Harukuni Tokuda

Production of bioactive triterpenes by Eriobotrya japonica calli

Callus tissue cultures induced from an axenic leaf of Eriobotrya japonica (Rosaceae) produced triterpenes in large amounts (ca. 50 mg/g dry wt). Nine triterpenes were characterized as ursolic acid, oleanolic acid, 2alpha-hydoxyursolic acid, maslinic acid, tormentic acid, 2alpha, 19alpha-dihydroxy-3-oxo-urs-12-en-28-oic acid, hyptadienic acid and a mixture of 3-O-cis-p-coumaroyltormentic acid and 3-O-trans-p-coumaroyltormentic acid. The triterpene composition in the callus tissues was noticeably different from that in intact leaves. The contents of tormentic acid with antidiabetic action, and 2alpha, 19alpha-dihydroxy-3-oxo-urs-12-en-28-oic acid with anti-HIV activity, were much larger than those in the intact leaves. All of the triterpenes isolated from the callus tissues showed an inhibitory effect comparable to (-)-epigallocatechin gallate (EGCG) of green tea on the activation of Epstein-Barr virus early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). 2alpha, 19alpha-Dihydroxy-3-oxo-urs-12-en-28-oic acid was the most potent inhibitor among them and caused a significant delay of two-stage carcinogenesis on mouse skin.

Cancer prevention by carotenoids

Various natural carotenoids seem to be valuable for cancer prevention, and these carotenoids may be more suitable in combinational use, rather than in single use. In fact, we have proven that combinational use of natural carotenoids resulted in significant suppression of liver cancer. Patients of viral hepatitis with cirrhosis were administered with beta-cryptoxanthin-enriched Mandarin orange juice, in addition to capsules of carotenoids mixture. Cumulative incidence of hepatocellular carcinoma development was compared with that in the group treated with carotenoids mixture capsules alone, or in the group without treatment (control group). In the data analysis at year 2.5, cumulative incidence of liver cancer in beta-cryptoxanthin-enriched orange juice with carotenoids mixture capsules-treated group was lower than that in the control group (p=0.05). Cumulative incidence of liver cancer in the group treated with carotenoids mixture capsules alone was also lower than that in the control group, but not statistically significant.

Chemoprevention of lung and skin cancer by Beta vulgaris (beet) root extract

The in vitro inhibitory effect of Beta vulgaris (beet) root extract on Epstein-Barr virus early antigen (EBV-EA) induction using Raji cells revealed a high order of activity compared to capsanthin, cranberry, red onion skin and short and long red bell peppers. An in vivo anti-tumor promoting activity evaluation against the mice skin and lung bioassays also revealed a significant tumor inhibitory effect. The combined findings suggest that beetroot ingestion can be one of the useful means to prevent cancer.

Chemoprevention of DMBA-induced UV-B promoted, NOR-1-induced TPA promoted skin carcinogenesis, and DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot.

Our previous studies identified the extract of Beta vulgaris (beetroot), commercially also known as betanin, as a potent cancer chemopreventive agent in both in vitro Epstein-Barr early antigen activation assay and in an in vivo two-stage mouse lung and skin carcinogenesis. To explore this issue further, we have now investigated its cancer chemopreventive potentials in three different chemical carcinogen initiation-promotion experimental tumor models in mice. Following tumor initiation with 390 nmol of 7,12-dimethylbenz(a)anthracene (DMBA) in 100 microl of acetone, the mouse skin tumor promotion with 3430 J/m(2) of ultraviolet light-B (UV-B) as well as splenomegaly was significantly inhibited by oral administration of 0.0025% betanin. At the same dose, betanin also afforded significant protection in the mouse skin cancer model following the topical application of 390 nmol of (+/-)-(E)-4-methyl-2-[(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) in 100 microl of acetone and promoted by topical administration of 1.7 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA). In the two-stage model of hepatocarcinogenesis in mice with N-nitrosodiethylamine (DEN, 30 mg/kg) as the initiator and phenobarbital as the promoter, oral administration of 0.0025% betanin also showed a very significant inhibition of both the incidence and multiplicity of the liver tumors. These findings along with our initial reports suggest that betanin which is a regularly consumed natural product colorant is an effective cancer chemopreventive agent in mice. The most interesting observation is that the cancer chemopreventive effect was exhibited at a very low dose used in the study and thus indicating that beetroot warrants more attention for possible human applications in the control of malignancy.

Constituents of Compositae plants III. Anti-tumor promoting effects and cytotoxic activity against human cancer cell lines of triterpene diols and triols from edible chrysanthemum flowers.

