Biological Evaluation of Some Selected Plant Species of Pakistan

Samia Inayatullah

Department of Biochemistry Quaid-i-Azam University Islamabad, Pakistan 2009 Biological Evaluation of Some Selected Plant Species of Pakistan

Submitted by

Samia Inayatullah

Thesis submitted to

The Department of Biochemistry

Quaid-i-Azam University Islamabad

In the partial fulfillment of the requirements for the degree of Doctor of Philosophy

Biochemistry/ Molecular Biology

Department of Biochemistry Quaid-i-Azam University Islamabad-Pakistan 2009

Certificate

This thesis submitted by Samia Inayatullah is accepted in its present form by the Department of Biochemistry, Quaid-i-Azam University Islamabad as satisfying the thesis requirement for the degree of Doctor of Philosophy in Biochemistry/ Molecular Biology.

Supervisor:………………………. Dr. Bushra Mirza

External Examiner: ………………

External Examiner :………………

Chairman: …………………..

Dated:…………….

Declaration

I hereby declare that the work presented in this thesis is my own effort except where other acknowledged and that the thesis is my own composition. No part of the thesis has previously been presented for any other degree.

Samia Inayatullah

In the name of Allah, the Beneficent, the Merciful

“With Him are the keys of the unseen, the treasures that none knoweth but He. He knoweth whatever there is on the earth and in the sea. Not a leaf doth fall but with His knowledge. There is not a grain in the darkness (or depths) of the earth, nor anything fresh or dry (green or withered) but is (inscribed) in a record clear (to those who can read).” (VI. 59)

To my beloved parents

List of Contents

Acknowledgements I List of tables III List of figures IV List of abbreviations VII Abstract IX Chapter 1 Introduction……………………………………………………………1 1: Drug discovery strategy………………………………………………………...2 1.1 : Selection of plant material…………………………………………………...3 1.2: Preparation of plant extracts………………………………………………....8 1.3: Biological screening of plant extracts…………………………………………9 1.3.1: Antimicrobial assays…………………………………………………….10 1.3.2: Toxicity assays…………………………………………………………...16 1.3.3: Antitumor potato disc assay…………………………………………….17 1.3.4: Antioxidant assays……………………………………………………….18 Determination of total phenolic contents………………………………...19 a. Antioxidant assays in aqueous system……………………………………....20 b. Antioxidant assays in lipid system…………………………………………..22

1.4: Bioassay guided fractionation of selected plant extracts .………………...... 23

1.5: Identification of components of plant extracts by analytical scale

HPLC equipped with UV-DAD as well as LC-MS………………………….23 1: Chromatography…………………………………………………………….23 2: Liquid chromatography-mass spectrometry (LC-MS)……………………32

Chapter 2 Antimicrobial, toxicity and antitumor activities……………………...34

Introduction………………………………………………………………………..34

Materials and methods……………………………………………………………...35 2.1: Collection of plant material…………………………………………………….35

2.2: Preparation of plant extracts………………………………………………...... 35

2.3: Antimicrobial assays…………………………………………………………….35 2.4: Toxicity assays…………………………………………………………………...38

2.5: Antitumor potato disc assay……………………………………………………39

2.6: Antibacterial assay against Agrobacterium tumefaciens……………………...40

Results…………………………………………………………………………………..41

2.1: Antimicrobial assays…………………………………………………………....41 2.2: Toxicity assays…………………………………………………………………..41 2.3: Antitumor potato disc assay…………………………………………………...46 2.4: Antibacterial assay against Agrobacterium tumefaciens……………………..46 Conclusion……………………………………………………………………...... 52 Chapter 3 Antioxidant activities…………………………………………………...... 53

Introduction…………………………………………………………………………...53

Materials and methods…………………………………………………………….....54

3.1: Determination of total phenolic contents……………………………………54 3.2: Antioxidant activity……………………………………………………………55 3.2.1: DPPH assay (1,1- Diphenyl-2-picryl-hydrazyl radical)………………..55

3.2.2: Calculation of EC50 value………………………………………………..56 3.2.3: ABTS+ Assay……………………………………………………………..56 3.2.4: TBARS (Thiobarbituric acid reactive substances) assay……………..57 3.3: DNA protection assay………………………………………………………...58 Results…………………………………………………………………………………...59 3.1: Determination of total phenolic contents……………………………………..59 3.2: Statistical analysis……………………………………………………………..59 3.3: DPPH scavenging activity…………………………………………………….63 3.4: ABTS+ assay…………………………………………………………………...63 3.5: TBARS (Thiobarbituric acid reactive substances) assay…………………..64 3.6: DNA protection assay………………………………………………………..74 Conclusion…………………………………………………………………………..74 Chapter 4 Semi-preparative HPLC………………………………………………….76 Introduction…………………………………………………………………………..76 Materials and methods……………………………………………………………….78 4.1: Preparation of samples……………………………………………………...78 4.2: Spectrophotometric scanning of hexane washes and extracts……………78 4.3: Semi-preparative HPLC…………………………………………………….78 4.3.1: Sample Preparation……………………………………………………….78 4.3.2: Method development for semi-preparative HPLC……………………...78 4.3.3: Optimization of conditions for semi-preparative HPLC of (L+F) S. nubicola………………………………………………………………….79 4.3.4: Method for fractionation………………………………………………...... 81 4.4: Determination of total phenolic contents of fractions……………………82 4.5: Antioxidant activity of fractions……………………………………….. …82 Results………………………………………………………………………….83 4.1: Spectrophotometric scanning of hexane washes and crude extracts….83 4.2: Semi-preparative HPLC…………………………………………………86 4.2.1: Method development for semi-preparative HPLC…………………..86 4.3: Determination of total phenolic contents for three fractions………...94 4.4: Antioxidant activity of fractions………………………………………..94 Conclusion………………………………………………………………….99 Chapter 5 Identification of phenolic compounds…………………………………100 Introduction……………………………………………………………………...100 Materials and methods……………………………………………………...... 101 5.1: Preparation of samples…………………………………………………...101 5.2: Preparation of standard……………………………………………...... 101 5.3: Analytical scale HPLC……………………………………………………101 5.3.1: HPLC Method…………………………………………………...... 101 5.3.2: Regression lines for five external standards………………………102 5.3.3: Quantification of various peaks……………………………………102 5.4: LC-MS (Liquid chromatography mass spectrometry)……………...... 103 5.5: Identification of phenolic components in crude plant extracts………...103 Results………………………………………………………………………….105 5.1: Analytical scale HPLC……………………………………………….105 5.2: Regression lines for five external standards by using analytical scale HPLC…………………………………………………………………112 5.3: Quantification of various peaks……………………………………..112 5.4: LC-MS (liquid chromatography mass spectrometry)……………...118 5.5: Identification of phenolic compounds………………………………126 Conclusion……………………………………………………………...135 Chapter 6 Discussion………………………………………………………………..136 Conclusion…………………………………………………………………..143 References…………………………………………………………………..144

ACKNOWLEDGEMENTS

I offer my humblest thanks to Almighty Allah who enabled me to make some material contribution to the preexisting ocean of knowledge and thoughts. All blessings and respects are for our beloved Prophet Muhammad (PBUH) whose teachings guide us towards light of knowledge. I am thankful to Higher Education Commission Pakistan for their scholarship throughout my research project and I am thankful to their IRSIP program which supported me while my visit to CSU, Australia. I want to offer my thanks to Dr. Fayyaz Ahmed Choudhary, Dean, Faculty of Biological Sciences for provision of research facilities in the department. I ardently extend my thanks to Dr. Waseem Ahmed, Chairman, Department of Biochemistry, Quiad-i-Azam University, for providing research facilities in the department. My reverent and gratitude is for my honourable supervisor Dr. Bushra Mirza, Associate Professor, Department of Biochemistry, Quaid-i-Azam University, whose dynamic supervision, keen interest, illustrious advice, philanthropic attitude and encouragement throughout my research work enabled me to achieve my goals. Dr. Bushra Mirza is that School for me who taught me confidence, enthusiasm and consistency. She has always been a very encouraging person. She always helped me while discussing research proposals. I am really inspired by her intelligence. I want to offer my thanks to Dr. Atta-ur-Rehman, Molecular Biologist, CSU, Australia who has been a very good host and supervisor during my visit to CSU Australia. I am thankful to his wife Alvina and all his children for giving me good company and hosting me. I have no words to pay my thanks to Dr. Paul Prenzlar, Senior lecturer, CSU, Australia whose constant help and encouragement helped me to achieve my goals during my visit. I want to offer my special thanks to Dr. Hassan Obeid, lecturer, CSU, Australia, whose guidance helped me to complete my research project in Australia. I am thankful to lab staff at Chemistry laboratory, CSU, Australia, and my friends April Cao, Long and Laura Rustioni who always gave me company and helped to extend my little knowledge.

I I offer my sincerest thanks to my friends and lab fellows and all others for their help and kind attitude. I wish to express my appreciation and sense of gratitude from the citadel of my heart to my parents, brother and sisters for their cooperation and encouragement. Samia Inayatullah

II List of Tables

Table 2.1 List of plant species with respective plant extracts 36

Table 2.2 Antibacterial activity of methanol extract of leaf and stem of

A. oblongifolium against six bacterial strains 42

Table 2.3 Percentage inhibition of crude extracts of five different species

against six fungal strains 44

Table 2.4 Illustration of % age mortality of brine shrimps at different

concentrations of extracts and respective ED50 value 45

Table 2.5 Average number of tumors produced at different

concentrations of extracts 50

Table 3.1 Total phenolic contents of crude extracts 62

Table 3.2 Result of DPPH assay, ABTS+ assay and TBARS 73

Table 4.1 Total phenolic contents and DPPH scavenging activity of fractions 97

Table 5.1 Result of analytical scale HPLC 117

III List of figures

Fig 1.1 Salvia nubicola (Lamiaceae) 4

Fig 1.2 Hedera nepalensis (Araliaceae) 5

Fig 1.3 Acer oblongifolium (Aceraceae) 6

Fig 1.4 Sorbaria tomentosa (Rosaceae) 7

Fig 1.5 Separation mechanisms of chromatography 26

Fig 2.1 Antibacterial activity of (L+S) A. oblongifolium 43

Fig 2.2 Effect of two different concentrations on root length 47

Fig 2.3 Radish seed phytotoxicity in terms of root length 48

Fig 2.4 Effect of methanol extracts on seed germination 49

Fig 2.5 Antitumor potato disc assay 51

Fig 3.1 Regression line with caffeic acid 60

Fig 3.2 Regression line with gallic acid 60

Fig 3.3 Regression line with rutin 61

Fig 3.4 Regression line with trolox 61

Fig 3.5 DPPH scavenging with three standards 65

Fig 3.6 Percentage inhibition with (L+F) S. nubicola 66

Fig 3.7 Regression line used to calculate EC50 66

Fig 3.8 Percentage inhibition with stem extract of S. nubicola 67

Fig 3.9 Regression line with stem extract of S. nubicola 67

Fig 3.10 Percentage inhibition with leaf and stem extract of H. nepalensis 68

Fig 3.11 Regression line with leaf and stem extract of H. nepalensis 68

Fig 3.12 Percentage inhibition with leaf and stem extract of A. oblongifolium 69

IV Fig 3.13 Regression line with leaf and stem extract of A. oblongifolium 69

Fig 3.14 EC50 value for four methanol extracts and standards 70

Fig 3.15 Percentage inhibition with Trolox in ABTS+ scavenging activity 71

Fig 3.16 Percentage inhibition with trolox in case of TBARS 72

Fig 3.17 DNA protection assay with different concentrations of crude extracts 75

Fig 4.1 Gradual removal of pigments 84

Fig 4.2 Result of scanning at 200 nm to 800 nm for crude extract 85

Fig 4.3 Method 1 and chromatogram 87

Fig 4.4 Method 2 and chromatogram 88

Fig 4.5 Method 3 and chromatogram 89

Fig 4.6 Method 4 and chromatogram 91

Fig 4.7 Method 5 and chromatogram 92

Fig 4.8 Chromatogram obtained for (L+F) S. nubicola 93

Fig 4.9 Pattern of absorbance of phenolic compounds at 765 nm 95

Fig 4.10 DPPH scavenging pattern for three fractions 96

Fig 4.11 DNA protection assay with different concentrations of fraction 98

Fig 5.1 Analytical scale chromatogram for (L+F) S. nubicola 107

Fig 5.2 Chromatograms for three fractions 108

Fig 5.3 Analytical scale chromatogram for (S) S. nubicola 109

Fig 5.4 Analytical scale chromatogram for (L+S) H. nepalensis 110

Fig 5.5 Analytical scale chromatogram for (L+S) A. oblongifolium 111

Fig 5.6 Analytical scale chromatogram for five external standards 113

Fig 5.7 Regression lines for five external standards 116 Fig 5.8 LC-MS chromatogram for (L+F) S. nubicola 120

V Fig 5.9 LC-MS chromatogram for (S) S. nubicola 121 Fig 5.10 LC-MS chromatograms for (L+S) H. nepalensis 122 Fig 5.11 LC-MS chromatograms for (L+S) A. oblongifolium 124 Fig 5.12 LC-MS chromatogram for five external standards 125 Fig 5.13 Mass spectrum for rosmarinic acid 127 Fig 5.14 Mass spectrum of peak from LC-MS chromatogram 128 Fig 5.15 Mass spectrum for chlorogenic acid 131 Fig 5.16 Chromatogram for chlorogenic acid standard 132 Fig 5.17 Mass spectrum for rutin 133 Fig 5.18 Analytical scale HPLC for standard rutin 134

VI List of abbreviations

(L+F) Leaf and flower (L+S) Leaf and stem (S) Stem A. oblongifolium Acer oblongifolium ABTS 2, 2/-Azinobis (3-ethylbenzothiazoline-6-sulfonate

ANOVA Analysis of variance

At A. tumefaciens

ATCC American type culture collection

BHA Butylated hydroxyanisole

BHT Butylated hydroxytoluene

DAD Diode array detectors

DMSO Dimethyl sulfooxide

DPPH 2-2-Diphenyl-1-picrylhydrazyl

EC50 Effective concentration at fifty percent inhibition

ED50 Effective dose at fifty percent mortality

GAE Gallic acid equivalent

GF/F Glass fiber filters

GLC Gas-liquid chromatography

H. nepalensis Hedera nepalensis HPLC High-performance liquid chromatography

KB cells A cell line derived from human carcinoma of the nasopharynx

KI Potassium iodide

LC-MS Liquid chromatography-mass spectrometry

VII LLC Liquid-liquid chromatography mg Milligram

MIC Minimum inhibitory concentration mM Millimolar nm Nanometer

NWFP North west frontier province

%age Percentage ppm Parts per million

S. nubicola Salvia nubicola S. tomentosa Sorbaria tomentosa S.E Standard error S.D Standard deviation SDA Sabouraud dextrose agar

TBA Thiobarbituric acid

TBARS Thiobarbituric acid reactive substances

TBHQ t-butyl hydroxyquinone

TEAC Trolox equivalent antioxidant capacity

TLC Thin layer chromatography

UV Ultraviolet

VIII

Abstract

Five methanol extracts from four different plant species [Salvia nubicola B.

(Laminiaceae), Hedera nepalensis K. (Araliaceae) Acer oblongifolium D. (Aceraceae)

and Sorbaria tomentosa L. (Rosaceae)] were evaluated for their antimicrobial activity (by

antibacterial and antifungal assays), toxicity activities (by brine shrimp cytotoxicity assay, radish seed phytotoxicity assay), antitumor activity (by potato disc assay) and

antioxidant activities (by DPPH scavenging assay, ABTS+ assay, DNA protection assay

and TBARS).

Leaf and stem extract of A. oblongifolium exhibited significant antibacterial activity

against all pathogenic strains tested, while none of the extract presented any antifungal

activity against six pathogenic strains tested. Two of the five extracts (L+S) A.

oblongifolium and (L+S) H. nepalensis revealed significant ED50 value i.e. 47.7 ppm and

226.8 ppm respectively in case of brine shrimp cytotoxicity assay. Growth inhibition was observed by all extracts in radish seed bioassay at high concentration (10,000 ppm). At

low concentration (1000 ppm) three extracts from two plant species (leaves and flower extract of S. nubicola, stem extract of S. nubicola and stem extract of H. nepalensis) presented stimulation of growth ranging from 3.5 to 43.2%. Inhibition of tumor formation ranged from 9 to 82.9% by all extracts in antitumor potato disc assay at three different concentrations tested (1000, 100, and 10 ppm). A positive correlation was observed in the results of three of the described assays (toxicity assays i.e. brine shrimp cytotoxicity assay and phytotoxicity assay and antitumor potato disc assay).

IX Four methanol extracts from three selected plant species i.e. Salvia nubicola (Lamiaceae),

Acer oblongifoium (Aceraceae) and Hedera nepalensis (Araliaceae)) were screened for their antioxidant potential. Antioxidant activities were investigated in aqueous system by using DPPH scavenging assay, ABTS+ radical scavenging assay and DNA protection assay while in lipid system by using TBARS (Thiobarbituric acid reactive substances).

Total phenolic contents were determined by using Folin-Ciocalteu reagent. Methanol extract of leaf and flower of S. nubicola showed the highest trolox equivalent values in case of DPPH scavenging assay i.e. 2484.08 ± 4.9 as well as total phenolic contents i.e.

342.08 ± 19.8. Fractionation of methanol extract of S. nubicola by semi-preparative

HPLC yielded three fractions (A, B and C). Fraction B was found to be the most active in

DPPH scavenging assay with highest phenolic contents as estimated by using Folin-

Ciocalteu reagent. Analytical scale HPLC and LC-MS results revealed presence of rosmarinic acid in fraction B of S. nubicola while chlorogenic acid and rutin were

identified as major antioxidants in methanol extract of H. nepalensis.

Chapter 1 Introduction

Medicinal plants contain active chemical constituents in any of their parts like root, stem, leaves, bark, fruit and seeds which produce a definite curing physiological response in the treatment of various ailments in human and other animals. Knowledge about medicinal plants has been gathered through trial and error based upon speculations and superstitions. Medicinal plants are primary source of health care throughout the world for thousand of years. In the middle of 20th century, researchers preferred to use synthetic

medicines over natural medicines for curing various diseases. However due to emergence

of various side effects of synthetic drugs, trend to use medicinal plants to cure various

diseases is becoming popular (Awal et al., 2004; Jiang et al., 2006). Natural products

from medicinal plants are known to be chemically balanced, effective and least injurious

with none or much reduced side effects as compared to synthetic medicines.

According to a report of world health organization (W.H.O), 70% of the world population

uses medicinal plants to cure diseases through their traditional practitioners. In sub-

continent, plant oriented drugs have been used extensively from a very long time.

According to a survey conducted by W.H.O, traditional healers treat 65% patients in

Srilanka, 60% in Indonesia, 75% in Nepal, 85% in Mayanmer, 80% in India, and 90% in

Bangladesh. In Pakistan, 60% of the population, especially in villages is getting health

care by traditional practitioners (Hakims), who prescribe herbal preparations (Gilani et

al., 2001; Ahmed et al., 2004).

Northern areas of Pakistan are well known for production of many useful medicinal plants. Ahmed et al., (2004) have described the ethnopharmacological survey of some

1 Chapter 1 Introduction

medicinally important plants of Galliyat areas of NWFP. Important ethnomedicinal herbs of Ayubia National Park, Abbottabad are described by Gilani et al., (2001) and Gilani et

al., (2007). Ethnobotanical profile of Utror and Gabral valleys, district Swat is described

by Hamayun et al., (2005). In another study by Hamayun et al., (2006), folk medicinal

knowledge and conservation status of some economically valued medicinal plants of district Swat, is described.

In order to get information about medicinal status of plant species, local healers provide

useful information. Local healers have traditional knowledge which is transmitted from

generation to generation.

1: Drug discovery strategy

Modern strategies for drug discovery emphasize on availability of some simple and in-

expensive biological assays to evaluate medicinal potential of plant species. Present study

demonstrates a modern strategy with combination of local and modern knowledge for

evaluation of biological activity of medicinal plant species. The main steps of this

strategy are as following

1.1. Selection of plant material

1.2. Preparation of plant extracts

1.3. Biological screening of plant extracts by simple bioassays

1.4. Bioassay guided fractionation of selected plant extracts using semi-preparative

HPLC

1.5. Identification of components of plant extracts by analytical scale HPLC equipped

with UV-DAD as well as LC-MS

2 Chapter 1 Introduction

1.1. Selection of plant material

Four different plant species were selected from Swat and Kalam district of NWFP,

Pakistan. Selection was based upon knowledge from folk healers.

1.1.1: Salvia nubicola (Lamiaceae)

Salvia nubicola (Fig 1.1) belongs to family Lamiaceae. Locally it is known as “khoropo” or “saag”.

The members of Lamiaceae are mostly shrubs or herbs comprising about 200 genera and

3,200 species, commonly with aromatic, herbage, quadrangular stems, and verticillate inflorescences. Flower bloom period is from April to November with yellow or maroon colour. Salvia is the largest genus of the family Lamiaceae having 800 species throughout the world.

Most of the plants of this genus are well known for their biologically active constituents, specifically ones with anti-tumor activity (Ali et al., 2005; Ali et al., 2006). A number of

Salvia species are used in folk medicine for treatment of dysentery, boils, fall injuries, hepatic problems, and cancer (Fujita and Node, 1984; Zhang and Li, 1994).

Salvia species like S. officinalis are an important source of antioxidants used in food

industry and have wider implications for the dietary intake of natural antioxidants (Kosar

et al., 2008). Tepe et al. (2007) have described antioxidant potential and rosmarinic acid levels of two Salvia species. A number of Salvia species have been investigated previously for antioxidant potential and their polyphenol constituents. Major antioxidants included carnosic acid, rosmarinic acid, flavone glycosides and salvianolic acid (Cuvelier et al., 1996; Lu and Foo, 2001).

3 Chapter 1 Introduction

1.1.2: Hedera nepalensis (Araliaceae)

Locally, H. nepalensis (Fig 1.2) is known as “Albumbar”. Members of family Araliaceae

are perennial climbers c. 30 m tall with aerial roots. Leaves simple, 2-15 cm long; lanceolate to ovate to variously lobed, glabrous; base cordate to rounded or cuneate; apex sub-acute to obtuse. Flowers yellow; pedicels 7-12 mm long. Pedicels and peduncles hairy. Calyx entire. Anthers 1-2 mm long. Stylar column c. 1 mm long, persistent. Fruit a berry, 5-7 mm long, 5-10 mm broad. They are distributed in west Asia, Japan,

Afghanistan and the Himalayas. The ‘Himalayan ivy’ grows well in moist soil and shaded localities from c. 1000-3000 m. Climbs extensively on walls, rocks, tree trunks by its aerial roots (Nasir, 1975).

Leaves of Hedera nepalensis are used traditionally for treatment of diabetes (Gilani et al.,

2007). According to another study by Hamayun et al., (2006), leaves are used to treat cancer. Phytochemically, there is frequent occurance of triterpenoid saponins and poly- ynes (possibly also oxalic acid) (Frohne and Pfander, 2004).

Fig 1.1: Salvia nubicola (Lamiaceae)

4 Chapter 1 Introduction

Fig 1.2: Hedera nepalensis (Araliaceae)

1.1.3: Acer oblongifolium (Aceraceae)

Locally A. oblongifolium (Fig 1.3) is known as “Kaeen”. Trees evergreen, 12 to 15 meter

tall. Bark smooth to wrinkled. Trunk irregularly buttressed at base. Twigs red-brown or

purplish, slender. Leaves ovate-lanceolate, acuminate, 5-18 cm long, 2-8 cm wide,

glabrescent, reddish when young, later dark green above, paler to glaucous beneath;

nerves pinnate in 6-8 pairs; base rounded to subacute; petioles slender, 2-10 cm long.

Inflorescence corymobose, pubescent on leafy terminal and lateral shoots, 5-15 cm long.

Pedicels pubescent. Flowers are 5-merous, 7-9 mm across, greenish-white. Sepals linear,

1-2 mm wide, acute, pubescent. Petals narrowly lanceolate, 1-2 mm wide. Stamens 8, inserted on disc. Ovary pubescent, styles free nearly to the base. Samaras glabrous, 2-3

5 Chapter 1 Introduction

cm long; wings veined, divergent, constricted at base; nutlets gibbous, locules white-

pubescent inside (Nasir, 1975).

Acer oblongifolium (Aceraceae) is known for its antitumor, cytotoxic and phytotoxic

potential (Inayatullah et al., 2007). A number of Acer species are reported to have

antioxidant activities including Acer albopurpurascens (Lee et al., 2005; Jiang et al.,

2006), Acer palmatum (Kim et al., 2005), Acer nikoense, Acer buerferianum (Hou et al.,

2003) and Acer saccharum (Berge and Perkins, 2007). Their major antioxidant

constituents have been identified as (+)-Rhododendrol, (+)-Catechin and Vitexin.

Fig 1.3: Acer oblongifolium (Aceraceae)

1.1.4: Sorbaria tomentosa (Rosaceae)

S. tomentosa (Fig 1.4) is known as “Karhee” locally. Genus Sorbaria presents plants which are mostly shrubs and deciduous. Branchlets are yellow to green when young, later dark reddish or yellowish brown, terete; buds are ovoid to cylindric, with several exposed, alternate scales, glabrous or slightly pubescent at apex. Leaves alternate,

6 Chapter 1 Introduction stipulate, pinnate; leaflets opposite, sessile or subsessile, doubly serrate. Inflorescence a large, terminal panicle. Flowers small, numerous. Hypanthium shallowly copular. Sepals

5, reflexed, short, broad, persistent. Petals 5, imbricate, white, ovate to orbicular, base cuneate, apex obtuse. Stamens 20-50, nearly equaling or longer than petals. Carpels 5, opposite sepals, basally connate, glabrous or subglabrous. Follicles glabrous, dehiscent along adaxial suture. Seeds several.

Fig 1.4: Sorbaria tomentosa (Rosaceae)

7 Chapter 1 Introduction

Flower bloom period is June to August and flower colour is white or near white. There are approximately 31 species, subspecies, varieties, forms and cultivars in this genus.

Many plants of family Rosaceae are of economic importance and contribute to people’s livelihoods. The Rosaceae contain a great number of fruit trees of temperate regions. The fruit contain vitamins, acids, and sugars and can be used both raw and for making preserves, jam, jelly, candy, various drinks, wine, vinegar, etc. Some plants in the genus

Rosa containing essential oils or with a high vitamin content are used in industry.

Rosaceae wood is used for making various articles, stems and roots are used for making tannin extract, and young leaves are used as substitute for tea. Numerous species are used for medical purposes or are cultivated as ornamentals (Bisby et al., 2007; Brands, 2000).

