Screening of selected plant species of Pakistan for their pharmacological activities


Doctoral Thesis / Dissertation, 2010
166 Pages

Excerpt

List of Contents

Acknowledgements

List of abbreviations

List of figures

List of tables

Abstract

Chapter 1 Introduction
1.1 Chrozophora hierosolymitana Spreng
1.2 Chrysanthemum leucanthemum
1.3 Ephedra gerardiana Wall. ex Stapf
1.4 Quercus dilatata
1.5 Bioassay guided fractionation:
1.6 Bioassays
1.6.1 Antimicrobial assay
1.6.2 Toxicity testing against brine shrimp
1.6.3 Potato disc antitumor assay
1.6.4 Phytotoxicity assay
1.6.5 Antioxidant assays
1.7 Phytochemicals:
1.7.1 Primary metabolites
1.7.2 Secondary metabolites
1.8 Separation and purification techniques:

Chapter 2 Biological assays of crude plant extracts Materials and methods
2.1 Plant material
2.2 Extraction
2.3 Antibacterial assay
2.4 Antifungal assay
2.5 Toxicity testing against brine shrimp
2.6 Antitumor assay
2.7 Radish seed bioassay
2.8. DPPH radical scavenging assay
2.9 Free radical induced oxidative DNA damage analysis
2.10 Phytochemical analysis
Results
2.11 Antibacterial activity of crude plant extracts
2.12 Antifungal assay
2.13 Toxicity testing against brine shrimp
2.14 Antitumor assay
2.15 Radish seed bioassay
2.16 DPPH free radical scavenging assay
2.17 Free radical induced oxidative DNA damage analysis
2.18 Phytochemical analysis

Chapter 3 Fractionation by solvent partitioning Materials and methods
3.1 Preparation of fractions
3.2 Antibacterial assay
3.3 DPPH radical scavenging assay
3.4 Free radical induced oxidative DNA damage analysis
3.5 Phytochemical Analysis
Results
3.6 Antibacterial activity of partitioned fractions
3.7 DPPH free radical scavenging assay
3.8 Free radical induced oxidative DNA damage analysis
3.9 Phytochemical analysis

Chapter 4 HPLC analysis of ethanol fraction Materials and methods
4.1 HPLC analysis of ethanol fraction
4.1.1 Anaytical HPLC
4.1.2 Preparative HPLC
4.2 Antibacterial assay
4.3 DPPH radical scavenging assay
4.4 Free radical induced oxidative DNA damage analysis
4.5 Characterization of the purified active component
Results
4.6 HPLC Fractionation
4.7 Antibacterial activity of the fractions
4.8 DPPH free radical scavenging assay
4.9 Free radical induced oxidative DNA damage analysis
4.10 HPLC analysis of active fractions
4.10.1 AM2 Fraction
4.10.2 AM3 Fraction
4.11 Antibacterial activity
4.12 DPPH Free Radical Scavenging Assay
4.13 Characterization of purified active component

Chapter 5 Discussion

References

Appendix

ACKNOWLEDGEMENTS

In the name of Allah, the most Beneficent, the most Merciful. All praises are for “Almighty Allah”. The most Benevolent, the only to be Praised, whose blessing and exaltations flourished my thoughts and enabled me to improve my knowledge up to this stage. I thank Allah that in spite of all problems, which I encountered during Ph D, enabled me to complete this dissertation. I offer my humble and sincerest words of thanks to His Holy Prophet Hazrat Muhammad (Peace Be Upon Him) who is forever a torch of knowledge and guidance for the humanity.

I would like to take this opportunity to pay my debt of gratitude to all those who helped me in one form or another to make it possible to bring this study to a successful completion. I am thankful to Higher Education Commission of Pakistan for financing my research work.

I wish to express my deepest regards to Professor Dr. Mir Ajab Khan, Dean, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, for providing me the research facilities.

I am deeply grateful to Professor Dr. Salman A. Malik, Chairman, Department of Biochemistry Quaid-i-Azam University, Islamabad, for providing access to facilities that ensured successful completion of this work.

It is a matter of immense pleasure to me to express my sincerest feelings of gratitude to Professor Dr. Wasim Ahmad, Ex Chairman, Department of Biochemistry Quaid-i-Azam University, Islamabad, for the encouragement and the assistance in the shape of providing access to facilities available in the department for completion of my work.

I take this opportunity to pay my reverent and profound gratitude to my honourable supervisor, Dr. Bushra Mirza, Associate Professor Quaid-i-Azam University, Islamabad for her dynamic supervision, able guidance, illustrious advice and encouragement during my study and research work present in this dissertation. I am particularly inspired by her affection and co-operative approach towards her students. Truly saying, I have no suitable words to express my wish.

I am thankful to Mr. Ihsan ul Haq for his help and guidance in my research work. Very special thanks to my friends Naila Safdar, Dr. Abida Yamin and Dr. Samia Inayatullah for their love, care, understanding and encouraging behavior. Without them it was not possible for me to enjoy my stay in the university.

I express my thanks to my lab fellows Rehana, Bushra, Farah, Samreen, Erum and Nabila for their caring and loving behavior.

How can I ignore my appreciation and deep sense of gratitude from the citadel of my heart to my sweet and affectionate parents who remembered me in their prayers and let me spare to complete my studies for a long time. I owe to thank my loving brother Shoaib who helped me a lot in my computer work, without him it was not possible to complete my thesis. I am thankful to my sisters Shazia, Fozia and Sana who shared my responsibilities and did not burden me during my Ph D.

