Exploration of Antioxidant and Anticancer Potential of Essential Oil of Salvia Plebeia Rbr

EXPLORATION OF ANTIOXIDANT AND ANTICANCER POTENTIAL OF ESSENTIAL OIL OF SALVIA PLEBEIA RBR.

A Dissertation submitted to the Central University of Punjab

For the Award of

Master of Philosophy

Environmental Science and Technology

Nandini Gautam

Supervisor: Dr. Sunil Mittal

Centre for Environmental Science and Technology School of Environment and Earth Sciences Central University of Punjab, Bathinda

September, 2012 CERTIFICATE

I declare that the dissertation entitled “EXPLORATION OF ANTIOXIDANT AND ANTICANCER POTENTIAL OF ESSENTIAL OIL OF SALVIA PLEBEIA RBR.” has been prepared by me under the guidance of Dr. Sunil Mittal, Assistant Professor, Centre for Environmental Science and Technology, School of Environment and Earth Sciences, Central University of Punjab. No part of this dissertation has formed the basis for the award of any degree or fellowship previously.

(Nandini Gautam) Centre for Environmental Science and Technology, School of Environment and Earth Sciences, Central University of Punjab, Bathinda - 151001.

Date:

ACKNOWLEDGEMENT Accomplishment of this work would not have been possible without the blessings of almighty God. I pay Him tribute whose blissful blessings enabled me to scale every problem that came in my path. I would like to thank my Supervisor, Dr. Sunil Mittal, Assistant Professor, Centre of Environmental Science and Technology, Central University of Punjab, Bathinda, under whose guidance the present work was carried out. I am grateful for his guidance, cooperation, immense support and valuable suggestions during the research work. I am highly obliged for the efforts of Dr. Sandeep Singh, who was there at every step to help, guide and critically analyse the difficulties encountered at every step of the accomplishment of this work. I would like to thank him from the core of my heart for teaching tissue culture techniques. Also, I would like to express my heartiest gratitude to Dr. Ajit Vikram who helped me in my work. I express my deep gratitude to Prof. P. Rama Rao, Dean Academic Affairs, Central University of Punjab, Bathinda for solving my queries, consistent support and guidance and for providing excellent laboratory facilities. I am highly thankful to Prof. R. G. Saini, COC Biosciences, Central University of Punjab, Bathinda for his immense support and guidance. I acknowledge Dr. Punita Pandy, Dr. Dhanya, Dr. Yogalakhshami, Dr. Nageder Babu, Dr. Vikas Jaitak and Dr. Raj Kumar for their encouragement and helping nature. I am highly grateful to Dr. Sanjeev Kumar Thakur, Assistant Professor, for his continuous support and helpful attitude while working in Tissue culture laboratory. I would like to acknowledge Dr. Bikram Singh, Head of the Natural Plant Products Division, IHBT, Palampur (H.P), for helping me in the GC-MS analysis of the essential oil. I am thankful to my seniors Mr. Gajendra Singh Vishwkarma, Mr.Prateek, Mr.Pushpender, Ms.Anamika, Ms.Upma and Mrs. Manohari for their immense support and help in day-to-day work. I have no words to thank my friends Shilpa, Gurpreet Kaur, Jaskiran and all others for their cooperation and support. I am thankful to all the non-teaching staff that helped me at various stages during this project but may not be listed here. I also offer big thanks to Mr. Gopal, Ms. Sona and Mr. Ashwani for their kind cooperation and support in the hour of need. I pay my sincere thanks to my parents, Mandakini Di, Ashu and beloved ones for their unconditional love, support, encouragement, trust and moral support. This work is dedicated to my sweet parents.

(Nandini Gautam)

iii

CERTIFICATE

I certify that NANDINI GAUTAM has prepared her dissertation entitled “EXPLORATION OF ANTIOXIDANT AND ANTICANCER POTENTIAL OF ESSENTIAL OIL OF SALVIA PLEBEIA RBR.” for the award of M.Phil. degree of the Central University of Punjab, under my guidance. She has carried out this work at the Centre for Environmental Science and Technology, School of Environment and Earth Sciences, Central University of Punjab, Bathinda.

(Dr. Sunil Mittal) Assistant Professor Centre for Environmental Science and Technology, School of Environment and Earth Sciences, Central University of Punjab, Bathinda - 151001.

Date:

ABSTRACT “Exploration of Antioxidant and Anticancer Potential of Essential Oil of Salvia plebeia RBr.”

Name of student : Nandini Gautam Registration Number : CUP/MPh-PhD/SEES/EVS/2010-2011/04 Degree for which submitted : Master of Philosophy Name of Supervisor : Dr. Sunil Mittal Centre : Centre for Environmental Science and Technology School of Studies : School of Environment and Earth Sciences Cancer; Anticancer Agents; Essential Oil; Salvia Key words : plebeia; Caryophyllene

Cancer is one of the major problems that accounts for millions of death worldwide per year. Breast cancer is the most commonly encountered cancer in the females. Chemotherapy and radiotherapy are the standard methods used in the treatment of cancer but these have various side effects also. Other complementary treatments based on plants and its products are used to lessen the side effects. Some essential oils are the potent source of the novel molecules and have been reported to have anticancer effect. In the present study, essential oil from Salvia plebeia has been screened for cytotoxic activity in vitro using MTT assay and has been observed to show inhibitory effect on the cancer cells with the increase in concentration against breast cancer cell lines, MCF-7 and MDAMB-231. The essential oil was observed to show IC50 at concentration 30µg/ml for MCF-7 cells and in MDAMB-231, IC50 was calculated to be 65µg/ml for 24 hours treatment. Essential oil was found to be effective against highly metatstatic MDAMB-231cells Exposure time effect was studied to know whether it has sustained effect or not and it was observed that essential oil had sustained effect at higher concentrations and longer exposure time was required to show sustained effect. Single cell gel electrophoresis (Comet assay) was used to find out its mode of action and it was observed to induce DNA damage in the cancer cells that might have resulted in cell death at various concentrations. Essential oil was observed to have antioxidant activity, which was studied using DPPH and hydroxyl radical scavenging assay. Various compounds like cayophyllene, alpha-humulene and cayophllene oxide were determined by GC-MS analysis. Cayophyllene was the major compound (46%). Some of the constituents might be responsible for anticancer activity of the oil. It has been concluded from the study that novel essential oil is very effective against breast cancer cells. This oil can be screened further against other cell lines and can be used as nutritional supplement.

(Nandini Gautam) (Dr. Sunil Mittal)

vii

TABLE OF CONTENTS

Sr. No. Contents Page No.

1. Introduction (Chapter-1) 1-5

2. Review of Literature (Chapter-2) 6-18

3. Objectives (Chapter-3) 19

4. Materials and Methods (Chapter-4) 20-26

5. Results and Discussion (Chapter-5) 27-42

6. Conclusion and Future Prospect 43-44

7. Summary 45-46

8. References 47-55

LIST OF TABLES

Table Table Description Page No. No. 1.1 Distribution and characteristics of the plant and its essential 4 oil

2.1 Studies related to various species of Salvia indicating its 9-11 anticancer properties

2.2 Essential oils as anticancer agents 14-15

4.1 Instruments used and the company 20

4.2 Treatments of the essential oil 24

5.1 Major compounds of essential oil of Salvia plebeia RBr. and 28 the data obtained by GC-MS analysis with their classification

LIST OF FIGURES

Figure Description of Figure Page No. No.

GC chromatogram of the essential oil obtained on the 5.2.1 27 DB-5 column DPPH radical scavenging ability of the essential oil of 5.3.1 29 Salvia plebeia

Effect of essential oil Salvia plebeia on hydroxyl radical 5.3.2 30 scavenging

Effect of essential oil of Salvia plebeia on MCF-7 cell 5.4.1 31 line for 12 and 24 hours treatments

Effect of essential oil of Salvia plebeia on MDAMB-231 5.4.2 32 cell line for 12 and 18 hours

Essential oil of Salvia plebeia showing significant 5.4.3 33 decrease in the viability of MCF-7 cells for 24 hours

Effect of 24 hours treatment of essential oil of Salvia 33 5.4.4 plebeia on MBAMB-231 cells

Effect of essential oil of Salvia plebeia on MCF-7 cells 5.4.5 34 with different exposure and chase times

Effect of essential oil of Salvia plebeia on MDAMB-231 5.4.6 35 cells with different exposure and chase time

Effect of the essential oil of Salvia plebeia on the individual MCF-7 cells showing the formation of the 5.4.7 36 comets in the treatment groups compared to control group observed at 40X of the fluorescent microscope

Histograms (A-F) showing the DNA damage induced by the different concentration determined by parameters 5.4.8 37 such as Head Intensity, Tail Intensity, % DNA in Head and Tail, Head diameter and Tail Length

CHAPTER-1

INTRODUCTION

Cancer has emerged as one of the most dreadful diseases in the last few decades throughout the world. It is a multifactorial disease leading to uncontrolled growth and invasion of the abnormal cells leading to the formation of tumour. The steep rise in the number of cancer cases may be attributed to change in food habits, use of tobacco and alcohol, chronic infections, exposure to harmful radiations and environmental pollution or more widely we can say due to change in lifestyle and environmental pollution (NCBI, 2010). As per International Agency for Research on Cancer, there were approximately 12 million cancer cases and these had accounted for 7.6 million deaths worldwide (around 13% of all deaths) in the year 2008. Moreover, cancer cases have been projected to increase over 13.1 million by 2030. As far India is concerned, the number of cancer cases has been calculated approximately to be broadly 24 lakhs until 2008 (IARC, 2010). However, the exact number may be much higher as there was no cancer registry programme in India (NCRP) before 1982. One of the important facts is that developing countries have half the number of cancer cases compared to the developed countries. However, survival rate is very poor for the people of economically less developed countries due to unavailability of timely care and lack of standard methods of treatment with cost being the major obstacle (Jemal et al., 2011). Hence, there is always search for new potent and cheap drugs as well as naturally occurring supplements.

Worldwide, breast cancer is the second most commonly found cancer and accounts for 10.9% of all the cancer (Ferlay et al., 2010). Breast cancer is heterogeneous both genetically and histopathologically. Hence, difficulties arise in its treatment. People in scientific arena have been circumventing these problems with the use of drugs. These drugs are designed to specifically target rapidly growing and dividing cells of tumour. But, these may also affect rapidly dividing normal cells in the body leading to certain other side effects. Also, chemotherapy used in cancer treatment leads to development of drug resistance (Naumovski et al., 1992) and multidrug resistance in cancer (Nooter and Herweijer, 1991). Plants are the best source of anticancer agents and can have potential for effective treatment with lesser side effects.

Natural plant products as anticancer agents

Plants have been used in traditional medicine since time immemorial as crude products. Various plant products have been used for the treatment of various diseases including diabetes and cancer (Amin et al., 2009). Also, natural products are regarded safe and most of the people use them for treating various diseases based on ancestral knowledge. Rural people mostly rely on the traditional medicine due to economic and traditional reasons. High cost of the widely used drugs and other health facilities in the treatment are the other reasons which force the common people to opt for these practices.

