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Advances in Medicinal Plant Research, 2007: 287-317 ISBN: 81-7736-255-0 Editors: Surya N. Acharya and James E. Thomas

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Research on Thai medicinal plants for cancer treatment

A. Itharat1 and B. Ooraikul2 1 Associate Professor, Applied Thai Traditional Medicine Center, Faculty of Medicine Thammasart University, Thailand; 2Professor, Department of Agricultural, Food and Nutritional Science, Faculty of Agriculture, Forestry and Home Economics, University of Alberta, Canada

Author Dr. Arunporn Itharat is an Associate Professor, Faculty of Medicine, Thammasart University, Thailand. She obtained BSc and MSc in Pharmacy from Chulalongkorn University, Thailand, a Certificate of Cytotoxicity on Medicinal Plant Extract and PhD in Pharmacognosy from Kings College, London, UK, in 1981, 1986, 1997 and 2002, respectively. She won a Student Award from Society of Phytochemistry, Switzerland, in 2001, and Best Community Research Award from Prince of Songklanakarin University in 2003. Dr. Itharat published 28 refereed papers on traditional medicine and medicinal plants. Her main research interests are in phytochemistry, pharmacognosy, ethnopharmacology, cytotoxicity, and anticancer drugs from natural products.

Abstract
This chapter reviews the research on anticancer effects of medicinal plants originating from Thailand. The selection of the plants for the research was based largely on their ethnomedical use by Thai folk doctors and on knowledge of Thai traditional medicine. The investigations focused primarily on the plants frequently used in preparations for cancer treatment.
Correspondence/Reprint request: Dr. A. Itharat, Associate Professor, Applied Thai Traditional Medicine Center, Faculty of Medicine, Thammasart University, Thailand. E-mail: iarunporn@yahoo.com

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The plants were tested for active components using National Cancer Institute (NCI) assays for anticancer compounds. In vivo assays were primarily used for initial screening of compounds for anticancer activity using L-1210 and P388 mouse leukemia models. In 1960 the NCI developed an in vitro assay using KB cells, which derived from a human carcinoma of the nasopharynx, as an anti-tumor assay for the screening of plant extracts. In vitro assays are more sensitive to most anti-tumor agents than in vivo assays, usually cost less and require less test material and time. They have contributed significantly to the discovery of many anticancer drugs. The potent active ingredients from plants after the initial screening were also evaluated for possible semi-synthesis in order to produce anticancer drugs. Plant extracts have been compared with whole plant preparations as well as combinations of many plants for their effectiveness as anticancer medicine. Generally, whole herbal preparations consisting of many plants were shown to have gentler effects on the human body due to the synergy of the plants that make up the preparations. The trend in cancer treatment research in Thailand is moving towards the holistic approach, which requires knowledge of both body and mind. Most of the current research on the subject in Thailand is on elucidation of biological activity of plant materials to confirm or disprove their use as practiced in the Thai folk medicine. Informal clinical trials have been conducted on some of the anticancer preparations, but most are unpublished. Also included in this chapter is a list of some Thai medicinal plants, which have been used in folk medicine for cancer treatment.

Introduction
Cancer or malignant disease is one of the major causes of death in humans. WHO (2002) reported that malignant neoplasm is the third (12.4%) leading cause of death worldwide, the first (30%) being cardiovascular disease, and the second (18.8%) being infectious diseases, which include HIV/AIDS (Mathers et al. 2001). Between 2000 and 2020, the total number of cases of cancer is predicted to increase by 73% in the developing world and by 29% in the developed world (Parkin 2001). Malignant neoplasm was the leading cause of death in Hong Kong during 1996 to 2001 (Stanley 2001). In Thailand the rate of people dying from cancer is still increasing every year and it is the first leading cause of death (National Statistical Office 2003). The disease affects men and women alike. Thus, research and development on anticancer agents has become a worldwide scientific effort in both private and public institutions. Cancer chemotherapy now plays a significant role in the treatment of many malignancies, either curative (by itself or as an adjuvant to surgery and/or radiation) or palliative care, depending upon the specific tumor situation (Carter 1982). The objective of cancer chemotherapy is to kill cancer cells with as little damage as possible to normal cells (Halliwell and Gutteridge 1988). Therefore, any discovery of anticancer agents must be related to novel molecular targets; i.e. they should be effective against specific types of cancer cells but less toxic to normal cells, or have a unique mechanism of action for specific types of cancer (Pezzuto 1997).

Discoveries of novel anticancer agents from plants used in traditional medicine

Nature has long been an important source of medicinal agents. An impressive number of modern drugs have been isolated or derived from natural sources, based on

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their use in traditional medicine (Cragg and Newman 2001). Plants have formed a basis for traditional medicine systems that have been used for thousands of years in countries with ancient civilizations such as China (Chang and But 1986), India (Kapoor 1990) and Thailand (Subchareon 1998a). The use of plants in traditional medicine systems of many other cultures has been extensively documented (Schultes and Raffauf 1990; Arvigo and Balick 1993; Gupta 1995; Ayensu 1981; Iwu 1993; Jain 1991). Plant-based systems continue to play an essential role in healthcare and it has been estimated by the WHO that approximately 80% of the worlds inhabitants rely mainly on traditional medicine for their primary healthcare (Farnsworth et al. 1985). Plant products also play an important role in the healthcare systems of the remaining 20% of the population who reside mainly in developed countries. Analysis of data on prescriptions dispensed from community pharmacies in the United States from 1959 to 1980 indicates that about 25% contained plant extracts or active principles derived from higher plants. Furthermore, at least 119 chemical substances derived from 90 plant species can be considered as important drugs currently in use in one or more countries (Farnsworth et al. 1985). About 74% of these 119 drugs were discovered as a result of chemical studies directed at isolation of the active substances from plants used in traditional medicine (Cragg et al. 1997). Ethnopharmacological or traditional use of plants often results in the discovery of new biologically active molecules (Houghton 1995). However, it is important that the investigators understand the principles of folk medicine or mode of action of folk herbs (Nakanishi 1999). Plants have a long history of use in the treatment of cancer (Hartwell 1982). However, many of the claims for the efficacy of such treatments should be viewed with some skepticism because cancer, as a specific disease entity, is likely to be poorly defined in terms of folklore and traditional medicine (Cragg et al. 1994). Of the plantderived anticancer drugs in clinical use, the best known is the so-called vinca alkaloids, which include vinblastine (1) and vincristine (2) (Fig. 1). These alkaloids are isolated from the Madagascar periwinkle, Catharanthus roseus (Linn.) G. Don, Apocynaceae, known in Thailand as Phaeng phuai farang. Vinblastine and vincristine were first discovered during an investigation of the plant for potential oral hypoglycemic agents. Therefore, their discovery as anticancer agents may be indirectly attributed to the observation of an unrelated medicinal use of the source plant. The two clinically-active agents, etoposide (3) and teniposide (4), which are semi-synthetic derivatives of the natural product epipodophyllotoxin, may be considered to be more closely linked to a plant originally used for the treatment of cancer. Epipodophyllotoxin is an isomer of podophyllotoxin (5), which was isolated as the active anti-tumor agent from the roots of various species of the genus Podophyllum (Berberidaceae). These plants possess a long history of medicinal use by early American and Asian cultures, including the treatment of skin cancers and warts (Cragg et al. 1994). From the time of Galen (about AD180), the juice expressed from woody nightshade (Solanum dulcamara L. Family Solanaceae) has been used to treat cancers, tumors and warts. The active tumor-inhibitory principle has been identified as the steroidal alkaloid glycoside -solamarine. Various lichens (e.g. species of Cladonia, Cetraria and Usnea) also have a history of use in folk medicine against cancer since about AD 970. These are all rich sources of usnic acid, a compound recognized for many years as an antibacterial and antifungal agent, but only more recently as an anti-tumor compound. Similarly, many centuries ago, the Druids claimed

