Phytochemical and Pharmacological

PHYTOCHEMICAL AND PHARMACOLOGICAL

INVESTIGATIONS ON FERULA NARTHEX BOISS.

Ph.D Thesis

MAHBOOB ALAM

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR, PAKISTAN

(2013)

PHYTOCHEMICAL AND PHARMACOLOGICAL

INVESTIGATIONS ON FERULA NARTHEX BOISS.

A THESIS SUBMITTED TO THE UNIVERSITY OF PESHAWAR IN

PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE

OF DOCTOR OF PHILOSOPHY IN PHARMACY

Ph.D Thesis

MAHBOOB ALAM

DEPARTMENT OF PHARMACY

UNIVERSITY OF PESHAWAR, PAKISTAN

(2013)

DEPARTMENT OF PHARMACY UNIVERSITY OF PESHAWAR

CERTIFICATE OF APPROVAL

It is certified that this thesis entitled “ Phytochemical and Pharmacological Investigations on Ferula narthex Boiss.” submitted by Mr. Mahboob Alam is hereby approved and recommended as partial fulfillment for the award of degree of “DOCTOR OF PHILOSOPHY IN PHARMACY”.

Dr. Shumaila Bashir ______Research Supervisor Associate Professor Department of Pharmacy University of Peshawar.

Prof. Dr. Salim Ullah Khan ______Professor & Chairman, Department of Pharmacy University of Swabi, Swabi. (External Examiner)

Prof. Dr. Zafar Iqbal ______Chairman, Department of Pharmacy, University of Peshawar.

Acknowledgement

Acknowledgement In the name of Allah, who has given me strength and courage to accomplish this PhD work in the benefit of mankind. I bow my head on thanks and gratitude to Allah for his countless blessings. My first debt of gratitude must go to my supervisor, Dr. Shumaila Bashir Associate Professor. She patiently provided the vision, encouragement and advise necessary for me to proceed through the PhD program and complete my dissertation.

My deepest regards to Prof. Dr. Muhammad Iqbal Choudhary S.I.T.I and Dr. Achyut Adhikare, International Centre for Chemical and Biological Sciences (ICCBS), HEJ Research Institute of Chemistry, University of Karachi, Karachi. I would like to thanks Prof. Dr. Zafar Iqbal, Chairman Department of Pharmacy University of Peshawar and ex-Chairman, Prof. Dr. Fazal Subhan, for timely providing me all the necessary facilities and administrative support. Perhaps I would not be able to present this work in present form without co-operation of Higher Education Commission (HEC) Pakistan for funding me through Indigenous PhD fellowship programme. Thanks to all teachers, students, friends and staff members of Department of Pharmacy, University of Peshawar for sharing expertise and for providing a friendly environment. I am blessed by having a friend like Akhtar Aman who always provided support and motivation to me. His love, encouraging behavior and help were always there where things didn’t seem to work. To everybody who had contributed directly or indirectly for the completion of this study. Special thanks to my sweet brothers Mujeeb Alam and Fakhr-e-Alam for their moral support and encouragement throughout the studies. In the last I wish to thank my parents and entire family (Inam Ullah), their love provided my inspiration and was my driving force. I owe them everything and wish I could show them just how much I love and appreciate them. Mahboob Alam

Summary

This PhD project is mainly focusing on phytochemical and pharmacological screening of medicinally important Ferula narthex, belongs to Umbelliferae family.

F. narthex has a widespread ethnobotanical uses; cough, asthma, toothache, gastric problems, constipation and in angina pectoris. Gum resin is used in hysteria, treatment of habitual abortion, whooping cough and scorpion sting.

The crude methanolic extract of F. narthex (MeFn), its various fractions, isolated pure compounds along with fixed oils obtained from roots and aerial part were screened for various biological / pharmacological activities (in-vivo, in-vitro) to justify the folkloric use of this plant scientifically. Chloroform (CHCl3) and ethyl acetate (EtOAc) fractions (roots) were explored for isolation of pure chemical compounds.

The crude MeFn extract and various fractions of roots and aerial parts showed significant antibacterial activity against tested bacteria. P. aeruginosa was found more susceptible to crude MeFn extract (86%), n-hexane (73%), CHCl3 (69%), BuOH (78%) and aqueous (73%) fractions, while EtOAc fraction showed no activity against this organism. EtOAc fraction showed significant antibacterial activity against S. typhi (81%) and E. coli (80%). The crude MeFn extract and various fractions of roots and fixed oils were showed low to moderate antifungal activity. The maximum (50%) phytotoxic effect was observed for n-hexane and aqueous fractions among all test samples of roots and aerial part at 500 μg/ml dose. Among test samples, only n-hexane fraction of roots showed good insecticidal activity (60%) against tested insects.

Summary

The crude MeFn extract and various fractions of roots were screened for antioxidant activity (DPPH). The BuOH (99%) fraction showed maximum radical scavenging activity followed by EtOAc (95%) and aqueous fractions. In oxidative burst assay the crude MeFn extract (IC50 value, 46.7), n-hexane (IC50 value, 54.9), CHCl3 (IC50 value, 60.2) and EtOAc (IC50 value, 95.0) fractions showed good activity against reactive oxygen species, while BuOH and aqueous fractions including pure compound (Conferol) were found inactive.

Fnarthexol (MA-4) showed good activity (74.8%) against β-glucuronidase enzyme while Conferol (MA-1) was found inactive. The crude MeFn extract and various fractions of roots along with pure compounds were tested for anticancer activity (PC3 cell-lines). The crude MeFn extract, n-hexane and CHCl3 fractions showed good anticancer activity with IC50 values of 7.317, 5.434 and 9.613 respectively, while moderate activity by MA-4 pure compound. The EtOAc, BuOH and aqueous fraction along with MA-1 showed low anticancer activity.

The crude MeFn extract and various fractions along with pure compounds (MA-1,

MA-3, MA-4 and ET-1) showed good leishmanicidal activity against Leishmania major.

The IC50 values observed for n-hexane (6.16), chloroform fraction (11.32) and for pure compound (Conferol, 11.51) respectively. The F. narthex was screened for urease inhibitory activity. The crude MeFn extract and various fractions of roots and aerial parts along with fixed oils and pure compounds showed moderate to good urease inhibitory activity. The pure compounds; Conferol and Fnarthexol showed weak xanthine oxidase inhibitory activity, while they were found inactive against carbonic anhydrase and acetylcholinesterase enzymes.

III

Summary

The crude MeFn extract was showed the presence of good analgesic, anti- inflammatory and antipyretic effect in a dose dependent manner without any toxic effect.

The F. narthex crude MeFn extract was also screened for GIT motility. Decrease in GIT motility was observed in a dose dependent manner. The crude MeFn extract was showed no antidepressant activity.

The chloroform (CHCl3) and ethylacetate (EtOAc) fractions of F. narthex roots were subjected to column chromatography and five pure compounds were isolated. These compounds were characterized with the help of modern spectroscopic techniques. Two new and three known compounds were obtained from F. narthex. The new compounds;

Fnarthexol (MA-4) and Fnarthexone (ET-1) and known compounds were Conferol (MA-

1), Conferone (MA-3) and Umbelliferone (MA-7). The known compounds were isolated for the first time from F. narthex.

New Compounds

Fnarthexol (MA-4) Fnarthexone (ET-1)

List of Abbreviations

Abbreviation Detail

MeFn Crude methanolic extract of Ferula narthex

WHO World Health Organization CI Chemical Ionization UV Ultra violet TLC Thin Layer Chromatography GC-MS Gas chromatography-mass spectrometry CC Column chromatography GC Gas chromatography Rf Relative flow DMSO Dimethyl Sulfoxide

LD50 Lethal Dose 50 MIC Minimum inhibitory concentration CFU Colony Forming Unit

IC50 Inhibitory concentration 50 DCM Dichloromethane HMBC Heteronuclear Multiple Bond Connectivity 2D-NMR Two Dimensional Nuclear Magnetic Resonance NOESY Nuclear Overhauser Effect Correlation Spectroscopy MS Mass Spectroscopy HMQC Heteronuclear Multiple Quantum Correlation FAB Fast Atom Bombardment 13C-NMR Carbon Nuclear Magnetic Resonance 1H-NMR Proton Nuclear Magnetic Resonance 2D-NMR Two Dimensional Nuclear Magnetic Resonance IR Infrared EI-MS Electron Impact Mass Spectrum EI Electron Impact DEPT Distortionless Enhancement by Polarization Transfer BB Broad Band CI Chemical Ionization

List of Abbreviations

FD Field Desorption ppm Parts Per Million HREI-MS High Resolution Electron Ionization Mass Spectrometry

CHCl3 Chloroform EtOAc Ethyl-Acetate BuOH n- Butanol GIT Gastro-intestinal tract COX Cyclo-oxygenase RSA Radical Scavenging Activity S. aureus Staphylococcus aureus P. aeruginosa Pseudomonas aeruginosa E. coli Escherichia coli S. typhi Salmonella typhi M. canis Mycosporum canis C. albicans Candida albicans T. longifusus Trichophyton longifusus F. solani Fusarium solani A. flavus Aspergillus flavus R. dominica Rhyzopertha dominica T. castaneum Tribolium castaneum C. glabarata Candida glabarata S. oryzae Sitophilus oryzae C. analis Callosbruchus analis

Table of Contents

ACKNOWLEDGEMENT ...... I

SUMMARY...... II

LIST OF ABBREVIATIONS ...... V

1. INTRODUCTION AND LITERATURE REVIEW ...... 1

1.2. THE FAMILY UMBELLIFERAE...... 9 1.3. THE GENUS FERULA ...... 10 1.3.1. Genus Ferula reported work ...... 10 1.3.2. Ethnopharmacology of Genus Ferula ...... 23 1.3.3. Biological studies on genus Ferula ...... 25 1.4. TAXONOMICAL POSITION OF FERULA NARTHEX BOISS...... 29 1.4.1. Plant Morphology ...... 29 1.4.2. Distribution ...... 30 1.4.3. Ethnobotanical uses ...... 30 1.4.4. Reported isolated compounds of F. narthex ...... 30

1.5. AIMS AND OBJECTIVES ...... 30

1.5.1. Screening for Biological activities...... 30 1.5.2. Phytochemical studies ...... 31 1.5.2.1. Identification of different groups of compounds...... 31 1.5.2.2. Isolation of biologically active constituents / compounds...... 31 1.5.2.3. Biological testing of pure isolated compounds...... 31

2. MATERIALS AND METHODS ...... 32

2.1. DRUGS AND REAGENTS ...... 32 2.2. GENERAL EXPERIMENTAL CONDITIONS ...... 33 2.3. PHYSICAL CONSTANTS ...... 33 2.4. SPECTROSCOPY ...... 33 2.4.1. UV Spectra ...... 33 2.4.2. IR Spectra ...... 33 2.4.3. Mass Spectra ...... 33 2.4.4. Nuclear Magnetic Resonance (NMR) ...... 34 2.4.5. Gas Chromatography and Gas Chromatography-Mass Spectrometry ...... 34 2.5. PHYTOCHEMICAL TESTS ...... 35 2.5.1. Screening for Different Groups of Compounds ...... 35 2.5.2. Alkaloids ...... 35 2.5.3. Flavonoids ...... 35

VII

Table of Contents

2.5.4. Saponins ...... 35 2.5.5. Tannins ...... 36 2.5.6. Steroids ...... 36 2.5.7. Coumarins ...... 36 2.5.8. Sterols ...... 36 2.6. ISOLATION AND PURIFICATION OF COMPOUNDS ...... 37 2.6.1. Column Chromatography (CC) ...... 37 2.6.2. Thin-layer Chromatography (TLC) ...... 37 2.6.3. Spray Reagents for Visualization of Spots ...... 37 2.7. PLANT MATERIALS ...... 39 2.7.1. Collection ...... 39 2.7.2. Extraction ...... 39 2.7.3. Fractionation ...... 39 2.7.4. Isolation of compounds ...... 42 2.8. CHARACTERIZATION OF ISOLATED PURE COMPOUNDS...... 46 2.8.1. Characterization of Fnarthexol (new compound) ...... 46 2.8.2. Characterization of Fnarthexone (new compound) ...... 47 2.8.3. Characterization of Conferol ...... 48 2.8.4. Characterization of Conferone ...... 49 2.8.5. Characterization of Umbelliferone ...... 50 2.9. IN-VITRO BIOLOGICAL ACTIVITIES ...... 51 2.9.1. Antibacterial activity ...... 51 2.9.2. Antifungal Activity ...... 52 2.9.3. Phytotoxic Activity ...... 53 2.9.4. Insecticidal Activity ...... 55 2.9.5. Antioxidant assay...... 56 2.9.6. β-Glucuronidase inhibition assay ...... 57 2.9.7. Cytotoxicity assay (anticancer activity) ...... 58 2.9.8. Antileishmanial assay ...... 59 2.9.9. Urease inhibition activity ...... 60 2.9.10. Xanthine oxidase (XO) inhibition assay ...... 61 2.9.11. Carbonic anhydrase inhibition assay ...... 62 2.9.12. Acetylcholinesterase inhibition assay ...... 63 2.10. IN-VIVO BIOLOGICAL ACTIVITIES...... 64 2.10.1. Experimental animals ...... 64 2.10.2. Acute toxicity ...... 65 2.10.3. Analgesic activity ...... 66

VIII

Table of Contents

2.10.4. Anti-inflammatory activity ...... 67 2.10.5. Antipyretic activity ...... 68 2.10.6. GASTROINTESTINAL TRACT (GIT) MOTILITY ...... 69 2.10.7. Antidepressant Activity ...... 70 2.10.8. Locomotor activity ...... 71

3. RESULTS AND DISCUSSION ...... 72

3.1. PHYTOCHEMISTRY OF FERULA NARTHEX BOISS ...... 72 3.1.1. Phytochemical screening (Qualitative) ...... 72 3.2. CHEMISTRY OF ISOLATED COMPOUNDS ...... 73 3.2.1. Structure elucidation of Fnarthexol (new compound) ...... 73 3.2.2. Structure elucidation of Fnarthexone (new compound) ...... 79 3.2.3. Structure elucidation of Conferol ...... 85 3.2.5. Structure elucidation of Conferone ...... 91 3.2.6. Structure elucidation of Umbelliferone...... 94 3.3. COMPOSITION OF FIXED OILS ISOLATED FROM F. NARTHEX...... 97 3.4. IN-VITRO BIOLOGICAL ACTIVITIES ...... 102 3.4.1. Antibacterial Activity of roots ...... 102 3.4.2. Antibacterial effect of Aerial part ...... 103 3.4.3. Antifungal activity ...... 114 3.4.4. Phytotoxic activity of roots and aerial parts ...... 119 3.4.5. Insecticidal activity ...... 123 3.4.6. Antioxidant studies of crude MeFn extract and its fractions ...... 125 3.4.7. Immunomodulatory activity (Oxidative Burst Assay) of roots and pure compounds ...... 128 3.4.8. β- glucuronidase inhibitory activity of Conferol and Fnarthexol ...... 131 3.4.9. Cytotoxic assay (PC3 Cell Lines) of Roots ...... 134 3.4.10. Antileishmanial assay (in vitro) ...... 137 3.4.11. Urease inhibitory activity of crude MeFn, fractions, oils and pure compounds ...... 142 3.4.12. Urease inhibitory activity of pure compounds ...... 145 3.4.13. Xanthine oxidase activity of Conferol and Fnarthexol ...... 148 3.4.14. Carbonic Anhydrase inhibitory Activity of Fnarthexol compound ...... 150 3.4.15. Acetylcholinesterase inhibitory activity of Conferol compound ...... 152 3.5. IN-VIVO BIOLOGICAL ACTIVITIES OF CRUDE MEFN EXTRACT ...... 153 3.5.1. Acute toxicity ...... 153 3.5.2. Analgesic activity of crude MeFn extract of roots ...... 155 3.5.3. Anti-inflammatory activity of crude MeFn extract...... 159 3.5.4. Antipyretic activity of crude MeFn extract of roots ...... 163 3.5.5. Gastrointestinal tract (GIT) Motility activity of crude MeFn extract ...... 168

Table of Contents

3.5.6. Antidepressant activity of crude MeFn extract ...... 171 3.5.7. Locomotor activity of crude MeFn extract (open field test)...... 173

CONCLUSION ...... 175

REFERENCES ...... 176

Introduction

1. Introduction and Literature Review Since pre-historic era, humans are in the search of plants for the treatment of various diseases [1]. In the beginning of life human beings used plants for various purposes like shelter, food and medicines, but with the passage of time they became more dependent on plants [2-3]. Throughout the world, nearly all civilizations are more dependent on plants than animals. Knowledge of medicines among different generations was developed through discoveries and experiments on a wide variety of plants [4].