Plants are rich source of natural antioxidants and novel molecules. Antioxidants counteract the oxidative damage caused by reactive oxygen species and free radicals, which lead to many physiological problems including cancer, one of the major problems that had become more prominent in the last few decades. Therefore, it is valuable to evaluate biological activity and efficacy of widely growing plant species and their potential applications in search of novel compounds. Also, methods and the technology applied for the extraction of the plant products has scaled new heights enabling better detection and separation of the plant products and their individual compounds. Moreover, natural products are the valuable choice in comparison to synthetic molecules used as drugs keeping in view the safety issues and many cancer drugs have been derived from plant origin (Amin et al., 2009). Hence, many researchers are focusing on the applicability of the natural plant products in the cancer treatment. Essential oils are one among the most valuable plant products used in the medicine and complementary treatment strategies.

Essential oils as anticancer agents

Essential oils are the secondary metabolites produced by aromatic plants in lower amounts and are concentrated hydrophobic liquids with specific aroma (Celiktas et al., 2007). These are also called volatile oils or ethereal oils. These are composed of terpenes, aromatic compounds and some other compounds of miscellaneous

2 origin. Terpenes are further divided into monoterpenes and sesquiterpenes. Aromatic compounds found in the oils are polyphenol derivatives. Essential oil is found in flowers, leaves, fruits and seeds of plants from many families like Lamiaceae, Astrales, Rutales etc. These are used for their specific aromas in perfumery and as flavouring agents in food products.

Essential oils have been used in aromatherapy for improving the health due to its sedative and stimulant properties. These are used for massage, bath and inhalation as relaxants and treatment in aromatherapies for various diseases. These can be used as antioxidants (Tepe et al., 2004; Yu et al., 2011), and antimicrobial agents (Cardile et al., 2009).

These have been reported to have active ingredients that are being exploited in medicine (Lancelle et al., 2009). Plant essential oils have gained interest of the researchers in the last decade (Sylvestre et al., 2006). Most of the preservative and medicinal properties of aromatic plants are attributed to their essential oils. To the much interest of scientific group, essential oils from some medicinal plants and spices possess anticancer properties (Yu et al., 2011) and plants from different regions show different cytotoxic effects due to variable composition. Variability in essential oil composition is attributed to occurrence of chemotypes, stocking the material for extraction, conservation of the oils, influence of environmental factors, cultivation practices and vegetative cycle (Cardile et al., 2009; Kamatou, 2006). Essential oils isolated from different geographical locations can therefore, be used as anticancer agents (Zu et al., 2010) and these have different mechanism of action which make them suitable for screening as anticancer agents.

No published study evidence the use of essential oils for treatment of cancer but are only used to improve the quality of life of the cancer patients in lowering the level of their agony (NCI). This therapy cannot be a substitute to the standard chemotherapy but is being used in cancer to decrease the side effects of the drugs. Essential oils can be used for improving the health of the cancer patients and as a source of novel anticancer compounds.

Plant selected for the study-Salvia plebeia RBr.

Plants of the lamiaceae family are very important source for treatment in Ayurveda. Various species of Ocimum have many chemical and biopharmacological properties that make them suitable for use in medicine. Essential oil found in the leaves of Ocimum gratissimum has been reported to be anti-inflammatory and analgesic (Sahouo et al., 2010). Many plant species belonging to this family have anticancer activity (Sukumaran et al., 1994). Similarly, various plants of Salvia genus belonging to the same family, have also been reported to have anticancer activity. Based on the wide importance of the plants of Salvia genus, we selected a novel plant Salvia plebeia for our study.

Salvia plebeia RBr. is a small herbaceous plant growing from January to May. The plant flowers in the month of March. Its inflorescence is highly aromatic.

Salvia plebeia Classification

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Lamiales

Family Lamiaceae

Genus Salvia

Species plebeia

Table 1.1: Distribution and characteristics of the plant and its essential oil.

S. No. Characteristic Salvia plebeia RBr.

1. Distribution Grows in Punjab, Haryana, Himachal Pradesh

2. Localisation Leaves, inflorescence

3. Quantity 0.06-0.09%

The essential oils from the plants have been exploited as anticancer agents. In view of the various reports for anticancer potential of many species of Salvia, it is useful to screen Salvia plebeia RBr, for its anticancer potential.

Essential oil of S. plebeia RBr. has not been used as anticancer agents and screened for its cytotoxic activity against breast cancer cells MCF-7 and MDAMB- 231 till date in any of the study in India. Its potential evaluation for counteracting cancerous growth can be beneficial for pharmaceutical preparations. Present work thus, aims at screening the essential oil of this plant in vitro against breast cancer cell lines.

CHAPTER-2

REVIEW OF LITERATURE

Wide array of plants used in traditional medicine may be exploited for their anticancer potential. Present review represents the potential of plants from Lamiaceae family as anticancer agents with main focus on the essential oils of Salvia genus.

Anticancer drugs used in chemotherapy have various side effects and multidrug resistance (MDR) to some anticancer drugs is an important problem that occurs in cancer patients (Nooter and Herweijer, 1991) and makes them unresponsive to further treatment. The damages caused by the reactive oxygen species at molecular level are one of the causative factors for the diseases like cancer (Waris and Ahsan, 2006), aging and diabetes. Reactive oxygen species and free radicals are one of the main causes of alteration of the biomolecules like DNA, proteins, amino acids and unsaturated lipids by their oxidation. Antioxidants protect cells against damage caused due to free radicals by interacting and stabilizing them. Synthetic antioxidants like Butylated Hydroxyl Anisole (BHA) and Butylated Hydroxyl Toluene (BHT), are used widely in food products and natural antioxidants are preferred over synthetic ones (Singh et al., 2009). Natural plant products have antioxidant activity and they can reduce the oxidative stress in living beings by quenching of the free radicals. Oxidative stress is the leading cause for the cancer. Antioxidants from natural origin are potent in prevention and treatment of cancer (Li et al., 2010a). Interests have increased in searching for the novel natural antioxidants during the last few decades.

Anticancer potential of plants

In order to overcome the side effects of chemotherapy, it becomes essential to search for new plants and novel bioactive molecules for treatment of cancer. Many studies have indicated the potential of plant extracts in treatment and prevention of cancer. Studies have been done on various in vitro and in vivo models for screening of anti-proliferative agents from plants. Cell lines are the best and widely used in vitro models for screening of plant extracts and their compounds as anticancer agents (Paik et al., 2005; Loizzo et al., 2007; Ozkan et al., 2010).

Plants from lamiaceae family are best known for their medicinal properties (Hussain, 2009). Ocimum is the most important reported genus belonging to family lamiaceae and has been used in the traditional medicine. Various studies authenticate its anticancer properties in the selected models. Antiproliferative activity had been demonstrated against Hela cells by its aqueous leaf extract and seed oil (Prakash and Gupta, 2000). Ocimum sanctum had been reported to have antiproliferative effect against lung cancer (A549) cells and antiproliferative activity in the cancer cells had been reported to be due to apoptosis (Magesh et al., 2009). Other species of this genus like Ocimum gratissimum also have anticancer properties (Chen et al., 2010). O. gratissimum have been reported to be effective against lung cancer cells in dose dependent manner but did not show cytotoxicity against normal BEAS-2B epithelial cells (Chen et al., 2010). Also, methanolic extracts of Teucrium genus of this family, have antiproliferative and proapoptotic activity against HCT-116 cells and dose dependent reduction in viability (Stankovic et al., 2011). Hence, plants from Lamiaceae family are of wide interest due to their anticancer potential.

Salvia is the largest genus of Lamiaceae family with about 900-1100 species distributed worldwide. Positive results had been obtained in the research related to the use of the extracts from this genus in cancer treatment. Table 2.1 gives the brief overview of the studies related to this genus. These are indicative of the fact that this genus is rich source of anticancer agents from natural origin due to the differences observed in their specificity against cancer cells. Salvia miltiorrhiza is commonly used plant with anticancer property used in Chinese medicine (Chang et al., 2002; Wang et al., 2004; Lee et al., 2010) and novel compound, Salvicine from this plant showed significant antitumor activity against various cell lines (Qing et al., 2001) and is under phase II clinical trials for cancer treatment therapy (Cai et al., 2007). Many other species of Salvia have been reported to have anticancer activities against various organ-specific cancer cell lines (Fiore et al., 2006; Fiore et al., 2012).

Various compounds like terpenoids isolated from methanolic extracts of species of Euphorbia have potential antineoplastic activity against various human cancer cell lines like gastric, pancreatic and colon along with different multidrug-resistant (MDR) variants of these cancer cell lines with over-expression of MDR1/P-gp or no

MDR1/P-gp expression (Lage et al., 2010). Diterpenes were reported to be effective against MDR resistant cells (Lage et al., 2010). This provides an important aspect in evaluating the role of natural compounds in cancer treatment as a remedy to the most common problem encountered in cancer treatment. Hence, Salvia and other plants can overcome this problem.

Table 2.1: Studies related to various species of Salvia indicating its anticancer properties

Plants used Cell lines used Conclusions Reference Salvia menthifolia Human glioblastoma cell line(DBTRG-05MG) Antiproliferative and apoptosis Fiore et al., 2012 Salvia miltiorrhiza HL-60, A549, and MCF-7 Anticancer, fatty acid synthase (FAS) Jang et al., inhibitory diterpenoids 2012 Salvia leriifolia Renal adenocarcinoma (ACHN), amelanotic Cytotoxicity with naringenin more Tundis et al., melanoma(C32), colorectal adenocarcinoma effective than vinblastine 2011 (Caco-2), lung large cell carcinoma (COR-L23), lung carcinoma (A549), malignant melanoma (A375) and hepatocellular carcinoma (Huh- 7D12) cells Salvia plebeia A549, LoVo, SGC-7901, DU-145, HepG2, HCT- Dose dependent Cytotoxicity Ren et al., 116, HeLa 2010 and K562 Salvia fruticosa and Human colon carcinoma-derived cell lines Antiproliferation and apoptosis Xavier et al., Salvia officinalis (HCT15 and CO115) attributed to inhibition of MAPK/ERK 2009 pathway Salvia africana- Breast adenocarcinoma (MCF-7), the Anticancer activity Kamatou, caerulea, S. africana- glioblastoma (SF-268) and the colon 2008

9 lutea, S. albicaulis, S. adenocarcinoma (HT-29) aurita, S. chamelaeagnea Salvia miltiorrhiza Human hepatoma cell line (HepG2) Dose dependent Cytotoxicity Lee et al., 2008 Salvia dominica, Salvia Human breast adenocarcinoma (MCF7) Maximum Cytotoxicity of S. dominicia Abu-Dahab Hierosolymitana, Salvia and Afifi, 2007 indica Salvia miltiorrhiza Human breast cancer (MCF-7) Apoptosis and antiproliferation Wang et al., Bunge 2005 Salvia miltiorrhizae Human oral epidermoid carcinoma (KB), Growth inhibition and cell cycle arrest, Chang et al., Bunge nasopharyngeal carcinoma (HONE-1), tubulin polymerisation inhibition and 2004 colorectal carcinoma (HT29), apoptosis in drug resistant cell lines nonsmall cell lung cancer(H460) and glioblastoma multiforme (DBTRG) vincristine- resistant cell line(KB vin10), VP-16–resistant cells line(KB 7D) and the paclitaxel-resistant cell line, (KB taxol-50) Salvia miltiorrhiza Mouse lymphoblast cells(P388) Cytotoxic Mosaddik, 2003

Salvia miltiorrhiza Human hepatocellular carcinoma cell line Apoptosis and antiproliferation Liu et al., 2000 (HepG2) Salvia santolinifolia, S. Bladder carcinoma(Fen), Cervix epitheloid Inhibitory effect Amirghofran et eremophil, S. carcinoma(Hela), myelogenous leukemia al., 2010 macrosiphon, S. (K562), T cell leukemia (Jurkat) and Burkitt's reuterana, Teucrium lymphoma (Raji) persicum, and Anvillea gracini

Essential oils as anticancer agents

Essential oils have been reported more effective antioxidants (Lee et al., 2008) and anticancer agents than natural extracts (Tepe et al., 2004). The essential oils from various plants differ in their composition. Moreover, composition of essential oil from the same plant varies with season and region (Hussain, 2009; Kamatou, 2006). Hence, antioxidant and biological properties of the essential oil also vary with plant type, genus, species, seasonal and regional variation.