1.2: Preparation of plant extracts

Preparation of plant extracts requires a suitable organic solvent. Solvents can be classified into polar and non-polar solvents. Both classes have ability to dissolve polar and non-polar components. Various strategies have been used previously for the preparation of plant extracts from plant material.

1.2.1: Organic solvents for preparation of plant extracts

Various organic solvents including methanol, dichloromethane, light petroleum ether, acetone, chloroform, ethanol and hexane etc have been in use previously for preparation of crude plant extracts.

8 Chapter 1 Introduction

1.2.2: Procedures used for preparation of plant extracts

(a). Maceration procedure.

Dried parts of plant material (leaves, roots and stem dried at room temperature) are

powdered and macerated with organic solvents for 3-7 days at room temperature.

Filtration of soaked material is done by using filter paper. Filtered material is

concentrated at low pressure and temperature by rotary evaporator to get the crude

extract. The aqueous part of the crude plant extract is dried by using a freeze-drier.

(b). Use of Soxhlet apparatus for extract preparation.

Soxhlet apparatus has been in use previously for preparation of crude plant extracts. A known mass of each ground plant is extracted continuously with organic solvent by liquid-solid extraction procedure.

(c). Preparation of aqueous extracts.

Most popular procedure for preparation of aqueous plant extracts is decoction. Plant material is ground and infused in boiling water for 15 minutes after which the mixture is filtered. The filtrate is evaporated to dryness under vacuum and then freeze dried.

1.3: Biological screening of plant extracts

Biological screening of plant extracts to evaluate their potential for various activities requires development of simple, rapid and inexpensive biological assays. Potent plant extracts then can further be fractionated for isolation and identification of biologically active constituents.

The commonly used biological assays can generally be described in the following way.

1.3.1. Antimicrobial assays

(a) Antibacterial assay

9 Chapter 1 Introduction

(b) Antifungal assay

1.3.2. Toxicity assays

(a) Phytotoxicity assay

(b) Brine shrimp cytotoxicity assay

1.3.3. Antitumor assay

1.3.3. Antioxidant assays

1.3.1: Antimicrobial assays

In order to check antimicrobial activity of purified antibiotics or crude extracts following methods can be used

(a) The disc diffusion method

(b) Agar well diffusion method

(d) Serum Killing power

(e) Automated methods

(a) The disc diffusion method

In the disc diffusion method, or Kirby-Bauer method, a standard quantity of the

causative is uniformly spread over an agar plate. Then several filter paper discs

impregnated with specific concentrations of selected chemotherapeutic agents are

placed on the agar surface. Finally, the culture with the antibiotic discs is incubated.

A new version of the diffusion test, called an E test uses a plastic strip containing a

gradient of concentration of antibiotic.

10 Chapter 1 Introduction

(b) Agar well diffusion method

In this method, wells are cut in seeded agar and the test sample is then introduced

directly into these wells. After incubation the diameter of the clear zones around each

well is measured and compared against zone of inhibition produced by solution of

known concentration of standard antibiotics. Five or six samples may be tested

simultaneously by the diffusion method.

In this method a constant quantity of microbial inoculum (specimen) is introduced

into a series of broth cultures containing decreasing concentrations of a

chemotherapeutic agent. After incubation (for 16 to 20 hours) the tubes or wells are

examined, and the lowest concentration of the agent that prevents visible growth

(indicated by turbidity or colony forming units) is noted. This concentration is the

minimum inhibitory concentration (MIC) for a particular agent acting on a specific

microorganism.

Samples from tubes that show no growth but that might contain inhibited organisms

can be used to inoculate broth that contains no therapeutic agent. In this test, the

lowest concentration of the therapeutic agent that yields no growth following this

second inoculation, or subculturing, is the minimum bactericidal concentration

(MBC).

11 Chapter 1 Introduction

(d) Serum killing power

This test is performed by obtaining a sample of a patient’s blood while the patient is

receiving an antibiotic. A bacterial suspension is added to a known quantity of the

patient’s serum (blood plasma minus the clotting factors). Growth (turbidity) in the

serum after incubation means that the antibiotic is ineffective. Inhibition of growth

suggests that the drug is working, and more quantitative determinations can be made

to identify the lowest concentration that still provides serum killing power.

(e) Automated methods

Automated methods are now available to identify the pathogenic organisms and to

determine which antimicrobial agents will effectively combat them. One such method

uses prepared trays with small wells into which a measured quantity of inoculum is

automatically dispensed. Trays are also available to determine the sensitivity of

organisms to a variety of antimicrobial agents.

The trays are inserted into a machine that measures microbial growth. Some machines

do this by using a beam of light to measure turbidity. Others utilize media containing

radioactive carbon. Organisms growing on such media release radioactive carbon

dioxide into the air, and a sampling device automatically detects it (Black, 2005).

Microbial agents

(a) Bacteria

(b) Fungi

(a). Bacteria

12 Chapter 1 Introduction

In 1884, a Danish scientist Hans Christian Gram developed the most frequently used differential stain, which now bears his name. Bacteria are classified on the basis of Gram staining of bacterial cell wall into two major categories.

1. Gram negative bacteria

2. Gram positive bacteria

Gram negative bacteria

After Gram-staining procedure, Gram-negative cells appear pink

The Gram negative bacteria used in this study include E. coli, S. setubal and B.

bronchiseptica.

Escherichia coli

E.coli are known to cause Urinary infection, wound infection and gastroenteritis

Salmonella setubal

S. seubal are known to cause Enteric fever (typhoid), food poisoning, bone infection

Bordetella bronchiseptica

Bordetella bronchiseptica have been involved in upper respiratory tract infections in

human and animals.

Gram negative bacteria

The thick cell wall of a Gram-positive organism retains the crystal violet dye used in

the Gram-staining procedure, so the stained cells appear purple under magnification.

. Gram positive bacteria used in this study include S. aureus, M. leuteus and B.

subtillus.

Staphyllococcus aureus. S. aureus is causative agent of Pneumonia, meningitis and

food poisoning.

13 Chapter 1 Introduction

Micrococcus leutus

M. leutus has been known to be involved in hepatic abcess (Andreopoulos et al.,

2000) as well as meningitis (Fosse et al., 1985).

Bacillus subtillus

B. subtillus is mostly involved in Urinary infection, wound, ulceration and septicemia

(b). Fungi

Evidence about fungi species suggests that 1.5 million species of fungi exist in the world.

Pathogenic fungi are responsible for a number of diseases in human, animals and plants.

Due to resistance to fungicidal drugs by plants and animals new means of drugs are required. Herbal plant species can provide a better source of antifungal components as compared to other chemical resources.

A number of pathogenic strains of fungi are found. The following describes some of the species.

Alternaria species

The Alternaria species is found in soil, seed and plants. It has been associated with hypersensitivity, pneumonitis and asthma hypersensitivity; type1 (Crissy et al., 1995) and asthma. Alternaria alternate produces tenuazonic acid and other toxic metabolites which is associated with disease in humans or animals.

Aspergillus Species

The genus Aspergillus includes over 185 species. Around 20 species have so far been reported as causative agents of opportunistic infections in man. Among these, Aspergillus fumigatus is the most commonly isolated species, followed by Aspergillus flavus and

Aspergillus niger.

14 Chapter 1 Introduction

Aspergillus spp. are well-known to play a role in three different clinical settings in man:

(i) opportunistic infections; (ii) allergic states; and (iii) toxicoses. Immunosuppression is

the major factor predisposing to development of opportunistic infections. These

infections may present in a wide spectrum, varying from local involvement to

dissemination and as a whole called aspergillosis. Among all filamentous fungi,

Aspergillus is in general the most commonly isolated one in invasive infections. It is the

second most commonly recovered fungus in opportunistic mycoses following Candida.

Mucar species

Mucor specie naturally occurs in soil, dead plant material, fruits and fruit juice. It is also

found in leather, meat, dairy products, animal hair, and jute. This species may cause

mucorosis in immune compromised individuals. The sites of infection are the lung, nasal

and sinus passage (Crissy et al., 1995).

Fusarium species

As well as being common plant pathogens, Fusarium spp. are causative agents of

superficial and systemic infections in humans. Infections due to Fusarium spp. are

collectively referred to as fusariosis. The most virulent Fusarium spp. is Fusarium solani

(Mayayo et al., 1999). Trauma is the major predisposing factor for development of

cutaneous infections due to Fusarium strains. Disseminated opportunistic infections, on

the other hand, develop in immunosuppressed hosts, particularly in neutropenic and

transplant patients (Austen et al., 2001, Boutati and Anaissie, 1997, Girmenia et al.,

1999, Vartivarian et al., 1993, Venditti et al., 1988). Fusarium infections following solid

organ transplantation tend to remain local and have a better outcome compared to those

15 Chapter 1 Introduction

that develop in patients with hematological malignancies and bone marrow

transplantation patients (Sampathkumar and Paya, 2001).

1.3.2: Toxicity assays

(a) Phytotoxicity assay

Herbicidal properties of plant extracts can be evaluated by using phytotoxic assays.

Chemicals used to kill weeds in crops are known as herbicides. Herbicides can be obtained from natural resources or synthesized chemically in the laboratory which can be used to improve the quality and yield of crops. Due to side effects of chemically synthesized herbicides and their environmental hazards, herbicides from natural resources

are preferred. Site of action of herbicide, their translocation in the plant and metabolism are keys, used to select proper herbicide for a specific crop.

Turker and Camper (2002) have described radish seed phytotoxicity assay as a general prescreening assay for phytotoxic evaluation of plant extracts. Raddish seeds are easily available and assay is easy to perform. Measurement of phytotoxicity in terms of root length and number of germinated seeds also provide simple means to use this assay.

Moreover phytotoxic evaluation is necessary in case of plant antitumor agents because those with growth inhibitory properties can not be used as antitumor agents against A. tumefaciens induced tumors.

(b) Brine shrimp cytotoxicity assay

Brine shrimp cytotoxicity assay is very simple bench-top assay used to measure cytotoxicity of plant extracts as well as synthetic compounds. Brine shrimp eggs are

16 Chapter 1 Introduction available commercially, and being used as fish food. Evaluation of natural products and synthetic compounds by using brine shrimp cytotoxicity assay describes not only cytotoxicity but also anticancer, antiviral, insecticidal and pesticidal potential (Sheikh et al., 2004). A good correlation has been found between brine shrimp cytotoxicity and cytotoxicity against KB cells (McLaughlin, 1991). Awal et al., (2004) has demonstrated toxicity of leaf and seed extracts of Cassia alata by using brine shrimp cytotoxicity assay while in another study by Mongelli et al., (2003), cytotoxic evaluation of components of

Bolax gummifera was demonstrated by using brine shrimp cytotoxicity assay. Brine shrimp assay has also been used by Chowdhury et al., (2004) while describing cytotoxic potential of extracts and purified components of Stachytarpheta urticaefolia .

1.3.3: Antitumor potato disc assay

The development of a possible strategy was required to screen potent plant extracts and products for anticancer activity. Although people were using cancer cell lines to evaluate anticancer potential of plant extracts, a less expensive strategy was required. Ferrigini et al., (1982) proposed the idea to use antitumor potato disc assay by the modified procedure described by Galsky et al., (1980). Moreover, a statistically significant correlation was found between antitumor potato disc assay and 3PS (P388) in vivo mouse leukemic system (Ferrigini et al., 1982). Antitumor potato disc assay requires use of wild type strain of Agrobacterium tumefaciens which can induce tumors on potato discs.

Anderson et al., (1988) have proposed that antitumor potato disc assay is convenient supplement to 9 KB and 3PS antitumor assays so sparing the requirement for cell cultures and serum.

17 Chapter 1 Introduction

Turker and Camper (2002) have described antitumor potato disc assay as major

prescreening assay to evaluate anticancer potential of mullein extracts. Coker et al.,

(2002) have demonstrated that antitumor potato disc assay is a general prescreening assay to determine antineoplastic activity of plant extracts and purified compounds regardless of the mechanism of drug action.

In potato disc assay, wild type strain of Agrobacterium tumefaciens is used to develop tumors on potato discs while various concentrations of plant extracts are used to check

their antitumor potential. Tumor inhibition is calculated by using following formula.

% age of tumor inhibition = 100 – ns/nc x 100

Where

ns = number of tumors in sample

nc = number of tumors in control

1.3.4: Antioxidant assays

Aerobic life on earth depends upon oxygen but it is also involved in a number of toxic chemical reactions. Auto-oxidation occurs when any organic molecule reacts with atmospheric oxygen. Auto-oxidation of food stuff results in oxidation of lipids and rancidity of food. Human physiology also involves a number of oxidation reactions.

Diseases such as atherosclerosis, cancer and tissue damage in rheumatoid arthritis involve oxidative stress (McDonald et al., 2001, Halliwell, 1994, Basu et al., 1999). Antioxidants are compounds responsible for balancing oxidation processes in our body. These compounds are mostly phenolic compounds which are oxidized very quickly and reduce the effect of oxidants.

There are two types of antioxidants available

18 Chapter 1 Introduction

(a). Synthetic antioxidants

(b). Natural antioxidants

(a) Synthetic antioxidants

Antioxidants which are synthesized under laboratory conditions are known as synthetic antioxidants. For inhibition/delaying of onset of oxidation processes in food stuff there is extensive use of synthetic antioxidants like butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and t-butyl hydroxyquinone (TBHQ) in food industry.

(b) Natural antioxidants

Antioxidants produced naturally by living organisms are called natural antioxidants.

Toxicity of synthetic antioxidants is obvious so there is decreased use of synthetic antioxidants in food industry. Evaluation of herbal extracts for naturally occurring antioxidants especially phenolic phytochemicals is need of the present medicine and food industry. Now many natural antioxidants like ascorbic acid, caffeic acid, gallic acid, quercetin and crude herb and spice-derived extracts are available in the market and regarded as safe, naturally occurring antioxidants (Kosar et al., 2003).

Determination of total phenolic contents

Total phenolic contents in crude extracts can be measured by Folin-Ciocalteu reagent by using the method of Singleton and Rossi (1965). Folin-Ciocalteu reagent is molybdotungsto phosphoric heteropolyanion reagent that can reduce phenols specifically.

A blue colour is developed which can be measured at 765 nm by using

19 Chapter 1 Introduction

spectrophotometer. Intensity of blue colour depends upon concentration of phenolic

compounds in the test substance. Gallic acid is used as standard and standard curve is

used to calculate GAE (gallic acid equivalent) value as mg of gallic acid per gram of

crude extract. Phenolic standards other than gallic acid can also be used.

Evaluation of natural and synthetic antioxidants requires antioxidant assays.

Antioxidant assays can be described in two systems

(a) Antioxidant assays in aqueous system

(b) Antioxidant assays in lipid system

(a) Antioxidant assays in aqueous system

In aqueous system three assays can be described.

1. DPPH assay

2. ABTS+ assay

3. DNA protection assay

1. DPPH (2-2-Diphenyl-1-picrylhydrazyl) assay

This assay measures reducing ability of antioxidants towards DPPH radical. DPPH

radical is available commercially in deep blue colour. Antioxidant capacity can be

measured by measuring the decrease in absorbance of methanolic solution of DPPH at

517 nm. Reaction is monitored by spectrophotometer (Prior et al., 2005). The percentage

of antioxidant capacity is measured by using following equation

20 Chapter 1 Introduction

% age of antioxidant capacity = (Ac – As/Ac) x 100

Where

Ac = Absorbace of negative control at 517 nm

As = Absorbance of sample at 517 nm

2. ABTS+ assay (2, 2/-Azinobis (3-ethylbenzothiazoline-6-sulfonate)

In this assay, ABTS+ is oxidized by peroxyl radicals or other oxidants to its radical

cation, ABTS.+, which is intensely coloured (dark green), and antioxidant capacity is

measured as the ability of the test compounds to decrease the colour reacting directly

with the ABTS.+ radical (Prior et al., 2003). Results of test compounds are expressed

relative to trolox. Decrease in absorbance by test compound and control is measured at

415 nm by using spectrophotometer.

3. DNA protection assay

Antioxidant/pro-oxidant activity of crude extracts or pure compounds can also be assessed by another assay in aqueous system. The assay is based upon Fenton reaction. In

2+ a Fenton reaction, Fe reacts with H2O2, resulting in the production of hydroxyl radical,

which is considered to be the most harmful radical to biomolecules (Meneghini, 1997).

Fe2+ is oxidized to Fe3+ in the Fenton reaction (Tian and Hua, 2005). With the attack of

-OH generated from the Fenton reaction, supercoiled plasmid DNA is broken into three

forms, including supercoiled (SC), open circular (OC) and linear form (Linear). The

degree of DNA protection can be assessed by the percentage of supercoiled form in gel

21 Chapter 1 Introduction

electrophoresis, and the antioxidant or prooxidant effect of tested sample is presented by

the ratio of supercoiled percentage of tested sample to that of the control (DNA treated

with FeSO4 and H2O2).

(b) Antioxidant assays in lipid system

Lipid oxidation of food results in food spoilage. Lipid oxidation is involved in a number

of physiological conditions so evaluation of antioxidant potential of natural and synthetic

compounds requires an assay in lipid system too.

TBARS (thiobarbituric acid reactive substances) assay

Most commonly used assay in lipid system is TBARS (thiobarbituric acid reactive

substances) assay. Linoleic acid is subjected to oxidation in presence of Copper chloride

as a result of which lipid oxidation products are formed. Melondialdehyde (MDA) is one

of the major products of lipid oxidation. These products react with TBA (thiobarbituric

acid) and give pink colour which can be measured at 532 nm by spectrophotometer.

Presence of any antioxidant of lipid in this system result in less products of lipid

oxidation and therefore less pink colour is developed (McDonald et al., 2001).

Percentage inhibition of oxidation can be measured by using following formula.

% age of antioxidant capacity = (Ac – As/Ac) x 100

Where,

Ac = Absorbance of negative control at 532 nm

22 Chapter 1 Introduction

As = Absorbance of sample at 532 nm

1.4: Bioassay guided fractionation of selected plant extracts using semi-preparative

HPLC

Crude plant extracts selected for a biological activity can be fractionated in various ways.

Semi-preparative HPLC is one of the best methods which can be used to get various

fractions in sufficient quantity. Method development requires various steps. Several

factors including solvent system, type of column, concentration of crude extract, flow

rate of solvent, injection volume and time required for a single run are very important.

The best method would require less time span for a single run by using high concentration of injection volume of crude extract. In this way, sufficient quantity of

fractions, in less number of runs can be obtained. Fractions obtained from semi-

preparative HPLC can further be tested for biological activity so that potent fraction for a

biological activity can be identified.

1.5: Identification of components of plant extracts by analytical scale HPLC

equipped with UV-DAD and LC-MS

Chemical analysis of crude plant extracts include two very interesting techniques i.e.

analytical scale HPLC equipped with UV-DAD and LC-MS. These techniques can help

to identify various components in crude plant extracts.

1: Chromatography

Chromatography can be defined as a physical process whereby components (solutes) of a

sample mixture are separated by their differential distribution between stationary and

mobile phases (Ullman and Burtis, 2006).

23 Chapter 1 Introduction

There are two forms of chromatography

1. Planar chromatography: In planar chromatography, the stationary phase is

coated on a sheet of paper (paper chromatography) or bound to a solid surface

(thin layer chromatography).

a. Paper chromatography: In paper chromatography, the stationary phase is a

layer of water or a polar solvent coated onto the paper fibers.

b. Thin layer chromatography (TLC): In thin layer chromatography, a thin

layer of particles of a material such as silica gel is spread uniformly on a glass plate or a plastic sheet. When the thin layer consists of particles with small diameters (4.5 µm), the technique is termed as high-performance, thin-layer chromatography (HPTLC).

2. Column chromatography: Depending upon type of mobile phase column

chromatography can either be gas chromatography or liquid chromatography

In column chromatography, the stationary phase can be pure silica or polymer, or it can be coated onto, or chemically bonded to, support particles. The stationary phase may be packed into a tube, or it is coated onto the inner surface of the tube. When the stationary phase in the liquid chromatography consists of small-diameter particles, the technique is called high-performance liquid chromatography (HPLC).

Chromatogram

In analytical scale gas chromatography and liquid chromatography, the mobile phase, or eluent, exits from the column and passes through a detector or series of detectors that

24 Chapter 1 Introduction

produces a series of electronic signals that are plotted as a function of time, distance or

volume. The resulting graphical display is called a chromatogram.

Separation mechanisms

Chromatographic separations are classified by the chemical or physical mechanisms used

to separate the solutes (Fig 1.5). They can be classified as follows.

Ion-exchange chromatography

Ion-exchange chromatography is based on an exchange of ions between a charged

stationary surface and mobile phase of the opposite charge. Depending upon the

conditions, solutes are either cations (positively charged) or anions (negatively charged).

They are separated depending on the differences in their ionic charge or the magnitude of

their ionic charges.

Partition chromatography

The differential distribution of solutes between two immiscible liquids is the basis for separation by partition chromatography. Operationally, one of the immiscible liquids serves as the stationary phase. To prepare this phase, a thin film of the liquid is adsorbed or chemically bonded onto the surface of support particles or onto the inner wall of a capillary column. Separation is based on differences in the relative solubility of solute molecules between the stationary and mobile phases.

Partition chromatography is categorized as either GLC (gas-liquid chromatography) or liquid-liquid chromatography (LLC).

25 Chapter 1 Introduction

Fig 1.5: Separation mechanisms of chromatography

26 Chapter 1 Introduction

Liquid-liquid chromatography is further categorized as either normal phase or reverse phase.

Normal phase liquid-liquid chromatography

For normal phase LLC a polar liquid is used as the stationary phase, and a relatively non- polar solvent or solvent mixture is used as the mobile phase.

Reversed-phase partition chromatography

In reversed-phase partition chromatography, the stationary phase is non-polar and the mobile phase is relatively polar. When particles of small diameter are used as the stationary-phase support, the technique is HPLC. Because column efficiency is inversely related to the column packing particle size and pressure drop is related to the square of the particle diameter, relatively high pressures are required to pump liquids through efficient HPLC columns. Consequently the technique has also been referred to as high- pressure liquid chromatography.

1. Adsorption chromatography

The basis of separation by adsorption chromatography is the differences between the

adsorption and desorption of solutes at the surface of a solid particle. Electrostatic,

hydrogen-bonding, and dispersive interactions are the physical forces that control this

type of chromatography.

27 Chapter 1 Introduction

2. Affinity chromatography

In affinity chromatography the unique and specific biological interactions of the

analyte and ligand is used for the separation. The specificity resulting from enzyme-

substrate, hormone-receptor, or antigen-antibody interactions has been used in this

type of chromatography.

3. Size-exclusion chromatography

Size-exclusion chromatography also known as gel-filtration, gel-permeation, steric-

exclusion, molecular-exclusion or molecular-sieve chromatography, separates solutes

on the basis of their molecular sizes. Molecular shape and hydration are also factors

in the process.

The basic components of a liquid chromatograph

a. Column

Advances in column technology have improved the selectivity, stability, and

reproducibility of liquid chromatography (LC) analytical columns. For example,

analytical columns are packed with a variety of stationary phases, providing

enormous versatility in the separation process.

28 Chapter 1 Introduction

b. Guard columns

A guard column is placed between the injector and the analytical column. It is packed

with the same or similar stationary phase as the analytical column. It collects

particulate matter and any strongly retained components from the sample and thus

conserves the life of the analytical column. After a predetermined number of

separations, a guard column is routinely replaced.

c. Solvent reservoir

Solvents used as the mobile phase are contained in solvent reservoirs. In their

simplest forms, the reservoirs are glass bottles or flasks into which “feed lines” to the

pump are inserted. To remove particles from solvents, inline filters are placed on the

inlets of the feed lines.

d. Pump

Both constant pressure and constant displacement pumps are used in liquid

chromatographs. The HPLC pump is operated in either an isocratic or gradient mode.

In the isocratic mode, the mobile phase composition remains constant throughout the

chromatographic run. This mode is usually used for simple separations and

separations of those compounds with similar structures and/or retention times.

Gradient elution is used for more complex separations. In this mode, mobile phase

composition is changed during the run in either a stepwise or continuous fashion.

29 Chapter 1 Introduction

e. Injector

To initiate an LC separation, an aliquot of sample (e.g., 0.2 to 50 µl) is first

introduced into the column via an injector. The most widely used type of injector is

the fixed-loop injector. In the fill position, an aliquot of sample is introduced at

atmospheric pressure into a stainless steel loop. In the inject mode, the sample loop is

rotated into the flowing stream of the mobile phase, and the sample is swept into the

chromatographic column. These injectors are precise, function at high pressures, and

can be programmed for use in automated systems.

f. Detectors

Examples of the detectors used in HPLC include UV photometer (fixed wavelength),

UV photometer (variable wavelength), diode array, fluorometer, refractometer and

electrochemical detectors.

Most commonly used detectors as HPLC detectors are diode array detectors.

Diode array detectors (DAD)

Diode arrays are used as HPLC detectors because they rapidly yield spectral data over

the entire wavelength range of 190 to 600 nm in about 10 milliseconds. During

operation the diode array detector passes polychromatic light through the detector

flow cell. The transmitted light is dispersed by a diffraction grating and then directed

to a photodiode array, where the intensity of light at multiple wavelengths in the

30 Chapter 1 Introduction

spectrum is measured. Such detectors have been helpful in the identification of drugs

in urine and serum.

g. Computers

The incorporation of computer technology into HPLC instrumentation has resulted in

costeffective, easy-to-operate automated systems with improved analytical

performances. In these systems, a computer provides both system control and data

processing functions.

Qualitative and quantitative analyses by HPLC

Chromatography is basically a separation technique. However it can be used for both

qualitative (identifying the analytes of interest) and quantitative analyses.

Analyte identification

The retention time or volume at which an unknown solute elutes from a column is

often matched to that of a reference compound.

Analyte quantification

The electronic signals from the detector are used to produce quantitative information.

Both external and internal calibrating techniques have been used. With external

calibration, reference solutions containing known quantities of analytes are processed

in a manner identical to the samples containing the analyte. A calibration curve of

31 Chapter 1 Introduction

peak height, peak are versus calibrator concentration is constructed and used to

calculate the concentration of the analyte in the samples.

With internal calibration, also called internal standardization, reference solutions of

known analyte concentrations are prepared, and a constant amount of a different

compound, the internal standard, is added to each reference solution and each sample.

By plotting the ratio of the peak height (or area) of the analyte to the peak height or

area of the internal standard versus the concentration of the analyte, a calibration

curve that corrects for systematic losses is constructed. This curve is then used to

compute the analyte concentration in the samples by interpolation.

2. Liquid chromatography-mass spectrometry (LC-MS)

Several interface techniques have been developed for coupling a liquid

chromatograph to a mass spectrometer, which has allowed HPLC-MS and HPLC-

MS/MS to be successfully applied to a wide range of compounds. In theory, as long

as a compound can be dissolved in a liquid, it can be introduced into an HPLC-MS

system. Thus polar and non-polar analytes and large molecular weight compounds,

such as proteins, can be monitored using this technique (Annesley et al., 2006).