Before biding farewell, I would also pay my thanks to all those researchers who sent me valuable research papers and articles to carry out my research work. I would also like to thank Mr. Mohammed Javed who helped me taking photographs and in editing them for my research results, to give the real picture of my research.

Maryam Jamil

LIST OF FIGURES

1.1 Chrozophora hierosolymitana
1.2 Chrysanthemum leucanthemum
1.3 Ephedra gerardiana
1.4 Quercus dilatata
1.5 Artemia nauplii brine shrimp
1.6 Ti Plasmid
1.7 Oxidative stress causing damage to body cells
1.8 Reduction of 1,1-Diphenyl-2-picrylhydrazyl into 1,1- Diphenylhydrazin
1.9 Restriction map of pBR322 (Fermentas) plasmid
1.10 Analytical HPLC with isocratic mode
1.11 Preparative HPLC with isocratic mode
1.12 Analytical HPLC with gradient mode

2.1 Antibacterial activity of (A) Q. diltata against Staphylococcus aureus
2.2 Antifungal activity of seven plant extracts against Alternaria sp.
2.3 Effect of (A) Q. dilatata at different concentrations, on inhibition of tumor formation, along with control for comparison
2.4 Average number of tumors at different concentrations of extracts
2.5 Radish seed phytotoxicity in terms of root length of (A) Q. dilatata
2.6 Effect of different concentrations (10,000 ppm, 1000 ppm, 100ppm and 10ppm) on root length
2.7 Effect of the plant extracts on seed germination as a function of incubation period of seeds at a. 7500 ppm b. 1000 ppm
2.8 Effect of (A) Q. dilatata at concentrations 25 ppm, 50 ppm and 100 ppm on DPPH free radical scavenging along with control for comparison
2.9 %age DPPH free radical scavenging activity of plant extracts at different concentrations (100 ppm, 50 ppm, and 25ppm)
2.10 Effect of the crude plant extracts on pBR322 Plasmid DNA

3.1 Scheme of fractions preparation
3.2 Antibacterial activity of partitioned fractions against S. aureus
3.3 Effect of the ethanol fraction at concentrations 25 ppm, 50 ppm and 100 ppm on DPPH free radical scavenging
3.4 %age DPPH free radical scavenging activity of partitioned fractions at different concentrations (100 ppm, 50 ppm, and 25 ppm)
3.5 Effect of the partition fractions on pBR322 Plasmid DNA

4.1 Chromatogram obtained for ethanol partitioned fraction to get seven fractions by using preparative HPLC
4.2 Antibacterial activity of fractions against Salmonella setubal.
4.3 Effect of AM2 fraction at concentrations 1 ppm, 5 ppm and 10 ppm on DPPH free radical scavenging
4.4 %age DPPH free radical scavenging activity of partition fractions at different concentrations (10 ppm, 5 ppm, and 1ppm)
4.5 Effect of the fractions on pBR322 Plasmid DNA
4.6 Chromatogram obtained for AM2 fraction to get four subfractions by using preparative HPLC
4.7 Chromatogram obtained for AM3 fraction to get three subfractions by using preparative HPLC
4.8 Antibacterial activity of subfractionsAM3b and AM3c against Salmonella setubal.
4.9 Effect of AM2b at concentrations 10 ppm on DPPH free radical scavenging
4.10 Absorption spectra of AM2b, AM3b and AM3c
4.11 Comparison of absorption spectrum of AM2b with that of standard compounds
4.12 Comparison of absorption spectrum of AM3b with that of standard compounds
4.13 Comparison of absorption spectrum of AM3c with that of standard compounds

LIST OF TABLES

2.1 List of plant species with respective plant extracts used for this study
2.2 Dilution prepared for antibacterial assay
2.3 Dilutions prepared for brine shrimp toxicity assay
2.4 Dilutions prepared for antitumor assay
2.5 Dilutions prepared for radish seed bioassay
2.6 Concentrations prepared for DPPH radical scavenging assay
2.7 Concentrations preparation for OH induced DNA damage analysis
2.8 Final concentrations prepared for OH induced DNA damage analysis
2.9 Control reaction mixture preparation (15 µl)
2.10 Activity of (A) Q. dilatata against six bacterial strains.
2.11 Activity of (R) C. hierosolymitana against six bacterial strains
2.12 Analysis of variance for factors affecting zone of inhibition
2.13 MIC of (A) Q. dilatata against six bacterial strains
2.14 Antifungal activity of crude plant extracts against seven fungal strains
2.15 Analysis of variance for factors affecting fungal growth
2.16 Illustration of percentage mortality of brine shrimps at different concentrations of extracts and respective ED50 value
2.17 Percentage inhibition of tumor formation at different concentrations
2.18 Analysis of variance of tumor inhibition of compounds
2.19 Percentage root growth inhibition or stimulation on 5th day
2.20 Analysis of variance for factors affecting radish seedling root length
2.21 Analysis of variance for factors affecting radish seed germination
2.22 IC50 value of plant extracts
2.23 Analysis of variance DPPH scavenging of crude plant extracts.
2.24 Phytochemical analysis of crude plant extracts.

3.1 Activity of (A) partition fractions against six bacterial strains.
3.2 Analysis of variance for factors affecting zone of inhibition
3.3 Analysis of variance DPPH scavenging of partitioned fractions.
3.4 Phytochemical analysis of partitioned fractions.

