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An antitumor promoter from Moringa oleifera Lam.
Mutation Research 440 Ž1999. 181–188
An antitumor promoter from Moringa oleifera Lam. Amelia P. Guevara a,) , Carolyn Vargas a , Hiromu Sakurai b, Yasuhiro Fujiwara b, Keiji Hashimoto b, Takashi Maoka b, Mutzuo Kozuka c , Yoshohiro Ito c , Harukuni Tokuda d , Hoyoku Nishino d a
d
Institute of Chemistry, College of Science, UniÕersity of the Philippines, Diliman, Quezon City, Philippines b Kyoto Pharmaceutical UniÕersity, Misasagi, Yamashina-ku, Kyoto 607, Japan c Research Institute for Production DeÕelopment, Kyoto 606, Japan Department of Biochemistry, Kyoto Prefectural UniÕersity of Medicine, Kawaramachi-hirokoji, Kamigyo-ku, Kyoto 602, Japan Received 11 November 1998; received in revised form 25 January 1999; accepted 2 February 1999
Abstract In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-Ž a-L-rhamnosyloxy.benzyl carbamate Ž1. together with seven known compounds, 4Ž a-L-rhamnosyloxy.-benzyl isothiocyanate Ž2., niazimicin Ž3., niazirin Ž4., b-sitosterol Ž5., glycerol-1-Ž9-octadecanoate. Ž6., 3-O-Ž6X-O-oleoyl-b-D-glucopyranosyl.-b-sitosterol Ž7., and b-sitosterol-3O-b-D-glucopyranoside Ž8.. Four of the isolates Ž2, 3, 7, and 8., which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen ŽEBV-EA. activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate ŽTPA.. All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin Ž3. was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenzŽ a.anthracene ŽDMBA. as initiator and TPA as tumor promoter. From these results, niazimicin Ž3. is proposed to be a potent chemo-preventive agent in chemical carcinogenesis. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Antitumor promoter; Two-stage carcinogenesis; Epstein–Barr virus-early antigen activation; O-ethyl-4-Ž a-L-rhamnosyloxy.benzyl carbamate; Moringa oleifera Lam.
1. Introduction Moringa oleifera Lam., a member of family Moringacae, is commonly known as horse radish. The tree was introduced to the Philippines from tropical Asia or Malaya in the prehistoric period w1x. )
Corresponding author.
The young leaves, flowers, and green pods are common vegetables in the Filipino diet. The medicinal value of the seeds and the different parts of the plant have long been recognized in folklore medicine w1–7x. Because of the many traditional uses of M. oleifera, it is not surprising that several investigations have been conducted to isolate bioactive compounds from various parts of the plant. Nitrile, mus-
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 0 2 5 - X
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tard oil glycosides and thiocarbamate glycosides have been isolated and found to be responsible for the hypotensive principles of the leaves w3–5x while 4-Ž a-L-rhamnosyloxy.benzyl thiocyanate Ž2. had been found to be responsible for the antimicrobial activity of the roots w6x and seeds w7x. A mutagen, 4-Ž a-L-rhamnosyloxy.benzyl acetonitrile Žniazirin 4., was isolated from the roasted seeds w8x. As part of our research program on the isolation of bioactive compounds from Philippine plants, the seeds of M. oleifera Lam. were examined for antigenotoxic and anti-inflammatory activities. In this
paper, we report the results of our investigations on the effect of our isolates on the short-term in vitro EBV-EA activation assay for antitumor promoters and present evidence on the role of niazimicin as inhibitor against the two-stage mouse tumorigenesis. Although of already known structure, it is the first time that the antitumor promoting activity of niazimicin is reported. In our study, we isolated a new carbamate rhamnoside Ž1. together with seven known compounds, 4Ž a-L-rhamnosyloxy.benzyl isothiocyanate Ž2. w6x, niazimicin Ž3. w3x, niazirin Ž4. w3,4x, b-sitosterol Ž5.,
Scheme 1.
A.P. GueÕara et al.r Mutation Research 440 (1999) 181–188
glycerol-1-Ž9-octadecanoate. Ž6. w9x, 3-O-Ž6X-Ooleoyl-b-D-glucopyranosyl.-b-sitosterol Ž7. w10x, and b-sitosterol-3-O-b-D-glucopyranoside Ž8. w11x. The structure of the new compound was determined from spectral data to be O-ethyl-4-Ž a-L-rhamnosyloxy. benzyl carbamate Ž1.. A primary screening for antitumor promoting activity was carried out on the ethanol extract and four of the isolates Ž2, 3, 7, and 8., which were obtained in relatively good yields. The screening process employed the in vitro synergistic assay, the Epstein–Barr virus-early antigen ŽEBV-EA. activation method w12–14x wherein the inhibitory effect of the isolates on the EBV-EA activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoylphorbol-13acetate ŽTPA., was observed. Since many compounds that inhibit in vitro EBV-EA activation have been found to be antitumor promoters in vivo w12x, we eventually tested the in vivo antitumor promoting activity of isolate 3 Žniazimicin. with the two-stage chemical carcinogenesis on mice skin. Niazimicin Ž3. was chosen for the in vivo assay because the structural type of this compound is not yet known as an antitumor promoter ŽScheme 1..