Other clinically relevant compounds that are amenable to HPLC-MS analysis include

all of the major immunosuppressants, antiretrovirals, homocysteine, biogenic amines,

methylmalonic acid, and hemoglobin variants.

MS is widely used in pharmaceutical development via the process of “high

throughput screening”. To most efficiently use available resources, research labs

32 Chapter 1 Introduction

synthesize large sets of diverse organic compounds or derivatives of starting core

structures. This is often called combinatorial chemistry. The same process of

identification is also applied to natural products as pharmaceutical candidates. These

vast set of compounds must be screened for unique structure and evaluated for

potential as candidates for further study. This requires high throughput screening of

large mixtures or isolates containing many compounds. MS has become the most

efficient technique to identify these types of compounds. One example is the use of

HPLC-MS to identify new taxanes in botanical extracts as effective anticancer

reagents.

Objectives of the present study

Several bioassys are available to screen crude plant extracts for their potential for a

specific biological activity. Present study was objected towards determination of

hidden potential of a crude plant extract for a specific biological activity. Selected

plant extract was fractionated and potent fraction for biological activity was selected.

Isolation and identification of biologically active components from crude plant

extracts using bioassay-guided fractionation procedure was major objective of the

present study. Pre-screening assays, analytical scale HPLC, semi-preparative HPLC

and LC-MS were the techniques used to select, analyze, isolate and identify the active

components.

Chapter 2 Antimicrobial, toxicity and antitumor activities

During the study of medicinal plants, prior to fractionation and structural elucidation of individual components of botanical extracts, it is necessary to evaluate their biological activity. Several bench top assays such as antimicrobial assays including antibacterial and antifungal assays, toxicity assays i.e. brine shrimp cytotoxicity assay and phytotoxicity assy and antitumor potato disc assay can be used as major prescreening assays in this regard.

Antimicrobial assays can provide means to detect antibacterial as well as antifungal potential of crude plant extracts. Due to emergence of side effects of synthetic drugs as well as microbial resistance against already known drugs, it is necessary to explore potent antimicrobial medicines from natural resources.

Brine shrimp lethality assay and phytotoxic evaluation of botanical extracts present predictions for various types of biological activities. Potent tumor inhibition in antitumor potato disc assay by crude botanical extracts confirms this potential and can open new ways towards discovery of anticancer drugs. Growth stimulation or inhibition in radish seed phytotoxicity assay can determine herbicidal or growth stimulatory potential of plant extracts tested. A positive correlation between these assays can determine pharmacological importance of medicinal plants.

Results included in this chapter elaborate antimicrobial (antibacterial and antifungal assays), toxicity (Brine shrimp cytotoxicity and radish seed phytotoxicity assays) and antitumor potential of crude botanical extracts.

34 Chapter 2 Antimicrobial, toxicity and antitumor activity

Materials and methods

2.1: Collection of plant material.

Plant material was collected from Northern areas (Swat and Kalam District, NWFP

Pakistan) and dried at room temperature. The dried material was crushed using a local

grinder and stored at -70oC. The plant samples were identified at Taxonomy laboratory,

Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan by Dr Mir

Ajab Khan, Professor, Department of Plant Sciences, Quaid-i-Azam University,

Islamabad, Pakistan and their voucher specimens were deposited to the Taxonomy

Laboratory.

2.2: Preparation of plant extracts

Five plant extracts (Table 2.1) from four plant species were prepared according to

maceration procedure. Plant material was ground and soaked in methanol for 7 days at

room temperature, filtered and concentrated in rotary evaporator at 40oC under low pressure. After concentrating plant extracts, they were freeze dried using a freeze dryer and stored at -20oC.

2.3: Antimicrobial assays

(a) Antibacterial assay

Antibacterial assay was performed by agar well diffusion method (Ansari et al., 2005)

and modified by Hanif et al., (2007). Antibacterial activity was studied against six

bacterial strains three Gram negative (E.coli, ATCC # 15224, Samonella setubal ATCC

# 19196 and Bordetella bronchiseptica ATCC # 4617) while three Gram positive

including Staphylococcus aureus ATCC # 6538, Bacillus subtillus ATCC # 6633 and

35 Chapter 2 Antimicrobial, toxicity and antitumor activity

Table 2.1: List of plant species with respective plant extracts

Family Plant species name Methanol extracts (L+F) S. nubicola (methanol extract of leaves and Salvia nubicola Lamiaceae flowers of S. nubicola) (S) S. nubicola (methanol extract of stem of S. nubicola) (L+S) H. nepalensis (methanol extract of leaves and Hedera nepalensis Araliaceae stem of H. nepalensis) (L+S) A. oblongifolium (methanol extract of leaves and Acer oblongifolium Aceraceae stem of A. oblongifolium) (L+S) S. tomentosa (methanol extract of leaves and stem Sorbaria tomentosa Rosaceae of S. tomentosa)

36 Chapter 2 Antimicrobial, toxicity and antitumor activity

Micrococcus leuteus ATCC # 10240. Single colony from each bacterial culture plate was

transferred to nutrient broth (Merck, 0.8% pH 7) and incubated at 37οC for 24 hours.

Eight concentrations of each of the extract (1mg/ml to 25mg/ml) were prepared by serial

dilution method. Agar plates were prepared by pouring 75ml of 2% sterilized nutrient agar medium (Merck, pH 7) seeded with respective bacterial strain in 14cm Petri plates.

Wells were prepared by using 8 mm borer, sealed with media and filled with 100 µl of respective concentration of each extract. Roxythromycin (1mg/ml) and cefixime

(1mg/ml) were used as standard drugs while DMSO was used as negative control. Plates were incubated at 37οC and zone of inhibition was measured after 24 hours and 48 hours.

Experiment was repeated in triplicate and data was presented as mean of three values

with S.E. S.E was calculated by following formula

S.E = S.D/√ number of observations

MIC (Minimum inhibitory concentration)

Minimum inhibitory concentration was determined by method described by Ansari et al.,

(2005). Extracts which revealed antibacterial activity at concentration of 1mg/ml were

further diluted by serial dilution method to get minimum zone of inhibition.

(b) Antifungal assay

Antifungal assay was performed as described by Khan et al., (2005). Antifungal activity was studied against six fungal strains (Mucor species, Fusarium solani, Fusarium moniliformes, Alternaria specie, Aspergillus flavous and Aspergillus fumigatus). All fungal strains were grown on 6.5 % SDA (Sabouraud dextrose agar, pH 5.7) at 28οC and preserved at 4οC in refrigerator. SDA slants of 100 mm length were prepared by adding

each extract at 400 µg/ml concentration. Terbinafine (200 µg/ml) was used as standard

37 Chapter 2 Antimicrobial, toxicity and antitumor activity

drug while DMSO was used as negative control. Each slant was inoculated with 4 mm

diameter piece of respective fungal strain and incubated at 28οC for 7-10 days. Fungal

growth was compared with negative control to get the % age inhibition by using the following formula

% age of fungal inhibition = 100 – Fungal growth (mm) in sample x 100

Fungal growth (mm) in control

2.4: Toxicity assays

(a) Brine shrimp cytotoxicity assay

Brine shrimp cytotoxicity assay was performed according to the standard procedure

described by McLaughlin, (1991). Three concentrations (1000, 100, and 10 ppm) of the

plant extracts were used in this assay. Brine shrimp larvae were hatched in a small

partitioned tank in artificial seawater. Illumination was provided on one side to attract

newly hatched larvae. Brine shrimp larvae with second instar stage were used in this

assay.

Plant extracts of respective concentrations were added to dram vials. To each dram vial

ten brine shrimp larvae were added. Negative control was prepared by evaporating 0.5 ml

of methanol in dram vials and then by adding sea salt solution to it. Following 24 h of

incubation, survivors were counted by using magnifying glass. The experiment was

repeated three times. Mortality data was transformed by probit analysis in finny computer

program to estimate ED50 value. Percentage of mortality was also calculated at all

concentrations.

38 Chapter 2 Antimicrobial, toxicity and antitumor activity

(b) Radish seed phytotoxicity assay

Experiment was conducted according to standard procedure described by Turker &

Camper, (2002). Experiment consisted of two parts. In part one, two concentrations

(10,000 and 1000 ppm) of the plant extracts were prepared in methanol. Filter papers

(Whatman # 1) were placed in Petri plates and 5 ml of each concentration was added.

Methanol was evaporated and 5 ml of distilled water was added. To each Petri plate 20

radish seeds surface sterilized with 0.1% mercuric chloride were placed. The plates were

sealed with parafilm to avoid moisture loss and incubated at 23 ± 2°C. In control plates 5

ml of methanol was added and evaporated. Root length was measured on 3rd and 5th day of incubation. The experiment was repeated in triplicate.

In the second part of the experiment two concentrations of the plant extracts (7500 and

1000 ppm) were used. Procedure for second part is similar to the first part except concentrations of the extracts and number of seeds. In the second part of the experiment

100 radish seeds were added to each plate. Germinated seeds were counted everyday from 1st to 5th day. Experiment was repeated in duplicate. Both experiments were carried

out in strict sterilized conditions. Results were statistically analyzed by using ANOVA.

Statistical analysis of radish seed bioassay data was performed only on 5th day data.

2.5: Antitumor potato disc assay

Antitumor potato disc assay was performed according to standard procedure described by

McLaughlin & Rogers, (1998). A 48 h bacterial culture of At 10 strain of Agrobacterium tumefaciens was used in this experiment. Inoculum with three concentrations of test

39 Chapter 2 Antimicrobial, toxicity and antitumor activity

samples (1000, 100, and 10 ppm) was prepared containing bacterial culture and autoclaved distilled water.

Red skinned potatoes were purchased from a local market and surface sterilized by using

10% bleach solution. A borer of 8 mm diameter was used to bore out potato cylinders.

Cylinders were cut into 2 mm discs. Autoclaved agar solution (1.5%) was poured in

petriplates and solidified. Ten discs were placed on agar surface of each plate and 50 µl

of inoculum was placed on the surface of each disc. The plates were sealed with parafilm

to avoid contamination and moisture loss. The plates were incubated at 28°C in incubator

in dark. Experiment was carried out in strict sterilized conditions and repeated in

triplicate. After 21 days of incubation, potato discs were stained with Lugol’s solution

(10% KI, 5% I2), and tumors were counted under dissecting microscope with side illumination. Tumor inhibition was calculated by using following formula

% age of tumor inhibition = 100 – ns/nc x 100

Where

ns = number of tumors in sample

nc = number of tumors in control

More than 20% tumor inhibition is considered significant (Ferrigini et al., 1982).

Data was statistically analyzed by using ANOVA.

2.6: Antibacterial assay against Agrobacterium tumefaciens

Antibacterial assay was performed according to standard agar well diffusion method as

described previously. The only difference was that a 24 h bacterial culture of At 10 strain

of A. tumefaciens was used in this experiment.

40 Chapter 2 Antimicrobial, toxicity and antitumor activities

Results

2.1: Antimicrobial assays

(a) Antibacterial assay

Leaf and stem extract of A. oblongifolium presented significant antibacterial activity

against the six bacterial strains at eight different concentrations tested (Table 2.2).

Highest activity was observed against B. bronchiseptica at all the concentrations (Fig

2.1). MIC (Minimum inhibitory concentration) value ranged from 0.8 mg/ml to 1 mg/ml

(Table 2.2). Other four extracts i.e. (L+F) S. nubicola, (S) S. nubicola, (L+S) S.

tomentosa and (L+S) H. nepalensis did not show any antibacterial activity against the six

bacterial strains.

(b) Antifungal assay

None of the extract showed significant antifungal activity against the six fungal strains.

However, moderate to low fungal inhibition was observed by all methanol extracts

against six fungal strains tested. Highest activity was observed by leaf and flower extract

of S. nubicola against Alternaria specie (Table 2.3).

2.2: Toxicity assays

(a) Brine shrimp cytotoxicity assay

Two of the five extracts i.e. (L+S) H. nepalensis and (L+S) A. oblongifolium, exhibited

potent cytotoxicity in brine shrimp cytotoxicity assay. ED50 in these extracts remained

highly significant as 47.7 ppm and 226.8 ppm respectively (Table 2.4).

Results for % age mortality of brine shrimp indicate that highest % age mortality

was observed at 1000 ppm by most of the extracts tested. At 100 ppm only one extract

(L+S) H. nepalensis presented significant mortality rate i.e. 56.6%.

41 Chapter 2 Antimicrobial, toxicity and antitumor activities

Table 2.2: Antibacterial activity of methanol extract of leaf and stem of A. oblongifolium against six bacterial strains. Difference in superscript letters indicate difference at p < .05

MIC Bacterial strains 1 mg/ml 2 mg/ml 5 mg/ml 7 mg/ml 10 mg/ml 15 mg/ml 20 mg/ml 25 mg/ml Roxythromycin Cefixime mg/ml E. coli 8.5 ± .14 10.5 ± .12 14.4 ± .24 15.4 ± .15xyz 16.4 ± .08uv 18.1 ± .05op 18.9 ± .05mn 19.1 ± .08m 19.3 ± .12m 37.8 ± .68d 0.8 S. setubal 0 ± 0 0 ± 0 15.3 ± .05xyz 15.8 ± .08wx 16.2 ± .05vw 17.5 ± .1qrs 23.1 ± .2j 23.6 ± .15ij 12.2 ± .06 35.1 ± .24e 1 S. aureus 0 ± 0 12.5 ± 0.17 15 ± .05z 15.4 ± .05xyz 16.3 ± .03uvw 17.16 ± .08rst 18.2 ± .14o 18.5 ± .03no 33 ± .49f 41.1 ± .23b 1 M. leuteus 10.1 ± .08 12.5 ± .05 15.6 ± .3xy 17.2 ± .08rst 18.5 ± .27no 19 ± .05mn 19.2 ± .06m 20.7 ± .1k 13.8 ± .14 41.1 ± .3b 0.8 B. bronchiseptica 10.3 ± .08 12.5 ± .18 17.5 ± .03qrs 19.9 ± .18l 20.8 ± .4k 23.9 ± .08i 28.5 ± .08h 30.5 ± .3g 44.8 ± .76a 17.6 ± .17pqr 0.8 B. subtillus 10.1 ± .08 13.1 ± .08 15.2 ± .12yz 16.8 ± .06tu 17 ± .06st 18 ± .05opq 18.2 ± .06o 19.2 ± .12 16.4 ± .17uv 40 ± .1c 0.8

Significant Level Zone Diameter Non significant 11-14 mm Low 15-17 mm Good 18-20 mm significant 20 mm

42 Chapter 2 Antimicrobial, toxicity and antitumor activities

B. bronchiseptica

Fig 2.1. Antibacterial activity of (L+S) A. oblongifolium at eight different concentrations against B. bronchiseptica.

43 Chapter 2 Antimicrobial, toxicity and antitumor activities

Table 2.3: Percentage inhibition of crude extracts of five different species against six fungal strains.

Extracts Mucor specie Fusurium solani Alternaria specie Fusarium moniliformes Aspergillus flavous Aspergillus fumigatus (L+F) S. nubicola 5.3 ± .3m 39.7 ± 1.4cd 47.9 ± 1.6a 41.9 ± 3.2bcd 19.04 ± .9jk 2 7± 1fgh (S) S. nubicola 1.6 ± 1.6m 40.8 ± 1.1bcd 47.4 ± .5a 37.6 ± 2.8de 21.6 ± .3ij 15.3 ± .3kl (L+S) H. nepalensis 45 ± 2.8ab 28.6 ± 4.1fg 41.8 ± 1.1bcd 34.4 ± 1e 22.7 ± .9hij 24.6 ± .8ghi (L+S) A. oblongifolium 1.6 ± 1m 11.5 ± 2.7l 29.5 ± .9f 4.3 ± 1m 38.8 ± .3de 15.6 ± .6kl (L+S) S. tomentosa 1.3 ± .6m 11.5 ± 2.7l 44.07 ± .5abc 34.4 ± 1e 1.8 ± .3m 3.3 ± 1.6m

Criteria for fungal inhibition

Significant 70% and above Good 60-70% Moderate 40-60% Low below 40%

44 Chapter 2 Antimicrobial, toxicity and antitumor activities

Table 2.4: Illustration of % age mortality of brine shrimps at different concentrations of extracts and respective ED50 value.

Methanol extracts 1000 ppm 100 ppm 10 ppm ED50 ( ppm )

(L+F) S. nubicola 13.3% 10% 10% >1000 ppm

(S) S. nubicola 10% 6.6% 6.6% >1000 ppm

(L+S) H. nepalensis 100% 56.6% 23.3% 47.7 ppm

(S) A. oblongifolium 66.70% 16.70% 6.70% 226.8 ppm

(L+S) S. tomentosa 13.40% 14% 10% >1000 ppm

45 Chapter 2 Antimicrobial, toxicity and antitumor activities

(b) Radish seed phytotoxicity assay

Effect of two different concentrations (10,000 and 1000 ppm) of the extracts was studied on root growth inhibition or stimulation of radish seedling. All extracts inhibited root growth at 10,000 ppm. In three of the extracts, root growth stimulation was observed at

1000 ppm (Fig 2.2). Highest percentage of inhibition was observed by leave and stem extract of Sorbaria tomentosa (Fig 2.3). Leave and stem extract of H. nepalensis presented highest stimulation of root length at 1000 ppm.

In second experiment, effect of two different concentrations of each extract (7500 and 1000 ppm) on seed germination was observed as a function of incubation period of seeds. A gradual increase in seed germination for all extracts was observed till 5th day of incubation. Effect of concentrations remained significant and inhibition of seed germination was observed in case of all extracts at 7500 ppm (Fig 2.4). Leave and stem extract of Sorbaria tomentosa showed highest inhibition of seed germination at 7500 ppm

2.3: Antitumor potato disc assay

All extracts exhibited tumor inhibition at three concentrations tested. Tumor inhibition was observed in concentration dependant mode. Statistical analysis by using ANOVA showed that the effect of concentration and extract was highly significant. Effect of interaction of concentration and extract factors is presented in table 2.5 with respective rank order obtained. Extract of leave and stem of A. oblongifolium presented highest percentage of tumor inhibition at all concentrations (Fig. 2.5).

2.4: Antibacterial assay against Agrobacterium tumefaciens

Effect of extracts on viability of A. tumefaciens was evaluated by using agar well diffusion method. None of the four extracts tested showed any significant effect on

46 Chapter 2 Antimicrobial, toxicity and antitumor activities

(a)

a 25 20 15 b c d d 10 e 5

Root length (mm) length Root 0

m l u o li tr lensis on a ifo C ep ng mentosa n .to F) S.nubicola (S) S.nubicola .oblo + A (L ) (L+S) H. S (L+S) S (L+

(b)

a 35 b 30 c cd d cd 25 20 15 10 5 Root length (mm) length Root 0

a is ol ola s en lium bic bic o pal Control nu .nu ngif S. .ne o F) ) H ) S.tomentosa (S) S S (L+ (L+ (L+S (L+S) A.obl

Fig 2.2: Effect of two different concentrations (a.10,000 ppm and b.1000 ppm) on root length. Values with similar letters do not show significant difference p > 0.05

47 Chapter 2 Antimicrobial, toxicity and antitumor activities

(a)

(b)

Fig 2.3. Radish seed phytotoxicity in terms of root length in case of (L+S) S. tomentosa at (a). 10,000 ppm and (b). 1000 ppm

48 Chapter 2 Antimicrobial, toxicity and antitumor activities

(a)

120 (L+F) S.nubicola 100 (S) S.nubicola 80 (L+S) H.nepalensis 60 (L+S) A.oblongifolium 40 (L+S) S.tomentosa 20 Number germinated seedsof Control 0 Days12345

(b)

120 (L+F) S.nubicola

100 (S) S.nubicola

80 (L+S) H.nepalensis 60 (L+S) A.oblongifolium 40 (L+S) S.tomentosa 20 Number of germinated seeds germinated Number of Control 0 Days12345

Fig 2.4: Effect of methanol extracts on seed germination at a. 7500 ppm and b.1000 ppm as a function of incubation period of seeds

49 Chapter 2 Antimicrobial, toxicity and antitumor activities

Table 2.5: Average number of tumors produced at different concentrations of extracts. Values with similar letters are not significantly different from each other at p > 0.05

Average number of % age of tumor Extracts Concentrations tumors per disc inhibition (L+F) S. nubicola 1000 ppm 3.4 ± 0.8 61.3 ± 0.6c 100 ppm 6 ± 0.9 31.8 ± 0.3i 10 ppm 8 ± 0.2 9 ± .03l (S) S. nubicola 1000 ppm 3.3 ± 0.4 62.5 ± 0.5c 100 ppm 5.1 ± 0.7 42.5 ± 0.6f 10 ppm 5.7 ± 0.5 35.2 ± 0.3h (L+S) H. nepalensis 1000 ppm 5.3 ± 1.07 39.1 ± 0.3g 100 ppm 6.7 ± 0.8 22.9 ± 0.3j 10 ppm 7.1 ± 1.1 20.2 ± 0.1k (L+S) A. oblongifolium 1000 ppm 1.5 ± 0.5 82.9 ± 0.6a 100 ppm 3.4 ± 0.6 61.3± 0.1c 10 ppm 3.6 ± 0.8 59 ± 0.08d (L+S) S. tomentosa 1000 ppm 2.3 ± 0.5 73.8± 0.5b 100 ppm 3.7 ± 0.6 57.9 ± 0.2d 10 ppm 4.8 ± 0.4 45.5 ± 0.7e Control 8.8 ± 0.9

50 Chapter 2 Antimicrobial, toxicity and antitumor activities

(a)

Tumor

(b)

Fig 2.5. (a). Antitumor potato disc assay at three different concentrations in case of

(L+S) A. oblongifolium (b). Controls used in antitumor potato disc assay

51 Chapter 2 Antimicrobial, toxicity and antitumor activities

viability of A. tumefaciens. One of the five extracts i.e. (L+S) S. tomentosa was slightly effective against A. tumefaciens at 1000 ppm (Zone size = 10 mm, MIC = 0.8 mg/ml).

Conclusion

Results for antibacterial activity illustrate that leaf and stem extract of A. oblongifolium has shown highly significant antibacterial activity against all the bacterial strains tested so this extract can be fractionated in future to get active components responsible for antibacterial activity.

Results of the present study indicate a positive correlation between three assays i.e. brine shrimp cytotoxicity assay, radish seed phytotoxicity assay and antitumor potato disc assay. A correlation between brine shrimp lethality assay and antitumor potato disc assay has been reported previously. Our two of the five extracts (leave and stem extract of A. oblongifolium and leave and stem extract of H. nepalensis) presented significant ED50 value in brine shrimp lethality assay and significant % age of tumor inhibition in potato disc assay. These results can lead to discovery of new anticancer drugs in future. All extracts used in the present study showed inhibition of root length and seed germination in radish seed phytotoxicity assay as well. An interesting aspect of the present study is less or no inhibition of growth in radish seed bioassay at low concentrations and antitumor activity in case of three plant extracts. Combinatorial effect of these extracts can be utilized to control crown gall disease in plants.

The overall results show that the crude botanical extracts evaluated in the present study can be fractionated in future and can lead to discovery of important chemotherapeutic agents.

Chapter 3 Antioxidant activities

The potentially toxic and beneficial properties of pro-oxidants and antioxidants have

made them the focus of many studies. Pro-oxidants may represent a threat to health,

whereas antioxidants may counteracts these effects by scavenging pro-oxidants.

Antioxidants are very important in industrial processes as well as in biological systems.

They are known to possess anti-inflammatory, anti-cardiovascular disease,

antineurogenerative and anticancer properties. Imbalances between pro-oxidants and

antioxidants in favor of the pro-oxidants may result in oxidative stress, which in turn

result in oxidative damage of cellular components in the form of lipid peroxidation,

protein denaturation or DNA conjugation. Oxidative stress has been associated with

many diseases like cancer, post-ischemic and neural degradation, Parkinson’s and

Alzheimer disease, AIDS, and aging and cardiovascular diseases (Kool et al., 2007).

Antioxidant assays were selected in two systems i.e. aqueous system and lipid system.

Three major assays selected in aqueous system were DPPH scavenging assay, ABTS+

scavenging assay, DNA protection assay while total phenolic contents were determined

by using Folin-Ciocalteu reagent. In lipid system TBARS (Thiobarbituric acid reactive

substances) was used to evaluate antioxidant activity of crude extracts.

Plant species were selected on the basis of previous screening (Inayatullah et al., 2007).

Four plant extracts i.e. (L+F) S. nubicola, (S) S. nubicola, (L+S) H. nepalensis and (L+S)

A. oblongifolium were selected on the basis of their antitumor and toxicity activities for further studies.

53 Chapter 3 Antioxidant activities

Materials and methods

3.1: Determination of total phenolic contents

Requirements: Folin-Ciocalteu reagent (2N, Sigma), Sodium carbonate (20%), Gallic acid (Sigma), Caffeic acid (Sigma), Rutin (Sigma), Trolox (S-6 methoxy-2, 5, 7, 8- tetramethoxy chromane-2-carboxylic acid, Sigma)

Procedure

Total phenolic contents were determined by modifying the method of Singleton and

Rossi (Singleton and Rossi, 1965).

Six millilitre of instrument grade water was added to 10 ml volumetric flask followed by addition of 100 µl of plant extract solution (initial concentrations 1000, 10,000 and 100,

000 ppm) in 100% methanol leading to final concentration of 10 ppm, 100 ppm and 1000 ppm. Folin-Ciocalteu reagent (Sigma, 500 µl 1:10 dilution of 2N solution) was added immediately with vigorous shaking. After one minute freshly prepared aqueous solution of Sodium carbonate (1.5 ml, 20%) was added while shaking constantly. Volume was made up to 10 ml and flasks were kept at room temperature for one hour. Absorbance was measured in 1cm cuvette at 760 nm by using spectrophotometer (Carry WinUV,

Varian). Gallic acid, caffeic acid, rutin and trolox were used as positive control and linear regression curves were drawn for each standard used. Data was expressed in terms of

GAE/gms, mg of caffeic acid/gram of extract, mg of rutin/ gram of extract as well as mM

Trolox/gram of extract. Data was analysed statistically by using ANOVA (SPSS version

11.1).

54 Chapter 3 Antioxidant activities

3.2. Antioxidant activity

In order to investigate antioxidant capacity of the plant extracts, four antioxidant assays were selected, three in aqueous system ( DPPH assay, ABTS+ assay and DNA protection assay) and one in lipid system (TBARS).

3.2.1: DPPH assay (1,1- Diphenyl-2-picryl-hydrazyl radical)

Requirements: DPPH (Sigma), Methanol (HPLC grade), Gallic acid, Caffeic acid,

Trolox

Procedure

DPPH assay was performed according to the procedure described by Kulisic et al.,

(2004) modified by Obeid et al., (2005).

