Screening for in vitro anti-tumor promoting activities of edible plants from Thailand

hncer Letters 95 ( 1995) 139-I 46

CANCER LETTERS

Screening for in vitro anti-tumor promoting activities of edible plants from Thailand Akira Murakamia,‘, Suratwadee Jiwajindab, Koichi Koshimizuc, Hajime Ohigashia.* “Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606-01. Japan bEnvironmental Science Unit, Central Laboratory and Greenhouse Complex, Kasetsart University, Nakorn-Pathom, 73140, Thailand =Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Iwade- Vchita, Wakayama, 649-64, Japan

Received 8 May 1995;revision received 19 June 1995;accepted20 June 1995

Abstract A total of 112 speciesof edible plants (122 samples)from Thailand were randomly collected, and their methanol extracts were screenedfor in vitro anti-tumor promoting activity using the inhibition test of Epstein-Barr virus (EBV) activation in Raji cells induced by 12-O-hexadecanoylphorbol-13-acetate(HPA, 40 rig/ml). It was found that 60% of these extracts inhibited EBV activation by 30% or more at a concentration of 200 mg/ml. Significantly, the ratio is markedly higher than that (26%) previously observed in common edible plants in Japan.Thus, physiological potentiality of edible Thai plants has been implied in terms of cancer chemoprevention. Keywords:

Cancer chemoprevention;Anti-tumor promotion; Epstein-Barr virus; Raji cells; Flavor; Condiments

1. Introduction Several ubiquitous phytochemicals, such as carotenoids, flavonoids, or anti-oxidative vitamins, are widely known to reduce cancer risk on the basis of epidemiological surveys as well as animal model studies [ 141.Cancerpreventiveeffectsof somenatural or synthetic chemicals are currently being tested in clinical trials [2,7]. In particular, searching for new * Corresponding author. Tel.: +81 75 7536281; Fax: +81 75 7536284. ’ Present address: Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Iwade-Uchita, Wakayama,649-64, Japan.

candidates from food items would have an advantage for their clinical application on account of low toxicity. Previously, anti-tumor promoting properties of common edible plants [4,6] and marine algae [12] in Japan and their active constituents have been studied using a short-term in vitro assay, the inhibitory test of EpsteinBarr virus (EBV) activation induced by tumor promoters [8,9]. Some active constituents in vitro were then identified, and ursolic acid and mokko lactone, isolated from green perilla and edible burdok, respectively, were proven to have anti-tumor promoting activity in ICR mice skin [5]. In recent years we have focused on the edible plants from Thailand [3,9,11,13]. They are rich in plant species that are used for non-nutrient purposes, e.g. fla-

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vors, condiments, and concurrently traditional medicines. Moreover, Thailand still has many speciesof vegetablesalmost never given to plant breeders,giving rise to the possibility that they may contain effective anti-tumor promoters not occurring in long-bred plants, most of which may have been bred in terms of economic benefits. In fact, in preliminary screening testsof the non-nutritionally consumedThai plants, the activity-exhibiting ratio was about three times higher than that in common edible plants in Japan[lo]. However, the data did not generally demonstratethat edible Thai plants, as a whole, possessmuch higher potentiality for cancerprevention than thoseof Japan,because we did not test for randomly selectedThai plants. And yet, there was a considerabledifference in the number of test plants between two tests, i.e. n = 133 (Japan) and n = 40 (Thailand) [6,10]. The presentstudy was conductedto discern whether the high potentiality of edible Thai plants for cancer prevention can be generally observed. 2. Materials and methods 2.1. Chemicals

12X)-Hexadecanoylphorbol-13acetate(HPA), used as an inducer of EBV activation, was isolated form Sapium sebiferum as previously reported [ll]. Hightiter early antigen (EA)-positive sera from nasopharyngeal carcinoma (NPC) were kindly given to us by Dr. T. Osato of Hokkaido University, and FITClabeled anti-human IgG was purchasedfrom Dako Co, Ltd. (Glostrup, Denmark). Other chemicals were obtained from Wako Pure Chemical Industries Co. Ltd (Osaka,Japan). 2.2. Sample preparation