4.1 Seven fractions obtained at different retention time.
4.2 Antibacterial activity of fractions against six bacterial strains .
4.3 IC50 value of fractions
4.4 Analysis of variance DPPH scavenging of fractions.
4.5 Four subfractions obtained at different retention time.
4.6 Three subfractions obtained at different retention time.
4.7 Antibacterial activity of subfractions against six bacterial strains .
4.8 MIC of active subfraction against six bacterial strains
4.9 %age DPPH free radical scavenging activity of subfractions at 10 ppm

LIST OF ABBREVIATONS

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Abstract

In this study four plants (Chrozophora hierosolymitana Spreng , Chrysanthemum leucanthemum L., Ephedra gerardiana Wall. ex Stapf and Quercus dilatata L.) collected from different regions of Pakistan were screened to identify any chemotherapeutic agents present in them. Seven crude extracts of these plants (leaf, stem and root extracts of C. hierosolymitana, aerial parts of C. leucanthemum, stem and root extracts of E. gerardiana and aerial parts of Q. dilatata) were examined for antimicrobial activity using agar diffusion method and agar tube dilution method, cytotoxicity using brine shrimp assay, antitumor activity using potato disc assay, phytotoxic activity using radish seed bioassay and antioxidant activity by using DPPH radical scavenging assay and free radical induced oxidative DNA damage assay.

Two plant extracts of C. hierosolymitana and Q. dilatata showed antibacterial activity. Two plant extracts of E gerardiana and C. leucanthemum showed antifungal activity. Two plant extracts i.e., leaf extract of C. hierosolymitna and root extract of E. gerardiana showed significant brine shrimp cytotoxicity activity (IC50 171.55 to 523.8 ppm). Six of the seven extracts exhibited tumor inhibition at all the three concentrations tested ranging from 10 to 80%. All extracts showed significant plant growth and seed germination inhibition at higher concentrations against radish seeds. Two extracts of C. hierosolymitana and Q. dilatata showed growth stimulating effects at lower concentrations. Two extracts of C hierosolymitana and Q. dilatata showed significant DPPH radical scavenging activity (IC50 10.52 to 45.9 ppm) . Three of the seven extracts i.e., (R) E. gerardiana, (A) Q. dilatata and (A) C. leucanthemum showed DNA protection activity at 100 and 10 ppm while at 1000 ppm showed no DNA protection activity while rest of the four extracts showed DNA protection activity at all the three concentrations tested. Phytochemical tests showed presence of alkaloids, saponins, anthraquinones, terpenoids, flavonoids, flavones, tannins, phlobatannins and cardiac glycosides at varying levels in these extracts.

The crude extract of the most active antibacterial plant extract (A) Q. dilatata was subjected to bio-guided fractionation. Six partitioned fractions of aerial parts of Q.

dilatata were tested for antibacterial and antioxidant activities. Phytochemical analysis of these partitioned fractions was also done. Ethanol fraction was selected on the basis of results of bioassays and phytochemical analysis. This fraction was analyzed by RP- HPLC and seven fractions were collected. Out of the seven fractions, AM2 showed antioxidant activity while AM3 showed antibacterial as well antioxidant activity. These two active fractions were again analyzed by RP- HPLC. The subfractions AM3b and AM3c showed antibacterial activity while AM2b showed antioxidant activity. Purified active subfractions were charaterized by comparing their absorption spectra with that of standard natural products isolated from the plants of same genus. The absorption spectra of the active fractions were different from that of the standard compounds previously isolated from the Quercus genus suggesting that these are newly isolated compounds from this genus.

Chapter 1 Introduction

Nature has been the source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources, many based on their use in traditional medicines. The traditional medicinal methods, specially the use of medicinal plants, still play a vital role to cover the basic health needs in developing countries. Moreover, the use of herbal remedies has risen in the developed countries in the last few decades. In this connection, plants continue to be a rich source of therapeutic agents. The active constituents of many drugs are found in plants or are produced as secondary metabolites. Secondary metabolites are biosynthesized in plants for different purposes including growth regulation, inter and intra-specific interactions and defense against predators and infections. Many of these natural products have been shown to present interesting biological and pharmacological activities and are used as chemotherapeutic agents or serve as the starting point in the development of modern medicines (Verpoorte, 1998 and 2000). The remarkable contribution of plants to the drug industry has been possible, because of the large number of phytochemical and biological studies all over the world. In fact, plants produce a diverse range of bioactive molecules, making them a rich source of different types of medicines. Higher plants, as sources of medicinal compounds, have continued to play a dominant role in the maintenance of human health since ancient times (Farombi, 2003). Over 50% of all modern clinical drugs are of natural product origin (Stuffness and Douros, 1982).

Pakistan has a strong tradition of herbal remedies and its rural population still depends mainly on indigenous system of medicine for their health related matters (Khattak et al., 1985). Pakistan is bestowed with a unique biodiversity, comprising of different climatic zones and a wide range of plant species. Pakistan has four phytogeographical regions, consisting of Irano-Turanian (46%), Sino-Himalayan (10%), Saharo-Sindian (9.5%), and Indian element (4.5%). The country has about 6,000 species of wild plants of which about 400 to 600 are considered to be medicinally important (Hamayun et al., 2003).

For the present study four plants were selected. The selection of these plants was based on the observation that these are being used by local healers intensively for treatment of various ailments. The details of these plants are as follows.

1.1 Chrozophora hierosolymitana Spreng

Chrozophora hierosolymitana (Fig 1.1) is commonly known as Dyer's croton. Synonyms are Chrozophora tinctoria (L.) Raf , Chrozophora verbascifolia Baill (Nasir and Ali, 1972). Chrozophora is genus of the family Euphorbiaceae. The Euphorbiaceae (spurge family) is among the larger families of flowering plants with c. 300 genera and 8000 species (Webster, 1994). Chrozophora is sole genus comprised in the subtribe Chrozophorinae. It comprises 11 or 12 species, which are monoecious herbs or undershrubs. They are found from Africa and the Mediterranean to Southeast Asia.

