Tumour promoter activity in Malaysian Euphorbiaceae.

Herbal medication has been practised by the rural Malaysian Malays for a long time. However, the long-term side-effects have never been studied. In the present study, 48 species of Euphorbiaceae were screened for tumour-promoter activity by means of an in vitro assay using a human lymphoblastoid cell line harbouring the Epstein-Barr virus (EBV) genome. Twenty-seven per cent (13 out of 48) of the species tested were found to be positive, and in four species, namely Breynia coronata Hk.f, Codiaeum variegatum (L) Bl, Euphorbia atoto and Exocoecaria agallocha, EBV-inducing activity was observed when the plant extracts were tested at low concentrations of between 0.2 and 1.2 micrograms ml-1 in cell culture. This observation warrants attention from the regular users of these plants because regular use of plants with tumour-promoting activity could well be an aetiological factor for the promotion of tumours among rural Malaysian Malays.

Epidemiological studies reveal a strong circumstantial link between plants possessing tumour-promoting principles and the increased incidence of human tumours in certain geographic regions of the world. Hirayama and Ito (1981) indicated that there is a strong circumstantial link between the high incidence of nasopharyngeal carcinoma (NPC) in the southern regions of China and the distribution of Euphorbiaceae and Thymelaeceae plants. Among these plants, extracts from Croton tiglium, Euphorbia lathylis, Aleurites fordii, Jatropha curcas, Euphorbia antiquorum, E. milii, E. pekinensis, E. kansui and Daphne odora all exert Epstein-Barr virus (EBV)-activating capacity. The active component of the classic tumour promoter of croton oil from Croton tiglium L was purified and its chemical structure defined as 12-0-tetradecanoyl phorbol-13-acetate (TPA) (Hecker, 1968).
A similar study also indicated a causal link between the regular intake of phorbol esters contained in 'Walensalii tea' prepared from Crotonflaven L. (Euphorbiaceae) and the high incidence of oesophageal cancer in Curacao Island of the Caribbean (Hecker, 1987).
Burkitt lymphoma (BL), an EBV-associated non-Hodgkin's malignant lymphoma, is endemic in an area of Africa known as the lymphoma belt. In East Africa (Kenya, Uganda and Tanzania), where BL is endemic, over 90 species of Euphorbiaceae plants are reported to be currently used as common folk remedies (Kokwaro, 1979). EBV-activating capacity has been demonstrated in extracts of some of these plants (Ito, 1986).
EBV-activating capacity has also been reported in 11 out of the 12 species of the Euphorbiaceae family commonly grown in the Cameroon, one of the endemic areas of BL (Ohigashi et al., 1985). In Malawi, E. tirucalli and other EBV-activating plants were found significantly more often in homes of BL patients than in those of controls (Van den Bosch et al., 1993). Recently, E tirucalli has been reported to induce the characteristic 8:14 translocation of endemic BL in EBV-infected lymphoblastic cell lines in vitro (Aya et al., 1991).
In Malaysia, herbal medicaments are widely used by the rural Malays. Several parts of the different plants are used. Dried parts of the plants such as roots and barks are usually boiled and the decoction taken internally. Fresh specimens are usually squashed and applied externally as a poultice.
Many of the indigenous plants used are also from the Euphorbiaceae and Thymelaeceae families. NPC, which is associated with EBV, is also common among the Malaysian Malays, although the prevalence is lower than in the Malaysian Chinese (Armstrong et al., 1979;Yadav et al., 1985). It would be interesting to study the incidence of NPC in relation to the use of these plants. However, in this study, we only screen the members of the Euphorbiaceae used by herbalists for their tumour-promoting activity.
It has now been established that latent EBV can be induced and activated from genome-carrying human lymphoblastoid cells in vitro by treatment with tumour-promoting phorbol and other diterpene esters (zur Hausen et al., 1979;Ito et al., 1981a), and indole alkaloid (Luka et al., 1979;Ito et al., 1981b;Yamamoto et al., 1981). In the present study, a short-term in vitro assay for detecting tumour-promoting activity using the activation of EBV antigen expression in EBV genome-carrying human lymphoblastoid cells (Raji) has been used as the screening assay (Ito et al., 1981b). This assay system is rapid and efficient for detecting EBV-active principles in the environment (Ito et al., 1981a,c).
It is also noteworthy that such EBV-activating principles can be considered to possess tumour-promoting capacity (Ito et al., 1981a). The assay can thus be used as a screening test in the search for promoter substances in nature.