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Figure 1

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Figure 1. Structures of some anti-tumor compounds of plant origin.

that mistletoe (Viscum album) could be used to cure cancer. Protein fractions with marked anti-tumor activity have been isolated from mistletoe extract. The benzophenanthridine derivatives are present in Chelidonium majus (Papaveraceae), a plant with substantial folklore history of use in the treatment of cancers (Dewick 2002).

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More recent additions to the armamentarium of the naturally derived chemotherapeutic agents are the taxanes and camptothecins. Paclitaxel (6) (Taxol) was initially isolated from the bark of the Pacific or American yew tree, Taxus brevifolia Nutt.(Taxaceae), collected in Washington State as part of a random collection program by the U.S. Department of Agriculture for the National Cancer Institute (NCI) (Cragg et al. 1993). The use of various parts of T. brevifolia Nutt. and other Taxus species (e.g., canadensis, baccata) by several Native American tribes for the treatment of some noncancerous conditions has been reported (Cragg et al. 1994). The leaves of T. baccata are used in the traditional Asiatic Indian (Ayurvedic) medicine system (Kapoor 1990), with one reported use in the treatment of cancer (Hartwell 1982). Paclitaxel, along with several key precursors (the baccatins), occurs in the leaves of various Taxus species, and the ready semi-synthetic conversion of the relatively abundant baccatins to paclitaxel, as well as active paclitaxel analogs, such as docetaxel (7) (Cortes and Pazdur 1995), has provided a major renewable natural source of this important class of drugs. Likewise, the clinically active agents, topotecan (hycamptamine) (8), irinotecan (9) (CPT-11), 9amino and 9-nitrocamptothecin (10, 11), are semi-synthetically derived from camptothecin (12), isolated from the Chinese ornamental tree, Camptotheca acuminate Decne (Family Cornacea) (Potmeisel and Pinedo 1995). Camptothecin (as its sodium salt) was advanced to clinical trials by the NCI in the 1970s, but was dropped because of severe bladder toxicity (Cragg 2001).

In vitro and in vivo assays used in the discovery of anticancer drugs

The search for natural products as potential anticancer agents dates back at least to the Ebers papyrus in 1550 B.C. However, the scientific period of this search is much more recent, beginning with the investigation by Hartwell and his co-workers on the application of podophyllotoxin and its derivatives as anticancer agents (Kingston et al. 1990). Plants offer scientists searching for novel bioactive compounds the added advantage of ethnobotanical observations, since many species are used in traditional medicine, principally in developing countries. The study of bioactive compounds from plants has required the development of bioassay techniques, especially in vitro methods which allow a large number of plant extracts to be screened for activity, especially cytotoxicity, against many types of cancer cell lines. In vitro assays are particularly useful for bioassay-guided fractionation of plant extracts, since it is not always possible to test against cancer in animal models. In vitro assays are more sensitive to most anti-tumor agents than in vivo assays and usually cost less and require less test material and time. The US National Cancer Institute (NCI) has had an established program for the development of methods for initial screening of anticancer compounds since 1955. The initial screening program or primary screening used the in vivo L-1210 and P388 mouse leukemia model for the selection of anticancer compounds. The NCI developed an in vitro assay using KB cells, which derived from a human carcinoma of nasopharynx, as an anti-tumor assay for the screening of plant extracts since 1960 (Purdue 1982). Plant extracts in the pre-screening assay are considered to have cytotoxic activity against KB cells when they show an IC50 of <30 g/ml. Thus, it might be appropriate to screen the

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materials first with this in vitro assay and choose only in vitro-active compounds or natural products for further in vivo tests. It took approximately five years to develop the in vitro cell line screening process (1985-1990). The process was implemented and became fully operational in 1990. It is capable of screening 20,000 compounds per year. The screening process utilizes 60 different human tumor cell lines, representing leukemia, melanoma and cancers of lung, colon, brain, ovary, breast, prostate, and kidney. Selective growth inhibition or cell killing of particular tumor cell lines indicates a positive result. The evaluation uses a pattern recognition algorithm to assign a putative mechanism of action for the compounds. In addition to characteristics associated with various cellular targets in the 60 cell lines, it may be possible to select compounds most likely to interact with a specific target (Boyd 1997). The in vitro screening has contributed to discoveries and developments of new clinically useful anticancer drugs. Tens of thousands of pure compounds from plant, marine, and microbial origins were tested initially in the in vitro primary screening; subsets were selected for in vivo preclinical follow up within 5 years (1990-1995) (Boyd 1997). A bioassay-directed fractionation procedure was, thus, designed for the isolation of active compounds (Pezzuto 1997). The terms cytotoxic, anti-tumor and anticancer were defined by the NCI program. A cytotoxic agent is toxic to tumor cells in vitro and if this toxicity transfers through to tumor cells in vivo, the agent is said to have anti-tumor activity. The term anticancer is reserved for materials that are toxic to tumor cells in clinical trial on humans.

