Chemical and Biological Evaluation of Medicinal

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CHEMICAL AND BIOLOGICAL EVALUATION OF

MEDICINAL PLANTS FOR THEIR ANTI

CARCINOGENIC ATTRIBUTES

AISHA ASHRAF

M.phil. (UAF)

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSPHY

CHEMISTRY

DEPARTMENT OF CHEMISTRY

FACULTY OF SCIENCES

UNIVERSITY OF AGRICULTURE, FAISALABAD

PAKISTAN

2015

Declaration

I hereby declare that contents of the thesis ―Chemical and biological evaluation of medicinal plants for their anti carcinogenic attributes are product of my own research and no part has been copied from any published source (except the references, standard mathematical or genetic modals/ equations/ formulae/ protocols etc). I further declare that this work has not been submitted for award of any other diploma/ degree. The University may take action if the information provided is found inaccurate at any stage (in case of any default, the scholar will be proceeded against as per HEC plagiarism policy).

Aisha Ashraf

2004-ag-414

Dedicated to

My Loving Mother

My Affectionate Father

My Worthy Hazrat Abdul

Haq Haqqani (Damut Brkaat Hm)

iii Acknowledgements

I have no words to thank Almighty ALLAH, The beneficent, The merciful, The most gracious, WHO is the entire and only source of knowledge and wisdom endowed to mankind and who blessed me with the ability to do this work. It is the blessing of Almighty ALLAH an His Prophet Hazrat Muhammad (Sallallaho Alaihe Wasallam) which enabled me to achieve this goal.

I would like to take this opportunity to convey my cordial gratitude and appreciation to my supervisor Dr. Raja Adil Sarfraz, Assistant Professor, Department of Chemistry and Biochemistry/ Incharge Central Hi-Tech Lab, University of Agriculture, Faisalabad. Without whose keen interest, constant help and vigilant guidance, the completion of this thesis was not possible. I am really indebted to him for his though provoking guidance, immense intellectual input, patience and sympathetic behavior.

I would like to pay my deepest gratitude to members of my supervisory committee, Dr. Muhammad Abid Rashid, Assistant Professor and Dr. Muhammad Shahid, Associate Professor, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, for their valuable suggestions and kind help during accomplishment of my Ph.D.

I am also very much thankful to Higher Education Commission (HEC), Pakistan, for awarding me the Indigenous PhD Fellowship and foreign scholarship under International Research Support Initiative Program (IRSIP) to carry out part my research work in The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, United States of America under kind supervision of Dr. Bharat B Aggarwal. Words are too debilitate to express my appreciation and deep sense of gratitude to Fouzoa Mohammad (Dental Hygenist) and Muhammad Abrar Sanaullah (Mechanical Engineer), Houston, Texas, for their loving, caring and understanding behviour with me during my IRSIP stay in USA. Their invigorating encouragement and affectionation can never be repaid. Truly saying, I have no suitable words to express my gratitude them. Special thanks to my fellows especially, Bazgah Ahmad, Qurat-ul-ain, Fiaza Nazir, Maryum Aslam, Nadia Noor, Moin ud Din and Ayesha Mahmood for their assistance, good company and friendly attitude. I am deeply grateful to my worthy Hazrat Abdul Haq Haqqani (Damut Barkaat Hm) for His prayers and strong moral support in my life matters and during the conduct of my Ph.D. I might not have courage to complete my degree without His magnificent moral support. Words seem to be meaningless to express my obligation to my Affectionate Parents, for their huge sacrifice, moral support, encouragement, patience, tolerance and prayers for my health and success who enabled me to achieve this goal. I owe to thank my loving brother Dr. Adnan Ashraf, for his moral support, encougement during conduct of my Ph.D and especially the prayers during foriegn evaluation and publication of my thesis. I am thankful to my younger borther Farhan Ashraf, who helped me a lot in my computer work, without him it was not possible to complete my thesis. I am thankful to my dearest sister Rabia Ashraf, who shared my responsibilities and did not burden me during my Ph D. My special regards for my aunt Vajiha Irum and Uncle Dr. Muhammad Naeem Akhtar for their encouragement during the studies.

Aisha Ashraf

Contents

Chapters Titles Page No. Chapter 1 Introduction 1-6 1.1 History of medicinal plants 1 1.2 Traditional uses of medicinal plants 1 1.3 Therapeutic compounds from medicinal plants in modern era 2 1.4 Oxidation reactions and their side effects 2 1.5 Antioxidants and their functions 2 1.6 Need for natural antioxidants 3 1.7 Alarming death rate due to infectious diseases 3 1.8 Limitation of modern antibiotics 3 1.9 Need for natural antimicrobial agents 3 1.10 Cancer 4 1.11 Motives of cancer 4 1.12 Conventional cancer therapies and their side effects 4 1.13 Use of plants in anticancer activity 5 1.14 Medicinal flora of Pakistan 5 1.15 Need for project 5 1.16 Amis and objectives 6 Chapter 2 Review of literature 7-35 2.1 Euclayptus camaldulensis 7 2.1.1 Ethno botanical description 7 2.1.2 Distribution 7 2.1.3 Ethno medicinal application 7 2.1.4 Chemical evaluation 8 2.1.5 Biological evaluation 11 2.2 Viola betonicifolia 13 2.2.1 Ethno botanical description 13 2.2.2 Distribution 13 2.2.3 Ethno medicinal application 13 2.2.4 Chemical evaluation 13 2.2.5 Biological evaluation 15 2.3 Euphorbia royleana 17 2.3.1 Ethno botanical description 17 2.3.2 Distribution 17 2.3.3 Ethno medicinal application 18 2.3.4 Chemical evaluation 19 2.3.5 Biological evaluation 20 2.4 Psidium guajava 21 2.4.1 Ethno botanical description 22 2.4.2 Distribution 22 2.4.3 Ethno medicinal application 22 2.4.4 Chemical evaluation 23 2.4.5 Biological evaluation 26 2.5 Ziziphus mauritiana 29 2.5.1 Ethno botanical description 29 2.5.2 Distribution 30 2.5.3 Ethno medicinal application 30 2.5.4 Chemical evaluation 30 2.5.5 Biological evaluation 31 Chapter 3 Materials and methods 36-50 3.1 Materials 36 3.1.1 Chemicals and reagents 36 3.1.2 Instruments 37 3.1.3 Collection of plant materials 37 3.1.4 Plants used in current study 38 3.1.5 Human cancer cell lines employed to access the anticancer potential of 39 plant extracts 3.1.6 Strains of microorganisms used to esetimate the antimicrobial activity 41 of plant extracts 3.2 Extraction of dried plant material 41 3.3 Extraction of fresh plant material 42 3.4 Chemical evaluation 42 3.4.1 Total phenolic contents 42 3.4.2 Total flavonoid contents 43 3.4.3 High performance liquid chromatography analysis 43 3.4.4 Gas chromatography mass spectrometry 44 3.5 Biological evaluation 44 3.5.1 Free radical scavenging activity (DPPH assay) 44 3.5.2 Antitumour activity (Potato disc assay) 45 3.5.3 Antimicrobial activity (Disc diffusion assay) 46 3.6 Anticancer attributes of plant extracts 47 3.6.1 Anticancer activity (MTT assay) 49 3.6.2 Anti-inflammatory activity (Electrophoretic mobility shift assay) 49 3.7 Statistical analysis 50 Chapter 4 Results and discussions 51-111 4.1 Euclayptus camaldulensis 51 4.1.(a) Chemical evaluation 51 4.1.1 Total phenolic acnd total flavnoid contents of E. camaldulensis 51 4.1.2 High performance liquid chromatographic (HPLC) analysis of E. 53 camaldulensis 4.1.3 Gas chromatography mass spectrometric (GC-MS) study of E. 54 camaldulensis 4.1.(b) Biological evaluation 57 4.1.4 Free radical (DPPH) scavenging activity of E. camaldulensis 57 4.1.5 Antitumor activity of E. camaldulensis 58 4.1.6 Antimicrobial activity of E. camaldulensis 60 4.2 Viola betonicifolia 61 4.2.(a) Chemical evaluation 61 4.2.1 Total phenolic acnd total flavnoid contents of V. betonicifolia 61 4.2.2 High performance liquid chromatographic (HPLC) analysis of V. 63 betonicifolia 4.2.3 Gas chromatography mass spectrometric (GC-MS) study of V. 64 betonicifolia 4.2.(b) Biological evaluation 69 4.2.4 Free radical (DPPH) scavenging activity of V. betonicifolia 69 4.2.5 Antitumor activity of V. betonicifolia 71 4.2.6 Antimicrobial activity of V. betonicifolia 72 4.2.7 Anticancer activity of V. betonicifolia 73 4.2.8 Anti-inflammatory activity of V. betonicifolia 76 4.3 Euphorbia royleana 78 4.3. (a) Chemical evaluation 78 4.3.1 Total phenolic acnd total flavnoid contents of E. royleana 78 4.3.2 High performance liquid chromatographic (HPLC) analysis of E. 80 royleana 4.3. (b) Biological evaluation 81 4.3.3 Free radical (DPPH) scavenging activity of E. royleana 81 4.3.4 Antitumor activity of E. royleana 82 4.3.5 Antimicrobial activity of E. royleana 83 4.4. Psidium guajava 85 4.4.(a) Chemical evaluation 85 4.4.1 Total phenolic acnd total flavnoid contents of P. guajava 85 4.4.2 High performance liquid chromatographic (HPLC) analysis of P. 86 guajava 4.4.3 Gas chromatography mass spectrometric (GC-MS) study of P. guajava 87 4.4. (b) Biological evaluation 90 4.4.4 Free radical (DPPH) scavenging activity of P. guajava 90 4.4.5 Antitumor activity of P. guajava 91 4.4.6 Antimicrobial activity of P. guajava 92 4.4.7 Anticancer activity of P. guajava 94 4.4.8 Anti-inflammatory activity of P. guajava 97 4.5 Ziziphus mauritiana 99 4.5.(a) Chemical evaluation 99 4.5.1 Total phenolic acnd total flavnoid contents of Z. mauritiana 99 4.5.2 High performance liquid chromatographic (HPLC) analysis of Z. 101 mauritiana 4.5.3 Gas chromatography mass spectrometric (GC-MS) study of Z. 102 mauritiana 4.5.(b) Biological evaluation 104 4.5.4 Free radical (DPPH) scavenging activity of Z. mauritiana 104 4.5.5 Antitumor activity of Z. mauritiana 106 4.5.6 Antimicrobial activity of Z. mauritiana 107 4.5.6 Anticancer activity of Z. mauritiana 108 Chapter 5 Summary 112-115 Refrences 116-167

List of Tables

Serial Title Page No. No. 2.1 Taxonomic classification of E. camaldulensis 9 2.2 Taxonomic classification of V. betonicifolia 14 2.3 Taxonomic classification of E. royleana 18 2.4 Taxonomic classification of P. guajava 23 2.5 Taxonomic classification of Z. mauritiana 32 3.1 Instruments used with their model and company 37 4.1.1 HPLC analysis of methanol, chloroform and hexane extracts of E. 54 camaldulensis for identification and quantification of phenolic compounds 4.1.2 Chemical components of methanol, chloroform and hexane extracts of E. 56 camaldulensis analyzed by GC-MS 4.1.3 Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform 58 and hexane extracts of E. camaldulensis 4.1.4 Antimicrobial activity of methanol, chloroform and hexane extracts of E. 61 camaldulensis 4.2.1 HPLC analysis of methanol, chloroform and hexane extracts of V. 64 betonicifolia for identification and quantification of phenolic compounds 4.2.2 Chemical components of methanol, chloroform and hexane extracts of V. 65 betonicifolia analyzed by GC-MS 4.2.3 Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform 70 and hexane extracts of V. betonicifolia 4.2.4 Antimicrobial activity of methanol, chloroform and hexane extracts of V. 72 betonicifolia 4.3.1 HPLC analysis of methanol, hexane and aqueous extracts of E. royleana 80 for identification and quantification off phenolic compounds 4.3.2 Scavenging (%) of stable free radicals (DPPH) by methanol, hexane and 82 aqueous extracts of E. royleana 4.3.3 Antimicrobial activity of methanol, hexane and aqueous extracts of E. 84 royleana 4.4.1 HPLC analysis of methanol, chloroform and hexane extracts of P. guajava 87 for identification and quantification off phenolic compounds 4.4.2 Chemical components of methanol, chloroform and hexane extracts of P. 88 guajava analyzed by GC-MS 4.4.3 Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform 91 and hexane extracts of P. guajava 4.4.4 Antimicrobial activity of methanol, chloroform and hexane extracts of P. 93 guajava 4.5.1 HPLC analysis of methanol, hexane and chloroform extracts of Z. 101 mauritiana for identification and quantification off phenolic compounds 4.5.2 Chemical components of methanol, chloroform and hexane extracts of Z. 103 mauritiana analyzed by GC-MS 4.5.3 Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform 105 and hexane extracts of Z. mauritiana 4.5.4 Antimicrobial activity of methanol, chloroform and hexane extracts of Z. 107 mauritiana

List of Figures

Serial Title Page

No. No.

3.1 Photographs of indigenous medicinal plants used in current study 39

3.2 Human cancer cell lines used in current research work 40

3.3 A typical agar plate showing the antimicrobial activity of plant extracts 47

4.1.1 Total phenolic and total flavonoid contents of methanol, chloroform and 52

hexane extracts of E. camaldulensis

Antitumor activity of methanol, chloroform and hexane extracts of E. 4.1.2 59

camaldulensis

Total phenolic and total flavonoid contents of methanol, chloroform and 62 4.2.1

hexane extracts of V. betonicifolia

Antitumor activity of methanol, chloroform and hexane extracts of 73 4.2.2

V.betonicifolia

Anticancer activity Of (A) methanol, (B) chloroform and (C) hexane 76 4.2.3

extracts of V. betonicifolia leaves against KBM5, SCC4 and HCT116 cells

4.2.4 Anti-inflammatory activity of chloroform extract of V. betonicifolia 77

Total phenolic and total flavonoid contents of methanol and hexane extracts of E. royleana 79 4.3.1

4.3.2 Antitumor activity of methanol and hexane extracts of E. royleana 83

4.4.1 Total phenolic and total flavonoid contents of methanol, chloroform and 86

hexane extracts of P. guajava

Antitumor activity of methanol, chloroform and hexane extracts of P. 92 4.4.2 guajava

Anticancer activity of (A) methanol, (B) chloroform and (C) hexane 96 4.4.3

extracts of P. guajava leaves against KBM5, U266 and HCT116 cells

4.4.4 Anti-inflammatory activity of hexane extract of P. guajava 98

4.5.1 Total phenolic and total flavonoid contents of methanol, chloroform and 100

hexane extracts of P. guajava

Antitumor activity of methanol, chloroform and hexane extracts of Z. 106 4.5.2

mauritiana

Anticancer activity of (A) methanol, (B) chloroform and (C) hexane 110 4.5.3 extracts of Z. mauritiana leaves against KBM5, U266 and SCC4 cells

Abstract

Cancer is terrible disease. It is second leading cause of mortality worldwide. Conventional cancer therapies burdened the disease crippled patient with toxic side effects and are also very expensive. Therefore, the demand to use alternative approaches in treatment of cancer is increasing. Plant derived compounds due to their unique structure and sophisticated mechanism of action play promising role in anticancer therapies. The main objective of current study was to evaluate anticancer potential of medicinal flora of Pakistan.

The plants used in current study are frequently utilized in folk medicines for treatment of many ailments in Pakistan. In present research work these traditional medicinal plants were scientifically examined for their antioxidant, antitumor, antimicrobial, anticancer and anti- inflammatory potential. Furthermore, chemical composition of the plant extracts was evaluated by state of art chromatographic techniques such as UV/Vis spectrophotometer, HPLC and GC- MS, revealed the presence of phenolics, flavonoids and broad range of other bioactive components in them. Antioxidant activity was estimated by free radical scavenging activity estimated by DPPH (1,1- diphenyl 2-picrylhydrazyl) assay. The outcome of antimicrobial activity assay showed that E. coli was the resistant strain to most of tested plant extracts. Overall, among the fungal strains, A. niger was the sensitive one. Furthermore, we examined antitumor activity by potato disc assay. Chloroform extract of V. betnocifolia (IC50= 38.13 µg/mL) exhibited maximum antitumor activity. We determined anticancer activity of different plant extracts against human cancer cell lines (KBM, mylegeous leukemia; U266, multiple myeloma; SCC4, tongue squamous carcinoma and HCT116, colon carcinoma cells) by MTT assay. The extracts with maximum anticancer activity against human mylegeous leukemia (KBM5) cells were examined for inhibition of inflammatory transcription factor, nuclear factor kappa B (NF- kB). Surprisingly all the plant extracts inhibited TNF-α induced NF-kB activation. CHAPTER 1 INTRODUCTION

1.1. History of medicinal plants

Plants are the earliest companion of mankind. They serve humanity chiefly by granting food, shelter, oxygen and medicinal compounds (Mamedov, 2012). They have been employed for medical issues since 60¸000 years ago. There are ample evidences from Indian, Arabian, Chinese, Roman and other traditions about the use of medicinal plants to fulfill health needs (Bensoussan et al., 1998; Saad et al., 2005; Mazid et al., 2012). This traditional herbal knowledge had been conveyed to next generations, mainly by oral communications (Shinwari, 2010). However, written documents, some original herbal medicines and preserved monuments revealed the use of herbal remedies by ancient civilizations (Petrovska, 2012). In India the first oldest written herbal recipes were found about 5000 years ago, engraved on Sumerian clay slab (Kelly, 2009).

250,000 species of medicinal plants persist on earth, out of which more than one thousand plant species are assessed to have significant anticancer activity (Mukherjee et al., 2001), but there is still huge amount of work to be done on these lines. Pivotal role of plants in health scenario is attributed to assets of bioactive compounds in them (Jagadish et al., 2009). Phytochemical components of plants include immense array of compounds which might impediment or inhibit the inception of degenerative ailments (Robard et al., 1999; Guilford and Puzetto, 2008; Mehta et al., 2010).

1.2. Traditional uses of medicinal plants

Traditionally plants had been used in natural remedies as teas, tinctures or powders (Samuelsson, 2004). Traditional therapies are still on peak practice in many regions of world (Abe and Ohtani, 2013). Most of the people in developing countries prefer herbal remedies, due to their low cost, easy availability and safe mode of action. World Health Organization (WHO) witnessed that 80% of population in Asian and African countries relies on herbal formulations to fulfill their health needs (Kim et al., 2012).

1.3. Therapeutic compounds from plants in modern era

In modern era potential therapeutic compounds such as vincristine, camptothecin, navellbine, etoposide (Pezzuto, 1997), taxol, vinblastine (Flescher, 2007), doxorubicin (Norris et al., 2000), mechlorethamine (Vonderheid et al., 1989), teniposide (Postmus et al., 2000), topotecan (Gordon et al., 2001), casticin (Shen et al., 2009), proanthocyanidin B2 (Avelar and Gouvea, 2012), cirsimaritin (Quan et al., 2010), protoapigenone (Chang et al., 2008), apigenin (Yin et al., 2001), androsacin, curcumin, xanthohuskiside A, genistein, resveratrol, gallocatechin gallate, lycopene, flavopiridol, anethole, anthroquinone, chrysin, eugenol, epicatechin, cinnamic acid, ellagitannin, catechol and many others have been isolated from medicinal plants and are used in targeted therapies against cancer, inflammation and microbial infections (Norris et al., 2000; Gupta et al., 2011). In addition to leading role in health scenario they are also used as potential agent in food preservation, neutraceuticals, pharmaceuticals, fragrances, natural therapies, functional foods, cosmetics (Dung et al., 2008).

1.4. Oxidation reactions and their side effects

Oxidation reactions in mitochondria bestow energy for biological processes that act as soul of cell life, but on the other hand oxidant and oxidation reactions are major contributors to induce many pathological conditions (Dalleau et al., 2013). When production of reactive oxygen species is higher in body as compared to enzymetic and non-enzymetic antioxidants then this imbalance leads to cell damage and escort a vast range of degenerative diseases including cancer, atherosclerosis, hypertension, AIDS, parkinsonism, inflammation, aging, cardiovascular diseases (Li et al., 2009; Makasci et al., 2010) and microbial infections (Samec et al., 2010; Steer et al., 2002; Peuchant et al., 2004).

1.5. Antioxidants and their function

Antioxidants are ubiquitous components that can hinder the oxidation process by adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides (Khadri et al., 2010). In addition to leading role in health scenario they are also used as potential agent in food preservation, neutraceuticals, pharmaceuticals, fragrances, functional foods, and cosmetics (Dung et al., 2008).

1.6. Need for natural antioxidants

Synthetic antioxidants such as butylated hydroxyltoulene (BHT), butylated hydroxylanisole (BHA), propyl gallate and citric acid are frequently used in processed foods and medicinal materials (Ebrahimabadi et al., 2010). Nutritionists and toxicologists dampen the use of synthetic antioxidants because of reported side effects such as liver enlargement, increase microsomal enzyme activity and carcinogenesis (Gulcin et al., 2004; Dung et al., 2008). Therefore, the quest for exploring antioxidants from natural sources have received great attention all over the world (Oktay et al., 2003; Dalal et al., 2010). Thus in current study effort was done to evaluate antioxidant potential of plants.

1.7. Alarming death rate due to infectious diseases

Infectious diseases are main cause of high mortality rate in developing countries. About 50,000 people are dying worldwide every day because of infectious disease (Ahmad and Beg, 2001).

1.8. Limitation of modern antibiotics

Although pharmaceutical industries have introduced wide range of novel antibiotics in last few years, but resistance to these antibiotics has been boosted by microorganisms and it has become alarming situation globally (Nascimento et al., 2000). The global emergence of multidrug bacteria is restraining the efficacy of modern drugs and ultimately causes the failure of treatment.

1.9. Need for natural antimicrobial agents

As the resistance to antibiotics had been increased by microorganisms, so there is an intense need to explore alternative antimicrobial agents. Affirmative effects of plant food are attributed chiefly to innovative profile of bioactive components in them (Cai et al., 2004). Plant derived bioactive compounds are used not only for treatment of infections but also to dampen microflora growth in food, and ultimately increase the expectation of life. Thus in present study attempts were made to investigate antimicrobial efficacy of plants.

1.10. Cancer

Cancer is lethal disease. It is second leading cause of mortality worldwide characterized by invasion, apoptotic death, angiogensis, overproliferation of cells, dysregulayion of cell signaling pathways and metastasis (Aggarwal et al., 2006). Cancer cells may assault tissues which are in close proximity and may broaden through blood flow and lymphatic scheme to other parts of body (Nagarani et al., 2011).

It causes more than six million deaths each year in the world (Nisa et al., 2011). Consistent with global cancer statistics issued by the American Cancer Society, daily 20,000 deaths were reported worldwide from cancer, in 2007. 27 million new cancer cases are predicted in the world up to 2050 (American Cancer Society, 2007).

1.11. Motives of cancer

Chief motives of cancer are viruses, environmental exposure (e.g. Ionizing and UV radiations), life style factors (e.g. high calorie diets, excessive use of tobacco), medication (e.g. alkylating agents, immunosuppressants) and genetic factors, e.g. inherited mutations, cancer causing genes (Dhanamani et al., 2011). There are ample evidences that inflammation particularly the chronic inflammation plays critical role in cancer (Elinav et al., 2013). In addition to inflammation, different NF-κB regulated genes are associated with cellular transformation, proliferation, angiogensis, invasion, tumor cell survival and metastasis (Aggarwal et al., 2004). Another study reported tumor promoting role of NF-kappaB in inflammation based cancer (Pikarsky et al., 2004). Thus suppression of NF-κB pathway will play distinctive role in cancer therapy (Gupta et al., 2011). The current research work was conducted to examine influence of potent plant extracts on NF-κB signaling pathways.

1.12. Conventional cancer therapies and their side effects

Conventional modalities for treatment of cancer such as radiation therapy, chemotherapy, immunodilution and surgery have limited success as evident by high morbidity, toxicity, indiscriminate nature, permanent disfigurement (Nawab et al., 2011). Therefore, the demand to use alternative approaches in treatment of cancer is increasing.

1.13. Use of plants in anticancer activity

Plant derived compounds due to their unique structure and sophisticated mechanism of action play promising role in anticancer therapies.114,000 extracts from 35,000 plants had been screened by National Cancer Institute (NCI) for anticancer drug discovery. Mounting evidence has indicated use of plant extracts in clinical trials (Chrubasik et al., 2004). The current study also deals with screening of plant extracts for their anticancer activity.