Many plants have been screened for the cytotoxic effects of their essential oils. Antiproliferation by the essential oil is one of the ways for cancer suppression. Essential oils of different plants from different families including lamiaceae have been reported to be cytotoxic to different cancer cell lines (Al-Kalaldeh et al., 2010; Sharma et al., 2010; Bostancıoglu et al., 2012). Essential oils of plants were tested against PC-3, A549 and MCF-7 cancer cell lines and thyme oil was found to be most effective (Zu et al., 2010). PC-3 cells were found to have strongest cytotoxic activity among all the tested cell lines (Zu et al., 2010). This indicates the more efficacy of the plant against a particular cell line. Manosroi et al. (2006) reported the anti-proliferative activity of essential oil from different plants against mouth epidermal carcinoma (KB) and murine leukemia (P388) cell lines and sweet Basil (Ocimum basilicum L.) oil gave the highest antiproliferative activity against P388 cell line.

Various studies in the table 2.2 report the anticancer properties of the essential oils from Salvia genus. Salvia officinalis oil was one of the essential oils used against breast cancer cell line, amelanotic melanoma, renal cell adenocarcinoma and hormone-dependent prostrate carcinoma using protein-staining sulforodamine B (SRB) assay. Essential oil of Salvia officinalis showed cytotoxic effect against melanoma and renal adenocarcinoma cells at IC50 at 100.70µg/ml while these were unable to show any action against human breast cancer cell line (Liozzo et al., 2007). 1,8-cineole, trans-caryophyllene and α-humulene are the constituents of essential oil and were observed in high amount in S. officinalis oil which have been reported to show anticancer activities in other studies some of which are listed in the table 2.2. Essential oil of S. officinalis was evaluated against breast cancer, colon cancer, and murine macrophage cell lines using MTT assay. α-humulene

12 and transcaryophyllene fractionated from S. officinalis have been reported to show cytotoxic activity against cancer cells and animal models (Hadri et al., 2010). Antiproliferative activities of the essential oil can be therefore, due to the presence of certain anticancer compounds.

Table 2.2: Essential oils as anticancer agents

Plants used Cell lines used Conclusion Reference Origanum onites 5RP7 (c-H-ras transformed rat embryonic Apoptotic Bostancıoglu et fibroblasts) cell lines al., 2012 Thymus vulgaris Oral cavity squamous cell carcinoma (OCSCC) Cytotoxic Sertel et al., 2011 Mentha spicata, Zingiber officinale, Human prostate carcinoma cell (PC-3), human More inhibition on PC-3 Zu et al., 2010 Citrus limon, C. paradisi, Jasminum lung carcinoma (A549), human breast cancer by Thyme oil grandiflora, Rosa damascena (MCF-7) cell lines lavender, Thymus vulgaris, Matricaria chamomilla and Cinnamomum zeylanicum Laurus nobilis, Origanum syriacum Human breast adenocarcinoma (MCF7) Antiproliferative Al-Kalaldeh et and S. triloba al., 2010 Carvacrol Human metastatic breast cancer cell line (MDA- Induction of apoptosis in Arunasree, MB 231) dose dependent manner 2010 Ocimum viride Human colorectal adenocarcinoma cells (COLO Time and dose Sharma et al., 205 cell line) dependent Cytotoxicity 2010 Salvia pisidica Hepatoma G2 cells (Hep G2) line and H1299 Protective effect against Ozkan et al.,

cell line H2O2 induced toxicity 2010

Rosmarinus officinalis and other Human breast cancer (MCF-7) and hormone Antiproliferative Hussain et al., plants of lamiaceae dependent prostate carcinoma 2009 Salvia leriifolia and Salvia COR-L23, C32, renal adenocarcinoma Synergistically Loizzo et al., acetabulosa antiproliferative 2009 Salvia rubifolia and S. bracteata Human melanoma cells (M14) S. rubifolia induced Cardile et al., more apoptosis than S. 2009 bracteata Salvia officinalis Sideritis perfoliata, Breast cancer, amelanotic melanoma, renal Laurus nobilis was Loizzo et al., Satureia thymbra, Laurus nobilis and adenocarcinoma and hormone dependent reported to be most 2007 Pistacia palestina prostrate carcinoma effective (75 µg/l) Ocimum sanctum, Alpinia Human mouth epidermal carcinoma (KB) and Anticancer activity Manosroi et al., officinarum, Cymbopogon citratus, C. murine leukemia (P388) cell lines 2006 longa

Induction of apoptosis by the essential oil by different pathways

Screening studies have been followed by the thrust to investigate the mechanism of action of the essential oils. Attempts have been made to study various mode of inhibition of cancer cell growth by the essential oils. Various studies have been done to study the mechanism of action on various in vitro and in vivo cancer models. Drugs used in cancer treatment target the cancer cells by inducing apoptosis or cell cycle inhibition. Hence, natural products causing apoptosis in the cancer cells are valuable in cancer suppression. Apoptosis or cell death plays an important role in studying the anticancer potential. Apoptosis can occur due to effect on various signalling pathways and other reasons. Yu et al. (2011) working on Bel-7402 cell line have reported that the glutathione level in the body regulates cell proliferation and reduction in glutathione leads to apoptosis. Cardile et al. (2009) in his study on melanoma reported that treatment of essential oil induces DNA damage in cancer cells, which is an indicator of apoptosis. Further, essential oil induces changes in Bcl-2 and Bax genes leading to release of cytochrome C into cytosol according to a study on KB human oral epidermoid carcinoma cells (Cha et al., 2010). This triggers the activation of caspase-9 leading to caspase-3 formation which in turn cleaves targets that causes apoptosis and increased phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase, and p38 MAPK (Cha et al., 2010). Essential oil-induced apoptosis involve mitochondrial and MAPKs pathways (Cha et al., 2010).

Studies have also been done on the mechanism of action of essential oil Salvia species indicating no effect on normal cells in comparison to cancer cells. Itani et al. (2008) attempted to study the mechanism of action of the anticancer potential of the components from essential oil of Salvia libanotica like linalyl acetate, terpeniol and camphor. These were reported to inhibit the growth of two isogenic human colon cancer cell lines HCT-116 (p53+/+ and p53-/-) synergistically while in contrast no effect was observed on normal human intestinal cells. Mitochondrial- mediated and caspase-dependent pathways were concluded to be responsible for apoptosis in p53+/+ cells, while in p53-/- cells, apoptosis was caspase independent.

Essential oil has also been reported to have anti-metastasis action (Loutrari et al., 2011) and these induce apoptosis by affecting intrinsic and extrinsic pathways. Essential oil from rosewood Aniba rosaeodora, was reported to induce apoptosis by increasing reactive oxygen species production (Soeura et al., 2011). Essential oil of Ocimum viride also has been found to induce apoptosis (Sharma et al., 2010). In another study, essential oil of Curcuma zedoaria has been tested in vitro and in vivo keeping in view anti- angiogenesis effect. It was reported to exhibit anti-proliferative activity against various cell lines and resulting in suppressing melanoma growth and lung metastasis. This action was reported to be attributed to the down-regulation of matrix metalloproteinases (Chen et al., 2011). Essential oils have different mechanism of action.

Essential oils are not only effective in cell line models but in animal models also. These can help in cancer prevention also. Studies on animal models also authenticate the use of essential oil in cancer treatment. Essential oils are found to have preventive effect on cancer treatment. This was shown by the study of Li et al. (2010a). Varying concentrations of the essential oil were administered to mouse having diethylnitrosamine-induced gastric cancer with basic diet and it affected phase I enzymes superoxide dismutase, catalase and glutathione peroxidase activities as the markers of oxidative stress. Essential oil was found to show protective effect against oxidative stress (Li et al., 2010a). Essential oil of Allium sativum has also been reported to be efficient in gastric cancer in mouse model (Li et al., 2010b). Dietary modifications can be useful in cancer prevention. Hence, essential oils can have wide therapeutic potential as these can act by two ways- chemoprevention and cancer suppression. These induce phase I and phase II enzymes which prevent the interaction of carcinogens with DNA therefore are chemopreventive effect (Li et al., 2010a; Li et al., 2010b; Ozkan, 2010). Antiproliferative action of the essential oils during the growth phase can be by the inhibition of the cancer cell growth and induction of cell death.

Importance of selection of Salvia plebeia for the study

Various plants from Salvia genus show anticancer properties (Chang et al., 2002; Wang et al., 2004; Abu-Dahab and Afifi, 2007). Extracts of Salvia plebeia plant used in the present study, were reported to have phenolic compounds like

17 hispidulin-glucuronide, hispidulin-7-O-d-glucoside, 6-methoxy-luteolin-7-glucoside, b-sitosterol, 20-hydroxy-50-ethoxybiochanin A and coniferyl aldehyde (Weng and Wang, 2000). Natural compounds isolated from S. plebeia were reported to show even higher antioxidant activity than Butylated Hydroxyl Toluene (BHT) and α- tocopherol (Weng and Wang, 2000). These compounds namely 6-methoxy- luteolin-7-glucoside b-sitosterol and 20-hydroxy-50-methoxybiochanin A can therefore, be beneficial for their use in the food products and for improving health of the people as antioxidants protect against diseases like cancer.

Extracts of Salvia plebeia had been reported to induce apoptosis in various cell lines like A549, LoVo, SGC-7901, DU-145, HepG2, HCT-116, HeLa and K562 in dose and time-dependent manner. Extracts were reported to be most effective cytotoxic agents against K562 cells having inhibitory concentration less than 15μg/ml for 3 days treatment (Ren et al., 2010). But, essential oils have been reported to be more effective anticancer agents than other extracts in some other studies (Tepe et al., 2004). Therefore, essential oil of Salvia plebeia is expected to be more effective than its extract. No study has been done on the essential oil of this plant against breast cancer cell line. Hence, novel essential oil of plant has been used in the present study.