In vitro cytotoxic assays

Bioassay systems for anti-tumor activity were established using the screening process to guide the fractionation and separation of crude extracts. The in vitro tests are usually used as initial or primary screening in cytotoxic assays of crude extracts at dilutions of less than 0.1% to as low as 1 ppm (Suffness and Douros 1982). It is more sensitive than the in vivo system, which requires high extract concentrations and often shows no activity although the material has been shown to be active in the in vitro assay. It is also considerably cheaper than the in vivo system. The in vitro assay can be divided into two main groups, a cellular assay and a molecular assay (Suffness and Pezzuto 1991). The cellular assay (cellbased assay or cytotoxicitybased bioassay) uses intact cells while the molecular assay (based on mechanism) aims for specific inhibition of the expression of an oncogene or oncoproducts or inhibition of a specific biochemical target at the subcellular level, such as enzymes and receptors. The cellular assay or cytotoxicity-based bioassay has been used to screen materials for anticancer agents in the NCI Natural Product Program. The aim is to determine whether the compounds, synthetic or natural, showing selective growth inhibition or killing of particular tumor cell lines, are worth further evaluation. Compounds showing differential cytotoxicity for particular tumor types will be followed up with in vivo testing using the same sensitive cell lines. The most important advantage of the new in vitro screening panel is the potential to identify tumor-type-selective compounds. The enhanced sensitivity of the in vitro screening will also enable the discovery of active constituents present in low concentrations in the plant extracts. This screening is unique

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in that the complexity of a 60 cell line dose response produced by a given compound results in a biological response pattern which can be utilized in pattern recognition algorithms. Using these algorithms, it is possible to assign a putative mechanism of action to a test compound, or to determine that the response pattern is unique and not similar to that of any of the standard prototype compounds included in the NCI database. In addition, following the characterization of various cellular molecular targets in the 60 cell lines, it may be possible to select compounds most likely to interact with a specific molecular target. The success of the in vitro cytotoxicity screening assay will be assessed by (a) the effectiveness for isolation of pure and selective cytotoxins from crude plant extracts and (b) the confirmation of their selective anti-tumor activity in the animal tumor models (Cassady et al. 1990; Boyd 1997). It is important to note that there are a number of advantages of in vitro testing that would save evaluation time and expense. These include species specificity of the analysis, the feasibility of using only small amounts of test substances and the possibility for doing mechanistic studies. However, the active extracts and compounds have to be further tested in vivo to confirm the anti-tumor activity because some of the active compounds, which have been tested in vitro, may be metabolized as non-active compounds by the in vivo test. The effects of compounds or drugs on animal organs and the symptoms displayed by the animal are detected only by in vivo tests.

The SRB and MTT assays

The NCI is implementing a large scale in vitro drug screening program that requires a very efficient automated assay of drug effects on tumor cell viability or growth. Many laboratories worldwide have adopted a micro-culture assay based on metabolic reduction of 3-(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). However, because of certain technical advantages in using the protein-binding dye sulforhodamine B (SRB) instead of MTT, the SRB is now used in large-scale screening. The principle of SRB, which is a bright pink aminoxanthene dye, is that it is an anionic protein stain containing two sulfonic groups which bind to protein basic amino acid residues in TCA-fixed cells under mildly acidic conditions. The protein-bound dye is then solubilized by weak base for spectrophotometry. This colorimetric assay can be used to estimate the cell number indirectly by providing a sensitive index of total cellular protein content which is linearly related to cell density (Skehan et al. 1990). This assay was found to give good results over a wide range of cell density (Freshney 1994). The MTT assay is based on metabolic reduction of colorless tetrazolium salt (MTT), by mitochondrial enzyme activity in viable cells, to formazan salt (blue), which can be quantified spectrophotometrically. It is particularly useful for assaying cell suspensions because of its specificity for living cells (Mosmann 1983).

In vivo study on antitumor assay

The in vivo assay is the secondary stage for screening; it is usually carried out in rats, mice or hamsters, which have been implanted subcutaneously. Induced tumor cells are applied depending on each type of tumor model. For example, L-1210 and P-388 were implanted intraperitonially (i.p), Lewis lung carcinoma was induced intravenously (i.v.) and colon 38 tumor cells were implanted subcutaneously (s.c.). The number of days

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for the treatment schedule depends on each tumor cell model. For example, the administration of test drug for P-388 begins at one day after implantation and continues for 5 days via the i.p. route. Anti-tumor activity has been defined in terms of a therapeutic index or the ratio of the 50% lethal dose to the ED90 (the dose required to reduce tumor mass by 90%) or the growth rate of the tumor cell. The therapeutic index is the packed cell volume or weight of the tumor cells of the test group/control group (Suffness 1989).

Some potent cytotoxic, antitumor and anticancer drug from Thai plants
Cancer chemotherapeutic agents on the market have four structural classes of plant-derived anticancer agents, represented by the Catharanthus (Vinca) alkaloids [vinblastine (1) vincristine (2) and vindesine (13)], the epipodophyllotoxins [etoposide (3), teniposide (4)], the taxanes [paclitaxel (6) and docetaxel (7)], and the camptothecin derivatives [camptothecin (12) and irinotecan (9)] (Figs. 1 and 2). Many anticancer agents from plants were discovered and developed into products for treatment of various forms of cancer by an extensive program set up by the NCI. Both of the anticancer drugs, taxol, isolated in 1971, and camptothecin, isolated in 1966, were discovered through the NCI screening program (Evans 2002). About 500 plants are collected each year from the tropical rain forest through the network of botanical cooperators. Non-polar and polar extracts are then prepared from the plant parts obtained, which are then evaluated in a broad range of cells and mechanisms based on the in vitro bioassay. The potential anticancer agents are novel cytotoxic compounds, which are isolated from Thai plants and structurally characterized by the NCI, as shown in Figure 2. For example; four new 1H-cyclopenta[b]benzofuran derivatives (14-17) were isolated from Aglaia elliptica Bl. (Meliaceae), a rain forest tree collected from Thailand (Lee et al. 1998). A number of the compounds isolated were previously known products from plants grown in Thailand such as the anthraquinone aloe-emodin (18) from Cassia spp., members of Caesalpiniaceae family, the naphthoquinones lapachol (19) extracted from the trees of Tabebuia spp. of Bignoniaceae family, and the alkaloid colchicine (20) from Glosiosa superba L. of Liliaceae family (known as Dong Dung Hua Kwan in Thai). In most cases the activity levels noted in the screens were not sufficient to justify further evaluation; e.g. lapachol entered phase 1 clinical trials but the lack of therapeutic response and blood anticoaglulant side effects resulted in termination of the trials. Indicine N-oxide (21) from Heliotropium indicum L. of Boraginaceae family (Ya Nguang Chang in Thai) showed substantial activity on acute leukemia but hepatotoxicity was more severe. Bruceantin (22), with a similar structure to the qussinoid group from Brucea or Brucea antidysenterica Merril (Rajchadad in Thai) of Simaroubaceae family, showed high antileukemic activity at a wide range of doses including low ones. It inhibited protein synthesis and had undergone clinical trials. No significant therapeutic activity has been noted but the research on the whole quassinoid related to bruceantin still continues. The Eastern Cooperative Oncology Group (ECOG) has conducted a Phase II trial of bruceantin on malignant melanoma. Twenty-two patients, thirteen without prior cytotoxic chemotherapy, were recruited. All patients were evaluated for

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Figure 2. Some potent cytotoxic compounds (13-17), antitumor agents (18-23) and anticancer drugs (13) of Thai plant origins.