1.14. Medicinal flora of Pakistan

Pakistan is the sixth most populous country in the world. Flora of Pakistan is rich in medicinal plants due to its diverse climatic and soil conditions and many ecological regions (Ali et al., 2001). The country has about 6,000 species of flowering plants of which about 400 to 600 are medicinally imperative (Hamayun et al., 2005). A vast range of medicinal plants is reported from different areas of Pakistan including Balochistan (Goodman and Ghafoor, 1992), Swat and Chitral (Sher, 2001), Utror and Gabral Valleys (Hamayun et al., 2005), Margalla Hills (Shinwari and Khan, 1999), Pirgarharh Hills, South Waziristan Agency (Badshah et al., 1996), Cholistan desert (Ahmed et al., 2014), Kadhi areas of Khushab (Qureshi et al., 2011), Nara Desert, Sindh (Qureshi and Bhatti, 2008) and many more areas (Malik et al., 1990; Hamayun et al., 2003; Ahmad et al., 2009).

1.15. Need for project

Although a heap of work is done to explore chemical and biological potential of medicinal plants, WHO is also spectator of 20,000 plant species studied for medicinal purposes (Gulluce et al., 2006), but data is still insufficient and further information is needed on multi functionalities of plants. Moreover, novel therapeutic applications of plant derived products have impelled many researchers to explore pharmacological potential of plants to increase life expectancy.

In continuation of our efforts (Ashraf et al., 2014; Ahmed et al, 2014) to explore ethnomedicinal, phytochemical and bioactive potential of flora of Punjab, Pakistan, the current study reports chemical and biological potential of plant materials indigenous to Punjab, Pakistan. To the best of our knowledge, our study is first report on chemical

5 composition, antioxidant, antimicrobial, antitumor, anticancer and anti-inflammatory activities of the investigated medicinal flora of Punjab, Pakistan.

1.16. Aims and objectives:

(i) Scientific assessment of traditional medicinal plants of Pakistan. (ii) Investigation of plants with respect to variation in extracting solvents. (iii) Chemical characterization of plant extracts using state of the art chromatographic and spectrophotometric techniques. (iv) Assessment of biological activities (antioxidant, antimicrobial and antitumor) of plant extracts. (v) Exploration of in vitro anticancer potential of selective plant extracts against different human cancer cell lines. (vi) Evaluation of potent extracts for inhibition of inflammatory nuclear factor kappa B (NF-κB), which is chief mediator of carcinogenesis.

CHAPTER 2 REVIEW OF LITERATURE

2.1. Euclayptus camaldulensis:

2.1.1. Ethno botanical description:

Euclayptus camaldulensis is commonly known as ―Safeda‖ in Pakistan. It is perennial, large, single-stemmed and evergreen tree. It is tall tree of 40 m height and 0.8 m diameter. It has irregular branches which spread widely.

Leaves are present in alternate form on branches. Leaves are alternate, glabrous and sickle like. They are 8-22 cm in length and 1-2 cm broad and are pale green in color. Roots are very dispersal and deep.

Flowers are 5-10. They have white thread like stamens which are 5-6 mm in length. Anthers are present in tiny encircling glands. Flowering intensity is changeable. Almost 45% of flowers not succeed to reach maturity level (Dexter, 1978; Bren and Gibbs, 1986).

2.1.2. Distribution:

It is distributed in Pakistan (Shahwar et al., 2012), Kenya, Australia, Turkey (Basak and Candan, 2010), North Africa (Ei-Ghorab et al., 2003), Senegal, Spain, Morocco, Arizona, Sierra Leone, Egypt (El-Mageed et al., 2011), Iran (Gharekhani et al., 2012), Argentina, California, Nigeria etc.

2.1.3. Ethno medicinal applications:

In Pakistan paste of E. camaldulensis stem and decoction of its leaves is traditionally used for treatment of fever, sneezing, malarial shivering and cough (Qasim et al., 2014).

In addition to Pakistan, it is used all over the world in traditional therapies for treatment of diarrhea, eczema, spasm, cold, hemorrhage, sore throat, stomach problems, cough, vermifuge, boils, bronchitis, bladder infections, oral thrush, sores, athletes foot, dysentery, skin infections, fever, asthma, laryngitis, trachagia, constipation and wound infections (Bala, 2006; Lassack and Maccarthy, 2006; Musa et al., 2011; Ozturk et al., 2013).

2.1.4. Chemical Evaluation:

(i) Triterpenoids:

Siddiqui et al., 2000, reported one new triterpenoid from fresh leaves of E. camaldulensis var. obtuse that was identified as amirinic acid. Maurya and Srivastava, 2012, identified two triterpenoids (uroslic acid lactone and uroslic acid) in leaves of Euclayptus tereticornis. Pentacyclic triterpenoids from other medicinal plants are also reported (Alqahtani et al., 2013).

(ii) Polyphenols:

(a) Leaves:

Conde et al., 1997, investigated polyphenols in leaves of Euclayptus camaldulensis, Euclayptus rudis and Euclayptus globulus and identified them as vanillin, ellagic acid, gallic acid, protocatechuic acid, quercetin-3-arabinoside, rutin, quercetin-3,7-dirhamnoside, kaempferol-3-arabinoside, and quercetin. Antibacterial activity of vanillin related hydrazone derivatives was estimated by disc diffusion assay (Govindasami et al., 2011). Ellagic acid exhibited photoprotective efficacy on collagen breakdown.Moreover, it prevents inflammation in UV-B irradiated human skin cells (Bae et al., 2010). Gallic acid is known for its antioxidant activities (Bhadonya et al., 2012).

Quercetin plays leading role against cancer, inflammation and allergy (Shaik et al., 2006; Jagtap et al., 2009). Furthermore, the presence of flavones like apigenin and luteolin was also examined. Apigenin plays promising role in cancer prevention (Shukla and Gupta, 2010).

(b) Bark:

Polyphenolic components from bark extracts of Euclayptus camaldulensis, Euclayptus rudis and Euclayptus globules were reported (Conde et al., 1996). Gallic acid, vanillic acid, protocatechuic acid, protocatechuic aldehyde and ellagic acid were found common in three investigated species of Euclayptus.

Protocatechuic acid is narrated for its antioxidant efficacy (Li et al., 2011). Protocatechuic aldehyde is documented for its anti-inflammatory, antioxidant and neuroprotective efficacies (Chang et al., 2011; Zhao et al., 2013).

Conde et al., 1995, studied the polyphenolic components of wood extracts of Euclayptus camaldulensis, Euclayptus rudis and Euclayptus globulus. For this purpose wood of each of three Euclayptus species was extracted with methanol. Frequent use of methanol for extraction is attributed to comparatively higher extraction of several bioactive components in it (Akowuah et al., 2002; Bae et al., 2012; Caunii et al., 2012).

Table. 2.1. Taxonomic classification of E. camaldulensis

Taxonomic classification

Kingdom Plantae

Subkingdom Tracheobionta

Super division Spermatophyta

Division Magnoliophyta Class Magnoliopsida

Subclass Rosidae

Order Myrtales

Family Myrtaceae

Genus Euclayptus

Species Euclayptus camaldulensis

Various compounds such as gallic acid, vanillin, vanillic acid, sinapaldehyde, quercetin, syringaldehyde, naringenin and ellagic acid were detected. Syringaldehyde has potent applications in food, cosmetic, pharmaceutical and textile industries (Ibrahim et al., 2012).

(ii) Flavonoid glycosides:

Abd-Alla et al., 1986, identified flavonoid glycosides from leaves of E. camaldulensis and named them as: kaempferol 3-glucoside, kaempfero 3-glucuronide, apigenin 7-glucuronide, quercetin 3-glucoside, 3-glucuronide, 3-rutinoside, 3-rhamnoside and quercetin 7-glucoside, luteolin 7-glucoside and luteolin 7-glucuronide. Flavonoid and phenolic components from leaves of E. camaldulensis Dehn were extracted by using microwave assisted extraction method (Gharekhani et al., 2012).

(iii) Volatile constituents:

Hydro distillation technique was used for isolation of volatile oil of fruit of E. camaldulensis var. brevirostris (El-Ghorab et al., 2002). The volatile oil isolated from E. camaldulensis were identified as aromadendrene, p-cymenene, cubenol,p-cymen-7-ol, thymol, α-gurjunene and α-pinene.

Francisco et al., 2001, isolated oils from E. camaldulensis by two techniques: supercritical CO2 extraction and hydro distillation. The oil attained by hydrodistillation technique presented higher amounts of globulol , α-pinene, p-cymene, β-pinene, terpinen-4- ol and 1,8-cineole. Previous study showed that p-cymene had efficacy against Escherichia coli (Kisko et al., 2005). Biological potential of α and β-pinenes is well documented (Silva et al., 2012).

(iv) Phytochemical components:

Adeniyi and Ayepola, 2008, screened Euclayptus torelliana and Euclayptus camaldulensis leaf extracts for their phytochemical components. Presence of tannins, cardiac glycosides and saponins was noticed in both the extracts.

Musa et al., 2011, reported the presence of tannin and saponin in water extract of E. camaldulensis. Pyhtochemical screening of E. camaldulensis leaves showed the presence of steroid, cardiac glycosides, volatile oils, saponin glycosides, tannins, phenols and saponins (Babayi et al., 2004). Previous studies indicated the use of saponins in preparation of folk medicines (Asl and Hosseinzadeh, 2008).

2.1.5. Biological evaluation:

(i) Antimicrobial activity:

Babayi et al., 2004, reported antimicrobial potential of E. camaldulensis leaf extract against Bacillus subtilis, Staphylococcus aureus and Candida albicans. Essential oil of Euclayptus citriodora was more effective against C. albicans than B. subtilis and S. aureus (Luqman et al., 2008). Antimicrobial potential of E. camaldulensis may be attributed to existence of bioactive components in it. Similar finding was reported by other scientists (Puupponen-Pimia et al., 2000; Nohvnek et al., 2006).

Adeniyi and Auepola, 2008, extracted leaves of Euclayptus camaldulensis and Euclayptus torelliana in methanol and examined antibacterial activity. Another research group (Tambehar and Dahikar, 2011) reported the similar antibacterial potential of Indian medicinal plants. Aljebourey, 2014, observed that Euclayptus microtheca extracts also have potent activity against P. aeruginosa.

Antibacterial activity of E. camaldulensis water extract at various concentrations 12.5, 25 and 50 mg/mL was also explored against six bacterial strains. A direct link was observed between concentration of plant extract and inhibition zone.

It is interesting to note that Akgul and Kaya, 2004, narrated the analogous behavior for Turkish medicinal plants. Among the tested bacterial strains Salmonella typhi was the most sensitive strain. These results were different from previous findings (Shuaib et al., 2013).

(ii) Anticancer activity:

Hrubik et al., 2012, extracted E. camaldulensis leaves of ethyl acetate, methanol, water and n-butanol. Maximum anticancer activity by MTT assay was observed against MDA-MB-231 and MCF-7 with IC50 values of 26.7 and 34.4 µg/ml ethyl acetate extract. Some previous studies also reported anticancer potential of natural products against MDA- MB-231 and MCF-7 cell lines (Rahman et al., 2011; Lin et al., 2013).

Anticarcinogenic potential of leaves of E. camaldulensis extracted with aqueous acetone was evaluated against MCF-7, HeLa, Hep-2, HCT-116, HepG-2 and Caco-2 carcinoma cells (Singab et al., 2011). The aqueous acetone extract decreased the growth of carcinoma cells in a dose-dependent mode.

Another research group (Parsad et al., 2012) reported the similar relationship between viability of cancer cells and dose of tested compound. (iii) Antioxidant activity:

Singab et al., 2011, explored antioxidant activity of aqueous acetone extract of E. camaldulensis leaves by DPPH, super oxide anion and hydroxyl radical scavenging tests. High stable free radical (DPPH) scavenging activity was examined in methanol fraction. These results were in accordance with previous findings (Sowndhararajan and Kang, 2013) in which methanol extract of medicinal flora had exhibited stronger free radical scavenging activities.

(iv) Spasmolytic activity:

Spasmolytic components of E. camaldulensis var obtuse leaves were reported by Begum et al., 2000. The outcome of study revealed the isolation of one new and four already known compounds. The new one was identified as triterpenoid camaldulin. The others were known as ursolic acid lactone acetate, betulinic acid and ursolic acid lactone evaluated for their spasmolytic potential.

Spasmolytic potential of other natural flora of Pakistan is also narrated in literature (Gilani et al., 2008). Betulinic acid also plays potent role in programmed cell death (Tan et al., 2003) human melanoma cells.

2.2. Viola betonicifolia:

2.2.1. Ethno botanical description:

Viola betonicifolia is known as ―Banafsha‖ in Pakistan. It is perennial herb which is 8 to 20 cm tall. It has no stem, leaves are basal, triangular, numerous with dark brownish stipules. They have a smaller lamina than petiole.

Leaves are arrow shaped and deep green in color. Leaves may be up to 10 cm in length. Flowers are light or deep purplish in color sometimes with lighter colored patches. The flowers stand on a slender stalk. They have a long pedicel. Roots are slender and have no branches. 2.2.2. Distribution:

It is distributed in Pakistan, Sri Lanka, India, Malaysia, Nepal, Australia and China (Muhammad et al., 2012).

2.2.3. Ethno medicinal applications:

It is used in traditional therapies in different parts of world. In Pakistan it is traditionally used as anticancer, astringent, febrifuge, diuretic, purgative, antipyretic and diaphoretic. Furthermore, it is also used for treatment of bronchitis, boils, nervous disorders, pneumonia, epilepsy, kidney diseases and lung troubles (Ilyas and Hamayun, 2005).

Traditional use of V. betonicifolia for treatment of kidney diseases, skin disorders, pharyngitis, pneumonia, blood disorders and sinusitis is reported from different parts of world (Bhatt and Negi, 2006; Tiwari et al., 2010).

2.2.4. Chemical evaluation:

Yang et al., 2011, identified fifteen chemical components of Viola tianshanica which represented 89.67% of oil. The main components in essential oil of V. tianshanica were hexadecanoate, dibutyl phthalate methyl, 2,3-pentanedione and palmitic acid. Muhammad et al., 2013, for the first time reported a cinnamic acid derivative from V. betonicifolia in form of off white needle. It was named as 2,4-dihydroxy, 5-methoxy-cinnamic acid.

Table. 2.2. Taxonomic classification of V. betonicifolia

Taxonomic classification

Kingdom Plantae

Subkingdom Tracheobionta

Super division Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Subclass Dilleniidae

Order Violales

Family Violaceae

Genus Viola

Species Viola betonicifolia

Another research group (Akhbari et al., 2011) reported twenty five compounds in essential oil of Viola odorat L. GC-MS study of Viola etrusca explored a wide range of chemical components in which α-pinene, 2-pentyl furan, β-pinene, p-cymene, 1,8-cineole, nonanal, camphor, borneol, methyl salicylate, farnesane and germacrene D were the potent one (Flamini et al., 2003).

(i) Phytochemical components:

Phytochemical screening of V. betonicifolia revealed the presence of alkaloids, flavonoids, saponins, tannins, phenolics and proteins in it (Muhammad et al., 2012). Total phenolic and flavonoid contents were higher in ethyl acetate fraction of V. betonicifolia followed by chloroform, butanol and aqueous fractions, respectively (Muhammad et al., 2011).

2.2.5. Biological evaluation:

(i) Antimicrobial activity:

Muhammad et al., 2013, described antimicrobial potential of crude methanol extract and other fractions of V. betonicifolia. Antibacterial potential was estimated against seven bacterial strains (Enterobacter aerogenes, Proteus mirabilis, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Salmonella typhi and Enterococcus fecalis).

However, in a recent scientific study, methanol extract of another specie of Violaceae was found more potent against respiratory tract bacteria (Gautam et al., 2012). Viola tricolor herb is also known for its antimicrobial efficacy (Witkowska-Banaszczak et al., 2005).

Aqueous fraction obtained from V. betonicifolia methanol extract was predominantly active against C. albicans. Literature survey showed that some natural products have efficacy to completely inhibit the growth of C. albicans (Manohar et al., 2001). C. albicans is very common funal pathogen. In individuals which have impaired immune function, the infections caused by C. albicans may be life threatening (Scherer and Magee, 1990).

(ii) Neuro pharmacological potential:

Muhammad et al., 2012, examined hexane extract of V. betonicifolia for its neuropharmacological potential. The consequence of staircase test showed that animals fed with hexane extract of V. betonicifolia showed significant anxiolytic activity. In addition to it, muscle relaxing, antidepressant and sedative potential of V. betonicifolia hexane extract was also demonstrated.

(iii) Analgesic and anti-inflammatory activity:

Muhammad et al., 2012, observed dose dependent activity of methanol extract of V. betonicifolia in different pain models. But analgesic activity of this extract was totally antagonized by antagonists injection.

Moreover, it was also observed that the mentioned extract considerably antagonized the histamine and carrageenan induced inflammation. In addition to V. betonicifolia, wide

15 range of medicinal plants of Pakistan is reported for their anti-inflammatory activity (Zaidi et al., 2012).

(iv) Larvicidal activity:

Larvicidal activity of V. betonicifolia extracts was assessed against Aedes aegypti vector, which explored that ranking of extracts for larvicidal activity was as: chloroform > ethyl acetate > methanol with LC50 values of 13.03, 16.00 and 61.30 µg/ml, respectively (Muhammad et al., 2011). Dengue fever is serious health problem in different parts of world and Aedes aegypti vector is liable for it propagation (Jawale et al., 2010).

(v) Antipyretic potential:

Pyrexia induced by yeast is termed as pathogenic fever and it may be due to synthesis of prostaglandins (Moltz, 1993). Reduction in synthesis of prostaglandins may be a approach for antipyretic activity. In addition to it different mediators of pyrexia and hindrance to these mediators may also be a potential factor for antipyretic efficacy (Rawlins, 1973). But Muhammad et al., 2012, administrated methanol extract of V. betonicifolia in yeast induced hyperthermia to estimate its antipyretic potential and it showed a dose dependent effect which was close to standard drug.

(vi) Antioxidant activity:

Antioxidant activity of V. betonicifolia was determined by DPPH assay in a dose dependent mode (Muhammad et al., 2011). Maximum scavenging activity was examined in chloroform fraction followed by ethyl acetate, butanol, water and hexane with IC50 values of 80, 82, 176, 496 and 500 ppm, respectively.

Ebrahimzadeh et al., 2010, reported IC50 value of 245.1 µg/mL for Viola odorata.

IC50 values of Viola tricolor evaluated by DPPH assay was in range of 13.46 to 284.7 µg/mL

(Goncalves et al., 2012). Nikolova et al., 2010, evaluated IC50 value of Turkish V. tricolor L. above 200 µg/mL.

(vii) Cytotoxic activity:

Cytotoxic activity of crude methanol extract and different solvent fractions of V. betonicifolia was estimated by brine shrimp lethality assay (Muhammad et al., 2012). Surprisingly, methanol and ethyl acetate extracts exhibited 100% cytotoxicity.

Cytotoxic cyclotides of V. tricolor are also reported (Svangard et al., 2004). Deng et al., 2013, investigated cytotoxic activities of another specie of Violaceae (Viola philippica) by MTT assay. Lindholm et al., 2002, isolated cytotoxic cyclotides from Vioal odorata L. and Viola arvensis Murr.

(viii) Antiglycation activity:

Cinnamic acid derivative (2,4-dihydroxy, 5-methoxy-cinnamic acid) of V. betonicifolia exhibited antiglycation activity with IC50 value of 355 ± 7.56 µM. Ramkissoon et al., 2013, examined a strong correlation between anti-glycation activities and bioactive components of medicinal herbs.

2.3. Euphorbia royleana:

2.3.1. Ethno botanical description:

Euphorbia royleana is commonly known as ―Thor‖ or ―Danda thor‖ or ―Dozkhi meva‖in different areas of Pakistan. It is well known as ―Cactus‖ in English. It is spiny, succulent, glabrous, readily deciduous long shrub or small tree usually up to 5 m tall. It has stout stalk with 5-7 cm thick fleshy branches with pairs of thorns on the margins.

Flowers are yellow green, 3-4 in clusters in leaf axils. E. royleana belongs to family Euphorbeaceae, which is one of the largest family among flowering vegetation. This family consists of more than 300 genera and almost 8000 species are reported from this family.

2.3.2. Distribution:

It is widely distributed in Pakistan (Sabeen and Ahmad, 2009), India (Bani et al., 2000), China (Li et al., 2009), Nepal (Kunwar et al., 2010), Bhutan, Burma. Many members of Euphorbiaceae are distributed in hot and dry climatic conditions, but some exist as herbs and rainforest tress.

2.3.3. Ethno medicinal applications:

In Pakistan it is used in folk medicines for treatment of bladder stone, earache, loose motions and paralysis. For this purpose little amount of plant extract mixed with salt, is drop wise fed to babies for treatment of loose motions.

Moreover, a part of main stem body is cut and for few minutes, it is kept on the fire. The extract from this process is used for treatment of earache. For removing urinary bladder stone, people remove the bark of plant and eat jelly like matter in it. The above mentioned recepies are used in folk medicines, indigenous to Abbotabad, Islamabad, Pakistan (Sabeen and Ahmad, 2009).

Table. 2.3. Taxonomic classification of E. royleana

Taxonomic classification

Kingdom Plantae

Subkingdom Tracheobionta

Super division Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Subclass Rosidae

Order Euphorbiales

Family Euphorbeaceae

Genus Euphorbia

Species Euphorbia royleana

Latex of E. royleana is irritant. In traditional phytotherapies E. royleana latex is used as purgative, however in large doses it may be acrid and emetic (Singh and Singh, 2012).

Different species of Euphorbiaceae are used in folk medicines for treatment of inflammations, swollen belly, arthritis, insanity, infections, neuralgia, asthma, convulsions, cough, rheumatism, bowel problems, pimples, tumors, wounds, gonorrhea, earache, toothache, warts, diarrhea, constipation, ring worms, dysentery and jaundice (Kapoor, 1989; Kirtikar and Basu, 1991; Cataluna, 1999; Damme, 2001; Elujoba, 2005; Ali and Qaiser, 2009; Hussain et al., 2010).

2.3.4. Chemical evaluation:

(i) Phytochemical components:

Sivaraj et al., 2012, screened the pytochemical components (terpenoids, flavonoids, phenols, cardio glycosides, amino acids, carbohydrates, proteins, tannins, steriods, alkaloids, sterols and saponins) of E. royleana and four other plant species.

Phytochemical profile of E. royleana and five more plants of Euphorbiaceae is also documented (Tanvir et al., 1994). Glycosides and alkaloids were present in all the investigated plants. Alkaloids are copious secondary metabolites in plants and stand for one of the most widespread class of compounds endowed with multiple pharmacological properties (Stevigny et al., 2005).

(ii) Bioactive components:

Husain et al., 1992, reviewed amyrin, succinic acid, ingenol, tetracosanol, taraxerol, sitosterol, luepol, terpenes, phenolics, ellagic acid, stigmasterol, campesterol, diterpene, octacosanol and cycloroylenol from E. royleana. Tiwari et al., 2008, isolated cycloart-24-en- 3β-ol from latex of E. royleana

Total phenolic contents determined (Shahwar et al., 2010) by Folin-Ciocaltu method showed that chloroform extract of E. royleana had highest amount of phenolic contents.

Another study (Rastogi and Meharotra, 1993) narrated that poisonous and medicinal properties of E. royleana latex are ascribed to presence of bioactive components like 7- methoxy-3,4-benzocoumarin, epitaraxerol, euphol, taraxerol, m-hydroxy benzoic acid, 2,7- dihydroxy3,4-benzocoumarin, ellagic acid, sitosterol, 7-hydroxy 3,4-benzocoumarin.

(ii) Diterpenes:

Li et al., 2009, reported twelve diterpenes from E. royleana aerial parts. Ten were ingol lathyrane diterpenes and were reported for the first time, while two were known ingenol derivatives. It was interesting to note that ingol type diterpenes were also examined in Euphorbia antiquorum (Qi et al., 2014).

However, Yang et al., 2013, showed the existence of Jatropholane-type diterpenes in Euphorbia sikkimensis. Euphorbeaceae is rich in diterpenes. Another specie of Euphorbeaceae (Euphorbia paralias) is also known for diterpenes (Jakupovic et al., 1998). Diterpenes from Euphorbia prolifera and Euphorbia microsciadia are also in literature records (Ghanadian et al., 2012; Xu et al., 2013).

2.3.5. Biological evaluation:

(i) Anti-inflammatory activity:

Anti-inflammatory potential of latex of E. royleana was investigated in chronic and acute test models in mice and rats (Bani et al., 2000). E. royleana latex extracted with ethyl acetate represented the considerable reduction of oedema in mice and rats.

It is surprising to note that Euphorbia splendens also exhibited anti-inflammatory activity by inhibition of oedema in mice and rats (Bani et al., 1996).

Furthermore, chemical components of Euphorbia kansui are known for their anti- inflammatory activity (Yasukawa et al., 2000). Euphorbia nerifolia Linn. leaf extract was described for their anti-inflammatory potential (Gaur et al., 2009).