DPPH solution was prepared by dissolving 32 mg in 1L 80% methanol. Three millilitre of DPPH solution was added to 1cm plastic cuvette followed by the addition of 200 µl of test sample of concentrations (150 ppm, 1500 ppm and 15000 ppm) leading to the final concentration of 10 ppm, 100 ppm and 1000 ppm. Mixture was shaken well and kept in dark at room temperature for one hour. Absorbance was measured at 517 nm by using spectrophotometer (Carry WinUV, Varian). Absorbance of 80% methanol was considered as blank while negative control (DPPH solution) was also run simultaneously.

Gallic acid, trolox and caffeic acid were used as positive controls.

TEAC (trolox equivalent antioxidant capacity) values were calculated by using the standard regression curve. GAE/gram of extract as well as mg of caffeic acid per gram of extract was also calculated by using the standard regression curves.

Percentage inhibition was measured according to the following formula

55 Chapter 3 Antioxidant activities

%age inhibition = (Ac-As/Ac) x 100 where

Ac = Absorbance of control

As = Absorbance of sample

3.2.2: Calculation of EC50 value

In order to calculate EC50 value, plant extract solution in methanol was further diluted and tested for DPPH assay to find out 50% inhibition. EC50 value was calculated by

graph method. EC50 value for gallic acid, caffeic acid and trolox was also calculated.

Data was analysed statistically by using ANOVA (SPSS version 11.1).

3.2.3: ABTS+ Assay

Requirements: ABTS (Aldrich), Potassium persulfate, monosodium phosphate

monohydrate, Disodium phosphate heptahydrate, Methanol (HPLC grade), Trolox

Procedure

ABTS+ assay was performed by modified method of Paixao et al., (2007). ABTS+ was

dissolved in water (7mM) to get the stock solution. ABTS radical cation was produced by

reacting the stock solution with 2.45 mM (final concentration) potassium persulfate

solution. Solution was kept in dark at room temperature for 12 hours prior to use.

Solution was diluted 50 fold with phosphate buffer (pH 8.04) and absorbance was set as

0.7 at 415 nm. Three millilitre of ABTS+ solution was added to 1 cm cuvette followed by

the addition of 3µl, 15µl and 30 µl of methanolic solution of plant extracts to get the final

concentration as 1 ppm, 5 ppm and 10 ppm respectively. Trolox was used as positive

control while ABTS+ solution served as negative control. Absorbance was measured at

415 nm. Data is expressed in terms of TEAC (Trolox equivalent antioxidant capacity).

Percentage inhibition was measured according to following formula

56 Chapter 3 Antioxidant activities

%age inhibition = (Ac-As/Ac) x 100 where

Ac = Absorbance of control

As = Absorbance of sample

Data was analysed statistically by using ANOVA (SPSS version 11.1)

3.2.4: TBARS (Thiobarbituric acid reactive substances) assay

Requirements: Thiobarbituric acid (TBA, Sigma), Copper chloride (Sigma), BHT (2-6- di-ter-butyl-4 methyl phenol), Linoleic acid (Sigma), HCl (concentrated), Butanol (HPLC grade), Methanol (HPLC grade)

Procedure

TBARS for four plant extracts was performed by the method described by Kishida et al.,

(1993).

Initial stock solution of methanol extracts were prepared as 10,000, 50, 000 and 100,000 ppm in 100% methanol. Three hundred microliter of CuCl2 solution (0.05 mM) was

added to each test tube followed by the addition of 50 µl of test solution and 100 µl of

linoleic acid. Mixture was vortexed for five seconds and incubated at 37°C in shaking

water bath for 20 hours. Reaction was stopped by the addition of BHT (20 µl of 10mM in

ethanol) to each test tube and freshly prepared solution of TBA (Thiobarbituric acid)

(3ml, 0.67% in 0.1 M HCl) was added. TBA was dissolved in 0.1 M HCl by sonication

and momentary heating. Mixture was vortexed for five seconds and tubes were kept in

boiling water bath for 10 minutes. Tubes were allowed to cool and pink aqueous layer

was transferred to another test tube containing 2.5 ml of 100 % butanol. Mixture was

vortexed for five seconds and allowed to settle. Absorbance of pink layer was measured at 532 nm by using spectrophotometer (Carry WinUV, Varian). Butanol served as blank

57 Chapter 3 Antioxidant activities while negative control (without any test substance) was run simultaneously. Trolox was used as positive control.

Standard curve for trolox was used to calculate trolox equivalent values.

Percentage inhibition was measured according to following formula

%age inhibition = (Ac-As/Ac) x 100 where

Ac = Absorbance of control

As = Absorbance of sample

Data was analysed statistically by using ANOVA (SPSS version 11.1)

3.3: DNA protection assay

Requirements: Plasmid DNA (pBR322, Fermentas), Phosphate buffer, Methanol (HPLC grade), DNA ladder (Hind III digest, Fermantas), Ferrous sulphate, Hydrogen peroxide,

Ethedium bromide, Bromophenol blue, Agarose (Sigma)

Pro-oxidant and antioxidant potential of plant extracts was investigated by modified version of Tian and Hua (2002) method. Plasmid DNA (pBR322 Fermentas) was diluted two fold with phosphate buffer (pH 7.6) and treated with three different concentrations of the plant extracts (10 ppm, 100 ppm and 1000 ppm) in the final reaction volume of 15 µl.

Fenton reaction was induced by addition of 30% H2O2 (4 µl) and 2mM FeSO4 (3 µl).

Four controls (untreated DNA, DNA treated with 2mM FeSO4, DNA treated with 30%

H2O2, DNA treated with 2mM FeSO4 and 30% H2O2) were run simultaneously. Mixture was incubated at 37°C in dark for one hour. Reaction was stopped by addition of 2 µl of

6X bromophenol blue. Reaction mixture was run on 1% agarose gel and visualized by

UV-transilluminator.

58 Chapter 3 Antioxidant activities

Results

3.1: Determination of total phenolic contents

Highly significant quantity of phenolic contents were found in crude extract of leaf and

flower of S. nubicola in terms of equivalents of caffeic cid, gallic acid and rutin (Table

3.1). Phenolic contents were quite high in case of methanol extract of leaf and stem of A.

oblongifolium too. While lowest phenolic contents were found in leaf and stem extract of

H. nepalensis. Equivalents of standards were calculated on the basis of standard

regression lines for caffeic acid (R2 = 0.987) (Fig 3.1), gallic acid (R2 = 0.987) (Fig 3.2)

and rutin (R2 = 0.981) (Fig 3.3). Order of total phenolics in terms of mM of trolox also remained the same i.e. highest in case of S. nubicola and lowest in case of H. nepalensis

(Table 3.1). Standard regression line of trolox was used to calculate TEAC (trolox

equivalent antioxidant capacity) (R2 = 0.992) (Fig 3.4).

3.2: Statistical analysis

Result of ANOVA presented highly significant difference in phenolic contents amongst

the four extracts tested in terms of equivalent of standards.

59 Chapter 3 Antioxidant activities

Regression line with Caffeic Acid

0.3 0.25 y = 0.001x - 0.0091 R2 = 0.9873 e 0.2 0.15 0.1

Absorbanc 0.05

0 -0.05 0 50 100 150 200 250 300 Concentration

Fig 3.1. Regression line with caffeic acid with Folin-Ciocalteu reagent

Regression Line with Gallic Acid

0.4 y = 0.0013x - 0.005 R2 = 0.9874 e 0.3

0.2

0.1

Absorbanc 0 0 50 100 150 200 250 300 -0.1 Concentration

Fig 3.2. Regression line with gallic acid with Folin-Ciocalteu reagent

60 Chapter 3 Antioxidant activities

Regression line for Rutin

0.14 0.12 y = 0.0005x - 0.0006 2 0.1 R = 0.9813 e 0.08 0.06 0.04 Absorbanc 0.02 0 -0.02 0 50 100 150 200 250 300 Concentration

Fig 3.3. Regression line with rutin with Folin-Ciocalteu reagent

Regression Line with Trolox

0.6 y = 5.5786x - 0.0008 R2 = 0.9924 0.5

0.4 e 0.3

0.2 Absorbanc

0.1

0 0 0.02 0.04 0.06 0.08 0.1 0.12 -0.1 Concentration (mM)

Fig 3.4. Regression line with trolox with Folin-Ciocalteu reagent

61 Chapter 3 Antioxidant activities

Table 3.1: Total phenolic contents in terms of mg of caffeic acid, mg of gallic acid and mg of rutin per gram of crude extracts.

Difference in superscript letters indicate significance level at p < .05

mg of crude Total phenolic contents extract per gm of dry Extracts weight Caffeic acid Gallic acid Rutin Trolox (L+F) S. nubicola 80 201.2 ± .32a 138.82 ± .22a 351.20 ± .58a 342.08 ± 19.8a (S) S. nubicola 30 120 ± .50c 82.53 ± .34c 205.80 ± .90c 187.50 ±1 2.80c (L+S) H. nepalensis 20 69 ± .37d 47.692± .25d 115.20 ± .66d 107.85 ± 3.829d (L+S) A. oblongifolium 200 151.5 ± .74b 104.35 ± .51b 262.53 ± 1.3b 233.03 ± 13.05b

62 Chapter 3 Antioxidant activities

3.2: DPPH scavenging activity

Standard regression line for trolox, gallic acid and caffeic acid were used to calculate

DPPH scavenging activity in terms of TEAC (trolox equivalent antioxidant capacity in

terms of mM of trolox per gram of extract) GAE (gallic acid equivalent i.e. mg of gallic

acid per gram of extract) and caffeic acid equivalent values (mg of caffeic acid per gram

of extract) (Fig 3.5). R2 values remained as 0.994, 0.997 and 0.991 for trolox, gallic acid and caffeic acid respectively. Leaf and flower extract of S. nubicola presented highest

TEAC, GAE and caffeic acid equivalent values (Table 3.2).

Percentage inhibition remained highest in case of methanol extract of leaf and flower

extract of S. nubicola (Fig 3.6). While lowest %age inhibition was observed in case of

leaf and stem extract of H. nepalensis (Fig 3.10). EC50 value ranged from 5.29 ± 0.04 to

25.1 ± 0.17 (Fig 3.14)). Highly significant EC50 value was obtained in case of leaf and

flower exract of S. nubicola. EC50 value remained as 16.59 and 14.31 in case of gallic

acid and caffeic acid respectively. EC50 value was calculated by using regression lines

for four crude extracts. Fig 3.7, 3.9, 3.11 and 3.13 present calculation of EC50 value for

(L+F) S. nubicola, (S) S. nubicola, (L+S) H. nepalensis and (L+S) A. oblongifolium

respectively. While Fig 3.8, 3.10 3.12 present pattern of DPPH scavenging in case of (S)

S. nubicola, (L+S) H. nepalensis and (L+S) A. oblongifoilium respectively.

Statistical analysis by using ANOVA revealed significant difference in EC50 value for

crude extracts and three standards tested (Fig 3.14).

3.3: ABTS+ ASSAY

Highly significant TEAC value was obtained in case of leaf and stem extract of A.

oblongifolium (Table 3.2). Standard regression line for trolox was used to calculate

63 Chapter 3 Antioxidant activities

TEAC value (R2 = .996) (Fig 3.15). Percentage inhibition gradually increased with

increase in concentration.

3.4: TBARS (Thiobarbituric acid reactive substances) assay

Significant difference was observed in TEAC value for TBARS (Table 3.2) for four

crude extracts tested. TEAC value remained in the range of 392.886 ± 3.42 to 462.094 ±

3.62. Standard curve for trolox was used to calculate TEAC value in case of TBARS (R2

= .911). Highest TEAC value in terms of mM of trolox per gram of crude extract was obtained in case of leaf and stem extract of A. oblongifolium i.e. 462.094. Overall result of the TBARS indicated potential of crude extracts to inhibit oxidation in lipid system.

Pattern of percentage inhibition with trolox is presented in Figure 3.16.

64 Chapter 3 Antioxidant activities

(a)

90 80 70 60 50 40 30 20

Percentage scavenging 10 0 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 Concentration

(b)

100 90 80 70 60 50 40 30 20

Percentage scavenging Percentage 10 0 0 50 100 150 200 250 300 Concentration

(c)

120

100

20 Percentage Scavengigng Percentage 0 0 50 100 150 200 250 300 Concentration

Fig 3.5: DPPH scavenging with three standards (a) trolox (b) gallic acid (c) caffeic acid

65 Chapter 3 Antioxidant activities

DPPH Assay with (L+F) S. nubicola

100

Percentage scavenging Percentage 0 024681012 Concentration (ppm)

Fig 3.6: Percentage inhibition with (L+F) S. nubicola in DPPH scavenging assay

EC50 value with (L+F) S. nubicola

60 n 50 y = 9.33x + 0.72 R2 = 0.999 40

10 Percentage inhibitio Percentage 0 01234567 Concentration (ppm)

Fig 3.7: Regression line used to calculate EC50 in case of DPPH scavenging by (L+F) S. nubicola.

66 Chapter 3 Antioxidant activities

DPPH assay with (S) S. nubicola

100

Percentage scavenging Percentage 0 0 50 100 150 Concentration

Fig 3.8: Percentage inhibition with stem extract of S. nubicola in DPPH scavenging assay

EC50 with (S) S. nubicola

80 y = 3.4615x + 0.9117 70 2 60 R = 0.9979 50 40 30 20 10

Percentage scavenging Percentage 0 0 5 10 15 20 25 Concentration (ppm)

Fig 3.9: Regression line with stem extract of S. nubicola used to calculate EC50 value in DPPH scavenging activity

67 Chapter 3 Antioxidant activities

DPPH assay with (L+S) H. nepalensis

100

Percentage Scavenging Percentage 0 0 20406080100120 Concentration

Fig 3.10: Percentage inhibition with leaf and stem extract of H. nepalensis in DPPH scavenging assay

EC50 with (L+S) H. nepalensis

90 80 y = 1.8907x + 3.728 70 R2 = 0.9823 60 50 40 30 20 10

Percentage scavenging Percentage 0 0 5 10 15 20 25 30 35 40 45 Concentration (ppm)

Fig 3.11: Regression line with leaf and stem extract of H. nepalensis used to calculate

EC50 value in DPPH scavenging activity

68 Chapter 3 Antioxidant activities

DPPH Assay with (L+S) A. oblongifolium

100 90 80 70 60 50 40 30 20

Percentage scavenging Percentage 10 0 0 50 100 150 Concentration

Fig 3.12: Percentage inhibition with leaf and stem extract of A. oblongifolium in DPPH scavenging assay

EC50 with (L+S) A. oblongifolium

60 y = 5.0807x + 4.1848 2 50 R = 0.9793 40 30 20

Percentage scavenging Percentage 0 024681012 Concentration (ppm)

Fig 3.13: Regression line with leaf and stem extract of A. oblongifolium used to calculate EC50 value in DPPH scavenging activity

69 Chapter 3 Antioxidant activities

EC50 for standards and crude extracts in DPPH assay

) 30 a 25 b c 20 d 15 e 10 g f 5 EC50 (ppm value 0

G C T (L (L (S (L a a ro + + ) + l ff l S li e o S) S) . F c ic x n ) a a A H u S ci c . o . b . d i b n ico n d l ep ub o a la i ng le co if n l o s a li is um

Standards and crude extracts

Fig 3.14: EC50 value for four methanol extracts and standards. Difference in letters present significance difference between EC50 value at p < .05.

70 Chapter 3 Antioxidant activities

Percentage scavenging with ABTS+ assay with trolox as standard

90 80 y = 1.0339x + 2.486 70 2 60 R = 0.9836 50 40 30 20 10

Percentage scavenging Percentage 0 0 102030405060708090 Concentration (ppm)

Fig 3.15: Percentage inhibition with Trolox in ABTS+ scavenging activity

71 Chapter 3 Antioxidant activities

Fig 3.16: Percentage inhibition with trolox in case of TBARS (thiobarbituric acid reactive substances) assay.

72 Chapter 3 Antioxidant activities

Table 3.2: Result of DPPH assay, ABTS+ assay and TBARS in terms of TEAC

(Trolox equivalent antioxidant capacity) in terms of mM of trolox per gram of

extract. DPPH scavenging is also expressed in terms of GAE (Gallic acid equivalents

i.e. mg of gallic acid per gram of extract) and equivalents of caffeic acid (mg of

caffeic acid per gram of extracts). Difference in superscript letters indicate

significance level at p < .05

ABTS+ assay TBARS DPPH scavenging activity Caffeic acid TEAC (mM) TEAC Plant extracts TEAC (mM) GAE (mg) (mg) (mM)

(L+F) S. nubicola 248.4 ± .49a 289.86 ± .51a 249.98 ± 1.6a 149.23 ± 3.5b 400.314 ± 6.94 c

(S) S. nubicola 66.9 ±. 075c 123.75 ± .112c 106.64 ± .09c 70.42 ± 2.6c 420.062 ± 8.90 b

(L+S) H. nepalensis 26.7 ± 0.18d 94.5 ± .14d 81.24 ± .9d 54.74 ± 1.6d 392.886 ± 3.42 d

(L+S) A. oblongifolium 140.4 ± 1.2b 176.76 ± .9b 149.85 ± .9b 160.23 ± 6.4a 462.094 ± 3.62 a

73 Chapter 3 Antioxidant activities

3.5: DNA protection assay

DNA protection activity was observed in case of leaf and flower extract of S. nubicola at

10 ppm and 100 ppm while at 1000 ppm pro-oxidant activity was obvious (Fig 3.17).

Stem extract of S. nubicola showed DNA protection activity at 100 ppm and 1000 ppm while at 10 ppm pro-oxidant activity was observed. Leaf and stem extract of H. nepalensis showed pro-oxidant activity at 1000 ppm. While leaf and stem extract of A. oblongifolium could not show DNA protection activity at any concentration tested.

Results of DNA protection assay are explained on the basis of Fenton reaction as described in chapter 1, page 21.

Conclusion

It is obvious that total phenolic contents by Folin-Ciocalteu reagent were higher in leaf and flower extract of S. nubicola and its DPPH scavenging activity also remained highest. (L+F) S. nubicola can further be selected for fractionation. Crude extract of A. oblongifolium showed highest potential for inhibition of lipid oxidation which can be considered in future to isolate active antioxidants in lipid system.

74 Chapter 3 Antioxidant activities

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Fig 3.17: DNA protection assay with different concentrations of crude extracts. Lane 1 is λ Hind III marker. Lane 2-5 presents controls (untreated DNA, DNA treated with 2mM FeSO4, DNA treated with 30 % H2O2, DNA treated with 2mM

FeSO4 and 30 % H2O2). Lane 6-8 presents DNA treated with crude extract of leaf and flower of S. nubicola at 10, 100 and 1000 ppm. Lane 9-11 is treatment of DNA with different concentrations (10, 100 and 1000 ppm) of crude extract of (S) S. nubicola . Lane 12-14 presents treatment of DNA with three concentrations (10, 100 and 1000 ppm) of crude extract of (L+S) H. nepalensis. Lane 15-17 presents DNA treatment with three different concentrations (10, 100 and 1000 ppm) of crude extract of A. oblongifolium.

Chapter 4 Fractionation by Semi-preparative HPLC

Evaluation of biological activities of crude plant extracts leads to selection of potent plant

extracts. In order to isolate active principal of a crude plant extract, fractionation is the

required step. Several traditional and modern fractionation techniques are in use to get sufficient quantity of fractions. Bioassay-guided fractionation is the technique mostly used to isolate active fraction from crude plant extracts.

Semi-preparative scale HPLC has been used previously to fractionate many different types of crude plant extracts. Semi-preparative HPLC involves several steps including selection of solvent system suitable for fractionation, selection of column, injection volume, flow rate, selection of method involving gradient system and concentration of crude extract. When a method with less time span and proper separation of peaks is developed, then total runs are calculated to get sufficient quantity of fractions.

Chromatogram obtained from semi-preparative HPLC determines total number of

fractions. Several peaks can be selected to isolate from the chromatogram obtained. Each

separated peak can further be assessed for purity by using analytical scale HPLC.

Sometimes false peaks appear in the chromatogram which can be detected by comparing the chromatogram of the sample with that of the blank. These false peaks are known as artifacts.

Evaporation of solvent present in the fraction can be done by using several methods which can involve use of rotary evaporator and nitrogen current for evaporation of organic solvents and evaporation of aqueous phase requires use of freeze drier.

Present chapter describes results of bioassay-guided fractionation for (L+F) S. nubicola.

Leaf and flower extract of S. nubicola was fractionated by using semi-preparative HPLC.

76 Chapter 4 Fractionation by Semi-preparative HPLC

For this purpose first of all conditions of semi-preparative HPLC were optimized and then fractions were obtained. In order to select most active fraction, total phenolic contents of each fraction were determined and two bioassays including DPPH scavenging assay as well as DNA protection assay were conducted.

77 Chapter 4 Fractionation by Semi-preparative HPLC

Materials and methods

4.1: Preparation of samples

Ten milligrams of the powdered plant extract were dissolved in 80% methanol. In order to remove chlorophyll and lipids, plant extracts were washed three times with n-hexane carefully. Samples were filtered using GF/F (Glass fiber filters) and 0.2µm syringe filters.

4.2: Spectrophotometric scanning of hexane washes and extracts

The plant extracts were analysed spectrophotometrically (Carry WinUV Varian,

Australia) at wave length range of 200 nm to 800 nm to get the spectrum. Spectrum was obtained before hexane washing and after washing to observe any difference between the peaks obtained. Hexane washes were also scanned by using spectrophotometer to observe the removal of carotenoids and chlorophyll.

4.3: Semi-preparative HPLC

In order to get various fractions of the methanolic extract, semi-preparative HPLC was performed for leaf and flower extract of S. nubicola.

4.3.1: Sample Preparation

Hundred milligram of the plant extract was dissolved in 100 ml of 80% methanol to get the final concentration of 1mg/ml. Solution was washed three times with n-hexane to remove the lipids and pigments. Following n-hexane washing, 100 ml of instrument grade water was added to get the final concentration of 0.5mg/ml. Sample was filtered using GF/F (Glass fiber filters) and later on 0.2µm syringe filters.

4.3.2: Method development for semi-preparative HPLC

Several preliminary steps were taken into account to develop a method for fractionation of leaf and flower extract of S. nubicola. Various runs were carried out using variable

78 Chapter 4 Fractionation by Semi-preparative HPLC

gradient systems and time spans. Following factors were taken into account for

development of semi-preparative method for fractionation.

a. Solvent composition

The solvent system in which sample is dissolved must relate with the initial composition

of mobile phase of HPLC gradient. It can help to get the semi-preparative chromatogram with proper resolution of peaks. Solvent A was a mixture of methanol, acetonitrile, and

acetic acid in the ratio of 90:10:1 while solvent B was a mixture of water and acetic acid

in the ratio of 100:1.

b. Sample concentration

Concentration of the sample to be fractionated was set to get the best resolution of peaks.

Fractions were collected after optimization of concentration.

c. Time span of method

Various gradient systems were applied to reduce the total time span of the method.

d. Gradient of mobile phase

Composition of mobile phase was optimized to obtain elution of various components

earlier.

4.3.3: Optimization of conditions for semi-preparative HPLC of (L+F) S. nubicola

a. Method 1

Method 1 consisted of six steps with total time span of 60 minutes (Fig 4.3). Initial

composition of gradient started as 90% solvent A and 10% solvent B which was changed

to 70% solvent A and 30% solvent B after 10 minutes. Gradient remained isocratic for

next five minutes. After 25 minutes composition of gradient changed to 60% solvent A

and 40% solvent B. Composition of gradient was changed to 50% solvent A and 50%

79 Chapter 4 Fractionation by Semi-preparative HPLC

solvent B for next 15 minutes. In next 10 minutes, composition of gradient was attained

as 0% solvent A and 100 % solvent B which remained isocratic for next 10 minutes.

b. Method 2

Method 2 consisted of five steps with total time span of 45 minutes (Fig 4.4). Gradient

was started as 70% solvent A and 30% solvent B. In next 10 minutes, gradient was

changed to 60% solvent A and 40% solvent B. After five minutes, composition of

gradient was 50% solvent A and 50% solvent B. Gradient composition of 40% solvent A

and 60% solvent B was attained in next 10 minutes. In next 10 minutes, gradient

composition of 30% solvent A and 70% solvent B was achieved. Gradient composition of

0% solvent A and 100% solvent B was attained in last 10 minutes.

c. Method 3

Method 3 consisted of four steps with total time span of 20 minutes (Fig 4.5). Gradient

composition was started as 55% solvent A and 45% solvent B. In next 5 minutes,

gradient was changed to 40% solvent A and 60% solvent B. Gradient remained isocratic

for next five minutes. Gradient composition of 30% solvent A and 70% solvent B was

attained in next five minutes. In last five minutes gradient composition of 0% solvent A

and 100% solvent B was attained.

d. Method 4

Method 4 consisted of three steps with total time span of 20 minutes. Starting gradient composition was 40% solvent A and 60% solvent B. Gradient remained isocratic for next

10 minutes. Gradient composition of 30% solvent A and 70% solvent B was attained in next five minutes. In last five minutes, gradient composition of 0% solvent A and 100% solvent B was attained.

80 Chapter 4 Fractionation by Semi-preparative HPLC

e. Method 5

Method 5 consisted of four steps with total time span of 20 minutes (Fig 4.7). Gradient

composition was started as 60% solvent A and 40% solvent B. In next five minutes,

gradient of 40% solvent A and 60% solvent B was achieved. Gradient was changed to

35% solvent A and 65% solvent B after 5 minutes. Composition of gradient was changed

to 30% solvent A and 70% solvent B in next 5 minutes. For the last five minutes, gradient

composition of 0% solvent A and 100% solvent B was attained.

4.3.4: Method for fractionation

In order to fractionate methanol extract of leaf and flower of S. nubicola to get three fractions A, B and C, a four-step method (Method 5, Fig 4.7) was developed by using semipreparative column (Alltech, 250 mm, ID 10 mm, C18 5µ) equipped with guard column (Phenomenex, Australia). Fractions were collected from column waste by using

UV-Vis detector Varian 9050 and Varian 9012 pump system. Flow rate of 2ml/min with

2 ml of injection volume was used. Gradient composition for each step in described above in method 5. System was equilibrated between runs for 10 minutes.

Fractions were collected at following retention times (Fig 4.8).

Fraction A: 0 min to 12.9 min

Fraction B: 13 min to 14 min

Frction C: 14.1 to 20 min

Organic solvents were evaporated by using rotary evaporator (Rotavapor, Buchi,

Switzerland) at 45°C under vacuum. Fractions were freeze dried for removal of aqueous

part of the fractions.