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response and toxicity. Dose limiting toxicity was found to result in hypotension during bruceantin infusion. Other prominent side effects were nausea, vomiting, anorexia, fever, chills, and weakness. Only minor hematologic toxicity was encountered. Two partial responses, both in previously treated patients, were observed (response rate of 9%). It was concluded that bruceantin had only limited activity against malignant melanoma and was unlikely to contribute to systemic therapy of this disease, either as a single agent or in combination with cytotoxic drugs. Elephantopin (23) as sesquiterpene lactone, an extract from Elepantopus elatus Bertoloni or Elephant's Foot (Asteraceae), was found to have the best in vitro active compound but showed little useful in vivo antitumor activity. Thus, the evaluation for study in clinical trials should carry over into in vivo systems, but the relatively narrow therapeutic index may make the compounds unpromising as therapeutic agents in clinical studies (Evans 2002). Although many plant-derived cytotoxic agents may prove toxic or inactive using in vivo evaluations, their structure is still of interest since they can be used as lead molecules for synthetic compounds and as tools for mechanism studies. The structures of these cytotoxic compounds are shown in Figure 2.

Anticancer agents from Thai traditional medicines for use in cancer treatment
Thai traditional medicine (TTM) is a cultural heritage and indigenous wisdom, which has helped take care of the health of Thai people for over a thousand years. Our ancestors had accumulated precious experiences of holistic health care in fighting against illnesses. The Thai medical concept is involved with Buddhism and natural survival. The basic principle of every branch of TTM is the knowledge of four elements or Dhatu (earth, water, wind and fire), their functions and their interrelations, which affect the health of the individual. The diagnostic method of TTM, which does not only put emphasis on diseases, but also the causes of diseases, can be organized into six categories. They are the variations of the body element, season, age, place, time and behavior. These variations bring about the imbalance and disharmony between the four body elements that is the cause of various diseases (Subchareon 1998b). The concept of cancer in TTM used by Thai traditional doctors (Itharat et al. 1998) is that it is caused by an abnormal earth element or the imbalance of too much earth element in the patient body and chronic ulcer disease. The main causes are bad eating habits (too much, too little, or bad quality), emotion (sadness, depression, anger), environment (rapid change of temperature and climate), exercise (imbalance action and work) and excretion (abnormal excretion). These causes, especially the excretion of waste products in the body, make the patient have more earth element, or imbalance of the element. Having these conditions for a long period of time, especially the incomplete excretion and accumulation of the waste products in the body, may cause the cells to differentiate into tumors, ulcers, or abscesses. There are two steps in the TTM diagnostic method for the treatment of cancer. The first step involves a review of the patients personal data, which includes his birth date for the calculation of his body element. It also includes his lifestyle and behavior, which leads to illness, as this information is used to determine the imbalance of the elements. The second step requires physical examination, which includes the measurement of pulse rate, heart rate, body

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temperature, blood circulation, and determination of the illness through meditation and an astrological chart. From the results of the diagnosis, folk doctors will calculate the medicinal formula to treat the patients. The curative method of the TTM is based on the prescription of herbal foods and medicine to readjust the bodys balance of elements and meditation for relaxation and reduction of pain. Preparations to treat the illness in the TTM constitute several herbs rather than a single herb. A preparation for cancer is composed of many plants to balance the four body elements and kill the cancer cells. The principle of the Thai medicinal plant combination in the preparation is that it must contain ingredients that have the ability to destroy the cancer cell. The folk doctors use the taste of the plant to indicate its action; a nauseating taste shows that the plant is almost certain to be toxic and, thus, could be used as an anticancer plant. The second group of plants in the preparation is considered auxiliary drugs whose functions are to support the main ingredients. Their actions are tonic and can reduce side effects or other symptoms (e.g. pain, fever, nausea, abscess) and laxative plants are thought to be a necessary part of the preparation. From the investigation of the frequency of Thai plants in 30 cancer preparations which Thai folk doctors used to treat cancer, 10 exhibited high frequencies (Itharat et al. 1998). These are Hua-Khao-Yen (Smilax corbularia Kunth, Smilax glabra Roxb. Liliaceae, Dioscorea membranacea Pierre, Dioscorea birmanica Burkill Dioscoreacea, Premna herbacea Roxb.- Verbenaceae), Khunthongpayabat (Gelonium multiflorum A. Juss - Euphorbiaceae or Siphonodon celastrineus Griff - Celastraceae), Khampang Jed Chan or Lolly Berry Vine (Salacia chinensis Linn. - Hippocrateaceae), Husakunted or Muichang or Lime Berry (Micromelum minutum Wight & Arn. Rutaceae), Hua-Roi-Ru or Ant Plant (Hydnophytum formicarum Jack - Rubiaceae), Thongpunchang or White Crane Flower (Rhinacanthus nasutus (Linn.) Kurz Acanthaceae), Ngungpramoo or Sea Holly (Acanthus ebracteatus Vahl Acanthaceae), Kaminooi or Zedoary or white turmeric, (Curcuma zedoaria Roscoe Zingiberaceae) Kui (Wullughbeia cochinchinensis Pierre Linn.), and Hnontaiyak (Stemmona burkilli Prain - Stemonaceae). Itharat et al. (2004) studied cytotoxicity of these Thai medicinal plants against cancer cell lines using the SRB assay. The extract procedures used were similar to those practiced by Thai traditional doctors (ethanolic and water extracts). The extracts were tested against human large cell lung carcinoma (COR-L23), human breast adenocarcinoma (MCF-7), human colon adenocarcinoma (LS-174T), and one normal human keratinocyte cell line (SVK-14). The results showed that three plants, Dioscorea membranacea Pierre, Dioscorea birmanica Burkill (Dioscoreaceae) and Siphonodon celastrineus Griff. (Celastraceae), exhibited high cytotoxic activity, showing a certain degree of selectivity against the different cell types. Thus, the ethnobotanical data of the investigated plants appear to corroborate the efficacy of the cancer treatment in Thai traditional medicine. The cytotoxicity screening of the aforementioned plants against the three cancer cells showed that the water extract of two species of Dioscorea exhibited high cytotoxic activity and Dioscorea membranacea rhizomes were the most cytotoxic against CORL3, LS-174T, and MCF-7. The water extract of Dioscorea burmanica Burkill was also cytotoxic against lung cancer COR-L23 but not against colon or breast cancer. The water extract of Dioscorea membranacea Pierre was the most effective against breast, colon

