the present study is devoted to the screening of medicinal plants with high antioxidant activities has been described in chapter II.

According to the studies taken up in chapter II B.aristata exhibited highest antioxidant activity. Activity guided isolation and quantification of phytochemicals from B.aristata was carried out in chapter III.

Biological activity of ethyl acetate fraction from B.aristata ethanolic extract against lipid and protein peroxidation and DNA fragmentation were described in chapter IV.

Further, purification and characterisation of antioxidant compounds from ethyl acetate fraction of B.aristata has been given in chapter V.

Chapter VI comprises in silico docking studies of the isolated compounds with the cancer regulating target molecules to study their interactions.

Anti-proliferative and anti-protease activities of the purified compounds have been described in chapter VII.

Chapter 2 Screening of Medicinal Plant Extracts for Antioxidant Activity and Phytochemicals

2.1 Introduction

Antioxidant protects the body from damage caused by free radical induced oxidative stress. Oxidative stress is one of the major sources of free radicals causing many chronic and degenerative diseases. The most effective way to eliminate free radical induced oxidative stress is supplementation of antioxidants. Currently, there is a growing interest towards natural antioxidants from medicinal plants since they are safe and inexpensive. Extraction of natural antioxidants from fresh or dried plant parts is usually carried out by using organic solvents or water (Starmans and Nijhuis, 1996). However, dried plant materials are better choice for extraction of antioxidants due to lack of processing and solubility problems (Eloff, 1998). The basic procedure for extraction includes pre-washing, drying and solvent extraction (Ong, 2004). Soxhlet extraction, percolation or extraction under reflux and steam distillation is an alternative choice for extraction of bioactive compounds from medicinal plants (Sukhdev Swami, 2008). Thus the present study is devoted to explore antioxidant activity of some Indian medicinal plants mentioned in (table 1).

2.2 Aim and Objectives

The chemistry responsible for antiradical activity is donation of electrons to ROS/RNS by antioxidants of medicinal plants which quench ROS/RNS to form more stable and less damaging species. The present chapter deals with the investigation of following objectives.

1. To determine the antioxidant activity of medicinal plant extracts by using DPPH, hydroxyl, superoxide radical scavenging methods and to calculate the IC50 values.
2. To determine the in-vitro inhibition of lipid peroxidation generated in liver homogenate by medicinal plant extracts.
3. To assess the total antioxidant ability of medicinal plant extracts by FRAP method.
4. To screen the medicinal plants extracts for phytochemicals by qualitative tests.

2.3 Materials and Methods

Plant materials

In the present study medicinal plants were selected based on their usage in the traditional medicine. All the plant parts were purchased from local herbal stores and authenticated by the experts, Department of Botany, Andhra University. Voucher specimens were deposited in the herbarium, Department of Botany, Andhra University, Visakhapatnam.

Chemicals

1, 1 diphenyl-2-picrylhydrazyl (DPPH), nitro blue tetrazolium (NBT), 2, 4, 6-tripyridyl-s-triazine (TPTZ) were purchased from Sigma-Aldrich, USA. Ascorbic acid, basic bismuth carbonate, butylated hydroxyl toluene (BHT), copper sulphate, deoxyribose, ethylenediaminetetra acetic acid (EDTA), ferric chloride, ferrous ammonium sulphate, gallic acid, hydroxylamine hydrochloride (HA), iodine crystals, mercuric chloride, picric acid, potassium chloride, potassium iodide, potassium dichromate, sodium dodecyl sulphate (SDS), sodium acetate, sodium carbonate, sodium hydroxide, sodium iodide, thiobarbituric acid (TBA), trichloroacetic acid (TCA), disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Merck chemicals Mumbai, India (AR grade). All solvents and acids used were analytical grade obtained from either Merck chemicals, Mumbai or from Qualigens, India.

2.3.1 Preparation of plant extracts

Plant parts were chopped into small pieces, shade dried and coarsely powdered using handheld grinder. The powdered materials were weighed and extracted separately with n-hexane, chloroform, ethyl acetate, ethanol and distilled water. The soaked material were kept for 7 days in tightly closed tubes at room temperature, protected from sunlight and mixed several times daily with sterile glass rod. The extracts were filtered through muslin cloth to remove the debris and further filtered with Whatman No. 1 filter paper. The extracts were subsequently concentrated under rotary evaporator. The dried residues were weighed and re-dissolved in aforesaid solvents to get appropriate concentrations and used for assaying radical scavenging activity.