It can be concluded that the plant extracts are one of the most important and promising avenue for anticancer therapies. Its importance increases manifold specially keeping in view that these compounds can be used in our daily diets by slight diet modifications and are extremely cost effective. Also, diet modifications can minimise the risks of cancer development. While on the other hand, in the cancer patients undergoing treatment, these slight diet modifications can act as combination therapies along with the modern chemo and radio therapeutics. Keeping this in the mind, it becomes extremely important to screen more naturally occurring plant essential oils for their anticancer properties. Hence, present work has been framed keeping in view the advances in screening of more essential oils from different plants for cancer treatment and the literature reviewed.

CHAPTER-3

OBJECTIVES

3.1 Objectives:

 To extract and analyse the essential oil of Salvia plebeia RBr.

 To estimate the antioxidant property of essential oil of Salvia plebeia RBr.

 To determine the effect of essential oil of Salvia plebeia on cell viability.

Breast cancer cell lines, MCF-7 and MDAMD-231 have been used as in vitro models to determine the anticancer potential of S. plebeia.

3.2 Plan of work:

 Collection of the plants of Salvia plebeia.

 Extraction of the essential oil from the inflorescence and leaves.

 Composition determination of the essential oil using GC-MS analysis.

 Determination of the antioxidant activity of the essential oil by DPPH and hydroxyl radical scavenging assays.

 Determination of the anticancer activity of the essential oil using MTT assay in time and dose dependent manner.

 Determination of the mode of action of the essential oil using single cell gel electrophoresis.

CHAPTER-4

MATERIALS AND METHODS

The present research work was carried out in the Tissue Culture and Environment Science Laboratories of the Central University of Punjab, Bathinda.

4.1 Materials

4.1.1 Instruments

Instruments used in the course of experimental work are listed below along with the company in the table 4.1.

Table4.1 Instruments used and the company

Name of the Instrument Manufacturing Company Analytical Balance TE214,S Sartorius Autoclave (vertical) NSW-227 Calton Company

CO2 Incubator New Brunswick, UK Comet Assay Electrophoresis Unit Sci. Plas, UK ELISA reader 642 Systronics Essential oil Distillation Unit (Clevenger Andel Works Apparatus) Fluorescent Microscope aided with computer Olympus Magnus Hot Plate Tarsons Inverted Microscope with live Imaging Facility Olympus Magnus Light Binocular Microscope with Digital Camera Olympus Magnus Attachment Spinwin Daikan Scientific Mini Centrifuge MC-02 Co. Ltd. pH meter Mettler Toledo Rectangular water bath New India Refrigerated Centrifuge 5430R Eppendorf, Germany Refrigerated Circular Water Bath Julabo, Germany UV-VIS double beam 2202 Spectrophotometer Systronics

4.1.2 Chemicals and Standard Compounds

2,2,-diphenyl-1-picrylhydrazyl, Butylated Hydroxy Toulene (BHT), ascorbic acid, Trichloro-acetic acid (TCA), Dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol)- 2,5- diphenyltetrazolium bromide (MTT) and other chemicals of analytical grade like anhydrous sodium carbonate, ferrous chloride, chloroform and methanol, used in this study were purchased from Loba, Sigma Eldrich and MP Biomedicals. Culture media, trypsin, heat inactivated foetal bovine serum (FBS, Gibco), antibiotic solution were purchased from Invitrogen and Himedia.

4.1.3 Collection of plant material

Salvia plebeia was selected for the present study on the basis of the review and its properties. There is no past study related to essential oil of this species of the plant in the present context. Aerial parts of the wildly growing plants were collected from Tira village and were identified by the herbarium-incharge of Punjab University, Chandigarh.

4.1.4 Cell lines used for determining anticancer potential of the essential oil of Salvia plebeia

Human breast cancer cell lines, MCF-7 and MDA-MB 231 were procured from National Centre for Cell Culture (NCCS), Pune.

MCF-7 is the breast cancer cell line isolated from a 69year old Caucasian female and has characteristics of mammary epithelium like estrogen receptors, progesterone receptors and ability to form dome or clusters. MDAMB-231 is a derived from 51year old Caucasian female and have epidermal growth factor transforming growth factor-alpha and is aneuploid (ATCC). MDAMB-231 is hormonal independent cell line and is highly metastatic.

4.2 Experimental methods

4.2.1 Extraction of essential oil- Plant material was collected and cleaned. Inflorescence and leaves were separated out. Essential oil of Salvia plebeia was extracted by the steam-distillation of fresh inflorescence and leaves of the herb using Clevenger apparatus in the laboratory.

4.2.2 Analysis of the oil

4.2.2.1 Physical properties- Colour of the essential oil and specific gravity was determined.

4.2.2.2 Gas chromatography/mass spectrometry (GC-MS) analysis– Essential oil sample was analysed at Institute of Himalayan Bioresource and Technology (IHBT), Palampur (Himachal Pradesh). Compounds were separated on DB-5 MS capillary column (30 m x 0.25 mm, film thickness 0.25 μm; made in USA). 30µl sample in Dichloromethane was injected using split mode. Injector temperature was set at 250oC. Column oven temperature was programmed from 40oC to 220oC at the rate of 4oCmin-1, initial and final temperatures were held for 4 and 15 minutes, respectively. Mass scanning range was 50 –800 m/z.

4.2.2.3 Identification of the compounds

The identification of the components was done by comparing calculated Kovats Index (KI) values of different compounds with that of compounds already present in NIST library (http://webbook.nist.gov/chemistry/).

4.2.3 Antioxidant activity:

4.2.3.1 DPPH assay (Liyana-Pathiranan and Shahidi, 2005) - Antioxidant property of the essential oil was measured spectrophotometrically using DPPH radical as a reagent. Hydrogen atom or electron donating abilities of essential oil and an antioxidant, Butylated Hydroxy Toulene (BHT) were measured from bleaching of the purple coloured methanolic solution of DPPH.

Procedure: Prepared various concentrations of the essential oil (20- 100µg/ml) in methanol. 2ml of the above prepared concentrations were added to 2ml of 0.135mM methanolic solution of DPPH. Mixture was shaken vigorously and was left to stand for 30 minutes in dark. Absorbance was recorded at 517nm against blank. Blank was prepared with the addition of DPPH in methanol. BHT was used as standard and similar procedure was followed for the standard. The ability to scavenge DPPH radical was calculated by the following equation:

DPPH radical scavenging activity (%) = [(Abscontrol - Abssample)/Abscontrol] × 100 where, Abscontrol is the absorbance of DPPH radical + methanol; Abssample is the absorbance of DPPH radical + sample extract/standard.

Decreased absorbance of the reaction mixture indicates stronger DPPH radical scavenging activity.

4.2.3.2 Hydroxyl Radical (OH) Scavenging Assay (Halliwell et al., 1987)

The hydroxyl radical scavenging of the essential oil was measured using the modified method of Halliwell et al. (1987). Stocks of EDTA (1mM), FeCl3 (10 mM), ascorbic acid (1mM), H2O2 (10mM) and 2-deoxyribose (10mM) were prepared in distilled deionized water. Added 0.1 ml of EDTA, 0.01 ml of FeCl3, 0.1 ml of H202, 0.36 ml of 2-deoxyribose, 1.0 ml of concentrations of essential oil, 0.33 ml of phosphate buffer (50 mM, pH7.4) and 0.1ml of ascorbic acid respectively. Incubated the mixture at 37°C for 1hour. Taken 1.0 ml of the incubated mixture and mixed with 1.0 ml of 10% TCA and 1.0 ml of 0.5% Thiobarbituric acid (TBA). Heated at 1000C for 20 minutes and measured absorbance at 532 nm with 2- deoxyribose and buffer as blank.

Hydroxyl scavenging activity (%) = [(Absblank - Abssample)/Absblank] × 100 where, Absblank is the absorbance of blank; Abssample is the absorbance of sample oil /standard.

4.2.4 in vitro assays for anticancer activity 4.2.4.1 Culturing of the cell lines – Breast cancer cell lines, MCF -7 and MDAMB-231were procured from National Centre for Cell Culture (NCCS), Pune. MCF-7 and MDAMB-231 were cultured and maintained in 25 cm2 (Tarsons) flasks containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1 µg/ml penicillin/streptomycin. The MCF-7 cells were incubated at 370C in a humidified atmosphere containing 5%

CO2 and 18% oxygen. MDAMB-231 cells were grown in CO2 free atmosphere at 370 C. 70% confluent flasks were used for sub culturing and further treatments.

4.2.4.2 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (modified method of Mosmann, 1983) – This is colorimetric assay that determines the viability of the cells by measuring the reduction of yellow 3-(4,5- dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product which is then solubilised with an organic solvent like DMSO and the released, solubilised formazan product is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells therefore, level of activity is a measure of the viability of the cells. Procedure: 100µl of 5-6×104 cells were plated per well in the microtitre (96 well) plate and incubated at 37oC for 24 hours followed by serum starvation to make cells at the same phase. Suspension of the essential oil in 0.25% methanol was prepared and further dilutions were prepared in the media. Treatment was given to the cells by adding 200µl per well of the prepared dilutions. Incubated for the required time and added 100µl MTT (5mg/10ml in PBS). After keeping in dark for 4 hours, added 100µl of DMSO to dissolve the formazan crystals. Reading was taken in ELISA reader at 490nm.

Table 4.2 Treatments of the essential oil. Treatment time Concentrations used 12 hours 30-105µg/ml

18 hours 30-105µg/ml

24 hours 30-105µg/ml

1 hour +23 hours chase time 30-105µg/ml

6 hours + 18 hours chase time 30-105µg/ml

12 hours + 12 hours chase time 30-105µg/ml

4.2.4.3 Single cell gel electrophoresis, SCGE or Comet assay (modified method of Olive and Banath, 2006)- Equipments: Horizontal gel electrophoresis chamber (Comet assay tank) and power supply and Fluorescence microscope aided with computer.

Reagents:

1. Low-gelling-temperature agarose (Hi-Media, India) 2. 10 mg/ml Ethidium Bromide (Stock solution) 3. Alkaline lysis solution for single-strand break detection 4. Alkaline rinse and electrophoresis solution

Procedure:

Agarose preparation- Water baths were set at 400C and 1000C and prepared 1% low-gelling-temperature agarose by mixing powdered agarose with distilled water in a glass beaker. Placed the beaker in microwave for 1 minute at low power for short time intervals. Avoided vigorous boiling of the agarose and ensured that whole agarose was dissolved. Placed beaker with agarose into a 40 0C water bath.

Slide pre-coating- Prepared agarose-precoated slides by pouring molten 1% agarose and allowed agarose to air-dry to form a thin film.

Sample preparation- Single-cell suspensions of the essential oil treated cells and control cells were prepared by trypsinisation. Cell density was adjusted to 30×105 cells/ml using haemocytometer. 1ml of 1% low-gelling-temperature agarose at 40°C was added to 1ml of the suspension prepared. Mixed and rapidly pipetted 1ml of cell suspension onto the agarose-covered surface of a pre-coated frosted slide and avoided producing bubbles. Prepared slides in triplicates for each treatment and control. Slides were allowed to dry for about 5 minutes.