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and lung cancerous cells but less active with normal cells (IC50 of this extract were 5.5, 15.6, 16.3 and 78.37 g/ml, respectively). IC50 of the water extract of Dioscorea burmanica Burkill showed low activity with all cells (Itharat et al. 2004). The activities of the extracts of Dioscorea membranacea Pierre against each cancer cell for the exposure time of 72 hours were compared with normal cells by the ratio of IC50 cancer cell/normal cell (SVK-14). The results showed that its activity against breast cancer compared to that against SVK-14 was highly significant (p<0.0001). Therefore, it was concluded that the water extract of Dioscorea membranacea had selective toxicity against cancer cells with little damage to normal cells, indicating suitability of the plant for cancer treatment (Halliwell and Gatteridge 1988). Ethanolic extracts of Dioscorea spp. were also most active against cancer cell lines. Studies with a range of dilutions showed that extracts of Dioscorea membranacea rhizomes were most active against lung, colon and breast cancer (IC50 at exposure time of 72 h = 6.2, 16.7, and 12.0 g/ml, respectively). The ethanolic extract of Dioscorea burmanica rhizomes was the second most effective against the three cancers (IC50 = 7.4, 22.6 and 16.3g/ml, respectively). The ethanolic extract of two Dioscorea species exhibited the most activity against lung cancer, followed by breast and colon cancers, respectively. Eight compounds were isolated by bioassay guild fractionation from the ethanolic extract of Dioscorea membranacea. They are two novel naphthofuranoxepins (Dioscorealide A [DMS1] and B [DMS2]), one novel 1,4-phenanthraquinone (Dioscoreanone [DMS3]), three steroids (-sitosterol [DMS4], stigmasterol [DMS5] and -D-sitosterol glucoside [DMS6]), and two steroid saponins (diosgenin 3-O--Lrhamnopyranosyl (12)- -D-glucopyranoside [DMS7] and diosgenin 3-O--Dglucopyranosyl (13)--D-glucopyranoside [DMS8) (Fig. 3). The cytotoxicity of all compounds was determined using the SRB assay (Skehan et al. 1994). The target cell lines were large-cell lung carcinoma COR-L23, colon adenocarcinoma LS-174T, breast adenocarcinoma MCF-7, and non-cancer human keratinocyte SVK-14. The results showed that Dioscorealide B, dioscoreanone and diosgenin 3-O--L-rhamnopyranosyl (12)- -D-glucopyranoside had cytotoxic activity against the three cancer cell lines and dioscorealide B showed selective cytotoxic activity against lung and breast cancer but less activity against two normal cells: keratinocyte and skin fibroblast cells, while DMS7 was toxic to all types of cells (Table 1 and Fig. 4). Diosgenin 3-O--Lrhamnopyranosyl (12)- -D-glucopyranoside or prosapogenin A of dioscin (DMS7) has been reported for its cytotoxicity against HL-60 human promyelocytic leukemia cells (IC50 = 1.8 g/ml) (Mimiki et al. 2001). Dioscoreanone also showed the highest antioxidant activity. The structural activity relationships (SAR) for dioscorealide A and B, and diosgenin 3-O--L-rhamnopyranosyl (12)- -D-glucopyranoside, diosgenin 3O--D-glucopyranosyl (13)--D-glucopyranoside were discussed (Itharat et al. 2005a). The effect of ethanolic extracts of Dioscorea membranacea was also studied using the prostate (PC3) cancer cell line and found to exhibit low cytotoxic activity (IC50 = 17.55 g/ml). Dioscoreanone isolated from the ethanolic extract of Dioscorea membranacea was cytotoxic against the prostate cancer cell line (IC50 8.14 M) but less toxic against a human fibroblast cell line (IC50= 21.4 M). Surprisingly, dioscorealide B had no toxicity against prostate cancer cells. This compound showed high selectivity

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Figure 3. Cytotoxic agents from Dioscorea membranacea DMS1 = Dioscorealide A ; DMS2 = Dioscorealide B ; DMS3 = Dioscoreanone ; DMS4 = -sitosterol ; DMS5 = Stigmasterol DMS6 = -sitosteryl--D-glucopyranoside DMS7 = diosgenyl-3-O--L-rhamnopyranosyl (12)- -Dglucopyranoside or protosaponin A of dioscin ; DMS8;, diosgenyl-3-O--D-glucopyranosyl (13)- -D-glucopyranoside.

against only breast and lung cancers and was not active against colon and prostate cancers. Diosgenin 3-O--L-rhamnopyranosyl (12)--D-glucopyranoside also showed high cytotoxic activity against prostate cancer cells (IC50=5.88 M) but was less active against normal cells (IC50 =38.05 M) (Saetung 2006).

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Table 1. IC50 (M)SEM of cytotoxic compounds from Dioscorea membranacea against cell lines at an exposure time of 72 h (n=3).