Fresh edible plants were collected at the marketsin Bangkok and Chaingmai in November, 1993. Each sample, cut into small pieces, was extracted with methanol at room temperature.After drying in vacua, the extract (2001(g/5~1 of DMSO) was subjectedto bioassay. 2.3. Inhibitory assayof Epstein-Barr virus (EBV) activation

In vitro anti-tumor promoting activity was evaluated by the inhibitory test of EBV activation as previously reported [4]. Human B-lymphoblastoid cells,

Raji, were incubated in 1 ml of RPM1 1640 medium (supplementedwith 10% fetal bovine serum) containing sodium n-butyrate (44Opg), HPA (40 ng), and the test compoundat 37°C under 5% CO, atmospherefor 48 h. EBV activation was measuredby detection of EA, stainedby a conventional indirect immunofluorescence method with high-titer EA-positive sera from NPC patientsfollowed by FIX-labeled IgG. The ratio of EA-induced cells was comparedto that of a control experiment only with sodium n-butyrate and HPA, in which the ratio of EA-induced cells was ordinarily around50%. 3. Results and discussion

A total of 112 species(122 test samples)of fresh edible plants from 43 plant families, were extracted with methanol. Then, in vitro anti-tumor promoting activity of each extract (2OOpg/ml) was measuredby inhibition of EBV activation induced by HPA (40 ngl ml), a phorbol ester-typetumor promoter closely related to 12-O-tetradecanoylphorbol13-acetate(TPA). The experimental conditions were kept strictly the sameas those used in the previous screeningtests for common edible plants in Japan161.The inhibitory effect (IE) of each test extract towards EBV activation was classified into four ranks by the inhibitory rate of EA (early antigen)-induced cells (%): +++, strongly active (IE 2 70%); ++, moderately active (70% > IE 2 50%); +, weakly active (50% > IE 230%); -, inactive (30% > IE). The results are shown in Table 1. Nine plants of the total (marked as ‘e’ in Table 1) were the same speciesas those in the preliminary test of the non-nutritionally consumedThai plants [lo]. Thirtynine plants were evaluated as being ‘strongly active (IE 170%)’ and 34 plants were found to be ‘weakly or moderately active (70% > IE 2 30%)‘. Each rate of strongly (IE 2 70%, 32% of the total), moderately (70% > IE 2 50, lo%), and weakly (50% > IE 2 30%, 18%) active plants was markedly higher than those previously reported in the screening test of common edible plants in Japan (Fig. 1) [4]. The activityexhibiting patternis similar with that in the preliminary non-nutritionally consumedThai plants (Fig. 1) [lo]. This result suggeststhat the potential of edible Thai plantsfor cancerpreventionmight be generally high. In the preliminary screeningtest of the Thai plants [lo], we indicatedthat the speciesin the plant families

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Table 1 In vitro anti-tumor promoting activities of edible plants from Thailand Family/species

Common name

Part tested

Activity

Pakkuudb

Leaves

-

Lentin& Hednangfahb

Whole part Whole part

-

Phak khuangb

Leaves

+++

AmaranthC

Leaves

++t

Mango Makoak’

Leaves Fruit

+++

Tare Pakpooyab

Stalks Flower

Loog Kumyarntb

Fruit

Indian spinach Leaves

Flower

Indian trumpet flower

Young pod

ttt

Pineapple

Fruit

-

Papaya

Fruit

-

Sage brush Garland Lettuce Pakpaab Flower

Leaves Leaves Leaves Leaves ++t

+tt +++ ++

Morning glory

Leaves, Stem

+

Cabbage Cabbage Kanaeng Kaluumb Raddish Malta

Leaves (green) Leaves (purple) Leaves Root” -

+++ ++

Benincasa hispidu Thunb.a Citrullus lunufus Thunb.a Coccinia indica Cucurbitu maxima

White gourd Watermelon Tumleongb

Fruit Fruit Fruit

-

Duch. ex. Lam

Winter squash Angled luffa Chayote Buab ngoob Bitter cucumber

Leaves Fruit Fruit Fruit Leaves

++ + t

Acenthaceae A.systasielh

nusiutu

Agaricaceae Berk (Fr.) Singer

Lentinus praergidus Pfeumtus sajor-caju

Alizoaceae Glinus cq7posit~foliius Amaranthaceae Amuranthus graciiis Desf. Anacardiaceae Mungiferu,foetida Lour. Spondius pinnata Kurz Araceae Colocusiu esculentu Schotta Lusia spinma Thw. Asclepiadaceae Atherolepis pierrei Cost. var. Glabra kerr Basellaceac Busellu

rubm

L.