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Fig 1.1 Chrozophora hierosolymitana

1.1.1 Distribution:

Eleven species are from Africa and the Mediterranean. In Malaysia single species is found, probably accidentally introduced into Central Java (Welzen, 1999). This species is native to a number of countries in Africa, (Algeria, Egypt, Libya, Morocco, Tunisia and Yemen) temperate and tropical Asia (Kuwait, Saudi Arabia, Afghanistan, Iran, Iraq, Israel, Jordan, Lebanon, Syria, Turkey, Kazakhstan, Turkmenistan, India and Pakistan), and Europe (Ukraine, Albania, Bulgaria, Greece, Italy, Malta, France, former Yugoslavia, Portugal and Spain) (Delazar et al., 2006).

C. hierosolymitana is found in Pakistan in Thal, Las Bela, Ormara, Sitana, South Wazirastan, Between Saidu sharif and Madian, Between Madian and Kalam (Nasir and Ali, 1972).

1.1.2 Morphology.

The plant is mainly found as a shrub or herb formation; non-laticiferous and without coloured juice. Plants succulent, or non-succulent. Herbs annual. Mesophytic, or xerophytic. Leaves small to medium-sized; alternate; spiral, or distichous; herbaceous, or leathery, or fleshy; petiolate; non-sheathing; gland-dotted, or not gland-dotted; simple. Leaf blades entire, or dissected (lobed); pinnately veined, or palmately veined. Leaves stipulate. Stipules filiform; caducous, or persistent. Leaves without a persistent basal meristem. Urticating hairs present, or absent. Nodes tri- lacunar, or unilacunar. Secondary thickening developing from a conventional cambial ring, or anomalous; from a single cambial ring. Fertile flowers functionally male, or functionally female. Unisexual flowers present. Plants monoecious. Male flowers without pistillodes. Entomophilous (Macfarlane et al., 2002).

1.1.3 Phytochemical studies

Usman et al., (2007) reported the presence of carbohydrates, tannins, saponins, sterols in Chrozophora. Alkaloids, coumarins, chromones (Mohamed, 2001) xanthones (Agrawal and Singh, 1988), diterpenoids (Mohamed et al., 1994 and 1995) phenylpropanoid glycosides (Mohamed, 2001) and chrozophorin (Delazar et al., 2006) have previously been reported from few species of the genus Chrozophora. Chrozophora tinctoria is well known for producing dye substances (Ba lar and Mert, 1999) and flavonoids (Hashim et al., 1990). Phytochemical studies on C. hierosolymitana have not been previously done.

1.1.4 Medicinal values

Harraz and Abdel-Aziz (1994) reported the central analgesic effect of Chrozophora sp. Chrozophora species are used in traditional medicine for the treatment of diverse ailments (Adam et al., 1999). Antifungal (Shahidi et al., 2004) and antiyeast (Shahidi, 2004) activities have been reported from the genus Chrozophora. The plant has been reported to serve as a remedy for intestinal pain, for conjunctivitis as well as cicatrizant (Tignokpa et al., 1986; Etkin, 1997). In Northern Nigeria, the plant is an astringent for diarrhea taken boiled with cereal foods and also to treat boils. It is chiefly used as a remedy for syphilis (Dalziel, 1955). C. tinctoria is used to treat warts, this plant has been used as an emetic, cathartic, and for the treatment of fever (Phytochemical and Ethnobotanical Databases, 2008). Medicinal value of C. hierosolymiatana has not been reported yet.

1.1.7 Toxicity

Chrozophora caused toxicity to wistar rats (Adam et al., 1999). Galal and Adam (1988) reported pathological effects of Chrozophora on goats and sheep. The main signs of Chrozophora poisoning in both species of ruminants were salivation, dyspnea, bloat, inappetence, dullness, diarrhea, paresis of the hind limbs, recumbency and lateral deviation of the head and neck. The main lesions were hemorrhage in the lungs, heart and kidneys, pulmonary cyanosis and edema, hepatic fatty change and depletion of glycogen, catarrhal enteritis, ascites, hydropericardium and serous atrophy of the cardiac fat and renal pelvis. An increase in the concentration of urea, ammonia and bilirubin and in the activity of GOT (glutamate oxaloacetate transa minase) and a decrease in total protein were detected in the serum. Hematological changes indicated the development of anemia. C. tinctoria is the poisonous plant found in cholistan (Khan http://www.weru.ksu.edu/symposium/proceedings/khan.pdf). Toxicity of C. hierosolymiatana has not been reported yet.

1.1.8 Other Uses

Red and blue dyes are obtained from the flowers, fruit and sap (Usher, 1974).

1.2 Chrysanthemum leucanthemum L

Chrysanthemum leucanthemum (Fig 1.2) is commonly known as ox eye daisy. Synonym is Leucanthemum vulgare - Lam. Chrysanthemum is a genus of about 30 species of perennial flowering plants in the family Asteraceae, native to Asia and northeastern Europe. These are herbaceous perennial plants. The family Asteraceae or Compositae (known as the aster, daisy, or sunflower family) is the largest family of flowering plants, in terms of number of species.

1.2.1 Distribution:

These hardy plants are natives of China, Japan, northern Africa, and southern Europe. In Pakistan C. leucanthemum is common in gardens and found in hill station like Murree, Nathia gali, Dunga gali and Swat (Nasir and Ali, 1972).