Generally it is believed that the use of medicinal plants in indigenous culture is based on traditional medicines [5]. The Indian subcontinent was the first, who reported the use of different plants for the treatment of various diseases. In between 4500 and 1600 B.C. a book “Rig Veda” was written by Aitareya Aranyaka, which was claimed to be the oldest book containing information regarding medicinal plants [6]. In 1550 B.C. the Egyptians enlightened in Ebers Papyrus the values of herbal remedies for different diseases. In

1000 B.C a book “Susruta Samhita” was written by Vagbhata, Charaka and Sushruta also contains detailed information on medicinal plants [7]. The “Charat Samhite” book was used by ancient Indians, which posses a detail knowledge of Materia Medica.

During Buddhist time a great work was done in field of herbal medicines and they also cultivated medicinal plants which were supervised by experts in field of herbal medicines [8]. The Indian Materia Medica was further strengthened due to close interaction between Indian, Romans, Persians, Greeks and Arabs. “Fongs” one of the

Chinese traditional pharmaceutical preparation was derived from plants. The traditional medicine system of China is linked with Shen Nong Ben Cao Jing (22-250 A.D) period.

A detail pharmacopeia (Ben Cao Gang Mu), containing knowledge of about 1894 herbal agents, was written by a famous naturalist and great physician, Li Shizhen. In 1956, Ben

Introduction

Cao Gang Mu book was published and still used in China for teaching as well as a reference book. Many Arab Muslims scientists also made remarkable contributions in this field. Ibn-Al-Baitar, a great botanist and pharmacist, has enlisted importance of different medicinal plants in his book “Kitab al-jami fi- al-Mufradat”. It refers to the work of some of 150 authors including 20 Greek scientists. In 1758, it was translated in Latin and was published. Al-Idrisi (1099-

1166 A.D.) mentioned in his book “Kitab al-jami lisifat ashtatal-nabata” about medicinal use of plants in six different languages [9].

Al-Tabari (883-870 A.D.) wrote a book “Firdous al-Hikma” which consists of seven parts. Specially part six deals with drugs and poisons. Ibne

Sina (980-1037), mentioned 679 herbal agents in his book “Qanun fi al-Tibb”.

Later on in West it was known as Canon. In that era it was believed as the most reliable Materia Medica. Mohammad Ibn Zakariaya Al-Razi (864-930 A.D), a great physician, chemist and philosopher, was the author of a very famous book

“Kitab-al-Mansoori” composed of ten volumes, in which Greco-Arab medicine were discussed in detail. In 15th century, it was translated in Latin. First time he used opium as an anesthetic substance[6].

In Western tradition, many authors, including well known personalities such as Diosocridies, Galen and Cilpeper in the 17th century described herbal remedies. Various herbal plants were used in different traditional system of medicines including Chinese system given in Table 1.1.

Introduction

Table 1.1: Use of herbal plants in various traditional systems of medicines [10]. S. No Chinese traditional American Ayurvedic African system traditional system traditional system system 1 Panax ginseng Peumus Elettaria Aloe vera (ren shen) boldus (boldo) cardamomum (north African (ela or origin) cardamomum) 2 Artemisia annua Psidium guajava Terminalia Artemisia afra (qing hao) (guava) species (African (myrobolan) wormwood)

3 Ephedra sinica (ma Ilex Harpagophytum Aloe ferox huang) paraguariensis procumbens (Cape aloes) (maté) (devil’s claw)

4 Paeonia lactiflora (bai Spilanthes Azadirachta Acacia senegal shao yao) acmella indica (neem) (gum Arabic) (Brazilian cress) Myroxylon 5 Angelica polymorpha Paullinia Santalum album Agathosma var. sinensis (dang gui). cupana (sandalwood) betulina (guarana) (buchu)

6 Rheum palmatum Cinchona Rauwolfia Catha edulis (da huang) pubescens serpentina (khat) (peruvian bark) (Indian snake root) 7 ------Tabebuia Centella Boswellia sacra mpetiginosa asiatica (gotu (frankincense) (lapacho) kola)

8 ------balsamum (tolu Withania Aspalanthus balsam) somnifera linearis (aswargandh (rooibos tea) 9 ------Erythroxylum Cinnamomum Commiphora coca (coca) camphora myrrha (myrrh) (camphor)

Introduction

The benzoic acid was the first pure chemical agent that was obtained from plant in 1560 [11]. Karl W. Scheele (1742-1786), was a German chemist, who isolated some simple chemicals like citric, lactic, tartaric and oxalic acid from plant and animal sources [12]. The search for useful drugs of known structures from plant kingdom however did not really begin until about 1806, when F. W. Serturner (1783-1841) separated morphine from dried exudates of opium (Papaver somniferum L.). After this in next 15 years, Caventou and

Pelletier, obtained caffeine, quinine, brucine, strychnine and cinchonine from different plant sources. Conine was the first alkaloid to have its structure established and later to be synthesized [13]. There are many agents which are obtained from plants include atropine, tubocurarine, aspirin, reserpine and morphine [14].

In 1997, Jiaxiang enlisted various species of plants, which possesses different pharmacological actions. These species of plants presented in Table 1.2.

Introduction

Table 1.2: Different plant groups and their therapeutic uses given in number [15].

S/No Name Number Angiosperm

1 a) Monocotyledones 676

b) Dicotyledones 3495

2 Gymnosperm 382

3 Thalophytes 230

4 Pteridophytes 382

5 Bryophytes 39

Total 4877

Introduction

Medicinal plants are used for different types of diseases ranging from stomachic to headache and also for different types of cuts and wounds [16]. World Health

Organization (WHO) giving more stress to encourage the use of folk medicines especially in third world countries. About 8,000 medicinal plants are used in South Asia for various ailments and 19% of these are found in Pakistan [17]. Using knowledge of traditional medicines, about 119 plant derived pharmaceuticals were launched in the market [18]. According to WHO throughout the world more than 20,000 medicinal plants are used in the traditional medicines system. It has now reached upto 70,000 throughout the world [19]. Currently more than 89 plants derived products are used in modern medicine was discovered through the knowledge of local peoples [20]. The use of herbal medicines are centuries old and it is clear from clinical results that these are as effective as currently available single agent for treating different ailments. These herbal products are enlisted in Table 1.3.

Introduction

Table 1.3: Common herbal medicines with their uses [21].

S. No. Common Botanical name Used for name

1 Garlic (Allium sativum) Blood cholesterol level reduction and lowering of heart attack risks. 2 Saw palmetto (Sarenoa serrulata), For enlarged prostate.

3 St.John’s wort Hypericum Treatment of mild to moderate perforatum depression with much fewer side effects and better patient compliance

4 Ginger (Zingber officinale) To relieve nausea and vomiting

5 Black Cohosh Actaea racemosa For menopausal symptoms

6 Ginkgo Ginkgo biloba, To improve mental performance in people with Alzheimer’s disease. 7 Horse chestnut, Aesculus For chronic venous insufficiency. hippocastanum

Introduction

In Pakistan approximately 1572 genera exist and out of which 5521 species of medicinal plants are found in hilly areas [2]. More than 80% population in the developing countries (Pakistan) preferably uses different medicinal plants for various diseases.

Folkloric herbal therapy is still used by the population of the rural areas due to its efficacy, unavailability of modern medicines and their cultural belief [5]. In Pakistan the knowledge regarding the medicinal plants is centuries old, the local communities knows about the folkloric uses of medicinal plants found in their surroundings and these information passed from generations to generations through verbal communication [22].

Introduction

1.2. The Family Umbelliferae

The family Umbelliferae comprises 275 genera and 2850 species, which are distributed throughout the world, especially in Afghanistan, Iran, India and Pakistan [23].

In 16th century when botanist started classifying plant, the Umbelliferae was the first family to be systemically studied and recognized. Most of the species belonging to this family are herbaceous, containing number of leaves and tiny flowers appeared in umbel shaped. Each flower contains five sepals and five petals. The fruits of these species are grooved and when ripe divide into two parts. Most of the plants belonging to this family are aromatic. The family Umbelliferae includes different spices and culinary herbs, like coriander, fennel, caraway and angelica, also some vegetables, like parsnip, celery and carrot [24]. Some genera of this family are also cultivated in gardens for ornamental purpose, including Astrantia, Aciphylla and Eryngium. A large number of plants from this family also have got importance to use for different purposes including, abortifacient, anticonvulsant and CNS stimulant [25]. Many species of Umbelliferae are present in Iran.

According to the report of Moscow State University Botanical Garden, about 114 genera and 363 species of Umbelliferae are present in Iran [26]. Among different genera the

Ferula consist of 170 species, Peucedanum consist of 100-120 species and Angelica 110 species [27]. This family produced different herbs, medicines, perfumes, food crops along with root crops including parsnips (Pastinica sativa) and carrots (Daucus carota).

Some other members of this family were also used for treatment of various diseases like gout, eczema and Alzheimer disease since medieval era. For commercial purpose still some plants like anise (Pimpinella anisum) and Dill (Anethum graveolens) belongs to this family are grown to obtain medicinal agents to treat ailments [28].

Introduction

1.3. The Genus Ferula

The genus Ferula is a perennial herb belongs to family Umbelliferae, consisting of 170 species. It is found in central Asia westward to Mediterranean region and in

Northern Africa, grows in dry climates [29]. These plants are perennial herbs and dicotyledonous, height ranges 1-4 m tall with hollow heavy stem poses unpleasant odor.

They produce yellow color flower in cluster pattern.

In central Asia, a large numbers of Ferula species are found; about 100 species are reported and among these 60 species are widespread. In Iran, Pakistan and in Canary

Island 20, 15 and 3 species of Ferula are exist respectively [30]. Locally Ferula is known as “Hing” in Hindi and Urdu. Some members of Ferula are famous as a rich source of oleo gum resins which is used as spice in cooking and as a medicine in different ailments in prehistoric time [24]. In India some species are used in pickles, sauces and as a flavoring agent [31]. Pectin is obtained from various species of Ferula, which is used in different food preparations [32]. Genus Ferula was investigated for phytochemical analysis, which results in the isolation of different volatile and non volatile agents like catechols, tannins, fructose, oils and glucose [33].

1.3.1. Genus Ferula reported work

Different species of genus Ferula were phytochemically investigated, which results in separation of a large number of compounds/ secondary metabolites. The results showed that it is a rich source of biological active substances like monoterpene [34-35] and sesquiterpene derivatives. Monoterpene coumarins also derived from Ferula [36].

During last twenty years Ferula was systematically studied for its chemical constituents and this screening was made possible with rapid advancement in structure elucidation

Introduction methods and also rapid improvement in purification methods. The genus were investigated for chemical moieties as early in 1935 [37].

In Ferula mainly Sesquiterpene coumarins and sulfur containing compounds are presents. From F. assafoetida, following compounds have been isolated. Sesquiterpene coumarins; gummosin, franesiferol A, tadshiferin, umbelliferone, assafoetidinol B, galbanic acid, microlobin, asacoumarin A, asacoumarin B, franesiferol B, conferol, franesiferol C, assafoetidinol A, foetidine, saradaferin, feselol, ligupersin A, epi- conferdione and polyanthinin [38-41]. From the roots of F. diversivittata; guaianolides,

Diversolides A–G, stigmasterol and diversin were isolated [42]. From F. szowitsiana; szowitsiacoumarin A (1) and szowitsiacoumarin B (2), galbanic acid and persicasulfide A were isolated [43]. From F. persica; persicaosides A–D and stigmasterol 3-O-b-glucoside are isolated compounds [44].

From F. assafoetida the isolated sulfur containing compounds include 2-butyl3-

(methylthio)-2-propenyl disulfide, 1-(methylthio)propyl 1-propenyl disulfide and 2-butyl

1-propenyl disulfide [45]. Some other polysulfide compounds including Foetisulfide (C) and Foetisulfide (A) are also identified in the oleo-gum resin [46]. Most commonly sesquiterpene coumarins are found in this genus. The previous research work on genus

Ferula presented in Table 1.4.