2. Materials and methods 2.1. General experimental procedures 1
H-NMR and 13 C spectra were recorded on a Varian XL-300 spectrometer in CDCl 3 using TMS as an internal standard. EI and FAB mass spectra were measured on a JEOL JMS-SX 102 A QQ mass spectrometer. Silica gel ŽMerck G60, 230 mesh. was used for rapid column flash chromatography w15x. Columns of 2–3 in. in height and of varying diameters depending on the amounts of extracts Žranging from a 2-in. diameter for about 12 g crude extracts to 1 in. diameter for a few grams of column fractions. were used. Preparative high-performance liquid chromatography ŽPrep. HPLC. was performed using Shimadzu LC 6AD liquid chromatograph and Shimadzu SPD-10A UV–VIS detector Ž250 nm. with a Shimpak PreSil column ŽShimadzu. Ž250 mm = 20 mm, i.d. 5 mm. and hexaner2-propanol Ž6:4. as mobile phase. Pre-coated silica gel plates ŽMerck, 0.25 mm. were used for thin layer chromatography ŽTLC..
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Compounds were visualized by spraying with vanillin-concentrated H 2 SO4 solution followed by heating. 2.2. Plant material, extraction and isolation of compounds 1–8 The seeds of M. oleifera Lam. were collected from Abucay, Bataan, Philippines in April and May. Decoated seeds were ground and soaked in distilled ethanol for a few days with occasional stirring. The ethanol extract was partitioned between CCl 4 and water and thence, the CCl 4 fraction was subjected to repeated and sequential flash column chromatography on silica gel using vacuum elution. A total of 12 g of the extract were loaded into 2–3 in. height= 2 in. diameter silica gel columns and eluted with hexane-ethyl acetate as the solvent system. A combination of gradient and isocratic elution was employed, starting with hexane and increasing the polarity of the eluting solvent with ethyl acetate by 10% increment until pure isolates were obtained ŽFig. 1.. The progress of separation was monitored by TLC. Successive purification of fractions D 21 and D 22 by high-performance liquid chromatography on silica gel afforded four isolates 1 Ž5 mg., 2 Ž15 mg., 3 Ž20 mg. and 4 Ž10 mg.. Isolates 1, 2, 3, and 4 were identified as O-ethyl-4-w a-L-rhamnosyloxy.benzylxcarbamate, 4-Ž a-L-rhamnosyloxy.benzyl isothiocyanate, niazimicin, and niazirin, respectively, by IR, NMR and MS data. Isolates 5 Ž50 mg., 6 Ž65 mg., 7 Ž30 mg., and 8 Ž10 mg. were also obtained from fractions B, C, D 1 and E, respectively ŽFig. 1.. These isolates were identified as b-sitosterol Ž5., glycerol-1-Ž9-octadecanoate. Ž6. w9x, 3-O-Ž6X-O-oleoyl-b-D-glucopyranosyl.-b-sitosterol Ž7. w10x, and b-sitosterol-3-O-glucopyranoside Ž8. w11x by NMR and MS data. Isolates 5, 7, and 8 each contained about 10% of the stigmasterol analog. 2.3. In Õitro bioassay for antitumor-promoting actiÕity: the Epstein–Barr Virus-Early Antigen (EBV-EA) actiÕation 2.3.1. Chemicals The cell culture reagents, n-butyric acid and Triton X100 were purchased from Nacalai Tesque, ŽKyoto, Japan.. TPA, 7,12-dimethylbenzw axanthra-
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Fig. 1.