1.2.2 Morphology:

Chrysanthemums are perennial or rarely annual herbs, with reddish tipped roots. Leaves alternate, entire, serrate, pinnatifid. Capitula radiate or discoid, pedunculate, solitary or 2-6 in lax corymbs, terminal. Involucre saucer-shaped, 3-4- seriate, phyllaries herbaceous, outer narrowly and inner broadly membranousmargined. Receptacle convex, distinctly alveolate, without paleae. Ray florets, when present, mostly uniseriate, female, fertile, with white, pink or occasionally yellow ligules. Disc florets and marginal florets, when ray florets absent, bisexual, with 5- lobed, basally spongy and swollen in fruit, yellow, corolla tube.

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Fig 1.2 Chrysanthemum leucanthemum

1.2.3 Phytochemical studies:

Search for the concerned active compounds has led to the isolation of several pyrethroids, sesquiterpenoids, flavonoids, coumarins, triterpenoids, steroids, phenolics, purines, lipids, aliphatic compounds and monoterpenoids from different plant parts of Chrysanthemum. Compounds isolated from Chrysanthemum plants are Pyrethroids (Essig and Zhao, 2001), Sesquiterpene lactones (Haruna et al., 1981), (+)-Sesamin (Yoshikawa et al., 2000), Sterols, Flavonoids (Wilkomirski and Dubielecka, 1996), Flavone, Flavanone (Hu et al., 1994) and Quinic acid (Chuda et al., 1996).

1.2.4 Medicinal values

The medicinal values of Chrysanthemum are different depending on the varieties. The white or yellow Chrysanthemum has been used for cold, headaches and inflamed eyes. The wild variety has been made into a decoction to treat retained menses, as a wash for infected and cancerous sores and as a fomentation for enlarged glands. Antivinous (alcohol detoxifying) properties were also ascribed to this plant (Smith and Stuart, 1973). Today wild Chrysanthemum is used as an antipyretic and anti-inflammatory herb and is prescribed to clear away heat, detoxicate, subdue swelling, dissolve lumps and treat respiratory infections (Dong et al., 1998). Hou and Jin (2005) reported the use of wild Chrysanthemum for dizziness, headaches, hypertension, fullness in the head and blood shot, swollen, and painful eyes due to hyperactivity of liver (Liver-fire).

1.2.6 Toxicity

Chrysanthemum showed allelopathic effects on germination and growth of several herbaceous plants (Kil and Youb, 1987). Furthermore, it had antifeeding, phagostimulating and toxic activity (Haouas et al., 2008). According to one report Chrysanthemums are particularly noted for their allergenic effects (Bowles, 2003).

1.2.7 Other uses:

Chrysanthemum plants have been shown to reduce indoor air pollution (Wolverton et al., 1983) .

1.3 Ephedra gerardiana Wall. ex Stapf

Ephedra gerardiana (Fig 1.3) is commonly known as Ma Huang. Local Name is Soon and Someni. Synonyms are E. vulgaris Rich. and E. wallichii stapf. Ephedra is a genus of non flowering seed plants belonging to the family Ephedraceae (Jointfir ). It comprises only one genus, Ephedra, with 35-45 species which are mostly dioecious shrubs, climbers or small tree (Green et al., 1990). In Pakistan 9 species are found (Nasir and Ali, 1972).

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Fig 1.3 Ephedra gerardiana

1.3.1 Distribution

The Ephedra sp. are xerophytes and in part also cold resistant. Most of the Ephedra species worldwide (Stevenson, 1993; Price, 1996) are shrubs adapted to semiarid and desert conditions (Pearson, 1929). The Eurasian distribution forms a broad belt from Canary Island and the Mediterranean through the arid subtropical regions of the inner Asia with outliers on the Arabian Gulf. In the New World, Ephedra is found in two areas; in the western USA and northern Mexico, and in South America; Ecuador to Patagonia and lowland Argentina (Green et al., 1990).

E. gerardiana is found in Pakistan in Urak, South Waziristan, Razmak, Chitral, Tirch Mir, Swat, Kalam, Gilgit, Baltistan, above Skardu, Shaksgan, Tog, Ladak, Zanskar, Char and Kashmir (Nasir and Ali, 1972).

1.3.2 Morphology

All species of Ephedra have rush like, equisetoid shoots, most are shrubs, some hanging or scandent. Many spread by means of rhizomes which grow from underground buds. In most species the leaves are opposite and decussate but in some they are in whorls of 3 or 4. Branching is basitonic, and the condensed whorls of lateral branches that result therefore lead to the shrubby habit of the plants. The leaves are minute scale and are often shed (Green et al., 1990).

1.3.3 Phytochemical studies

Ephedrine, the active alkaloid in Ephedra sinica, was first isolated, and characterized by a Japanese chemist, N.M. Nagai in 1887. The leaves and stems of Ephedra sinica contain many potentially active compounds such as tannins, saponin, falvone, and volatile oils, but it is the protoalkaloids (ephedrine, pseudoephedrine, norpseudoephedrine) which have been isolated (Chevallier, 1996). Miyazawa (1998) reported that the oil of E. sinica contained 146 volatile components of which 38.9% were terpenoids. The main constituents were -terpineol (13.0%), tetramethylpyrazine (3.9%), terpinen-4-ol (3.9%), linalol (3.2%), 2,3-dihydro-2- methylbenzofuran (3.1%) and cis-p- menth-2-en-7-ol (3.1%).