Anti-inflammatory efficacy of Euphorbia hirta was also in literature records (Das et al., 2010). Antiarithritic and immunosuppressive potential of E. royleana latex was also reported (Bani et al., 2000; Bani et al., 2005).

(ii) Antiangiogenic activity:

Angiogensis play main role in development of cancer (Carmeliet and Jain, 2000). Ingenol derivatives isolated from E. royleana aerial parts (Li et al., 2009) exhibited antiangiogenic effects on zebra fish model.

Ingenol derivatives may inhibit angiogensis by stimulating different protein such as angiostatin, platelet factor 4, prolactin 16 kd fragment, interferon, tissue inhibitor of metalloproteinase-1, -2, -3 and endostatin (Nishida et al., 2006).

(iii) Antioxidant activity:

Shahwar et al., 2010, examined antioxidant potential of different solvent extracts of E. royleana. Antioxidant activity determined by DPPH assay explored that ethyl acetate extract (34.7± 0.8%) had maximum potential. Comparatively higher DPPH scavenging activity (72.9.4± 0.78%) was observed in Euphorbia hirta leaf extract (Basma et al., 2011).

Other members of Euphorbiaceae (Euphorbia helioscopia, Euphorbia neriifolia, Euphorbia resinifera, Euphorbia hetrophylla, Euphorbia tirucalli) also had potent antioxidant potential (Jyothi et al., 2008; Sharma et al., 2011; Maoulainine et al., 2012; Okeniyi et al., 2012; Nadia and Benmahdi, 2013).

(iv) Toxic effects:

Parsad et al., 2010, narrated toxic effect of latex of E. royleana on Heteropneustes fossilis (cat fish), which is dwelling of fresh waters. Singh and Singh, 2012, evaluated muricidal and piscicidal potential of Euphorbia royleana latex against Channa punctatus (Snakehead fish) and Mus musculus (Swiss albino mice).

Molluscicidal efficacy of various fractions of Euphorbia royleana was estimated against Lymnaea (Radix) acuminata Lamarck which is dwelling of fresh waters (Tiwari et al., 2005).

Tiwari et al., 2008, isolated cycloart-24-en-3β-ol from latex of E. royleana and its piscicidal potential was explored by using a fish (Channa punctatus), dwelling of fresh water.

2.4. Psidium guajava:

2.4.1. Ethno botanical description:

Psidium guajava (family Myrtaceae) is common guava known as ―Amrood‖ in Pakistan. It is a fruit tree generally 3-10m high and has many branches.

(a) Leaves:

The leaves of P. guajava are opposite with short petiole (3-10 mm) and have prominent veins.

(b) Fruit:

The P. guajava fruit (3-6 cm long; 5cm diameter) is ovoid that resides a fleshy pericarp, juicy pulp and a seed cavity with several number of white kidney shaped or flattened hard seeds (Gutierrez et al., 2008; Okunrobo et al., 2010; Rai et al., 2010).

(c ) Root:

It has superficial root system with numerous deep roots, but lacking any discrete taperoot. Root system is very wide and extends beyond the canopy (Shruthi et al., 2013).

(d) Flower:

P. guajava has white flowers which are fragrant. The flowers have incurved petals with yellow anthers. P. guajava is a hard tree and can sustain in broad range of temperature, however the average temperature of 23-28ºC is optimum for its yield. Rai et al., 2010, reported that a tropical atmosphere and bursting sun shine is required for its healthy growth.

2.4.2. Distribution:

It is widely distributed in Pakistan, India, China, Mexico, Brazil, United States of America, Bangladesh, Thiland, Malaysia, Africa, Peru, Europe and many other countries of the world (Pathak and Ojha, 1993; Gutierrez et al., 2008; Elekwa et al., 2009; Joseph and Priya, 2011).

2.4.3. Ethnomedicinal applications:

It has wide ethno medicinal applications, while anti-diarrheal is the major one (Gutierrez et al., 2008). In Pakistan, it is traditionally used for treatment of cancer, pain, microbial infection, inflammation, diabetes and coughs (Sabeen and Ahmad, 2009).

In district Chakwal of Pakistan, P. guajava leaves are boiled with water and this decoction was used for treatment of high blood pressure (Sultana et al., 2006).

It is traditionally used in different forms for treatment of diarrhea, stomach ache, gastroenteritis, insomnia, asthma, dysentery, skin sore, menstrual disorders, ulcers, vertigo, edema, hysteria, swelling, nephritis, epilepsy, bowel disorders, cholera, convulsions, scabies, hemorrhoids, dyspepsia, vomiting, wounds, piles and other ailments in all over the world (Lutterodt, 1988; Nadkarni and Nadkarni, 1991).

Table. 2.4. Taxonomic classification of P. guajava

Taxonomic classification

Kingdom Plantae

Subkingdom Tracheobionta

Super division Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Subclass Rosidae

Order Myrtales

Family Myrtaceae

Genus Psidium

Species Psidium guajava

2.4.4. Chemical evaluation:

(i) Phenolic compounds:

P. guajava leaves have broad spectrum of phenolic components. Many phenolic compounds such as gallic acid, protocatechuic acid (Okuda et al., 1984), caffeic acid (Liang et al., 2005), ferulic acid (Zhu et al., 1997), chlorgonic acid (Qian and Nihorimbere, 2004; Liang et al., 2005), ellagic acid (Misra and Seshadri, 1968) and guavin B (Okuda et al., 1984; Zhu et al., 1997) are isolated from P. guajava leaves.

(b) Flavonoids:

Literature reports (Nakarni and Nadkarni, 1999; Arima and Dano, 2002; Michael et al., 2002; Liang et al., 2005; Prabu et al., 2006) showed the isolation of many flavonoids including quercetin, leucocyanidin, kaempferol, querecetin3-α-L-arabinofuranoside, guaijaverin, mecocyanin from leaves and other parts of P. guajava plant.

Arima and Danno, 2002, reported four flavonoids from P. guajava leaves and identified them as quercetin, morin-3-O-α-L-arabopyranoside, guaijavarin and morin-3-O-α- L-lyxopyranoside.

Vargas et al., 2006, showed the significant quantities of quercetin, apigenin, luteolin, myricetin and kaempferol in floral buds of P. guajava.

(ii) Carotenoids:

An other research group (Mercandante et al., 1999) isolated many carotenoids such as β-carotene, lycopene, rubixanthin, lutein, neochrome, β-cryptoxanthin, phytofluene and criptoflavin from P. guajava leaves and fruit. Reddy et al., 2012, explored β-carotene contents (40,000µg) of P. guajava leaves.

(iii) Triterpenes:

P. guajava is marvelous source of triterpenes. Different triterpenes such as oleanolic acid (Siddiqui et al., 2002), uroslic acid, β-sitosterol, 2α-hydroxyuroslic acid, guavacoumaric acid, guavenoic acid, asiatic acid, 2-α-hydroxyuroslic acid, 2α-hydroxy-3β-p-E- coumaroyloxyurs-12,18-dien-28-oic acid (Begum et al., 2002), arjunolic acid and jacoumaric acid (Salib and Michael, 2004) are isolated from P. guajava leaves, fruit and other parts.

Sesqui-terpenes, triterpenoids, flavonoida, alcohols and minerals are reported in twigs (Okwu and Ekeke, 2003).

(iv) Nutritive profile:

Conway, 2002, described the P. guajava contain 5 times higher vitamin C contents than oranges. Nutriative evaluation of P. guajava fruit showed the presence of moisture, carbohydrates, crude fiber , fats , protein , ash , calcium , iron, Vitamin A , thiamin , niacin and riboflavin (Fujita et al., 1985; Conway, 2002; Kamath et al., 2008).

Nadkarni and Nadkarni, 1999, reported the presence of Mn in this plant combined with malic, phosphoric and oxalic acids. Moreover, skin of P. guajava fruit is rich in ascorbic acid (Charles et al., 2006). Aminu et al., 2012, reported the presence of vitamin C in stem bark extract of P. guajava.

(v) Chemical composition essential oil:

Iwu, 1993, indicated the presence of β-sitosterol, nerolidiol, uroslic, guayavolic and crategolic acids in essential oil of P. guajava leaves. An extensive array of bioactive components including copaene, azulene, β-caryophyllene, limonene (Li et al., 1999), euclayptol (Oliver-Bever, 1986), octanol, 3-phenylpropanol (Kenneth et al., 1970), α- humulene, benzaldehyde, obtusinin (Jordan et al., 2003), ascorbic acid, amritoside (Fujita et al., 1985), asorbigen (Radha and Chandrasekaran, 1997), meocyanin (Salib and Michael, 2004) were isolated from essential oil of P. guajava. Paninandy et al., 2000, described the presence of 3-caryophyllene, caryophyllene oxide, 3-phenylpropyl acetate and nerolidol in essential oil of P. guajava fruit.

(vi) Other bioactive components:

Reddy et al., 2012, reported alkaloids, tannins, phenolics, flavonoids, reducing sugars, steroids, saponins, terpenoids in different extracts of P. guajava leaves. Shruthi et al., 2013, described the existence of tannis in bark of P. guajava tree.

Michel et al., 2002, reported the presence of quercetin-3-O-β-D-(2"- Ogalloyglucoside)-4'-O-vinylpropionate in guava seeds. Some other research groups

(Dweck, 2001; Hwang et al., 2002) narrated the existence of glykosen, protein and saccharose in P. guajava fruits.

Nagar and Rao, 1981, reported cytokinins from P. guajava plant. Ascorbic acid (34 mg) and glutathione (50.4 mMoles) contents of P. guajava leaves are also in literature records (Reddy et al., 2012).

Different active components such as phenol, 1,2-benzenedicarboxylic acid, diethyl phthalate, phthalic acid, phytol, 2,5-bis(1,1-dimethylethyl), asarone, butyldodecyl ester and mono(2-ethylhexyl) ester were isolated from ethyl acetate fraction of P. guajava root bark (Velmurugan et al., 2012).

2.4.5. Biological evaluation:

(i) Antioxidant activity:

Stem bark extract of P. guajava exhibited free radical (DPPH) scavenging activity (Aminu et al., 2012). Ogunlana and Ogunlana, 2008, evaluated antioxidant potential of P. guajava by scavenging superoxide, hydrogen peroxide and stable free (DPPH) radicals.

Reddy et al., 2012, narrated maximum stable free (DPPH) radicals scavenging activity in aqueous extract of P. guajava. However, another group of researcher (He and Venanat, 2004), showed that inhibition of free radicals (DPPH) in ethanol extract of P. guajava leaves were higher than aqueous extract.

Siow and Hui, 2013, demonstrated that antioxidant potential of fresh P. guajava is higher than oven dried one. Zahidah et al., 2013, investigated that ferric reducing antioxidant power (FRAP) and free radical (DPPH) scavenging ability of P. guajava leaves was greater than P. guajava seeds.

Antioxidant efficacy of P. guajava may be attributed to bioactive components like quercetin, gallic, ferulic, protocatechuic, ascorbic and caffeic acids in it (Thaipong et al., 2005).

(ii) Antimicrobial activity:

Zahidah et al., 2013, evaluated antimicrobial activity of P. guajava leaves and seeds against various microorganisms. In another study (Pandey and Shweta, 2011), antifungal activity of different extracts of P. guajava fruits and leaves was assessed by agar well diffusion assay.

Arima and Danno, 2002, isolated four compounds from P. guajava and evaluated their antibacterial activity against Salmonella enteritidis and Bacillus cereus. S. enteritidis was found more sensitive to isolated compounds. Velmurugan et al., 2012, studied antiviral activity of ethyl acetate fraction of P. guajava root bark.

Sato et al., 2000, reported that methanolic extract of ripe P. guajava fruit had fungicidal activity against Chaetomium funicola and Arthrinium sacchari. Ethanolic extract of P. guajava ripe fruit showed significant activity against Escherichia coli and Streptococcus mutans (Neira and Ramirez, 2005). Fungistatic activity of bark tincture of P. guajava was observed against Candida albicans (Dutta and Das, 2000).

(iii) Anticancer activity:

(a) Prostate cancer:

Chen et al., 2007, studied inhibition in growth of prostate cancer (DU-145) cells by aqueous extract of P. guajava leaves.

(b) Murine leukemia:

Manosroi et al., 2006, observed that essential oil of P. guajava leaves has potent activity against murine leukemia (P388) cells with IC50 value of 0.037 mg/mL. The efficacy of essential oil might be on the basis of monoterpenes in it (Cito et al., 2003).

Acetone extract of P. guajava branches exhibited antiproliferative efficacy against human colon carcinoma (HT-29) cells (Lee and Park, 2010). Ampasavate et al., 2010, narrated that P. guajava leaves extracted with ethanol had no effect on promyeloid (HL60), lymphoblastic (Molt4), erythroid (K562) and monocytic (U937) cells.

(d) Leukemia and ovarian cancer:

Levy and Carley, 2012, described the decrease in growth of Kasumi-1 leukemia and OV2008 ovarian cancer cells by hexane extract of P. guajava leaves.

Another group of scientist (Fernandes et al., 1997) studied anticancer activity of methanol extract of P. guajava against mice induced cancer that was inoculated B16 melanoma cells.

(e) Osteosarcoma, breast cancer and cervical cancer:

Sulain et al., 2012, extracted P. guajava leaves with different solvents and evaluated their anticancer activity against breast cancer (MDA-MB-231), osteosarcoma (MG-63) and cervical cancer (HeLa) cells. However, Kaileh et al., 2007, described that methanolic extract of P. guajava is effective against human breast cancer (MCF-7) and murine ficrosarcoma (L929sA).

A group of researchers (Sato et al., 2010) showed that anticancer activity of P. guajava was due to potential bioactive components in its bark, fruit and leaves.

(iv) Antidiabetic activity:

Diabetes mellitus is becoming a stern risk to human health. Some researcher has reported anti diabetic activity of fruit, leaf and bark extracts of P. guajava. Mukhtar et al., 2004, screened the decoction P. guajava leaves for hypoglycemic potential on alloxan- induced rats.

Moreover, significant reduction on LDL glycation by P. guajava aqueous leaf extract was observed in a dose-dependant mode. Some research groups (Ojewole, 2005; Wang et al., 2005) have narrated that tannins, pentacyclic triterpenoids, quercetin, flavonoids, guiajaverin and other bioactive components present in P. guajava plant are responsible for hypotensive and hypoglycemic effects.

(v) Antinociceptive activity:

Santos et al., 1998, examined that essential oil of P. guajava leaf and its component α-pinene had considerable antinociceptive activity. Shaheen et al., 2000, extracted the P. guajava leaves with different solvents (methanol, ethyl acetate and hexane) and evaluated their antinociceptive potential on central nervous system of mice.

2.5. Ziziphus mauritiana:

2.5.1. Ethno botanical description:

Ziziphus mauritiana (Rhamnaceae) is commonly known as ―Ber‖ in Pakistan. It is known as Indian jujube in English. It exists as small spreading tree (9-15 m long) or in form of large shrub with densely, drooping branches (Marwat et al., 2009).

(i) Leaves:

Leaves of Z. mauritiana are ovate or eliptic oblong. Leaves (2.5 to 6 cm long; 2 to 4cm wide) grow on alternate sides of branches. The leaves have dark green glossy look on the upper side with fine tooth on the margins (Marwat et al., 2009; Goyal et al., 2012).The leaf has three conspicuous, depressed and longitudinal veins.

(ii) Fruit:

The fruit has variable size usually 1.25-2.5 cm long. Under favorable conditions fruit may reach up to 6.25 cm in length and 4.5 cm in width. Fruit is ovate to oblong with fleshy, juicy, crispy and delicious pulp.

The color of fruit is yellowish or light green. It is of red brown color in ripe (Mahajan and Chopda, 2010). It has a single central seed which is very hard and oval in shape.

(iii) Seed:

The seed has two elptic kernals, each of them is 6 mm long (Anonymous, 1989).

(iv) Flower:

Z. mauritiana has 5 petteled greenish white small flowers with acrid smell. Flowers are produced in axillary cyme or small clusters (7-20 flowers). They had lobes in calyx.

2.5.2. Distribution:

Z. mauritiana is extremely drought and hardy tree. It is restricted to drier tropics and is natural vegetation of desert and rocky area in Pakistan and India (Goyal et al., 2012). It is cultivated and self sown in different areas of word including China, Afghanistan, Malaysia, Australia, Africa, Southern Florida, Colombia, Guatemala, Venezuelan, Belize, Ceylon.

2.5.3. Ethno medicinal applications:

Z. mauritiana is used in folk medicines all over the world. In Pakistan it is traditionally used for treatment of dysentery, wounds, fever, abscesses and intestinal worms. It is also used as blood purifier in form of herbal teas (Jabeen et al., 2009).

In Khushab, Punjab, Pakistan, women boil its leave with water and apply it on hairs to get smooth, healthy, long and shining hairs.

Moreover, in the same area people use leaves of Z. mauritiana to remove spines from any body part. For this purpose leaves are combined with wheat flour, oil and turmeric and are somewhat warmed over fire to make poultice which is applied externally on the skin to remove spines (Qureshi et al., 2011).

In North Western areas of Pakistan, people use leaves of Z. mauritiana for treatment of abscesses. For this purpose fresh crushed leaves are mixed with little quantity of soap and powdered loaf sugar and this paste is used for dressing of abscesses. The dressing is changed after one day and this treatment is practiced for 3 to 4 days. As a consequence of this treatment new abscesses will vanish and the older ones will burst (Marwat et al., 2009).

In addition to Pakistan, various segments of Z. mauritiana plant are used in folk medicines in different areas of world for treatment of cough, headache, asthma, diarrhea, biliousness, leucorrhoea, fever, smallpox, nausea, eye troubles, hoarseness of throat, constipation, pulmonary ailments, urinary infections, dysentery, ulcers, abdominal pains in pregnancy, vomiting and liver troubles (Nadkarni, 1986; Anonymous, 1989; Oudhia, 2003; Mahajan and Chopda, 2009).

2.5.4. Chemical evaluation:

(i) Alkaloids:

Chebouat et al., 2013, analyzed the crude alkaloidal extract of Z. mauritiana and lead to identification of ten alkaloids. Out of ten, three alkaloids (4-Methoxyquinoline, 2-Fluoro- 3-(1-hydroxy-2-(methylamino)ethyl) phenol, (2-(4-Amino-2-methylphenyl)-7-methyl-1H- indol-3-yl)(4-chlorophenyl)methanone) were identified in flowers.

Schmeller and Wink, 1998, reviewed the use of alkaloids in modern medications. Literature survey indicates potent role of vinca alkaloids in treatment of cancer (Noble, 1990; Hill et al., 1993).

Panseeta et al., 2011, narrated that alkaloids isolated from Z. mauritiana root extracts had antimycobacterial and antiplasmodial activities.

Srivastava and Srivastava, 1979, isolated zizogenin from stem of Z. mauritiana. Jossang et al., 1996, studied mauritine J in Z. mauritiana root bark. Panseeta et al., 2011, examined five alkaloids (mauritine L, nummularine B, hemsine A, nummularine H and maurtine M.) from methanol extract of Z. mauritiana roots. Bhatt and Dhyani, 2013, quantified alkaloids (2.2%) from Z. mauritiana.

(ii) Phenolic acids:

Memon et al., 2012, studied the phenolic acid profile of Z. mauritiana fruit extract. They quantified ten phenolic acids including protocatechuic, vanillic, p-coumaric, chlorgonic ,ferulic and caffeic acids. In this study bound and free phenolic acids were examined, but in previous literature surveys (San and Yildirim, 2010) only free phenolic acids were reported.

The variation in phenolic acid contents of different natural foods may be attributed to difference in geographical factors (Alstyne et al., 1998; Kumar et al., 2013).

Samee et al., 2006, extracted sliced fruit of Z. mauritiana with different solvents and demonstrated phenolics (148.0± 36.6 µg GAE/g) and ascorbic acid ( 488.3 ±7.3 µg/g) contents. However, Choi et al., 2011, examined lower content of phenolics in fruit and seed of sister Ziziphus species (Ziziphus jujuba).

(iii) Nutritive profile:

Nutritive evaluation (Morton, 1987; Pareek and Dhaka, 2008; Pareek et al., 2009) of Z. mauritiana fruit indicated the presence of fat (0.07 g), carbohydrates (17g), ash (0.3- 0.59g), niacin 90.7-0.0873 mg), total sugars (1.4-6.2 g), Ca (25.6mg), flouride (0.1-0.2 ppm), thiamine (0.02-0.024 mg), iron (0.76-1.8mg), protein (0.8 mg), phosphorus (26.8 mg), fiber (0.60 g) and other components. Another research group (Gupta et al., 2012) examined total ash, volatile matter, crude fiber and moisture contents in the Z. mauritiana leaves.

(iv) Nortriterpenes:

Ji et al., 2012, isolated six nortriterpenes from root extract of Z. mauritiana. Out of six three were known (Ceanothic acid, betulinic acid and ceanothenic acid) and three were novel (Zizimauritic acids A,B,C) nortriterpenes.

Table. 2.5. Taxonomic classification of Z. mauritiana

Taxonomic classification

Kingdom Plantae Subkingdom Tracheobionta

Super division Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Subclass Rosidae

Order Rhamnales

Family Rhamnaceae

Genus Ziziphus

Species Ziziphus mauritiana

Previously, various cyclopeptide alkaloids, triterpenes, aliphatic components, steroids and flavonoids are reported from this Ziziphus (Jossang et al., 1996; Singh et al., 2007; Pandey et al., 2008).

(v) Other bioactive components:

Goyal et al., 2012, reviewed phytocomponents of Z. mauritiana including jujubosides, betulonic acid, zizyberanalic acid, zizyberenalic acid, 2- hydroxybetulinic acid and leanonic acid.

Gupta et al., 2012, reported the presence of alakloids, proteins, carbohydrates, amino acids, phenolics, triterpenoids, saponins, glycosides and flavonoids in different extracts of leaves of Z. mauritiana. Phytochemical analysis revealed the presence of glycosides, tannins, phenol and saponins in Z. mauritiana leaves (Nijafi, 2013).

Bhatt and Dhyani, 2013, quantified saponins (92.65%), flavonoids (25.8%) and tannins (5 µg/ml) and saponins (92.65%) from fruit of Z. mauritiana. These bioactive components play leading role against cancer, AIDS, HIV infection, cardiovascular diseases and wide range of other ailments (Mcmahon et al., 1995; Urquiaga and Leighton, 2000; Mahajan and Chopda, 2009).

Flavonoids, phenolics and ascorbic acid contents of ripe and green Z. mauritiana fruits were demonstrated (Das, 2012). Literature survey indicated antimicrobial efficacy of flavonoiods, phenolics and ascorbic acid (Cushnie and Lamb, 2005; Tajkarimi and Ibrahim, 2011; Alves et al., 2013).

2.5.5. Biological evaluation:

(i) Antimicrobial activity:

Najafi, 2013, reported antimicrobial potential of Z. mauritiana leaves against two bacterial strains in a dose dependant mode. Many recent studies on antimicrobial potential of plant extracts are also narrated in dose dependent mode (Kuber et al., 2013; Ahmed et al., 2014).

Antimicrobial potential of Z. mauritiana leaves and bark against five bacterial and three fungal strains was evaluated (Mahesh and Singh, 2008). Antimicrobial activity of ripe and green Z. mauritiana fruit against panel of eight microorganisms was described by Das, 2012. Abalaka et al., 2010, extracted Z. mauritiana leaves with ethanol and evaluated their antimicrobial activity. An Indian study demonstrated that E. coli is less susceptible to Z. mauritiana leaves (Mahesh and Satish, 2008).

Ziziphus lotus exhibits considerable activity against E. coli (Naili et al., 2010). Cyclopeptide alkaloids from the root bark extract of Z. mauritiana are documented for antimycobacterial activites (Panseeta et al., 2011).

(ii) Antioxidant activity:

Antioxidant activity of two varieties (Narikeli kul and Local) of Z. mauritiana fruit was assessed by DPPH assay (Bhuiyan et al., 2009).

The antioxidant potential may be attributed to broad spectrum of phenolic and flavonoid contents in the Z. mauritiana fruit (Memon et al., 2012).

Lamien-Meda et al., 2008, determined antioxidant activity of wild Z. mauritiana fruit along with other thirteen edible fruit species. Z. mauritiana fruit variety from Thiland is also known for scavenging activity on DPPH radicals (Samee et al., 2006).

Antioxidant activity of five different solvent extracts of Z. mauritiana was evaluated by reduction of NBT and inhibition of nitric oxide in a dose dependant mode (Gupta and Singh, 2008). Previously some other research groups also used the dose dependant mode to evaluate antioxidant efficacy of plant extracts (Sim et al., 2010). Olajuyigbe and Afolayan, 2011, reported potent antioxidant potential of Ziziphus mucronata bark extracts.