Introduction

Table 1.4: List of isolated compounds from genus Ferula

S # Plant name Compound name Mol. Formula Mol.wt References

1 F. assafoetida Umbelliprenin C24H30O3 366 [47]

2 F. assafoetida 5-Hydroxy- Umbelliprenin C24H30O4 382 [47]

3 F. assafoetida 8-Hydroxy- Umbelliprenin C24H30O4 382 [47]

4 F. assafoetida Tadshiferin C24H30O4 382 [47-48] F. tadshikorum

5 F. assafoetida Asacoumarin A C24H30O5 398 [49]

6 F. assafoetida 8-Acetoxy-5-Hydroxy- Umbelliprenin C26H32O6 440 [47]

7 F. tadshikorum Deacetyl-tadshikorin C24H30O5 398 [50]

8 F. tadshikorum Tadshikorin C26H32O6 440 [51]

9 F. cocanica Cocanicin C24H30O3 366 [52]

10 F. aitchinossii Karatavicinol C24H32O5 400 [34, 53] F. karatavica F. sinaica

11 F. sinaica 6,7-Dihydroxy- karatavicinol C24H34O7 434 [34]

12 F. kirialovii Reoselin C36H52O15 742 [54] F. pseudooreoselinum

13 F. karatavica Karatavicin C26H34O6 442 [55]

14 F. korshinskyi Feroside C32H44O11 604 [56]

15 F. assafoetida Farnesiferol B C24H30O4 382 [57] F. gummosa F. kopetdagensis

Introduction

F. szovitisiana

16 F. lehmanni Lehmferin C24H30O4 382 [55]

17 F. assafoetida Assafoetidin C24H30O4 382 [47]

18 F. assafoetida Farnesiferol C C24H30O4 382 [58] F. caspica F. kopetdagensis

19 F. gummosa Kopetdaghin C24H30O4 382 [55] F. kopetdagensis

20 F. kopetdagensis Fekolin C26H32O5 424 [59]

21 F. kopetdagensis Fekolone C24H28O4 380 [59]

22 F. gummosa Kopeolin C24H32O5 400 [52] F. kopetdagensis

23 F. gummosa Kopeoside C30H42O10 562 [52] F. kopetdagensis

24 F. kopetdagensis Kopeolone C24H30O5 398 [52]

25 F. krylovii Fekrol C24H32O5 400 [60]

26 F. polyantha Feropolol C24H34O6 418 [61] F. vicaria

27 F. polyantha Feropolin C26H36O7 460 [61]

28 F. polyantha Feropolone C24H32O6 416 [61] F. vicaria

29 F. Folisa Foliferin C24H34O6 418 [61] F. schtschurowskiana F. vicaria

Introduction

30 F. assafoetida Asacoumarin B C24H30O5 398 [62]

31 F. assafoetida Galbanic acid C24H30O5 398 [63] F. gummosa F. szovitsiana

32 F. microloba Methyl galbanate C25H32O5 412 [58] F. szovitsiana

33 F. aitchinossii Karatavic acid C24H28O5 396 [64] F. karatavica

34 F. krylovii Fekrynol C24H32O4 384 [65]

35 F. krylovii Fekrynolacetate C26H34O5 426 [65]

36 F. assafoetida Farnesiferol A C24H30O4 382 [49] F. linczevskii F. samarcandica F. badrakema

37 F. kokanica Polyanthin C26H32O5 424 [66] F. polyantha

38 F. mogoltavica Mogoltadone C24H28O4 380 [61, 67] F.schtschurowskiana F. vicaria

39 F. assafoetida Gummosin C24H30O4 382 [68-69] F. gummosa F.schtschurowskiana

Introduction

40 F. polyantha Polyanthinin C26H32O5 424 [66]

41 F. badrakema Badrakemin C24H30O4 382 [70-71] F. kokanica F. lehmanni F. linczevskii F. teterrima F. tuberifera

42 F. badrakema Badrakemin acetate C26H32O5 424 [71-72] F. kokanica F. linczevskii F. teterrima F. tuberifera

43 F. arrigonii Badrakemone C24H28O4 380 [73-74] F. badrakema F. kokanica F. linczevskii F. nevskii F. teterrima

44 F. arrigonii Colladonin C24H30O4 382 [75-76] F. coummunis F. linczevskii F. tingitana

45 F. arrigonii Colladin C26H32O5 424 [77]

Introduction

F. coummunis

47 F. loscossi Colladonin isovalerate C29H38O5 466 [78]

48 F. conocaula Cauferin C24H30O5 398 [79]

49 F. conocaula Cauferoside C30H40O10 560 [80]

50 F. conocaula Cauloside C36H50O15 722 [81]

51 F. conocaula Feterin C26H32O6 440 [82-83] F. iliensis F. incisoserrata F. teterrima

52 F. conocaula Cauferidin / Kauferidin C24H28O4 380 [84] F. foetidissima

53 F. lehmanni Lehmferidin C24H28O4 380 [72] F. iliensis

54 F. sumbul Sumferin C24H30O5 398 [85]

55 F. assafoetida Foetidin C24H30O4 382 [86]

56 F. concaula Feselol C24H30O4 382 [87-89] F. diversivittata F. korshinskyi F. pallida F. pseudooreoselinum F. sumbul F. tingitana

Introduction

F. iliensis F. foetidissima F. moschata

57 F. diversivittata Feselol angelate C29H36O5 464 [88]

58 F. mogoltavica Mogoltacin C24H30O4 382 [90]

59 F. polyantha Feropolidin C24H30O4 382 [61, 91] F. vicaria

60 F. assafoetida Conferol C24H30O4 382 [92-93] F. concaula F. foetidissima F. incisoserrata F. iliensis F. korshinskyi F. lipskyi F. moschata F. pallida F. persica F. sumbul F. tuberifera

61 F. badrakema Conferol acetate C26H32O5 424 [71, 84] F. incisoserrata F. tuberifera

Introduction

62 F. concaula Conferone C24H28O4 380 [94-95] F. foetidissima F. iliensis F. incisoserrata F. korshinskyi F. persica F. sumbul F. teterrima

63 F. conocaula Ferocaulin C24H28O5 396 [96]

64 F. conocaula Conferdione C24H26O5 394 [97]

65 F. conocaula Ferocaulinin/ Ferocolinin C24H28O5 396 [96]

66 F. conocaula Conferoside C30H38O10 558 [80]

67 F. conocaula Conferin C26H30O6 438 [48]

68 F. conocaula Ferocaulidin C24H28O5 396 [96]

69 F. assafoetida Ferocaulicin C26H30O6 438 [96, 98] F. conocaula

70 F. mogoltavica Mogoltavin C26H32O6 440 [99]

71 F. mogoltavica Mogoltavinin C29H36O6 480 [99]

72 F. iliensis Samarcandin C24H32O5 400 [100-101]

Introduction

F. nevskii F. persica F. samarcandica F. schtschurowskiana F. sinaica

73 F. lipskyi Samarcandin acetate C26H34O6 442 [102-103] F. persica F. pseudooreoselinum F. teterrima

74 F. samarcandica Samarcandone C24H28O5 398 [34, 100] F. sinaica

75 F. badrakema Isosamarkandin C24H32O5 400 [34, 104] F. microloba F. sinaica

76 F. arrigonii Isosamarcandin angelate C29H38O6 482 [104-105] F. diversivittata F. microloba F. pseudooreoselinum F. tingitana

77 F. assafoetida Episamarcandin C24H32O5 400 [92, 106] F. neveskii

78 F. assafoetida Acetylepisamarcandin C26H34O6 442 [107]

79 F. neveskii Nevskone C24H30O5 398 [106]

80 F. sinaica Isosamarcandin C29H38O6 482 [34]

Introduction

81 F. kokanica Feshurin C24H32O5 400 [61, 108] F. lipskyi F. schtschurowskiana

82 F. kokanica Kokanidin C26H34O6 442 [109]

83 F. kopetdagensis Ferukrin C24H32O5 400 [110-111] F. krylovii

84 F. kopetdagensis Ferukrin acetate C26H34O6 442 [110]

85 F. foetidissima Ferukrin isobutyrate C28H38O6 470 [112]

86 F. foetidissima Ferukrinone C24H30O5 398 [113-114]

87 F. kelleri Deacetylkellerin C24H32O5 400 [109] F. kokanica

88 F. kelleri Kellerin C26H34O6 442 [109] F. kokanica

89 F. pallida Fepaldin C24H32O5 400 [81]

90 F. mogoltavica Mogoltavidin C24H32O5 400 [68]

91 F. mogoltavica Mogoltavicin C26H34O6 442 [68]

92 F. conocaula Cauferinin C24H32O6 416 [80]

93 F. foliosa Kamolol C24H32O4 384 [115-116] F. iliensis F. krylovii1

Introduction

F. penninervis

94 F. foliosa Kamolone C24H30O4 382 [115, 117] F. gummosa F. iliensis F. krylovii F. penninervis

95 F. microcarpa Fecarpin C26H34O5 426 [118]

96 F. microloba Microlobin C24H30O5 398 [104]

97 F. assfoetida Kamolonol C24H30O5 398 [119]

98 F. microloba Microlobidene C24H28O4 380 [120]

99 F. sinaica p-Coumaroyllancerodiol C24H30O5 398 [121]

100 F. aitchinossii Tavicone C23H26O4 366 [122] F. karatavica

101 F. communis Ferprenin C24H28O3 364 [123-124]

102 F. communis Isoferprenin C24H28O3 364 [125]

103 F. communis Ferchromone C24H30O4 382 [35]

104 F. communis Ferchromonol C24H30O5 398 [35]

105 F. communis Fercoprenol C24H30O5 398 [35]

106 F. communis Fercoprolone C18H18O5 314 [35]

Introduction

177 F. communis Ferulenol C24H30O3 366 [126]

108 F. communis FerulenoloxyFerulenol C48H58O7 746 [127]

109 F. microloba Auraptene C19H22O3 298 [128] F. szovitisiana

110 F. diversivittata Diversinin C19H20O4 312 [129]

111 F. litwinowiana Diversin (E) C19H20O4 312 [129] F. diversivittata Diversin (Z)

112 F. diversivittata Diversoside C25H34O10 494 [130]

113 F. ferulago Ferulagol A C19H22O4 314 [131]

114 F. ferulago Ferulagol B C19H22O4 314 [131]

Introduction

1.3.2. Ethnopharmacology of Genus Ferula

The ethnopharmacological importance of Ferula is mainly due to production of oleo-gum resin, obtained from roots of different species. This oleo-gum resin is also known as Asafoetida. The major source is F. assafoetida, while several other species including F. narthex, F. alliacea, F. foetida and F. rigidula are accounts for production of asafoetida [45]. In Iran asafoetida is known with name of “Anghouzeh” while in Pakistan it is known as “Anjadana / Kama”. From centuries asafoetida is used in folkloric medicines and also as a spice. In India it is used in different foods as a flavoring agent to enhance the taste. In Nepal it is used as a diuretic, sedative and aphrodisiac agent [89,

132].

F. gummosa and F. badrakema they are used in the treatment of neurological diseases, as a tonic, as a decongestant and also used in hysteria [133]. The Ferula is also a rich source of essential oils, they are also known as Ferula oils. F. assafoetida, Ferula gummosa and F. badrakema contain essential oils. Due to presence of oils these plants possess strong aromatic smell. Ferula oils have antifungal and antibacterial activities

[134]. These oils are rich in alpha-pinene and beta-pinene [135-136].

The Table 1.5 given below represents the uses of asafoetida in traditional medicines of different countries.

Introduction

Table 1.5: Ethnobotanical uses of genus Ferula (Asafoetida) in various countries

S # Country Part used Route of administration Conditions treated Ref. 1 Afghanistan Extract of dried gum oral Ulcers, Hysteria, Whooping cough [137] 2 Brazil Extract of dried leaf oral Aphrodisiac [138] and stem As a nerve tonics 3 China Plant decoction Oral Vermifuge [139] 4 Egypt Dried gum Vagina (Topical use) As a contraceptive [86] Dried roots extract Oral Analgesic, Antispasmodic, Diuretic and Vermifuge 5 Fiji Dried resin Paste Applied on chest, Oral Whooping cough, Stomach problems [140] Extract of dried resin 6 India Dried extract Oral Abortifacient, Expectorant, Antispasmodic [141- Dried gum extract Oral Antibacterial agent 143] 7 Iran Dried gum resin Oral Antispasmodic, Anticonvulsant and Carminative [144] 8 Malaysia Gum Orally Chewed Amenorrhea [145] 9 Morocco Gum Orally Chewed Antiepileptic [146] 10 Nepal Resin extract Oral Anthelmintic, Sedative and Diuretic [41] 11 Rome Gum resin Orally used in food Culinary spice [147] 12 Saudi Arabia Dried gum Oral Whooping cough, Bronchitis & Asthma [148] 13 United States Resin extract Oral Emmenagouge, Anthelmintic, Aphrodisiac, [149] Expectorant, Antispasmodic, Nerve and Brain stimulant

Introduction

1.3.3. Biological studies on genus Ferula

The oleo-gum resin obtained from Ferula was meticulously studied scientifically.

Various pharmacological activities have been reported from Ferula. F. assafoetida has antidiabetic, anti-fertility, antifungal, antispasmodic, antiviral, antiulcerogenic, cancer chemopreventive and molluscicidal activity [150-157]. From F. gummosa, spasmolytic

[158] and anticonvulsant [159] activities have been reported. F. heuffelii Griseb possesses antimicrobial and spasmolytic activities [160]. F. hermonis is famous for its aphrodisiac effect, in the treatment of impotence [161-162] and also having insecticidal and antibacterial activities [163]. F. ovina has antispasmodic, antihistaminic and anticholinergic effects [164]. Decoction made from dried root of F. glauca has antiseptic effect [165]. The biological activities reported from Ferula are presented in Table 1.6.

Introduction

Table 1.6: Reported biological activities of genus Ferula

Activity Part used/ Dosage form Route of Adm. Ref.

Allergenic activity Dry Powder of Oleo-gum Topical [166]

resin

Antibacterial Oleo-gum resin On Agar plates [167]

Anticancer Oleo-gum resin Oral [168]

Cholesterol lowering effect Resin Oral [169]

Blood thinning effect Water extract of resin I/V [170]

Antifertility effect Dried oleo-gum resin Oral [171]

Antifungal effect Dried gum extract On Agar plates [172-173]

Essential oils (roots)

Hepatoprotective effect Dried oleo-gum resin Gastric intubation [174]

Antihyperglycemic effect Water extract of resin Gastric intubation [175]

Blood-pressure lowering Water extract of resin I/V [176] effect

Anti-inflammatory effect Ethanolic extract Oral [146]

Anti-mutagenic effect Dried oleo-gum resin On Agar plates [177]

Anti-oxidant effect Oleo-gum resin Oral [168]

Anti-parsitic effect Oleo-gum resin of roots [89]

and stem

Anti-spasmodic effect Gum extract Isolated guinea pig [178]

ileum

Anti-cancer effect Dried oleo-gum resin Gastric intubation [179]

Anti-ulcer effect Solution of Gum Oral [180]

Anti-apoptosis Sod. ferulate [181]

Introduction

Anticarcinogenic Gum Oral [182]

Cardiac depressant effect Gum tincture Perfusion [92]

Chemomodulatory effect Dried oleo-gum resin Oral [168]

CNS effect Ethanol extract Oral [183]

Cytotoxic effect Whole plant ethanol Administration to cell [184]

extract culture

Inhibition of digestive Dried oleo-gum resin Oral [185] enzymes

Inhibition of DNA synthesis Whole plant ethanol [184]

extract

Fibrinolytic effect Gum extract Oral [186]

Smooth muscle relaxing Tincture Oral [187] effect

Cancer chemopreventive Oleo-gum extract Topical [188]

(acetone)

Mulluscicidal effect Dried gum powder [189]

Antidiabetic effect Gum extract IP [190]

Antiviral effect Oleo-gum [89]

Introduction

Picture of Ferula narthex Boiss Plant.

Introduction

While going through the literature survey we have selected F. narthex belonging to family Umbelliferae. In 1872, Pierre Edmond Boissier was the first who described F. narthex.

1.4. Taxonomical position of Ferula narthex Boiss.

Kingdom: Plants- Plantae

Subkingdom: Vascular plants- Tracheobionta

Superdivision: Seed plants- Spermatophyta

Division: Flowering plants- Magnoliophyta

Class: Dicotyledons- Magnoliopsida

Subclass: Rosidae

Order: Apiales

Family: Umbelliferae / Apiaceae- Carrot family

Genus: Ferula L.

Species: Ferula narthex Boiss.

1.4.1. Plant Morphology

F. narthex plant is a perennial herb. Height of the plant range from 1.5 - 2 m.

Roots are fusiform and thick. Base of the stem is fibrous. Plant consists of large leaves, which are bipinnate with oblong segments. Leaves are distributed opposite to one another. Leaf margin ranges from entire to sinuate. Flowering season is July. Flowers are in umbels, hermaphrodite and evenly distributed on whole plant. Approximately 30 rays are present. Flower consists of 5 petals yellow in color and deciduous. Size of fruit range from 10-12 mm in length. F. narthex Boiss also consists of 1furrows and 2 vittate, which

Introduction

are large and branched. Commissure is 4 with 6 vittate, which are variable and unequal.

The plant likes to grow in moist soil and in sunny area. It also prefers well drained soil.