+

Bingnoniaceae Orwylum

indicum

Vent.

Bromeliaceae Anunas comosus c L.” Caricaceae Carica pupaya L.”

Compositae Artemisiu luct@~ra var. gennina Chrywnfhemum coronurium L.a Lactuca sutiva L.” Pluchea euputorioldes

Convolvulaceae lpomeu purpureu

L.

Cruciferae Brassica

oleruceu

L.

var. capitata” var. capitata” var. capitata” Ruphanus

sativus

L.

Cucurbitaceae

Lgflir acutangula L. Secium edulr SW. Tricisunthus anguina L.e Momnrdica c.haruntia L.a

tt+

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Table 1 (continued) Family/species

Common name

Part tested

Mara-thee-nokb Mara-cheenb Gnetaceae Gnetum gnemon L. Gramineae Cymbopogon citrutus Stapt.e Zea mays L.” Petercom Guttiferae Gurciniu utroviridis Gurciniu co~u Roxb. Gurciniu schmburgkiuna Labiatae Menthu cordifoliu Ocimum basilicurn L.*se Ocimum curturn Sims Ocimum grutissimun L. Lauraceae Perseu americana MilLa Leguminosae Cussia siumia Lam.

Fruit Fruit

+ -

Spinach joint fir

Leaves

+++

Lemon grass Maize Fruit

Leaves Fruit (boiled)

+++

Somkaekb Paksompongb Ma doneb

Dried fruit Leaves Fruit

+ +++ +

Saranaeb Common basila Hairy basil Tree basil

Leaves Leaves Leaves Leaves

++

Avocado

Fruit

Bombay blackwood Leave8 White popinac Nitta tree Common bean Garden bean Madras thorn Loogneangb Indies goa bean Ma paepb Tamarind Fruit (ripe) Leaves Swamp pea Flower SesbaniaC

Flower + Fruit Seeds Seeds Seeds Fruit Seeds Fruit Pod Fruit (unripe) +

++

Leaves ++ Flower

+++

Levant garlic Eschalot Onion Leek Chinese chive Asparagus

Bulb Bulb Bulb Leaves Stem, Flower Stem

Okura Roselle

Fruit Flower

+ -

Water clover

Leaves

+++

Doakkruangb

Flower

+++

Prayongb

Leaves

-H-f

Leucuenu leucocephalu De wit Purkiu speciosa Hassk. Phaseolus vulgaris SL.a Pisum sutivum L.a Pithecellobium dulce Benth Pithecellobium jiringu Psophocurpus tetragonolobus DC. Rhynchosiu bructeuta Low. Tumarindus indica L.

Sesbunia grund$oru

Desv.

Sesbuniu juvunicu Liliaceae Allium ampeloprasum L. Allium usculonicum L.a Allium cepu L.a Allium porrum L.a Ailium tuberosum Rottl. ex Spreng Aspuragus officinufis L.a Malvaceae Abelmoschus esculentus Moencha Hibiscus subduriffa I,. Marsileaceae Mursileu crenatu Presl Melastomataceae Diplectriu burbata Meliaceae Agiaiu odorutu Lour.d

Activity

+++ +-k-k +++

A. Murakami et al. I Cancer Letters 95 (1995) 139-146

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Table 1 (continued) Family/species Awdirachta

indica Juss.