81 Chapter 4 Fractionation by Semi-preparative HPLC

4.4: Determination of total phenolic contents of fractions

Total phenolic contents of three fractions were determined by using Folin-Ciocalteu reagent by the method as described previously for crude extracts in chapter 3 (page no

54) (Singleton and Rossi, 1965). Statistical analysis was carried out using ANOVA. LSD

(least significant difference) between values was determined by Duncan’s multiple range test. Data was expressed in terms of TEAC (trolox equivalent antioxidant capacity) i.e. mM of trolox per gram of fraction.

4.5: Antioxidant activity of fractions

Antioxidant potential of three fractions (A, B and C) from leaf and flower extract of S. nubicola was investigated by two assays in the aqueous system (DPPH scavenging activity, DNA protection assay).

(a) DPPH Scavenging activity: DPPH scavenging assay was performed by using

method of Kulisic et al., (2004) modified by Obeid et al., (2005) as described

previously (Chapter 3, page no 55). Data was expressed in three ways i.e. EC50 value,

%age scavenging of DPPH at different concentrations of fractions and mM of trolox

per gram of fractions i.e. TEAC (trolox equivalent antioxidant capacity). Statistical

analysis was performed by using ANOVA for TEAC values and EC50 values. LSD

(least significant difference) was calculated by using Duncan’s multiple range test.

(b) DNA protection assay: DNA protection assay for three fractions was carried out

by using modified method of Tian and Hua (2002). As described previously

(Chapter 3, page no 58). DNA protection assay for fraction A and fraction C was

performed at five concentrations i.e. 10, 20, 30, 40 and 50 ppm while for fraction

B, assay was performed at 1, 3, 5, 7 and 9 ppm.

82 Chapter 4 Fractionation by Semi-preparative HPLC

Results

4.1: Spectrophotometric scanning of hexane washes and crude extracts

Before coducting HPLC tasks, it was necessary to remove chlorophyll contents and carotenoids from crude plant extracts. In order to check the integrity of phenolic compounds, crude plant extracts were scanned before and after hexane washing. As different classes of compounds show absorbance at specific wave lengths. Another aspect of spectrophotometric scanning of crude plant extract was identification of group of compounds present in crude plant extracts.

(a) Scanning of hexane washes of crude extracts

Scanning of hexane washes of crude extracts revealed removal of carotenoids and chlorophyll A contents gradually (Fig 4.1). Spectrum for hexane washes of three extracts

((S) S. nubicola, (L+S) H. nepalensis, (L+S) A. oblongifolium) revealed removal of pigments after three washes while methanolic extract of (L+F) S. nubicola required more than three washes to remove the pigments.

(b) Spectrophotometric scanning of crude extracts

Spectrophotometric scanning of crude extracts before and after hexane washing revealed that verbescosides (absorbance at wave lengths 328 nm) as well as hydroxycinnamic acid derivatives (absorbance at wave lengths 280 nm) may be present in four extracts tested

(Obied et al., 2007). Photometric scanning of (L+F) S. nubicola before and after hexane washing is presented in Fig 4.2. Data revealed that there is no decrease in phenolic contents after hexane washing.

83 Chapter 4 Fractionation by Semi-preparative HPLC

(a)

1 2

(b)

(c)

Fig 4.1: Gradual removal of pigments including carotenoids (1) and chlorophyll A (2) from leaf and stem extract of H. nepalensis following hexane washing

84 Chapter 4 Fractionation by Semi-preparative HPLC

1 2

(b)

Fig 4.2: Result of scanning at 200 nm to 800 nm for crude extract before (a) and after hexane washing (b) at 100 ppm of (L+F) S. nubicola. Major peaks (1) at 280 nm and (2) at 328 nm may indicate presence of conjugated hydroxycinnamic acid analogue and verbascoside analogues respectively.

85 Chapter 4 Fractionation by Semi-preparative HPLC

4.2: Semi-preparative HPLC

During method development for fractionation of leaf and flower extract of S. nubicola several steps were applied by using different gradient systems and time spans .

4.2.1: Method development for semi-preparative HPLC

Following methods were applied before fractionation of leaf and flower extract of S. nubicola.

(a) Method 1

Result of method 1 showed several peaks. Major peak was at retention time of 47.5 minutes and there were many small peaks (Fig 4.3). This method had a large time span of

60 minutes because of six steps involved. Another method was developed to minimize the total run time and to elute the main peak earlier than 47.5 minutes.

(b) Method 2

As a result of method 2 (Fig 4.4) elution time for main peak was shifted to 26.9 minutes.

Total run time for this method was 45 minutes which needed to be reduced further.

In order to reduce the time span further to elute the main peak earlier, a five step method with time span of 20 minutes was developed. Gradient was started as 55% solvent A and

45% solvent B. Chromatogram showed many small peaks (Fig 4.5). As a result of this method main peak was eluting at retention time of 11.2 minutes but was eluting with other peaks. Separation between peaks was not proper so another method was developed.

86 Chapter 4 Fractionation by Semi-preparative HPLC

Method 1 Time (min) % composition Solvent A % composition Solvent B 0 90 10 10 70 30 15 70 30 25 60 40 40 50 50 50 0 100 60 0 100

Fig 4.3. Method 1 and chromatogram obtained by application of method in semi-preparative HPLC

87 Chapter 4 Fractionation by Semi-preparative HPLC

Method 2

Time (min) % composition Solvent A % composition Solvent B 0 70 30 10 60 40 15 50 50 25 40 60 35 30 70 45 0 100

Fig 4.4. Method 2 and chromatogram obtained by application of method in semi-preparative HPLC

88 Chapter 4 Fractionation by Semi-preparative HPLC

Method 3 Time (min) % composition Solvent A % composition Solvent B 0 55 45 5 40 60 10 40 60 15 30 70 20 0 100

Fig 4.5. Method 3 and chromatogram obtained by application of method in semi-preparative HPLC

89 Chapter 4 Fractionation by Semi-preparative HPLC

(d) Method 4

A four step method was adapted to get better separation of peaks. Gradient was started as

40% solvent A and 60% solvent B. Total run time was 20 minutes. Main peak was eluting at retention time of 13.3 minutes (Fig 4.6) but without any improvement in separation of peaks.

(e) Method 5

In order to get better separation of peaks, a final method was selected (Fig 4.7). Method included total run time of 20 minutes and five steps. As a result of method five, all peaks were eluting separately. While retention time of main peak was 13.3 minutes. Following semi-preparative HPLC, three fractions denoted as Fraction A, Fraction B and Fraction C were obtained (Fig 4.7). Each 100 mg of extract yielded 72 mg of fraction A, 4 mg of fraction B and 14 mg of fraction C.

Three fractions obtained from (L+F) S. nubicola were separated by semi-preparative

HPLC. Three fractions are presented in Fig 4.8 with respective retention times

90 Chapter 4 Fractionation by Semi-preparative HPLC

Method 4 Time (min) % composition Solvent A % composition Solvent B 0 40 60 10 40 60 15 30 70 20 0 100

Fig 4.6. Method 4 and chromatogram obtained by application of method in semi-preparative HPLC

91 Chapter 4 Fractionation by Semi-preparative HPLC

Method 5 Time (min) % composition Solvent A % composition Solvent B 0 60 40 5 40 60 10 35 65 15 30 70 20 0 100

Fig 4.7. Method 5 and chromatogram obtained by application of method in semi-preparative HPLC

92 Chapter 4 Fractionation by Semi-preparative HPLC

Fraction B

Fraction A Fraction C

Fig 4.8: Chromatogram obtained for (L+F) S. nubicola to get three fractions A, B and C by using semi-preparative HPLC.

93 Chapter 4 Fractionation by Semi-preparative HPLC

4.3: Determination of total phenolic contents for three fractions (A, B and C)

Total phenolic contents expressed in terms of mM of trolox/gram of fraction indicated

highly significant value in case of fraction B i.e. 2524.4 ± 3.9 (Table 4.1). Standard

regression line for trolox was used to calculate TEAC value (R2 = 0 .992) (Fig 3.4 chapter

3, page 61). Absorbance pattern for total phenolic contents of three fractions is indicated

in Fig 4.9.

4.4: Antioxidant activity of fractions

Antioxidant activity of three fractions was tested by using three assays in aqueous system

4.4.1: DPPH scavenging activity

Significant EC50 value was obtained in case of fraction B i.e. 4.08 ± 0.14 as compared to

fraction A and fraction C. DPPH scavenging activity in terms of mM of trolox/gram of

fraction is expressed in table 4.1 while DPPH scavenging pattern is elucidated in Fig 4.10 for three fractions. Linear regression line for trolox was used to calculate the value

(R2=0.994) (Fig 3.4, chapter 3, page 65).

4.4.2: DNA protection assay for three fractions

DNA protection activity was obvious by all the fractions tested, although there was

variability in the level of activity at different concentrations of the three fractions tested.

In case of fraction A, variable level of DNA protection was observed at different concentrations (4.11 (a)) while in case of fraction B DNA protection was higher at two concentrations i.e. 1 ppm and 3 ppm compared to higher concentrations of 5, 7 and 9 ppm (Fig 4.11 (b)). However there was no change in the level of activity of fraction C at

all tested concentrations (10, 20, 30, 40 and 50 ppm) (Fig 4.11 (c)).

94 Chapter 4 Fractionation by Semi-preparative HPLC

(a)

0.2 y = 0.0072x + 0.0023 0.18 R2 = 0.9949 0.16 0.14 0.12 0.1 0.08 Absorbance 0.06 0.04 0.02 0 0 5 10 15 20 25 30 Concentration (ppm)

(b)

0.4 y = 0.07x + 0.0039 0.35 R2 = 0.9983 0.3

0.25

0.2

0.15 Absorbance 0.1

0.05

0 0123456 Concentration (ppm)

(c)

0.25 y = 0.0155x + 0.0129 2 0.2 R = 0.9643

0.15

0.1 Absorbance

0.05

0 0 2 4 6 8 10 12 14 Concentration (ppm)

Fig 4.9: Pattern of absorbance of phenolic compounds at 765 nm for three fractions

(a. Fraction A, b. Fraction B, c. Fraction C by using Folin-Ciocalteu reagent.

95 Chapter 4 Fractionation by Semi-preparative HPLC

(a)

90 80 70 60 50 40 30 20

Percentage inhibitionPercentage 10 0 -10 0 10203040506070 Concentration (ppm)

(b)

100

20 Percentage inhibition 0 012345678910 -20 Concentration (ppm)

(c)

120

100

20 Percentage inhibitionPercentage 0 0 5 10 15 20 25 30 -20 Concentration (ppm)

Fig 4.10: DPPH scavenging pattern for three fractions a. Fraction A, b. Fraction B, c. Fraction C.

96 Chapter 4 Fractionation by Semi-preparative HPLC

Table 4.1. Total phenolic contents and DPPH scavenging activity of three fractions

(A, B and C). Difference in letters present significance at p < .05.

Total phenolic contents DPPH scavenging activity

Fractions mM Trolox/gram of extract mM Trolox/gram of fraction EC50 (ppm) Fraction A 1279.174 ± 0.717c 73.3 ± .046c 40.33 ± 1.97a Fraction B 2524.4 ± 3.9a 367.87 ± .08a 4.08 ± .14bc Fraction C 1395.61 ± 3.58b 173.68 ± .09b 11.6 ± .5b

97 Chapter 4 Fractionation by Semi-preparative HPLC

(a) 1 2 3 4 5 6 7 8 9 10

(b) 1 2 3 4 5 6 7 8 9 10

Fig 4.11 (a). DNA protection assay with different concentrations of fraction A (a), B

(b) and C (c) of crude extract of (L+F) S. nubicola. Lane 1 is λ Hind III marker.

Lane 2-5 presents controls (untreated DNA, DNA treated with 2mM FeSO4, DNA

treated with 30 % H2O2, DNA treated with 2mM FeSO4 and 30 % H2O2). Lane 6-10

presents treatment of DNA with five different concentrations (10, 20, 30, 40 and 50 ppm) of fraction A (a) and C (c). While in lane 6-10 present treatment of DNA with

five different concentrations (1, 3, 5, 7 and 9 ppm) of fraction B (b).

98 Chapter 4 Fractionation by Semi-preparative HPLC

Conclusion

Optimized method for semi-preparative HPLC was method 5. This method lead to isolation of three fractions. Fraction B showed highest phenolic contents as well as antioxidant and DNA protection activity suggesting presence of important phenolic antioxidants.

Chapter 5 Identification of phenolic compounds

In order to identify various constituents in crude plant extracts, as well as in purified fractions some modified techniques are required. Several hyphenated techniques have been in use previously including HPLC-UV and LC-MS to investigate molecular weight and structure of various constituents in plant extracts.

HPLC coupled with UV-diode array detector has been used for the last two decades successfully to get information about the constituents of crude plant extracts. UV spectra of various constituents in plant extracts provide useful information about components.

Moreover, UV spectra of known metabolites can be stored in data base and comparison between known and new metabolites can be easily made.

LC-MS is another technique providing useful information about molecular masses and structure of the constituents of crude plant extracts. Several interfaces are used for this purpose due to opposite conditions required for HPLC and MS techniques. Mostly four interfaces including thermospray (TSP), continuous flow FAB (CF-FAB), atmospheric pressure chemical ionization (APCI) and electro-spray ionization have been in use for chemical screening of crude plant extracts (Hostttmann et al., 2001).

Present chapter describes results of analytical scale HPLC of the purified fraction.

Furthermore, HPLC-DAD and LC-MS analysis were carried out to identify purified fraction as well as any additional compound present in the crude plant extract.

100 Chapter 5 Identification of phenolic compounds

Materials and methods

5.1: Preparation of samples and purified fractions

Ten milligrams of powdered plant extract were dissolved in 80% methanol. In order to

remove chlorophyll and lipids, plant extracts were washed three times with n-hexane

carefully. Samples were filtered using GF/F (Glass fiber filters) and 0.2cm syringe filters.

Three purified fractions of (L+F) S. nubicola obtained by semi-preparative HPLC were also analyzed by using analytical grade HPLC. One mg of each fraction was dissolved in

100% methanol to get final concentration of 1mg/ml. Sample was filtered using 0.2um syringe filters.

5.2: Preparation of standard

Five milligram of each of standard (Homo vanillyl alcohol, Caffeic acid, Quercetin,

Catechin hydrate, Gallic acid) was dissolved in 5 ml of 80% methanol to prepare 1 mg/ml concentration. Before use, solution was diluted to 0.1 mg/ml and filtered by using 0.2µm syringe filters.

5.3: Analytical scale HPLC

In order to analyze samples for antioxidant components, analytical grade HPLC was performed. Analytical scale HPLC was performed for purified fractions of (L+F) S. nubicola, four crude extracts as well as five external standards.

5.3.1: HPLC Method

Data was obtained at 280 nm by using UV-DAD (UV diode array detector). Data consisted of various peaks for crude extracts with their respective retention times.

Analytical grade HPLC (high performance liquid chromatography) was performed on a

Varian 9021 solvent delivery system equipped with Varian 9065 Polychrom UV-diode

101 Chapter 5 Identification of phenolic compounds array detector (190-367 nm). Data was processed by Star Polychrom version 5.2. The system was maintained in a controlled room temperature at 21±10°C. A flow rate of

1ml/minute and injection volume of 10 µl were used. Sample analysis was performed by gradient elution on a 150 mm x 4.6 mm i.d., 5uM, Luna C-18(2) column (Phenomenex,

Australia) with guard column (Phenomenex, Australia). The mobile phases were freshly prepared and degassed under vacuum using Phenomenex nylon 45 µm membranes and sonicated in a sanophon ultrasonic bath (Ultrasonic Industries Pty. Ltd, Sydney,

Australia) for 15 minutes prior to HPLC analysis.

Solvent A was a mixture of 90:10:1 methanol/acetonitrile/acetic acid (V/V/V) while solvent B was a mixture of 100:1 water/acetic acid (V/V).

A six step gradient analysis for a total run time of 60 min was used (Method 1, chapter 4, page No, 78) to get proper separation of peaks.

5.3.2: Regression lines for five external standards

Five external standards were used at five different concentrations i.e. 100, 200, 300, 400 and 500 ppm for each standard to get calibration curves. Use of external standards can determine stability of method. Equation for calibration can be used to get quantity of various peaks in chromatograms of different crude extracts.

5.3.3: Quantification of various peaks

Various peaks in chromatograms obtained for four crude extracts were quantified in terms of equivalents of five external standards. Data was expressed in terms of mg of standard per gram of crude extract for a specific peak in a chromatogram.

102 Chapter 5 Identification of phenolic compounds

5.4: LC-MS (Liquid chromatography mass spectrometry)

Liquid chromatography–mass spectrometry (LC–MS) of the methanolic extracts of all

plant species was performed on a Micromass Quattro micro tandem quadrupole mass

spectrometer (Waters, Manchester, UK). LC separation was attained by a Waters liquid

chromatograph (Waters, Milford, USA), consisting of a 2695 Separation Module and

2487 dual wavelength UV detector operated at 240 and 280 nm. Columns and gradients

were same as described previously for analytical scale HPLC. An injection volume of 10

µL and a constant flow of 1 mL/min was used for each analysis. The entire flow from the

LC was directed into the mass spectrometer. Data was acquired by the Masslynx data

system for both the MS and UV data. The mass spectral data was acquired for four

alternative scans; Scan 1: Positive ion mode, cone voltage 35 V; Scan 2: Positive ion mode, voltage 70 V; Scan 3: Negative ion mode, cone voltage 30 V; Scan 4: Negative ion mode, cone voltage 70 V.

5.5: Identification of phenolic components in crude plant extracts

Phenolic contents in crude plant extracts were identified on the basis of following criteria.

(a) Molecular mass from LC-MS data

LC-MS chromatograms obtained for four crude extracts were analysed by Masslynx

to get spectra of various peaks. Spectra were recorded in negative and positive ion

mode to estimate molecular masses of various components. Only major peaks in each

chromatogram were analysed for determination of molecular masses. Determination

of molecular mass from LC-MS data can be a useful tool to identify components on

the basis of molecular masses.

103 Chapter 5 Identification of phenolic compounds

(b) Retention time of standards

In order to confirm retention times of components in crude extracts, standards were

run simultaneously with crude extracts on analytical column. Peaks in the crude

extract with same retention time of standards show same compounds.

Fragmentation data of various groups in a compound can give information about

structure of a compound. Fragmentation data of known compounds was compared to

the data already available.

104 Chapter 5 Identification of phenolic compounds

Results

5.1: Analytical scale HPLC

Analytical scale HPLC equipped with UV-DAD was carried out for four crude extracts, three purified fractions of (L+F) S. nubicola and five external standards. Results are described below.

(a). Analytical scale chromatogram for crude extracts

Results for analytical scale chromatography for four crude extracts ((L+F) S. nubicola,

(S) S. nubicola, (L+S) H. nepalensis, (L+S) A. oblongifolium) revealed presence of several peaks. Moreover, purity of three fractions from (L+F) S. nubicola obtained by semi-preparative HPLC was also assessed by analytical scale HPLC.

(1). Analytical scale chromatogram for (L+F) S. nubicola

Analytical scale chromatogram for (L+F) S. nubicola revealed presence of many small peaks and one main peak at retention time of 27.7 min (Fig 5.1).

Analytical scale HPLC for three fractions

Three fractions of (L+F) S. nubicola separated by semi-preparative HPLC were run on analytical scale HPLC to determine purity of fractions. Chromatograms for three fractions indicated purity of fractions (Fig 5.2).

(2). Analytical scale chromatogram for (S) S. nubicola

Analytical scale chromatogram for (S) S. nubicola is similar to chromatogram for (L+F)

S. nubicola. But main peak height is smaller as compared to peak height in case of (L+F)

S. nubicola (Fig 5.3).

105 Chapter 5 Identification of phenolic compounds

(3). Analytical scale chromatogram for (L+S) H. nepalensis

Analytical scale chromatography for leaf and stem extract of H. nepalensis revealed

many peaks (Fig 5.4). Main peaks eluting at retention time of 9.8 min, 12.3 min, 22.8

min, 23.8 min, 27.5 and 28.6 min.

(4). Analytical scale chromatogram for (L+S) A. oblongifolium

Analysis of leaf and stem extract of A. oblongifolium by analytical scale chromatography

revealed many small and large peaks. Three main peaks were eluting at retention times of

9.3 min, 11.7 min and 28.1 minutes (Fig 5.5).

106 Chapter 5 Identification of phenolic compounds

6 I:8 I:1 W W mAU d:\work at csu\505(l+f ).run File: d:\work at csu\505(l+f ).run Channel: 1 = 280.00 nm Results 27.709 600 Last recalc: NA

500

400

300 54.389

200 51.367

100 51.739 11.369 1.767 25.962 24.340 52.164 5.312 52.656 12.167 53.059 53.522 47.827 53.263 1.583 55.294 22.259 30.101 44.799 45.183 48.065 54.093 32.546 47.582 50.868 47.284 48.395 57.985 1.896 2.411 9.965 15.836 43.979 44.331 46.196 46.419 46.794 46.999 X: 3.4922 Minutes 57.661 2.495 27.028 30.769 45.815 46.064 48.777 49.035 55.606 55.935 56.232 9.619 33.457 49.393 49.598 50.012 50.174 8.272 21.281 10.523 50.416 50.606 1.483 6.660 7.544 17.046 12.992 13.235 31.532 31.613 34.488 35.271 37.717 59.438 2.116 17.387 18.735 19.802 20.087 13.928 14.013 36.610 36.723 38.363 38.887 40.199 40.883 41.882 0.908 1.075 2.845 3.492 5.945 8.972 18.096 14.621 39.243 39.417 41.354 4.358 0 Y: -9.17 mAU Peak Name: Result: 0.299 Area: 119 mAU*sec Width: 0.000 sec -79 10 20 30 40 50 Minutes

Figure 5.1: Analytical scale chromatogram for (L+F) S. nubicola

107 Chapter 5 Identification of phenolic compounds

(a)

Artefact

(b)

Main peak

(c)

Artefact

Fig 5.2: Chromatograms for three fractions separated by semi-preparative HPLC (a. Fraction A, b. Fraction B, c. Fraction C) and analysed by analytical scale HPLC

108 Chapter 5 Identification of phenolic compounds

mAU 6 I:8 I:1 W W 400 d:\work at csu\505(s).run File: d:\work at csu\505(s).run Channel: 1 = 280.00 nm Results Last recalc: NA 27.702

300

200

Artefact

100 52.041 25.917 11.523 52.323 1.757 55.178 52.763 52.880 53.185 53.310 53.898 54.534 1.625 24.290 45.064 2.436 58.216 46.193 56.141 44.765 21.278 32.477 33.361 44.097 44.339 45.437 46.000 46.397 47.571 47.861 9.744 13.087 22.315 43.638 45.665 46.755 47.353 48.182 12.329 1.880 29.978 46.936 48.396 8.387 12.157 12.536 57.347 26.907 29.625 22.669 8.984 9.244 5.439 59.234 59.704 24.809 31.769 35.246 40.106 48.810 10.562 15.845 50.214 51.351 34.443 35.854 35.989 36.217 37.595 38.348 39.050 40.865 18.441 18.587 18.886 13.930 14.726 41.382 41.830 42.146 17.455 17.517 17.781 17.914 19.333 19.699 19.979 20.106 20.372 20.470 20.540 49.037 49.324 6.458 6.847 7.526 15.361 X: 0.6795 Minutes 16.403 16.699 16.899 17.039 49.691 50.994 6.010 2.112 3.055 3.606 4.218 4.738 0.117 0.178 0.325 0.631 0.679 Y: 0.0146 mAU 0 Peak Name: Result: 0.004 Area: 1.10 mAU*sec Width: 0.000 sec -45 10 20 30 40 50 Minutes

Figure 5.3: Analytical scale chromatogram for (S) S. nubicola

109 Chapter 5 Identification of phenolic compounds

mAU

:8 200 WI d:\work at csu\503(l+s).run File: d:\work at csu\503(l+s).run Channel: 1 = 280.00 nm Results Last recalc: NA 9.856 150

100 51.964 49.894

50 27.563 22.851 23.841 12.368 28.689 48.931 52.524 1.753 52.700 52.898 55.251 10.677 53.530 55.430 9.136 53.323 56.114 56.247 53.913 54.154 54.434 54.319 54.583 25.805 51.050 34.297 54.961 56.914 6.837 51.668 11.681 11.802 12.871 57.972 24.390 58.590 8.611 48.462 14.914 45.005 45.366 47.016 47.643 58.316 43.881 45.726 45.922 46.215 46.472 46.906 47.270 47.364 48.660 49.143 29.454 29.725 32.025 44.176 44.272 44.410 13.920 17.817 24.875 47.880 47.986 15.861 21.277 30.297 30.379 30.452 30.663 30.772 30.854 31.286 31.378 31.475 32.875 36.826 2.514 13.508 14.382 15.548 18.500 18.545 33.341 33.550 33.685 37.532 37.633 38.646 42.839 42.912 43.031 49.434 2.419 15.245 17.001 19.967 20.037 21.980 22.082 35.509 35.603 35.675 35.801 35.919 36.010 36.070 36.325 36.473 37.187 38.000 38.103 38.261 38.942 39.423 39.502 39.697 39.989 40.161 40.262 40.341 40.576 40.735 40.839 40.931 41.012 41.147 41.248 41.324 41.515 41.672 41.759 41.872 42.049 42.092 42.208 42.314 50.375 7.685 7.781 16.707 19.062 20.314 1.603 16.353 19.162 19.266 19.420 1.883 6.095 3.128 5.661 4.457 4.580 1.470 3.576 3.740 3.940 4.028 4.193 4.908 4.981 5.345 2.129 0 0.494 0.972 1.078 1.165 X: 3.1275 Minutes Y: 3.09 mAU Peak Name: Result: 1.308 Area: 555 mAU*sec Width: 0.000 sec -34 10 20 30 40 50 Minutes

Fig 5.4: Analytical scale chromatogram for (L+S) H. nepalensis

110 Chapter 5 Identification of phenolic compounds

mAU d:\work at csu\507(l+s).run File: d:\work at csu\507(l+s).run Channel: 1 = 280.00 nm Results

28.134 Last recalc: NA 125

100 51.868

75 9.339 45.138 43.804 3.664

50 52.474 11.716 1.751 1.854 56.051 23.401 23.977 9.849 55.079 52.793 27.185 53.047 56.298 53.592 12.889 25.809 53.923 53.286 53.416 25 54.393 15.163 54.180 11.186 51.299 13.900 54.738 15.832 37.800 12.248 13.445 14.328 57.096 22.645 19.203 26.670 10.430 10.620 10.771 32.910 50.669 57.871 58.445 4.191 51.596 50.382 29.451 30.175 45.784 2.517 51.475 46.333 2.586 46.181 29.778 29.832 6.506 24.875 16.609 16.656 44.580 6.677 50.868 6.912 47.036 8.504 46.570 46.836 33.458 47.430 35.602 47.742 47.915 20.430 1.599 31.054 31.434 31.700 7.512 17.603 20.324 20.753 20.834 20.970 32.080 21.490 21.691 2.424 5.441 8.009 48.245 48.487 34.510 5.983 34.049 36.777 36.828 38.871 42.279 36.422 38.690 38.767 38.949 39.582 39.673 39.849 49.150 49.831 50.128 40.165 40.356 40.425 41.578 41.660 41.742 40.726 40.892 40.968 41.075 49.591 3.048 1.468 2.119 0.649 1.137 1.226 0 X: 11.1862 Minutes Y: 17.7 mAU Peak Name: Result: 1.164 Area: 536 mAU*sec Width: 0.000 sec -20 10 20 30 40 50 Minutes

Fig 5.5: Analytical scale chromatogram for (L+S) A. oblongifolium

111 Chapter 5 Identification of phenolic compounds

(b) Analytical scale chromatogram for five external standards

Analytical scale chromatogram for mixture of five external standards was carried out by using HPLC-DAD. Five peaks at retention time of 4.1 min, 9.5 min, 10.9 min, 12.5 min and 38.5 min for gallic acid, catechin hydrate, homo vanillyl alcohl, caffeic acid and quercetin were obtained respectively. Chromatogram is shown in Fig 5.6.