1.4.2. Distribution

Locally F. narthex is known as Raw in Chitral and found in various localities of

Pakistan like Gilgit, Chitral (Kamari, Damusar. Chillim, Gudai, Astore and also found at

the hill of Majini Harai) [191].

1.4.3. Ethnobotanical uses

F. narthex has widespread ethnobotanical uses such as used for cough, asthma,

toothache, gastric problems and also used in constipation, angina pectoris. Gum resin of

F. narthex is used in hysteria, treatment of habitual abortion, whooping cough and

scorpion sting [142, 191-193]. Some investigator discovered its pharmacological uses

like anticancer activity [152], antidiabetic activity [194] and anti-fertility effect [195].

1.4.4. Reported isolated compounds of F. narthex

Umbelliprenin [196], Umbelliferone [197] and Ligupersin A [46] were isolated

from the oleo gum resin of F. narthex.

1.5. Aims and Objectives

Based on folkloric use and medicinal importance of F. narthex, the following

objectives were set for the present study.

1.5.1. Screening for Biological activities

The present study will be focused mostly on the pharmacological screening of

the crude methanolic extract of F. narthex (MeFn) and various fractions. Both in-vitro

(antibacterial, antifungal, insecticidal, leishmanicidal, cytotoxic and phytotoxic activity)

Introduction and in-vivo (anti-inflammatory, analgesic, antipyretic and anti-depressant activity) pharmacological screening will be performed to provide scientific background of the folkloric use of F. narthex.

1.5.2. Phytochemical studies

1.5.2.1. Identification of different groups of compounds.

The crude methanolic extract of F. narthex (MeFn) will be tested for the presence of different groups of natural products like; flavonoids, alkaloids, saponins and tannins.

1.5.2.2. Isolation of biologically active constituents / compounds

Based on initial phytochemical and pharmacological screening the active fractions of F. narthex will be processed for the isolation pure compounds with the help of column chromatography. For structure determination the modern techniques like; 1H-

NMR, ESI/MS, 13C-NMR, NOSY, COSY and UV will be used.

1.5.2.3. Biological testing of pure isolated compounds.

Based on the fair amount of pure isolated compounds, these will be tested for various biological activities.

Materials and Methods

2. MATERIALS AND METHODS

2.1. Drugs and reagents

Different drugs and other chemicals of commercial grade were used in various experiments given in Table 2.1. In distilled water and normal saline dose of crude MeFn extract was prepared for various biological activities. Distilled water and normal saline were also served as a negative control. Different commercial grade organic solvents; n- hexane, chloroform, methanol, butanol, acetone and ethyl acetate were used.

Table 2.1: List of drugs / chemicals used

S.No. Drugs / Chemicals Source

1 Silca gel Sigma Chemical Co, St Louis, MO, USA

2 Carrageenan Sigma Chemical Co, St Louis, MO, USA

3 Diclofenac sodium Sigma Chemical Co, St Louis, MO, USA

4 Paracetamol Alfa Aesar - A Johnson Matthey Company

5 Brewer’s yeast Vahine Professional, France

6 Pentylenetetrazole Vahine Professional, France

7 Dragendorff's reagent Searle pharmaceuticals Pakistan Limited

8 Fluoxetine Osaka Pharmaceuticals Pakistan

9 Diazepam Sigma Chemical Co, St Louis, MO, USA

10 Acetic acid Sigma Chemical Co, St Louis, MO, USA

11 Ceric sulphate Merck, Darmstadt, Germany

Materials and Methods

2.2. General Experimental Conditions

Chemical, biological and instrumental studies were performed in Department of

Pharmacy, Centre of Biotechnology and Microbiology (COBAM), University of

Peshawar and International Centre for Chemical and Biological Sciences (ICCBS),

University of Karachi.

2.3. Physical Constants

With the help of Buchi 535 melting apparatus, the melting points of compounds were determined. For the determination of optical rotation the digital Polari meter of

JASCO DIP-360 model was used.

2.4. Spectroscopy

2.4.1. UV Spectra

The UV spectra of compounds were determined with the help of Hitachi

Spectrophotometer model U-3200 (fully automated).

2.4.2. IR Spectra

The IR spectra were taken on potassium bromide (KBr) disc, with the help of

Infrared Spectrometer, JASCO 302-A model.

2.4.3. Mass Spectra

For the determination of low-resolution electron impact spectra of the compounds, the mass spectrophotometer was used (model MAT311A linked with computer system of PDP11/34).

Materials and Methods

For the determination of High resolution (HR) mass and Fast atom Bombardment mass (FAB Positive and FAB Negative), the Jeol mass spectrometer model JMS HX 110 was used.

2.4.4. Nuclear Magnetic Resonance (NMR)

Bruker AM-300, AM-400 and AMX-500 nuclear magnetic resonance spectrometer was used for the determination of 1H-NMR spectra of compounds. The spectra were taken at 300, 400, or 500 MHz, for internal reference TMS was used.

Distortionless Enhancement by Polarization Transfer (DEPT) experiments were

o o performed at 90 and 135 for CH, CH2 and CH3 moieties. The Quaternary carbons were analyzed with 13C-NMR.

2.4.5. Gas Chromatography and Gas Chromatography-Mass Spectrometry

With the help of Varian Mat 312 and Jeol JMS -600H linked with GC and Jeol

JMS HX 110 mass spectrometer the qualitative and quantitative GC and GC-MS spectra were taken for oils.

Materials and Methods

2.5. Phytochemical Tests

2.5.1. Screening for Different Groups of Compounds

The crude methanolic extract of Ferula narthex (MeFn) was screened for different classes of compounds according to the procedures of Ayoola [198]. All these different qualitative analysis are given below:

2.5.2. Alkaloids

Plant material (2.5g) was extracted with methanol. 5 ml of hydrochloric acid

(HCl, 2N) was added and heated (40oC) in water bath. The mixture was cooled, filtered and divided into two parts. Wagner’s reagent was added to one part and Mayer’s reagent was added to another part and was checked for the presence of precipitation or turbidity.

Slight turbidity represented by (+), complete turbidity represented by (++), while (+++) represented precipitation in the sample. Formation of reddish brown precipitation after treatment with Draggendorff’s reagent represents presence of alkaloids.

2.5.3. Flavonoids

The plant material (1g) was dissolved in methanol (5ml) for determination of flavonoids. To this mixture Magnesium (5g) and concentrated HCl (few drops) were also added. Appearance of pink color showed the presence of flavonoids in the sample.

2.5.4. Saponins

Plant material was extracted with boiling water in a test tube. The mixture was cooled at room temperature and shake thoroughly until froth was formed. The test tube was placed in a stand for 15 min and following results were observed:

Materials and Methods

Negative (-) represent no froth, one plus sign (+) represent froth less than 1 cm,

(++) represent more than 2 cm froth and strongly positive represented by (+++).

2.5.5. Tannins

Plant material (0.5g) was taken in a test tube along with hot distilled water (10ml).

It was filtered and 2ml of gelatin solution (1%) was added to it. Tannins forms precipitate upon addition of gelatin solution, which confirms the presence of tannins. It was further confirmed with addition of ferric chloride (FeCl3, 2ml) solution to test tube, which forms precipitation in test tube.

2.5.6. Steroids

For screening of steroids 0.25 g of methanolic extract was taken in a test tube and

1 ml of acetic anhydride was added to it. Change of color to green or blue shows the presence of steroidal compounds in sample.

2.5.7. Coumarins

For determination of coumarins, plant crude extract was taken in a test tube and it was boiled. On top of test tube a piece of filter paper previously moistened with NaOH was kept. Under UV light the yellow color on filter paper indicates presence of coumarins.

2.5.8. Sterols

For screening of sterols, CHCl3 and crude MeFn extract was mixed together.

Concentrated H2SO4 (3ml) was added to this mixture and mixed. It was kept undisturbed for few mins. The appearance of red color in the lower layer confirmed the presence of sterol.

Materials and Methods

2.6. Isolation and Purification of Compounds

From different fractions of F. narthex various pure compounds were isolated with the help of various chromatographic techniques.

2.6.1. Column Chromatography (CC)

Column chromatography was carried out using silica gel as a stationary phase.

Silica gel of 70-230 mesh size and flash silica gel of 230-400 mesh size (E-Merck) was used. Different mobile phases were used including n-hexane, chloroform, ethyl acetate, dichloromethane and methanol for column chromatography.

2.6.2. Thin-layer Chromatography (TLC)

For thin layer chromatography precoated silica gel cards (PF 254, 0.25mm,

Merck) were used. Precoated preparative silica gel plates (0.5mm thickness, 20 x 20cm) were used as preparative TLC for purification of compounds.

2.6.3. Spray Reagents for Visualization of Spots

Different chemical reagents; ceric sulphate, vanillin, Dragendorff’s and iodine solution were used for locating and visualization the spots on TLC plates. These chemical reagents were sprayed with the help of a spray gun on TLC plate. The spots on TLC plate were visualized with the help of UV light at short (254nm) and long (365nm) wavelength.

Vanillin solution

To prepare vanillin solution, 1 g of vanillin was dissolved in 50% aqueous phosphoric acid. The presence of terpenes and steroids were confirmed by spraying

Materials and Methods vanillin solution and heating (100-110 oC) the TLC. The appearance of pink or deep purple color indicates the presence of terpenes and steroids.

Ceric sulphate solution

For the preparation of ceric sulphate solution, ceric sulphate was dissolved in 65%

H2SO4. This solution was used to visualize the oxidizable constituents in plant materials on TLC plate. After spraying the reagent on TLC plate and upon heating the appearance of pink color confirms the presence of terpenoids, while the appearance of blackish or light yellow color without heating represents the presence of alkaloids.

Dragendorff’s solution

For the preparation of Dragendorff’s solution 8 g of potassium iodide (KI) was dissolved in distilled water (20 ml). Bismith nitrate (0.85 g) was dissolved separately in

20% of acetic acid (A.A) and distilled water. These were mixed together to prepare stock solution. 5 ml from stock solution was diluted with acetic acid (10 ml) and distilled water

(90 ml). The appearance of blackish brown or light pink color indicates the presence of alkaloids.

Iodine solution

In TLC tank few crystals of iodine were placed and for few min it was warmed (40-

50oC). Upon exposure of TLC plate to tank spots will appear.

Materials and Methods

2.7. Plant Materials

2.7.1. Collection

The F. narthex (roots and aerial part) was collected from Chitral Gol National

Park, Chitral, Khyber Pakhtunkhwa, Pakistan, in June-July 2010 and was identified by

Professor Dr. Abdurasheed, Plant Taxonomist, Department of Botany, University of

Peshawar. A specimen with voucher number BOT.20002 (PUP) was submitted in the herbarium.

2.7.2. Extraction

The collected plant materials were shade dried at room temperature. The dried roots of plant were processed with electric grinder for powder formation. The powder material (12kg) was soaked in commercial grade methanol (25L) for 15 days at room temperature with occasional shaking. After 15 days, with colorless white thin cloth it was filtered and the methanol soluble residue obtained was concentrated with rotary evaporator at 40 oC and in this way we obtained 900 g of crude methanolic extract

(MeFn). About 100 g of crude MeFn extract was reserved for various biological activities

(in-vivo and in-vitro).

2.7.3. Fractionation

800 g of crude MeFn extract of F. narthex was suspended in 400 ml distilled water and partitioned with n-hexane (3 x 400ml), CHCl3 (3 x 400ml), EtOAc (3 x 400ml) and BuOH (3 x 400ml) to obtain; n-hexane (100g), CHCl3 (210g), EtOAc (150g), BuOH

(100g) and aqueous (190g) fractions [Scheme 1].

As stated above, the aerial part was also processed in same way to obtain the

Materials and Methods crude MeFn extract and various fractions [Scheme 2].

Roots powder 12 kg

Extraction

Methanolic Extract (900 g)

For actvities (100 g) Fractionation

n-hexane Fraction EtOAc Fraction H2O Fraction (100 g) (150 g) (190 g)

BuOH fraction CHCl3 Fraction (210 g) (100 g)

Scheme 1: Extraction and Fractionation of F. narthex roots.

Materials and Methods

Powdered Leaves (4 Kg)

Extraction

Biological activities Methanolic Extract 50 g (450g)

Fractionation

n-hexane Fraction EtOAc Fraction H2O Fraction (75 g) (100 g) (100 g)

CHCl3Fraction (125 g)

Scheme 2: Extraction and Fractionation of F. narthex aerial part.

Materials and Methods

2.7.4. Isolation of compounds

Isolation of compounds from CHCl3 fraction of roots

The CHCl3 fraction was selected for isolation of compounds. Slurry was prepared with silica gel and was subjected to column chromatography. Using n-hexane and EtOAc solvent system in increasing order of polarity, it was further fractionated into fifteen sub fractions (A-O) [Scheme 3].

Conferol pure compound was obtained from sub fraction (D) using n-hexane /

EtoAc (6.5:3.5) solvent system. Conferone was isolated from sub fraction (C) eluting column with n-hexane / EtoAc (7.5:2.5) solvent system. Fnarthexol (new compound) was obtained from sub fraction (D) subjected to n-hexane / acetone (0.9:9.1) solvent system.

Umbelliferone was isolated from sub fraction (D).

Materials and Methods

Chloroform fraction (260 g)

EtOAc/n-hexane EtOAc/n-hexane 2.5:7.5 (C) 3.5:6.5 (D)

hexane

hexane hexane -

n n

0.5:9.5 (iii) 0.5:9.5 0.9:9.1 (v) 0.9:9.1

Conferol (vii) 2.0:8.0

Acetone/ Acetone/ Acetone/ 1.5 g

Conferone Fnarthexol Umbelliferone (75 mg) (75 mg) (75 mg)

Scheme 3: Isolation of pure compounds from CHCl3 fraction of roots

Materials and Methods

Isolation of compounds from EtOAc fraction of roots

The EtOAc fraction was also processed as CHCl3 fraction. 10 sub fractions were obtained and sub fraction (6) was selected for isolation [Scheme 4]. Fnarthexone (new compound) was obtained from sub fraction (D) eluting with n-hexane / EtOAc solvent system (8.5:1.5).

Materials and Methods

EtOAc fraction (150 g)

EtOAc/n-hexane

Sub fractions (1-10)

EtOAc/n-hexane 1.5:8.5 (6)

Fnarthexone (30 mg)

Scheme 4: Isolation of Fnarthexone from EtOAc fraction of roots

Materials and Methods

2.8. Characterization of isolated pure compounds

2.8.1. Characterization of Fnarthexol (new compound)

The sub fraction (D) obtained from CHCl3 fraction was re-chromatographed and using n-hexane / acetone (0.9:9.1) solvent system Fnarthexone was obtained in amorphous form.

Table 2.2: Characterization of Fnarthexol

S # Parameters Observations 1 Physical state White amorphous form

2 Molecular formula C24H30O4 26 3 [] D -29.8 (c 0.2, CHCl3) 4 UV activity On TLC UV active 5 Rf. value 0.58 (n-hexane/acetone) 6 Yield 75 mg 7 Solubility at room temperature Methanol, Acetone

8 UV max (MeOH) 325 nm (3.6) and 243 nm (2.4) 9 IR spectrum, cm-1 hydroxyl (3534 cm−1), aromatic (1613 cm−1)

1 10 H-NMR (CDCl3: 400 MHz) (See Table 3.2) 13 11 C-NMR (CDCl3:400 MHz) (See Table 3.2)

12 HR ESI-MS (m/z) 383.2222 (C24H30O4+H)

Materials and Methods

2.8.2. Characterization of Fnarthexone (new compound)

The EtOAc sub fraction (6) was subjected to column chromatography using silica gel. The Fnarthexone was isolated with n-hexane / EtOAc (8.5:1.5) solvent system.