(iii) Anticancer activity:

Mishra et al., 2011, explored anticancer activity of seeds of Z. mauritiana against three human carcinoma (cervical (HeLa), lymphoblastic leukemia (Molt-4), promyelocytic leukemia (HL-60)) and one normal (human gingival fibroblast (HGF) cell line by MTT

34 assay. Furthermore, the result of flow cytometric analysis explored that Z. mauritiana extract cause induction of apoptosis in HL-60 cells. Huang et al., 2007, examined that chloroform fraction of Z. jujube not only induce apoptosis but also G2/M arrest in HepG2 cells.

Betulinic acid isolated from Z. mauritiana roots (Panseeta et al., 2011) is narrated for its role against neck, head, lung, cervical and ovarian carcinoma (Pisha et al., 1995). Slight modification of betulinic acid structure can generate valuable derivatives, which may play significant role in development of antitumor drugs (Kim et al., 1998).

CHAPTER 3 MATERIAL AND METHODS

The research work demonstrated in this dissertation was carried out in the Central Hi Tech Laboratory, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan; Department of Experimental Therapeutics, Cytokine Research Laboratory, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, United States of America.

3.1. Materials:

3.1.1. Chemicals and reagents:

Aluminium chloride hexahydrate (Fluka chemicals, GmbH), potassium ferricyanide (DAEJUNG Chemicals and metals, Korea), ferric chloride (BDH laboratory supplies, UK). Trichloroacetic acid, 2,2′- diphenyl-1-picrylhydrazyl (DPPH), gallic acid (GA), quercitrin, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and various reference chemicals (gallic acid, vanillic acid, caffeic acid, syrengic acid, sinapic acid, ferulic acid, m- coumaric acid, p-coumaric acid and chlorogenic acid etc.) and rifampicin were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Methanol, chloroform and hexane were purchased from Merck KGaA (Darmstadt, Germany). Nutrient agar was obtained from Oxoid, Hampshire, UK. Nutrient Broth was acquired from LAB M, Limited UK. Potato dextrose agar and Sabarose Dextrose Broth were obtained from Applichem, Dermastadt, Germany. 0.45 µm filter paper was obtained from Biotech, Germany. Terbinafine was generously provided by Saffron pharmaceuticals, Faisalabad, Pakistan. Folin-Ciocalteu’s phenol reagent, sodium carbonate were acquired from Applichem, GmbH, Darmstadt, Germany. Sodium dodecyl sulfate and N,N-Dimethylformamide were provided by Fisher Scientific, USA. DPBS (Dulbeco’s phosphate buffered saline), Trypsin EDTA, 1X (0.25% trypsin, 2.2 Mm EDTA), DMEM (Dulbeco’s modification of Eagle’s medium, with 4.5 g/L glucose L- glutamine and sodium pyruvate), RPMI 1640with L-glutamine, Antibiotic-antimycotic solution (10,000 I.U./mL Penicillin, 10,000 µg/mL Streptomycin, 25 µg/mL Amphotericin B) were purchased from Mediatech Inc. Manassas, VA, USA. Ferric chloride was attained from BDH laboratory supplies, England. Fetal bovine serum was obtained from Atlanta Biologics. Human recombinant TNF-α purified from bacterial cells to homogeneity with a

36 specific activity of 5 × 107 units/mg was provided by Genentech (South San Francisco, CA). Optical density of cells was measured by ELISA (Dynex Technologies, USA)

3.1.2. Instruments:

The instruments used for different analyses during the study along with their company identification are listed in Table. 3.1.

Table. 3.1. Instruments with their model and company

Equipment Made Electric balance Shimadzu, Japan Blender Singer, FP-500 Rotary shaker Gallenkamp, England Water bath Memmertt, Japan Vacuum drying oven Memmertt, Germany Stereomicroscope Olympus, Japan Autoclave Omron, Japan Ultra low freezer Sanyo, Germany Laminar air flow Dalton, Japan Hot air oven Memmertt, Germany Spectrophotometer Lambda 25, Perkin Elmer, USA Phosphor-Imager imaging device Molecular Dynamics, Sunnyvale, CA ELISA Dynex Technologies, USA GC-MS (Model 6890GC-597MSD) Phenomenex, Torrance Simple microscope Nikon, Japan Centrifuge (GR G/2) Jouan, USA

CO2 incubator Thermo scientific HPLC LC-10A, SHIMADZU, JAPAN

3.1.3. Collection of plant materials:

Ethno botanical surveys were undertaken in various areas of Punjab, Pakistan, where herbal therapies are in common practice.

The plants were selected and collected on the basis of frequent use in traditional medicines for treatment of various ailments. Ethno medicinal information was basically collected from herbal practitioners of different areas of Punjab, Pakistan, including Lathianwala, Chuhdary wala and Faisalabad. In addition to it, dialogues, meetings, discussions and interviews with rural knowledgeable people about traditional use of plants, also provided substantial information. Ethno medicinal importance of selected plants was further confirmed from literature surveys (Sabeen and Ahmad, 2009). The collected plant materials were identified by by authentic herbarium techniques.

3.1.4. Plants used in current study:

(i) Euclayptus camaldulensis

(ii) Viola betonocifolia

(iii) Euphorbia royleana

(iv) Psidium guajava

(v) Ziziphus mauritiana

Euclayptus camaldulensis

Viola betonicifolia Euphorbia royleana

Psidium guajava Ziziphus mauritiana

Figure. 3.1. Photographs of selected Indigenous plants used in current study

3.1.5. Human cancer cell lines employed to access the anticancer potential of medicinal plants:

KBM5 cells HCT116 cells

SCC4 cells U266 cells Figure. 3.2. Human cancer cell lines (KBM5, myelogenous leukemia; HCT116, colon carcinoma; SCC4, tongue squamous carcinoma and U266 multiple myeloma) used in current study

KBM5 (Human myelogenous leukemia cells), U266 (Human multiple myeloma cells), SCC4 (Human tongue squamous carcinoma cells) and HCT116 (Human colon

40 carcinoma cells) used in current study were obtained from American Type Culture Collection (Manassas, VA) by The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA, where cell culturing experiments were carried out.

KBM5 and U266 cells were maintained in RPMI-1640. DMEM (Dulbeco’s modification of Eagle’s medium) was used for sustaining SCC4 and HCT116. Both the media were supplemented with 10% fetal bovine serum (Atlanta Biologicals), and antibiotic (10,000 I.U/mL). Cultures were maintained in 75cm2 flasks in humidified (95% air) incubator at 37ºC with 5% CO2.

3.1.6. Strains of microorganisms used to estimate antimicrobial activity of plant extracts:

(i) Escherchia coli (ii) Bacillus subtilis (iii) Pasterula multocida (iv) Aspergillus niger (v) Fusarium solani

The pure cultures of bacterial and fungal strains were procured from Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad and characterized from Department of Veterinary Microbiology, University of Agriculture, Faisalabad, Pakistan. These microbial strains were utilized to demonstrate antimicrobial activity of selected plant extracts.

3.2. Extraction of dried plant materials:

Fresh leaves of Euclayptus camaldulensis, Viola betonicifolia, Psidium guajava and Ziziphus mauritiana were rinsed with distilled water to remove dust and any particulate matter. Leaves were spread separately on paper sheets in well ventilated room.

Dried leaves were ground with help of food processor (Singer, FP-500) into fine powder. The powder was passed through seiver (0.25 mm). Sieved powdered material was stored in tightly packed glass jars. Extraction was carried out according to reported method (Sultana et al., 2009) with slight modification.

10 gm of leaf powder was extracted with 100 mL methanol on a rotary shaker at 350 rpm for 6 hour. Filtration was made with the help of Buchner funnel and Whatman No. 1 filter paper. Filtrate was evaporated in vacuum drying oven at 65°C to dryness. Dried extract was scratched with the help of sterilized spatula. Dried extract was transferred to extract vials and stored at -4°C for further use. Similar procedure was repeated with hexane and chloroform. Temperature of vacuum drying oven was adjusted at 62°C and 70°C for chloroform and hexane, respectively.

3.3. Extraction of fresh plant material:

Fresh plant material of Euphorbia royleana was washed with distilled water and cut into cubes of 2X2X2 cm3 with stain less steel knife (GLOBAL, Japan). Cubes of fresh plant material (stem) were immediately used for extraction according to modified method in literature (Shofian et al., 2011).

20 gm of cubes of fresh plant material were placed in 500 mL flask, mixed with 200 mL of methanol, plugged with cotton swab and tightly wrapped with aluminum foil. Extraction was carried out by using an orbital shaker at 350 rpm for 72 hours. After 72 hours filtration was done with the help of Whatman No.1 filter paper. The filtrate was evaporated at 65°C using vacuum drying oven (Memmert GmbH, Germany) to get dry extract. Solvent free extract was transferred to extract vials and stored at 4°C for further use. Similar practice for extraction was done with hexane and water.

3.4. Chemical evaluation:

3.4.1. Total phenolic content:

Total phenolics were determined by Folin-Ciocalteu process (Slinkard and Singleton, 1997; Jagadish et al., 2009). Briefly, one mL of plant extract solution (800 µg/mL) was mixed with 7.5 mL of double deionized water. Then 500 µL of Folin-ciocalteu reagent and one mL of 5% (W/V) Na2CO3 solution was added and mixed thoroughly.

Mixture was incubated for 90 min at room temperature. Measurement of absorbance was carried out at 760 nm by using UV-Vis spectrophotometer (Lambda EZ 201, Perkin

Elmer, USA). The same procedure was repeated for all the standard gallic acid solutions and a standard curve was obtained with following equation:

Absorbance = 0.005 µg gallic acid + 0.074 (R2 = 0.99)

Total phenols of each extract, as gallic acid equivalents, were determined using its absorbance measured at 760 nm as input to the obtained standard curve and its equation. All tests were carried out in triplicate.

3.4.2. Total flavonoid contents:

Total flavonoids were determined by the AlCl3 method. Briefly, 2 mL of plant extract solution (800 µg/mL) was mixed with 2 mL of aqueous AlCl3.6H2O (0.1 mol/L). Mixture was incubated at room temperature for 10 min and absorbance was measured with UV-Vis spectrophotometer at 417 nm.

The same procedure was repeated for all the standard quercetin solutions and a standard curve was obtained with following equation:

Absorbance = 0.040 µg quercetin + 0.051 (R2 = 0.99)

Total flavonoids of each extract, as quercetin equivalents, were determined using its absorbance measured at 417 nm as input to the obtained standard curve and its equation. All tests were carried out in triplicate with modified method (Lamaison and Carnat, 1990; Quettier-Deleu et al., 2000).

3.4.3. High Performance Liquid Chromatography (HPLC) analysis:

Gallic acid, caffeic acid, vanillic acid, chlorogenic acid, syringic acid, sinapic acid, m- coumaric acid, p-coumaric acid, ferulic acid, p-Hydroxy benzoic acid, catechin and quercetin were used as standard.

HPLC analysis was performed by hydrolysis of the test samples (50 mg) of each plant extract dissolved in 24 mL methanol and was homogenized. 16 mL distilled water was added followed by 10 mL of 6M HCl. The mixture was then thermostated for 2 hr at 95oC. The

43 final solution was filtered using 0.45 µm nylon membrane filter (Biotech, Germany) prior to high performance liquid chromatography (HPLC) analysis (Pak-Dek et al., 2011).

The separation of plant samples on gradient was performed using shim-pack column.

The chromatographic separation was carried out using as mobile phase gradient : A (H2O: Acetic acid- 94:6, pH = 2.27), B (acetonitrile 100%), 0-15 min = 15% B, 15-30 min = 45% B, 30-45 min = 100% B with 1 mL/min flow rate using UV- visible detector at 280 nm wavelength at room temperature at injection rate of 20 µL. The identification of each compound was established by comparing the retention time and UV-Visible spectra of the peaks with those previously obtained by injection of standards. The quantification was performed by external calibration.

3.4.4. Gas Chromatography Mass Spectrometry (GC-MS) study:

The analysis of plant extracts (0.5 mg/mL) was performed using a GC-MS that had an electron energy of 70Ev, ion source temperature of 230ºC and electron emission of 34.6 µA. The temperature of analyzer was maintained at 150ºC. Helium was used as carrier gas at flow rate of 1mL/min. The injector and interface temperature was set at 290ºC and 360ºC, respectively. The oven temperature was programmed as 50ºC (1 min) to 310ºC (20 min), at increasing rate of 6ºC/min. Compounds were identified on the basis of relative retention time and comparison of their mass spectrum with the spectrum of the known components stored in National Institute Standard and Technology (NIST) database of GC/MS system.

3.5. Biological Evaluation:

3.5.1. Free radical (DPPH) scavenging activity (DPPH assay):

Free radical scavenging capability of each extract solution on was determined on DPPH (2, 2’- diphenyl 1-picrylhydrazyl) radicals. A stock solution of 1000 µg/mL was prepared of each tested extract. Different dilutions (25, 50, 100, 200, 500 and 800 µg/mL) of each extract were prepared from respective stock solution of that extract.

Briefly, 4 mL of of DPPH (0.1mM) was mixed with 1mL of each of plant extract solution at different concentrations (25, 50, 100, 200, 500 and 800 µg/mL). Incubation of reaction mixture was carried out in dark room for 30 minutes and the free radical scavenging

44 ability was estimated by measuring the absorbance at 515nm with UV-Vis spectrophotometer.

The reaction was carried out in capped glass test tubes which were tightly wrapped with aluminum foil from top to bottom. The DPPH radical stock solution was freshly prepared every day for the reaction, and precautionary measures were taken to reduce the loss of free radical activity during the experiment. All experiments were carried out in triplicate with modified method (Ozturk et al., 2011). The inhibition percentage of DPPH radicals were calculated as:

Inhibition (%) of DPPH radicals = Ac – As/ Ac X 100

Where Ac is absorbance of control reaction (reaction in which all reagents participate except plant extract) and As is absorbance of sample (plant extract).

3.5.3. Antitumor activity (Potato disc assay):

Antitumor activities of each plant extract was evaluated by potato disc assay (McLaughlin and Rogers, 1998; Mahmood et al., 2012). The growth medium was prepared by adding 0.5 g sucrose, 0.8 g nutrient Nutrient Broth and 0.1 g yeast extract in 100 mL of distilled water and autoclaved. Medium was allowed to cool and one loop of Agrobacterium tumefaciens from storage culture was inoculated in growth medium using aseptic techniques. The culture was vigorously shaken and then placed on orbital shaker for 48 hrs at 28°C.

Red skinned potatoes were surface sterilized in 10% sodium hypochlorite for 20 minutes. Ends of potatoes were removed and again immersed in sodium hypochlorite for 10 minutes. Cylinders of potatoes were made using sterile cork borer. These cylinders were extensively washed with autoclaved distilled water and cut into discs with the help of surgical blades sterilized by gamma irradiation (2.5 M Rads). Agar was prepared 1.5% by dissolving 1.5g plane agar powder in 100 mL distilled water and autoclaved. Agar (25 mL) was poured into sterilized petri plates and allowed to solidify in laminar air flow. 8 potato discs were placed on each agar plate with the help of sterilized forceps (Rolzem international, Pakistan). 50µL of inoculums was placed on surface of each disc and allowed to diffuse for

10 to 20 minutes. DMSO was used as negative control. Plates were wrapped with para film and incubated at 27ºC for 21 days.

After 21 days staining of discs was made with Lugol solution (10 % KI+5 % I2) for 20 minutes and tumors were counted on each disc by using stereo microscope. The area where no tumors were found became brown or blue because starch of potatos had absorbed the dye, while the area of disc possessing tumor could not be stained and thus appeared creamy white. Percent inhibition was calculated by the formula (Kanwal et al., 2010).

Percentage inhibition = 1- Number of tumors in the sample / Number of tumors in negative

control X 100

3.5.4. Antimicrobial activity:

3.5.4. (a). Microbial culture preparation:

The plant extracts were individually tested against panel of microorganisms. Bacterial strains were cultured in Nutrient Broth and kept incubation at 37°C for 24 hours (Dhale and Markandeya, 2011). Fungal strains were grown in Sabouraud Dextrose Broth at 28°C for 48 to 72 hours. The turbidity in broth medium showed the growth of fungus. The cultures were stored in refrigerator at 2-8°C for further analysis.

3.5.4. (b): Disc diffusion assay:

The agar disc diffusion method (NCCLS, 1997) was used for determination of diameters of inhibition zones made by each plant extract solution against tested bacterial and fungal strains. The petri plates were washed and autoclaved. Briefly, 100 µL of suspension containing 108 colony forming units (CFU/ mL) of bacteria cells and 104 spore/mL of fungi were spread on petri plates containing Nutrient agar (NA) and Potato dextrose agar (PDA) medium (50 mL media/ plate). Sterile filter discs (4 mm) of wicks sheets were prepared and impregnated with 50 µL of sample solution (20 mg/mL) of tested plant extract and were placed in inoculated petri plates with the help of sterile forceps. Rifampicin (100 µg/mL) and Terbinafine (100 µg/mL) were used positive control in bacterial and fungal inoculated plates, respectively.

DMSO was used as negative control. The plates were incubated at 37°C for 24 hours and at 27°C for 48 hours for maximum bacterial and fungal growth, respectively. Antibacterial and antifungal activities were evaluated by measuring diameter (Millimeter) of inhibition zones with the help of digital zone reader.

Figure. 3.3. A typical agar plate showing antimicrobial activity in form inhibition zones

3.6. Anticancer attributes of plant extracts:

3.6. (a). Media preparation:

All the media components were heated at 37°C before media preparation. 5 mL of Antibiotic-antimycotic solution (10,000 I.U./mL Penicillin, 10,000 µg/mL Streptomycin, 25 µg/mL Amphotericin B) and 50 mL of FBS (Fetal Bovine Serum) were added into 500 mL of DMEM (Dulbeco’s modification of Eagle’s medium) and RPMI 1640, individually. The media were stored at 2-8°C till further used.

3.6. (b): Defrosting of cells:

DMEM and RPMI-1640 were prewarmed up to 37°C at least one hour before use. Cells (KBM5, U266, SCC4 and HCT116) were removed from liquid nitrogen and placed in an incubator at 37°C. Contents were transferred from vial to sterile centrifuge tube. Volume was made up to 10 mL by adding DMEM for SCC4 and HCT116 cells and RPMI-1640 for KBM5 and U266 cells, respectively.

Cells were centrifuged for 5 min at 700 rpm. Supernatant was removed and pallet of each cell type was suspended in 8 to 10 mL of respective media. Suspension of each cell type was transferred into 25 mL of cell culture flasks, individually.

Flasks were labeled with cell type and date initials. Flasks were incubated (95% humidified air 5% CO2 at 37ºC) for overnight.

3.6. (c). Trypsinisation of cells:

Cells (HCT116 and SCC4) were trypsinized when they were 80-100% confluent. Media was removed and cells were washed twice using 5 mL of PBS (Phosphate Buffer Saline) to partially remove dead cells as they are non-adherent to flask surface.

Pre-warmed trypsin-EDTA (1mL per 25 mL flask) was added and incubated (95% humidified air 5% CO2 at 37ºC) for 1-2 min. Flask was tapped gentle to dislodge the cells and then viewed under microscope. After the cells were completely disassociated from the flask, 4 Ml of prepared media (DMEM) was added into flask. Media was pipetted up and down several times to break up any cell lumps. Solution was divided into two fresh flasks. DMEM media about 5 mL was added to each flask.

Flasks were labeled and kept in incubator (95% humidified air 5% CO2 at 37ºC). After 24 hour media was removed from each flask to remove trypsin and fresh media was added.

3.6. (d). Cell counting:

10 µL of cell suspension and 10 µL of trypan blue were mixed by pipetting up and down few times. 10 µL of suspension was added to the prepared hameocytometer slide. Cells were counted in counted in each of 5 squares and mean was calculated.

3.6.1. Anticancer activity (MTT assay):

Anticancer assay was performed as described previously (Prasad et al., 2010). Briefly, 5,000 cells of HCT116, SCC4, KBM5 and U266 were seeded individually in Biolite

96-well (Thermo Scientific, Korea) plates and incubated (95% humidified air 5% CO2 at 37ºC) for 24 hr. Then cells were treated with different concentrations (10, 25, 50, 100 and

200 µg/mL) of plant extracts and incubated (95% humidified air 5% CO2 at 37ºC) for 72 hr. Then 20 µL of 5 mg/mL MTT was added to each well.

After 2 hour incubation each well was supplemented with 100µL of lysis buffer. Cells were further incubated for 6 hrs and optical density values were measured at 570 nm using an

MRX Revelation 96-well multiscanner (Dynex Technologies, USA). The IC50 values (concentration at which 50% of cells were killed) were calculated from the graph plotted concentration against percent cell viability.

3.6.2. Anti-inflammatory activity (Electrophoretic Mobility Shift Assay):

To assess the anti-inflammatory potential of potential extracts against KBM5 (Human myelogenous leukemia) cells, we have determined the NF-κB activation in cancer cells pretreated with respective plant extract. We isolated nuclei from treated-, untreated-, and induced-cells and performed electrophoretic mobility shift assay (EMSA) as described previously (Chainy et al., 2000).

In brief, nuclear extracts prepared from cancer cells were incubated with 32P end- labeled 45-mer double-stranded NF-κB oligonucleotide (15 μg of protein with 16 fmol of

DNA) from the HIV long terminal repeat (5′-TTGTTACAAGGGACTTTC CGCTG GGGACTTTC CAGGGA GGCGT GG-3′, with NF-κB-binding sites) for 30 min at 37 °C. The resulting protein-DNA complex was separated from free oligonucleotides on 6.6% native polyacrylamide gels. The dried gels were visualized by Phosphor-Imager imaging device (Molecular Dynamics, Sunnyvale, CA).

3.7. Statistical Analysis:

Three samples of each plant extract were assayed. Each sample was analyzed in triplicate and data is reported as Mean ± S.D. Minitab software version 16 was applied to perform analysis of variance (ANOVA) and to determine significant differences (P < 0.05).

CHAPTER 4 RESULTS AND DISCUSIONS

In this study five plants from four different families were collected from Punjab, Pakistan. These plants were examined for their chemical components by using various spectroscopic and chromatographic techniques such as, ultraviolet-visible (UV/Vis) spectrometry, high performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC-MS). Biological activities (antioxidant, antitumor, antimicrobial, anticancer and anti-inflammatory) of these plants were observed by different in vitro assays.

4.1. Euclayptus camaldulensis:

4.1. (a). Chemical Evaluation:

4.1.1. Total phenolic and flavonoid contents of E. camaldulensis :

Phenolic compounds are health benefactors. They as antioxidative agents (Ozturk et al., 2010). In the current study Folin-Ciocalteu (FC) method was used to estimate total phenolic contents of Euclayptus camaldulensis leaf extracts (methanol, chloroform and hexane). FC method is inexpensive, reproducible and rapid method, that is extensively used for measurement of total phenolic contents of plant extracts and is also narrated in various pharmacopoeias (Cicco et al., 2009; Blainski et al., 2013). The results obtained are represented in Figure. 4.1.1.

In FC method reaction of phosphomolybdate and phosphotungstate take place with the phenolic compounds present in the investigated sample (Ramirez-Sanchez et al., 2010). As a result of this reaction a blue pigment is generated that had extensive absorption of light at 760 nm (Schofield et al., 2001).

Total phenolic components of E. camaldulensis leaf extracts were solvent dependent and expressed as microgram gallic acid equivalents (µg GAE) per milligram of plant extract. Figure. 4.1.1. shows that total phenolic contents in different extracts (methanol, chloroform and hexane) varied widely ranging from 39.13 ± 0.30 to 148.68 ± 3.15 µg GAE/ mg of plant extract. Methanol extract exhibited the highest total phenolic contents (148.68 ± 3.15 µg

GAE/ mg of plant extract), followed by chloroform (75.75 ± 2.87 µg GAE/ mg of plant extract) and hexane (39.13 ± 0.30 µg GAE/ mg of plant extract) extracts. Our results are in agreement with earlier study in which extracting solvents (methanol, chloroform and hexane) exhibited the similar order (methanol > chloroform > hexane) for extraction of phenolic compounds (Yeboah and Majinda, 2009). We observed higher extent of phenolics in E. camaldulensis leaf extracts (methanol, chloroform and hexane), than previous reports on E. camaldulensis wood and bark extracts for the same compounds (Conde et al., 1995; Conde et al., 1996). It is interesting to note that Singab et al., 2011, demonstrated comparatively higher level of phenolics in methanol fractions of E. camaldulensis leaves.