Lysis and electrophoresis Lysis and electrophoresis was performed in alkaline conditions to detect the combination of DNA single-strand breaks, double-strand breaks and alkali-labile sites in the DNA. After agarose got gelled, submerged slides in a covered dish containing alkaline lysis solution (1.2 M NaCl, 100 mM

Na2EDTA, 0.1% SDS, 0.26 M NaOH, pH > 13) gently by holding slides horizontally and lower into solution. Lysed samples overnight (18−20 h) at 4°C in the dark. After overnight lysis, carefully removed slides and submerged in room temperature (18−25°C) rinse solution for 20 min. Repeated two times to ensure removal of salt and detergent. After two rinses, submerged slides in fresh rinse solution in an electrophoresis chamber. The chamber was filled with a consistent volume of buffer that was about 1–2 mm above the top of the agarose. Electrophoresis was done in rinse solution (0.03 M NaOH, 2 mM Na2EDTA, pH ∼12.3) for 30 min at a voltage of 15 V and 40 mA current.

Slide staining- Removed slides from electrophoresis chamber and rinsed and neutralized with distilled water. Pipetted about 500μl of a 0.5µg/ml solution (prepared from 10mg/ml stock) of ethidium bromide directly onto the slide and incubate for 20 min. Placed slides in distilled water to remove excess stain.

Slide analysis- Analyzed cells by examining at 50 comet images from each slide under fluorescent microscope. Scoring of the comets obtained was done by using Cometscore15 software Version 1.0.1.0.

4.3 Statistical analysis- Results obtained by the experimentation were analysed by one way ANOVA by applying Tukey’s test for antioxidant assays and Dunnett’s test for multiple comparisons with control for MTT assays. All statistical analysis was performed by SigmaPlot Version 11.0 software.

CHAPTER-5

RESULTS AND DISCUSSION

RESULTS

5.1 Extraction of essential oil- Essential oil was extracted by steam distillation method using Clevenger apparatus and was pale orange in colour with specific gravity of 0.928. Yield of the essential oil was 0.05% approximately.

5.2. Composition of the essential oil of Salvia plebeia-

Various components of the essential oil have been identified by the GC-MS analysis which indicates that caryophyllene (45.93%) was the most abundant component in the essential oil of S. plebeia. Other components like caryophyllene oxide (5.85%), α-humulene (2.6%), aromadendrene (1.02%), alloaromadendrene (0.27%), gemacrene B (1.62%), alpha terpenyl acetate (1.94%), beta-elemene (0.6%) were also present. Major compounds identified are listed in Table 5.1 by comparing the calculated values of the Kovats index with the values of the compounds in NIST library and figure 5.2.1 shows the chromatogram obtained.

Figure 5.2.1: GC chromatogram of the essential oil obtained on the DB-5 column Compounds identified by the GC-MS analysis constitute 68.44% of the total compounds of the essential oil.

Table 5.1: Major compounds of essential oil of Salvia plebeia RBr. and the data obtained by GC-MS analysis with their classification.

% Area RI ( calculated) Compound Classification

7.6 991.04761 Beta-Myrcene Monoterpene

1.94 1350.31392 alpha terpenyl acetate Monoterpene

0.6 1390.6963 Beta-elemene Sequiterpene

45.93 1424.9621 Caryophyllene Sesquiterpene

1.02 1439.6671 Aromadendrene Sesquiterpene

2.68 1457.5189 alpha-humulene Sequiterpene

0.27 1461.1195 Alloaromadendrene Sesquiterpene

0.18 1490.59 beta-Selinene Sesquiterpene

0.33 1520.8692 delta-Cadinene, Sesquiterpene

1.62 1562.1923 Germacrene B, Sesquiterpene

5.85 1589.5493 Caryophyllene oxide Sesquiterpene

0.42 1760.4609 Spathulenol Sesquiterpene

5.3 Antioxidant activity of the essential oil-

5.3.1 DPPH assay- Lower concentrations of the essential oil in methanol were observed to have very low antioxidant activity. Essential oil was observed to show 50% inhibition at the approximately 625µg/ml which is very high in comparison to BHT which showed the same inhibitory concentration at 45µg/ml. Figure 5.3.1 depicts the comparative account of the antioxidant activity of the essential oil with BHT. DPPH scavenging activity increased in the concentration dependent manner in all the concentrations above 50µg/ml showing significant increase in the activity. DPPH scavenging activity for the 100µg/ml was observed to be 7% and 97% for

28 essential oil of S. plebeia and BHT respectively. Hence, essential oil has antioxidant property.

Figure 5.3.1: DPPH radical scavenging ability of the essential oil of Salvia plebeia. Data has been analyzed by one way ANOVA, multiple comparison Tukey’s test. Means followed by the same letter on same line are not significantly different by Tukey’s test (p value<0.05).

5.3.2 OH radical scavenging assay- Concentration dependent scavenging of the hydroxyl radicals was observed. Antioxidant activity of the essential oil was very low in comparison to BHT as shown by the figure 5.3.2, in the concentration varying from 20-100µg/ml. Inhibitory concentration was about 60% for BHT at 100µg/ml while only 15% for the same concentration of essential oil. Figure 5.3.2 shows the clear demarcation between the efficiency of the BHT with respect to essential oil showing increase in the antioxidant activity of the essential oil with the increase in the concentration resulting from the ability to scavenge Hydroxyl radical. Essential oil was found to have IC50 at 375µg/ml.

Figure 5.3.2: Effect of essential oil Salvia plebeia on hydroxyl radical scavenging. Data has been analyzed by one way ANOVA, multiple comparison Tukey’s test. Means followed by the same letter on same line are not significantly different by Tukey’s test (p value<0.05).

5.4 Anticancer activity of the essential oil-

Essential oil of S. plebeia was tested for cytotoxic activity against breast cancer cell lines, MCF-7 and MDAMD-231 at various concentrations. Effectiveness of the essential oil of S. plebeia on viability of cancer cells was studied by the MTT assay as shown by the results.

5.4.1 Effect of essential oil on cell viability- Cell viability was observed to reduce in dose dependent and time dependent manner with significant differences among the control and treatment groups. The results shown for the two breast cancer cell lines clearly depict the cytotoxic activity.

The essential oil treatment for 12 and 18 hours to the MCF-7 cell line resulted in the inhibition of the growth of MCF-7 cells with increase in concentration from 30- 105µg/ml. Rapid decrease in viability of the cancer cells was observed between 30-40µg/l and decreased at higher rates at the higher concentrations (figure 5.4.1). The values were significant at higher concentrations and the viability of the cancer cells was observed to reduce with essential oil treatment shows the effectiveness of essential oil against the breast cancer cells. %viability was observed to decrease from 80% at 30µg/ml to 43% at 60µg/ml and then reached 21% (approximately) at 105µg/ml for 12 hour treatment. Treatment of the essential oil for 18 hours resulted in 77% to 38% viability reduction of the cancer cells at 25

30 and 75µg/ml concentrations decreasing to 13%(approximately) at higher concentration of 105 µg/ml.

110 100 90 a 80 70

SEM) b

± 60 12hrs 50 b 18hrs a a

(Mean b 40 b a 30 a b a

viability viability 20 b

% % 10 0 Control 30 45 60 75 90 105 Essential oil conc. (µg/ml)

Figure 5.4.1: Effect of essential oil of Salvia plebeia on MCF-7 cell line for 12 and 24 hours treatments. Data has been analysed by one way ANOVA followed by Dunnett’s test for multiple comparison with control. a (p<0.05) and b (p<0.05) represent statistically significant values in comparison to respective controls for 12 hours and 18 hours treatments respectively.

Similar results were observed in the MDAMB-231 cell line. Dose and time dependent effect of essential oil on the cell viability was observed to decrease but differences in the 45µg/ml treatment were not significant with respect to time and viability was found to increase for 18 hours treatment in comparison to 12 hours treatment as shown in figure 5.4.2. Viability percentage was observed to decrease from 91% to 6% for 12 hours treatment and from 98% to 4% for 18hours treatment for the 30µg/ml to 105µg/ml concentrations respectively. Significant decrease in cell viability was observed with concentration at higher concentrations but variability in response with respect to treatment time in both the cell lines. However, overall treatment response was higher for the more long-lasting treatment.

110 100

90 80

SEM) a

± 70 60 12hrs 50 a 18hrs 40 b 30

% viability (Mean viability % 20 a b a b a 10 b 0 Control 30 45 60 75 90 105 Essential oil conc. (µg/ml)

Figure 5.4.2: Effect of essential oil of Salvia plebeia on MDAMB-231 cell line for 12 and 18 hours. Data has been analysed by one way ANOVA followed by Dunnett’s test for multiple comparison with control. a (p<0.05) and b (p<0.05) represent statistically significant values in comparison to respective controls for 12 hours and 18 hours treatments respectively.

Keeping in view the variability observed in the viability of the cancer cells for the 12 and 18 hours treatments, treatment time interval was increased to 24 hours for both the cell lines which resulted the most effective response. There was significant decrease in the viability of the cells with the concentration in MCF-7

(Figure 5.4.3) and MDAMB-231 (Figure 5.4.4). IC50 of MCF-7 was approximately

30µg/ml while MDAMB-231 has higher IC50 at 65µg/ml. Data showed high significant difference in comparison to control at higher concentrations of the oil and indicates the effectiveness of the essential oil treatments. In comparison to untreated cells, there was 10% and 80% call death at the doses of 30 and 70µg/ml respectively in MDAMB-231 cells after 24 hours treatment.

120

100

SEM)

± 80

60 a a 40 a

a a % viability (Mean viability % 20 a

0 Control 30 45 60 75 90 105 Essential oil conc. (µg/ml)

Figure 5.4.3: Essential oil of Salvia plebeia showing significant decrease in the viability of MCF-7 cells for 24 hours. Data has been analysed by one way ANOVA followed by Dunnett’s test for multiple comparison with control. Data with same letters indicate that results are statistically significant (p<0.05) in comparison to control for 24 hours treatments.

120

100

SEM, n=3) SEM,

± 60

40 a a a

20 % viability (Mean (Mean % viability

0 control 30 45 60 75 90 105 Essential oil conc. (µg/ml)

Figure 5.4.4: Effect of 24 hours treatment of essential oil of Salvia plebeia on MDAMB-231 cells. Data has been analysed by one way ANOVA followed by Dunnett’s test for multiple comparison with control. Means with same letters indicate that results are statistically significant (p value<0.05) in comparison to control for 24 hours treatments.

Effect of different exposure time of the essential oil on MCF-7 and MDAMB-231 cell line with different chase time showed significant differences in the viability of the cells. Various treatments were given to the breast cancer cells to determine the efficacy of the inhibition of the cell viability. Treatment with the essential oil followed by varied time interval of the normal growth (chase time) showed sustained exposure time-effect of the essential oil. Essential oil treatment response varied with different concentrations. Inhibitory effect was observed on the cell viability to the concentration of 30µg/ml for 1 hour treatment followed by 23 hours of chase time with viability but at higher concentrations cell viability was found to increase rapidly and then ultimately decreased. This may be due to effective inhibitory effect of the essential oil in the low exposure time. This increased viability was observed at 75 µg/ml concentration in MCF-7 cells while at 60 µg/ml concentration in MDAMB-231 cells. This has been depicted in the figure

5.4.4 and 5.4.5.