1.3.5 Medicinal values

Plants of the Ephedra genus have traditionally been used by indigenous people for a variety of medicinal purposes, including treatment of asthma, hay fever, and the common cold (Abourashed et al., 2003). The shrub E. breana is used in decoction as diuretic, cholagogue, antiinflammatory and vulnerary. Other species of the same genus have been used to cure urinary infections (Ratera and Ratera, 1980). It is often taken to relieve chills, fevers, coughs and wheezing, and also is taken in powder form in combination with Rehmannia (Rhemannia glutinosa) to treat kidney yin deficiency (Chevallier, 1996). Ephedra is currently used as herbal medicine in Western countries in a decoction, for asthma, hay fever, acute onset of colds/flu, to raise blood pressure, and possibly to initiate menstruation. It increases sweating, dilates bronchioles (antiasthmatic use), diuretic, central nervous system stimulant. Also it is used in tincture form to alleviate rheumatism and for aches and pains (Chevallier, 1996). Almost all commercial applications of Ephedra extracts derive from the ephedrine alkaloids found in the stems in many Eurasian species. The best-documented drug made from Ephedra is Ma-huang, used in Chinese medicine for 5000 yr as a treatment for fever, nasal congestion and asthma (Zhu, 1998). Ma-huang is also an effective respiratory sedative and cough remedy. Ma-huang was traditionally obtained from the dried stems of species found in the drier regions of China, North West India, and Pakistan (Qazilbach, 1971; White et al., 1997; Zhu, 1998). Ephedroxane, an anti- inflammatory drug is related in structure to ephedrine (Konno et al., 1979).

1.3.6 Toxicity

Adverse central nervous system effects associated with ephedrine toxicity include anxiety, insomnia, restlessness, psychosis and seizures. These effects are especially common when multiple stimulants are involved. Additional signs of ephedrine toxicity include nausea, vomiting, headache, flushing, paresthesias, difficulty in micturation and precordial pain (Morton, 1977). It has also been suggested that sympathomimetic drugs may precipitate thyroid storm in individuals with thyrotoxicosis (Wilson, 1993).

1.3.7 Other uses

It is used locally for fuel (Gamble, 1972). This herb is a good ground cover plant for dry soils (Brickell, 1990).

1.4 Quercus dilatata L

Quercus dilatata (Fig 1.4) is commonly known as holly oak. Local name is Barungi. Synonym is Q. leucorticophora. Quercus is a plant genus of the family Fagaceae. The Fagaceae (beech family) is the small family of monoecious trees and shrubs comprising 6-8 genera and about 800 species (Shah et al., 2005). It comprises 400-450 species, which are monoecious, evergreen or deciduous trees, rarely shrubs

1.4.1 Distribution

Over 400 species of Quercus are distributed in America, temperate Europe, Asia, and sub-tropical Africa. In Pakitan this evergreen tree is found in the Himalayas mountains specifically in Dir, Chitral, Swat, Hazara, Tirah, Kurram Agency, Murree Hills, and Azad Kashmir (Nasir and Ali, 1972).

1.4.2 Morphology

Tree up to 20 m tall. Leaves elliptic-ovate to broadly lanceolate, coriaceous, 4- 12 x l.6-5.5 cm, entire to spiny toothed, acute or obtuse; nerves 9-12 pairs, forked at the extremities, both surfaces green, glabrous, glaucous, base often oblique; petiole 0.3-l cm long, glabrous. Male flowers in lax catkins, up to 5 cm long; bract lanceolate, c. l.5 mm long, shorter than the perianth, tomentose; stamens 4-8, sub-sessile; anthers glabrous. Styles 3-5. Cupule 2-2.4 cm broad, covering half the nut, pubescent. Nut ovoid, brownish, glabrescent, tipped with an umbo.

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Fig 1.4 Quercus dilatata

1.4.4 Phytochemical studies

The chemical composition of heartwood from the Spanish oaks includes a wide range of volatile compound families i.e., volatile phenols, phenolic aldehydes, furanic compounds, lactones, phenyl ketones (Cadahia et al., 2007). Yuan and Sun (1998) isolated four compounds from Quercus sp and identified as friedelin, glutinol, lupeo and beta-sitosterol. Fourteen glycosides and acylated triterpene saponin have been isolated from leaves of another species of Quercus (Romussi et al., 2006). Quercus species consist of phenolic compounds (Ohemeng et al., 1993) such flavonoids and tannins (Zhentian et al., 1999; Meng et al., 2001; Hideyuki et al., 2002). Five yellow compounds isolated from Quercus dentata are Kaempferol 3-O- - D-glucopyranoside, quercetin 3-O- -D-glucopyranoside, Kaempferol 3-O-(6-trans-p- coumaroyl)- -D-glucopyranoside, Kaempferol 3-O-(2-6-di-trans-p-coumaroyl)- -D- glucopyranoside and Kaempferol 3-O-(2-4-di-acetyl-3-cis-p-coumaroyl-6-trans-p- coumaroyl)- -D-glucopyranoside (Meng et al., 2001). The constituents of galls comprise a large amount of tannins, gallic acid, syringic acid, ellagic acid, sitosterol, amentoflavone, hexamethyl ether, isocryptomerin, methyl betulate, methyl oleanate and hexagalloyl glucose (Hwang et al., 2000; Dar et al., 1976; Ikram and Nowshad, 1977).