160 Total phenolic contents (µg 140 GAE/mg of plant extract)

120 Total flavonoid contents (µg QE/mg of plant extract) 100

0 Methanol Chloroform Hexane E. camaldulensis extracts

Figure. 4.1.1. Total phenolic and total flavonoid contets of E. camaldulensis extracts (methanol, chloroform and hexane). Values are Mean ± SD of triplicate determinations. (P < 0.05). Total phenolic contents are expressed as microgram gallic acid equivalents per milligram of plant extract (µg GAE/ mg of plant extract). Total flavonoid contents are expressed as microgram quercetin equivalents per milligram of plant extract (µg QE/ mg of plant extract)

Flavonoids (polyphenolic plant compounds) are known for pharmacological activities (Robard et al., 1999). In current study flavonoid contents were determined using aluminum chloride colorimetric assay. In this method reaction of aluminum chloride with adjacent keto or hydroxyl groups of flavones or flavonols generates acid stable complexes (Chang et al., 2002), which showed maximum absorption at 417 nm.

Flavonoid contents of different E. camaldulensis leaf extracts are mentioned in Figure. 4.1.1. Significant difference (P < 0.05) was observed among flavonoid contents of methanol, chloroform and hexane extracts. The content of flavonoids expressed as (µg QE/ mg of plant extract) varied from 6.09 ± 0.96 to 20.45 ± 1.74 µg QE/ mg of plant extract, as shown in Figure. 4.1.1.

It is depicted from the results (Figure. 4.1.1) that methanol is the successful solvent for extraction of flavonoid contents from E. camaldulensis leaves. This is similar to previous findings in which methanol was used for extraction of flavonoids from medicinal plants (Stankovic, 2011). Our results in current study are in strong agreement with another scientific study (Hossain et al., 2011) who reported similar hierarchy (methanol > chloroform > hexane) of solvents for extraction of flavonoids. As compared to our finding, Abu-Qatouseh et al., 2013, explored relatively higher amount of flavoniods in another Euclayptus species.

4.1.2. High performance liquid chromatography (HPLC) analysis of E. camaldulensis:

The results of HPLC analysis of E. camaldulensis leaf extracts (methanol, chloroform and hexane) are shown in Table. 4.1.1. Amount of phenolic compounds in three extracts ranged from 0.12 ± 0.05 ppm to 5.86 ± 0.23 ppm. Syringic acid (0.63 ± 0.07 ppm) was the major component of hexane extract.

Among the investigated phenolic compounds, gallic acid was the dominating component of methanol and chloroform extracts. Maximum amount of gallic acid was examined in methanol extract (5.86 ± 0.23 ppm).

Extent of gallic acid in chloroform extract (2.21 ± 0.36 ppm) was greater than hexane extract (0.42 ± 0.15 ppm). Our results in current study are in accordance with previous finding in which predominant amount of gallic acid was observed in ethanolic extract of

Egyptian E. camaldulensis (El-Ghorab et al., 2003). Furthermore, Sasikumar et al., 2001, confirmed the presence of gallic acid in fresh leaves of E. camaldulensis.

Table. 4.1.1. High performance liquid chromatographic (HPLC) study of methanol, chloroform and hexane extracts of E. camaldulensis for identification and quantification of phenolic compounds (ppm)

Phenolic compounds Methanol extract Chloroform extract Hexane extract

Gallic acid 5.86 ± 0.23b 2.21 ± 0.36d 0.42 ± 0.15c

Syringic acid 1.75 ± 0.30d 1.97 ± 0.02d 0.63 ± 0.07c

p-Coumaric acid Nd 0.12 ± 0.05 Nd

Vanillic acid 4.53 ± 0.01ab Nd Nd

Quercetin 0.69 ± 0.11e Nd Nd

p-Hydroxy benzoic acid 2.91 ± 0.01c 1.33 ± 0.04a 1.01 ± 0.09b

Catechin 0.44 ± 0.07e Nd Nd

Nd = Not detected. Values are Mean ± SD of triplicate determinations. (P < 0.05)

The least existing component was p-coumaric acid with chemical contribution of 0.12 ± 0.05 ppm. Existence of p-coumaric acid is previously reported in specie of Euclayptus (Rashwan, 2002). Some research groups demonstrated antioxidant and antibacterial properties of p-coumaric acid (Lou et al., 2012; Kilic and Yesiloglu, 2013).

Katsuragi et al., 2010, reported biotransformation of p-coumaric acid by plant cell cultures of Euclayptus perriniana. Quercetin, an importrant flavonoid, was identified in methanol (0.69 ± 0.11 ppm) extract. Euclayptus globules is previously reported for existence of quercetin (Proestos and Komaitis, 2013).

4.1.3. Gas chromatography mass spectrometry (GC-MS) study of E. camaldulensis:

The outcome of GC-MS study of E. camaldulensis leaf extracts (methanol, chloroform and hexane) is presented in Table. 4.1.2. Chemical investigation showed

54 significant difference in composition of hexane extract from methanol and chloroform extracts. Only palmitic acid was common in three extracts with maximum contribution (31.06 %) in hexane extract. Considerable amount of palmitic acid was examined (Rencoret et al., 2007) in other Euclayptus species (Euclayptus globulus, Euclayptus maidenii, Euclayptus nitens, Euclayptus dunnii and Euclayptus grandis). Plamitic acid known as n- hexadecanoic acid is also observed in other medicinal plants (Al-Shammari et al., 2012; Rajeswari et al., 2012; Dubal et al., 2013). Aparna et al., 2012, evaluated anti-inflammatory potential of palmitic aicd (n-hexadecanoic acid). An in vivo study reported that palmitic acid regulates hypothalamic insulin resistance (Benoit et al., 2009). Al-Shammari et al., 2012, showed that essential oil rich in hexadecanoic acid exhibits significant antimicrobial activity.

Furthermore, we examined that eucalyptol, 5-Hydroxymethylfurfural, spathulenol, β- eudesmol, stearic acid and eicosane were common in methanol and chloroform extracts, as shown in Table. 4.1.2. In comparison to previous study on essential oil of E. camaldulensis fruit we obtained significantly higher amount of eucalyptol in both (methanol and chloroform) the investigated extracts. Euclayptol is also an important component of other aromatic plants (Vincenzi et al., 2002). Another study reported that eucalyptol plays considerable role in reduction of contractile activity in cardiac muscles of experimental organisms (Soares et al., 2005).

Chemical contribution of β-eudesmol in methanol and chloroform extract was 7.61 % and 1.24 %, respectively. β-eudesmol is investigated for in vitro and in vivo antiangiogenic efficacy (Tsuneki et al., 2005). Li et al., 2013, reported that β-eudesmol induces apoptosis in HL60 cells.

Moreover, pyrogallol, o-cymene, heneicosane, L-lyxose, alloaromadendrene and many other components were present in the investigated extracts (methanol, chloroform and hexane) at varying degrees. o-cymene is also examined in essential oils (Romanenko and Tkachev, 2006; Custodio et al., 2010). Pyrogallol is known for its antimicrobial potential (Kocacaliskan et al., 2006).

Reports on chemical composition of E. camaldulensis leaves are scant in literature. To the best of knowledge, our study is first report to explore chemical composition of

55 methanol, chloroform and hexane extracts of E. camaldulensis leaves. It is interesting to note that each extract (methanol, chloroform and hexane) of E. camaldulensis leaves had good profile of bioactive components with pharmacological potential.

Table. 4.1.2. Chemical constituents of methanol, chloroform and hexane extracts of E. camaldulensis analyzed by Gas chromatography mass spectrometry (GC-MS)

Components Retention Methanol Chloroform Hexane time extract extract extract Composition (%) Sec. isoamyl alcohol 5.28 ---- 6.61±0.12 ---- Eucalyptol 9.86 31.86±0.03 45.94±0.001 ---- o-cymene 9.69 ---- 0.63±0.04 ---- Catecholborane 13.33 ------10.56±0.09 5-Hydroxymethylfurfural 13.52 2.05±0.10 4.30±0.07 ---- Pyranone 14.47 ------1.17±0.04 o-methxy-p-vinylphenol 15.20 ------15.27±0.002 Pyrogallol 16.12 11.91±0.01 ------Aromandendrene 17.53 7.69±0.20 ------Succinamic acid 17.70 ------2.81±0.002 D-(+)-galactosamine 17.75 ------1.26±0.15 Alloaromadendrene 17.89 2.49±0.03 ------Epiglobulol 19.44 1.43±0.14 ---- 1.92±0.03 Lactose 19.59 1.07±0.03 ------Spathulenol 19.70 3.58±0.02 1.37±0.01 ---- Ledol 19.82 11.88±0.02 ---- 0.73±0.23 L-Lyxose 19.83 ------3.58±0.03 β -lactic acid 19.85 ------2.75±0.07 Viridiflorol 19.96 3.07±0.03 ------β-eudesmol 20.40 7.61±0.01 1.24±0.11 ----

56 d-Gulopyranose 22.16 2.22±0.04 ------Hentriaconatane 22.34 ---- 1.28±0.26 ---- Carhydrine 22.56 4.42±0.13 ------3-Heptadecene 23.13 ---- 2.25±0.19 ---- D(+)-Raffinose pentahydrate 24.33 1.07±0.06 ------Palmitic acid 27.27 4.38±0.13 6.94±0.01 31.06±0.03 Stearic acid 31.15 1.34±0.05 1.32±0.03 ---- Docosene 31.73 ---- 0.58±0.22 ---- Palmityl chloride 32.60 ---- 1.93±0.01 ---- Eicosane 38.32 1.71±0.11 1.18±0.04 ---- Benzisothiazolinone 38.57 ------2.22±0.13 Heneicosane 40.97 ---- 6.82±0.001 ---- Fatty alcohol 42.60 ---- 1.49±0.02 ---- Flunixin 43.38 ---- 1.16±0.17 ---- Stearyl iodide 43.49 ---- 1.21±0.09 ---- ( ---- ) = not detected. Compounds are identified on the basis of comparison of retention time and mass spectra in NIST data

4.1.(b). Biological Evaluation:

4.1.4. Free radical (DPPH) scavenging activity of E. camaldulensis :

DPPH assay is robust method to assess antioxidant potential of plant extracts or pure compounds (Gawron-Gzella et al., 2012). This assay is aimed at measuring the capacity of plant extract to scavenge purple colored 2,2-diphenyl-1-picryl hydrazil and converting it to yellow colored diphenylpicrylhydrazin (Saha et al., 2008).

Greater the extent of antioxidant compounds in plant extract, greater will be extent of yellow colored diphenylpicrylhydrazin molecules in the test solution and higher will be the antioxidant efficacy (Tepe et al., 2005).

In the current study we investigated antioxidant activity of E. camaldulensis leaf extracts (methanol, chloroform and hexane) in concentration dependant mode (25 to 800 µg/ml). Increase in free radical scavenging activity (%) was observed with increase in concentration of each extract (methanol, chloroform and hexane).

Table. 4.1.3. Scavenging (%) of free radicals (DPPH) by methanol, chloroform and hexane extracts of E. camaldulensis

E. camald- Scavenging (%) of free radicals at different concentrations (µg/mL) IC 50 ulensis (mg/mL) extracts 25 50 100 200 400 800 e a c d f b Methanol 23.64±1.92 37.51±1.26 55.66±1.51 68.93±0.21 79.22±0.95 87.61±2.03 0.08 a c e f b a a b f c e d Chloroform 21.74±1.06 30.28±2.49 35.95±1.97 57.11±5.13 72.48±2.70 74.93±3.24 0.15 b d d a d c c a a d b e Hexane 13.12±2.32 20.28±1.83 20.66±0.92 28.51±1.48 45.03±4.81 63.29±3.62 0.54 d f b e c e Values are Mean ± SD (Standard deviation) of triplicate determinations. Mean with different superscript letters in the same row indicate significant difference (P < 0.05) among concentrations tested.

Minimum percent inhibition (%) values for three extracts (methanol, chloroform and hexane) were examined at 25 µg/mL, while maximum rate of inhibition (%) was noticed at 800 µg/mL. Gallic acid that was used as positive control in free radical (DPPH) scavenging assay exhibited excellent free radical scavenging activity with IC50 value of 0.02 mg/mL. It is depicted from the results (Table. 4.1.3) that among the three extracts, methanol extract was examined as leading one with respect to inhibition (%) values.

Overall, among three extracts moderate free radical scavenging activity was observed in chloroform extract. Hexane extract exhibited minimum scavenging of stable free radicals at various doses (25 to 800 µg/mL). These results are in strong agreement with previous finding (Yusri et al., 2012) in which hexane extract of a Malaysian medicinal plant was reported as poor candidate for scavenging stable free radicals (DPPH) as compared to chloroform and methanol extracts. Our results are in accordance with the fact that different geographical conditions are innovative factor to effect phytochemical components and related pharmacological properties of medicinal plants (Edoga et al., 2005).

4.1.5. Antitumor activity of E. camaldulensis (Potato Disc assay):

Agrobacterium tumefaciens induces neoplastic disease in plants (Galsky et al., 1981). The consistency of this assay is based on the observation that some tumorigenic mechanisms are same in animals and plants. Galsky et al., 1981, showed that inhibition of crown gall tumor

58 in potato tissues had good correlation with compounds and extracts active in 3PS leukemic mouse assay.

The results of antitumor activity are shown in Figure. 4.1.2. Antitumor activity of three extracts varied from 17.06 ± 1.20% to 90.09 ± 0.70%.

100 Methanol Chloroform Hexane 90

(%) 50

40 Inhibition of tumors of Inhibition

0 25 50 100 200 400 800 Concentartion (µg/mL)

Figure. 4.1.2. Antitumor activity of methanol, chloroform and hexane extracts of E. camaldulensis, estimated by potato disc assay. Data is represented as Mean ± SD for three samples of each extract (methanol, chloroform and hexane) analyzed individually in triplicates. (P < 0.05).

Maximum antitumor activity was observed in methanol extract (IC50 = 59.68 µg/mL) followed by chloroform (IC50 = 112.5 µg/mL) and hexane (IC50 = 191.7 µg/mL) extracts. The activity of three extracts (methanol, chloroform and hexane) was significantly (P < 0.05) different from each other. This is in accordance with previous investigation in which

59 significant effect of extracting solvents was observed on A. tumefaciens induced tumors (Mazid et al., 2011).

Mclaughlin and Rogers (1998) narrated that potato disc assay is inexpensive, animal- sparing, rapid, statistically reliable and safe technique for preliminary screening of antitumor agents. Many researchers used this technique for screening of antitumor potential of plants (Islam et al., 2009; Bibi et al., 2011).

4.1.6. Antimicrobial activity of E. camaldulensis:

The methanol, chloroform and hexane extracts obtained from E. camaldulensis leaves were tested against five microorganisms in order to assess their antimicrobial efficacies. The results are shown in Table. 4.1.4. Among the bacterial strains, B. subtilis (21.78 ± 2.60 mm) was more susceptible, while E. coli (10.06 ± 0.19 mm) was the resistant one. Hexane extract showed no activity against E. coli and A. niger.

Tefsen et al., 2005, observed that outer membrane of gram negative bacteria like E. coli is bestowed with lipopolysachrides which provide them protection by conferring a negative charge on the surface. So the E. coli is less susceptible to examined extracts (methanol, chloroform and hexane).

In case of fungi, F. solani (18.56 ± 0.05 mm) was found to be more sensitive to methanol extract than A. niger (11.49 ± 0.86 mm). This finding is in line with previous study, in which methanol extract of a plant was found to be more potent against F. solani than A. niger (Alagesaboopathi, 2012). Significant antimicrobial activity of methanol extract might be attributed to higher level of bioactive components in it, which have strong correlation with antimicrobial activity (Si et al., 2006).

It is interesting to note that chloroform extract was more potent against A. niger (16.16 ± 0.07 mm) as compared to F. solani (9.27 ± 1.46 mm). These results are different

Table. 4.1.4. Antimicrobial activity of methanol, chloroform and hexane extracts of E. camaldulensis

Diameter of inhibition zones (mm) E. Bacterial Strains Fungal Strains camaldulensis Escherichia Bacillus Pasteurella Aspergillus Fusarium leaf extracts coli subtilis multocida niger solani

Methanol 10.06±0.19a 21.78±2.60c 15.21±0.34f 11.49±0.86a 18.56±0.05d

Chloroform 12.67±0.98b 18.43±1.54a 0 ± 0 16.16±0.07d 9.27±1.46a

Hexane 0 ± 0 14.84±0.03f 17.99±0.76e 0 ± 0 13.32±2.09c

*Rifampicin 21.66±1.41c 24.66±0.47b 23.33±1.69c Nt Nt

**Terbinafine Nt Nt Nt 25.66±1.69c 24.00±0.82b

Nt = not tested. Data is represented as Mean ± SD of triplicate determinations of each extracts (methanol, chloroform and hexane) against each microbial strain. Mean with different superscript letter in the same column indicate significant difference ( P < 0.05) among solvents tested. *Standard antibiotic for bacteria; * *Standard antibiotic for fungi. from previous finding (Shirurkar and Wahegaonkar, 2012) in which Euclayptus oil exhibited equivalent activity against A. niger (19 mm) and F. solani (19 mm). Methanol and hexane extracts were more effective against F. solani than A. niger. These results are in agreement with previous investigation, in which a potent medicinal plant (Chromolaena odorata) was found more active against F. solani than A. niger at the highest tested dose of extract (Llondu, 2014).

4.2. Viola betnocifolia

4.2. (a). Chemical Evaluation:

4.2.1. Total phenolic and flavonoid contents of V. betonicifolia extracts:

Total phenolic and total flavonoid contents in methanol, chloroform and hexane extracts of Viola betonicifolia leaves were demonstrated by using the Folin-Ciocalteu and

61 aluminum chloride colorimetric methods, respectively. The results are presented in Figure. 4.2.1.

Total phenolic contents of three solvents ranged from 29.35 ± 1.66 (µg GAE /mg of plant extract) to 155.78 ± 4.12 (µg GAE /mg of plant extract). Total flavonoid contents were in range of 2.06 ± 0.70 (µg QE / mg of plant extract) to 16.30 ± 1.27 (µg QE /mg of plant extract).

180 Total phenolic contents (µg GAE/mg of plant extract) 150 Total flavonoid contents (µg QE/mg of plant extract) 120

0 Methanol Chloroform Hexane

V. betnocifolia extracts

Figure. 4.2.1. Total phenolic and total flavonoid contents of V. betnocifolia extracts (methanol, chloroform and hexane). Values are Mean ± SD of triplicate determinations. (P < 0.05). Total phenolic contents are expressed as microgram gallic acid equivalents per milligram of plant extract (µg GAE/ mg of plant extract). Total flavonoid contents are expressed as microgram quercetin equivalents per milligram of plant extract (µg QE/ mg of plant extract).

It is evident from the results (Figure. 4.2.1) , that we observed higher extent of total phenolic contents in each extract as compared to total flavonoid contents. This observation is in line with scientific studies (Kaur and Mondal, 2014; Saeed et al., 2011) in which higher extent of total phenolic contents was observed in medicinal plants as compared to flavonoid contents.

It was observed that total phenolic and flavonoid contents vary significantly (P < 0.05) with variation in extracting solvent. These results are in strong agreement with previous findings (Shabir et al., 2011) where significant difference (P < 0.05) was observed in total phenolic and flavonoid contents for different extracts.

It was depicted from the results that three extracts shared the similar ranking (methanol > chloroform > hexane) in case of Folin-Ciocalteu and aluminum chloride colorimetric assays. These findings are in comparison with previous scientific studies in which identical hierarchy of extracting solvents (methanol > chloroform > hexane) was examined for measurement of total phenolic and total flavonoid contents (Yeboah and Majinda, 2009). In contrast to previous literature (Muhammad and Saeed, 2011) on Viola betonicifolia, we observed comparatively higher phenolic and lower flavonoid contents in current study.

4.2.2. High performance liquid chromatography (HPLC) analysis of V. betonicifolia extracts:

The extracts obtained from V. betonicifolia were investigated for the presence of phenolic compounds by HPLC analysis, as presented in Table. 4.2.1. For sinapic acid, methanol extract was the most rich (6.61 ± 0.40 ppm), followed by chloroform (3.20 ± 0.02 ppm) extract. Sinapic acid was absent in hexane extract. The extent of ferulic acid, gallic acid and p-coumaric acid ranged from 0.37 ± 0.001 ppm to 4.52 ± 0.15 ppm for the three extracts. Quercetin, was also detected in methanol extract (2.65 ± 0.003 ppm).

All the investigated compounds had distinct pharmacological properties. Zhao and Hu, 2013, observed that gallic acid increases death rate of human cervical carcinoma cells. Chemopreventive effects of gallic acid in mice model were also observed (Raina et al.,

2008). Antioxidant efficacy of p-coumaric acid is documented in literature records (Zang et al., 2000).

Picone et al., 2013, reported that ferulic acid is potential candidate for inhibition of oxidative stress. Some research group showed that ferulic acid and caffeic acid inhibit tumor promotion in mouse skin (Huang et al., 1988).

Siger et al., 2013, narrated antioxidant potential of sinapic acid derivatives. Thus, the biological activities (antioxidant, antimicrobial, anticancer, and anti-inflammatory) of V. betonicifolia evaluated in current study may be correlated to potential phenolic compounds in these extracts. To the best of our knowledge, phenolic compounds of V. betonicifolia are not previously reported in literature. Table. 4.2.1. High performance liquid chromatographic (HPLC) analysis of methanol, chloroform and hexane extracts of V. betonicifolia for identification of phenolic compounds (ppm)

Phenolic compounds Methanol extract Chloroform extract Hexane extract

Gallic acid 4.52 ± 0.15b Nd 0.29 ± 0.08c

Sinapic acid 6.61 ± 0.40cd 3.20 ± 0.02be Nd p-coumaric acid 0.93 ± 0.03 Nd Nd

Ferulic acid 3.28 ± 0.21a 0.37 ± 0.001b Nd

Quercetin 2.65 ± 0.003 Nd Nd

Nd = Not detected. Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicate significant difference (P < 0.05) among solvents (methanol, chloroform and hexane) used.

4.2.3. Gas chromatography mass spectrometry (GC-MS) study of V. betonicifolia extracts:

The results of GC-MS analysis of methanol, chloroform and hexane extracts of V. betonicifolia leaves were expressed in percentage (%) and are shown in Table. 4.2.2. It is

64 evident from the results that 24 compounds were identified in methanol extract. Total number of compounds in chloroform and hexane extract were 37 and 31 respectively.