120

100

80 control b

SEM, n=3)SEM, 30

c ± 45 60 c

d 60 (Mean d a 75 b d 40 b 90

%viability a b 105 b c c 20 c d d d

0 1hr 6hrs 12hrs 24hrs Exposure time(hours)

Figure 5.4.5: Effect of essential oil of Salvia plebeia on MCF-7 cells with different exposure and chase times. Data has been analysed by one way ANOVA followed by Dunnett’s test for multiple comparison with control. Mean values with same letters indicate that results are statistically significant (p value<0.05) in comparison to control for different treatments.

140

120

100

control SEM, n=3) SEM, d 30 ± 80 45 a c

60 60 (Mean (Mean 75 40 a b b b c a c d d d 90 20 %viability %viability 105

0 1hr 6hrs 12hrs 24hrs Exposure time (hours)

Figure 5.4.6: Effect of essential oil of Salvia plebeia on MDAMB-231 cells with different exposure and chase times. Data has been analysed by one way ANOVA followed by Dunnett’s test for multiple comparison with control. Mean values with same letters indicate that results are statistically significant (p value<0.05) in comparison to control for different treatments.

Exposure time plays an important role in the antiproliferative action of the essential oil against two different breast cancer cell lines. With the increase in the exposure time (24 hours) cell viability was observed to show regular decrease with the increasing concentration. Essential oil treatments were observed not to have significant differences in the cell viability values at low concentrations in comparison to control. This means that cells were killed immediately after treatments but treatments have stimulated proliferation in the surviving cells as this trend is observed in the same concentrations after different treatments or adaptation have occurred in the cells. Variability with concentration and exposure time may have resulted in difference in the viability percentage due to different activity of the constituent components of the essential oil which can have synergistic or antagonistic effects. Hence, higher dose and exposure time had sustained effect of the oil on the breast cancer cell lines.

5.4.2. Single cell gel electrophoresis- Antiproliferative activity of the essential oil was observed to be the result of DNA damage caused due to essential oil

35 treatment against breast cancer cells. This was shown by the results of the comet assay (Figure 5.4.7).

Figure 5.4.7: Effect of the essential oil of Salvia plebeia on the individual MCF-7 cells showing the formation of the comets in the treatment groups compared to control group observed at 40X of the fluorescent microscope.

In order to access DNA damage by the essential oil treatment comet assay was performed on MCF-7 cells. Treatment concentrations were selected on the basis of MTT assay (25, 30 and 45µg/ml) of essential oil on MCF-7 cells for 24 hours. Variation was observed in the comets formed with concentration and maximum number of the cells in the particular treatment showed similar structures. Various parameters selected were head intensity, tail intensity, %DNA in tail, % DNA in head, tail length and head diameter. Figure 5.4.8 shows the variation of the selected parameters with concentration.

A B

C D

E F

Figure 5.4.8: Histograms (A-F) showing the DNA damage induced by the different concentrations determined by parameters such as Head Intensity, Tail Intensity, % DNA in Head and Tail, Head diameter and Tail Length. All values are expressed as mean ± SEM (n=3).

DNA in head (expressed in %) was found to decrease with the concentration in comparison to control cells which indicates the more migration of the DNA fragments on electrophoresis leading to formation of the comets while % DNA in tail conversely increased in tail. Similar trends were observed for head diameter and tail length but tail length was observed to have less variability at 45µg/ml. DNA fragments have not moved further at this concentration however, tail intensity has increased at the same concentration indicative of more fragments in tail of the comets formed. These parameters suggest the damage induced in the DNA with concentrations. From the data available after comet scoring, tail DNA and tail length of the MCF-7 cells exposed for 24 hours increased rapidly at 25-45 µg/ml concentration of the essential oil indicating DNA damage caused by the action of essential oil. This suggests that the oil used in the treatment have induced cell death as DNA fragmentation occurs during cell death.

Our results show the cytotoxic activity of the essential oil as observed from the MTT assay and was observed to induce DNA damages as the cause of the cell death. Essential oil was found to have antioxidant activity and its composition analysis indicate the compounds that can have anticancer properties.

DISCUSSION

Cancer is one of the major health problems in the present day and accounts for millions of death per year worldwide. There are various types of cancer like lung cancer, prostate cancer and colon cancer based on organ of the origin. Breast cancer is the major cancer type found in females. Chemotherapy and radiation therapy are the most effective and widely used methods in the cancer treatment but these treatment methods have various side effects including hair loss, nausea and drug resistance (Nooter and Herweijer, 1991; Naumovski et al., 1992). Naturotherapy and other treatments are therefore becoming popular as complementary treatments along with standard therapies. Improved nutrition and healthy lifestyle play an important role in the improvement of the health of cancer patients.

Essential oils are widely used as food additives and nutritional supplements and have been reported to have anticancer activities attributed to the its components. In our study, various components of the essential oil of S. plebeia were observed to be caryophyllene, caryophyllene oxide, alpha- humulene, aromadendrene, alloaromadendrene, gemacrene B, alpha-terpenyl acetate and beta-elemene. Studies support the occurrence of alpha-humulene (10.3%) as the major component of essential oil of S. plebeia which is present in only 2.6% in our results (Thoppil, 1993) but the detailed account of the components was not available and hence, some of the major compounds have been identified. Some components of essential oils have been reported to have anticancer activity (Carnesecchi, 2001; Chang et al.; 2004; Cha et al., 2010 and Hadri et al., 2010) against the cancer cells. Caryophyllene and alpha-humulene present in the oil had been reported to have anticancer activity (Hadri et al., 2010) which may be responsible for antiproliferative activity of the oil in the study. This study also shows the antioxidant activity of the essential oil at the concentration higher than 100µg/ml in the DPPH assay and hydroxyl radical scavenging. Free radical scavenging capacity of the essential oil has been found to be comparatively lower than the synthetic standard BHT used in the food products. Our results are supported by the study of Kamatou (2006) in which low antioxidant activity of the essential oils of various species of Salvia has been reported and active anti-inflammatory activity was observed. Low antioxidant activity in comparison to BHT has been

39 reported in S. officinalis with these showing IC50 for the essential at the concentrations of 62.3 and 9.9µg/ml respectively (Hussain, 2009).

Hence, antioxidant activity of the essential oil cannot be directly correlated to its antiproliferative activity but it may be one of the factors for this. Salvia plebeia extracts have been reported to have anticancer activity (Ren et al., 2010) but the studies suggest the more efficiency of the essential oil in comparison to the extracts used for various species of the same genus (Kamatou, 2006). Complementary treatment methods for cancer widely use essential oils due to their stress relieving properties. No study have been done in this respect on the essential of Salvia plebeia and in this study, essential oil of S. plebeia have been shown to have anticancer property against the breast cancer cell lines as shown by the results of the MTT assay. Inhibitory effect was observed on the viability of breast cancer cell lines, MCF-7 and MDAMB-231 with different concentrations of essential oil. Cell viability was observed to decrease in both the cancer cell lines with increase in concentration of the essential oil and 0.25% methanol used as vehicle control was not found to have any inhibitory effect. MCF-7 and MDAMB- 231 responded differently to the different concentrations with MCF-7 cancer cell line being more susceptible to the oil. This can be due to difference in the properties of the cell lines. Dose and time dependent effect on the cancer cell viability was concluded from the study. Treatment time played an important role in the determination of inhibition of the cancer cell growth by the essential oil. Concentration dependent inhibition with irregular trend was observed at lower concentrations. But treatment time of 24 hours showed significant dose dependent inhibition. MCF-7 was observed to show IC50 at concentration 30µg/ml and in

MDAMB-231, IC50 was calculated to be 65µg/ml for 24 hours treatment. Significant decrease in the viability was observed in comparison to the control indicating the effectiveness of the essential oil against breast cancer cell lines. These results show the more efficacy of the essential oil of S. plebeia in comparison to S. officinalis which was reported to show 50% inhibition of the viability at very high concentration of 320µg/ml (Hussain, 2009) with former is having IC50 at 30µg/ml against the breast cancer cell line MCF-7 for 24 hour treatment that was used for the study. Biological properties of the natural substances are the result of their bioactive components. Essential oil cytotoxicity is attributed to the bioactive

40 compounds present which may act synergistically or antagonistically to produce the effects. Exposure time effect of the essential oil was evaluated in order to know the effect of exposure to the essential oils as these are also used as food products. Various treatments were given to the breast cancer cells to determine the efficacy of the inhibition of the cell viability. Treatment with the essential oil followed by varied time interval of the normal growth (chase time) were given to determine the exposure time and sustained time effect of the essential oil. Essential oils were found to respond differently at different concentrations. Inhibitory effect was observed on the cell viability to the concentration of 30µg/ml for 1 hour treatment followed by 23 hours of chase time but at higher concentrations cell viability was found to increase rapidly and then ultimately decreased. This may be due to effective cytotoxic effect of the essential oil in the initial stages and then surviving cells may have adapted to the treatment which enhanced the cell viability while at higher concentrations, cells were susceptible to the treatment. Cell viability was observed to decrease for 12 hour treatments with 12 hours of the chase time. This suggests that essential oil require time to act on the breast cancer cells and cell viability further decreased on treating cells with essential oils for 24 hours reaching to the maximum. Essential oils are used in food products also and hence from this study we have found that these oils can show the anticancer activity on exposure for long time durations.

From the above results we concluded that the essential oil has anticancer property and to determine the mechanism of action of essential oil, alkaline single cell gel electrophoresis (comet assay) was performed to analyse the changes in the nuclear DNA at the individual cell level. With this assay we can differentiate apoptotic and normal cells and the necrotic cells also. This test is widely used for genotoxicity testing as an indicative of DNA damage caused by direct genotoxicity of the substances but can also indicate the DNA fragmentation caused by membrane and mitochondrial damage due to apoptosis or necrosis (Olive and Banath, 2006). Hence, this assay has been performed to determine the effect of essential oil on the DNA as a method to show its mechanism of action. Comet assay performed on MCF-7 cells showed changes at the DNA level. The parameters like increase in tail length, tail intensity, tail moment and decrease in head diameter, head intensity considered suggest the damage induced in the DNA

41 with concentrations. This suggests that the oil used in the treatment have induced apoptosis as DNA fragmentation occurs during apoptosis (Choucroun et al., 2001). Study of Cardile et al. (2009) support the present study in which two other species namely Salvia rubifolia and S. bracteata studied against the melanoma and has been reported to be antiproliferative and induced apoptosis which was determined by single cell gel electrophoresis. They also observed the increased tail moment and DNA in the tail as the indicators of apoptosis. This study supports the results of increased DNA in tail and decrease in head along with other parameters considered after scoring of the comets. Comet assay was used to determine the effect of essential oil of Salvia at DNA level as an indicator of apoptosis induction.