2.3.2 DPPH radical scavenging assay

DPPH radical scavenging activity was measured by the modified method of Koleva et al., (2002). To 5ml of methanolic solution of DPPH1 (0.004%) plant extract of concentration (10-200mg/ml) was added. The reaction mixture was incubated at 37 0C for 30 min in dark and the absorbance was measured at 517 nm using spectrophotometer (Systronic UV-visible). BHT was used as standard and in control, plant extract was replaced by suitable solvent. The percent of inhibition was calculated from following equation.

A0-A/A0x100

A0 is absorbance of control and A is absorbance of the plant extract. IC50 value was determined using regression analysis which denotes the concentration of the plant extract required to scavenge 50% of DPPH radicals which is determined by regression analysis.

1. DPPH reagent: 4mg of DPPH dissolved in 100ml of methanol.

2.3.3 Superoxide radical scavenging assay

Superoxide radical scavenging activity was determined by method of Beauchamp and Fedovich (1971) with some modification. The method is based on the reduction of NBT by superoxide radicals generated from hydroxylamine-EDTA system in presence of oxygen. The superoxide anions were generated in a reaction mixture containing 1.0ml of sodium carbonate (125mM), 0.4ml NBT (24µM) and 0.2ml of EDTA (0.1mM). The reaction was initiated by adding 0.4ml of hydroxylamine hydrochloride (1mM) and plant extract ranging from 10-200mg/ml concentration. The reaction mixture was incubated at room temperature for 5 min and the absorbance was measured at 560nm. In control, plant extract was replaced by suitable solvent. As standard BHT (1mg/ml) was used. The percent of inhibition of superoxide radical was calculated as

A0-A/A0x100.

The capacity of the plant extract to inhibit 50% of superoxide radical is considered as IC50.

2.3.4 Hydroxyl radical scavenging assay

Hydroxyl radical scavenging capacity of medicinal plant extracts was assayed by Kunchandy and Rao (1990) method with some adaptations. The hydroxyl radicals were generated using Fe3+ / Ascorbate /EDTA/ H2O2 system (Fenton reaction).

The reaction mixture contained plant extract concentration (10-200mg/ml), 500µl of each (0.6mM) of deoxyribose in phosphate buffer (20mM, pH 7.4), ferric chloride (0.1mM) , EDTA(0.1mM), and ascorbic acid (0.1mM), 100µl of H 2O 2 (1mM) and 800µl of phosphate buffer in a final volume of 3ml. After incubation at 37 0C for 1hr, 1.0ml of each of TCA (2.8%) and TBA (1%) were added and kept in water bath at 100 0C for 20 min. The reaction mixture was allowed to cool to room temperature and centrifuged at 4000rpm for 15 min. The absorbance of the supernatant was measured at 532nm. In control, plant extract was replaced by suitable solvent. BHT (1mg/ml) was used as standard. The percent of inhibition was calculated from following equation

A0-A/A0x100.

A0 is absorbance of control and A is absorbance of sample. IC50 value denotes the concentration of plant extract required to scavenge 50% of hydroxyl radicals which is determined by regression analysis.

2.3.5 In-vitro inhibition of lipid peroxidation

Lipid peroxidation is an autocatalytic process, which is a common consequence of cell death. This process may cause peroxidative tissue damage. Lipid peroxidation induced by FeSO4 –Ascorbate system in sheep liver homogenate (Bishayee and Balasubramaniyam, 1971) was adopted to determine lipid peroxidation inhibitory activity of medicinal plant extracts and the formed thiobarbituric acid reactive substance (TBARS) was estimated by Ohkawa et al., (1979). Briefly, sheep liver obtained from slaughter house was washed several times with normal saline and homogenized in Tris-HCl buffer (40mM) pH 7.0 (KCl (30mM); ferrous ammonium sulphate (0.16mM), ascorbic acid (0.06mM)). To 0.1ml of homogenate (25%), plant extract of concentration (10-200mg/ml) was added and incubated at 37 0C for 1hr. After incubation 0.4ml of reaction mixture was taken and treated with 0.2ml of SDS (8.1%), 1.5ml of acetic acid (20%), 1.5ml of TBA (0.8%) and incubated in water bath at 92 0C for 1hr. The reaction mixture was allowed to cool, and 1ml of distilled water and 5ml of butanol: pyridine (15:1) was added. The contents were centrifuged at 4000 rpm for 15 min and the absorbance of organic layer (upper layer) was measured at 532 nm. In control, plant extract was replaced by suitable solvent. BHT (1mg/ml) was used as standard. The percentage of inhibition was calculated from following equation A0-A/A0x100. A0 is absorbance of control and A is absorbance of sample. IC50 value denotes the concentration of plant extract required to scavenge 50% of generated radicals.