cene ŽDMBA., and ribonuclease ŽRNase. were obtained from Sigma ŽSt. Louis, MO, USA.. EBV-EA positive serum from a patient with nasopharyngeal carcinoma ŽNPC. was a gift from Prof. H. Hattori, Department of Otorhinolaryngology, Kobe University and used for the immunofluorescence test. 2.3.2. Cells The EBV genome carrying lymphoblastoid cells ŽRaji cells derived from Burkitt’s lymphoma. were cultured in RPMI-1640 medium ŽNissui, Tokyo,
Japan. under the conditions as described previously w12–14x. Spontaneous activation of EBV-EA in the subline Raji cells was less than 0.1%. 2.3.3. Experimental procedure The indicator cells ŽRaji, 1 = 10 6rml. were incubated at 378C for 48 h in 1 ml of medium containing 4 mM n-butyric acid Žinducer., TPA Ž32 pmol. solution in 2 ml dimethylsulfoxide ŽDMSO.x, and various amounts of the test compounds dissolved in 5 ml of DMSO w12x. Smears were made from the cell
A.P. GueÕara et al.r Mutation Research 440 (1999) 181–188
suspension. The EBV-EA inducing cells were stained with high titer EBV-EA positive sera from NPC patients and detected by an indirect immunofluorescence technique w16x. In each assay, at least 500 cells were counted and the number of stained cells Žpositive cells. among them was recorded. Triplicate assays were performed for each data point. The EBV-EA inhibitory activity of the test compound was expressed by making a comparison with that of the positive control experiment which was carried out with n-butyric acid Ž4 mM. plus TPA Ž32 nM.. In the experiments, the EBV-EA induction was ordinarily at around 35% and these values were taken as the positive control Ž100%.. Inhibition of EBV-EA activation Ž Percentage inhibition s 100% y percentage to positive control. was taken as an indication for potential antitumor promoting activity. An amount of 4 mM n-butyric acid alone induced 0.1% EA-positive cells. Less than 20 ml of DMSO per milliliter of medium did not show toxicity against our assay system. The viability of treated Raji cells was assayed by the trypan-blue staining method. A high viability of the Raji cells is necessary for the in vitro assay. 2.4. In ÕiÕo bioassay for antitumor-promoting actiÕity: two-stage carcinogenesis test on mouse skin papillomas 2.4.1. Animals Specific pathogen-free female ICR mice Ž6 weeks old. were obtained from Nippon SLC ŽHamamatsu, Japan.. 2.4.2. Experimental procedure Specific pathogen-free female ICR mice Ž6 weeks old. were housed, five per polycarbonate cage, in a temperature-controlled room at 24 " 28C and given food and water ad libitum. The backs of mice were shaved with surgical clippers and the mice were topically treated with DMBA Ž100 mg, 390 nmol. in acetone Ž0.1 ml.. One week after the initiation, carcinogenic growth was promoted twice a week by the application of TPA Ž1 mg, 1.7 nmol. in acetone Ž0.1 ml. on the skin. The animals were then divided into two experimental groups, of 15 mice per group. Group I received TPA treatment alone while group II received topical applications of compound 3
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Žniazimicin. Ž85 nmol. in acetone Ž0.1 ml. at 1 h before each TPA treatment. The incidence and number of papillomas were observed weekly for 20 weeks, as described previously w17,18x.
3. Results and discussion 3.1. In Õitro screening for potential antitumor promoting actiÕity Various assay methods are currently being used to detect tumor promoters as defined by the two-step hypothesis of skin carcinogenesis. Such methods include the classical protocol of skin painting in mice, activation of protein kinase C, induction of ornithine decarboxylase w22x and others w23x. From our previous studies on the induction of EBV-genome-carrying human lymphoblastoid cells, it was reported that TPA and other well-known tumor promoters are all efficient EBV-inducers w24x. This led to our assumption that the EBV-inducing system could be utilized as an in vitro assay for the detection of such tumor promoter substances. Thus, the ethanol extract and isolates 2, 3, 7, and 8 were tested for their potential antitumor promoting activity by observing their inhibitory effect on the induction of EBV-EA activation. Their inhibitory effects at various concentrations, the corresponding viability of the Raji cells and the calculated IC 50 for the pure isolates are summarized in Tables 1 and 2. At a concentration of 100 mgrml, the ethanol extract inhibited the induction of EBV-EA activation by 73%. However, upon dilution by 100-fold, such inhibitory effect was reduced to 11%. No activity was observed at 0.1 mgrml. These inhibitory effects
Table 1 Inhibitory effect of ethanol extract of seeds from M. oleifera Lam. on the induction of EBV-EA activation Concentration Žmgrml.
Percentage inhibition of induction of EBV-EA activation Ž% cell viability a .
100 10 1 0.1
72.9 Ž60. 40.4 Ž )80. 11.2 Ž )80. 0 Ž )80.
a
Values in parentheses are the percentage viability of Raji cells.
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Table 2 Inhibitory effects of isolates from M. oleifera Lam. on induction of EBV-EA activation Isolates
4-Ž a-L-rhamnosyloxy.benzyl isothiocyanate Ž2. Niazimicin Ž3. X 3-O-Ž6 -O-oleoyl-b-D-glucopyranosyl.-b-sitosterol Ž7. b-sitosterol-3-O-b-D-glucopyranoside Ž8. a b
Percentage inhibition Ž% cell viability. at different concentrationsa 1000 mol ratio
500 mol ratio
100 mol ratio
10 mol ratio
100 Ž60. b 100 Ž60. 100 Ž60. 100 Ž60.