Table 2.3: Characterization of Fnarthexone

S # Parameters Observations 1 Physical state White amorphous form

2 Molecular formula C24H26O4 26 3 [] D -27.0 (c 0.2, CHCl3) 4 UV activity On TLC UV active 5 Rf. value 0.54 (n-hexane/EtoAc) 6 Yield 30 mg 7 Solubility at room temperature Methanol, Acetone

8 UV max (MeOH) 324 nm (3.8) and 246 nm (2.3) 9 IR spectrum, cm-1 ketone (1711 cm−1), aromatic (1618 cm−1) 1 10 H-NMR (CDCl3: 400 MHz) (See Table 3.3) 13 11 C-NMR (CDCl3:400 MHz) (See Table 3.3)

12 HR ESI-MS (m/z) 379.1909 (C24H26O4+H)

Materials and Methods

2.8.3. Characterization of Conferol

Conferol was obtained from sub fraction (D) of CHCl3 fraction. With the help of column chromatography, using n-hexane / EtOAc (6.5:3.5) solvent system the Conferol was obtained in white crystalline form.

Table 2.4: Characterization of Conferol

S # Parameters Observations 1 Physical state White crystalline form

2 Molecular formula C24H30O4 3 UV activity On TLC UV active 4 Rf. value 0.58 (n-hexane/EtoAc) 5 Yield 1.5 g 6 Solubility at room temperature Methanol, Acetone

7 UV max (MeOH) 326 and 246 nm 8 IR spectrum, cm-1 hydroxyl (3534 cm−1), aromatic (1613 cm−1)

1 9 H-NMR (CDCl3: 400 MHz) (See Table 3.4 ) 13 10 C-NMR (CDCl3:400 MHz) (See Table 3.4 )

11 HR ESI-MS (m/z) 383.2110 (C24H30O4+H)

Materials and Methods

2.8.4. Characterization of Conferone

Conferone was obtained from sub fraction (C) of CHCl3 fraction. Sub fraction C was eluted with acetone / n-hexane solvent system (0.5:9.5).

Table 2.5: Characterization of Conferone

S # Parameters Observations 1 Physical state White amorphous powder

2 Molecular formula C24H28O4 3 UV activity On TLC UV active 4 Rf. value 0.56 (acetone/n-hexane) 5 Yield 75 mg 6 Solubility at room temperature Methanol, Acetone

7 UV max (MeOH) 326 and 246 nm 8 IR spectrum, cm-1 ketone (1711 cm−1), aromatic (1618 cm−1) 1 9 H-NMR (CDCl3: 400 MHz) (See Table 3.5) 13 10 C-NMR (CDCl3:400 MHz) (See Table 3.5)

11 HR ESI-MS (m/z) 381.1011 (C24H28O4+H)

Materials and Methods

2.8.5. Characterization of Umbelliferone

CHCl3 sub fraction D was loaded to column and eluted with acetone / n-hexane solvent system (2.0:8.0), Umbelliferone was isolated in white powder form.

Table 2.6: Characterization of Umbelliferone

S # Parameters Observations 1 Physical state White powder form

2 Molecular formula C9H6O3 3 UV activity On TLC UV active 4 Rf. value 0.56 (acetone/n-hexane) 5 Yield 75 mg 6 Solubility at room temperature Methanol, Acetone 1 7 H-NMR (CDCl3: 400 MHz) (See Table 3.6) 13 8 C-NMR (CDCl3:400 MHz) (See Table 3.6) 9 Mass (m/z) 162.2

Materials and Methods

2.9. In-vitro biological activities

Crude MeFn extract and various fractions of F. narthex were screened for different in-vitro biological activities. The following activities were performed.

2.9.1. Antibacterial activity

The crude MeFn extract of roots, aerial parts and various fractions were screened against Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus,

Salmonella typhi, Klebsella pneumoniae, Streptococcus pneumonia and Escherichia coli available at Centre of Biotechnology and Microbiology, University of Peshawar, using agar well diffusion method [199-200]. From nutrient broth 18 hrs old bacterial culture was taken and spread on sterile agar plates to prepare a bacterial lawn. Metallic cork borer (6 mm) was used for formation of wells in agar plates. In Dimethyl Sulfoxide

(DMSO) stock solution of test samples was prepared at a concentration of 3 mg/ml. 100

µl from each stock solution was transferred to their respective well in agar plate and were incubated for 24 hrs at 37 oC.

Amoxicillin was used as positive control, while DMSO as negative control. Zone of inhibition was measured for standard as well as for crude MeFn extract of roots, aerial parts and its fractions. Antibacterial activity was determined by zone of inhibition of test samples and it was compared with standard drug. Percent (%) antibacterial activity was calculated by comparison with positive control

Formula:

Materials and Methods

2.9.2. Antifungal Activity

Antifungal activity of crude MeFn extract of roots, aerial part, various fractions and fixed oils were analyzed using agar tube dilution method [201-202]. Stock solution of test sample was prepared in DMSO at a concentration of 24 mg/ml. The fungal strains were refreshed using Sabouraud Dextrose Agar (SDA). For preparation of slants about 5 ml of SDA medium was taken in each test tube and autoclaved. Then 66.6 µl of sample from stock solution was transferred to test tube and also a 7 days old fungal culture was introduced. The test tubes were incubated for 7 days (25±1oC). After 7 days, the linear growth of the test fungi was noted and percent inhibition was determined by comparison with positive control. Amphotericin-B was used as a standard drug for Candida albicans while for other fungi Miconazole was used [203-204] .

Percent growth inhibition was determined with formula given below:

Materials and Methods

2.9.3. Phytotoxic Activity

The crude MeFn extract of roots, aerial parts and various fractions of F. narthex was screened for phytotoxic activity using Lemna bioassay protocol [201, 205]. E- medium was prepared by dissolving different ingredients (Table 2.7) and was autoclaved for 15 min at 121oC. The pH (5.5-6.5) of the medium was adjusted with potassium hydroxide (KOH). Stock solution of test sample was prepared in methanol at concentration of 20 mg/ml. From stock solution 500, 50 and 5 µg/ml was taken in flasks and was kept for the evaporation of solvent. In each flask 20 ml of the E-medium and ten healthy Lemna minor plants (three fronds/ plant) were transferred. The flask with only medium and L. minor served as negative control while the flask with standard (Paraquat) served as positive control. In growth chamber all these flasks were kept for seven days

(27oC±1oC). After seven days the phytotoxic activity of test samples was determined by recording number of dead fronds in each flask.

The percent growth regulation was determined with the formula given below:

Materials and Methods

Table 2.7: Composition of E-medium for Phytotoxic activity [204].

S. No. Constituents Formula Concentration

(mg/ml)

1 Potassium nitrate KNO3 1515

2 Manganous chloride MnCl2.4H2O 3.62

3 Ferric chloride FeCl3.4H2O 5.40

4 Sodium molybdate Na2MoO4.2H2O 0.12

5 Magnesium sulphate MgSo4.7H2O 492

6 Calcium nitrate Ca(NO3)2.4H2O 1180

7 Zinc sulphate ZnSO4.5H2O 0.22

8 Potassium dihydrogenphosphate KH2PO4 680

9 Ethylene diaminetetraacetic acid EDTA 11.20

10 Boric acid H3BO3 2.86

11 Copper sulphate CuSO4.5H2O 0.22

Materials and Methods

2.9.4. Insecticidal Activity

Crude extract of root, aerial part and various fractions of F. narthex were screened for insecticidal activity against Callosbruchus analis, Tribolium castaneum and

Rhyzopertha dominica. Stock solution was prepared by dissolving 200 mg of test sample in 3 ml of acetone. A 90 mm filter paper was placed in petri dishes and loaded with test sample (1019.10μg/cm2). It was left for 24 hrs to evaporate the volatile organic solvent.

Next day 10 active insects were transferred to each petri plate and incubated for 24 hrs

(27oC±1oC). Permethrin (239.50μg/cm2) was used as positive control and acetone as negative control. Percent mortality was calculated by comparison of result (test sample) with positive control [205-209].

Percentage mortality was determined by using below formula:

Materials and Methods

2.9.5. Antioxidant assay

The isolated compounds of F.narthex were screened for its antioxidant potential according to the reported procedure of Shaheen et al; 2005 (DPPH radical scavenging assay). Analytical grade chemicals (Sigma, USA) were used in this assay. Test samples were dissolved in DMSO and the reaction mixture was prepared by taking 5 µl of test samples and DPPH 95 µl in ethanol. It was then transferred into 96-well micro plate and incubated for 30 min (37oC). With multiple readers spectrophotometer (Spectra-Max

3400) the absorbance (515nm) of test samples was recorded. Ascorbic acid was served as a positive control. The radical scavenging activity (percent) of test samples was determined by comparing it with positive control.

RSA (%) = 100 (Optical Density test well /Optical Density control) ×100

Materials and Methods

2.9.6. β-Glucuronidase inhibition assay

Isolated compounds were screened for inhibition of β-Glucuronidase enzyme. The inhibitory effect was noted by spectrophotometric method. For the activity of enzyme, as a substrate the p-nitrophenyl-β-D-glucuronide was used. Formation of p-nitrophenol from substrate was confirmed by absorbance at 405 nm. A total of 250 µl of substrate was used in this reaction and added to 96-well micro plate [210]. Composition of reaction mixture consists of acetate buffer (0.1m, 185µl), enzyme solution (10µl), and test sample (10 µl) all these were dissolved in DMSO. The reaction mixture was incubated for 30 min at 37 oC. To reaction mixture, p-nitrophenyl-β-D-glucuronide (0.4mM, 50µl) was added and for 30 min change in absorbance was continuously measured at 405 nm with Spectra Max spectrophotometer.

With the below given formula the percent inhibition was calculated;

% inhibition = 100 – (O.D. of S/ O.D. of B) x 100

Where S is the activity of enzyme with test sample and B is the activity of enzyme without test sample.

Materials and Methods

2.9.7. Cytotoxicity assay (anticancer activity)

The pure compounds were screened for cytotoxic activity using MTT standard colorimetric protocol with 96–well micro plates (flat-bottom). In Modified Dulbecco’s eagle’s medium, Prostate Cancer Cell (PC-3) was cultured, with addition of serum (fetal bovine 5 %), Penicillin (100 IU/ml) and Streptomycin (200 µg/ml) in three flasks. At 37 o C these flasks were incubated in 5 % CO2 incubator. Then growing cells (exponentially) were harvested by diluting with a specific medium and counted with the help of haemocytometer. At a concentration of 1 x 105 cells/ml cell culture was prepared and transferred to micro plate (96-well), each well containing 100 µl of culture. The plate was incubated overnight and medium was replaced by freshly prepared medium (200 µl) for various concentration of compounds (1-100 µM).

After 72 hrs, to each well 2 µg/ml was added, which was further incubated for 4 hrs followed by addition of DMSO (100 µl). ELISA micro plate reader (Molecular

Devices, Spectra Max Plus, USA) was used for calculation of MMT reduction with respect to formazan within the cell; at 570 nm absorbance was measured. At a concentration causing 50 % growth inhibition the cytotoxicity was recorded.

Materials and Methods

2.9.8. Antileishmanial assay

The crude MeFn extract, various fractions and pure compounds were tested for leishmanicidal effect using Leishmania major. Promastigotes of Leishmania were grown in NNN biphasic medium which was early modified and for this purpose normal physiological saline was used. RPMI medium (1640 Sigma) was used for culturing parasite. The medium was also supplemented with inactivated foetal bovine serum (FBS,

10 %). At log phase of growth the L. major parasites were harvested and centrifuged at

3000 rpm for 10 min. With normal saline these were washed thrice, maintaining same experimental conditions. The parasites final density (106 cells/ml) was achieved with the addition of freshly prepared culture medium. About 180 µl medium and test sample (20

µl) was added to each well of 96-well micro plate and serially diluted. The prepared

Leishmania culture (100 µl) was added to all wells. One row served as negative control containing only media and leishmania culture, while another row was supplemented with positive control; Pantamidine (ICN) and amphotericin B (Fluka). After incubation for 72 hrs (22oC) microscopically the numbers of living parasites were counted using neubauer chamber. The IC50 value was determined by Ezfit 5.03 software [211].

Materials and Methods

2.9.9. Urease inhibition activity

The crude MeFn and its subsequent fractions along with fixed oils and pure compounds were screened for urease inhibitory activity using the reported procedure of

Weatherburn [212]. The reaction solution consists of urease enzyme (Jack bean 25 µl) and buffer solution (55 µl) and urea (100 mM). The reaction mixture along with each test sample (5 µl, 1mM) was incubated in 96-well plate for 15 min at 30oC. The indophenol’s procedure was used for the measurement of ammonia produced during reaction. To each well 5 µl of 1 % w/v phenol reagent, sodium nitroprusside (0.005 % w/v) and also alkali reagent (70 µl) was added. After 50 min of incubation with the help of micro plate reader the increasing absorbance was measured at 630 nm. The Soft-Max Pro software was used for recording of change (per min) in absorbance. The pH was kept at 8.2 with the help of

LiCl2 (0.01 M), K2HPO4.3H2O (0.01M) and Ethylene diamine tetra acetic acid (EDTA,

1.0mM). The experiment was performed in triplicate. The standard drug was Thiourea.

The percent inhibition was determined by formula given below.

inhibition 100 (Optical Density test well /Optical Density control) ×100

Materials and Methods

2.9.10. Xanthine oxidase (XO) inhibition assay

The pure compounds were tested for XO inhibitory activity by spectrophotometric method according to the reported protocol of Filha et al. with slight modification. The compounds were dissolved in 1 % methanol-phosphate buffer, keeping final concentration of 100 µg/ml. The reaction mixture contain 100 µl of test samples, 300 µl of phosphate buffer (0.2M, pH 9) and 100 µl of enzyme solution prepared in phosphate buffer (0.2 U/ml). At room temperature the reaction mixture was incubated for 2 min.

Then XO solution (500 µl) prepared in phosphate buffer at a concentration of 0.15 mM was added to reaction mixture, to start the reaction [213]. The absorbance (205 nm) was noted for 2 min at room temperature using spectrophotometer. In this assay Allopurinol

(100 µg/ml) was used as a positive control. The percent XO inhibition was calculated with formula given below.

% inhibition of XO = ((A –B) x 100) / A

Where A represent change in absorbance without test sample, while B represent change in the absorbance of assay with test sample.

Materials and Methods

2.9.11. Carbonic anhydrase inhibition assay

Pure compounds isolated from F. narthex were also screened for carbonic anhydrase inhibition assay. In this assay upon hydrolysis there is production of colorless acetate (4-nitrophenylacetate) and yellow color 4-nitrophenol was measured [214]. The reaction was carried out in buffer solution at 25-28 oC. Buffer solution composed of Tris-

HEPES at a concentration of 20mM (pH 7.2-7.9). In each sample tube solution 140µl of

Tris-HEPES and 20µl aqueous solution of purified erythrocytes (bovine) was added. In

DMSO (10 %) the pure compounds and 4-nitrophenylacetate substrate (20µl, 0.6-0.8mM) were dissolved and diluted in ethanol. Using 96- well micro plate the reaction was carried out. With the help of spectrophotometer (Spectra Max 340) at 400 nm the reaction product was observed for 30 min keeping 1 min interval.