5.2: Regression lines for five external standards by using analytical scale HPLC

Five external standards including gallic acid, caffeic acid, homo vanillyl alcohl, catechin hydrate and quercetin were used at five different concentrations (100, 200, 300, 400 and

500 ppm) for analytical scale HPLC to get five calibration curves with equation for linear regression. Five regression lines are presented in figure 5.7. Standard curves showed R2 valueas 0.752, 0.940, 0.893, 0.920 and 0.995 for gallic acid, caffeic acid, catechin hydrate, homo vanillyl alcohl, and quercetin respectively.

5.3: Quantification of various peaks in terms of equivalents of five external standards

Main peaks in crude extracts i.e. (L+F) S. nubicola, (S) S. nubicola, (L+S) H. nepalensis and (L+S) A. oblongifolium were quantified in terms of five external standards. Data was expressed in terms of mg of standard per gram of crude extracts. Data was analysed statistically by using ANOVA. Highest quantity of peak in terms of equivalents of all standards at retention time of 27.7 min in case of (L+F) S. nubicola was obtained (Table

5.1).

112 Chapter 5 Identification of phenolic compounds

mAU 250

:8 :16 :8 :4 WI WI WI WI d:\work at csu\standard.run File: d:\work at csu\standard.run Channel: 2 = 280.00 nm Results Last recalc: NA 200 Gallic acid Caffeic acid Dirt 12.601 4.142

150 51.355 51.829

100 Catechin hydrate

Homo vanillyl Quercetin

alcohl 10.909 50 53.757 53.114 54.477 53.256 9.534 54.850 38.587 50.778 50.204 50.412 50.082 1.789 49.190 48.509 48.080 47.130 47.469 47.696 46.325 46.603 44.845 45.211 45.273 45.403 45.614 45.750 44.203 44.284 44.440 40.173 40.287 42.724 42.978 43.090 43.305 24.535 24.709 24.798 24.893 26.403 27.007 27.183 27.364 27.457 27.725 27.81827.853 28.362 28.505 28.554 28.709 28.857 28.953 29.111 29.292 29.566 29.629 29.707 29.760 29.922 30.020 30.124 30.358 30.420 30.553 33.578 35.053 37.970 40.884 40.999 41.119 41.245 41.573 41.690 41.885 42.087 42.223 23.507 23.699 23.796 23.986 24.100 25.309 25.540 25.647 25.727 25.806 26.028 26.287 26.590 26.694 26.817 27.600 28.034 30.924 31.054 31.159 31.253 31.287 31.381 31.449 31.802 31.986 32.057 32.247 32.327 32.387 32.457 32.650 32.757 33.034 33.935 34.100 34.382 34.459 35.509 35.612 35.743 35.951 36.046 36.121 36.199 36.331 36.624 36.810 36.981 37.146 21.258 21.657 21.932 22.020 22.146 22.234 22.640 23.072 11.531 11.747 11.963 12.093 13.720 14.083 14.187 18.237 18.376 18.433 18.677 18.889 18.960 19.013 19.182 19.260 19.408 19.533 19.618 19.749 19.996 20.241 20.434 20.604 20.786 20.893 20.993 22.420 14.268 14.556 14.627 14.760 14.870 15.213 15.397 15.576 16.021 16.103 16.171 16.366 16.549 16.647 16.793 16.989 17.502 17.593 17.733 17.809 10.161 10.356 2.435 55.680 56.425 56.678 7.690 7.874 8.308 8.553 8.611 8.805 8.863 57.168 5.012 5.220 5.525 5.992 6.455 6.688 6.997 7.177 7.412 2.520 3.413 58.073 58.140 58.887 58.997 59.086 2.989 59.426 59.621 59.697 59.869 0.273 0.627 1.003 1.350 1.438 2.165 1.930 X: 10.3565 Minutes 2.018 0 Y: 2.90 mAU Peak Name: Result: 0.003 Area: 1.19 mAU*sec Width: 0.000 sec -33 10 20 30 40 50 Minutes

Fig 5.6: Analytical scale chromatogram for five external standards

113 Chapter 5 Identification of phenolic compounds

(a)

Regression line for gallic acid

600 m 500 y = 0.7934x + 128.81 2 R = 0.7525 400

300

200

100 Absorbance at 280 n Absorbance at 0 0 100 200 300 400 500 600 Concentration (ppm)

(b)

Regression line for caffeic acid

1000

m 900 800 y = 1.6086x + 101.86 700 2 R = 0.9409 600 500 400 300 200

Absorbance at Absorbance280 n at 100 0 0 100 200 300 400 500 600 Concentration (ppm)

114 Chapter 5 Identification of phenolic compounds

(c)

Regression line for Homo vanillyl alcohl

350 m 300 y = 0.5189x + 39.952 2 250 R = 0.9209 200 150 100 50 Absorbance at 280 n Absorbance at 0 0 100 200 300 400 500 600 Concentration (ppm)

(d)

Regression line for catechin hydrate

180

m 160 y = 0.278x + 27.243 140 R2 = 0.8934 120 100 80 60 40 20 Absorbance at Absorbance280 n at 0 0 100 200 300 400 500 600 Concentration (ppm)

115 Chapter 5 Identification of phenolic compounds

(e)

Regression line for Quercetin

400 m 350 y = 0.6896x + 10.61 300 R2 = 0.9952 250 200 150 100 50 Absorbance at 280 n Absorbance at 0 0 100 200 300 400 500 600 Concentration (ppm)

Fig 5.7: Regression lines for five external standards used to calculate quantity of various peaks in case of analytical scale chromatograms for four crude extracts.

116 Chapter 5 Identification of phenolic compounds

Table 5.1: Result of analytical scale HPLC. Quantity of various peaks in terms of mg of standards per gram of crude extract.

Difference in superscript letters indicate level of significance at p < 0.05.

Plant extracts Peaks Gallic acid Catechin hydrate Homo vanillyl alcohl Caffeic acid Quercetin (L+F) S. nubicola Peak at 28.8 min 11.6 ± .02a 52.4 ± .09a 158.3 ± .3a 37.9 ± .07a 153.3 ± .2a (S) S. nubicola Peak at 28.8 min 7.9 ± .02b 37.3 ± .09b 107.6 ± .3b 26.5 ± .07b 95.1 ± .2b (L+S) H. nepalensis Peak at 9.8 min 2.5 ± .09c 15.05 ± .3c 32.1 ± 1.2c 9.5 ± .28c 38.7 ± .9c (L+S) A. oblongifolium Peak at 27.1 min .75 ± .004d 7.85 ± .01d 7.80 ± .06d 4.06 ± .01d 20.05 ± .04d

117 Chapter 5 Identification of phenolic compounds

5.4: LC-MS (liquid chromatography mass spectrometry).

Liquid chromatography mass spectrometry was performed for four crude extracts and

five external standards. Data was acquired at four cone voltages i.e. 30 V (negative ion

mode), 35 V (positive ion mode), 70 V in negative and positive ion mode. Mass to charge

ratio was obtained by using MassLynx for various peaks. Mass to charge ratio i.e. m/z in negative and positive ion mode was obtained to get idea about molecular mass of a compound in case of crude extracts.

(a) LC-MS for (L+F) S. nubicola

LC-MS data for (L+F) S. nubicola was obtained by using mass spectrometer. LC-MS scan remained helpful to identify main peak in crude extract of leaf and flower of S. nubicola. This main peak was actually purified fraction B from the crude extract by semi- preparative HPLC (Chapter 4, page No, 80) Five scans were carried out. One scan was obtained at 280 nm by using UV-DAD. Remaining four scans were carried out at four cone voltages i.e. in positive ion mode at cone voltage of 35, in negative ion mode at cone voltage of 30, in negative ion mode at cone voltage of 70 and in positive ion mode at cone voltage of 70. In all scans peak at retention time of 28 min is obvious (Fig 5.8).

(b) LC-MS for (S) S. nubicola

Five scans in case of stem extract of S. nubicola by LC-MS data revealed one main peak at retention time of 28 min (Fig 5.9).

LC-MS data at four cone voltages revealed presence of few main peaks in case of crude extract of H. nepalensis (Fig 5.10). At all cone voltages, peak at retention time of 9.9 min

118 Chapter 5 Identification of phenolic compounds is obvious. At cone voltage of 70 in negative ion mode, four peaks were obvious i.e. at retention time of 23.2 min, 24.2 min, 27.8 min and 28.8 min (Fig 5.10).

119 Chapter 5 Identification of phenolic compounds

040208_0205 Scan EI+ 53.92 TIC 100 (a) 9.28e5

% 50.47 52.17 28.02 1.82 0 040208_0204 Scan ES- 28.05 51.74 TIC 100 51.08 53.89 55.07 (b) 48.93 57.00 61.81 62.481.26e7 47.08 % 1.70 44.86 41.90 5.62 24.58 34.94 0 040208_0203 Scan ES- 53.95 TIC 100 55.20 (c) 59.50 1.06e7 52.09 52.83 58.53 61.05 28.04 47.36 48.54 % 1.68 45.80 34.77 33.81 41.36 42.10 3.24 5.61 11.75 14.79 22.78 25.0026.78 0 040208_0202 Scan ES+ 53.85 59.63 TIC 100 52.82 55.11 58.59 (d) 47.56 62.51 1.64e8 45.78 48.97 % 1.22 28.02 1.66 0 040208_0201 Scan ES+ 49.24 TIC (e) 100 52.43 53.98 61.09 1.20 56.57 62.05 1.99e8 43.84 46.73 47.54 % 1.65 28.00 41.18

0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00

Fig 5.8: LC-MS chromatogram for (L+F) S. nubicola (a) Scan at cone voltage 70 in positive ion mode (b) Scan by UV-DAD at 280 nm (c) Scan in negative ion mode at cone voltage 30 (d) Scan in positive ion mode at cone voltage 35 (e) Scan in positive ion mode at cone voltage 70

120 Chapter 5 Identification of phenolic compounds

040208_0305 Scan EI+ 53.88 100 TIC (a) 9.33e5 % 52.15 28.02 1.82 0 040208_0304 Scan ES- 52.11 100 28.05 48.86 51.52 52.78 TIC 56.5558.55 61.66 62.481.21e7 (b) 1.70 47.01 % 45.53 1.33 26.28 35.24 0 040208_0303 Scan ES- 53.87 55.13 100 TIC 56.54 1.04e7 (c) 48.69 52.09 58.61 60.6862.46 1.68 28.04 47.88 % 46.10 64.01 35.4439.1440.25 3.31 11.75 25.67 26.41 29.74 31.81 34.11 0 040208_0302 Scan ES+ 53.85 100 52.82 56.52 TIC 47.49 58.66 62.22 1.61e8 46.97 49.04 (d) % 44.38 1.66 28.02 0 040208_0301 Scan ES+ 49.24 TIC 100 52.43 53.91 56.6558.57 62.20 1.94e8 (e) 43.84 46.73 47.47 % 1.792.61 27.93 3.57 0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00

Fig 5.9: LC-MS chromatogram for (S) S. nubicola (a) Scan at cone voltage 70 in positive ion mode (b) Scan by UV-DAD at 280 nm (c) Scan in negative ion mode at cone voltage 30 (d) Scan in positive ion mode at cone voltage 35 (e) Scan in positive ion mode at cone voltage 70

121 Chapter 5 Identification of phenolic compounds

040208_0405 Scan EI+ 53.87 TIC 100 9.33e5

(a) % 52.15 1.82 9.94 0 040208_0404 Scan ES- 52.1153.89 55.07 100 48.78 51.37 TIC 47.30 56.48 58.55 61.14 1.14e7 (b) 1.78 62.48 % 9.92 44.27 1.33 24.21 27.8328.87 12.29 23.24 0 040208_0403 Scan ES- 53.87 100 55.13 TIC 49.13 56.54 1.01e7 1.68 47.36 52.10 52.84 58.68 62.09 (c) 45.21 62.90 % 40.84 43.80 1.31 9.97 27.81 37.14 2.65 6.42 23.22 0 040208_0402 Scan ES+ 53.85 TIC 100 47.49 52.74 55.11 58.59 62.44 46.75 60.89 1.50e8 (d) 50.52 % 1.22 1.66 9.96 0 040208_0401 Scan ES+ 49.24 100 52.43 53.91 55.02 TIC 56.57 58.5761.83 1.87e8 47.39 (e) % 1.20 43.84 1.79 9.94 0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00

Fig 5.10: LC-MS chromatograms for (L+S) H. nepalensis (a) Scan at cone voltage 70 in positive ion mode (b) Scan at 280 nm by UV-DAD (c) Scan at cone voltage 30 in negative ion mode (d) Scan at cone voltage 70 in positive ion mode (e) Scan at cone voltage 35 in positive ion mode 122 Chapter 5 Identification of phenolic compounds

(d) LC-MS for (L+S) A. oblongifolium

LC-MS data at three cone voltages i.e. 35 V (positive ion mode), 30 V (negative ion mode), 70 V (negative ion mode) revealed presence of one main peak at retention time of

27.8 min (Fig 5.11).

(e) LC-MS for five external standards

LC-MS data for five external standards was obtained at four cone voltages. At cone voltage of 30 (negative ion mode) and at cone voltage of 70 (negative ion mode), four peaks for four standards were visible. Four standards i.e. gallic acid, catechin hydrate, caffeic acid and quercetin showed peaks at retention times of 3.85 min, 8.73 min, 11.7 min and 36.05 min respectively (Fig 5.12). While at cone voltage of 35 (positive ion mode) and at cone voltage of 70 (positive ion mode) three peaks were visible for three standards. Three standards including catechin hydrate, caffeic acid and quercetin showed peaks at three retention times i.e. 8.7 min, 11.7 min and 36 min respectively (Fig 5.12).

123 Chapter 5 Identification of phenolic compounds

040208_0505 Scan EI+ 53.87 100 32.88 TIC 9.45e5 52.15 (a) % 1.82 0 040208_0504 Scan ES- 51.6752.11 100 53.89 56.5558.55 TIC 48.78 60.1161.96 1.00e7 1.78 47.30 (b) % 1.33 27.98 45.82 9.40 11.84 23.98 0 040208_0503 Scan ES- 53.87 55.13 100 TIC 58.53 61.0561.50 8.48e6 52.09 52.84 1.68 % 48.32 2.42 11.82 14.79 (c) 1.31 3.90 9.38 13.08 17.67 21.37 23.45 27.96 43.73 44.99 64.68 0 040208_0502 Scan ES+ 52.0053.85 56.52 TIC 100 58.59 60.9661.40 47.49 1.48e8 (d) % 27.94 1.66 0 040208_0501 Scan ES+ 52.43 53.9155.02 100 49.24 TIC 56.50 58.5761.83 1.65e8 (e) 43.9147.02 47.54 % 1.20 1.65 28.00 0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00

Fig 5.11: LC-MS chromatograms for (L+S) A. oblongifolium (a) Scan at cone voltage 70 in positive ion mode (b) Scan at 280 nm by UV-DAD (c) Scan at cone voltage 30 in negative ion mode (d) Scan at cone voltage 70 in positive ion mode (e) Scan at cone voltage 35 in positive ion mode

124 Chapter 5 Identification of phenolic compounds

040208_0105 Scan EI+ (a) 53.88 TIC 100 11.70 9.40e5 3.85 % 52.13 8.77 36.05 1.82 0 040208_0104 Scan ES- 36.05 TIC 100 (b) 8.73 51.45 52.1153.89 56.48 1.55e7 49.23 60.9261.37 62.03 11.70 % 47.30 45.23 3.85 0 040208_0103 Scan ES- 53.87 55.13 TIC 100 11.75 62.24 (c) 52.10 52.84 58.54 61.13 1.08e7 3.83 8.79 36.03 46.32 48.54 % 44.91 25.00 27.07 35.22 41.36 1.83 10.27 28.26 32.40 0 040208_0102 Scan ES+ 52.82 53.85 55.11 TIC (d) 100 58.5259.63 47.56 62.59 1.66e8 46.82 50.74 % 36.01 8.77 11.73 0 40208_0101 Scan ES+ 49.24 TIC 100 52.43 53.98 (e) 56.57 58.57 62.35 2.00e8 43.84 46.14 47.54 % 35.99 0.61 8.75 11.71 0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00

Fig 5.12: LC-MS chromatogram for five external standards (a) Chromatogram at 280 nm by UV-DAD (b) Scan at cone voltage 70 in negative ion mode (c) Scan at cone voltage 30 in negative ion mode (d) Scan at cone voltage 35 in positive ion mode (e) Scan at cone voltage 70 in positive ion mode

125 Chapter 5 Identification of phenolic compounds

5.5: Identification of phenolic compounds

Phenolic components were identified in crude extracts on basis of some evidences.

Results of analytical scale HPLC and LC-MS played important role in this regard.

Results for identification of some of the identified compounds are described below.

5.5.1: Evidences for identification of rosmarinic acid from (L+F) S. nubicola and (S)

S. nubicola

Main peak (fraction B from (L+F) S. nubicola) was identified as rosmarinic acid.

Rosmarinic acid was identified from (L+F) S. nubicola and (S) S. nubicola on the basis of following evidences.

(a) Molecular mass from LC-MS

LC-MS data was analysed by using MassLynx. Peak at retention time of 28 min was scanned in positive and negative ion mode. In negative ion mode it showed m/z ratio of

359 while in positive ion mode it showed m/z ratio of 361. Which indicated molecular mass of 360 specific for rosmarinic acid (Fig 5.13).

(b) Fragmentation pattern from mass spectrum

Fragmentation pattern for molecule of rosmarinic acid was obtained in negative ion mode. Data is exactly similar to the data available previously (Mehrabani et al., 2005), which clearly indicates presence of rosmarinic acid in (L+F) S. nubicola and (S) S. nubicola (Fig 5.14).

126 Chapter 5 Identification of phenolic compounds

040208_0302 379 (28.018) Cm (377:384-(327:357+410:446)) Scan ES+ 89 5.12e6 100

163 117 135 94 361 383 399 631 730 759 850 960 1051 1389 164 219 249275 301 421 482 525 579 609 686 743 804 834 907 919 999 1103 11191128 1169 1208 13581366 0 040208_0303 379 (28.036) Cm (375:383) Scan ES- 359 6.57e5 100

% 119 161 360

101 197 221 719 741 381 419 441 141 257 313 631 742 877 1054 357 457 524 554 607 698 773823 834 916 957 968 1079 11311140 1226 1304 13511358 1398 1487 1497 0 Da/e 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

Fig 5.13: Mass spectrum from (L+F) S. nubicola for rosmarinic acid indicating m/z

ratio of 361 in positive ion mode while m/z ratio of 359 in negative ion mode.

127 Chapter 5 Identification of phenolic compounds

133 100 161 Mass Spectrum peaks in negative ion mode 135 72.9933 123.0450 132.0216 133.0291 134.0354 135.0448 161.0240 161.2165 162.0276 % 179.0350 197.0454 359.0767 123 179 360.0808

132 197 359

221 105109 136 162 89 97 151 180 195 201 219 360 381 119 124 222 81 150 153 163 257 261 279 295299 313 379 382 202 229 262 309 324 339 341 353 362 397 399 0 Da/e 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

Fig 5.14: Mass spectrum of peak from LC-MS chromatogram at 28 min in negative ion mode indicating fragmentation pattern of rosmarinic acid from (L+F) S. nubicola.

128 Chapter 5 Identification of phenolic compounds

5.5.2: Evidences for identification of chlorogenic acid from (L+S) H. nepalensis

Molecular mass of 354 in crude extract of leaf and stem of H. nepalensis indicated presence of chlorogenic acid.

(a) Molecular mass from LC-MS data

Molecular mass of chlorogenic acid was determined by scanning peak at 9.92 min from

LC-MS chromatogram. In negative ion mode m/z ratio of 353 while in positive ion mode m/z ratio of 355 was obtained, which indicated molecular mass of 354 (Fig 5.15).

(b) Retension time of standard peak and peak in crude extract by analytical scale

HPLC

In order to make comparison between retention time of chlorogenic acid standard and chlorogenic acid from crude extract, analyticl scale HPLC was carried out by using the same method. Retention time of 10.4 min was obtained in case of standard run (Fig 5.16) while retention time of 9.8 min was obtained in case of crude extract of leaf and stem of

H. nepalensis (Fig 5.4).

5.5.3: Evidences for identification of rutin from (L+S) H. nepalensis

Rutin was identified from (L+S) H. nepalensis. Following evidences were used to indicate rutin in the crude extract.

(a) Molecular mass from LC-MS data

Molecular mass of 610 was determined by scanning peak at 24.41 min from LC-MS chromatogram in negative and positive ion mode. In negative ion mode, m/z ratio of 609 was obtained while in positive ion mode m/z ratio of 611 was obtained (Fig 5.17).

(b) Retension time of standard peak and peak in crude extract by analytical scale

HPLC

129 Chapter 5 Identification of phenolic compounds

In order to make comparison between retention time of standard molecule of rutin and

peak in the crude extract, analytical scale HPLC was performed. Analytical scale HPLC

result indicated retention time of 25.7 min in standard run (Fig 5.18) while retention time of 27.5 min was obtained in analytical scale run for crude extract (Fig 5.4).

130 Chapter 5 Identification of phenolic compounds

040208_0403 135 (9.974) Cm (129:139-(90:118+154:184)) Scan ES- 353 1.38e5 100 191

119 354

375 97 192 339 435 451 707 729 120 163 221 251 309 376 482 519 889 1009 1087 1494 575 619 664 741 814 865 911 963 991 1105 1160 1263 1369 1409 1425 1439 0 040208_0402 135 (9.955) Cm (127:143-(162:201+67:91)) Scan ES+ 89 1.28e6 100

% 117

135 163

164 299 355 377 395 521 649 747 976 1048 1083 1493 219 245 279 410 441 541 598 628 726 731 769 800 872 907 913962 1065 1143 1155 1364 1430 1471 0 Da/e 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

Fig 5.15: Mass spectrum for chlorogenic acid from (L+S) H. nepalensis indicating m/z ratio of 355 in positive ion mode while m/z ratio of 353 in negative ion mode

131 Chapter 5 Identification of phenolic compounds

Fig 5.16: Chromatogram for chlorogenic acid standard by analytical scale HPLC

132 Chapter 5 Identification of phenolic compounds

040208_0401 328 (24.224) Scan ES+ 89 1.87e6 100 99 133 117

303 % 177 159

181 305 465 349 221 261 504 611 633 697 265 319 393 437 525 652 741 1349 1473 569 701 808 818 828 939943 1118 12431251 1315 1406 1446 1499 0 040208_0404 326 (24.132) Scan ES- 300 1.77e5 100

301 119 609 %

302 141 179 103 610 271 347 604 161 217 255 367 428 445 485 516 671 812 998 1013 1084 1144 395 523 633 718 761 771 874 885 939 951 1114 1225 1238 1297 1367 14091416 14841491 0 Da/e 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

Fig 5.17: Mass spectrum from (L+S) H. nepalensis for rutin indicating m/z ratio of 611 in positive ion mode while m/z ratio of

609 in negative ion mode

133 Chapter 5 Identification of phenolic compounds

Fig 5.18: Analytical scale HPLC for standard rutin

134 Chapter 5 Identification of phenolic compounds

Conclusion

Three phenolic compounds were identified from four crude plant extracts on the basis of

results of analytical scale HPLC equipped with UV-DAD and LC-MS. One phenolic

compound i.e. rosmarinic acid was identified from leaf and flower extract of S. nubicola

and stem extract of the same specie. Rosmarinic acid is purified fraction B from (L+F) S.

nubicola by semi-preparative HPLC (Chapter 4, page No 80). Rosmarinic acid was

identified on the basis of molecular mass from LC-MS data and fragmentation pattern of the compound. Moreover rosmarinic acid quantity was found highly significant as compared to quantity of other compounds from other species. Fraction B (rosmarinic acid) remained the most active fraction while studying antioxidant activity (Chapter 4).

Two phenolic compounds including chlorogenic acid and rutin were identified on the basis of their molecular masses from LC-MS data and standard run by analytical scale

HPLC. The overall results of the present study indicate that methanolic extract of leaf and flower of S. nubicola is a good source of rosmarinic acid, a known antioxidant.

Previously rosmarinic acid is reported in various Salvia species however it is first report of presence of rosmarinic acid in S. nubicola.

135

Chapter 6 Discussion

Medicinal plants have been in use all over the world to treat various diseases including infections, cancer, inflammation, heart diseases etc. The use of natural products for treatment of all kind of diseases is due to their less harmful effects as compared to drugs synthesized in the laboratory. In all areas of the world, locally used herbal treatments are common and have been under investigation to get active principal of these remedies.

Pakistan is rich in natural resources and plants in Northern areas are well known for their medicinal importance. Plants from Northern areas of Pakistan have been in use by local

healers to treat many diseases including diseases of digestive system, skin diseases and infectious diseases as well. For research purpose, knowledge from local healers play most important role to get information about medicinal nature of various plant species.

Many extraction procedures have been in use previously for preparation of crude plant

extracts including maceration, use of soxhlet apparatus and decoction preparation

method.

Methanol was used as extraction solvent for preparation of all the five extracts (Table

2.1). Methanol is a polar solvent which can extract mostly polar constituents in it.

Methanol has been used previously for extraction of plant constituents and many active

compounds have been isolated. Chowdhury et al., (2002) evaluated cytotoxic and antitumor activity of Amoora rohitaka stem bark extract and found significant activity in methanol extract. Methanol extract of Stachytarpheta urticaefolia also showed significant bioactivity and an active compound (Ipolamiide) has been isolated (Chowdhury et al.,

2004). In another study from Iran, Kianbakht and Jahaniani. (2003) demonstrated

antibacterial activity of methanolic extract of Tribulus terrestris L.