Fifteen pentacyclic triterpene diols and triols, consisting of: six taraxastanes, faradiol (1), heliantriol B0 (2), heliantriol C (3), 22alpha-methoxyfaradiol (4), arnidiol (5), and faradiol alpha-epoxide (6); five oleananes, maniladiol (7), erythrodiol (8), longispinogenin (9), coflodiol (10), and heliantriol A(1) (11); two ursanes, brein (12) and uvaol (13); and two lupanes, calenduladiol (14) and heliantriol B2 (15), isolated from the non-saponifiable lipid fraction of the edible flower extract of chrysanthemum (Chrysanthemum morifolium) were evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, in Raji cells as a primary screening test for anti-tumor-promoters. All of the compounds tested showed inhibitory effects against EBV-EA activation with potencies either comparable with or stronger than that of glycyrrhetic acid, a known natural anti-tumor-promoter. Evaluation of the cytotoxic activity of six compounds, 1-3 and 5-7, against human cancer cell lines revealed that compound 5 possesses a wide range of cytotoxicity, with GI50 values (concentration that yields 50% growth) of mostly less than 6 microM.

Anti-tumor-promoting activity of lignans from the aerial part of Saussurea medusa.

In the course of our continuing search for novel cancer chemopreventive agents from natural sources, several kinds of Compositae plants were screened. Consequently, the lignans, arctiin (ARC) and arctigenin (ARC-G), were obtained from the aerial part of Saussurea medusaas active constituents. These compounds exhibited the remarkable anti-tumor-promoting effect on two-stage carcinogenesis test of mouse skin tumors induced by 7, 12-dimethylbenz[a]anthracene as an initiator and 12-O-tetradecanoyl phorbol-13-acetate as a promoter by both topical application and oral administration. Furthermore, ARC-G exhibited potent anti-tumor-promoting activity on two-stage carcinogenesis test of mouse pulmonary tumors induced by 4-nitroquinoline-N-oxide as an initiator and glycerol as a promoter.

Cancer chemopreventive agents, 4-phenylcoumarins from Calophyllum inophyllum

In a search for anti-tumor-promoting agents, we carried out a primary screening of ten 4-phenylcoumarins isolated from Calophyllum inophyllum L. (Guttiferae), by examining their possible inhibitory effects on Epstein--Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. All of the compounds tested in this study showed inhibitory activity against EBV, without showing any cytotoxicity. Calocoumarin-A (5) showed more potent activity than any of the other compounds tested. Furthermore, calocoumarin-A (5) exhibited a marked inhibitory effect on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. The results of the present investigation indicate that some of these 4-phenylcoumarins might be valuable as potential cancer chemopreventive agents (anti-tumor-promoters).

Chalcones, coumarins, and flavanones from the exudate of Angelica keiskei and their chemopreventive effects

From an ethyl acetate-soluble fraction of the exudate obtained from the stems of Angelica keiskei (Umbelliferae), 17 compounds, viz. five chalcones (1-5), seven coumarins (6-12), three flavanones (13-15), one diacetylene (16), and one 5-alkylresorcinol (17), were isolated. These compounds were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, which is known to be a primary screening test for antitumor-promoters. With the exception of three compounds (10, 16, and 17), all other compounds tested showed potent inhibitory effects on EBV-EA induction (92-100% inhibition at 1x10(3)mol ratio/TPA). In addition, upon evaluation of these compounds for the inhibitory effects against activation of (+/-)-(E)-methyl-2-[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor, as a primary screening test for antitumor-initiators, two chalcones (2 and 3) and six coumarins (6-11) exhibited potent inhibitory effects.

Inhibitory effects of dihydroagarofuran sesquiterpenes on Epstein-Barr virus activation

To search for possible antitumor promoters, we carried out a primary screening of thirty-seven dihydroagarofuran sesquiterpenes from Tripterygium wilfordii Hook fil. var. regelii Makino and Euonymus sieboldianus Blume, using their possible inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation which is induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Some of these sesquiterpenes, triptofordin F-2 (Takaishi et al., 1988), 1,2,6,8,15-pentaacetoxy-9-benzoyloxy-4-hydroxy-beta-dihydroagarofuran and triptogelin A-1 (Takaishi et al., 1990) were observed to significantly inhibit the EBV-EA activation at low doses. Based on the results, the structural requirements for the activity of these compounds were discussed [corrected].

Cancer chemopreventive activity of Achyranthes aspera leaves on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis.

Achyranthes aspera leaves have been assessed for chemopreventive activity. The MeOH extract, alkaloid, non-alkaloid and saponin fractions exhibited significant inhibitory effects (concentration 100 microg) on the Epstein-Barr virus early antigen activation induced by the tumor promotor 12-O-tetradecanoylphorbol-13-acetate in Raji cells. In this in vitro assay the non-alkaloid fraction containing mainly non-polar compounds showed the most significant inhibitory activity (96.9%; 60% viability). In the in vivo two-stage mouse skin carcinogenesis test the total methanolic extract possessed a pronounced anticarcinogenic effect (76%). The present study suggests that A. aspera leaf extract and the non-alkaloid fraction are valuable antitumor promotors in carcinogenesis.