Materials and Methods

2.9.12. Acetylcholinesterase inhibition assay

The pure isolated compounds of F.narthex were screened for acetyl choline esterase (AChE) inhibitory potential. The assay was carried out according to the procedure of Lopez et al. with slight modification [215]. The reaction mixture contain 50 mM Tris-Hcl with pH 8.0, (200 µl), BSA buffer (1%), test sample (100µl) keeping final concentration at 100 µg/mL. Before the addition of acetylthiocholine iodide (ATCI) substrate (100µl, 15mM), 5,5 V- dithiobis [2-nitrobenzoic acid] 500 µl (DTNB 3 mM) and incubated for 2 min at room temperature. The yellow color was measured after 4 min at 405 nm. Galantamine (final conc. 100 µg /ml) was used as positive control. The AChE percent inhibition was calculated by below given formula;

% AChE inhibition = ((A-B) x100) / A

Where A represent change in absorbance without test sample, while B represent change in the absorbance with test sample.

Materials and Methods

2.10. In-vivo biological activities

For the screening of various in-vivo biological activities, the crude MeFn extract obtained from roots of F. narthex was used.

2.10.1. Experimental animals

During pharmacological experiments the animals were used include two different strains of mice, one was BALB/C and second was NMRI of either sex. BALB/C mice were obtained from Department of Pharmacy, University of Peshawar, while the NMRI mice were obtained from the Hussain Ebrahim Jamali institute (HEJ), University of

Karachi, Pakistan. To keep these animals healthy, the guidelines given by institute of laboratory animal resources, Commission on life sciences, National Research Council were followed throughout the experiments [216].

Materials and Methods

2.10.2. Acute toxicity

The acute toxicity was determined by using crude MeFn extract of roots at various doses ranging from 500 mg/kg (body weight of mice) upto 2.5 gm/kg. The mice were distributed equally in different groups and administered with different doses of plant crude MeFn extract. Negative control group was treated with distilled water at a dose of

10 ml/kg. After treatment of the animals with test doses, they were kept under observation for 24 hrs. For the first 4 hrs the animals were observed for acute toxicity effect. The percent death was calculated after 24 hrs [216].

Materials and Methods

2.10.3. Analgesic activity

The below protocol was used to know about antinoceciptive / analgesic effect of crude MeFn extract.

2.10.3.1. Acetic acid induced writhing

The crude MeFn extract was screened for the presence of analgesic effect. For this purpose BALB/C mice of either sex 18-22g body weights were selected. These animals were divided into five different groups (n=6). Group I and II were served as negative and positive control groups respectively. Normal saline at a dose of 10 ml/kg (Body weight) was administered to Group I, while Diclofenac sodium at a dose of 10 mg/kg (Body weight) was administered to Group II. They were fed according to recommended guidelines, but food supply was stopped 2 hrs before the start of activity [216]. The crude

MeFn extract was administered to the remaining groups, III, IV and V at a dose of 50,

100 and 200 mg/kg (Body weight). After 30 min of the above mentioned treatments 1% acetic acid was administered to all groups through intra-peritoneal route. Then after 5 min of acetic acid injection abdominal writhes (constrictions) were started to count for next

10 min. The analgesic effect (percent) was calculated according to the formula given below.

Materials and Methods

2.10.4. Anti-inflammatory activity

Crude MeFn extract was evaluated for the presence of anti-inflammatory effect.

For this carrageenan induced edema protocol was used.

2.10.4.1. Carrageenan induced paw edema

The crude MeFn extract was screened for the presence of anti-inflammatory effect. For this purpose BALB/C mice of both sex 25-30g body weights were selected.

These animals were divided into five different groups. Each group of animals were consists of 6 mice (n=6). Group I and II were served as negative and positive control respectively. Normal saline at a dose of 10 ml/kg (Body weight) was administered to

Group I, while Diclofenac sodium at a dose of 10 mg/kg (Body weight) was administered to group II. The crude MeFn extract was administered to the remaining groups, III, IV and V at a dose of 50, 100 and 150 mg/kg (Body weight) respectively. After 30 min of the above mentioned treatments 1% Carrageenan was injected in the right hind paw subplanter tissue of each mouse. With the help of Plethysmometer (LE 7500 plan lab S.L) the anti-inflammatory effect was measured for 5 hrs (0, 1, 2, 3, 4 and 5 hrs) after the injection of Carrageenan [217]. With the help of formula given below the percent inhibition of edema was calculated.

Where A represents edema volume of negative control, while B as paw edema of tested group.

Materials and Methods

2.10.5. Antipyretic activity

2.10.5.1. Pyrexia induction with Brewer’s yeast

For screening of antipyretic effect of crude MeFn extract NMRI mice (30–35g) of either sex were used. They were kept on fasting for 12 hrs providing water only. These animals were divided into five different groups (n=6). Group I and II were served as negative and positive control respectively. Normal saline at a dose of 10 ml/kg (Body weight) was administered to group I, while Paracetamol at a dose of 150 mg/kg (Body weight) was administered to group II respectively. The crude MeFn extract was administered to the remaining groups, III, IV and V at a dose of 50, 100 and 200 mg/kg

(Body weight) respectively. With digital thermometer normal temperature of each animal was recorded. For induction of pyrexia aqueous suspension of Brewer’s (15%) was subcutaneously administered at a dose of 10 ml/kg to each mouse. Rise in body temperature for each mouse was recorded with digital thermometer after 24 hrs. Those animals were selected for experiment which showed at least 0.5 oC increase in body temperature, while all other animals which showed less than 0.5 oC increase in temperature were excluded [218]. Above stated doses were injected through i.p route to all groups. Regularly at 1, 2, 3, 4 and 5th hrs the rectal temperature of each mouse was recorded for all groups after administration of crude extract and drug. The percent reduction in body temperature was calculated according to formula given below.

Where A represent normal body temperature; B is body temperature after 24 hrs and C is body temperature at 1,2,3,4 and 5 hrs of treatment.

Materials and Methods

2.10.6. Gastrointestinal tract (GIT) Motility

2.10.6.1. Charcoal meal protocol

The crude MeFn extract was tested for its effect on GIT motility. BALB/C mice

25-30 g were selected. These animals were divided into five different groups [219].

Group I and II served as negative and positive control respectively. Normal saline at a dose of 10 ml/kg was administered to group I, while castor oil (0.1 ml/kg) as a standard drug was administered to group II. The crude MeFn extract was administered to the remaining groups, III, IV and V at a dose of 50, 100 and 200 mg/kg through i.p route.

After 15 min of the above mentioned treatments, charcoal suspension (aqueous) at a dose of 0.3 ml, p.o. was administered to each mouse. Then animals were killed by cervical dislocation after 30 min of charcoal treatment. Through dissection the small intestine was removed and movement of charcoal in small intestine was noted by calculating percent

G.I.T motility with formula given below.

Percent Motility = 100 - Distance covered / total length of intestine × 100.

Materials and Methods

2.10.7. Antidepressant Activity

2.10.7.1. Using forced swimming test (FST) model

Forced swimming test (FST) was used for evaluation of antidepressant activity of crude extract of F. narthex. A water tub (bath) of 42 x 19 x 19 cm dimension was used for this purpose. All animals were divided into five different groups. Each group of animals consisted of 6 mice (n=6). The group I and II served as negative and positive control respectively. Normal saline at a dose of 10 ml/kg was administered to Group I, while fluoxetine at a dose of 15 mg/kg was administered to group II through i.p route

[220]. The crude extract was administered to the remaining groups, III, IV and V at a dose of 50, 100 and 150 mg/kg. One day before performing the experiment, all animals were trained in the water tub for swimming. The temperature of the water was maintained at 25 ± 2 oC. According to recommended protocol animals were properly handled and recommended laboratory environment (sound proof and red dim light) was maintained.

All groups were treated with above stated doses of crude extract and drug. After 30 min each mouse was exposed to water tub for swimming and allowed to swim for 360 s while immobility time was noted during last 240 s.

Materials and Methods

2.10.8. Locomotor activity

The locomotor activity was assessed using a wooden apparatus of 150 cm in length which was divided in 19 equal squares. Mice were divided in five groups, each group consists of 6 mice (n=6). The group I and II served as negative and positive control respectively [221]. The negative control (group I) treated with normal saline (10 ml/kg) and positive control (group II) was injected diazepam at a dose of 0.5 mg/kg i.p. the remaining groups of mice were administered the crude MeFn at a dose of 50, 100 and

200 mg/kg through i.p. route. After 30 min of administration of extract, standard drug and normal saline, the number of lines crossing for each mouse was noted for 10 min by placing the animal in the center of apparatus.

Results and Discussion

3. RESULTS AND DISCUSSION

3.1. Phytochemistry of Ferula narthex Boiss

3.1.1. Phytochemical screening (Qualitative)

The crude MeFn extract was screened for the presence of various phytochemical constituents as presented in Table 3.1. The crude MeFn extract gives positive results for alkaloids, carbohydrates, diterpenoids, coumarins, proteins, flavenoids, steroids, saponins, sterols and terpenes while negative result for anthraquinines.

Table 3.1: Qualitative Phytochemical screening of crude MeFn extract

S.No. Phytochemical Test Result

1 Carbohydrates Positive

2 Alkaloids Positive

3 Proteins Positive

4 Diterpenoids Positive

5 Flavonoids Positive

6 Steroids Positive

7 Anthraquinones Negative

8 Coumarins Positive

9 Terpenes and Sterols Positive

10 Tannins Positive

11 Proteins Positive

Results and Discussion

3.2. Chemistry of isolated compounds

3.2.1. Structure elucidation of Fnarthexol (new compound)

Compound Fnarthexol was isolated as white amorphous from chloroform fraction of methanolic extract of Ferula narthex Boiss. Its molecular formula C24H30O4, was established by the help of 13C-NMR and HR ESIMS, which showed pseudo molecular

+ ion peak [M+H] at 383.2260 (Calc. for C24H30O4 +H = 383.2222). EI MS Spectrum showed [M]+, at m/z 382 and fragments peaks at m/z 220, 203, 147, 119 and 55. Its IR spectrum exhibited absorption bands for the hydroxyl (3534 cm−1) and aromatic (1613 cm−1) functionalities. UV absorptions at 324 and 246 nm indicated the presence of coumarin [36].

Compound Fnarthexol was also determined to be a sesquiterpene coumarin by the presence of diagnostic peaks in the 1H-and 13C- NMR spectra (Table 3.2). The 1H- and

13 C- NMR spectra of Fnarthexone displayed signals due to five aromatic methines at δH/

δC [{6.21 (d, J = 9.6 Hz)/113.0, CH-3}, {6.80 (br. d, J = 8.4 Hz)/113.1, CH-6}, {6.78 (br. s)/101.3, CH-8}, {7.33 (br d, J = 8.4 Hz)/128.7, CH-5} and {7.61 (d, J = 9.3 Hz)/144.4,

CH-4}], and four quaternary carbons at δC 112.5/C-10, 155.9/C-9, 161.2/C-2 and

161.9/C-7, typical of an umbelliferone moiety. For the sesquiterpene portion, signals corresponding to tan olefinic methines at δH/δC [{5.54 (br s)/132.6, CH-6′], an oxygenated methine at δH/δC {3.26 (dd, J = 8.5 and 3.6 Hz), whereas oxygenated methylene at δH/δC {3.99 (d, J = 9.0 Hz), 4.14 (d, J = 9.0 Hz)/67.0, CH2- 11′}, and four methyl groups at δH/δC [ {0.87 s/15.2, CH3-15′}, {0.89 s/14.8, CH3-13′},{0.99 s/28.0,

CH3-14′}, {1.67 s/21.6, CH3-12′] clearly indicated the presence of sesquiterpene. The structure of the compound was further confirmed by using 2D NMR spectra such as

Results and Discussion

COSY, HSQC, HMBC and NOESY. Absence of methyl group at C-10′, was inferred by

COSY correlations between H-5′ (δ1.25) and H-10′ (δ 2.20) as well as H-10′ (δ 2.20) and

H2-1′(δ 2.02, 1.68) which was further supported by HMBC spectrum. Presence of Me-

15′ at C-9′ was inferred by HMBC correlations of Me-15′ (δ 0.87) to C-9′ (δ 35.8) and C-

11′ (δ 67.0), HMBC correlations of Me-12′ (δ 1.67) to C-9′ (δ 35.8) further supported the same. Resonance of H2-11′{3.99 (d, J = 9.0 Hz), 4.14 (d, J = 9.0 Hz)}, instead of double doublet in case of Fnarthexone compound, supported that C-9′ is an quaternary carbon, which gives clear evidence for the above supposition of substitution of Me-15′ at C-9′. A further HMBC correlation between the singlet methyl at δ 1.67 (Me-12′) and the carbon resonances at δ 132.8 (C-8′), 123.7 (C-7′), and 35.8 (C-9′) revealed that the double bond is in C-7′/C-8′ which was further confirmed from the COSY spectrum, which showed a correlation between the proton at δ 1.25 (H-5′) and the proton at δ 1.66, 2.01 (H2-6′), which, in turn, was coupled to the olefinic proton at δ 5.44 (H-7′).

Stereochemistry in compound Fnarthexol was determined by using coupling constant value and NOESY spectrum, H-3′ ( 3.26 dd, J = 8.5, 3.6 Hz), was pseudo axially oriented with the help of its coupling constant value. NOESY correlations of  3.26 (H-

3′) with δ 1.25 (H-5′) and  0.87 (H3-15′) with δ 1.68 (Hax-1′) confirmed the β-orientation of H-5′ and Me-15′, whereas α-orientation of H-10′ was determined from NOESY correlation between δ 2.20 (H-10′) and δ 3.99/4.14 (H2-11′). Key NOESY correlations in compound Fnarthexol is shown in Figure 3.2.

Results and Discussion

Figure 3.1: Structure of Fnarthexol

Results and Discussion

Table: 3.2: 13C- and 1H-NMR Chemical shift values of Fnarthexol

Fnarthexol (new compound)

C. No. C H (J, Hz)

2 161.2 - 3 113.0 6.21 d (9.3) 4 143.4 7.61 d (9.3) 5 128.7 7.33 br d (8.4) 6 113.1 6.80 br d (8.4) 7 161.9 - 8 101.3 6.78 br S 9 155.9 - 10 112.5 - 1’ 23.3 2.02, 1.68 m 2’ 27.2 1.69. 0.98 m 3’ 78.8 3.26 dd (8.5, 3.6) 4’ 38.7 - 5’ 49.3 1.25 m 6’ 37.8 2.01, 1.66 m 7’ 123.7 5.54 br s 8’ 132.2 - 9’ 35.8 - 10’ 53.7 2.20 br s 11’ 67.0 3.99 d (9.0), 4.14 d (9.0) 12’ 21.6 1.67 s 13’ 14.8 0.89 s 14’ 28.0 0.99 s 15’ 15.2 0.87

Results and Discussion

Figure 3.2: HMBC Interaction of compound Fnarthexol

Results and Discussion

Figure 3.3: Key COSY and HMBC correlations in compound Fnarthexol

Results and Discussion

3.2.2. Structure elucidation of Fnarthexone (new compound)

Compound Fnarthexone was obtained as an amorphous, white solid, and its

13 molecular formula, C24H26O4, was established by the help of C-NMR and HR ESIMS,

+ which showed pseudo molecular ion peak [M+H] at 379.1899 (Calc. for C24H26O4 +H =

379.1909). EI MS Spectrum showed [M]+, at m/z 378 and fragments peaks at m/z 217,

203, 175, 119 and 105. Its IR spectrum exhibited absorption bands at 1711 cm−1 (ketone) and 1618 cm−1 (aromatic moiety). UV absorptions at 324 and 246 nm indicated the presence of coumarin [36].