2.3.6 Total antioxidant assay by FRAP method

Ferric chloride reducing ability of plasma was used to determine the total antioxidant capacity of plant extracts as described by method of Benzie and Strain (1996). In this assay, the ability of reduction of ferric tripyridyltriazine (Fe (III)-TPTZ) to ferrous tripyridyltriazine (Fe (II)-TPTZ) by plant extracts was considered as total antioxidant capacity of plant extract. Briefly, in the presence of antioxidants, Fe +3 –TPTZ complex is reduced to Fe +2 –TPTZ complex which gives an intense blue colour with maximum absorbance at 595nm.The calibration curve was prepared using ammonium ferrous sulphate concentration ranging from 100-1000mM. To 1.5ml of FRAP1 reagent, plant extract of concentration (10-200mg/ml) was added and the absorbance was measured at 595nm. The results were expressed as Ascorbic acid Equivalent Antioxidant Capacity (AEAC) in terms of mM.

1. FRAP reagent: FRAP reagent is prepared by adding 2, 4, 6-tripyridyl-s-trizine (TPTZ) and ferric chloride.

2.3.7 Screening of medicinal plant extracts for phytochemicals (Ashok Kumar et al., 2009).

2.3.7.1 Tests for alkaloids

In separate test tubes small quantities of plant extracts were taken and treated with few drops of dilute HCl and filtered.

1. Mayer’s reagent: 1.36g of mercuric chloride and 5g of potassium iodide were dissolved in 100ml of water.
2. Dragendroff’s reagent: Stock solution was prepared by boiling 14g of sodium iodide and 5.2g of basic bismuth carbonate in 50ml of glacial acetic acid, filtered and 40ml of filtrate mixed with 160ml of ethyl acetate and 1ml of water. Working solution was prepared by mixing stock solution, acetic acid and water in the ratio of 1:2:7.
3. Wagner’s reagent: 1.27g of iodine and 2g of potassium iodide were dissolved in 100ml of distilled water.
4. Hager’s reagent: Saturated aqueous solution of picric acid.

Mayer’s test: To 1ml of filtrate, two drops of Mayer’s reagent1 was added along the walls of the test tube. If the test is positive, it gives creamy coloured precipitate.

Dragendroff’s test: To 1ml of filtrate, two drops of Dragendroff’s reagent2 was added along the walls of the test tube. If the test is positive, it gives orange brown coloured precipitate.

Wagner’s test: To 1ml of filtrate, two drops of Wagner’s reagent3 was added along the walls of the test tube. If the test is positive for alkaloids, it gives reddish brown coloured precipitate.

Hager’s test: To 1ml of filtrate, two drops of Hager’s reagent4 was added along the walls of the test tube. A prominent yellow precipitate indicates positive test.

2.3.7.2 Tests for flavonoids and polyphenolics

Wiefferering test: 2% copper sulphate solution and few drops of Conc. HCl were added to plant extracts. Formation of reddish-violet precipitate indicate positive test.

Bargellini test: Sodium amalgam and Conc. Sulphuric acid were added to the plant extracts. Formation of green colour precipitate indicates the positive test.

Shinoda’s test: Plant extracts were treated with Conc. HCl and metal magnesium and boiled. Formation of tomato red colour indicates positive test.

Lead acetate test: Plant extracts were treated with basic lead acetate. Formation of yellow colour precipitate indicates positive test.

Ferric chloride test: Plant extracts were treated with alcoholic ferric chloride. Brown colour precipitate indicate positive test.