Table. 4.2.2. Chemical constituents of methanol, chloroform and hexane extracts of V. betonicifolia analyzed by gas chromatography mass spectrometry (GC-MS) aComponents bRT Methanol Chloroform Hexane extract extract extract Composition (%)

Butanone dimethyl acetal 4.34 0.80 1.9 0.31

1,3-Dihydroxyacetone dimer 6.79 1.27 ------

Dicyclohexyl oxalate 8.41 ---- 0.49 ----

Glycerin 8.68 ---- 0.55 ----

Cyclopentanone,dimethylhydrazone 10.55 0.57 ------

4,5-Diamino-6-hydroxypyrimidine 10.76 ---- 0.85 ----

2-Hydroxy-2,3-dimethylsuccinic acid 10.95 ---- 0.76 ----

Geraniol 11.13 ------0.22

2,5-dihydropyrrrol 11.31 6.37 ------

Pyranone 12.02 1.18 ------

4H-Pyran-4-one, 2,3-dihydro-3,5- 12.14 ---- 0.94 ---- dihydroxy-6-methyl Coumaran 13.32 0.52 ------

5-Hydroxymethylfurfural 13.51 ---- 1.71 ----

1-Piperazineethanamine, 4-methyl- 13.56 0.72 ------

2-Methoxy-4-vinylphenol 15.20 ---- 1.01 ----

Eugenol 15.93 ---- 0.12 ----

Piperazine 17.41 ---- 0.74 ----

Octadecane 18.08 ---- 0.11 ----

2,4-Di-t-butylphenol 18.35 ------0.10

Nonadecyl pentafluoropropionate 18.53 ------0.15

Tetracosane 18.77 ------4.57

Lauric acid 18.96 ---- 0.53 ----

(E)-Stilbene 19.24 ---- 0.88 ----

Cetene 19.50 ------0.08

1-Methyl-4-amino-4,5(1H)-dihydro- 19.82 ---- 1.15 ---- 1,2,4-triazole-5-one Butanoic acid, 2-hexylimino- 19.95 ---- 0.83 ----

Ar-tumerone 20.86 ------0.72

Patchouli alcohol 21.22 ---- 0.33 ----

Curlone 21.56 ------0.37

Octacosyl trifluoroacetate 22.08 ------0.13

Mristic acid 22.31 1.02 2.07 0.69

Cis-pinane 24.33 0.55 ------

3-Nonen-1-ol, (Z)- 24.34 ---- 2.25 ----

Phytol, acetate 25.51 ---- 0.68 0.26

Methyl palmitate 26.54 ---- 1.21 0.38

Palmitic acid 27.29 25.67 19.48 22.52

Heptadecyl heptafluorobutyrate 28.07 ------0.41

Cyclohexanecarboxylic acid, cyclopentyl 28.87 ------0.18 ester

L-(+)-Ascorbic acid 2,6-dihexadecanoate 29.43 0.16 ------

Linoleic acid ethyl ester 30.10 ---- 0.64 ----

Methyl linolenate 30.22 ------1.62

Phytol 30.42 1.07 2.43 0.18

Linoleic acid 30.74 16.99 0.63 19.61

α-linolenic acid 30.87 18.48 9.72 13.63

Stearic acid 31.16 6.65 5.48 ----

1-Heptacosanol 31.74 ------1.536 p-Menth-8(10)-en-9-ol, cis- 32.80 ---- 0.82 ----

Eicosane 33.33 ------0.15

Butyl 9,12,15-octadecatrienoate 34.28 ------0.17

Funixin 35.47 ------0.10

Pentacosane 35.98 ------0.30

Eicosane 37.59 ------0.13

1-chloroheptacosane 38.32 0.51 ------

Octacosane 39.64 ------0.35

Squalene 40.22 ------0.10

Benzo quinoline, 2,4-dimethyl- 40.73 ---- 0.63 ----

2-Ethylacridine 41.00 ---- 22.15 ----

Silicic acid, diethyl bis(trimethylsilyl) 42.22 ---- 3.65 ---- ester 2,4 Dimethylbenzoquinoline 42.29 0.18 ------bis(trimethylsilyl) diethyl silicate 42.35 0.70 ------

β-Tocopherol 43.01 ------0.08

Thymol- TMS 43.38 1.24 ------

α-Tocopherol 44.16 ------1.53

Trimethyl[4-(1-methyl-1 45.19 ---- 0.24 ---- methoxyethyl)phenoxy]silane 5-Methyl-2-trimethylsilyloxy- 46.55 ---- 0.90 ---- acetophenone 4-Methyl-2-trimethylsilyloxy- 46.59 ---- 0.13 ---- acetophenone Silicic acid, diethyl bis(trimethylsilyl) 46.77 ---- 0.09 ---- ester 2,4 DimethylBenzoquinoline 47.32 1.80 ------

Stigmasterol 47.34 0.76 ------

γ-Sitosterol 47.68 3.42 ------

Fucosterol 47.97 0.19 1.92 ----

Arsenous acid, tris(trimethylsilyl) ester 48.43 ---- 0.38 ----

β-Amyrin 48.67 ------5.58

α-Amyrin 49.66 ------7.35

β-Sitostenone 50.62 5.45 ------

Tris(tert-butyldimethylsilyloxy)arsane 52.09 ---- 1.59 ----

( ---- ) = not detected. Compounds are identified on the basis of comparison of retention time and mass spectra in NIST data

Various chemical components such as α-linolenic acid, lactose, stearic acid, geraniol, eugenol, tetracosane, linoleic acid ethyl ester, cetene, patchouli alcohol, 2,6- dihexadeconate, ascorbic acid, methyl linoleate, phytol, heptacosanol, funixin, α-amyrin, β-amyrin, β- sitostenone, fucosterol, curlone, stigmasterol,α-tochopherol, β-tochopherol and many others are observed at varying degrees in the investigated extratcts.

Extent of linoleic acid ethyl ester (0.64 %) in chloroform extract of V. betonicifolia was lower than previously reported value (4.13 %) in an Indian medicinal plant (Gopalakrishan et al., 2011). Linoleic acid ethyl ester is reviewed for its anti-inflammatory,

68 hepatoprotective, insectifuge, antieczemic, antiandrogenic, antiacne, antiarthritic, nematicide and anticoronary activities.

α-amyrin and β-amyrin are potent triterpenes and are known for their antinociceptive prospective (Otuki et al., 2005). Amount of β-amyrin (5.58 %) in hexane extract of V. betonicifolia is different from previously reported value of β-amyrin (15.62 %) in petroleum ether extract of an Egyptian medicinal plant (Ibrahim, 2012).

Singh et al., 2009, reported in vivo antihyperglycaemic potential of α-amyrin acetate. Santos et al., 2012, examined in vivo hypolipidemic and antihyperglcemic efficacies of α- amyrin and β-amyrin. We also examined 7.35 % of α-amyrin in hexane extract of V. betonicifolia.

β-sitostenone (5.45 %) was potent component of methanol extract of V. betonicifolia. Chen and Wang, 2010, isolated β-sitostenone from an important Chinese medicinal plant along with other steroids.

Lee et al., 2003, isolated fucosterol from marine algae and evaluated it for hepatoprotective and antioxidant activities. We also observed fucosterol in methanol and chloroform extracts of V. betonicifolia as shown in Table. 4.2.2. Khanavi et al., 2012, narrated fucosterol fraction of marine algae for cytotoxic activities against colon and breast cancer cells.

Stilbenes are known for their antimicrobial attributes (Albert et al., 2011). Existance of (E)- stilbene (0.88 %) in chloroform extract of V. betonicifolia was examined.

GC-MS analysis of chloroform extract revealed the presence of silicic acid, diethyl bis(trimethylsilyl) ester. Extent of Silicic acid, diethyl bis(trimethylsilyl) ester is in comparison with previously reported value for an Indian medicinal plant (Tyagi and Sharma, 2014).

4.2. (b). Biological Evaluation:

4.2.4. Free radical (DPPH) scavenging activity of V. betonicifolia extracts:

The free radical scavenging activity of methanol, chloroform and hexane extracts of Viola betonicifolia was recorded in terms of percent inhibition (%) and results are shown in Table. 4.2.3. It is evident from the results that this activity has linear correlation with concentration (25 to 800 µg/mL) of each extract.

At higher concentration greater the extent of antioxidant components and higher will the rate of percent inhibition (%) of free radicals (Saha et al., 2008). In current study the three extracts (methanol, chloroform and hexane) effectively scavenged the DPPH radicals. The chloroform extract exhibited the highest free radical scavenging activity, with percent inhibition of (70.88 ± 0.45%) at the highest tested dose (800 µg/mL).

This is in accordance with previous study in which chloroform extract of Viola betonicifolia was examined for maximum inhibition of stable free radicals (Muhammad and Saeed, 2011). Lowest free radical scavenging activity was examined in hexane extract at various tested doses (25 to 800 µg/mL). Similar observation had already been reported for hexane extract of different medicinal plants of Pakistan (Bukhari et al., 2008; Khan et al., 2012). Statistical analysis revealed significant difference (P < 0.05) in free radical scavenging activities of different extracts at different concentrations.

Table. 4.2.2. Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform and hexane extracts of V. betonicifolia

V. betonici- Scavenging (%) of free radicals at different concentrations (µg/mL) IC 50 folia (mg/mL) extracts 25 50 100 200 400 800 a c d f e b Methanol 12.35±0.56 20.70±0.82 24.26±0.67 25.70±1.93 40.41±2.71 42.02±0.22 b c f e d c >1 f e c b d a Chloroform 19.44±0.08 30.16±3.73 36.75±2.21 41.12±2.36 58.46±1.51 70.88±0.45 0.23 c d c f b e b d f a c e Hexane 8.51 ±0.78 9.17 ±1.59 12.22±0.90 16.28±0.24 30.68±0.37 39.47±0.69 >1 f a b b e d Values are Mean ± SD (Standard deviation) of triplicate determinations. Mean with different superscript letters in the same row indicate significant difference (P < 0.05) among concentrations tested.

4.2.5. Antitumor activity of V. betonicifolia extracts (Potato Disc Assay):

It is evident from the literature that some plant extracts exhibit a cell-type antitumor efficacy (Rehman et al., 2009). In current study antitumor potential of methanol, chloroform and hexane extracts of Viola betonicifolia was evaluated by potato disc assay, as shown in Figure. 4.2.2. The three extracts were able to significantly (P < 0.05) inhibit the growth of A. tumefaciens at varying extents.

100 Methanol Chloroform Hexane 90

(%) 50

40 Inhibition of tumors of Inhibition

0 25 50 100 200 400 800 Concentration (µg/mL) Figure. 4.2.2. Antitumor activity of methanol, chloroform and hexane extracts of V. betonicifolia. Data is represented as Mean ± SD for three samples of each extract (methanol, chloroform and hexane) analyzed individually in triplicates. (P < 0.05).

We examined that inhibition of tumors (%) increase linearly with increase in dose of extracts. This observation is strongly supported by previous study (Mahmood et al., 2012) in which increase in crown gall tumor inhibition (%) was observed with increase in dose of extracts. Maximum antitumor activity (IC50 = 38.13 µg/mL) was examined in chloroform

extract. Antitumor activity of methanol extract (IC50 = 120.9 µg/mL) was greater than hexane

extract (IC50 = 196.4 µg/mL).

4.2.6. Antimicrobial activity of V. betonicifolia extracts:

The in vitro antimicrobial activity of methanol, chloroform and hexane extracts of V. betonicifolia against employed microorganisms was assessed by disc diffusion assay. As can be seen from Table. 4.2.4, the two extracts (methanol and hexane) illustrated considerable in vitro antimicrobial potential against all the investigated microorganisms (Escherichia coli, Bacillus subtilis, Pasteurella multocida, Aspergillus niger and Fusarium solani). However, chloroform extract was inactive against E. coli, P. multocida and F. solani.

Table. 4.2.4. Antimicrobial activity of methanol, chloroform and hexane extracts of V. betonicifolia

Diameter of inhibition zones (mm) V. Bacterial Strains Fungal Strains betonicifolia Escherichia Bacillus Pasteurella Aspergillus Fusarium extracts coli subtilis multocida niger solani

Methanol 9.97±0.57ab 13.23±1.52cd 11.05±0.83a 0 ± 0 10.46±1.24e

Chloroform 0 ± 0 8.74 ±0.07ae 0 ± 0 12.74±0.92cd 0 ± 0

Hexane 16.65±1.30bd 20.10±2.26c 7.68±0.09f 17.37±0.15cd 0 ± 0

*Rifampicin 21.66±1.41e 24.66±0.47b 23.33±1.69cd Nt Nt

**Terbinafine Nt Nt Nt 25.66±1.69af 24.00±0.82ac

Nt = Not tested. Data is represented as Mean ± SD of triplicate determination of each extracts (methanol, chloroform and hexane) against each microbial strain. Mean with different superscript letter in the same column indicate significant difference ( P < 0.05) among solvents tested. *Standard antibiotic for bacteria; * *Standard antibiotic for fungi.

The diameter of inhibition zones ranged from 7.68 ± 0.09 mm to 20.10 ± 2.26 mm. It is evident from the results that hexane extract showed good activity against most of tested

72 microbial strains. Potent antimicrobial activity of hexane extract might be attributed to presence of α and β amyrin in it, as represented in GC-MS analysis (Table. 4.2.2).

Johann et al., 2007, explored that α and β amyrin and their derivatives have distinctive antifungal activities. It was examined that α and β amyrin, α and β amyrin forminate and α and β amyrin acetate exhibited potential antifungal activities against all the investigated Candida species.

Surprisingly, α and β amyrin forminate was found as effective as fluconazole in inhibiting the adhesion of C. albicans to buccal epithelial cells. A. niger was found more sensitive to hexane extract than F. solani. These results are in line with previous study in which A. niger was examined more susceptible to Carica papaya extracts than F. solani (Llondu, 2011). However, methanol extract was effective against F. solani than A. niger. This observation is in strong agreement with earlier scientific study in which aqueous exrtact of an important plant of Pakistan was found more active against F. solani than A. niger (Ahmed et al., 2014).

4.2.7. Anticancer activity of V. betonicifolia extracts:

The relationship between doses of V. betonicifolia leaf extracts (methanol, chloroform and hexane) and their cell growth inhibiting effects on KBM5, myelogenous leukemia; SCC4, tongue squamous carcinoma and HCT116, colon carcinoma cells was evaluated by MTT assay.

The incubation of cell lines (KBM5, SCC4 and HCT116) with V. betonicifolia leaf extracts decreased the viability of these cell lines. Similar observation was noticed in previous study in which decrease in viability of lung (A549), colon (HCT116), breast (MCF7) and liver (HepG2) cancer cells was noticed with increase in concentration of Suadi plant extracts (Elsharkawy and Alshathly, 2013).

Based on the results of MTT assay the cell survival rate (%) was calculated for each concentration of extracts (Figure. 4.2.3). Overall the three extracts of V. betonicifolia leaves were active against the tested cell lines (KBM5, SCC4 and HCT116). The results in Figure. 4.2.3. (A) revealed that the methanol extract had poor effects on viability (%) of cancer cell

73 lines (KBM5, SCC4 and HCT116) as compared to chloroform and hexane extracts, whose results are represented in Figure. 4.2.3. (B) and Figure. 4.2.3. (C), respectively. The chloroform extract revealed remarkable decrease in viability of KBM5 cells with

IC50 value of 0.001 mg/mL followed by hexane (IC50 = 0.002 mg/mL) and methanol (IC50 = 0.067 mg/mL) extracts.

In case of SCC4 cells maximum anticancer activity was observed in hexane (IC50 =

0.096 mg/mL) extract followed by chloroform (IC50 = 0.103 mg/mL) and methanol (IC50 =

0.155 mg/mL) extracts. HCT116 cells were more susceptible to chloroform extract (IC50 =

0.063 mg/mL), while resistant to methanol extract (IC50 = 0.183 mg/mL). Moderate anticancer activity was observed by hexane extract (IC50 = 0.094 mg/mL) against HCT116 cells.

120 KBM5 SCC4 HCT116

100 A

Cell viability (%)viability Cell 40

0 0 10 25 50 100 200 Dose (µg/mL) 120 KBM5 SCC4 HCT116

100 B

60 Cell viability (%)viability Cell 40

0 0 10 25 50 100 200 Dose (µg/mL)

120 KBM5 SCC4 HCT116

100 C

60 Cell viability (%)viability Cell

0 0 10 25 50 100 200

Dose (µg/mL) Figure. 4.2.3. Anticancer activity of (A) methanol (B) chloroform and (C) hexane extracts of V. betonicifolia against KBM5 (Human myelogenous leukemia), SCC4 (Human tongue squamous carcinoma) and HCT116 (Human colon carcinoma) cell lines. Values are represented as Mean ± SD of quadruplicate determinations. (P < 0.05).

4.2.8. Anti-inflammatory activity of V. betonicifolia chloroform extract:

The inflammatory transcription factor is an important target for prevention of cancer (Gupta et al., 2011). Many investigations had demonstrated that plant extracts play promising role for in vitro NF-κB inhibition. In current study we examined the NF-κB inhibitory potential of chloroform extract of V. betonicifolia leaves.

Figure. 4.2.3. Anti-inflammatory activity of chloroform extract of V. betonicifolia. Lane 1 and lane 2 show unstimulated Myelogenous leukemia (KBM5) cells. In lane 3 cells were treated with TNF-α alone. In other lanes (4-6) cells were pre-incubated with chloroform extract of V. betonicifolia (10, 25 and 50 µg/mL) for 12 hours and then treated with 0.1 nM TNF-α for 30 minutes. Nuclear extracts were prepared and assayed for NF-κB activation by EMSA.

In this assay cells were treated with increasing doses of V. betonicifolia chloroform extract, and subsequently stimulated with TNF-α. Then equal amounts of nuclear extracts from cells (Human mylegenous leukemia) were investigated by EMSA for NF-κB activity.

As indicated in Figure. 4.2.4, activation of NF-κB almost completely inhibited by pretreatment of mylogenous leukemia cells with 50 µg/mL. It was postulated that anti- inflammatory potential of chloroform extract might be attributed to unique profile of bioactive components in it, as shown in Table. 4.2.2. Various components (Table. 4.2.2) of chloroform extract are known for their anti-inflammatory activity (Srinivasan et al., 2007; Boots et al., 2008; Chao et al., 2009).

4.3. Euphorbia royleana:

4.3.(a). Chemical Composition:

4.3.1. Total phenolic and flavonoid contents of E. royleana:

Total phenolic contents (TPC) of methanol, aqueous and hexane extracts of fresh E. royleana ranged from 30.65 ± 0.46 and 63.68 ± 0.43 µg GAEs/ mg of plant extract, respectively. Surprisingly, highest extent of phenolic compounds (63.68 ± 0.43 µg GAEs/mg of plant extract) was observed in hexane extract followed by methanol extract. Our results in current study are in strong agreement with previous findings in which higher phenolic contents were observed for hexane extract followed by methanol extract (Aris et al., 2009). A recent scientific study (Rahman et al., 2013) narrated that phenolic contents of hexane extract of an Indian medicinal plant was higher than that of methanolic extract.

It is interesting to note that FC reagent may also react with some non phenolic substances in reaction mixture (Ikawa et al., 2003; Everette et al., 2010) and results in unusuall higher extent of total phenolic contents. It is worthy to note that phenolics include diverse range of compounds which are often polar in nature, however, due to non polar linkages they are extracted with less polar and non polar solvents (Mokhtarpour et al., 2014). Furthermore, steric hindrance in polar compounds, awards them non polar naturte. In contrast to previous studies on phenolic contents of Euphorbiaceace, we observed comparitvely higher extent of phenolic contents in hexane extract of fresh Euphorbia royleana (Shahwar et

78 al., 2010). To the best of our knowledge, there is no literature report about phenolic contents of fresh Euphorbia royleana.

Methanol, hexane and aqueous extracts of fresh E. royleana exhibited significant (P < 0.05) amount of flavonoid contents as represented in Figure. 4.3.1. Maximum amount of flavonoids (µg QE/ mg of plant extract) was examined in hexane extract (47.47 ± 0.71). This is in line with previous investigation, in which maximum amount of flavonoids was observed in hexane extract of an endemic Indian medicinal plant (Fidrianny et al., 2014). Flavonoid contents of fresh Euphorbia royleana extracts are in comparison with other medicinal flora of Pakistan (Naz and Bano, 2013).

90 Total phenolic contents (µg 80 GAE/mg of plant extract) Total flavonoid contents (µg 70 QE/mg of plant extract)

0 Methanol Hexane E. royleana extracts

Figure. 4.3.1. Total phenolic and total flavonoid contents of E. royleana extracts (methanol, hexane and aqueous). Values are Mean ± SD of triplicate determinations. (P < 0.05). Total phenolic contents are expressed as microgram gallic acid equivalents per milligram of plant extract (µg GAE/ mg of plant extract). Total flavonoid contents are expressed as microgram quercetin equivalents per milligram of plant extract (µg QE/ mg of plant extract).

4.3.2. High performance liquid chromatography (HPLC) analysis of E. royleana:

HPLC is frequently used technique for identification of phenolic compounds (Brum et al., 2013). The identification of phenolic compounds in methanol, hexane and aqueous extracts of E. royleana was done by comparing the HPLC chromatograms of these compounds with HPLC chromatogram of standard compounds at the same conditions. The data is shown in Table. 4.3.1.

Methanol extract was found to be the highest in ferulic acid (4.59 ± 0.23 ppm). Another specie of Euphorbeaceae (Euphorbia Tirucalli) is also known for existence of ferulic acid (Araujo et al., 2014).

Table. 4.3.1. High performance liquid chromatographic (HPLC) analysis of methanol, and hexane extracts of E. royleana for identification and quantification phenolic compounds (ppm)

Phenolic compounds Methanol extract Hexane extract Aqueous extract

Gallic acid 2.62 ± 0.18bd 1.01 ± 0.51a 2.13 ± 0.01bd

Chlorogenic acid 1.08 ± 0.40ab 0.16 ± 0.001bc 1.49 ± 0.02ab

Ferulic acid 4.59 ± 0.23cf 0.51 ± 0.09ad 5.13 ± 0.05cf

Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicate significant difference

(P < 0.05) among solvents (methanol and hexane) used.

In addition to ferulic acid, gallic acid and chlorogenic acid were also detected in two extracts at varying degrees. Our results in current finding are in accordance with previous scientific studies in which presence of phenolic acids was reported in different members of Euphorbeaceae (Loh et al., 2009; Liu et al., 2011; Rakhimov et al., 2011; Jahan et al., 2013).

It is interesting to note that extent of phenolic compounds in current study is different from those detected in other members of Euphorbeaceae. This might be attributed to the fact that different plant species within the same family own a unique chemical profile (Cai et al., 2004).

Phenolic compound profile of hexane extract of E. royleana is in agreement with current scientific studies (Hussain et al., 2014; Sharma and Vig, 2014) which reported the existence of phenolic acids in hexane extracts of medicinal flora. To the best of our knowledge, our study is first report on phenolic profile of Euphorbia royleana.

4.3. (b). Biological Evaluation:

4.3.3. Free radical (DPPH) scavenging activity of E. royleana:

The stable free radical (DPPH) has been extensively used as tool to assess antioxidant efficacies of plant extracts or other antioxidant substances (Szabo et al., 2007). The two extracts (methanol and hexane) showed the significant (P < 0.05) free radical scavenging activity through all concentrations, as shown in Table. 4.3.2.

Minimum scavenging activities by methanol (18.22 ± 1.11%) and hexane (16.09 ± 0.51%) extracts were examined at 25 µg/mL. However, when concentration increased, free radical scavenging activities were also increased and maximum scavenging activities by methanol (56.78 ± 1.37%) and hexane (60.52 ± 0.39%) extracts were exhibited at 800 µg/mL.

It is surprising to note that hexane extract exhibited higher percent inhibition (%) values than methanol extract. These results were in strong agreement with previous finding in which hexane extract of an important medicinal plant was examined for the higher inhibition (%) of DPPH radicals as compared to methanol extract (Sim et al., 2010). Another study by Dhar et al., 2013, demonstrated that hexane extract of phytococktail had high free radical (DPPH) scavenging activity than methanol extract. Our observation was supported by some other group of scientists who narrated high free radical (DPPH) scavenging activity of hexane extract (Bae et al., 2012; Rahman et al., 2013).

The free radical scavenging activities for methanol and hexane extracts determined in the current study were in comparison with that reported for chloroform, n-butanol and ethyl acetate extracts of Euphorbia royleana (Shahwar et al., 2010). Some research groups (Turkmen et al., 2006; Zhao et al., 2006) reported that nature of extracting solvent plays critical role in demonstrating the antioxidant potential of plants.

Table. 4.3.2. Scavenging (%) of stable free radicals (DPPH) by methanol and hexane extracts of E. royleana

IC 50 E.royleana Scavenging (%) of free radicals at different concentrations (µg/mL) (mg/mL) extracts 25 50 100 200 400 800 a b c d e f Methanol 18.22±1.11 23.46±0.64 28.22±1.46 33.51±1.30 46.71±1.25 56.78±1.37 0.58 c d a f b e d f b e c a Hexane 16.09±0.51 24.89±0.68 31.19±0.38 39.38±1.19 50.96±0.44 60.52±0.39 0.38 c d c a e f e a f c d b 17.47±1.49 20.18±0.96 25.95±1.51 36.68±0.29 51.46±0.46 Aqueous 11.18±0.77a b d e f c 1.13 Values are Mean ± SD (Standard deviation) of triplicate determinations. Mean with different superscript letters in the same row indicate significant difference (P < 0.05) among concentrations tested. Mean with different subscript letters in the same column indicate significant difference (P < 0.05) among solvents tested.

4.3.5. Antitumor activity of E. royleana:

Euphorbia royleana is traditionally used for the treatment of paralysis, bladder stone, earache, inflammation and other diseases (Sabeen and Ahmad, 2013). However, antitumor potential of this plant was not scientifically reported. So in current study, antitumor activity of fresh Euphorbia royleana was examined by potato disc assay.

To achieve the goal, different doses (25 to 800 µg/mL) of methanol, hexane and aqueous extracts of fresh Euphorbia royleana were evaluated against Agrobacterium tumefaciens growth. The results (Figure. 4.3.2) of this assay showed that tumor inhibition (%) linearly increased with increase in dose of each extract. This trend was in agreement with previous finding (Kanwal et al., 2011).

We observed comparatively higher tumor inhibition in hexane extract at all the tested doses (25 to 800 µg/mL). Maximum antitumor activity was observed in hexane extract (IC50 = 267.5 µg/mL). These results are in strong agreement with previous finding (Mazid et al.,

2011) in which hexane extract (IC50 = 200 µg/disc) of an important medicinal plant was reported for higher inhibition of A. tumefaciens induced tumors as compared to methanol extract (IC50 > 400 µg/disc). Effect of extracting solvents and doses on tumor inhibition was statistically significant (P < 0.05).

100 Methanol Hexane 90

(%) 50

40 Inhibition of tumors of Inhibition

0 25 50 100 200 400 800 Concentartion (µg/mL)

Figure. 4.3.2. Antitumor activity of methanol, aqueous and hexane extracts of E. royleana. Data is represented as Mean ± SD for three samples of each extract (methanol and hexane) analyzed individually in triplicates. (P < 0.05).

4.3.6. Antimicrobial activity of E. royleana:

The results of antimicrobial potential of methanol and hexane extracts of fresh Euphorbia royleana are presented in Table. 4.3.3.