Compound Fnarthexone was determined to be a sesquiterpene coumarin by the presence of diagnostic peaks in the 1H-and 13C- NMR spectra (Table 3.3). The 1H- and

13C- NMR spectra of Fnarthexone displayed signals due to five aromatic methines at

δH/δC [{6.21 (d, J = 9.6 Hz)/113.6, CH-3}, {6.96 (dd, J = 6.0, 2.3 Hz)/113.7, CH-6},

{7.01 (br. s)/102.0, CH-8}, {7.58 (d, J = 8.4 Hz)/130.1, CH-5} and {7.90 (d, J = 9.6

Hz)/144.5, CH-4}], and four quaternary carbons at δC 113.5/C-10, 156.9/C-9, 162.7/C-2 and 162.8/C-7, typical of an umbelliferone moiety. For the sesquiterpene portion, signals corresponding to two olefinic methines at δH/δC [{6.34 (dd, J = 10.2, 3.0 Hz)/132.6, CH-

6′ and {5.77 (d, J = 10.2 Hz)/128.4, CH-6′}], an olefinic methylene at δH/δC {5.04 (br s),

5.08 (br s)/113.1, CH2-12′ } , an oxygenated methylene at δH/δC {4.38 (dd, J = 10.2, 7.2

Hz), 4.88 (dd, J = 10.2, 3.6 Hz)/67.4, CH2- 12′}, and three tertiary methyl groups at δH/δC

[ {1.02 s/14.8, CH3-15′}, {1.07 s/21.7, CH3-13′},{1.14 s/26.9, CH3-14′}] were evident.

HMBC correlations between the protons signal at δ 5.06/5.08 (CH2-12′) and the carbon resonance at δ 52.2 (C-9′) and between the protons at δ 4.38/ 4.88 (CH2-11′) with the same carbon C-9′ revealed the location of a terminal methylene group (CH2-12′) at C-8′.

Results and Discussion

A further HMBC correlation between the singlet methyl at δ 1.02 (Me-15′) and the carbon resonances at δ 34.8 (C-1′), 37.9 (C-10′), and 52.2 (C-9′) revealed that the singlet methyl is substituted at C-10′. The remaining methyl groups were determined to be at C-

4′ from the HMBC correlations between the proton signals at δ 1.07 (Me-13′) and 1.14

(Me-14′) and the carbon resonances at δ 47.5 (C-4′), 54.9 (C-5′), and 214.5 (C-3′). The locations of the olefinic protons at C-6′ and C-7′ were deduced from the COSY spectrum, which showed a correlation between the proton at δ 2.69 (H-5′) and the proton at δ 6.34

(H-6′), which, in turn, was coupled to the olefinic proton at δ 5.77 (H-7′).

Stereochemistry in compound Fnarthexone was determined by using NOESY spectrum, NOESY correlations between δ 1.07 (H3-14′) and δ 1.07 (H3-15′) indicated the

β-orientation of Me-15, whereas α-orientation of H-5′ was confirmed from NOESY correlation between δ 2.69 (H-5′) and δ 2.42 (Hax-1′). Key NOESY correlations in compound Fnarthexone is shown in Figure 3.5.

Results and Discussion

Figure 3.4: Structure of Fnarthexone

Results and Discussion

Figure 3.5: HMBC Interaction of compound Fnarthexone

Results and Discussion

Figure 3.6: Key COSY and HMBC correlations in compound Fnarthexone

Results and Discussion

Table: 3.3: 13C- and 1H-NMR Chemical shift values of Fnarthexone

Fnarthexone (new compound) 13 C. No. C-NMR (C) H (J, Hz)

2 162.7 - 3 113.6 6.21 d (9.6) 4 144.5 7.90 d (9.6) 5 130.1 7.58 d (8.4) 6 113.7 6.96 dd (8.4, 2.4) 7 162.8 - 8 102.0 7.01 br s 9 156.9 - 10 113.5 - 1’ 34.8 2.42, 2.72 m 2’ 36.5 2.18, 1.94 m 3’ 214.5 - 4’ 47.5 - 5’ 54.9 2.69 br s 6’ 132.6 6.34 dd (10.2, 3.0) 7’ 128.4 5.77 br d (10.2) 8’ 144.4 - 9’ 52.2 2.63 br m 10’ 37.9 - 11’ 67.4 4.38 dd (10.2,7.2) 4.88 dd (10.2, 3.6) 12’ 113.1 5.06 br s, 5.08 br s 13’ 26.9 1.14 s 14’ 21.7 1.07 s 15’ 14.8 1.02 s

Results and Discussion

3.2.3. Structure elucidation of Conferol

Conferol was obtained in the form of white crystals, and its molecular

13 formula,C24H30O4 was establish by the help of C NMR and HR-ESI-MS, which showed

+ pseudo molecular ion peaks [M+H] at m/z 383.2 [Calculated for C24H30O4]. Its IR spectrum exhibited absorption bands for the hydroxyl (3534 cm−1) and aromatic (1613 cm−1) functionalities. UV absorption at 326 and 246 nm indicated the presence of coumarin [36].

Conferol was determined to be like sesquiterpene coumarin by the presence of diagnostic peaks in the 1H and 13C NMR spectra (Table 3.4). The 1H and 13C NMR spectra of Conferol displayed signals due to five aromatic methines at δH/δC [{6.83 (d, J

= 9.4 Hz)/113.10, CH-3}, {7.32 (d, J = 9.0 Hz)/143.34, CH-4}, {7.62 (d, J = 6.0

Hz)/128.65, CH-5}, {6.82 (d, J = 9.0 Hz)/112.93, CH-6}, {6.23 (s)/101.37, CH-8}, and four quaternary carbons in the coumarin moiety at δC 161.15/C-2, δC 112.44/C-10, δC

155.92/C-9 and δC 162.08/C-7, typically of an umbelliferone moiety presented in Table

3.4.

While for sesquiterpene portion of the compound showed 8 signals of protons, corresponding to four olefinic methines at δH/δC [{1.66 (d, J = 6.4 Hz) and 1.67 (d, J =

8.0 Hz)/25.14, CH-1′}, {1.60 (d, J = 6.4 Hz) and 1.61 (d, J = 8.0 Hz)/31.79, CH-2′},

{1.93 (d, J = 8.0 Hz) and 1.94 (d, J = 8.0 Hz)/23.19, CH-6′}, {4.00 (d, J = 9.0 Hz) and

4.10 (d, J = 8.0 Hz)/67.08, CH-11′}, and {3.51 (s)/77.26, CH-3′}, {1.73 (s)/43.37, CH-5′},

{5.52 (d, J = 6.0 Hz)/123.79, CH-7′} and {2.31 (d, J = 6.0 Hz)/53.54, CH-9′}, whereas, four tertiary methyl groups at δH/δC [ {0.96 (s)/23.19 CH-14′}, {0.95 (s)/22.30 CH-13′},

{1.02 (s)/21.66 CH-12′}, {0.97 (s)/14.76 CH-15′}, and three quaternary carbons in the

Results and Discussion sesquiterpene moiety at δC 37.17/C-4′, δC 132.46/C-8′ and δC 35.62/C-10′].The spectroscopic data was compared with reported spectroscopic data of Conferol.

Results and Discussion

Table 3.4: 1H and 13C-NMR spectra Conferol

C. No Multiplicity 13C-NMR 1H (J = Hz) (DEPT) 1 O ------2 C=O 161.15 ------3 CH 113.10 6.83 (d, J = 9.4 Hz) 4 CH 143.34 7.32 (d, J = 9.0 Hz) 5 CH 128.65 7.62 (d, J = 6.0 Hz) 6 CH 112.93 6.82 (d, J = 9.0 Hz) 7 -C- 162.08 ------8 CH 101.37 6.23 (s) 9 -C- 155.92 ------10 -C- 112.44 ------1′ CH2 25.14 1.66 (d, J = 6.4 Hz) and 1.67 (d, J = 8.0 Hz) 2′ CH2 31.79 1.60 (d, J = 6.4 Hz) and 1.61 (d, J = 8.0 Hz) 3′ CH 77.26 3.51 (s) 4′ -C- 37.17 ------5′ CH 43.37 1.73 (s) 6′ CH2 23.19 1.93 (d, J = 8.0 Hz) and 1.94 (d, J = 8.0 Hz) 7′ CH 123.79 5.52 (d, J = 6.0 Hz) 8′ -C- 132.46 ------9′ CH 53.54 2.31 (d, J = 6.0 Hz) 10′ -C- 35.62 ------11′ CH2 67.08 4.00 (d, J = 9.0 Hz) and 4.10 (d, J = 8.0 Hz) 12′ CH3 21.66 1.02 (s)/ 13′ CH3 22.30 0.95 (s) 14′ CH3 23.19 0.96 (s) 15′ CH3 14.76 0.97 (s)

Results and Discussion

Figure 3.7: Structure of Conferol

Results and Discussion

3.2.4. X-ray crystallographic study Conferol

A colorless crystal of 0.50 x 0.12 x 0.08 mm was mounted on Bruker SMART

Apex II diffractometer at 273 (2) K. Cu K radiations of 0.7 Å with a graphite monochromator were used to collect the diffraction pattern. Conferol was crystallized in orthorhombic system with P212121 space group. The cell dimensions were a = 6.1621(7)

Å, b = 18.3914 (19) Å, c = 18.621(2) Å, = 90o,  = 90o,  = 90o and the volume was found to be 2110.3(4) Å3. The final cell parameters were determined from full matrix least square refinement of 12098/3720 reflections, with R = 0.0423. Full-matrix least- square method on F2 was used to refinement of the data. Crystallographic data (CCDC

825584) can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html.

Results and Discussion

Figure 3.8: Crystallography of Conferol

Results and Discussion

3.2.5. Structure elucidation of Conferone

Conferone was obtained in the form of white solid powder, and its molecular

13 formula, C24H28O4 was establish by the help of C NMR and HR-ESI-MS, which showed pseudo molecular ion peaks [M+H]+ at m/z 381.1. Its IR spectrum exhibited absorption bands at 1711 cm−1 (ketone) and 1618 cm−1 (aromatic moiety). UV absorption at 326 and 246 nm indicated the presence of coumarin [36].

Conferone was determined to be like sesquiterpene coumarin by the presence of diagnostic peaks in the 1H and 13C NMR spectra (Table 3.5). The 1H and 13C NMR spectra of Conferone displayed signals due to five aromatic methines at δH/δC [{6.83 (d,

J = 9.4 Hz)/113.22, CH-3}, {7.32 (d, J = 9.0 Hz)/143.32, CH-4}, {7.62 (d, J = 6.0

Hz)/128.77, CH-5}, {6.82 (d, J = 9.0 Hz)/113.05, CH-6}, {6.23 (s)/101.34, CH-8}, and four quaternary carbons in the coumarin moiety at δC 161.10/C-2, δC 112.65/C-10, δC

155.94/C-9 and δC 216.04/C-7, typically of an umbelliferone moiety given in Table 3.5.

While for sesquiterpene portion of the compound showed 7 signals of protons, corresponding to four olefinic methines at δH/δC [{1.66 (d, J = 6.4 Hz) and 1.67 (d, J =

8.0 Hz)/25.25, CH-1′}, {1.60 (d, J = 6.4 Hz) and 1.61 (d, J = 8.0 Hz)/35.81, CH-2′},

{1.93 (d, J = 8.0 Hz) and 1.94 (d, J = 8.0 Hz)/23.88, CH-6′}, {4.00 (d, J = 9.0 Hz) and

4.10 (d, J = 8.0 Hz)/66.68, CH-11′}, {1.73 (s)/51.13, CH-5′}, {5.52 (d, J = 6.0

Hz)/123.66, CH-7′} and {2.31 (d, J = 6.0 Hz)/53.06, CH-9′}, whereas, four tertiary methyl groups at δH/δC [ {0.96 (s)/23.88 CH-14′}, {0.95 (s)/22.30 CH-13′}, {1.02

(s)/21.54 CH-12′}, {0.97 (s)/14.50 CH-15′}, and four quaternary carbons in the sesquiterpene moiety at δC 161.79/C-3′, 47.48/C-4′, δC 132.33/C-8′ and δC 38.44/C-10′]

The spectroscopic data was compared with reported spectroscopic data of Conferone.

Results and Discussion

Table 3.5: 1H and 13C-NMR spectra of Conferone

C. No Multiplicity 13C-NMR 1H (J = Hz) (DEPT) 1 O ------2 C=O 161.10 ------3 CH 113.22 6.83 (d, J = 9.4 Hz) 4 CH 143.32 7.32 (d, J = 9.0 Hz) 5 CH 128.77 7.62 (d, J = 6.0 Hz) 6 CH 113.05 6.82 (d, J = 9.0 Hz) 7 -C- 216.04 ------8 CH 101.34 6.23 (s) 9 -C- 155.94 ------10 -C- 112.65 ------1′ CH2 25.25 1.66 (d, J = 6.4 Hz) and 1.67 (d, J = 8.0 Hz) 2′ CH2 35.81 1.60 (d, J = 6.4 Hz) and 1.61 (d, J = 8.0 Hz) 3′ C=O 161.79 ------4′ -C- 47.48 ------5′ CH 51.13 1.73 (s) 6′ CH2 23.88 1.93 (d, J = 8.0 Hz) and 1.94 (d, J = 8.0 Hz) 7′ CH 123.66 5.52 (d, J = 6.0 Hz) 8′ -C- 132.33 ------9′ CH 53.06 2.31 (d, J = 6.0 Hz) 10′ -C- 38.44 ------11′ CH2 66.68 4.00 (d, J = 9.0 Hz) and 4.10 (d, J = 8.0 Hz) 12′ CH3 21.54 1.02 (s)/ 13′ CH3 22.30 0.95 (s) 14′ CH3 23.88 0.96 (s) 15′ CH3 14.50 0.97 (s)

Results and Discussion

Figure 3.9: Structure of Conferone

Results and Discussion

3.2.6. Structure elucidation of Umbelliferone

Umbelliferone was obtained as an white powder, and its molecular formula, C9H6O3 was establish by the help of 13C NMR and HR-ESI-MS, which showed pseudo molecular

+ ion peaks [M+H] at m/z 163.2 [Calculated for C9H6O3]. The IR spectrum indicated the presence of an amide carbonyl (1660 cm-1), and benzene ring (1625 and 1391 cm-1). UV absorption at 326 and 246 nm indicated the presence of coumarin [36].

Umbelliferone was determined to be like coumarin by the presence of diagnostic peaks in the 1H and 13C NMR spectra (Table 3.6). The 1H and 13C NMR spectra of

Umbelliferone displayed signals due to five aromatic methines at δH/δC [{6.07 (d, J =

9.0 Hz)/113.39, CH-3}, {7.79 (d, J = 9.4 Hz)/143.50, CH-4}, {7.57 (d, J = 6.0

Hz)/128.95, CH-5}, {6.70 (d, J = 9.0 Hz)/111.60, CH-6}, {6.67 (s)/102.60, CH-8}, and four quaternary carbons in the coumarin moiety at δC 162.37/C-2, δC 155.60/C-9, δC

111.37/C-10 and δC 161.22/C-7, typically of an umbelliferone moiety.