1.4.5 Medicinal values

This herb is used to treat diarrhea, dysentery and bleeding and if used externally, the bark or leaves are boiled and then applied to bruises, swollen tissues, wounds that are bleeding, and varicose veins. (http://www.ayurveda-herbal- remedy.com/herbal-encyclopedia/ayurveda-encyclopedia-o.html). It is used as an astringent and antidiarrhoeal (Gorunovi and Luki , 2001). The stem bark is used to clean foul sores. The seeds are used in the treatment of diarrhoea, menorrhagia and gastrointestinal hypertrophy. The cupule (seed cup) is astringent (Duke and Ayensu, 1985). In Pakistan, this herb is used localy as astringent and diuretic, used in diarrhea, indigestion and asthma. It has been reported that different species of Quercus possess antibacterial actiivty (Güllüce et al., 2004; Andrensek et al., 2004), antioxidant activity (Al-Mustafa and Al-Thunibat, 2008; Chevolleau et al., 1993, McCune and Johns, 2002) and gastroprotective effect (Gharzouli et al., 1999). Any galls produced on the tree are strongly astringent and can be used in the treatment of haemorrhages, chronic diarrhoea, dysentery etc (Grieve, 1984). The galls of Quercus are used for the treatment of wounds or burns associated with bacterial infections (Umachigi et al., 2008). The galls of Q. infectoria have a great medicinal value and have pharmacologically been deciphered to be astringent, antidiabetic, antitremorine, local anaesthetic, antipyretic and antiparkinsonian (Hwang et al., 2000; Dar et al., 1976; Dar and Ikram, 1979).

1.4.6 Toxicity

There is no toxicity data for Quercus species in humans. Two outbreaks of oak poisoning in cattle in South Africa have been reported, with clinical signs that included severe weakness with a swaying gait, diarrhea and dehydration. Upon post mortem examination of three animals there was a non-suppurative interstitial nephritis accompanied by edema and ulceration of the cecum and colon (Neser et al., 1982).

1.4.7 Other uses

A mulch of the leaves repels slugs, grubs etc, though fresh leaves should not be used as these can inhibit plant growth. Oak galls are excrescences that are sometimes produced in great numbers on the tree and are caused by the activity of the larvae of different insects. The insects live inside these galls, obtaining their nutrient therein. When the insect pupates and leaves, the gall can be used as a rich source of tannin that can also be used as a dyestuff (Grieve, 1984). A black dye is obtained from the seed cups. Wood used for boat building and construction (Wilson, 1917). The wood is also used for fuel and charcoal (Bean, 1981).

1.5 Bioassay guided fractionation

Different approaches to drug discovery using higher plants can be distinguished: random selection followed by chemical screening; random selection followed by one or more biological assays; follow-up of biological activity reports (e.g., ecology based); follow-up of ethnomedical (traditional medicine) use of plants. The latter approach includes plants used in organized traditional medical systems; herbalism, folklore and shamanism; the use of databases (Fabricant and Farnsworth, 2001). The objective is the targeted isolation of new bioactive plant products, i.e., lead substances with novel structures and novel mechanisms of action.

Bioassay-guided isolation integrates the processes of separation of compounds in a mixture, using various analytical methods, with results obtained from biological testing. The process begins with testing an extract to confirm its activity, followed by crude separation of the compounds in the matrix and testing the crude fractions. Further fractionation is carried out on the fractions that are determined to be active, at a certain concentration threshold, whereas the inactive fractions are set aside or discarded. The process of fractionation and biological testing is repeated until pure compound(s) are obtained. Structural identification of the pure compound then follows. This methodology precludes overlooking novel compounds that are often missed in studies that only identify those compounds with which the investigator is familiar. Moreover, the possibility of discovering an unknown molecular site of action is maximized (Duke et al., 2000).

There are several reports of successful isolation of active components by using bioassay guided fractionation. Some of these studies are reported here. Malheiros et al., (2005) determined antifungal activity of drimane sesquiterpenes from Drimys brasiliensis using bioassay-guided fractionation. The bioassay-guided fractionation of the antifungal dichloromethane extract from the roots of Vernonanthura tweedieana (Baker) H. Rob., using an agar overlay bioautographic method, allowed the isolation of one active sesquiterpene (Portillo et al., 2005). Zhang et al., (2007) isolated the components having antifertility actiivty of castorbean (Ricinus communis L.) seed extracts using bioassay-guided fractionation. In order to isolate and identify the active principles possibly responsible for the antimitotic activity of the extract from roots of Harpalyce brasiliana using sea urchin egg development, a bioassay-guided fraction was performed and six bioactive compoundds were isolated (Militão et al., 2007). Using a bioassay-guided fractionation technique, Unno et al., (2004) isolated two active compounds (Xanthine oxidase inhibitors) from the aqueous extracts of the Lagerstroemia speciosa leaves, namely valoneic acid dilactone (VAD) and ellagic acid (EA).

1.6 Bioassays

A combination of chemical screening with biological screening is the fastest way to arrive at new medicinal compounds from plants. For this purpose, relatively simple biological or pharmacological tests are available in order to localize the specific activities in the plant extracts or in the numerous fractions resulting from the different purification steps which lead from the plant to the pure active constituents. These tests are very sensitive because the active substances are present in the plants at very low concentrations (Hostettmann and Marston, 2002). A biological assay is a procedure for determining the concentration, purity, and/or biological activity of a substance (e.g., vitamin, hormone, plant growth factor, antibiotic, enzyme or any other chemical) by measuring its effect on an organism, tissue, cell, enzyme or receptor preparation compared to a standard preparation (Linton, 1983) . Bioassays offer a special advantage in the standardization and quality control of heterogeneous botanical products. These products are mixtures of bioactive components. Most often a desired biological response is due to not one but a mixture of bioactive components the relative proportions of single bioactive compounds can vary from batch to batch while the bioactivity still remains within tolerable limits. Thus, physical or chemical analysis of a single component in such mixtures is not completely satisfactory. The use of bioassays is adoptable to the purpose of standardization or quality control of bioactive components in such heterogeneous botanicals and to aid “drug discovery” work with botanicals (McLaughlin et al., 1998).