Collection ofplants
The Euphorbiaceae species were collected from various places in Malaysia. Some of the plants were collected from the University of Malaya botanical garden, Rimba Ilmu. The rest of the plants were collected during field trips conducted in the Malaysian states of Selangor, Pahang, Kedah and Kelantan. The list of plants collected is shown in Table   I.
Preparation of crude extract Crude ether extracts of dried, powdered plant material were prepared as previously described (Yadav et al., 1989). Ten grams of the powdered material was extracted with ether (10 ml) at room temperature for 48 h. After evaporating the solvent, the resultant residue was weighed and dissolved in dimethyl sulphoxide (DMSO) (Sigma) as a stock solution of 20 mg ml-' and kept at -20°C until use. lymphoblastoid Raji cells were incubated with plants extracts in the presence of 4 mm sodium butyrate. The cells were incubated for 72 h in a humidified incubator at 37°C with 5% carbon dioxide in air. The positive control consisted of Raji cells treated with optimal concentration (1O ng ml-') of TPA (Sigma) and 4 mM n-butyrate (Sigma). The negative control consisted of untreated culture media. After 72 h, the cells were harvested and checked for the presence of EBV early antigen by means of the indirect immunofluorescence assay. Nasopharyngeal carcinoma EBV-positive sera were used to detect the presence of EBV early antigen. The assay was repeated once in order to obtain consistent results.

Results
Twenty-seven per cent (13 out of 48) of the Euphorbiaceae screened were found to be positive for tumour-promoting activity when tested at concentrations of 10-40 ,g ml-' cell culture medium (see Table II). (a) Pounded root is rubbed into syphilitic soresa (b) Leaves are used to poultice the abdomen of children with urinary problemsa (a) A decoction of the leaves is used as a post-partum protective medicine and a treatment for colic or diarrhoeab (b) Leaves prescribed in medicinal bath to treat fever and as an application for ulcersa Not documented (a) It is made into poultices for sores on legs caused by the marine worm. The latex is dropped into the eyes for conjunctivitis and ulcerated corneaa (b) It has a reputation as a remedy for bronchitis and asthma, and is slightly narcotic, hence helping patients to sleep.a (a) Roots and stems are used as a poultice applied to ulceration of the nose, haemorrhoids, swellings of the nose and painful partsa Not documented (a) The latex is used in some places as a caustic in the treatment of obstinate ulcerb (b) A little of the bark chewed causes vomiting and purgingin and may be resorted to in constipationb (a) Oiled leaves are heated and applied to the stomach to treat feverb

Discussion
Since plant diterpene esters in Euphorbiaceae and Thymelaeceae plants have been found to exert EBV-inducing activity (Ito et al., 1981b;Zeng et al., 1983), only ether extracts were tested in the present study. However, the EBV-activating property has also been reported in methanol and water extracts of plants (Zeng et al., 1983). It is to be hoped that other plant extracts will be tested in the future.
To date, no studies have been carried out to relate the use of indigenous plants with cancer in Malaysia. Therefore, the link between tumour promoters and cancers in Malaysia is unknown. From this study, it is disturbing to note the high prevalence of EBV-inducing activity in the species of Euphorbiaceae tested, especially since nasopharyngeal carcinoma, which is associated with EBV, is common among Malaysians (Armstrong et al., 1979;Yadav et al., 1985).
Since many of the EBV inducers are tumour promoters (Ito et al., 1981b;Zeng et al., 1983), the hypothesis that these positive plants are the aetiological factors for NPC is valid not only for NPC but also for other malignant tumours. Among the Malays, these plant extracts are either taken internally as a decoction or a concoction or used externally as a poultice. In both ways, there is direct contact between the plant extract and the skin or mucous membrane of the digestive tract. Plant extracts with EBV-activating capacity have been -reported to retain their capacity to induce EBV complex in viral genome-carrying human lymphoblastoid cells (Raji) even after heat treatment at 120°C for 2 h or at 100°C for 12 h (Yanase and Ito, 1984). The medicinal plants with tumour-promoting activity could well be an important aetiological factor in the promotion of tumours among Malays who use these plants regularly in folk medication.
Detailed chemical studies on the positive EBV-activating plants identified in this study have not been carried out.
However, the active compounds of J. podagrica and J. gossypyfolia have been reported to be polyunsaturated esters of the tigliane-type diterpenoids 16-hydroxyphorbol and 12deoxy-16-dydroxyphorbols respectively (Adolf et al., 1984). Highly irritant and promoting principles of E. tirucalli have been characterised as 4-deoxyphorbol diesters (Kinghorn, 1979). The active tumour-promoting principle of E. agallocha has been shown to be the piscicidal daphnane-type orthoester 'excoecaria toxin', and the structures of the individual daphne-type irritant factors have been elucidated (Wikiyachitra, 1985). Active principles of the other positive plants identified in this study have not been documented.
EBV-activating principles have been documented to be present in C. variegatum (Ohigashi et al., 1985), E. hirta, E. splendes and P. tithymaloides . However, EBV-activating principles in B. coronata, C. argyratus, E. atoto, H. populneus and S. angustifolia have not been documented in the literature. E. pulcherrima did not exhibit EBVactivating principles and a similar observation has been reported previously .
The multifactorial aetiology of cancer is complex. However, the most important task of basic cancer research always was and still is the development of generally valid criteria for the detection and classification of environmental carcinogens. In Malaysia, good epidemiological studies regarding an association between the use of plants with tumour-promoting activity and the prevalence of cancer (tumours) are clearly needed. More plants should be screened for this activity, and further studies on tumour-promoting activity in Malaysian plants are necessary.