136 Chapter 6 Discussion

Antibacterial activity of methanolic extract of stem bark of Tridesmostemon omphalocarpoides was reported by Kuete et al., (2006) while in another study by Santos et al., (2006), sedative and anxiolytic effects of methanolic extracts from leaves of

Passiflora actinia was demonstrated. Fungitoxic activity of the hexane and methanol extracts of Copaiba plant leaves was evaluated by Amorim et al., (2003) in which methanol extract exhibited significant activity. Methanolic extracts of Eucalyptus camaldulensis and Terminalia catappa showed antimicrobial activity against some pathogenic strains (Babayi et al., 2004) while in another study by Souza et al., (2003), methanolic fractions of Hyptis ovalifolia presented antimicrobial activity towards

dermatophytes. Eftekhar et al., (2005) also used methanol as extraction solvent while

studying antimicrobial activity of Datura innoxia and Datura stramonium.

Drug discovery strategy involved various steps to be followed. Initially, prescreening assays were performed to select potent plant extracts. Potent plant extracts were fractionated and screened for biological activities. Active fractions were identified on the basis of analytical scale HPLC and constituents in the plant species were identified on the basis of LC-MS data.

Present study involved use of four plant species including S. nubicola, H. nepalensis, A. oblongifolium and S. tomentosa. Medicinal importance of these plant species is well

known for a long time as local healers use these plants for treatment of various diseases.

In order to investigate active principal of these plant species, methanolic extracts were

prepared by using various parts of plants.

137 Chapter 6 Discussion

6.1: Prescreening assays

6.1.1: Antimicrobial assays

Due to emergence of antibiotic resistant strains as well as side effects of synthetic drugs, search for new antimicrobial drugs from natural resources has been an objective of researchers and investigators.

Antibacterial assay was performed by standard agar well diffusion method described by

Ansari et al., (2005) and modified by Hanif et al., (2007). One of the five extracts i.e. leaf

and stem extract of A. oblongifolium has presented significant antibacterial activity

against all pathogenic strains tested. The result suggests that crude extract of A.

oblongifolium can be a good source of antimicrobial products. In a study from Canada,

Omar et al., (2000) demonstrated antibacterial activity of crude extracts of A. rubrum and

A. saccharum against S. aureus, B. subtillus, Mycobacterium phlei and Enterococcus faecalis while Nickell, (1959) has described antibacterial activity of many species of Acer genera previously.

6.1.2: Cytotoxicity, phytotoxicity and antitumor activity

Medicinal plant species all over the world have been playing a vital role in drug discovery efforts. Present study involves evaluation of crude botanical extracts for their potent cytotoxicity in brine shrimp cytotoxicity assay, herbicidal or growth stimulation activity by radish seed phytotoxicity assay and inhibition of tumor formation in antitumor potato disc assay.

Brine shrimp lethality assay has been considered as a prescreening assay for antimicrobial, antitumor, antimalarial, antifungal, and insecticidal activities. Two of the five extracts presented significant cytotoxicity. Highest rate of lethality to brine shrimp

138 Chapter 6 Discussion

was observed in case of leave and stem extract of A. oblongifolium (ED50 47.7 ppm)

followed by leaf and stem extract of H. nepalensis (ED50 226 ppm) indicating

pharmacological potential of these two plants. Toxicity is pharmacology at lower doses

that is why medicinal plant extracts are tested for cytotoxicity. A number of previous studies indicated potent cytotoxicity in case of methanol extracts of several plant species.

Kanegusuku et al., (2001) reported cytotoxicities of methanol extracts and ethyl acetate fraction from Rubus imperialis C. (Rosaceae). In another study from Brazil, sixty medicinal plant species were evaluated for their cytotoxicity to brine shrimps (Maria et

al., 2000). Only 10% of the species presented ED50 < 1000 ppm. Jacques et al., (2003)

demonstrated the screening of 226 methanol and water extracts for lethality towards

larvae of brine shrimp and identified several cytotoxic plant species.

Antitumor potato disc assay is a valuable tool, which indicates antitumor activity of test

compounds by their inhibition of formation of characteristics crown galls induced in

wounded potato tissues by A. tumefaciens. Present study revealed moderate to high rate

of inhibition of tumor formation in case of all methanol extracts tested (Table 2.5). Leave

and stem extract of S. tomentosa presented significant inhibition of tumor formation

however this extract slightly affected the viability of A. tumefaciens strain too. Therefore

antitumor activity observed in case of S. tomentosa could partially or completely be because of its ability to kill A. tumefacienc and not because of any antitumor potential present in this extract. Turker and Camper (2002) screened biological activity of common

mullein by antitumor potato disc assay but no effect on viability of A. tumefaciens strain

was observed. Coker et al., (2002) indicated antitumor potato disc assay as an acceptable

139 Chapter 6 Discussion tool to primarily screen antineoplastic activity of various crude extracts as well as purified fractions regardless of mode of inhibitory action on tumor formation.

Radish seeds have been used in general toxicity studies because of their sensitivity to phytotoxic compounds (Einhellig and Rasmussen, 1978) and as a standard assay in alleopathic studies (Patterson, 1986). All extracts exhibited toxicity in radish seed bioassay at high doses. A very interesting feature of the present study is growth stimulation effect of three extracts (leave and flower extract of S. nubicola, leave and stem extract of H. nepalensis and stem extract of S. nubicola) at low concentration (1000 ppm). Ali et al., (2005) also revealed growth stimulation effect of one of the fractions

(3α-hydroxy-20-oxo-30-norlupane) of S. nubicola at 5 ppm in Lemna minor

phytotoxicity assay. Similar types of results were obtained by Tsao et al., (2002). They

studied different fractions of Ailanthus altissima and observed growth stimulation effect

at low doses while growth inhibitory effect at higher doses. Good antitumor and no

phytotoxic activity indicate use of the plant extract to control crown gall disease in plants.

6.1.3: Antioxidant assays

A lot of antioxidant work on crude extracts of Salvia specie has been done by using

DPPH scavenging assay (Bozan et al., 2002, Gulcin et al., 2004, Orhan et al., 2006).

Highly significant DPPH scavenging activity was observed in case of methanol extract of

leaf and flower of S. nubicola ( Fig 3.6) that is comparable to study of 14 Salvia species

from Turkey (Orhan et al., 2006). They described that DPPH scavenging activity of ethyl

acetate and methanol fractions remained highly significant.

Leaf and flower extract of S. nubicola showed DNA protection activity at low

concentrations (10 ppm and 100 ppm) which is higher as compared to other crude

140 Chapter 6 Discussion extracts tested. DNA protection activity at low concentration is consistant with the results of other assays for (L+F) S. nubicola. Phenolic contents remained highly significant in

case of leaf and flower extract of S. nubicola. Salvia species are known for various

phenolic contents previously (Akkol et al., 2008, Kosar et al., 2008). Particularly S.

nubicola is an known anticancer and antitumor plant specie. It is also obvious that

phenolic compounds are responsible for antioxidant activities in crude extracts.

H. nepalensis is known anticancer and cytotoxic plant specie as reported by Inayatullah et

al., (2007). Present study reveals its lowest antioxidant potential in different antioxidant

assays. Two phenolic compounds (Chlorogenic acid and rutin) were identified in leaf and

stem extract of H. nepalensis. Table 5.1 presents quantity of chlorogenic acid in terms of

mg of standards which is lower as compared to quantity of other phenolic compounds in other plant species tested. The extract showed lowest activity in assays in aqueous system however its activity in TBARS is comparable to activity of other species.

A quite high rate of ABTS+ scavenging was observed in case of A. oblongifolium. Leaf

and stem extract of A. oblongifolium also showed antioxidant activity in case of DPPH

scavenging assay and its total phenolic contents are significantly higher as compared to

stem extract of S. nubicola and leaf and stem extract of H. nepalensis. Previously, a

number of Acer species are known for antioxidant activities and various phenolic

compounds responsible for antioxidant activities were isolated (Jiang et al., 2006).

6.2: Fractionation of crude extract and identification of phenolic compounds

Fractionation of most potent plant extract i.e. leaf and flower extract of S. nubicola by

semi-preparative HPLC resulted in three fractions i.e A, B and C. Fraction B remained

highly active in case of DPPH scavenging activity and its total phenolic content is the

141 Chapter 6 Discussion highest. Fraction B showed DNA protection activity at low concentrations which is also very important with respect to biological activities.

Analytical scale HPLC and LC-MS revealed the presence of rosmarinic acid (fraction B) as a major antioxidant in methanol extract of (L+F) S. nubicola as well as (S) S. nubicola.

Rosmarinic acid has been identified previously in many Salvia species as major antioxidant and its quantity is comparable to other species tested (Akkol et al., 2008,

Kosar et al., 2008). Although rosmarinic acid is major phenolic compound in case of two extracts of S. nubicola i.e. leaf and flower extract and stem extract but varying activity can be attributed to variable quantity of rosmarinic acid. Moreover, presence of other phenolic compounds in case of leaf and flower extract of S. nubicola may be one of the reasons of such a high activity.

For identification of rosmarinic acid LC-MS data played most important role. Mass spectrum of rosmarinic acid in positive and negative ion mode revealed m/z ratio of 361 and 359 respectively indicating molecular mass of 360. Fragmentation data of rosmarinic acid from mass spectrum in negative ion mode was compared to fragmentation data from literature, which provided clear evidence for presence of rosmarinic acid in leaf and flower extract of S. nubicola as well as stem extract of S. nubicola.

Two important phenolic compounds i.e. chlorogenic acid and rutin were identified from leaf and stem extract of H. nepalensis. Molecular mass was estimated from the result of

LC-MS while standard compounds were run on analytical scale HPLC with crude extract to confirm the presence of these compounds in crude extract.

142 Chapter 6 Discussion

Conclusion

Investigation of crude botanical extracts has revealed potent biological activities of variable level. Antioxidant potential of crude extracts described another screening strategy to select active extracts. Leaf and flower extract of S. nubicola has presented

antitumor, phytotoxic and antioxidant activities. Fractionation of (L+F) S. nubicola by

using semi-preparative HPLC yielded three fractions. Fraction B (rosmarinic acid)

remained most active in two antioxidant assays. Furthermore, chemical analysis by

analytical scale HPLC and LC-MS revealed presence of rosmarinic acid in fraction B of

(L+F) S. nubicola which is already known antioxidant and commercially available.

Present study describes first report of identification of rosmarinic acid in S. nubicola.

Present study also revealed presence of chlorogenic acid and rutin in (L+S) H. nepalensis.

Another important aspect of present study is highest antibacterial and significant antioxidant activity of (L+S) A. oblongifolium which can be fractionated in future to isolate active components responsible for antibacterial as well as antioxidant activity.

Result of crude extracts indicated presence of important phenolic compounds as well as antimicrobial compounds. Above mentioned potent plant extracts can further be fractionated in future to identify and isolate active components, which can provide important anticancer and antimicrobial drugs in future.

143

Ahmed E, Arshad M, Ahmad M, Saeed M, Ishaque M (2004). Ethanopharmacological

survey of some medicinally important plants of Galliyat areas of NWFP, Pakistan. Asian

J Plant Sci, 3(4): 410-415

Akkol EK, Goger F, Kosar M, Baser KHC (2008). Phenolic composition and biological

activities of Salvia halophila and Salvia virgata from Turkey. Food Chem, 108: 942-949

Ali MS, Ahmed S, Ibrahim SA, Tareen RB (2005). Characterization and bioscreening of

a new triterpenoid and a flavanone isolated from Salvia nubicola. Chem Biodivers, 2:

910-916

Ali MS, Ahmed W, Armstrong AF, Ibrahim SA, Ahmed S, Parvez M (2006).

Guaianolides from Salvia nubicola (Lamiaceae). Chem Pharm Bull, 54(9): 1235-1238

Amorim AC, Cardoso MD, Pinto JE, Souza PE, Filho ND (2003). Fungitoxic activity

avaliation of the hexane and methanol extracts of copaiba plant leaves. Cienc agrotec

Lavras, 28(2): 314-322

Anderson JE, Chang CJ, McLaughlin JL (1988). Bioactive components of Allamanda

Schottii. J Nat Prod, 51(2): 307-308

Andreopoulos T, Papanikolaou G, Politou M, Konstantopoulos K, Stefanou J,

Loukopoulos D (2000). M. leutus, a putative cause of hepatic abscess. Panminerva Med,

42(3): 231-232

Annesley T, Rockwood AL, Sherman NE (2006). Mass spectrometry In: TIETZ text

book of clinical chemistry and molecular diagnostics (Burtis CA, Ashwood ER, Bruns

DE eds), pp 165-190, Elsevier India.

Ansari FL, Nazir S, Noureen H, Mirza B (2005). Combinatorial synthesis and

antibacterial evaluation of an indexed chalcone library. Chem. Biodivers, 2: 1656-1664

144 Austen B, McCarthy H, Wilkins B, Smith A, and Duncombe A (2001). Fatal

disseminated fusarium infection in acute lymphoblastic leukaemia in complete remission.

J Clin Pathol, 54:488-490

Awal MA, Nahar A, Hossain MS, Bari MA, Rahman M, Haque ME (2004). Brine shrimp

toxicity of leaf and seed extracts of Cassia alata Linn. and their antibacterial potency. J

Med Sci, 4(3): 188-193

Babayi H, Kolo I, Okogun JI, Ijah UJ (2004). The antimicrobial activities of methanolic

extracts of Eucalyptus camaldulensis and Terminalia catappa against some pathogenic

microorganisms. Biokemistri, 16(2): 106-111

Basu TK, Temple NJ, Garg ML (1999). Antioxidants in human health and disease. New

York: CABI Publishing.

Berg AK and Perkins TD (2007). Contribution of anthocyanins to the antioxidant

capacity of juvenile and senescing sugar maple (Acer saccharum) leaves. Funct plant biol, 34: 714-719

Bisby FA, Roskov YR, Ruggiero MA, Orrell TM, Paglinawan LE, Brewer PW, Bailly N,

Van Hertum J (2007). Species 2000 and ITIS Catalogue of Life: 2007 Annual Checklist.

Species 2000: Reading, U.K.

Black JG, (2005). Antimicrobial therapy In: Microbiology, Principles and explorations

(edis), John Wiley and sons, Inc, USA, pp, 363-365

Boutati EI, and Anaissie EJ (1997). Fusarium, a significant emerging pathogen in patients with hematologic malignancy: Ten years' experience at a cancer center and implications for management. Blood, 90:999-1008

145 Bozan B, Ozturk N, Kosar M, Tunalier Z, Base KHC (2002). Antioxidant and free radical scavenging activities of eight Salvia species. Chem Nat comp+, 38(2): 198-200

Brands SJ, (2000). The Taxonomicon. Universal Taxonomic Services, Amsterdam, The

Netherlands.

Chowdhury R, Rashid M, Khan OF, Hassan CM (2004). Bioactivity of extractives from

Stachytarpheta urticaefolia . Pharm Biol, 42(3): 262-267

Chowdhury R, Rashid RB, Hasan CM (2002). Cytotoxicity and antitumor activity of

Amoora rohituka stem bark. Bangladesh Pharmaceutical Journal, 12(3): 8-10

Coker PS, Radecke J, Guy C, Camper ND (2002). Potato tumor induction assay: A

multiple mode of drug action assay. Phytomedicine, 10: 133-138

Crissy JT, Lang H, Parish LC (1995). Manual of Medical Mycology Cambridge.

Massachusett: Blackwell Science. Pp, 263.

Cuvelier ME, Richard H, Berset C (1996). Antioxidant activity and phenolic composition

of pilot-plant and commercial extracts of sage and rosemary. J Am Oil Chem Soc, 73:

645-652.

Eftekhar F, Yousefzadi M, Tafakori V. (2005). Antimicrobial activity of Datura innoxia

and Datura stramonium. Fitoterapia, 76:118-120

Einhellig FA, Rasmussen JA (1978). Synergistic inhibitory effects of vanillic and p-

hydroxybenzoic acids on radish and grain sorghum. J Chem Ecol, 4: 425-436

Ferrigini NR, Putna JE, Anderson B, Jacobsen LB, Nichols DE, Moore DS, McLaughlin

JL (1982). Modification and evaluation of the potato disc assay and antitumor screening

of Euphorbia seeds. J Nat Prod, 45: 679-686.

146 Fosse T, Toga B, Peloux Y, Granthil C, Bertrendo J, Sethian M. (1985). Meningitis due to M. leutus. Infection, 13(6): 280-281

Frohne D, Pfander HJ, (2004). Araliaceae In: Poisnous plants, Manson publishing, UK,

PP, 75

Fujita E, Node M (1984). Diterpenoids of Rabdosia species. Prog Chem Nat Prod, 46:

77-157

Galsky AG, Wilsey JP, Powell RG (1980). Crown gall tumor disc bioassay. Plant

Physiol, 65: 184-185

Gilani SA, Qureshi RA, Farooq U (2001). Ethnobotanical studies of Ayubia national park district Abbottabad, Pakistan. Online J Biol Sci, 1: 284-286

Gilani SA, Qureshi RA, Gilania SJ. (2007). Indigenous uses of some important ethnobotanical herbs of Ayubia national park, Abbottabad, Pakistan.

Girmenia C., A. P. Lori, F. Boecklin, A. Torosantucci, P. Chiani, P. De Fabritiis, F.

Taglietti, A. Cassone, and P. Martino. (1999). Fusarium infections in patients with severe aplastic anemia: review and implications for management. Haematologica, 84:114-118

Gulcin I, Ugus M, Oktay M, Beydemir S, Kufrevioglu O (2004). Evaluation of the antioxidant and antimicrobial activities of Clary Sage (Salvia sclarea L). Turk J Agric

For, 28: 25-33

Halliwell B (1994). Free radicals and antioxidants: a personal view. Nutrition Reviews,

52: 253-265

Hamayun M, Khan MA, Hayat T (2005). Ethanobotanical profile of Utror and Gabral valleys, district Swat Pakistan. http://www.siu.edu/~ebl/leaflets/swat.htm.

147 Hamayun M, Khan SA, Sohn EY, Lee IJ. (2006). Folk medicinal knowledge and

conservation status of some economically valued medicinal plants of district Swat,

Pakiatan. Lyonia, 11(2): 101-113

Hanif M, Hussain M, Ali S, Bhatti MH, Ahmed MS, Mirza B, Evans HS (2007).

Synthesis, spectroscopic investigation, crystal structure, and biological screening,

including antitumor activity of organotin (IV) derivatives of piperonylic acid. Turk J

Chem, 31: 349-361

Hostettmann K, Wolfender JL, Terreaux C, (2001). Modern screening techniques for plant extracts. Pharml Biol, 39:18-32

Hou W, Lin R, Cheng K, Hung Y, Cho C, Chen C, Hwang S, Lee M (2003). Free radical- scavenging activity of Taiwanese native plants. Phytomedicine, 10(2-3): 170-175

Inayatullah S, Irum R, Rehman A, Chaudhary MF, Mirza B (2007). Biological evaluation of some selected plant species of Pakistan. Pharm Biol, 45: 397-403

Jacques EL, Pohlit AM, Nunomura SM, Da AC, Mustafa EV, Reis SK, Alecrim AM,

Brito BR, De CS, Finney EK, Oliveira ED, Santos KD, Pereira LC, Castro LD, Rosha

LF, Andrade MM, Henrique MC, Santos MD, Souza PD, Silva SG (2003). Screening of plants in Amazon state for lethality towards brine shrimp. Acta Amaz, 33: 93-104

Jiang C, Chang M, Wen C, Lin Y, Hsu F, Lee M (2006). Natural products of cosmetics:

Analysis of extracts of plants endemic to Taiwan for the presence of tyrosinase- inhibitory, melanin reducing and free radical scavenging activities. J Food Drug Anal,

14(4): 346-352

148 Kanegusuku M, Benassi JC, Pedrosa RC, Yunes RA, Filho VC, Maia AA, Marcia M.,

Monache FD, Niero R (2001). Cytotoxic, hypoglycemic activity and phytochemical analysis of Rubus imperialis (Rosaceae). Z. Naturforsch C, 57c: 272-276

Khan T, Ahmad M, Khan H, Khan MA (2005). Biological activities of aerial parts of

Paeonia emodi Wall. Afr J Biotechnol, 4: 1313-1316

Kianbakht S, Jahaniani F. (2003). Evaluation of antibacterial activity of Tribulus terrestris L. growing in Iran. IJPT, 2(1): 22-24

Kim JH, Lee BC, Kim JH, Sim GS, Lee DH, Lee KE, Yun YP, Pyo HB (2005). The isolation and antioxidative effects of vitexin from Acer palmatum. Arch Pharm Res,

28(2): 195-202

Kishida E, Tokumaru S, Ishitani Y, Yamamoto M, Oribe M, Iguchi H, Kojo S (1993).

Comparison of the formation of melonaldehyde and thiobarbituric acid-reactive

substances from autoxidized fatty acids based on oxygen consumption. J Agric Food

Chem, 41: 1598-1600

Kool JMS, Liempd V, Harmsen S, Schenk T, Irth H, Commandeur J, Vermeulen N

(2007). An on-line post-column detection system for the detection of reactive-oxygen-

species-producing compounds and antioxidants in mixtures. Anal Bioanal Chem,

388:871-879

Kosar M, Dorman HJ, Bachmayer O, Baser KHC, Hiltunen R. (2003). An improved on-

line HPLC-DPPH method for the screening of free radical scavenging compounds in

water extracts of Lamiaceae plants. Chem Nat Comp+, 39(2): 161-166

Kosar M, Goger F, Baser K H C (2008). In vitro antioxidant properties and phenolic composition of Salvia virgata Jacq. From Turkey. J Agric Food Chem, (in press).

149 Kuete V, Tangmouo JG, Beng VP, Ngounou FN, Lontsi D (2006). Antimicrobial activity

of the methanolic extract from the stem bark of Tridesmonstemon omphalocorpoides

(Sapotaceae). J Ethnopharmacol, 104: 5-11

Kulisic T, Radonic A, Katalinic V, Milos M. (2004). Use of different methods for testing

antioxidative activity of oregano essential oil. Food Chem, 85:633-640

Lee M, Jiang C, Juan S, Lin R, Hou W (2005). Antioxidant and heme oxygenase-1 (HO-

1) induced effects of selected Taiwanese plants. Fitoterapia, 77(2): 109-115.

Lu Y, Foo LY (2001). Antioxidant activities of polyphenols from sage (Salvia officinalis). Food Chem, 75: 197-202.

Maria T, Silva AF, Brandao M, Mesquita TS, Fatima ED, Junior AS, Zani CL (2000).

Biological screening of Brazilian medicinal plants. Mem Inst Oswaldo Cruz, 95: 367-373

Mayayo E, Pujol I and Guarro J (1999). Experimental pathogenicity of four opportunist

Fusarium species in a murine model. J Med Microbiol, 48:363-366

McDonald S, Prenzler P, Antolovich M, Robards K (2001). Phenolic contents and antioxidant activity of olive extracts. Food Chem, 73: 73-84

McLaughlin JL (1991). Crown gall tumors on potato discs and brine shrimp lethality: two single bioassays for plant screening and fraction. In: Hostetsmann, K, ed., Methods in

Plant Biochemistry. Academic Press, London, pp. 1-31

McLaughlin JL, Rogers LL (1998). The use of biological assays to evaluate botanicals.

Drug Inf J, 32: 513-524

Mehrabani M, Ghassemi N, Sajjadi E, Ghannadi A, Shams-Ardakani M (2005). Main phenolic compounds of petals of Echium Amoenumfisch and C.A.Mey, a famous medicinal plant of Iran, Daru, 13:65-69

150 Meneghini, R. (1997). Iron homeostasis, oxidative stress, and DNA damage. Free radical

Bio Med, 23:783-792

Mongelli E, Martianez J, Anaya J, Grande C, Grande M, Torres P, Pomilio AB.( 2003).

Bolax gummifera: Toxicity against Artemia sp. of Bornyl and iso-Bornyl esters. Mol Med

Chem, 1:26-29

Nasir E, (1975). Flora of Pakistan In: Stewart Herbarium, Gordon College, Rawalpindi

and Department of Botany, University of Karachi, Karachi, pp, 2-3

Nickell LG (1959). Antimicrobial activity of vascular plants. Economic Botany, 13(4):

281-318

Obied HK, Allen MS, Bedgood DR, Prenzler PD, Robards K (2005).Investigation of

Australian Olive Mill Waste for Recovery of Biophenols. J Agric Food Chem, 53: 9911-

9920

Obied HK, Bedgood DR, Prenzler PD, Robards K (2007). Bioscreening of Australian

Olive Mill Waste extracts: Biophenol contents, antioxidant, antimicrobial and molluscicidal activities. Food Chem Toxicol, 45: 1238-1248

Omar S, Lemonnier B, Jones N, Ficker C, Smith ML, Neema C, Towers GHN, Goel K and

Arnason JT (2000). Antimicrobial activity of extracts of eastern North American hardwood trees and relation to traditional medicine. J Ethnopharmacol, 73:161-170

Orhan I, Kartal M, Naz Q, Ejaz A, Yilmaz G, Kan Y, Konuklugil B, Sener B, Choudhary

I (2006). Antioxidant and anticholinesterase evaluation of selected Turkish Salvia

species. Food Chem, 103: 1247-1254

151 Paixao N, Perestrelo R, Marques JC, Camara S (2007). Relationship between antioxidant

capacity and total phenolic content of red, rose and white wines. Food Chem, 105: 204-

214

Patterson DT (1986): Allelopathy. In: Camper, ND, ed., Research Methods In Weed

Science. 3rd edition Champaign, IL: Southern Weed Science Society, pp. 125-126

Prior R L, Hoang H, Gu L, Wu X, Bacchiocca M, Howard L, Hampsch-Woodill M,

Huang D, Ou B, Jacob R (2003). Assays for hydrophilic and lipophilic antioxidant

capacity (oxygen radical absorbance capacity (ORACFL)) of plasma and other biological

and food samples. J Agric Food Chem, 51: 3273-3279.

Prior R L, Wu X, Schaich K (2005). Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem,

53: 4290-430

Sampathkumar P and Paya CV (2001). Fusarium infection after solid-organ

transplantation. Clin Infect Dis, 32:1237-1240

Santos KC, Kurtz SM, Muller D, Biavatti MW, Oliveira RM, Santos CA. (2006).