In evaluating the pharmacological activities of a sample, to reach to an appropriate conclusion, the use of a number of bioassays and careful comparison of all the data is required (Linton, 1983). In this present study, the biological assays used to evaluate the biological activities of methanol extracts of the above mentioned plants were antibacterial assay, antifungal assay, brine shrimp lethality test, crown gall antitumor assay, radish seeds phytotoxic assay, DPPH scavenging assay and DNA protection assay.

1.6.1 Antimicrobial assay

Development of multi-drug resistance in pathogenic microbes and parasites and non-availability of safe antifungal drugs for systemic mycoses necessitates a search for new antimicrobial substances from other sources, including plants. Traditionally used medicinal plants produce a variety of compounds of known therapeutic properties (Chopra et al., 1992; Bruneton, 1995). The substances that can inhibit pathogens and have little toxicity to host cells are considered candidates for developing new antimicrobial drugs. Many infectious diseases are known to be treated with herbal remedies throughout the history of mankind. Even today, plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries (Zakaria, 1991). There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases (Rojas et al., 2003). Therefore, researchers are increasingly turning their attention to folk medicine looking for new leads to develop better drugs against microbial infections (Srinivasan et al., 2001; Benkeblia, 2004).

The fungal and bacterial strains used in this study were chosen primarily based on their importance as opportunistic human pathogens.

Bacterial strains

Staphylococcus aureus

Staphylococcus aureus is a spherical facultatively anaerobic, gram-positive coccus, frequently found in the nose and skin of a person. S. aureus can cause a range of illnesses from minor skin infections, such as pimples, impetigo (may also be caused by Streptococcus pyogenes), boils, cellulitis folliculitis, furuncles, carbuncles, scalded skin syndrome and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis endocarditis, Toxic shock syndrome (TSS), and septicemia.

Escherichia coli

Escherichia coli is a facultative anaerobic, gram negative bacterium that is commonly found in the lower intestine of warm-blooded animals. Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis.

Micrococcus leuteus

Micrococcus leuteus is an obligate aerobe, gram positive, spherical, saprotrophic bacterium and is found in soil, dust, water and air, and as part of the normal flora of the mammalian skin. The bacterium also colonizes the human mouth, mucosae, oropharynx and upper respiratory tract. It is non-pathogenic and is usually regarded as a contaminant, it should be considered as an emerging nosocomial pathogen in immunocompromised patients.

Bordetella bronchiseptica

Bordetella bronchiseptica is a gram-negative bacterium that colonizes the respiratory tracts of mammals. B. bronchiseptica causes an uncommon infection in humans that generally produce a "whooping cough" like syndrome in immunocompetent individuals. However, B. bronchiseptica has been associated with endocarditis, peritonitis, meningitis and wound infections. In some cases, a direct connection to animals is obvious. B. bronchiseptica is being isolated increasingly from immunocompromised hosts with respiratory tract infections ranging from sinusitis to pneumonia/pleuritis.

Bacillus subtilis

Bacillus subtilis is a rod-shaped gram-positive bacterium commonly found in soil. B. subtilis has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions.

Salmonella setubal

Salmonella is a Gram-negative facultative rod-shaped bacterium. Salmonellae live in the intestinal tracts of warm and cold blooded animals. Some species are ubiquitous. Other species are specifically adapted to a particular host. In humans, Salmonella are the cause of two diseases called salmonellosis: enteric fever (typhoid), resulting from bacterial invasion of the bloodstream, and acute gastroenteritis, resulting from a foodborne infection/intoxication.

Fungal species

Aspergillus niger:

Aspergillus niger is less likely to cause human disease than some other Aspergillus species, but if large amounts of spores are inhaled, a serious lung disease, aspergillosis can occur. A. niger is one of the most common causes of otomycosis (fungal ear infections), which can cause pain, temporary hearing loss and, in severe cases, damage to the ear canal and tympanic membrane.

Aspergillus flavus:

Aspergillus flavus is a fungus. It is a common mold in the environment. It is a human pathogen, associated with aspergillosis of the lungs and sometimes causing corneal, otomycotic, and nasoorbital infections. Many strains produce significant quantities of aflatoxin (Klich, 2007), a carcinogenic and acutely toxic compound. A. flavus spores are allergenic. A. flavus may invade arteries of the lung or brain and cause infarction.

Aspergillus fumigatus

Commonly found on soil, grains, fruits, and vegetables. It grows with temperatures higher than 40C. Aspergillus fumigatus causes both invasive and allergic aspergillosis in immune compromised patients. It also causes skin, pulmonary and ear infection (Hawksworth et al., 1995).

Fusarium solani

Fusarium solani (seed born pathogen) is commonly considered as contaminant, but disease has been reported in individuals. This species is quite easily recognized based upon colonies are woolly to cottony with cream to white aerial mycelium, long monophialides, and microconidia in false heads only. Fusarium solani is the most common Fusarium species recovered in humans and animals. It is an etiologic agent in cutaneous infections, burn patients, mycetoma, onychomycosis, sinusitis, pulmonary disease, endocarditis and septic arthritis (Summerbell and Schroers, 2002).

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Title
Screening of selected plant species of Pakistan for their pharmacological activities
Author
Year
2010
Pages
166
Catalog Number
V175245
ISBN (eBook)
9783640965014
ISBN (Book)
9783640965212
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3041 KB
Language
English
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screening, pakistan
Quote paper
Maryam Jamil (Author), 2010, Screening of selected plant species of Pakistan for their pharmacological activities, Munich, GRIN Verlag, https://www.grin.com/document/175245

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