Common name

Part tested

Neem tree (fresh) (boiled)

Inflorescence ++ +++

Jackfruit Fig Yuak kuayb

Fruit Leaves Stem

+ ++

Hindu lotus

Nuts

-

Water lily

Stem

Betelnut palm Dreid seeds

Freshseeds ++

Pandanwangi

Leaves

Pepper Pepper Betel pepper

Fruit Leaves Leaves

Shiitake (mushroom)

Whole

Knoyweed

Leaves

Indian mulberry

Leaves

Bitter orange Seeds Peal Ma kwenb

Leaves

+++

+++ Dried fruit

+++

Baikuntongb

Leaves

+++

Pakkayaengb

Leaves

Chilli pepper Chilli pepper Bell pepper Tomato Minitomato Egg plant Egg plant Egg plant Egg plant Egg plant Egg plant Potato

Fruit Fruit Fruit Fruit Fruit Fruit Fruit Pruit Fruit Fruit Fruit Root

Water chestnutC

Seeds

Celery

Leaves

Activity

Moraceae Artocarpus heterophyllus Lam. Ficus lacer Buch. Muss sp.

Nelumbonaceae Nelumbo nuc~era Gaertn. Nymphaeaceae Nymphaea lotus L.. var. pubescenshook. f. & th. Palmae Areca catechu L. Pandanaceae Pandanus odorus Ridl.

Piperaceae Piper n&rum L.a Piper sarmentosum Roxb. Piper betel L.

Pleutotaceae Lentinus edodes

Polygonaceae Polygonum odoratum Lour.

Rubiaceae Morinda citrifolia’:

Rutaceae Citrus hystrix D.C.

Zanthoxylum limonella

Saruraceae Houttuynia cordata Thunb.

Scrophulariaceae Limnophila aromatica Merrill

Solanaceae Cupsicum annuum L.a*e(green) Capsicum annuum L.‘(red) Capsicum grossum Sendt.a Lycopersicon esculentum MilLa Lycopersicon esculentum Milla Solanum melongena L. Solanum sp. (purple, large) Solanum sp. (purple, slender) Solarium stramonifolium Solanum torvum SW. Solanum trilobatum L. Solanum tuberosum L.a

Trapaceae Trapa bicornus Osbeck

Umbelliferae Apium graveolens var. Dulce Pers.L.a

+++

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Table 1 (conrinued) Family/species Centella asiutica Urban Coriandrum sativum L. Coriumdrum sp. Duucus curotu L.” Duucus carotu L.a Erygium foetidum L. Truchyspermum roxburghianum

Craib Zingiberaceae Boesenbergia

Part tested

Activity

Indian pennywort Coriander Pakshelaosb carrot Minicarrot Pakshe farang b

Leaves Leaves Leaves Root Root Leaves

++-I+++ ++

Hoomyaeb

Leaves

+++

Kra chaib Great galangal Ginger (immature) Ginger (matured)

Root Rhizome Rhizome -I-+ Rhizome +++

+++ +++

+++

pundurutu

Home .!LUQUUS gulunga Zingiber oficinule Ziqiber

Common name

@cinale

Swartze

Roscoe” Roscoea

a Occurring also in Japan. b Thai name. ’ Name of plant genus. d Used only for local medicine. e Previously reported in Ref. [4].

Rutaceae and Zingiberaceae were promising sources for effective anti-tumor promoters. The plants in Labiatae (one with ‘++’ and three with ‘+++’ of four species examined) and Piperaceae (three with ‘+++’ of 3 species examined) are additionally pointed out to presumably contain potent anti-tumor promoters. The plants listed in Table 1 include some species that are widely cultivated and consumed in the world.

Japan [4] (random setection, W133) m++t

Thaiand (random selection, Il=122) m+t

m+

mahd

We classified the plants into two groups to compare the inhibitory activity; (A) edible plant species occurring also in Japan (marked as ‘a’ in Table l), and (B) those mostly found in Thailand or other southeast Asian countries. The numbers of species in groups (A) and (B) are 34 and 88, respectively, and their activityexhibiting patterns are shown in Fig. 2. While the ratio of activity-exhibiting plants (IE 2 30%) of group (B) was 69%, that of group (A) was limited by 35%. The pattern in group (A) was rather closer to that obtained

[lo]

(deelgneted eeleclion, ll=40) Group

(A) (n=34)

Group(B)

(n=88)

o-

Fig. 1. Proportions of inhibitory activities toward EBV activation of the extracts from Japanese (left), Thai (middle) common edible plants, and edible Thai plants with medicinal effects (right). (EBV was activated by sodium n-butyrate (440yg/ml) and HPA (40 “g/ml). Each test sample was added at a concentration of 2OOpg/mI. Inhibitory effects (IE) were divided as follows: +++, IE t 70%; ++, 70% > IE 2 50%; +, 50% > IE 2 30; -,30% > IE.