The 2D HMBC and COSY disclosed many of the correlations and the elucidated structure from these data was.

Results and Discussion

Figure 3.10: Structure of Umbelliferone

Results and Discussion

Table 3.6: 1H and 13C-NMR spectra of Umbelliferone

C. No Multiplicity 13C-NMR 1H (J = Hz) (DEPT) 1 O ------

2 C=O 162.37 ------

3 CH 113.39 6.07 (d, J = 9.0 Hz)

4 CH 143.50 7.79 (d, J = 9.4 Hz)

5 CH 128.95 7.57 (d, J = 6.0 Hz)

6 CH 111.60 6.70 (d, J = 9.0 Hz)

7 -C- 161.22 ------

8 CH 102.60 6.67 (s)

9 -C- 155.60 ------

10 -C- 111.37 ------

Results and Discussion

3.3. Composition of Fixed oils isolated from F. narthex

The fixed oils isolated from F. narthex through column chromatography were identified with the help of GC-MS instrument. In 21_B sample total of fourteen components were identified presented in Table 3.7. In ET-17 fraction twenty eight components were identified as given in Table 3.8.

Results and Discussion

Table 3.7: Composition of 21_B fixed oils

S.No. Chemical name Formula Mass m/z Base Peak RT Ions

1 Oxirane,diethylboryloxymethyl- C7H15BO2 142.1 83.3 83.3 3.927 26

2 2-t-Butylthio-1,2dimethylthiovinyl C8H15ClS 178.1 178.3 178.3 16.243 29

chloride

3 1,2-dihydrocyclobutabenzene C8H8 104.1 104.3 104.3 25.505 31

4 Hexadecanoic acid, methyl ester (CAS) C17H34O2 270.3 74.3 74.3 25.754 35

Methyl palmitate Uniphat A60

5 n-Hexadecanoic acid Hexadecanoic acid C16H32O2 256.2 73.3 73.3 26.529 70

n-Hexadecoic acid Palmitic acid

6 9-Octadecenoic acid (Z)-, methyl ester C19H36O2 296.3 55.4 55.4 29.586 91

(CAS) Methyl oleate

7 Z,E-7,11-Hexadecadien-1-yl acetate E,Z- C18H32O2 280.2 81.3 81.3 30.258 69

7,11-Hexadecadien-1-ol acetate

8 9-Octadecenoic acid (Z)- (CAS) Oleic C18H34O2 282.3 55.4 55.4 30.38 86

acid Red oil Oelsauere

Results and Discussion

9 2,6-Diphenyl-4-piperidone 3,5-Dimethyl- C19H21NO 279.2 278.5 278.5 30.672 50

2,6-diphenylpiperidin-4-one

10 (1S*,2R*,5R*,7S*)-2,4-Dimethyl-7-ethyl- C10H16O2 168.1 149.3 149.3 38.613 19

6,8-dioxabicyclo[3.2.1]-oct-3-ene

11 7-Bromo-2,3,3a,8b-tetrahydro-3a- C13H15BrO2 282 282.4 282.4 44.718 13

methoxy-6-methyl-1H-

cyclopenta[b]benzofuran

12 Umbelliprenin C24H30O3 366.2 69.4 69.4 48.919 42

13 cis-2-Cyclohexyl-3-(4-methylphenyl)-4- C21H25NO2S 355.2 208.3 208.3 51.163 10

phenyl-1,2-thiazetizine 1,1-dioxide

14 3-Methyl-2,4,5-triphenyloxazolidine C22H21NO 315.2 208.3 208.3 55.728 11

isomer

Results and Discussion

Table 3.8: Composition of ET-17 fixed oils

S.No. Chemical name Formula mass m/z Base Peak RT Ions

1 Phenol, 2,4-bis(1,1-dimethylethyl)- (CAS) 2,4-Di- C14H22O 206.2 191.5 191.5 19.171 13 tert-butylphenol 2 Benzene, 1,2,3-trimethoxy-5-(2-propenyl)- C12H16O3 208.1 208.3 208.3 20 43 Benzene, 5-allyl-1,2,3-trimethoxy- 3 2-Hydrazinomethyl-1,5-oxazepin-4(5H)-one C10H11N3O2 205.1 189.4 189.4 22.416 39

4 .ALPHA.-BISABOLOL .alpha. - bisabolol C15H26O 222.2 109.4 109.4 22.942 52

5 1-[2',3',4',5'-bis(Trifluoromethylphenyl)propan-1- C9H8F4O 208.1 179.3 179.3 24.673 9 ol 6 5-Methoxy-3-[N-(2'- C16H25NO2S 295.2 147.4 147.4 26.799 37 methylthioethyl)propiylamino]chroman 7 Hexadecanoic acid, methyl ester (CAS) Methyl C17H34O2 270.3 74.3 74.3 28.454 39 palmitate Uniphat A60 8 1-(4-tert-Butyl)phenyl-2,3,3-trimethyl-2-butanol C17H28O 248.2 147.4 147.4 29.013 32

9 (E)-1[(4-tert-Butylbenzyl)oxy]-2,3-epoxyoctane C19H30O2 290.2 147.4 147.4 29.181 46

10 1,2-Benzenedicarboxylic acid, butyl 2- C16H22O4 278.2 149.3 149.3 29.315 7 methylpropylester (CAS) 11 1,2-dihydrocyclobutabenzene C8H8 104.1 104.3 104.3 30.655 27

12 1,2-dihydrocyclobutabenzene C8H8 104.1 104.3 104.3 31.847 29

13 9,12-Octadecadienoic acid (Z,Z)-, methyl ester C19H34O2 294.3 81.4 81.4 32.289 89 (CAS) Methyl linoleate 14 9-Octadecenoic acid (Z)-, methyl ester (CAS) C19H36O2 296.3 55.4 55.4 32.43 101 Methyl oleate 15 9-Octadecenoic acid, methyl ester, (E)- (CAS) C19H36O2 296.3 55.4 55.4 32.562 36 Methyl elaidate FAME 18:1

100

Results and Discussion

16 Octadecanoic acid, methyl ester (CAS) Methyl C19H38O2 298.3 74.3 74.3 33.016 52 stearate Kemester 9718

17 1,2-dihydrocyclobutabenzene C8H8 104.1 104.3 104.3 33.629 25

18 2,4,7-trimethylperhydroisoxazolo[2,3-a]-pyridine- C10H19N3O3 229.1 240.4 240.4 34.636 31 2,7-dicarbadehyde dioxime

19 (2R,3R)-1-(Benzyloxy)-2-phenylhex-5-en-3-ol C19H22O2 282.2 104.3 104.3 35.092 32

20 (1S*,2R*,5R*,7S*)-2,4-Dimethyl-7-ethyl-6,8- C10H16O2 168.1 149.3 149.3 41.59 19 dioxabicyclo[3.2.1]-oct-3-ene

21 2-(6-Heptenyl)-4-methyl-2,5-dihydrofuran C12H20O 180.2 83.3 83.3 41.706 11

22 Phenol, 2,4-bis(1,1-dimethylethyl)- (CAS) 2,4-Di- C14H22O 206.2 191.5 191.5 19.171 13 tert-butylphenol

23 3,4-dihydro-7-methoxy-N-methylisoquinoline-8- C11H13NO2 191.1 253.3 253.3 51.047 7 one

24 1-Aza-5,6-(2,3a-furyl)bicyclo[5.3.0]decan-11-one C11H13NO2 191.1 96.3 96.3 51.937 10

25 2-[N,N'- C43H53N3O9 755.4 91.3 91.3 54.085 11 bis(1",1"Dimethylethoxycarbonyl)hydrazino-N- (2"-hydroxy-1"-methyl-2...

26 2-(2,4-Dinitrobenzenesulfinyl) pyrrole C10H7N3O5S 281 281.4 281.4 56.094 8

27 3,4-dihydro-7-methoxy-N-methylisoquinoline-8- C11H13NO2 191.1 267.3 267.3 56.589 9 one

28 Methyl (2E,4Z) and (2E,4E)-4-methoxycarbonyl- C12H13NO4S 267.1 208.3 208.3 60.496 5 (2-thiazolyl)-2,4-hexadienoate

101

Results and Discussion

3.4. In-vitro biological activities 3.4.1. Antibacterial Activity of roots

The crude MeFn extract and various fractions of roots were tested against Gram positive; S. epidermidis, S. aureus, S. pneumoniae, and Gram negative; E. coli, S. typhi,

P. aeruginosa and K. pneumonia. Zone of inhibition was compared with Amoxicillin (10

µg/disc) and percent growth inhibition was calculated. Against S. epidermidis the following percent zone of inhibition was observed; n-hexane and CHCl3 fractions (64%), followed by crude MeFn (48%), BuOH (44%) and EtOAc (40%) fractions respectively.

Against S. pneumoniae all fractions showed good antibacterial effect, for crude MeFn

(72%) followed by n-hexane and aqueous (60%), BuOH (56%), EtOAc (44%) and CHCl3 fraction (40%) respectively. The antibacterial activity against S. aureus was recorded for n-hexane fraction (60%) while crude MeFn, CHCl3, EtOAc, BuOH and aqueous fractions

(52%) showed moderate antibacterial activity. The crude MeFn extract showed significant antibacterial activity (86.95%) while good activity showed by BuOH (78%), n-hexane (73.91%) and aqueous (73.91%) and CHCl3 fraction (69%) respectively, while

EtOAc fraction was inactive against P. aeruginosa as shown in Figure 3.11-3.17. The

EtOAc fraction showed significant antibacterial activity against E. coli (80%) and good activity was showed by aqueous fraction (73%) and moderate activity by crude MeFn extract (46%) while n-hexane, CHCl3 and BuOH fractions were inactive against E. coli.

The significant antibacterial activity against S. typhi was noted for EtOAc fraction (81%) and BuOH fraction (75%) showed good anti bacterial activity, while crude MeFn extract

(56%) showed moderate activity against S. typhi. The n-hexane, CHCl3 and aqueous fractions were inactive against S. typhi. All the test samples showed good antibacterial

102

Results and Discussion activity against K. pneumonia. Moderate antibacterial effect was showed by n-hexane fraction (53%), EtOAc (50%), CHCl3 (43%) and low activity showed by crude MeFn extract (33%), BuOH (33%) respectively as shown in Table 3.9.

3.4.2. Antibacterial effect of Aerial part

Crude MeFn extract and various fractions of the aerial parts of F. narthex were also screened for antibacterial activity. Zone of inhibition of crude MeFn extract and various fractions was compared with Amoxicillin (10 µg/disc) standard antibacterial agent and percent growth inhibition was calculated as mentioned in Table 3.10. All test samples showed antibacterial activity against S. epidermidis except EtOAc fraction. Good antibacterial effect was recorded for CHCl3 fraction (64%), while moderate activity by n- hexane (52%), crude MeFn extract (44%) and aqueous fraction (44%). Against S. pneumoniae all test samples were active, significant antibacterial activity was recorded for EtOAc fraction (80%) followed by crude MeFn extract (60%), CHCl3 (60%) and aqueous fractions (60%) with good antibacterial activity and moderate activity for n- hexane fraction (48%) were observed respectively. The n-hexane (56%) and aqueous fraction (56%) followed by crude MeFn extract (47%) and CHCl3 fraction (47%) showed moderate activity against S. aureus while EtOAc fraction was inactive against it. Against

E. coli, the EtOAc fraction showed significant activity (86%) and crude MeFn extract possess good activity (66%) while rest of the fractions were found inactive against E. coli. The EtOAc fraction showed significant activity (81%) against S. typhi. Moderate antibacterial activity (50%) was observed for the crude MeFn extract against S. typhi while CHCl3, EtOAc and aqueous fractions were found inactive against S. typhi. Against

P. aeruginosa, the crude MeFn extract (78%), EtOAc (73.91%), aqueous (73.91%) and

103

Results and Discussion

CHCl3 (69%) fractions showed good antibacterial active. Against K. pneumonia good antibacterial effect was observed for n-hexane fraction (66.66%) and moderate activity for CHCl3 fraction (40%) while crude MeFn extract (33%) showed low antibacterial activity against K. pneumonia. The EtOAc and aqueous fractions were found inactive against K. pneumonia as given in Figure 3.18-3.24.

The antibacterial activity result shows that the crude MeFn and various fractions can consider being a rich source for antibacterial agents and can be used in various pathological conditions. This result supports the folkloric use of this plant in various ailments like bronchitis and sinusitis [191].

104

Results and Discussion

Table 3.9: Antibacterial activity of crude MeFn and various fractions of roots of F. narthex

Name of Bacteria Crude MeFn n-hexane CHCl3 EtOAc BuOH Aqueous extract

standard

Zone of Zone of Zone of Zone of Zone of Zone of Zone

Inhibition (%) Inhibition

Zone of inhibition (mm) of (mm)of inhibition of Zone

inhibition (mm) inhibition (mm) inhibition (mm) inhibition (mm) inhibition (mm) inhibition (mm) inhibition

Inhibition (%) Inhibition (%) Inhibition (%) Inhibition (%) Inhibition (%) Inhibition E. coli 15 07 46.66 ------12 80 -- -- 11 73.33

S. epidermidis 25 11 44 16 64 12 48 10 40 11 44 -- --

S. typhi 16 09 56.25 ------13 81.25 12 75 -- --

S. pneumoniae 25 18 72 15 60 10 40 11 44 14 56 15 60

S. aureus 23 12 52.17 14 60.86 12 52.17 12 52.17 -- -- 12 52.17

P. aeruginosa 23 20 86.95 17 73.91 16 69.59 -- -- 18 78.26 17 73.91

K. pneumonia 30 13 43.33 16 53.33 14 46.66 15 50 10 33.33 10 33.33

Standard= Amoxicillin (10µg/Disc)

105

Results and Discussion

Table 3.10: Antibacterial activity of crude MeFn extract and various fractions of the aerial part, of F. narthex

Name of Bacteria Crude MeFn extract n-hexane CHCl3 EtOAc Aqueous

inhibition of of inhibition

standard

Zone of Zone

Zone of Zone (mm) inhibition (%) Inhibition of Zone (mm) inhibition (%) Inhibition of Zone (mm) inhibition (%) Inhibition of Zone (mm) inhibition (%) Inhibition of Zone (mm) inhibition (%) Inhibition E. coli 15 10 66.66 ------13 ------

S.epidermidis 25 11 44 13 52 16 64 -- -- 11 44

S.typhi 16 8 50 ------13 81.25 -- --

S.pneumoniae 25 15 60 12 48 15 60 20 80 15 60

S.aureus 23 11 47.82 13 56.52 11 47.82 -- -- 13 56.52

P.aeruginosa 23 18 78.26 -- -- 16 69.56 17 73.91 17 73.91

K. pneumoniae 30 10 33.33 20 66.66 12 40 ------

Standard= Amoxicillin(10µg/Disc)

106

Results and Discussion

100 90 80 70 60 50 40 30 % zone inhibition of % 20 10 0

Figure 3.11: Antibacterial activity of crude MeFn extract and various fractions of roots against E. coli

100 90

80 70 60

50 40 30

% zoneinhibition of % 20 10 0

Figure 3.12: Antibacterial activity of crude MeFn extract and its various fractions of roots against S.epidermidis

107

Results and Discussion

100 90

80 70 60 50 40 30