The three extracts exhibited poor activities against Escherichia coli and Pasteurella multocida. Our results in current study were found better than previous finding which demonstrated that Euphorbia trigona extracts had no effect on growth of E. coli and B. subtilis (Upadhyay et al., 2013).

Minimum antibacterial was examined against E. coli (Gram negative) with inhibition zone of 5.67 ± 0.57 mm. These results are in agreement with previous finding in which similar inhibition zone (5 mm) was observed against gram negative bacteria by methanol extract of vegetable (Ullah et al., 2013). Hexane extract strongly inhibited the growth of Bacillus subtilis. The most susceptible fungal strain was Aspergillus niger (14.00 ± 1.00 mm). Despite of the antifungal activity of our extracts, chloroform extract from the aerial parts of Euphorbia trigona also exhibited considerable antifungal activity (Upadhyay et al., 2013).

Table. 4.3.2. Antimicrobial activity of methanol, aqueous and hexane extracts of E. royleana

Diameter of inhibition zones (mm)

E. royleana Bacterial Strains Fingal Strains

extracts Escherichia Pasteurella Bacilus Aspergillus Fusarium

coli multocida subtilis niger solani

Methanol 5.67±0.57b 8.33 ±0.57c 10.67±0.57b 11.66±0.57c 10.00±1.00a

Hexane 7.00±1.00b 10.33±0.57c 12.00±0.57b 14.00±1.00b 10.33±0.57a

Aqueous 6.00±1.00b 8.00±1.00c 8.33±0.00c 9.33±0.57a 8.66±0.57a

*Rifampicin 21.66±1.41a 23.33±1.69b 24.66±0.47a Nt Nt

**Terbinafine Nt Nt Nt 25.66±1.69d 24.00±0.82b nt = Not tested. Data is represented as Mean ± SD of triplicate determination of each extracts (methanol, hexane and aqueous) against each microbial strain. Mean with different superscript letter in the same column indicate significant difference ( P < 0.05) among solvents tested.

*Standard antibiotic for bacteria; * *Standard antibiotic for fungi.

Overall the order of antimicrobial activities of two extracts was as: hexane > methanol. Hexane extract exhibited distinctive antimicrobial activity, as compared to methanol extract. Higher antimicrobial activity of hexane extract was also in line with literature records (Akhter, 2008; Alaribe et al., 2011; Zakaria et al., 2011).

In another study, Akinyele et al., 2011, screened crude n-hexane extract of a husk against forty five strains of Vibrio pathogens and twenty five other bacterial isolates. Surprisingly, hexane extract exhibited activity against 21 bacterial and 38 of test Vibrio species. Extracts from other Euphorbia species (Euphorbia hirta, Euphorbia milii, Euphorbia nerifolia, Euphorbia heterophylla) were also found active against some bacterial and fungal strains (Bakkiyaraj and Pandiyaraj, 2011; Ekundayo and Ekekwe, 2013; Venkatanagaraju and Goli, 2014).

4.4. Psidium guajava:

4.4. (a). Chemical Evaluation:

4.4.1. Total phenolic and flavonoid contents of P. guajava extracts:

Phenolic and flavonoid compounds act as antioxidants by scavenging free radicals, chelating metal ions, donating hydrogen and quenching singlet oxygen (Aminu et al., 2012). In addition to it they play major role against cancer, microbial infections and vast range of degenerative ailments (Quettier-Deleu et al., 2000; Jagadish et al., 2009). So while investigating pharmaceutical activities of P. guajava leaves, it was quite necessary to explore the extent of these bioactive components. Total phenolic and flavonoid contents (TPC and TFC) of three extracts (methanol, chloroform and hexane) were represented as µg gallic acid equivalent/mg of plant extract and µg qurecetin equivalents/mg of plant extract, respectively. The results are shown in Figure. 4.4.1.

The results showed that extraction of TPC with three solvents was as follows: methanol (83.34 ± 0.49) > chloroform (71.49 ± 0.48) > hexane (53.24 ± 2.05), while the order of solvents in case of TFC was methanol (53.39 ± 0.89) > chloroform (32.76 ± 1.15) > hexane (21.26 ± 1.49). Difference in amount of phenolics and flavonoids in different solvents is acknowledged to nature of solvent (Bae et al., 2012).

Total phenolic and flavonoid contents of methanol extract were higher than previous study (Aminu et al., 2012). However, Qian and Nihorimbere, 2004, investigated significantly higher phenolic contents in polar extract of P. guajava leaves.

100 Total phenolic contents (µg GAE/mg of plant extract)

80 Total flavonoid contents (µg QE/mg of plant extract)

0 Methanol Chloroform Hexane P. guajava extracts

Figure. 4.4.1. Total phenolic and total flavonoid contents of P. guajava extracts (methanol, chloroform and hexane). Values are Mean ± SD of triplicate determinations. (P < 0.05). Total phenolic contents are expressed as microgram gallic acid equivalents per milligram of plant extract (µg GAE/ mg of plant extract). Total flavonoid contents are expressed as microgram quercetin equivalents per milligram of plant extract (µg QE/ mg of plant extract).

4.4.1. High performance liquid chromatography (HPLC) analysis of P. guajava extracts:

Phenolic compounds detected in methanol, chloroform and hexane extracts of P. guajava are represented in Table. 4.4.1. The common phenolic compound of three extracts (methanol, chloroform and hexane) was quercetin. Caffeic acid, ferulic acid and vanillic acid were also examined. Gallic acid was observed in methanol (5.62 ± 0.29 ppm) and chloroform (3.21 ± 0.57 ppm) extracts.

Previously, Verza et al., 2008, quantified considerably higher extent of gallic acid in P. guajava leaves. Wu et al., 2009, examined that gallic acid present in P. guajava leaves, exhibit inhibitory effects on glycation process. Another study (Kato et al., 2001) showed that gallic acid derivative of P. guajava stimulated production of cytokines by Th2 cells. Lin and Yin, 2012, determined that P. guajava extract rich in quercetin, caffeic acid and ferulic acid exhibited significant in vivo renal protective effects. Rattanachaikunsopon and Phumkhachorn, 2010, studied that quercetin isolated from P. guajava leaves exhibited significant antibacterial activity against E. coli, P. fluorescens, V. cholerae, S. aureus, L. monocytogenes, B. hermosphacta, S. enterica and B. stearothermophilus.

Table. 4.4.1. High performance liquid chromatographic (HPLC) study of methanol, chloroform and hexane extracts of P. guajava for identification and quantification of phenolic compounds (ppm)

Phenolic compounds Methanol extract Chloroform extract Hexane extract

Gallic acid 5.62 ± 0.29bc 3.21 ± 0.53d Nd

Caffeic acid 0.70 ± 0.14 Nd Nd

Chlorogenic acid 2.35 ± 1.03 Nd Nd

Ferulic acid 1.00 ± 0.19d 1.17 ± 0.26e Nd

Vanillic acid 2.72 ± 0.11 Nd Nd

Quercetin 11.84 ± 0.002be 5.09 ± 0.08ac 3.61 ± 0.47d

4.4.3. Gas chromatography mass spectrometry (GC-MS) study of P. guajava extracts:

Chemical components of methanol, chloroform and hexane extracts are shown in Table. 4.4.2. Pyrogallol (35.18%), vitamin E (30.70%) and palmitic acid (30.73%) were the major chemical constituents of methanol, hexane and chloroform extracts, respectively.

Pyrogallol has wide range of pharmacological properties, including inhibition of proinflammatory cytokines (Nicolis et al., 2008).

Table. 4.4.2. Chemical components of methanol, chloroform and hexane extracts of P. guajava analyzed by gas chromatography mass spectrometry (GC-MS) aComponents bRT Methanol Chloroform Hexane extract extract extract Hydroxydimethylacetic acid 5.19 ---- 3.018 ----

Tridecyl trifluoroacetate 13.88 ---- 2.543 ---- n-cetane 14.47 ---- 2.229 ----

Farnesan 15.59 ---- 3.396 ----

Pyrogallol 16.10 35.18 ------

α-Copaene 16.42 11.92 ---- 2.18

Caryophyllene 17.21 6.36 ---- 8.43

Aromandendrene 17.35 3.48 ------

Dodecyliodide 17.83 ---- 3.170 ----

Heneicosane 17.83 ------6.28

Alloarmadendrene 17.89 2.52 2.182 ----

γ-Muurolene 18.04 2.30 ------

Eicosane 18.08 ---- 3.637 ----

Tetracosane 18.09 ------2.77

β-Bisabolene 18.41 2.23 ------

β –chamigrene 18.58 ---- 6.492 ----

α-Calacorene 19.11 ---- 3.260 ----

Cetene 19.50 ---- 2.238 ----

Caryophyllene oxide 19.83 19.71 ------

α-Bulnesene 19.83 ------11.37

Epiglobulol 20.13 2.64 ------

Ledol 20.14 ------2.38 cis-Thujopsene 20.40 3.56 ------

Copaene 20.69 ------3.27

Culmorin 21.15 1.43 ------

E-15-Heptadecenal 23.13 ---- 17.78 ---- cis-Z-α-Bisabolene epoxide 23.48 1.86 ------

Palmitic acid 27.27 5.05 30.73 ----

Stearic acid 31.15 1.76 8.86 ----

Hexachlorodisiloxane 32.55 ---- 2.95 ----

Squalene 40.22 ------15.36

Vitamin E 44.12 ------30.70

γ-Sitosterol 47.63 ------17.26

( ---- ) = not detected. Compounds are identified on the basis of comparison of retention time and mass spectra in NIST data Vitamin E also plays significant role against cancer (Constantinou et al., 2008). Carr et al., 2000, reported antiatherogenic potential of vitamin E. Singh et al., 2005, narrated that vitamin E is potential candidate against inflammation. Effectiveness of vitamin E against atherosclerosis is also evidenced by literature (Upston et al., 2003). Extent of palmitic acid (n-hexadecanoic acid) in methanol and chloroform extracts of P. guajava leaves is higher than previously reported value for P. guajava essential oil (Khadhri et al., 2014).

Other main components were caryophyllene oxide, copaene, alloaromadendrene, caryophyllene, palmitic acid, sitosterol, α-bulnesene, α-copaene and squalene. Caryophyllene oxide is used as preservative in cosmetics, food stuff and drugs. Antifungal potential of caryophyllene oxide is also documented (Yang et al., 1999).

Existence of caryophyllene oxide in P. guajava hexane extract is previously described Presence of α-copaene in fruit extract of P. guajava is reported in a scientific study (Meckes et al., 1998). β-caryophyllene oxide induce apoptosis in human prostate cancer cells (Ryu et al., 2012). Li et al., (1999), reported comparatively higher level of copaene and caryophyllene in essential oil from P. guajava leaves.

α-bulnesene (11.37%) an effective component of hexane extract of P. guajava as indicated in Table. 4.4.2. Some research groups (Hsu et al., 2006; Tsai et al., 2007) examined inhibitory prospective of α-bulnesene on platelet-activating factor.

Our results differ with regard to chemical constituents from previously reported results for hexane extract of P. guajava from India (Nisha et al., 2011). Analysis of methanol, chloroform and hexane extracts of P. guajava leaves by rapid and reliable technique (GC-MS) led to identification of bioactive components with therapeutic background.

4.3. (b). Biological evaluation: 4.4.2. Free radical (DPPH) scavenging activity of P. guajava extracts:

It is evident from our results (Table. 4.4.3) that inhibition of free radicals (%) is elevated with rise in concentration of investigated extracts (methanol, chloroform and hexane). This observation is supported by previous study in which a linear relationship was observed between inhibition (%) of free radicals (DPPH.) and dose of plant extract (Sim et al., 2012).

The free radical (DPPH.) scavenging activity of methanol extract was observed to be higher than that of chloroform and hexane extracts, respectively. Maximum free radical scavenging activity (93.19 ± 1.43%) seen in methanol extract of P. guajava was found to be higher than alcoholic extract of P. guajava root bark (75.6%) (Kuber et al., 2013). This

90 difference might be attributed to different geographical conditions; the key factor to effect phytochemical components and related pharmacological properties of medicinal plants

(Edoga et al., 2005). Scavenging activity of methanol extract (IC50 = 0.09 mg/mL) is in comaprison with gallic acid (IC50 = 0.02 mg/mL) that was used as positive control.

Table. 4.4.3. Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform and hexane extracts of P. guajava

P.guajajva Scavenging (%) of free radicals at different concentrations (µg/mL) IC 50 extracts (mg/mL) 25 50 100 200 400 800 d a c a f f Methanol 25.35±0.88 27.45±1.03 52.37±3.02 71.34±1.70 92.67±0.99 93.19±1.43 0.09 b d f c e a a b b d e c Chloroform 20.34±2.67 31.03±4.42 31.72±1.89 50.97±3.23 54.54±2.15 75.31±1.14 0.20 d c d a b f f e b c a d Hexane 16.23±0.63 18.10±0.66 29.43±1.93 38.05±1.51 50.09±4.87 59.90±3.31 0.42 a b c e d b Values are Mean ± SD (Standard deviation) of triplicate determinations. Mean with different superscript letters in the same row indicate significant difference (P < 0.05) among concentrations tested. Means with different subscript letters in the same column indicate significant difference (P < 0.05) among solvents tested.

Scavenging efficacy of hexane extract on DPPH stable free radicals was lower than chloroform and methanol extracts at all the tested doses (25 to 800 µg/ml). This finding is in agreement with previous study in which hexane extract of a Pakistani medicinal plant was reported for poor free radical scavenging activity than methanol extract (Iqbal et al., 2013). However, contrary to our observation, Fidrianny et al., 2102, reported comparatively different values of DPPH inhibition in hexane extract of P. guajava leaves.

4.4.3. Antitumor activity of P. guajava extracts: In this trial different concentrations (25 to 800 µg/mL) of methanol, chloroform and hexane extracts of P. guajava were evaluated for their antitumor activity by bench top potato disc assay (Figure. 4.4.2).

The results indicated that percent tumor inhibition was elevated with rise in concentration of P. guajava extracts (methanol, chloroform and hexane). These results are in accordance with previous scientific reports (Hussain et al., 2007; Rehman et al., 2009) on antitumor activity of medicinal flora of Pakistan, which showed decline in A. tumefacien

91 induced tumors with increase in concentration of plant extracts. The order of antitumor activity of three extracts was as: hexane (IC50 = 65.02 µg/mL) > chloroform (IC50 = 160.7

µg/mL) > methanol (IC50 = 337.4 µg/mL). This observation is in agreement with previous finding in which hexane extract was reported for higher anticancer activity as compared to polar extracts (Shylesh et al., 2005).

100 Methanol Chloroform Hexane 90

(%) 50

40 Inhibition of tumors of Inhibition

0 25 50 100 200 400 800 Concentartion(µg/mL)

Figure. 4.4.2. Antitumor activity of methanol, chloroform and hexane extracts of P. guajava. Data is represented as Mean ± SD for three samples of each extract (methanol, chloroform and hexane) analyzed individually in triplicates. (P < 0.05).

4.4.5. Antimicrobial activity of P. guajava extracts:

The results of antimicrobial activity assay are given in Table. 4.4.4. The maximal inhibition zones for microbial strains sensitive to P. guajava extracts (methanol, chloroform and hexane) were in range of 10.42 ± 0.91 mm to 19.33 ± 0.18 mm. Overall, the methanol

extract of P. guajava exhibited stronger antimicrobial potential than chloroform and hexane extracts, respectively. Hexane extract was inactive against the tested bacterial strains (E. coli, B. subtilis and P. multocida).

Table. 4.4.4. Antimicrobial activity of methanol, chloroform and hexane extracts of P. guajava

Diameter of inhibition zones (mm)

P. guajava Bacterial Strains Fungal Strains

extracts Escherichia Bacillus Pasteurella Aspergillus Fusarium

coli subtilis multocida niger solani

Methanol 13.74±1.11b 10.42±0.91bc 12.66±1.52d 15.21±0.14cf 0 ± 0

Chloroform 11.91±0.34d 13.00±1.25c 18.19±0.07b 19.33±0.18d 0 ± 0

Hexane 0 ± 0 0 ± 0 0 ± 0 11.36 ±0.73e 12.02 ±0.06c

*Rifampicin 21.66±1.41e 24.66±0.47a 23.33±1.69c Nt Nt

**Terbinafine Nt Nt Nt 25.66±1.69a 24.00±0.82f

Data is represented as Mean ± SD of triplicate determination of each extracts (methanol, chloroform and hexane) against each microbial strain. Means with different superscript letter in the same column indicate significant difference ( P < 0.05) among solvents tested.

*Standard antibiotic for bacteria; * *Standard antibiotic for fungi.

We observed that B. subtilis was more susceptible to chloroform extract than E. coli. These results are consistent with previous study (Kuber et al., 2013) on root bark extract of P. guajava in which B. subtilis was reported as sensitive strain while E. coli was the resistant one. Furthermore, according to another report (Zahidah et al., 2013) P. guajava leaves had distinctive antimicrobial potential against B. subtilis as compared to E. coli. It is interesting to note that methanol extract was more effective against E. coli (13.74 ± 1.11 mm) as compared to B. subtilis (10.42 ± 0.91 mm) and P. multocida (12.66 ± 1.52 mm). This observation in strong agreement with previous finding (Zubair et al., 2011) in which

93 methanol extract of an important medicinal plant was reported as successful candidate against E. coli than B. subtilis and P. multocida, respectively.

In case of fungi, methanol and chloroform extracts of P. guajava were more efficient against A. niger than F. solani. These results are in accordance with previous study (Leeja and Thoppil, 2007) in which methanol extract of an Indian medicinal plant exhibited greater antifungal activity against A. niger as compared to F. solani.

Hexane extract was more efficient against F. solani (12.02 ± 0.06 mm) than A. niger (11.36 ± 0.73 mm). This is in agreement with another scientific report on medicinal plant of Pakistan, (Jabeen et al., 2008) citing F. solani as more sensitive strain than A. niger. Antimicrobial activity of P. guajava leaves might be attributed to presence of potent antimicrobial compounds (morin-3-O-α-L- arabopyranoside, quercetin, morin-3-O-α-L- lyxopyranoside and guaijavarin) in them (Arima and Danno, 2002).

4.4.4. Anticancer activity of P. guajava extracts:

Antiproliferative activity of methanol, hexane and chloroform extracts of P. guajava against four different cell lines (KBM5, U266 and HCT116) was evaluated by MTT assay (Figure. 4.4.3) whereby mitochondrial dehydrogenase enzyme in metabolically active cells converts yellow tetrazolium salt to dark blue formazan and the intensity of blue color predicts cell viability (Mena-Rejon et al., 2009).

Hexane extract exhibited maximum decrease in cell viability (%) with IC50 values of 0.022, 0.020 and 0.005 mg/mL for KBM5, U266 and HCT116 cells, respectively. These results are in line with previous finding (Bibi et al., 2011) in which hexane extract (IC50 = 1.10 mg/mL) of a potent medicinal plant of Pakistan was found more effective against human malignant melanoma (HT144) cells as compared to methanol extract (IC50 = 10.69 mg/mL). Maximum anticancer activity of hexane extract might be attributed to presence of tetracosane, α-copaene, γ-sitosterol, vitamin E and squalene in it, as shown in Table. 4.4.2.

120 KBM5 U266 HCT116

100 A

Cell viability (%)viability Cell 40

0 0 10 25 50 100 200 Dose (µg/mL)

120 KBM5 U266 HCT116

100 B

Cell viability (%)viability Cell 40

0 0 10 25 50 100 200 Dose (µg/mL)

120 KBM5 U266 HCT116

100 C

60 Cell viability (%)viability Cell

0 0 10 25 50 100 200

Dose (µg/mL)

Figure. 4.4.2. Anticancer activity of (A) methanol (B) chloroform and (C) hexane extracts of P. guajava against KBM5 (Human myelogenous leukemia), U266 (Human multiple myeloma) and HCT116 (Human colon carcinoma) cell lines. Values are represented as Mean ±SD of quadreplicate determinations. (P < 0.05)

These vital bioactive components exhibit anticancer potential by different mechanisms including suppression of signaling pathways, apoptosis induction and cell cycle arrest (Ryu et al., 2012; Sundarraj et al., 2012).

Jurasz et al., 2004, showed that tumor cell induced platelets aggregation is necessary for the survival of tumor cells and its successful metastasis. α-bulnesene present in hexane extract inhibits platelet aggregation (Hsu et al., 2012). IC50 values lower than 0.03 mg/mL enabled the hexane extract to fulfill criteria of American National Cancer Institute, to attain attention for purification (Suffness and Pezzuto, 1990).

Chloroform extract exhibited maximum cytotoxic activity against HCT116 (IC50 =

0.006 mg/mL) followed by U266 (IC50 = 0.026 mg/mL) and KBM5 (IC50 = 0.041 mg/mL) cells Anticancer activity of chloroform extract may be attributed to significant amount of palmitic acid (30.98%) in it. Harada et al., 2002, reported apoptotic effect of palmitic acid in human leukemic cells.

Palmitic acid is also known for DNA topoisomerase I inhibition in human lung adenocarcinoma epithelial cells (Karna et al., 2012). The ranking of anticancer activity in case of methanol extract was as: HCT116 (IC50 = 0.019 mg/mL) > KBM5 (IC50 = 0.051 mg/mL) > U266 (IC50 = 0.089 mg/mL). IC50 values of methanol extract were comparable with previously reported values for methanol extract of P. guajava from Malaysia (Sulain et al., 2012).

It is interesting to note that, we observed similar ranking of extracts (hexane > chloroform > methanol) is same in case of anticancer (Section. 4.4.7) and antitumor (Section. 4.4.5) assays. This observation is supported by findings of some research groups (Galsky et al., 1980; Galsky et al., 1981; Karpas, 1982) that strongly correlated the antitumor activity assessed by potato disc to in vitro or in vivo anticancer activity.

4.4.8. Anti-inflammatory activity of P. guajava hexane extract:

In this section we investigated whether P. guajava hexane extract can exhibit anti- inflammation and modulate the NF-κB activation. Our results indicated that TNF-α induced the NF-κB activation, whereas P. guajava extract alone has no effect.

The pretreatment of KBM5 cells with this extract suppressed TNF-α-induced NF-κB activation in a dose-dependent manner (Fig. 4.4.4). TNF-α-induced NF-κB activation was completely inhibited at 25 μg/mL dose of P. guajava hexane extract. Treatment with hexane extract under these conditions had little effect on cell viability.

To the best of our knowledge, this is the first report to suggest that P. guajava hexane extract can down-modulate the NF-κB activation induced by proinflammatory cytokines in cancer cells.

Figure. 4.4.4. Anti-inflammatory activity of hexane extract of P. guajava. Lane 1 and lane 2 showed unstimulated Myelogenous leukemia (KBM5) cells. In lane 3 cells were treated with TNF-α alone. In other lanes (4-6) cells were pre-incubated with chloroform extract of P. guajava (10, 25 and 50 µg/mL) for 12 hours and then treated with 0.1 nM TNF-α for 30 min. Nuclear extracts were prepared and assayed for NF-κB activation by EMSA.

Chainy et al., 2000, reviewed that cancer patients have higher levels of proinflammatory cytokines and activated NF-κB, which is a major mediator of inflammatory pathways. Therefore, anti-inflammatory agents that can modulate the NF-κB activation and inflammatory pathways can play a major role in cancer treatment.

There are more than 80 % drugs derived from natural products that are serving mankind for thousands of years (Kim et al., 2012). Significant amount of Vitamin E (Table. 4.4.2) in hexane extract may be responsible for inhibition of NF- κB activation (Calfee-Mason et al., 2002). Similarly garcinol, anethole and many other plant derived components have been shown to inhibit NF-κB activity and to modulate inflammatory pathways (Chainy et al., 2000; Kim et al., 2012).

4.5. Ziziphus mauritiana:

4.5.(a). Chemical Evaluation:

4.5.1. Total phenolic and flavonoid contents of Z. mauritiana:

Phenolic and flavonoids are filed as potent antioxidants (Duthie and Morrice, 2012). They possess wide spectrum of biological and pharmacological activities (Huang et al., 2010). They are also documented as successful candidates against oxidative stress based diseases, microbial infections and malignancy (Pandey and Rizvi, 2009).

Total phenolic and flavonoid contents (TPC and TFC) of methanol, chloroform and hexane extracts of Z. mauritiana leaves were presented in Figure. 4.5.1. Significant differences (P < 0.05) were observed among TPC and TFC of three extracts. Chloroform extract presented the highest extent of total phenolics (84.69 ± 0.92 µg GAE/mg of plant extract). Overall the total phenolic contents of three extracts were in following order: chloroform (84.69 ± 0.92 µg GAE/mg of plant extract) > methanol (77.88 ± 1.10 µg GAE/mg of plant extract) > hexane (56.22 ± 1.46 µg GAE/mg of plant extract). This finding is in accordance with previous scientific study on a potent medicinal plant, in which Shahriar et al., 2013, examined maximum extent of phenolics in chloroform extract followed by methanol and hexane extracts, respectively. Yusri et al., 2012, also observed

99 the identical ranking of solvents (chloroform > methanol > hexane) for extraction of phenolic compounds.