CORL-23
160 140 120

LS-174T

MCF-7

SVK-14

HF

IC50 value (micromole)

100 80 60 40 20 0

DMS2

DMS3

DMS6

Figure 4. Comparison of IC50 values (M) of cytotoxic compounds of Dioscorea membranacea against all cell lines at an exposure time of 72 h

Dioscoreanone and diosgenin 3-O--L-rhamnopyranosyl (12)--D-glucopyranoside as cytotoxic compounds were tested for apoptosis using the TUNEL assay. The results showed that 10 g/ml dioscoreanone induced 7.75% apoptosis in a lung cancer cell line. Dioscoreanone and diosgenin 3-O--L-rhamnopyranosyl (12)--D-glucopyranoside at 5 and 10 M induced 3.37%, 3.71%, 3.43% and 7.29% apoptosis, respectively, in

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prostate cancer cell lines. Interestingly, diosgenin 3-O--L-rhamnopyranosyl (12)-D-glucopyranoside induced cell death via apoptosis only in prostate cancer cell lines (Saetung 2006). Chemical investigation and cytotoxicity determination of D. membranacea rhizome extracts supported the indigenous knowledge from traditional medicine. The ring systems, especially of the naphthofuranoxepins, might originate from a rather simple heptaketide precursor. The potent and selective cytotoxicity for tumor cells yet, lack of toxicity for normal cells demonstrates the strong potential of this plant; further studies with an in vivo assay are required to identify the mechanism of action for these compounds and, to develop this plant as a new source of compounds for use in cancer chemotherapy in the future. The ethanolic extract of Dioscorea birmanica rhizome showed high and selective cytotoxic activity against human large cell lung carcinoma (COR-L23), a colon cell line (LS-174T) and a breast cancer cell line (MCF-7) (IC50 = 7.04, 22.6 and 16.3 g/ml, respectively) using the SRB assay (Skehan et al. 1994). A known steroid saponin glycoside, gracillin (Diosgenin-3-O---D-glucopyranoside(13)[-L-rhamnosyl (12)--D-glucopyranoside) was isolated from it (Fig. 5). This compound showed specific cytotoxicity against colon and lung cancer cell lines (IC50=4.34 and 6.07 g/ml, respectively) but was less active against breast cancer cell lines (IC50= 30.64g/ml) (Itharat et al. 2005b).

Figure 5. Diosgenin-3-O---D-glucopyranoside (13) [L-rhamnosyl (12) --D-glucopyranoside (Gracillin)

In the case of the genus Dioscorea, cytotoxic activities have been reported for some species found in Thailand; e.g. Dioscorea bulbifera exhibited cytotoxicity against Hela cell (Kosuge et al. 1985). Ethanolic extracts from Siphonodon celastrineus leaves showed cytotoxic activity with breast cancer, especially with an exposure time of 48 h (IC50 = 17.1g/ml) and low activity against lung cancer (IC50 = 28.2g/ml) but no activity against colon cancer (IC50

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> 50 g/ml). Nevertheless, the growth of cancer cells, which were inhibited at an exposure time of 48 h could be recovered at an exposure time of 72 h as indicated by higher IC50 values at a 72 h exposure than at 48 h for breast and lung cancers. Stem extracts from Siphonodon celastraneus Griff. showed no activity against any cells (Itharat et al. 2004). Ethanolic extracts from Rhinacanthus nasutus roots used in anticancer preparations showed high cytotoxic activity against prostate, lung and normal skin fibroblasts (PC3, CORL-23 and 10FS) (IC50 = 2.1, 5.05 and 10.1 g/ml, respectively) (Saetung et al. 2005). Many Rhinacanthus nasutus Linn. derived compounds were effective against P388, HL-60, KB, HT-29 and A549; all compounds killed all cells although no specific cytotoxic activity was observed (Wu et al. 1988 a,b). Rhinacanthin B, which is naphoquinone isolated from the methanolic extracts of Rhinacanthus nasutus root, showed cytotoxic activity against nasopharyn cancer cells (KB) (Wu et al. 1988a). Ethanolic extracts of Curcuma zedoaria Roscoe showed specific activity against lung cancer cell lines, but were less cytotoxic against prostate cancer cells and normal fibroblast cells (IC50 6.05, 17.84 and 55.50 g/ml, respectively), using the SRB assay (Saetung et al. 2005). There is one report on cytotoxic compounds from Curcuma zedoaria Roscoe against human ovarian cancer cells, but no report on lung and prostate cancers (Syu et al. 1998). Other reports on Curcuma zedoaria extracts pertaining to cancer are related to its antimutagenic activity (Lee and Lin 1988), anti-tumor activity (Kim et al. 2000), anti-inflammatory activity (Hong et al. 2002; Jang et al. 2001; Lee et al. 2002; Yoshioka et al. 1998) and antihepatotoxic activity (Matsuda et al. 1998; Matsuda et al. 2001; Morikawa et al. 2002). They showed that cucuminoid from Curcuma zedoaria root was cytotoxic against hepatoma cells at IC50 = 33 g/ml (Matthes et al. 1980). Water extracts of Curcuma. zedoaria Roscoe had no cytotoxic activity against lung cancer cell lines and were less cytotoxic against prostate cancer cells (Saetung et al. 2005) but, showed high antitumor activity against Ehrlich ascites carcinoma induced in the wistar rat (Yokota et al. 1986). The seven plants which showed high frequency of effects on cancer cells (Smilax corbularia Kunth, Smilax glabra Roxb., Premna herbacea Roxb., Gelonium multiflorum A. juss, Salacia chinensis Linn., Micromelum minutum Wight et Arn and Hydnophytum formicarum Jack) had no cytotoxic activity (Itharat et al. 2004), but have been reported to have other activities related to cancer. For example, ethanolic extracts of Smilax glabra Roxb. showed anti-inflammatory activity (Jiang et al. 1997) and an ability to prevent immunological hepatocyte damage (Chen et al. 1999). Ethanolic extracts of S. corbularia Kunth showed cytotoxic activity against CA-KB, anti-tumor activity in rat (IP) and immunostimulant activity (Pornsiriprasert et al. 1986). Premna herbacea Roxb. or Pygmaeopremna herbacea (Roxb.) Moldenke, which is called Hua Khao Yen and is used for treatment of cancer and rheumatism in Thailand (Boonyaratanakornkit and Chantaptawan 1993; Itharat et al. 1998) showed no cytotoxicity against CA-KB (Dhar et al. 1973) but, showed good anti-inflammatory activity, analgesic activity and antipyretic activity (Narayanan et al. 2000). Ethanolic extracts of Hua Roi Ru (Hydnophytum. formicarum Jack) showed a high antioxidant activity but, less cytotoxicity against all types of cancer cell lines (breast, lung, colon and nasopharynx) (Dejadisai and Itharat 1999).