Fig. 2. Comparison of the activity-exhibiting pattern of Thai edible plants which also occur in Japan (group (A), left) and mostly found in Thailand or other southeast countries (group (B), right). Experimental conditions and activity classification are the same as those in Fig. 1.

A. Murakami et al. I Cancer Letters 95 (1995) 139-146 Table 2 Edible plantsin Thailandand Japanshowingmarked in anti-tumorpromotingactivity in vitro

Plant

Thailand

Apium graveolens (celery) Brassica oleracea (Chinesecabbage) Colocasia esculenta (tare) Ocimum basilicurn (basil) Persesa americana (avocado) Raphanus sativus (radish) Zingiber ojfkinale (ginger)

+++

differences Japana

+++ +++

+++ -u-t

++ ++

a SeeRef.[4]. from the common edible plants of Japan (Figs. I, 2). Therefore, edible plants mostly found in Thailand or other southeast Asian countries would appear to have greater potential in terms of cancer preventive effects. Furthermore, it is also worth noting that most of the strongly active plants in group (A) can be characterized in terms of flavor and/or condiments, i.e. Chrysunthemum coronarium (garland chrysanthemum), Momordica charantia (bitter cucumber), Ocimum basilicum (basil), Piper nigrum (pepper), Apium grareolens (celery), and Zingiber oficinale (ginger) (Fig. 2). The criteria for plant selection, directed to their flavor or taste, would be satisfactory to search for promising cancer preventive agents. In the present tests, 20 species were the same as those tested in common Japanese edible plants 141. Although the experiments were carried out by the same conditions in both tests, the activities of 9 out of these 20 species were ranked differently. Seven species, whose ranks are remarkably different between the two tests, are listed in Table 2. One of the reasons for such activity variation may come from the difference in the cultivar. More supportable causes may be harvest time, preservation or cultivation conditions such as temperature, humidity, or light. Concerning this discussion, it is worth noting that matured ginger possess strong activity (IE > 99%), while immature Zingiber oficinale (ginger) exhibited moderate activity (IE! = 50%). A similar tendency was shown in Areca catechu (betelnut palm), in which the fresh seed was inactive (0.1% > IE), while the dried one was evidently active (IE = 62%). These results suggest that the chemical characteristics of the active constituents or their contents could be changed by some environmental factors.

14.5

Hence, plant breeding, taking the content of such active constituents into account, is desired in order to obtain edible plants highly effective for cancer prevention. Most naturally occurring cancer preventive agents have thus far been derived from vegetables or fruits widely consumed throughout the world. Southeast Asia can be characterized as a point of great floral diversity. People in this area must daily ingest a variety of vegetables. Recent epidemiological studies revealed that the age-standardized rate of cancer mortality in Thailand is less than half that of Japan or the USA [ 1,141, even though some people in Southeast Asia, especially in the provinces, are thought to be still suffering from exposure to carcinogenic mycotoxins such as aflatoxin in crops [ 161. The traditional style of food in ‘Thailand may contribute to the reduction of cancer incidence to a certain degree. To demonstrate the beneficial effects of vegetables in this area, identification of the active constituents, in accordance with detailed epidemiological studies on diet and cancer, should be indispensable.

Acknowledgements This study was supported partly by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan, and the Japan Society for the Promotion of Science (A.M.) and by subsidies from Takeda Food Products, Ltd., and also the Yamaz.aki Foundation. We also thank Mr. Charae Sadakorn of the Ministry of Agriculture and Cooperatives of Thailand for plant identification.

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