The phenolic contents for Z. mauritiana leaf extracts observed in our study were higher than those of previously reported for Z. mauritiana fruit (Memon et al., 2012). Another study reported lower extent of phenolics in seed extract of Z. mauritiana (San et al., 2013). However, Dureja and Dhiman, 2012, reported higher level of total phenolic and flavonoid contents in fruits of Z. mauritiana.

100 Total phenolic contents (µg GAE/mg of plant extract)

80 Total flavonoid contents (µg QE/mg of plant extract)

0 Methanol Chloroform Hexane Z. mauritiana extracts

Figure. 4.5.1. Total phenolic and total flavonoid contents of Z. mauritiana extracts (methanol, chloroform and hexane). Values are Mean ± SD of triplicate determinations. (P < 0.05). Total phenolic contents are expressed as microgram gallic acid equivalents per milligram of plant extract (µg GAE/ mg of plant extract). Total flavonoid contents are expressed as microgram quercetin equivalents per milligram of plant extract (µg QE/ mg of plant extract).

100

In contrast to our study recently another research group (Lamien-Meda et al., 2008) reported high phenolic and flavonoid contents in Z. mauritiana fruit extracts. Variation in phenolic and flavonoid contents might be attributed to changes in variety and climate.

4.5.2. High performance liquid chromatographic (HPLC) analysis of Z. mauritiana extracts:

HPLC technique was used to identify and quantify the individual phenolic compounds in three solvent extracts (methanol, chloroform and hexane) of Z. mauritiana leaves. The results are shown in Table. 4.5.1.

Caffeic acid was the most abundant phenolic compound in the tested extracts (methanol, chloroform and hexane). Moreover, chlorogenic acid, ferulic acid, p-coumaric acid and sinapic acid were also detected at varying degrees in three extracts.

Table. 4.5.1. High performance liquid chromatographic (HPLC) study of methanol, chloroform and hexane extracts of Z. mauritiana for identification and quantification of phenolic compounds (ppm)

Phenolic Methanol Chloroform Hexane compounds extract extract extract

Caffeic acid 4.41 ± 0.11ab 2.13 ± 0.00ef 1.92 ± 0.02ce

Chlorogenic acid 2.39 ± 1.03be 1.22 ± 0.16d 0.17 ± 0.10a

Sinapic acid 0.78 ± 0.19 Nd Nd p-coumaric acid 1.16 ± 0.07b 1.04 ± 0.13b Nd

Ferulic acid 0.89 ± 0.12 Nd Nd nd = Not detected. Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicate significant difference (P < 0.05) among solvents (methanol, chloroform and hexane) used.

Our results were in strong agreement with previous findings; Muchuweti et al., 2005, detected the presence of p-coumaric acid, caffeic acid and ferulic

101 acid in Z. amuritiana fruit segment. Memon et al., 2012, reported the presence of p-coumaric acid, o-coumaric acid and ferulic acid in fruit extract of Z. mauritiana, but they did not find other phenolic compounds i.e. caffeic acid sinapic acid and chlorogenic acid detected in current research work. All of the identified phenolic compounds have good pharmacological properties (Zang et al., 2000; Raina et al., 2008; Zhao and Hu, 2013).

4.5.3. Gas chromatography mass spectrometry (GC-MS) study of Z. mauritiana extracts:

The data for GC-MS analysis is shown in Table. 4.5.2. It is indicated from the results that methyl sterate (15.59%) and α-Linolenic acid (26.45%) were the major components of methanol and hexane extracts, respectively. Other, significant components in methanol extract were palmitic acid (13.57%), squalene (12.09%), phytol (9.78%), methyl palmitate (7.81%), linoleic acid methyl ester (5.98%) and vitamin E (2.35%). Considerable quantities of palmitic acid (16.26%), squalene (12.83%), α-tochopherol (3.92%), γ-sitosterol (2.72%), phytol (2.52%), trans-geranylgeraniol (2.34%), octacosane (2.04%), methyl palmitate (1.01%) and myristic acid (0.73%) were observed in hexane extract. We also observed considerable quantities of α-tochopherol (10.01%), stearic acid (5.82%), vitamin E (5.41%), uneicosane (4.79%), α- nonadecylene (3.77%), bacchotricuneatin C (3.48%), myristic acid (2.80%) and lauric acid (1.66%) in chloroform extract.

Reports on chemical composition of Z. mauirtiana leaf extracts are scarce in literature. However, similar to our results, Memon et al., 2012, reported the presence of palmitic acid and lauric acid in fruit extracts of Z. mauritiana. Furthermore, palmitic acid, lauric acid, myristic acid and stearic acid were also detected in Z. mauritiana flowers collected from Brazil (Alves et al., 2005). All the mentioned fatty acids were also reported in current study.

On the other hand, Chebouat et al., 2013, presented absolutely different chemical components in crude alkaloidal extract of Z. mauritiana plant. Squalene which was present in considerable amount in methanol and hexane extracts was also reported from seed oil of Z. mauritiana fruit (Memon et al., 2012). Squalene is known for its chemo preventive effects against colon carcinoma cells (Rao et al., 1998).

102

Previous studies reported vitamin E from fruit of Z. mauritiana (Nyanga et al., 2013). Presence of vitamin E in methanol and chloroform extracts of Z. mauritiana leaves is also examined (Table. 4.5.2). Anticancer effect of vitamin E analogues has been demonstrated in many in vivo and in vitro studies (Anderson et al., 2005). Saturated isomer of vitamin E suppress proliferation in wide range of carcinoma cells including colon, breast, lung, liver, stomach, prostate and pancreas (Sakai et al.,2006; Comitato et al., 2010; Yang et al., 2010).

Table. 4.5.2. Chemical components of methanol, chloroform and hexane extracts of Z. mauritiana analyzed by Gas chromatography mass spectrometry (GC-MS) aComponents bRT Methanol Chloroform Hexane extract extract extract Composition (%) Diglycerol 8.68 0.30±0.01 ------2,3 dihydro benzofuran 13.31 0.60±0.12 ------1,2 diacetate glycerol 13.81 1.44±0.09 ------Uneicosane 18.76 ---- 4.79±0.34 ---- Lauric acid 19.00 ---- 1.66±0.05 ---- Myristic acid 22.35 ---- 2.80±0.13 0.73±0.51 E-15-Heptadecenal 23.13 ---- 12.31±0.01 ---- Phytol acetate 23.34 ------1.02±0.16 Methyl palmitate 26.55 7.81±0.11 2.83±0.25 1.01±0.43 Palmitic acid 27.28 13.57±0.02 38.55±0.08 16.26±0.01 Hentriaconate 27.57 ---- 3.25±0.14 ---- Linoleic acid, methyl ester 30.08 5.98±0.40 ---- 0.45±0.38 Phytol 30.14 9.78±0.19 ---- 2.52±0.15 Methyl state 30.19 15.59±0.01 2.31±0.07 0.53±0.06 Linoleic acid 30.73 4.75±0.08 ---- 1.37±0.04 α-Linolenic acid 30.87 14.21±0.32 ---- 26.45±0.01 Stearic acid 31.21 1.94±0.45 5.82±0.28 ---- α-Nonadecylene 31.73 ---- 3.77±0.04 ---- Archidic acid methyl ester 33.65 1.60±0.02 ------

103

Carbromal 33.78 0.76±0.05 ------Bacchotricuneatin C 34.14 ---- 3.48±0.16 ---- 3-methyl piperidine 34.21 0.48±0.07 ------o-methyl delta-tochopherol 36.71 ------0.47±0.09 Octacosane 38.33 ------2.04±0.08 Cyclobarbital 38.95 0.61±0.13 ------Squalene 40.23 12.09±0.35 ---- 12.83±0.26 Trans-Geranylgeraniol 41.74 ------2.34±0.35 2,4- Dimethyl Benzoquinoline 42.47 ------2.28±0.03 α-tochopherol 44.16 ---- 10.01±0.16 3.92±0.01 Vitamin E 44.17 2.35±0.09 5.41±0.22 ---- 4-Chloro-2- 45.93 ------1.74±0.01 trifluoromethylbenzoquinoline Thymol TMS 47.51 1.26±0.03 ------γ-sitosterol 47.65 ------2.72±0.48 17-Hydroprogesterone 54.12 ------3.42±0.02 ( ---- ) = not detected. Compounds are identified on the basis of comparison of retention time and mass spectra in NIST data

4.5.(b). Biological Evaluation:

4.5.4. Free radical (DPPH) scavenging activity of Z. mauritiana extracts:

Free radicals are major contributor of vast range of degenerative diseases (Pham-Huy et al., 2008). Pharmacological evaluation of extracts is incomplete without assessment of their free radical scavenging activity. Thus in the present study stable free DPPH radicals were used to explore free radical scavenging activity of methanol, chloroform and hexane extracts of Z. mauritiana leaves.

In our findings, trend for free radical scavenging activity of three extracts was as: methanol > chloroform > hexane (Table. 4.5.3) which agreed with previous scientific report anothter group of researcher (Yusri et al., 2012).

It is depicted from the results (Table. 4.5.3) that Z. mauritiana methanol extract had free radical scavenging power with value of 91.04 ± 15.6% at 800 µg/mL. Moderately high

104 free radical scavenging activity (54.49 ± 1.11%) as assessed by DPPH assay was examined in chloroform extract of Z. mauritiana leaves at highest investigated dose (800 µg/mL), while lowest value of antioxidant activity was found in hexane extract at various tested doses (25 to 800 µg/mL). Overall the order of antioxidant activity of three extracts estimated by DPPH assay was as: methanol > chloroform > hexane.

Our results differed from previously reported (Bhuiyan et al., 2009; Abalaka et al., 2011) values of antioxidant powers for Z. mauritiana. This difference might be due to genotypic differences in different verities grown under different geological and climatic conditions (Capocasa et al., 2008).

Furthermore, we used dose dependent (25 to 800 μg/mL) manner to estimate DPPH scavenging efficacy of three extracts.

Table. 4.5.3. Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform and hexane extracts of Z. mauritiana

Z. mauritia- Scavenging (%) of free radicals at different concentrations (µg/mL) IC 50 na extracts (mg/mL) 25 50 100 200 400 800 c a e d b e Methanol 25.13±0.04 29.10±0.74 42.04±1.72 59.79±0.25 85.41±0.84 91.04±1.56 0.11 f e a e b c a d b f e b Chloroform 18.62±1.13 20.57±0.89 26.72±2.68 41.3±1.50 43.17±1.05 54.49±1.11 0.63 b d c e a f d b a d f c Hexane 14.89±1.42 17.02±3.27 18.53±0.95 22.43±1.08 35.65±1.91 48.99±0.88 > 1 c f e a c e Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicate significant difference (P < 0.05) among concentrations tested. Mean with different subscript letters in the same column indicate significant difference (P < 0.05) among solvents tested.

It was examined that antioxidant activity was directly associated with dose of Z. mauritiana leaf extracts. This is in accordance with previous scientific studies in which highest antioxidant activity was reported at the maximum concentration of plant extract (Gawron-Gzella et al., 2012).

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4.5.5. Antitumor activity of Z. mauritiana extracts:

Bench top potato disc assay was used to assess potential of methanol, chloroform and hexane extracts of Z. mauritiana leaves against plant pathogen A. tumefaciens which is involved in induction of neoplastic disease (Crown gall tumor) in dicotyledonous plants. It is depicted from the results (Fig. 4.5.2) that three extracts significantly decreased the growth of A. tumefaciens.

Tumor inhibition by three extracts was observed in dose dependent mode (25 to 800 µg/mL). We observed minimum tumor inhibition in methanol, chloroform and hexane extracts at 25 µg/mL.

100 Methanol Chloroform Hexane 90

(%) 50

40 Inhibition of tumors of Inhibition

0 25 50 100 200 400 800 Concentartion(µg/mL)

Figure. 4.5.1. Antitumor activity of methanol, chloroform and hexane extracts of Z. mauritiana. Data is represented as Mean ± SD for three samples of each extract (methanol, chloroform and hexane) analyzed individually in triplicates. (P < 0.05).

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Overall the order of antitumor activity of three extracts was as: chloroform (IC50 =

70.74 µg/mL) > hexane (IC50 = 188.5 µg/mL) > methanol (IC50 = 596.4 µg/mL). Variation in antitumor approach of different extracts may be due to difference in secondary metabolites in each extract (Kuete et al., 2008).

4.5.6. Antimicrobial activity of Z. mauritiana :

Antimicrobial activity of Z. mauritiana leaf extracts was investigated by disc diffusion assay and results are summarized in Table. 4.5.4. It is evident from the results that three extracts exhibited significant (P < 0.05) antimicrobial potential against all the investigated strains.

Table. 4.5.4. Antimicrobial activity of methanol, chloroform and hexane extracts of Z. mauritiana

Diameter of inhibition zones (mm)

Z. mauritiana Bacterial Strains Fungal Strains

leaf extracts Escherichia Bacillus Pasteurella Aspergillus Fusarium

coli subtilis multocida niger solani

Methanol 12.3±0.1b 21.7±0.3d 0 ± 0 23.4±1.3e 16.7±0.1b

Chloroform 0 ± 0 0 ± 0 10.8±0.2b 19.5±1.0d 0 ± 0

Hexane 9.0±0.0c 13.3±1.6e 0 ± 0 0 ± 0 0 ± 0

*Rifampicin 21.7±1.4e 24.7±0.5f 23.3±1.7d Nt Nt

**Terbinafine Nt Nt Nt 25.7±1.7a 24.0±0.8c

Diameter of inhibition zones ranged from 9.00 ± 0.00 mm to 23.39 ± 1.25 mm. Among all the bacterial strains largest inhibition zone (21.66 ± 0.33 mm) was observed against B. subtilis. However, Nagumanthri et al., 2012, reported comparatively smaller

107 inhibition zone against B. subtilis by methanol extract of Indian Z. mauritiana. In contrast to our results, Abalaka et al., 2012, reported larger zone of inhibition against E. coli by Z. mauritiana leaves. These differences might be attributed to the difference in extraction techniques used to prepare the extracts and different geological conditions as well (Turkmen et al., 2007; Edoga et al., 2005). Furthermore, among fungal strains we observed larger zone of inhibition (23.39 ± 1.25 mm) by methanol extract against A. niger as compared to previous study (Das, 2012).

4.5.7. Anticancer activity of Z. mauritiana:

The anticancer activity of Z. mauritiana leaf extracts (methanol, chloroform and hexane) against cultured KBM-5 (myelogenous leukemia), U266 (multiple myeloma) and SCC-4 (tongue squamous) cancer cells was estimated by using MTT assay. Cells were exposed to increasing doses of Z. mauritiana leaf extracts (methanol, chloroform and hexane) ranging from 10 to 200 µg/mL for 72 hour at 37ºC. Then viability of cancer cells (KBM5, U266 and SCC4) was assessed and described in terms of relative absorbance of treated cells, in contrast to control cells.

The results are expressed in Figure. 4.5.3. It is evident from the results that viability of the three investigated cell lines (KBM5, U266 and SCC4) decreased with increase in dose of Z. mauritiana leaf extracts (methanol, chloroform and hexane). It was examined that Z. mauritiana leaf extracts (methanol, chloroform and hexane) were active against all these cell lines.

Maximum death of myelogenous leukemia (KBM5), multiple myeloma (U266) and tongue squamous carcinoma (SCC4) cells in each extract (methanol, chloroform and hexane) was observed at 200 µg/mL.

The chloroform extract showed pronounced anticancer activity with IC50 values of 0.016, 0.022 and 0.043 mg/mL for myelogenous leukemia (KBM5), multiple myeloma (U266) and tongue squamous carcinoma (SCC4) cells, respectively.

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120 KBM5 U266 SCC4

100 A

40 Cell viability (%)viability Cell

0 0 10 25 50 100 200 Dose (µg/mL) 120 KBM5 U266 SCC4

100 B

40 Cell viability (%)viability Cell 20

0 0 10 25 50 100 200 Dose (µg/mL)

109

120 KBM5 U266 SCC4

100

C 80

60 Cell viability (%)viability Cell

0 0 10 25 50 100 200 Dose (µg/mL)

Figure. 4.5.3. Anticancer activity of (A) methanol (B) chloroform and (C) hexane extracts of Z. mauritiana against KBM5 (Human myelogenous leukemia), U266 (Human multiple myeloma cells) and SCC4 (Human tongue squamous carcinoma) cell lines. Values are represented as Mean ±SD of quadreplicate determinations. (P < 0.05).

Potent anticancer activity of chloroform extract might be acknowledged to remarkably high concentration of phenolics in it as shown in, Figure. 4.5.1. Literature survey has indicated that these bioactive components exhibited anticancer activities by apoptosis induction, anti-angiogenesis, topoisomerase inhibition, upregulation of p53, cell cycle arrest and various other pathways (Luk et al., 2005; Sagar et al., 2006; Han et al., 2008).

Hexane extract exhibited moderate anticancer activity with IC50 values of 0.1, 0.069 and 0.094 mg/mL for KBM5 (myelogenous leukemia), U266 (multiple myeloma) and tongue squamous carcinoma (SCC4) cells. Poor anticancer activity was investigated in methanol extract with potent IC50 values for proliferating cells (KBM5, 0.148; U266, 0.046 and SCC4,

110

0.163 mg/mL). Similar results by Z. mauritiana polar extract against HL-60 were studied previously by Mishra et al., 2011.

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CHAPTER 5 SUMMARY

The present research work was carried out in the Central Hi Tech Laboratory, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan. A total of five species from four families were collected from different areas of Pakistan to explore chemical composition (total phenolic, total flavonoid contents, phenolic compounds and chemical profile) and biological activities (antioxidant, antimicrobial, antitumor, anticancer and anti-inflammatory) of investigated medicinal plants. Efforts were made to evaluate effect of extracting solvents on chemical composition and biological activities of tested medicinal plants.

Among the investigated plant extracts, chloroform extract of V. betonicifolia presented the maximum amount of total phenolic contents (155.78 ± 4.12 µg GAE/ mg of plant extract). Aluminium complexation assay used for assessment of total flavonoid contents revealed that methanol extract of P. guajava (53.39 ± 0.89 µg QE/ mg of plant extract) is leading one, while hexane extract of E. camaldulensis (6.09 ± 0.96 µg QE/ mg of plant extract) was at least position.

In HPLC analysis, a total of nine standard phenolic compounds (Gallic acid, caffeic acid, vanillic acid, chlorogenic acid, syringic acid, sinapic acid, m-coumaric acid, p-coumaric acid and ferulic acid) were run. However, all (nine) of the analyzed phenolic compounds were not detected in the same sample. The result of HPLC analysis showed the existence of different phenolic compounds at varying degrees in different plant extracts. In E. camaldulensis gallic acid (5.86 ± 0.23 ppm) was examined at maximum extent, while p- coumaric acid (0.12 ± 0.05 ppm) was the minor one. Sinapic acid (6.61 ± 0.40 ppm) was the major phenolic compound of V. betonicifolia examined in methanol extract. Ferulic acid (0.37 ± 0.001 ppm) noticed in chloroform extract of V. betonicifolia was the minimum contributor of phenolic profile. Among all the investigated phenolic compounds, quercetin was observed as major constituent of P. guajava phenolic profile. Extent of phenolic acids in E. royleana ranged from 0.16 ± 0.001 ppm to 4.59 ± 0.23 ppm. HPLC analysis of Z. mauritiana demonstrated the chlorogenic acid of hexane extract as the poor contributor of phenolic acid profile. Overall the major extent of phenolic acids was observed in highly polar solvent (methanol).

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The out come of GC-MS study revealed the innovative chemical spectrum of each extract. Chemical constituent of methanol, chloroform and hexane extracts of E. camaldulensis ranged from 0.58% to 45.94%. Euclayptol was the major and common constituent of chloroform and methanol extracts with chemical contribution of 45.94% and 31.86%, respectively. Ledol (0.73%) was the least existing component of hexane extract of E. camaldulensis. Palmitic acid (25.67%) was the major component of V. betonicifolia, while the β-tochopherol (0.08%) and cetene (0.08%) were the least existing components with equal chemical contribution. Chemical constituents of P. guajava leaf extracts (methanol, chloroform and hexane) varied from 1.43% to 35.18%. Pyrogallol (35.18%) of methanol extract was the leading constituent of P. guajava. Minor components of methanol, chloroform and hexane extracts of P. guajava were culmorin (1.43%), alloaromadendrene (2.18%) and α-copaene (2.18%), respectively. GC-MS analysis of Z. mauritiana leaf extracts indicated palmatic acid as major constituent and diglycerol (0.30%) as minor one.

The analyzed plant extracts showed good free radical scavenging activities. Maximum free radical scavenging activity (91.04 ± 1.56 %) was observed in methanol extract of Z. mauritiana. Hexane extract of V. betonicifolia exhibited poor free radical scavenging activity (39.47 ± 0.69 %).

Antimicrobial potential of the tested medicinal plant extracts was evaluated by disc diffusion assay. Among the bacterial strains maximum inhibition zone was observed by methanol extract of E. camaldulensis against B. subtilis. Minimum antibacterial activity was observed in methanol extract of E. royleana against E. coli (5.67 ± 0.57 mm). Maximum antifungal efficacy was observed in methanol extract of Z. mauritiana against A. niger (23.39 ± 1.25 mm). Overall, among the bacterial strains, maximum activities were observed by most of plant extracts against B. subtillis (Gram positive) and minimum against E. coli (Gram negative). In case of fungi, A. niger was the sensitive strain while F. solani was the resistant one against most of plant extracts.

Antitumor activity estimated by potato disc assay showed that tested extracts had potential to inhibit the growth of Agrobacterium tumefacien. Maximum antitumor activity

(IC50 = 38.13 µg/mL) was observed in chloroform extract of V. betnocifolia, while methanol extract of E. royleana (IC50 = 485.1 µg/mL) was poor candidate. Among three extracts

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(methanol, chloroform and hexane) of Z. mauritiana chloroform extract exhibited maximum ability to inhibit the growth of tumors (IC50 = 70.74 µg/mL). In case of E. camaldulensis methanol extract (IC50 = 59.68 µg/mL ) was the potential candidate. Antitumor activity of P. guajava ranged from (IC50 = 65.02 µg/mL ) to (IC50 = 337.4 µg/mL ).

The out come of MTT assay showed that human myelogenous leukemia (KBM5) cells were the most sensitive to V. betonicifolia chloroform extract (IC50 = 0.001 mg/mL).

Hexane extract (IC50 = 0.020 mg/mL) of P. guajava was successful candidate against human multiple myeloma (U266) cells. Maximum death of human tongue squamous carcinoma cells

(IC50 = 0.043 mg/mL) was observed by chloroform extract of Z. mauritiana. Human colon carcinoma cells (HCT116) exhibited maximum susceptibility to hexane extract of P. guajava

(IC50 = 0.005 mg/mL). Methanol extract of V. betonicifolia was least effective (IC50 = 0.183 mg/mL) against colon carcinoma (HCT116) cells. Methanol extract of Z. mauritiana (IC50 = 0.148 mg/mL) was poor candidate against human myelogenous leukemia (KBM5) cells.

Human multiple myeloma (U266) cells were most resistant (IC50 = 0.089 mg/mL) to methanol extract of P. guajava. Overall chloroform extract of most of plants was the best candidate against cancer. Methanol extract of most of investigated plants exhibited poor anticancer activity.

Plant extracts with potent anticancer activity against human myelogenous leukemia (KBM5) cells were tested for inhibition of inflammatory transcription factor, nuclear factor kappa B (NF-κB). The results of electrophoretic mobility shift assay (EMSA) demonstrated that the investigated extracts had excellent anti-inflammatory activity. Hexane extract of P. guajava proved best anti-inflammatory agent. It exhibited complete inhibition of TNF-α induced NF-κB activation at 25 µg/mL in human myelogenous leukemia (KBM5) cells. Chloroform extracts of V. betonicifolia also showed complete inhibition of TNF-α induced NF-κB activation at 50 µg/mL.

It is evident from our results, that current study provided valuable information about chemical composition, antioxidant, antimicrobial, antitumor and anticancer activities of traditional medicinal plants of Punjab, Pakistan. The knowledge of phenolic composition in different solvent extracts will help to explore their potential as source of natural medicinal agent particularly the antioxidants. Moreover, the comparison of phenolic composition and

114 other chemical compounds among solvents of variable polarity will help to optimize the solvent for extraction of phenolic compounds. The considerable antioxidant, antimicrobial, antitumor and anticancer activities of different extracts may assist in preparation of herbal drugs for treatment of microbial infections, cancer and oxidative stress related disorders. The future work will be focused on the isolation of bioactive compounds from the potential extracts followed by exploration of their mechanism of action against carcinogenesis.

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