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Saetung et al. (2005) studied a preparation for cancer treatment based on an in-depth review of Itharat et al. (1998) and examination of follow-up cancer patients treated by southern Thailand folk doctors. The cancer preparation was composed of twelve Thai plants. They were leaves of Bridelia ovata Decne- Euphorbiaceae (Maga in Thai), rhizomes of Curcuma zedoaria (Berg) Roscoe-Zingiberaceae, stems from Derris scandens (Roxb.) Benth Fabaceae (Thaowan Priang in Thai), rhizomes from Dioscorea membranacea Pierre, rhizomes from Drynaria quercifolia Linn. Polypodiaceae (Oak-Leaf-Fern, Kratae Teimai in Thai), stems from Erythrophleum teysmannii Craib Fabaceae (Zak or Punchad in Thai), bark from Moringa oleifera Lamk. Moringaceae (Horseradish-tree, Ben-oil tree, Drumstick-tree or Marum in Thai), flowers from Nardostachys jatamansi DC Varelianaceae (Muskroot, Indian Spikenard or Kod Chada Munksri in Thai), roots from Rhinacanthus nasutus (L.) Kurz. Acanthaceae (White crane flower or Thongpunchang in Thai), fruit from Sapindus rarak DC - Sapindaceae (Makumdeekwai in Thai), rhizomes from Smilax corbularia KunthSmilacaceae, and seeds from Strychnos nux vomica L - Loganiaceae (Strychnine tree, Vomit nut or Godgagling in Thai). Cytotoxicity was tested against two types of human cancer cell lines, (i.e., large cell lung carcinoma (COR L-23) and prostate cancer cell lines (PC3)) and one type of normal human cell line, (i.e., fibroblast cells (10FS)) using the SRB assay. Water and ethanolic extraction procedures used were similar to those practiced by Thai traditional doctors. The ethanolic extracts of six plants (Bridelia ovata, Curcuma zedoaria (Berg) Roscoe, Derris scandens (Roxb.) Benth, Dioscorea membranacea Pierre, Nardostachys jatamansi DC and Rhinacanthus nasutus (L.) Kurz. showed cytotoxicity (IC50 <30 g/ml) against lung and prostate cancer cell lines. Dioscorea membranacea Pierre roots showed the highest cytotoxic activity against a lung cancer cell line (IC50 = 4.6 g/ml) but, low activity against a prostate cancer cell line (IC50 = 20.8 g/ml) and, less activity against a normal cell line (skin fibroblast cells) (IC50 = 66.05 g/ml). Curcuma zedoaria (Berg) Roscoe showed cytotoxicity against COR L-23 and PC3 cells but, less activity against 10FS cells (IC50 = 6.05, 17.84 and 66.05 g/ml, respectively). Rhinacanthus nasutus root extracts showed the highest cytotoxic activity against PC3 cells (IC50 = 2.1 g/ml) and also high activity against COR L-23 and 10FS lines (IC50 = 5.1 and 10.1g/ml, respectively). Water extracts from all of the plants exhibited no activity against all types of human cells. Ethanolic extracts of Dioscorea membranacea Pierre and Curcuma zedoaria (Berg) Roscoe showed specific activity against lung cancer cell lines and less activity against normal cells. The previous data also revealed that Dioscorealide B, isolated from the ethanolic extracts of Dioscorea membranacea Pierre, was active against the lung cancer cell line COR-L23 (Itharat et al. 2004). It could be used as a marker for the analysis of preparations when produced as a cancer preparation. These results lend support to the use of these Thai folk medicine preparations for the treatment of cancer patients (Itharat et al. 1998). The three active plants (Dioscorea membranacea Pierre, Curcuma zedoaria (Berg) Roscoe and Rhinacanthus nasutus (L.) Kurz) used in these preparations also corroborate those reported in folk medicine textbooks (Vimonkunakorn 1979; Itharat et al. 1999; Pongboonrod 1976). Some plant extracts, (e.g., Derris scandens (Roxb.) Benth and Strychnos nux vomica L.) used in this cancer preparation, which had no cytotoxic activity, have been reported to have other activities related to cancer; e.g., anti-inflammation and analgesic effects (Laupattarakasem et al. 2003; Yin et al. 2003).

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Investigation of indigenous wisdom on cancer treatment of Thai traditional doctors by Itharat et al. (1998) revealed that a Benjakul preparation, which is composed of five Thai medicinal plants (Piper longum Linn., Piper sarmentosum Roxb., Piper interruptum Opiz., Plumbago indica Linn. and Zingiber officinale Roscoe) has been used as an adaptogen drug for cancer patients. SRB and MTT assays were used to test the cytotoxicity of the Benjakul preparation and its components against two types of human prostate cancer cell lines (PC3 and LnCAP). The extraction procedures used were similar to those practiced by Thai traditional doctors (ethanolic and water extracts). The results showed that all ethanolic extracts except that of P. sarmentosum exhibited cytotoxic activity against PC3 cells, but none of the extracts showed any activity against the LnCAP cell line. The results showed selectivity against different cell types of prostate cancer cell lines. Ethanolic extracts from Plumbago indica Linn. showed the highest cytotoxic activity against PC3 (IC50 = 9.30 g/ml). Water extracts from these plants showed no activity against the two types of cell lines. Water extracts from Plumbago indica Linn. and ethanolic extracts from Zingiber officinale Roscoe exhibited the highest antioxidant activity (EC50 = 2.59 and 3.80 g/ml, respectively). The Bejakul preparation showed cytotoxic activity against lung cancer (CORL23), androgen independent prostate cancer (PC3) and androgen dependent prostate cancer (LnCaP) (IC50=19.8, 29.8 and >50g/ml, respectively) (Ratanasuwan et al. 2005). Other cancer-related activities associated with the Benjakul preparation and its components, e.g. antibacterial activity, were also tested by Kummee et al. (2004). The results showed that ethanolic extracts of the Benjakul and other plants in the preparation were active against two types of gram-positive bacteria (MIC of Benjakul against S. aureus and B. subtilis = 4 and 2 mg/ml, respectively). The ethanolic extracts of Plumbago indica Linn were the most active against S. aureus, B. subtilis, E. coli and Candida albicans (1, 1, 8, 2.5 mg/ml, respectively). Only one of the water extracts of P. indica exhibited activity against S. aureus, B. subtilis, and C. albicans (MIC = 2, 2, 1 mg/ml, respectively). These results support use of the Benjakul mixture as an adaptogen preparation in the treatment of cancer by Thai folk doctors; it also showed cytotoxic effects against cancer cells. Benjakul extracts showed no toxicity and no body tissue and biomaterial changes when tested by a sub-chronic toxicity method (Chauvaltthamrong et al. 1996). Another cancer preparation of a Thai folk doctor from Singhburi Province is composed of five plants, i.e. Canna indica L. (Cannaceae) rhizome, Smilax corbularia rhizome, all parts of Polygala chinensis L. (Polygalaceae), Ludwigia hyssopifolia (G. Don) Exell (Onagraceae) and Clinacanthus siamensis Brem. (Acanthaceae). This preparation was used more than 22 years ago and was tested in vivo in female wistar albino rats treated with 1% carcinogen DMBA (7,12-dimethylbenantracene) to induce breast cancer. The result showed that the diameter of tumors was reduced by more than 32.5% at 1500 mg/kg and a T/C = 145% in the first 4 weeks of treatment (Nabanchang 1987). This preparation was tested for acute and chronic toxicity and was found to have no toxicity for tissues and produced no change in biochemical indicators when treatments with the drug were given for six months (Pornsiriprasert 1986). Currently, this preparation has been produced as a capsule by the Government Pharmaceutical Organization of Thailand and is being clinically tested as a cancer treatment. From the ethnopharmacolgical approach of Thai folk doctors in the treatment of cancer, a search for bioactive compounds against cancer cells has been reviewed and summarized in Table 2.