2.3.7.3 Test for saponins

Foam and froth test: To 1ml of extract 9ml of distilled water was added. The suspension was shaken for 15 min. A two cm layer of foam which is stable for 10 min indicates presence of saponins .

2.3.7.4 Test for tannins

Potassium dichromate test: To the plant extracts few drops of water was added and filtered. The filtrate was treated with 10% aqueous potassium dichromate. Formation of yellow brown precipitate indicates the presence of tannins.

2.4 Statistical analysis

Statistical analysis was carried out to find the significance difference between the two means by Student’s t’ test. Two way ANOVA was carried out to find out the variance among the plant species and with different concentrations of the plant extracts. Correlation was used to measure the degree of relationship between the two variables (‘x’ variable is concentration and ‘y’ variable is absorbance). Regression analysis was used to draw correlation between the average values of one variable for specified value of other variable. All the data was reported as the mean + S.D of five measurements (Daniel and Wayne., 1998).

2.5 Results and Discussion

Antioxidant activity of medicinal plant extracts

As part of metabolic reactions, a series of highly reactive free radicals are formed which causes damage to biomolecules (Klaus Apel, 2004). In addition, free radicals play crucial role in causing many diseases like arthritis, Parkinson’s disease, strokes, bovine spongiform encephalopathy (BSE), cancer and ageing (Harman, 1956; Halliwell and Gutteridge, 1997). In recent past, consumption of natural antioxidants has been increasing due to awareness provided by scientific evidence emphasising its importance to fight against various diseases (Temple, 2000). Previously, antioxidant ability of various medicinal plants was reported by several scientific communities. Some important medicinal plants whose antioxidant ability has been demonstrated are as follows. Nagulendran et al., (2007) reported the importance of Cyperus rotundus roots as a source antioxidant. Kumaran et al., (2007) studied the radical scavenging activity of ethyl acetate fraction of Coleus aromaticus . Effat et al., (2008) reported antioxidant activity of Biebresteinia multifida and Polypodium vulgare against linoleic acid induced peroxidation. Argyrei speciosa reported to exhibit antiradical activity against lipid peroxidation in rats (Ali et al., 2011). However, still there is lot of scope for exploration of antioxidants from medicinal plants.

Several assay methods have been evolved to estimate the antioxidant capacity of plant extracts (Leong and Shui, 2002). DPPH radical scavenging assay is widely used method for investigating radical scavenging capacity of natural products (Anuradha et al., 2008). DPPH free radical is a stable radical giving violet colour in methanol and decolourises on reduction in presence of antioxidants. It is highly stable and a reproducible method (Joseph, 2003; Kriengsak, 2006). DPPH assay is a method used to evaluate antioxidant activities in a relatively short time compared to other methods. Moreover, DPPH radical is not affected by metal chelation and enzyme inhibition reactions which are major disadvantages with biological free radical estimations (Cengiz et al., 2009).

DPPH radical scavenging activities of different solvent extracts of medicinal plants (10mg/ml) was given in Table. 2. The results show that highest radical scavenging activity was observed with ethanolic extract of B.aristata (87.8+0.62%) followed by M.fragrans (26.5+0.14), N.jatamansi (23.5+0.27), T.ammi (19.2+0.53), M.indica (16.9+0.18), G.glabra

Abbildung in dieser Leseprobe nicht enthalten

Table 2: Effect of medicinal plant extracts on DPPH radical scavenging activity.

DPPH radical scavenging activity of medicinal plant extracts at concentration of 10mg/ml. The results were expressed as inhibition in percent control. Values were mean +SD. The values are average of five experiments (n=5). Statistical analysis was performed by two way ANOVA and Student’s‘t’ test at significance at P>0.05.