These results clearly indicate the induction of apoptosis on the treatment of essential oil to the breast cancer cells. This seems to suggest DNA damage caused cell death. We have shown in the study, the efficiency of the essential oil as anticancer agent and effect of the exposure time on breast cancer cells. Detailed study is required to show the mechanism of action of the essential oil.

CONCLUSION AND FUTURE PROSPECT

Conclusion

In the present study, composition, antioxidant properties and the effect of essential oil of S. Plebeia on breast cancer cell lines, MCF-7 and MDAMB-231 were studied and the following conclusions were drawn from the results obtained:

 Essential oil of S. plebeia was composed of monoterpenes and sesquiterpenes with caryophyllene as the major compound.

 Antioxidant property was observed in the essential oil.

 Viability of the breast cancer cells was reduced with the increasing concentration of the essential oil. Dose and time dependent relation on the cytotoxic activity was observed.

 Exposure time was observed to show the direct effect on the cell viability. Increased exposure time showed sustained inhibitory effect on the growth of the cancer cells.

 Essential oil leads to DNA damage in the cancer cells which results in cell death.

 MCF-7 cells were observed to be more responsive than MDAMB-231 cells at comparatively low concentrations of the essential oil used. However,

differences were not so high at the concentrations above the IC50 for both the cell lines.

Future Prospect

Results of the present study indicates novel essential oil as anticancer agent. DNA damage has been found in cells treated with essential oil. This indicates that cell death was caused by essential oil in the breast cancer cells. Further, extensive studies are required to evaluate the mechanism of action and screening across different types of cancer can be done. Normal cells must be tested for the evaluation of its cytotoxic effects to find its efficacy. In vivo models can further be used to find the efficacy of essential oil. Significant results can ensure simple diet modifications to be introduced as supplementary therapy in the cancer patients.

On the other hand, normal people may also have beneficial effects of the essential oil. Study of the effect of the constituents of the essential oil is of prime importance to determine the novel compounds for the cancer treatment.

SUMMARY

Cancer is one of the dreadful diseases leading to increased mortality rate. Based on the origin in the organs, there are many types of cancer and breast cancer is one of the major threats to the females. Chemo and radio therapies are used as the standard methods for the treatment of cancer but these have many side effects. Naturotherapy and other complementary therapies are the promising methods to avoid the side effects. Natural plant products are valuable source of novel molecules with ability to prevent and treat cancer. Many cancer drugs have been derived from plant origin like paclitaxel. Essential oils are the volatile compounds present in various plant parts with specific aroma and have valuable medicinal properties. These hold wide potential in the cancer treatment and prevention. Keeping in view the value of natural products, we have screened a novel essential oil against breast cancer cells to determine its anticancer potential. There are many studies in which essential oil and its components have been shown to have anticancer activity. Essential oil of the Salvia plebeia was observed to be very effective against the breast cancer cell lines MDAMB-231 and MCF-7 at low concentrations leading to the reduced viability. MCF-7 was observed to show

IC50 at concentration 30µg/ml and in MDAMB-231, IC50 was calculated to be 65µg/ml for 24 hours treatment indicating the effectiveness of the essential oil. The oil was observed to show cell death at the higher concentrations used in the treatments. Time dependent response was also studied which showed that 24 hours treatment was the most effective treatment time for both the cell lines. MCF- 7 and MDAMB-231 cell lines responded differently at the lower concentrations with MCF-7 being more susceptible to the inhibitory effect of the essential oil of Salvia plebeia. Exposure time was found to have important role in the essential oil treatments to the cancer cells. At lower time interval, cancer cells responded variably while the increased exposure time was observed to have sustained effect on the inhibition of the cell growth. Comet assay was used to determine the DNA damage induction by the essential oil as it is the fast and effective way to determine the damages at the DNA level to the individual cells. It was observed to cause DNA damage and this indicates the DNA damage leading to cell death as one of the mechanism of action of the essential oil as anticancer agent. Essential oil of Salvia plebeia contains the compounds like caryophyllene and alpha-

humulene which constitute the major amount of the essential oil. These compounds have been reported to have anticancer properties. The cytotoxic activity of the essential oil can be attributed to the presence of these compounds. Also, essential oil was observed to have antioxidant property which was less in comparison to the synthetic Butylated Hydroxyl Toluene (BHT). Being a natural antioxidant, it can contribute to the improvement in the health of cancer patients. Novel essential oil as the anticancer agent has been screened in our study.

REFERENCES

Abu-Dahab, R. and Afifi, F. (2007). Antiproliferative activity of selected medicinal plants of Jordan against a breast adenocarcinoma cell line (MCF7). Scientia Pharmaceutica 75(3): 121-136.

Al-Kalaldeh, J.Z., Abu-Dahab, R. and Afifi, F.U. (2010). Volatile oil composition and antiproliferative activity of Laurus nobilis, Origanum syriacum, Origanum vulgare, and Salvia triloba against human breast adenocarcinoma cells. Nutrition Research 30(4): 271–278. Amin, A., Gali-Muhtasib, H., Ocker, M. and Schneider-Stock, R. (2009). Overview of major classes of plant-derived anticancer drugs. International Journal of Biomedical Science 5(1): 1-11.

Amirghofran, Z., Zand, F., Javidnia, K. and Miri, R. (2010). The cytotoxic activity of various herbals against different tumor cells: an in vitro study. Iranian Red Crescent Medical Journal 12(3): 260-265.

Arunasree, K.M. (2010). Anti-proliferative effects of carvacrol on a human metastatic breast cancer cell line, MDA-MB 231. Phytomedicine 17(8-9): 581–588.

[ATCC] ATCC Advanced Catalog Search. . Accessed on 2011Sept, 25.

Bostancıoglu, R.B., Kurkcuoglu, M., Baser, K.H.C. and Koparal, A.T. (2012). Assessment of anti-angiogenic and anti-tumoral potentials of Origanum onites L. essential oil. Food and Chemical Toxicology 50(6): 2002–2008. Cai, Y., Lu, J., Miao, Z., Lin, L., and Ding, J. (2007). Reactive oxygen species contribute to cell killing and P-glycoprotein downregulation by salvicine in multidrug resistant K562/A02 cells. Cancer Biology and Therapy 6(11): 1794-1799. Cardile, V., Russo, A., Formisano, C., Rigano, D., Senatore, F., Arnold, N.A. and Piozzi, F. (2009). Essential oils of Salvia bracteata and Salvia rubifolia from Lebanon: Chemical composition, antimicrobial activity and inhibitory effect on human melanoma cells. Journal of Ethnopharmacology 126(2): 265-272.

Celiktas, O.Y., Kocabas, E.E., Bedir, E., Sukan, F.V., Ozek, T. and Baser, K.H.C. (2007). Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations. Food Chemistry 100(2): 553-559.

Cha, J.D., Kim, Y.H. and Kim, J.Y. (2010). Essential oil and 1, 8-cineole from Artemisia lavandulaefolia induces apoptosis in KB cells via mitochondrial stress and caspase activation. Food Science and Biotechnology 19(1): 185-191.

Chang, R. (2002). Bioactive polysaccharides from traditional Chinese medicine herbs as anticancer adjuvants. The Journal of Alternative & Complementary Medicine 8(5): 559-565.

Chen, H.M., Lee, M.J., Kuo, C.Y., Tsai, P.L., Liu, J.Y. and Kao, S.H. (2010). Ocimum gratissimum aqueous extract induces apoptotic signalling in lung adenocarcinoma cell A549. Evidence-Based Complementary and Alternative Medicine 2011:7.

Chen, W. Lu, W. Gao, M. Wu, J. Wang, A. and Shi, R. (2011). Anti-angiogenesis effect of essential oil from Curcuma zedoaria in vitro and in vivo. Journal of Ethnopharmacology 133(1): 220–226.

Choucroun, P., Gillet, D., Dorange, G., Sawicki, B., and Dewitte, J. (2001). Comet assay and early apoptosis. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 478(1-2): 89-96.

Ferlay, J., Shin, H.R., Bray, F., Forman, D., Mathers, C. and Parkin, D.M. (2010). Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. International Journal of Cancer. 127(12): 2893–2917.

Fiore, G., Massarelli, P., Sajeva, M. and Franchi, G.G. (2012). Anti-tumor activity of the methanolic extracts of Salvia menthifolia. Revista Brasileira de Farmacognosia 22(2): 381-387.

Fiore, G., Nencini, C., Cavallo, F., Capasso, A., Bader, A., Giorgi, G. and Micheli, L. (2006). In vitro antiproliferative effect of six Salvia species on human tumor cell lines. Phytotherapy Research 20(8): 701-703.

Hadri, A., Gómez del Río, M.A., Sanz, J., González Coloma, A., Idaomar, M., Ribas Ozonas, B., Gonzalez, J.B. and Sánchez Reus, M.I. (2010). Cytotoxic activity of α-humulene and trans-caryophyllene from Salvia officinalis in animal and human tumor cells. Anales de la Real Academia Nacional de Farmacia 76(3): 343-356.

Halliwell, B., Gutteridge, J.M.C. and Aruoma, O.I. (1987). The deoxyribose method: a simple "test tube" assay for determination of rate constants for reactions of hydroxyl radicals. Analytical Biochemistry 165(1): 215‐219.

Hussain, A.I. (2009). Characterization and biological activities of essential oils of some species of lamiaceae. Ph.D. thesis, University of Agriculture, Faisalabad. [IARC] Globocan 2008, IARC, 2010 - GLOBOCAN: Country Fast Stat. . Accessed 2011 Dec, 5. Itani, W.S., El-Banna, S.H., Hassan, S.B., Larsson, R.L., Bazarbachi, A. and Gali- Muhtasib, H.U. (2008). Anticolon cancer components from Lebanese sage (Salvia libanotica) essential oil. Cancer Biology & Therapy 7(11): 1765-1773.

Jang, T.S., Zhang, H.Y., Kim, G.J., Kim, D.W., Min, B.S., Kang, W., Son, K.H., Na, M.K. and Lee, S.H. (2012). Bioassay-guided isolation of fatty acid synthase inhibitory diterpenoids from the roots of Salvia miltiorrhiza Bunge. Archives of Pharmacal Research 35 (3): 481-486.

Jemal, A., Bray, F., Center, M.M., Ferlay, J.F., Ward, E. and Forman, D. (2011). Global cancer statistics. CA: A Cancer Journal for Clinicians 61(2): 69–90.

Kamatou, G.P.P. (2006). Indigenous Salvia species − an investigation of their pharmacological activities and Phytochemistry. Ph. D thesis, University of the Witwatersrand, Johannesburg.

Kamatou, G.P.P., Van Zyl, R.L., Davids, H., Van Heerden, F.R., Lourens, A.C.U. and Viljoen, A.M. (2008). Antimalarial and anticancer activities of selected South African Salvia species and isolated compounds from S. radula. South African Journal of Botany 74(2): 238-243.

Lage, H., Duarte, N., Coburger, C., Hilgeroth, A. and Ferreira, M.J.U. (2010). Antitumor activity of terpenoids against classical and atypical multidrug resistant cancer cells. Phytomedicine 17(6): 441–448.