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Table 2. Medicinal plants used by Thai folk doctors in traditional preparations for cancer treatment.

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Table 2. Continued

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Table 2. Continued

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Table 2. Continued

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Table 2. Continued

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Reuengrungsri et al. (1993) researched anticancer agents from 108 samples of plants collected from 21 provinces of Thailand also based on their ethnomedical use for cancer treatment. They were extracted with 95% ethanol and tested against 388 lymphocytic leukemia cells (P388), human epidermoid carcinoma of nasopharynx (KB) cells and genetically engineered vinblastine resistant (KB-VI) cell lines. The results showed that 25 plant extracts were effective against KB cells (23.1%), 10 extracts against KB-VI cells (9.3%) and 28 extracts against P388 cells (25.9%). Studies were continued on 24 plant extracts for active compounds against cancer. For example, five cytotoxic alkaloids (tetrandrine, limacine thalrugosine, homoaromaline and cycleapeltine) were separated from the ethanolic extract of Cyclea bartata Mier (Krungkamao, in Thai), which had shown high cytotoxic activity against KB, KB-V1 and P388 cells (IC50 = 3.6, 12 and 1.5 g/ml, respectively) (Guinaudeau et al. 1993). Stephania erecta Criab. (Buabok) showed specific activity against only P388 (IC50= 3.23 g/ml), was less active against KB cells (IC50= 10.7 g/ml) and possessed no activity against KB-VI. From it, 13 alkaloids were isolated which showed high cytotoxicity against 11 types of cancer cell lines (Likhitvitthayawut et al. 1993). From an ethanolic extract of Garcinia hanburyi HooK.f. (Rongthong) resin three xanthones (gamboic acid, isogamboic acid and isomorellinol) were isolated. These and the ethanolic extract of Clitoria macrophylla roots (Hnontaiyak) (6-deoxyclitoriacetal) showed high cytotoxic activity against P388 cells (IC50 = 0.031 g/ml) (Reuengrungsri et al. 1993). Mahidol et al. (2000) investigated the plant Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae), locally known as Look Tai Bai, for cytotoxic activity. From it two lignan compounds (phyllanthin and hypophyllanthin) were isolated and these had enhanced cytotoxic responses in cultured multidrug resistant cells. She also investigated Gloriosa superba L. for anticancer activity. The plant is locally known as Dong Dueng Hua Kwan and is widely distributed in tropical parts of Asia and Africa. The active compound of Gloriosa superba is colchicine, which is obtained from the dried tubers. Four tropolone alkaloids were also isolated from the tuber (Engprasert 1995; Capraro and Brossi 1984; Bently 1998). All alkaloids were tested against a cholangiocarcinoma cell line (HuCCA-1), a form of bile duct cancer, which is globally a rare type of cancer but highly prevalent in Thailand and many other Asian countries. The cause of the disease is infestation with Opisthorchis viverrini or liver fluke. Colchicine and colchicine derivatives were tested against HuCCA-1 and KB cells using the SRB assay. The methanolic extract showed activity against HuCCA-1 cells (IC50 = 2.5 g/ml), while IC50 for the colchicine and its derivative (3-demethyl-N-formyl-N-deacetylcolchicine) were 0.2 and 0.625 g/ml, respectively. Surprisingly, these values were two times higher than the IC50 values for KB cells (0.01 and 0.03125 g/ml, respectively). Various analogues of colchicine have been synthesized with the aim of improving the therapeutic index of the target compounds by enhancing the potency of these analogues. The results of the biological tests with the colchicine analogues against the cholangiocarcinoma cell line showed a very low biological activity compared to colchicine. Derris reticulate was also investigated by Mahidol et al. (1997). This plant Cha-am Thai has been used for thirst relief and as an expectorant. Its isolate contains upinifoline as a major constituent as well as flavanone and three new pyranoflavonone compounds (epoxylupinifoline, dereticulatin and hydroxylepoxylupinifoline). They were tested against a P388 cell line and showed inhibition at 0.40.5 g/ml, but were inactive against a KB cell line (Mahidol et al. 2000)

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Conclusion
Most investigations of anticancer agents from Thai medicinal plants were based on their ethnomedical use by Thai folk doctors and on knowledge of Thai traditional medicine. Some potent active ingredients were studied for semi-synthesis by Thai chemists in order to produce potent anticancer drugs. However, some Thai researchers have still investigated the use of whole plant preparations (a combination of many plants within the preparation) by studying each plant in the preparation and comparing the results with extracts from the whole preparation. Subsequently, they determined the components of the plants, which were active against cancer cells and used them as markers in the analysis. The concept of cancer treatment in Thailand has changed considerably, based on the fact that using chemotherapy for cancer treatment causes major suffering to the patients. In contrast, using whole herbal preparations has produced gentler effects on the human body because of the synergy of the plants which make up the preparations. It is conceivable that Thai anticancer drugs in the future will consist of an anticancer formula composed of many medicinal plants. Each plant will contribute to the treatment of the disease by enhancing the immune response and by providing pharmacological effects related to cancer treatment; e.g., antibacterial, antiinflammatory, antioxidant, etc. effects. The trend for cancer treatment research will include the holistic approach, which requires knowledge of both mind and body. For example, meditation has shown positive effects on the mind by reducing the stress level, and knowledge of human behavior, especially related to food and exercise is very useful in cancer treatment. Nevertheless, investigations of herbal drugs in cancer treatment continue, especially the clinical trials of these drugs, which have been encouraged and supported by the Thai Traditional Medicine Institute, Ministry of Public Health. Research on novel drugs is very complex and time consuming, and clinical trials are extremely costly. Therefore, it is unlikely that in the foreseeable future there will be a major breakthrough in anticancer drugs in Thailand. In the meantime, prevention and treatment of cancer in the kingdom will continue to rely on the holistic approach of Thai traditional medicine.

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