G.glabra (12.7+0.39) , S.chirayata (11.6+0.85) , M.fragrans (10.6+0.97) , C.carvi (8.8+0.87) , B.aristata (7.9+0.43) , F.vulgare (7.6+0.95) , Z.officinale (5.8+0.86) and M.indica. (4.4+0.78%). The percent of inhibition of DPPH radicals by n-hexane extracts of N.jatamansi, M.fragrans, T.ammi, S.chirayata, G. Glabra, M.indica, F.vulgare, Z.officinale, B.aristata and C.carvi was 9.2+0.56, 9.0+0.12, 8.2+0.75, 7.9+0.69 , 7.3+0.34 , 7.2+0.67 , 5.4+0.32, 3.8+0.53 , 1.1+0.23 and 1.0+0.45%, respectively. On performing two way ANOVA analysis, the P (>0.05) for the means of different plant species and among solvents used n-hexane, ethyl acetate, ethanol and water. As the ethanolic extracts exhibited highest anti-radical activity, it can be further considered as suitable solvent to extract antioxidants from medicinal plants.

Further, to study the effect of increasing concentration of medicinal plant extracts on DPPH radical scavenging activity, different concentrations of the ethanolic extracts were used (10, 50, 100, 150 and 200mg/ml). The results show that the DPPH radical scavenging activity of ethanolic extract of B.aristata was 87.8+0.62% at 10mg/ml and with further increasing concentration from 10 to 200mg/ml, the activity was increased to 95.9+0.37%. M.fragrans exhibited 26.5+0.14% inhibition of DPPH radical at 10mg/ml, but significantly increased to 91.8+0.23% with increasing the concentration from 10 to 200mg/ml. T.ammi showed 19.2+0.53% inhibition of DPPH radical at 10mg/ml and with increasing concentration from 10 to 200mg/ml, it is increased to 75.1+0.24% (Table. 3).

Table 3: Effect of different concentrations of ethanolic extracts on DPPH radical scavenging activity

Abbildung in dieser Leseprobe nicht enthalten

BHT was used as standard antioxidant exhibited 76+0.42% DPPH radical scavenging activity at 1mg/ml concentration.

DPPH radical scavenging activity with different concentrations of medicinal plant extracts. The results were expressed as inhibition in percent control. Values were mean +SD; (n=5). ANOVA was significant P>0.05. % of inhibition of B.aristata was compared (10mg/ml) with other medicinal plants used in the present study by student’s‘t’ P<0.001.

Significantly, less inhibition of DPPH radical was observed at 200mg/ml with ethanolic extracts of N.jatamansi (68.7+0.21) compared to B.aristata (95.9+0.37%) and M.fragrans (91.8+0.23%), however, inhibition of DPPH radical was 23.5+0.27% at 10mg/ml. The ethanolic extracts of G.glabra (13.4+0.4 to 68.6+0.7), M. indica, (16.9+0.1 to 69.4+0.9), S.chirayata (11.2+0.2 to 41.4+1.1), Z. officinale (10.8+0.1 to 39.5+0.7), C. carvi (10.8+0.1 to 38.6+0.9) and F.vulgare (10.2+0.8 to 34.2+0.2) exhibited significantly less percent of inhibition of DPPH radical with increasing concentration from 10 and 200mg/ml. Further, DPPH radical scavenging activity of medicinal plants of present study was compared with BHT (1mg/ml), a known standard antioxidant, which exhibited 76+0.42%. These results indicate that significant DPPH radical scavenging activity was noticed with ethanolic extract of B.aristata at 10mg/ml concentration, compared to other medicinal plant extracts. Vinay et al., (2010) reported DPPH radical scavenging activity of methanolic extract of Hibiscus cannbinus fruits (65%) and stem (75%) at concentration of 10mg/ml. He also reported that leaf extracts of Caloptropis procera and Kigellia pinnata exhibited 88 and 82% of DPPH radical scavenging activity, respectively.The antioxidant potential is inversely propositional to IC50 value, which was calculated from the linear regression of the percent of antioxidant activity verses concentration of the extracts. The results indicate that the IC50 value of B.aristata, M.fragrans, T.ammi, N.jatamansi, G.glabra, M.indica, S.chirayata, Z.officinale, C.carvi and F.vulgare were 5.6+0.15, 19+0.18, 60+0.53, 72+0.16, 100+0.14, 144+0.17, 243+0.10, 253+0.12, 263+0.11 and 294+0.15 mg/ml, respectively. However, IC50 value of BHT was 0.65mg/ml. Safaa et al., (2010) reported that garlic an important ingredient of food exhibited IC50 value of 6.4mg/ml (Fig. 1). The results indicate that higher DPPH radical scavenging activity was associated with lower IC50 value.

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