Sedative and anxiolytic effects of methanolic extract from the leaves of Passiflora actinia. Braz Arch Biol Techn, 49(4): 565-573

Sheikh C, Hossain MS, Easmin MS, Islam MS, Hossain MA, Rashid M. (2004). New

Coordination complexes of Chromium as cytotoxic and antimicrobial agents. Pakistan J

Biol Sci, 7(3): 335-339

Singleton VL, Rossi JA (1965). Colorimetry of total phenolics with phosphomolybdic- phosphotungstic acid reagents. Am J Enol Viticult. , 16:144-158

152 Souza LK, Oliveira CM, Ferri PH, Junior JG, Junior AH, Fatima OD, Fernandes L, Silva

MD (2003). Antimicrobial activity of Hyptis ovalifolia towards dermatophytes. Mem Inst

Oswaldo Cruz, 98: 963-965.

Tepe B, Eminagaoglu O, Akpulat HA, Aydin E (2007). Antioxidant potentials and rosmarinic acid levels of the methanolic extracts of Salvia verticillata (L.) subsp. verticillata and S. verticillata (L.) subsp. amasiaca (Freyn and Bornm) Bornm. Food

Chem, 100(3): 985-989.

Tian B, Hua Y. (2002). Concentration dependence of prooxidant and antioxidant effects of aloin and aloe-emodin on DNA. Food Chem, 91: 413-418

Tsao R, Romanchuk FE, Peterson CJ, Coats JR (2002). Plant growth regulatory effect and insecticidal activity of the extracts of the Tree of Heaven (Ailanthus altissima L.).

BMC Ecol 2:1

Turker AU, Camper ND (2002). Biological activity of common mullein, a medicinal plant. J Ethnopharmacol, 82: 117-125

Ullman MD, Burtis CA (2006). Chromatography In: TIETZ text book of clinical chemistry and molecular diagnostics (Burtis CA, Ashwood ER, Bruns DE eds), pp 141-

163, Elsevier India

Vartivarian SE, Anaissie EJ, and Bodey GP (1993). Emerging fungal pathogens in immunocompromised patients: classification, diagnosis, and management. Clin Infect.

Dis,17:S487-91

Venditti M, Micozzi A, Gentile G, Polonelli L, Morace G, Bianco P, Avvisati G, Papa

G, and Martino P (1988). Invasive Fusarium solani infections in patients with acute

leukemia Rev Infect Dis,10:653-660

153 Zhang HJ, Li LN (1994). Salvianolic acid 1: A new depside from Salvia cavaleriei.

Planta Med, 60: 70-72

154 Pharmaceutical Biology 2007, Vol. 45, No. 5, pp. 397–403

Biological Evaluation of Some Selected Plant Species of Pakistan

Samia Inayatullah1, Rukhsana Irum1, Ateeq-ur-Rehman1, M. Fayyaz Chaudhary2, and Bushra Mirza1

1Department of Biochemistry, Quaid-i-Azam University Islamabad, Pakistan; 2Department of Microbiology, Quaid-i-Azam University Islamabad, Pakistan

Abstract Seven methanol extracts from five different plant species species of Acer genera are used traditionally in the treat- [Salvia nubicola B. (Laminiaceae), Acer oblongifolium D. ment of cancer, polio, and dysentery (Moerman, 1998). (Aceraceae), Sorbaria tomentosa L. (Rosaceae), Hedera Hamayun et al. (2005) described Sorbaria tomentosa L. nepalensis K. (Araliaceae), and Artemisia fragrans W. (Rosaceae) as a medicinal plant. Hedera nepalensis L. (Asteraceae)] were evaluated for brine shrimp cytotoxi- (Araliaceae) is considered an antidiabetic in folk medi- city, antitumor potato disc, and radish seed phytotoxicity cine (Gilani et al., 2001). Ahmed et al. (2004) described activity. Four of the seven extracts revealed significant the ethnobotanical importance of Artemisia fragrans ED50 value ranging from 11.9 to 226.8 ppm. Inhibition W. (Asteraceae). According to their report, leaf extract of tumor formation ranged from 9 to 82.9% by all of Artemisia fragrans has anthelmintic activity and is also extracts in antitumor potato disc assay at three different used against wounds, earache, toothache, and asthma. concentrations tested (1000, 100, and 10 ppm). Growth During the study of medicinal plants, prior to frac- inhibition was observed by all extracts in radish seed tionation and structural elucidation of individual compo- bioassay at high concentration (10,000 ppm). At low con- nents of botanical extracts, it is necessary to evaluate centration (1000 ppm), three extracts from two plant spe- their biological activity. Several bench top assays, such cies (leaf and flower extract of S. nubicola, stem extract as brine shrimp cytotoxicity assay, antitumor potato disc of S. nubicola, and stem extract of H. nepalensis) pre- assay, and radish seed phytotoxicity assays, can be used sented stimulation of growth ranging from 3.5 to as major prescreening assays in this regard. 43.2%. A positive correlation was observed in the results The brine shrimp cytotoxicity assay is a rapid, inex- of three of the described assays. pensive assay requiring no special technical training. A positive correlation between brine shrimp toxicity and KB (human nasopharyngeal carcinoma) has been Keywords: Antitumor, bioassays, chemotherapeutic, found (McLaughlin & Rogers, 1998). Moreover, this cytotoxic, phytotoxicity. assay has been used successfully to biomonitor the isolation of cytotoxic (Siqueira et al., 2001), antineoplastic Introduction (Badaway & Kappe, 1997), antimalarial (Perez et al., 1997), insecticidal (Oberlies et al., 1998), and anti-feedant For this study, medicinally important plant species of (Labbe et al., 1993) compounds from plant extracts. Northern areas of Pakistan were selected on the basis Crown gall is a neoplastic disease of plants induced of traditional knowledge, i.e., they are used by local hea- by specific strains of the Gram-negative bacterium, lers for the treatment of various diseases. Agrobacterium tumefaciens. Galsky et al. (1980) demon- Salvia nubicola B. belongs to the family Laminiaceae. strated that inhibition of crown gall initiation on potato A number of Salvia species are used in folk medicine for discs showed apparent agreement with compounds and the treatment of dysentery, boils, fall injuries, hepatic plant extracts known to be active in 3PS (in vivo, mouse problems, and cancer (Fujita & Node, 1984; Zhang & leukemia) antitumor assay. Coker et al. (2002) reported Li, 1994). Acer oblongifolium L. (Aceraceae) is also a the inhibition of tumor induction by means of different plant species evaluated in the present study. Several antineoplastic drugs. They further demonstrated that

Accepted: November 5, 2006. Address correspondence to: Bushra Mirza, Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. Tel.: 092-51-9219809; E-mail: [email protected]

DOI: 10.1080/13880200701215182 # 2007 Informa Healthcare 398 S. Inayatullah et al.

A. tumefaciens-induced potato disc assay was an effective Brine shrimp cytotoxicity assay indicator of antitumor activity regardless of the mech- Brine shrimp cytotoxicity assay was performed according anism of drug action. to the standard procedure described by McLaughlin Evaluation of herbicidal or growth stimulation (1991). Three concentrations (1000, 100, and 10 ppm) properties of plant extracts requires another simple of plant extracts were used in this assay. Brine shrimp benchtop assay. Radish seed bioassay has been used larvae were hatched in a small partitioned tank in arti- previously for phytotoxic evaluation of plant extracts ficial seawater. Illumination was provided on one side (Turker & Camper, 2002). Radish seeds are easily avail- to attract newly hatched larvae. Brine shrimp larvae with able, and the assay does not require special technical second instar stage were used in this assay. training. Plant extracts of respective concentrations were added Brine shrimp lethality assay and phytotoxic evalu- to dram vials. To each dram vial, 10 brine shrimp larvae ation of botanical extracts present predictions for various were added. Negative control was prepared by evaporat- types of biological activities. Potent tumor inhibition in ing 0.5 ml of methanol in dram vials and then adding sea antitumor potato disc assay by crude botanical extracts salt solution to it. Following 24 h of incubation, survi- confirms this potential and can open new avenues toward vors were counted using a magnifying glass. The experi- the discovery of anticancer drugs. Growth stimulation or ment was repeated three times. Mortality data was inhibition in radish seed phytotoxicity assay can deter- transformed by Probit analysis in a finny computer pro- mine the herbicidal or growth stimulatory potential of gram to estimate ED value. Percentage of mortality the plant extracts tested. A positive correlation between 50 was also calculated at all concentrations. these assays can determine pharmacological importance of medicinal plants. Major objectives of the present study involve evalu- Antitumor potato disc assay ation of biological activities of crude plant extracts and Antitumor potato disc assay was performed according to determination of any possible correlation between three standard procedure described by McLaughlin and types of biological assays. Rogers (1998). A 48 h bacterial culture of At 10 strain of Agrobacterium tumefaciens was used in this experiment. Inoculum with three concentrations of test samples Materials and Methods (1000, 100, and 10 ppm) was prepared containing bac- Plant species were collected from northern areas of terial culture and autoclaved distilled water. Pakistan (Swat and Kalam districts). Identification was Red-skinned potatoes were purchased from a local carried out in the taxonomy laboratory of the Depart- market and surface sterilized by using 10% bleach sol- ment of Plant Sciences, Quaid-i-Azam University, ution. A borer of 8-mm diameter was used to bore out Islamabad, and voucher specimens were deposited here. potato cylinders. Cylinders were cut into 2-mm discs. Autoclaved agar solution (1.5%) was poured into Petri- plates and solidified. Ten discs were placed on the agar Preparation of methanol extracts surface of each plate, and 50 ml of inoculum was placed Methanol extracts were prepared by maceration pro- on the surface of each disc. The plates were sealed with cedure. Plant material was separated into different parts parafilm to avoid contamination and moisture loss. (leaves, stems, and roots), dried under shade, and, The plates were incubated at 28C in an incubator in finally, powdered. Powdered material was soaked in the dark. The experiment was carried out in strict steri- methanol for 7 days, then filtered and evaporated in a lized conditions and repeated in triplicate. After 21 days rotary evaporator (Buchi Rotavapor R-200, Switzer- of incubation, potato discs were stained with Lugol’s sol- land). Extracts were stored at 20 C. Various extracts ution (10% KI, 5% I2) and tumors were counted under a used in the present study are described in Table 1. dissecting microscope with side illumination. Tumor

Table 1. List of plant species with respective plant extracts.

Plant species Family name Methanol extracts

Salvia nubicola Laminiaceae (L þ F) S. nubicola (methanol extract of leaves and flowers of S. nubicola) (S) S.nubicola (methanol extract of stem of S. nubicola) Acer oblongifolium Aceraceae (L þ S) A. oblongifolium (methanol extract of leaves and stem of A. oblongifolium) Sorbaria tomentosa Rosaceae (L þ S) S. tomentosa (methanol extract of leaves and stem of S. tomentosa) Hedera nepalensis Araliaceae (L þ S) H. nepalensis (methanol extract of leaves and stem of H. nepalensis) Artemisia fragrans Asteraceae (L þ F) A. fragrans (methanol extract of leaves and flower of A. fragrans) (R) A. fragrans (methanol extract of roots of A. fragrans) Evaluation of selected plants of Pakistan 399 inhibition was calculated using following formula: In the second part of the experiment, two concentrations of the plant extracts (7500 and 1000 ppm) were % age of tumor inhibition=100 - ns/nc 100 used. The procedure for the second part is similar to that where ns ¼ number of tumors in sample, nc ¼ number of of the first part except for the concentrations of extracts tumors in control. and number of seeds. In the second part of the experi- More than 20% tumor inhibition is considered signifi- ment, 100 radish seeds were added to each plate. Germi- cant (Ferrigini et al., 1982). Data was statistically nated seeds were counted every day from the first to the analyzed using ANOVA. fifth day. The experiment was repeated in duplicate. Both experiments were carried out in strict sterilized conditions. Results were statistically analyzed by using ANOVA. Statistical analysis of radish seed bioassay data Antibacterial assay against Agrobacterium tumefaciens was performed only on fifth-day data. Antibacterial assay was performed according to standard agar well-diffusion method. A 24 h bacterial culture of At 10 strain of A. tumefaciens was used in this experiment. Results Bacterial culture was mixed with autoclaved nutrient Brine shrimp cytotoxicity assay agar medium, poured in a Petri plate, and solidified. Wells were prepared by using an 8-mm borer and sealed Four of the seven extracts tested exhibited ED50 less than with nutrient agar medium. A volume of 100 ml of the 1000 ppm in brine shrimp assay, indicating potent cyto- plant extract at 1000 ppm concentration was used. Roxy- toxic activity of these extracts. ED50 in these extracts ran- thromycin (1000 ppm) and cefixime (1000 ppm) were ged from 11.9 to 226.8 ppm (Table 2). used as positive control simultaneously. The plates were Results for percentage mortality of brine shrimp indi- incubated at 28C for 48 h, and results were recorded. cate that the highest percentage mortality was observed Minimum Inhibitory Concentration (MIC) was determ- at 1000 ppm by most of the extracts tested. At ined by using serial dilution method (Ansari et al., 2005). 100 ppm, only three extracts (L þ F) A. fragrans, (R) A. fragrans, and (L þ S) H. nepalensis, presented a significant mortality rate, i.e., 66.6, 60, and 56.6% respectively. Radish seed phytotoxicity assay Antitumor potato disc assay The experiment was conducted according to standard procedure described by Turker and Camper (2002). It All extracts exhibited tumor inhibition at the three con- consisted of two parts. In part one, two concentrations centrations tested. Tumor inhibition was observed in a (10,000 and 1000 ppm) of the plant extracts were pre- concentration-dependent mode. Statistical analysis using pared in methanol. Filter papers (Whatman #1) were ANOVA showed that the effect of concentration and placed in Petri plates, and 5 ml of each concentration extract was highly significant. The effect of interaction was added. Methanol was evaporated, and 5 ml of of concentration and extract factors is presented in distilled water was added. Twenty radish seeds surface Table 3 with the respective rank order obtained. Extract sterilized with 0.1% mercuric chloride were placed in of leaves and stem of A. oblongifolium presented the high- each Petri plate. The plates were sealed with parafilm est percentage of tumor inhibition at all concentrations. to avoid moisture loss and incubated at 23 2C. In control plates, 5 ml of methanol was added and evapo- Antibacterial assay against Agrobacterium tumefaciens rated. Root length was measured on the third and fifth day of incubation. The experiment was repeated in The effect of extracts on viability of A. tumefaciens was triplicate. evaluated by using the agar well-diffusion method. None

Table 2. Illustration of percentage mortality of brine shrimp at different concentrations of extracts and respective ED50 values.

Methanol extracts 1000 ppm (%) 100 ppm (%) 10 ppm (%) ED50 (ppm)

(L þ F) S. nubicola 13.3 10 10 >1000 (S) S. nubicola 10 6.6 6.6 >1000 (S) A. oblongifolium 66.70 16.70 6.70 226.8 (L þ S) S. tomentosa 13.40 14 10 >1000 (L þ S) H. nepalensis 100 56.6 23.3 47.7 (L þ F) A. fragrans 100 66.6 46.6 11.99 (R) A. fragrans 100 60 46.6 19.7 400 S. Inayatullah et al.

Table 3. Average number of tumors produced at different concentrations of extracts. Values with similar letters are not significantly different from each other at p > 0.05.

Extracts Concentrations (ppm) Average number of tumors per disc Percentage of tumor inhibition

(L þ F) S. nubicola 1000 3.4 0.8i 61.3 100 6 0.9e 31.8 10 8 0.2b 9 (S) S. nubicola 1000 3.3 0.4i 62.5 100 5.1 0.7g 42.5 10 5.7 0.5f 35.2 (L þ S) A. oblongifolium 1000 1.5 0.5k 82.9 100 3.4 0.6i 61.3 10 3.6 0.8i 59 (L þ S) S. tomentosa 1000 2.3 0.5j 73.8 100 3.7 0.6i 57.9 10 4.8 0.4h 45.5 (L þ S) H. nepalensis 1000 5.3 1.07fg 39.1 100 6.7 0.8d 22.9 10 7.1 1.1c 20.2 (L þ F) A. fragrans 1000 4.7 0.3h 46.4 100 5.4 0.2f 38.5 10 6.9 0.3c 20.4 (R) A. fragrans 1000 5.7 0.3f 34.6 100 6.6 0.4d 24.4 10 7.4 0.3c 15.7 Control 8.8 0.9a

Superscript letters ranging from a to k indicate respective Least Significant Difference rank orders of mean values. of the six extracts tested showed any significant effect on Discussion viability of A. tumefaciens. One of the seven extracts, i.e., Medicinal plant species all over the world have been (L þ S) S. tomentosa, was slightly effective against playing a vital role in drug discovery efforts. The present A. tumefaciens at 1000 ppm (zone size ¼ 10 mm, MIC ¼ 0.8 mg=ml). study involves the evaluation of crude botanical extracts for their potent cytotoxicity in brine shrimp cytotoxicity assay, for herbicidal or growth stimulation activity by Radish seed phytotoxicity assay radish seed phytotoxicity assay, and for inhibition of The effect of two different concentrations (10,000 and tumor formation in antitumor potato disc assay. 1000 ppm) of the extracts was studied on root growth inhi- Brine shrimp lethality assay has been considered as a bition or stimulation of radish seedling. All extracts inhib- prescreening assay for antimicrobial, antitumor, antima- ited root growth at 10,000 ppm. The highest percentage of larial, antifungal, and insecticidal activities. Four of the inhibition was observed by leaf and stem extract of Sor- seven extracts presented significant cytotoxicity. The baria tomentosa. In three of the extracts, root growth highest rate of lethality to brine shrimp was observed in stimulation was observed at 1000 ppm (Fig. 1). Leaf and the case of leaf and flower extract of Artemisia fragrans. stem extract of Hedera nepalensis presented the highest A number of previous studies indicated potent cytotoxi- stimulation of root length at 1000 ppm. city in the case of methanol extracts of several plant spe- In a second experiment, the effect of two different cies. Kanegusuku et al. (2001) reported cytotoxicities of concentrations of each extract (7500 and 1000 ppm) on methanol extracts and ethyl acetate fraction from Rubus seed germination was observed as a function of incu- imperialis C. (Rosaceae). In another study from Brazil, bation period of seeds. A gradual increase in seed germi- 60 medicinal plant species were evaluated for their cyto- nation for all extracts was observed until the secnod day toxicity to brine shrimp (Maria et al., 2000). Only 10% of of incubation. The effect of concentrations remained the species presented ED50 < 1000 ppm. Jacques et al. significant, and inhibition of seed germination was (2003) demonstrated the screening of 226 methanol and observed in the case of all extracts at 7500 ppm (Fig. water extracts for lethality toward larvae of brine shrimp 2). Leaf and stem extract of Sorbaria tomentosa showed and identified several cytotoxic plant species. the highest inhibition of seed germination at 7500 ppm Antitumor potato disc assay is a valuable tool that and stimulated seed germination at low concentration indicates antitumor activity of test compounds by their (1000 ppm). inhibition of formation of characteristic crown galls Evaluation of selected plants of Pakistan 401

Figure 2. Effect of methanol extracts on seed germination at (a) 7500 ppm and (b) 1000 ppm as a function of the incubation period of seeds.

Figure 1. Effect of two different concentrations [(a) (Einhellig & Rasmussen, 1978) and are a standard assay 10,000 ppm and (b) 1000 ppm] on root length. Values with simi- in allelopathic studies (Patterson, 1986). All extracts lar letters do not show significant difference; p > 0.05. exhibited toxicity in radish seed bioassay at high doses. A very interesting feature of the present study is the growth-stimulation effect of three extracts (leaf and induced in wounded potato tissues by A. tumefaciens.The flower extract of S. nubicola, leaf and stem extract of present study revealed a moderate-to-high rate of inhibition H. nepalensis, and stem extract of S. nubicola) at a low of tumor formation in the case of all methanol extracts concentration (1000 ppm). Ali et al. (2005) also revealed tested (Table 3). Leaf and stem extract of S. tomentosa pre- the growth-stimulation effect of one of the fractions (3a- sented significant inhibition of tumor formation, however, hydroxy-20-oxo-30-norlupane) of S. nubicola at 5 ppm in this extract also slightly affected the viability of the A. tume- a Lemna minor phytotoxicity assay. Similar types of faciens strain. Turker and Camper (2002) screened the bio- results were obtained by Tsao et al. (2002). They studied logical activity of common mullein by antitumor potato different fractions of Ailanthus altissima and observed a disc assay, but no effect on the viability of the A. tumefa- growth-stimulation effect at low doses and a growth ciens strain was observed. Coker et al. (2002) indicated anti- inhibitory effect at higher doses. tumor potato disc assay as an acceptable tool to primarily screen antineoplastic activity of various crude extracts as well as purified fractions regardless of the mode of inhibi- Conclusion tory action on tumor formation. Radish seeds have been used in general toxicity studies Crude botanical extracts evaluated in the present study because of their sensitivity to phytotoxic compounds can be fractionated in the future and can lead to the 402 S. Inayatullah et al. discovery of important chemotherapeutic agents. Results Ferrigini NR, Putna JE, Anderson B, Jacobsen LB, Nichols of the present study indicate a positive correlation DE, Moore DS, McLaughlin JL (1982): Modification between three assays. A correlation between brine shrimp and evaluation of the potato disc assay and antitumor lethality assay and antitumor potato disc assay has been screening of Euphorbia seeds. J Nat Prod 45: 679–686. reported previously. Four of our seven extracts (leaf and Fujita E, Node M (1984): Diterpenoids of Rabdosia species. stem extract of Acer oblongifolium, leaf and stem extract Prog Chem Nat Prod 46: 77–157. of Hedera nepalensis, leaf and flower extract of Artimisia Galsky AG, Wilsey JP, Powell RG (1980): Crown gall fragrans, and root extract of Artimisia fragrans)presented tumor disc bioassay. Plant Physiol 65: 184–185. significant ED50 value in brine shrimp lethality assay and Gilani SA, Qureshi RA, Farooq U (2001): Ethnobotanical a significant percentage of tumor inhibition in potato disc studies of Ayubia national park district Abbottabad, assay. All extracts used in the present study showed inhi- Pakistan. Online J Biol Sci 1: 284–286. bition of root length and seed germination in radish seed Hamayun M, Khan MA, Hayat T (2005): Ethanobotanical phytotoxicity assay as well. These results can lead to the profile of Utror and Gabral valleys, district Swat discovery of new anticancer drugs in the future. An inter- Pakistan. http://www.siu.edu/~ebl/leaflets/swat.htm. esting aspect of the present study is the low or no inhi- Accessed on September 30, 2006. bition of growth in radish seed bioassay at low Jacques EL, Pohlit AM, Nunomura SM, Da AC, Mustafa concentrations and antitumor activity in the case of three EV, Reis SK, Alecrim AM, Brito BR, De CS, Finney plant extracts. The combined effects of these extracts can EK, Oliveira ED, Santos KD, Pereira LC, Castro be utilized to control crown gall disease in plants. LD, Rosha LF, Andrade MM, Henrique MC, Santos MD, Souza PD, Silva SG (2003): Screening of plants in Amazon state for lethality towards brine shrimp. Acta Amaz 33: 93–104. Acknowledgments Kanegusuku M, Benassi JC, Pedrosa RC, Yunes RA, Filho VC, Maia AA, Marcia M, Monache FD, Niero R The authors are grateful to Professor Staton Gelvin, (2001): Cytotoxic, hypoglycemic activity and phyto- Department of Biological Sciences, Purdue University, chemical analysis of Rubus imperialis (Rosaceae). Z West Lafayette, IN, USA for providing A. tumefaciens Naturforsch 57c: 272–276. wild-type strains to our Laboratory for antitumor potato Labbe C, Castillo M, Connoly JD (1993): Mono and sesqui- disc assay and to the H.E.C (Higher Education Com- terpenoids from Satureja gilliesii. Phytochemistry 34: mission) of Pakistan for providing funds throughout 441–444. the research project. Maria T, Silva AF, Brandao M, Mesquita TS, Fatima ED, Junior AS, Zani CL (2000): Biological screening of Brazilian medicinal plants. Rio de Janeiro 95: 367–373. References McLaughlin JL (1991): Crown gall tumors on potato discs and brine shrimp lethality: Two single bioassays for Ahmed E, Arshad M, Ahmad M, Saeed M, Ishaque M plant screening and fraction. In: Hostetsmann K, ed., (2004): Ethnopharmacological survey of some medicin- Methods in Plant Biochemistry. London, Academic ally important plants of Galliyat areas of NWFP, Press, pp. 1–31. Pakistan. Asian J Plant Sci 3: 410–415. McLaughlin JL, Rogers LL (1998): The use of biological Ali MS, Ahmed S, Ibrahim SA, Tareen RB (2005): Charac- assays to evaluate botanicals. Drug Inf J 32: 513–524. terization and bioscreening of a new triterpenoid and a Moerman D (1998): Native American Ethnobotany. Oregon, flavanone isolated from Salvia nubicola. Chem Biodivers Timber Press, pp. 453–459. 2: 910–916. Oberlies NH, Rogers LL, Martin JM, McLaughlin JL (1998): Ansari FL, Nazir S, Noureen H, Mirza B (2005): Combina- Cytotoxic and insecticidal constituents of the unripe fruit torial synthesis and antibacterial evaluation of an of Persea americana. J Nat Prod 61: 781–785. indexed chalcone library. Chem Biodivers 2: 1656–1664. Patterson DT (1986): Allelopathy. In: Camper, ND, Badaway A-Sams, Kappe T (1997): Potential antineoplastics. ed., Research Methods in Weed Science, 3rd edition. Synthesis and cytotoxicity of certain 4-chloro-3-(2-chlor- Champaign, IL, Southern Weed Science Society, oethyl)-2-methylquinolines and related derivatives. Eur J pp. 125–126. Med Chem 32: 815–822. Perez H, Diaz F, Medina JD (1997): Chemical investigation Coker PS, Radecke J, Guy C, Camper ND (2002): Potato and in vitro antimalarial activity of Tubebuia ochracea tumor induction assay: A multiple mode of drug action ssp. neochrysantha. Int J Pharmacog 35: 227–231. assay. Phytomedicine 10: 133–138. Siqueira JM, Ziminiani MG, Resende UM (2001): Estudo fito- Einhellig FA, Rasmussen JA (1978): Synergistic inhibitory quimico das cascas do caule de Duguetia glabriuscula– effects of vanillic and p-hydroxybenzoic acids on radish Annonaceae, biomonitorado pelo enasaio de toxicidade and grain sorghum. J Chem Ecol 4: 425–436. frente a Artemia salina Leach. Quim Nova 24: 185–187. Evaluation of selected plants of Pakistan 403

Tsao R, Romanchuk FE, Peterson CJ, Coats JR (2002): Turker AU, Camper ND (2002): Biological activity of common Plant growth regulatory effect and insecticidal activity mullein, a medicinal plant. J Ethnopharmacol 82: 117–125. of the extracts of the Tree of Heaven (Ailanthus Zhang HJ, Li LN (1994): Salvianolic acid 1: A new depside altissima L.). BMC Ecol 2:1. from Salvia cavaleriei. Planta Med 60: 70–72.