Lancelle, H.G., Giordano, O.S., Sosa, M.E. and Tonn, C.E. (2009). Chemical composition of four essential oils from Eupatorium spp: Biological activities toward Triboliusm castaneum (Coleoptera: Tenebrionidae). Revista de la Sociedad Entomológica Argentina 68(3-4): 329-338.

Lee, S.J., Umano, K., Shibamoto, T. and Lee, K.G. (2005). Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chemistry 91(1): 131- 137. Lee, W.Y.W., Chiu, L.C.M. and Yeung, J.H.K. (2008). Cytotoxicity of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on HepG2 cells in relation to glutathione perturbation. Food and Chemical Toxicology 46(1): 328-338.

Lee, W.Y.W., Cheung, C.C.M., Liu, K.W.K., Fung, K.P., Wong, J., Lai, P.B.S. and Yeung, J.H.K. (2010). Cytotoxic effects of tanshinones from Salvia miltiorrhiza on doxorubicin-resistant human liver cancer cells. Journal of Natural Products 73(5): 854-859.

Li, R., Chen, W., Wang, W., Tian, W. and Zhang, X. (2010a). Analysis of the essential oil of magnolia and its effect on antioxidant enzyme activity, sCD40L and phosphorylate-activate Akt protein levels in gastric cancer mice. Medicinal Chemistry Research 19(9): 1203-1209.

Li, R., Chen, W., Wang, W., Tian, W. and Zhang, X. (2010b). Extraction of essential oils from garlic (Allium sativum) using ligarine as solvent and its immunity activity in gastric cancer rat. Medicinal Chemistry Research 19(9): 1092–1105. 50

Liyana- Pathiranan, C.M. and Shahidi, F. (2005). Antioxidant activity of commercial soft and hard wheat (Triticum aestivum L) as affected by gastric conditions. Journal of Agricultural and Food Chemistry 53(7): 2433-2440.

Loizzo, M.R., Menichini, F., Tundis, R., Bonesi, M., Conforti, F., Nadjafi, F., Statti, G.A., Frega, N.G. and Menichini, F. (2009). In vitro biological activity of Salvia leriifolia Benth essential oil relevant to the treatment of Alzheimer’s disease. Journal of Oleo Science 58(8): 443-446.

Loizzo, M.R., Tundis, R., Menichini, F., Saab, A.M., Statti, G.A. and Menichini, F. (2007). Cytotoxic activity of essential oils from Labiateae and Lauraceae families against in vitro human tumor models. Anticancer Research 27(5A): 3293-3299.

Loutrari, H., Magkouta, S., Papapetropoulos, A. and Roussos, C. (2011). Mastic oil inhibits the metastatic phenotype of mouse lung adenocarcinoma cells. Cancers 3(1): 789-801.

Magesh, V., Lee, J.C., Ahn, K.S., Lee, H.J., Lee, H.J., Lee, E.O., Shim, B.S., Jung, H.J., Kim, J.S., Kim, D.K., Choi, S.H., Ahn, K.S. and Kim, S.H. (2009). Ocimum sanctum induces apoptosis in A549 lung cancer cells and suppresses the in vivo growth of Lewis lung carcinoma cells. Phytotherapy Research 23(10): 1385-1391.

Manosroi, J., Dhumtanom, P. and Manosroi, A. (2006). Anti-proliferative activity of essential oil extracted from Thai medicinal plants on KB and P388 cell lines. Cancer Letters 235(1): 114-120.

Mosaddik, M.A. (2003). In vitro cytotoxicity of Tanshinones isolated from Salvia miltiorrhiza Bunge against P388 lymphocytic leukemia cells. Phytomedicine 10(8): 682–685.

Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65(1-2): 55-63.

Naumovski, L., Quinn, J.P., Miyashiro, D., Patel, M., Bush, K., Singer, S.B., Graves, D., Plazkill, T. and Arvin, A.M. (1992). Outbreak of ceftazidime 51

resistance due to a novel extended-spectrum beta-lactamase in isolates from cancer patients. Antimicrobial Agents and Chemotherapy 36(9): 1991-1996.

[NCBI] National Center for Biotechnology. . Accessed on 2011Dec, 16.

[NCI] National Cancer Institute: PDQ® Aromatherapy and Essential Oils. . Accessed 2012 Feb, 2.

[NCRP] National Cancer Registry Programme. . Accessed 2012 Jan, 2.

[NIST] National Institute for Standards and Technology webbook. . Accessed 2012 Jan, 25.

Nooter, K. and Herweijer, H. (1991). Multidrug resistance (mdr) genes in human cancer. British journal of cancer 63(5): 663-669.

Olive, P.L. and Banath, J.P. (2006). The comet assay: a method to measure DNA damage in individual cells. Nature Protocols 1(1): 23-29. Accessed on 2011 Sept, 10.

Ozkan, A., Erdogan, A., Sokmen, M., Tugrulay, S. and Unal, O. (2010). Antitumoral and antioxidant effect of essential oils and in vitro antioxidant properties of essential oils and aqueous extracts from Salvia pisidica. Biologia 65(6): 990-996. Paik, S.Y., Koh, K.H., Beak, S.M., Peak, S.H. and Kim, J.A. (2005). The Essential Oils from Zanthoxylum schinifolium pericarp induce apoptosis of HepG2 human hepatoma cells through increase production of reactive oxygen species. Biological and Pharmaceutical Bulletin 28(5): 802-807. Prakash, J. and Gupta, S.K. (2000). Chemopreventive activity of Ocimum sanctum seed oil. Journal of Ethnopharmacology 72(1-2): 29-34. Qing, C., Jiang, C., Zhang, J.S. and Ding, J. (2001). Induction of apoptosis in human leukemia K-562 and gastric carcinoma SGC-7901 cells by salvicine, a novel anticancer compound. Anti-cancer Drugs 12(1): 51-56.

Ren, J., Pan, S., Lu, X., Zhou, M. and Hu, K. (2010). Antitumor activity of dichloromethane extract from Salvia plebeia and induction of apoptosis on k562 cells. Chinese Herbal Medicines 3(1): 36-40. Sahouo, G.B., Tonzibo, Z.F., Boti, B., Chopard, C., Mahy, J.P. and N’guessan, Y.T. (2010). Anti-inflammatory and analgesic activities: Chemical constituents of essential oils of Ocimum gratissimum, Eucalyptus citriodora and Cymbopogon giganteus inhibited lipoxygenase L-1 and cyclooxygenase of PGHS. Bulletin of the Chemical Society of Ethiopia 17(2):191-197.

Sertel, S., Eichhorn, T., Plinkert, P.K., and Efferth, T. (2011). Cytotoxicity of Thymus vulgaris essential oil towards human oral cavity squamous cell carcinoma. Anticancer Research 31(1): 81-87.

Sharma, M., Agrawal, S.K., Sharma, P.R., Chadha, B.S., Khosla, M.K. and Saxena, A.K. (2010). Cytotoxic and apoptotic activity of essential oil from Ocimum viride towards COLO 205 cells. Food and Chemical Toxicology 48(1): 336–344.

Singh, H.P., Mittal, S., Kaur, S., Batish, D.R. and Kohli, R.K. (2009). Chemical composition and antioxidant activity of essential oil from residues of Artemisia scoparia. Food Chemistry 114(2): 642–645.

Stankovic, M.S., Curcic, M.G., Zizic, J.B., Topuzovic, M.D., Solujic, S.R. and Markovic, S.D. (2011). Teucrium plant species as natural sources of novel anticancer compounds: antiproliferative, proapoptotic and antioxidant properties. International Journal of Molecular Sciences 12(7): 4190-4205. . Accessed on 2011 Dec, 25.

Sukumaran, K., Unnikrishnan, M. and Kuttan, R. (1994). Inhibition of tumour promotion in mice by eugenol. Indian Journal of Physiology and Pharmacology 38(4): 306-308. Soeur, J. Marrot, L. Perez, P. Iraqui, I. Kienda, G. Dardalhon, M. Meunierb, J.R. Averbeck, D. and Huang, M.E. (2011). Selective cytotoxicity of Aniba rosaeodora essential oil towards epidermoid cancer cells through

induction of apoptosis. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 718(1-2): 24–32.

Sylvestre, M., Pichette, A., Longtin, A., Nagau, F. and Legault, J. (2006). Essential oil analysis and anticancer activity of leaf essential oil of Croton flavens L. from Guadeloupe. Journal of Ethnopharmacology 103(1): 99-102.

Thoppil, J.E. (1993). Chromosome studies and exploration of chemical constituents in some members of South Indian Lamiaceae. Ph.D. thesis. Mahatma Gandhi University, Kerala, India.

Tepe, B., Donmez, E., Unlu, M., Candan, F., Daferera, D., Vardar-Unlu, G., Polissioud, M. and Sokmen, A. (2004) Antimicrobial and antioxidative activities of the essential oils and methanol extracts of Salvia cryptantha (Montbret et Aucher ex Benth.) and Salvia multicaulis (Vahl). Food Chemistry 84(4): 519-525. Tundis, R., Loizzo, M.R., Menichini, F., Bonesi, M., Colica, C. and Menichini, F. (2011). In vitro cytotoxic activity of extracts and isolated constituents of Salvia leriifolia Benth. against a panel of human cancer cell lines. Chemistry & Biodiversity 8(6): 1152-1162.

Wang, X., Bastow, K.F., Sun, C.M., Lin, Y.L., Yu, H.J., Don, M.J., Wu, T.S., Nakamura, S. and Lee, K.H. (2004). Antitumor agents. 239. Isolation, structure elucidation, total synthesis, and anti-breast cancer activity of neo-tanshinlactone from Salvia miltiorrhiza. Journal of medicinal chemistry 47(23): 5816-5819.

Waris, G. And Ahsan, H. (2006). Reactive oxygen species: role in the development of cancer and various chronic conditions. Journal of Carcinogenesis 5:1-14. . Accessed on 2012, Feb 01.

Weng, X. and Wang, W. (2000). Antioxidant activity of compounds isolated from Salvia plebeia. Food Chemistry 71(4): 489-493.

Xavier, C.P.R., Lima, C.F., Fernandes-Ferreira, M. and Pereira-Wilson, C. (2009). Salvia fruticosa, Salvia officinalis, and rosmarinic acid induce apoptosis

and inhibit proliferation of human colorectal cell lines: the role in MAPK/ERK pathway. Nutrition and Cancer 61(4): 564-571.

Yu, J.Q., Lei, J.C., Zhang, X.Q., Yu, H.D., Tian, D.Z., Liao, Z.X. and Zou, G. 2011). Anticancer, antioxidant and antimicrobial activities of essential oil of Lycopus lucidus Turz. var. hirtus Regel. Food Chemistry 126(4): 1593- 1598. Zu, Y., Yu, H., Liang, L., Fu, Y., Efferth, T., Liu, X. and Wu, N. (2010). Activities of ten essential oils towards Propionibacterium acnes and PC-3, A-549 and MCF-7 cancer cells. Molecules 15(5): 3200-3210.