83.1 Ž) 80. 79.3 Ž) 80. 65.3 Ž) 80. 93.5 Ž) 80.
52.6 Ž) 80. 47.2 Ž) 80. 15.2 Ž) 80. 62.7 Ž) 80.
20.3 Ž) 80. 18.1 Ž) 80. 0.0 Ž) 80. 11.4 Ž) 80.
IC 50 Žmgrml. 32.7 35.3 70.4 27.9
Mol RatiorTPA Ž20 ng s 32 pmolrml.. Values in parentheses are the percentage viability of Raji cells.
suggested that the ethanol extract had moderate antitumor promoting activity. All the isolates tested showed inhibitory effects on the induction of EBV-EA activation without significant cytotoxicity on Raji cells. Of the tested compounds, the isothiocyanate rhamnoside Ž2., niazimicin Ž3., and sitosterol glucoside Ž8. showed potent inhibitory activity in which about 50%, 80–90%, and 100% inhibitions of activation were observed at 100, 500, and 1000-fold mol ratio to 32 pmol of TPA, respectively, and preserved the high viability of Raji cells even at the highest concentration Ž1000 mol ratiorTPA.. On the other hand, steroid 7 was considered moderately active with about 15%, 65% and 100% inhibition at 100, 500, and 1000 mol ratiorTPA, respectively. These inhibitory activities for isolates 2, 3, 7, and 8 were higher than those of previously reported potent antitumor promoters like glycyrrhetic acid w12,19x, b-carotene w13x, and euglobals w12x. In our previous work, an intractable mixture of acylglucosylsterols, 3-O-w6X-O-palmitoyl-b-D-glucosylx-b stigmasta-5, 25-diene and its stearoyl derivative, isolated from the fruits of Momordica charantia Linn. w20x, was found to reduce the number of micronucleated polychromatic erythrocytes induced by mitomycin C, indicating the antimutagenic activity of the mixture of acylglucosylsterols. Considering the related structure of isolate 7, the antimutagenic activity of the acylglucosylsterol Ž7. isolated from M. oleifera may be worth testing, too. Also, in our previous work on the seeds of Leucaena leucocephala w21x, a mixture of 3-O-b-D-glucopyranosyl-b-sitosterol Ž8. and its stigmasterol analog was found to exhibit inhibitory activity on the EBV-EA activation as well as the antimutagenic
activity against mitomycin C on the micronucleus test. 3.2. In ÕiÕo two-stage carcinogenesis test on mouse skin Since the inhibitory effect on the induction of EBV-EA activation is well-correlated with antitumor-promoting activity in vivo w12x, the above results suggested that these test compounds, especially, compounds 2, 3, and 8 were potent antitumor promoters. Thus, on the basis of the results of the in vitro assay, the inhibitory effect of niazimicin Ž3. on the two-stage carcinogenesis on mouse skin using DMBA as an initiator and TPA as a promoter was examined. Although the EBV-EA inhibitory activity of 8 was higher than 2 and 3, and the activity of 2 was about the same as 3, niazimicin Ž3. was chosen for the in vivo assay because the structural type of this compound is not known as an antitumor promoter till now. Also, not enough of 2 and 8 were available for the in vivo assay at the time of investigation. Isolation of more samples of isolates 2 and 8 is underway. The inhibitory activities on tumor promotion were evaluated by the ratio Ž%. of papilloma-bearing mice ŽFig. 2A. and the average numbers of papillomas per mouse ŽFig. 2B.. In the positive control, papillomas started to appear on the 6th week of promotion with 20% of the mice already bearing papillomas. By the 10th week, all control mice already bore papillomas with an average of 6.2 papillomas per mouse. After 20 weeks of promotion, the number of papillomas increased to an average of 9.5 per mouse. On the other hand, when niazimicin was applied before each TPA treat-
A.P. GueÕara et al.r Mutation Research 440 (1999) 181–188
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Fig. 2. All mice were initiated with DMBA Ž390 nmol. and promoted with 1.7 nmol of TPA given twice weekly starting 1 week after initiation. ŽA. Percentage of mice bearing papillomas. ŽB. Average number of papillomas per mouse. v, control TPA alone; `, TPA q 85 nmol of niazimicin. Each symbol represents the mean for 15 animals.
ment Žgroup II., the formation of papillomas in mouse skin was delayed from the 6th week to the 9th week and the incidence and the mean numbers of papillomas per mouse were reduced. Only 20% and 93.3% of mice bore papillomas at 10 to 20 weeks of promotion with 0.9 and 5.8 papillomas per mouse detected, respectively. Thus, niazimicin Ž3. exhibited 50% delay in the promotion of tumors and decreased the incidence of papilloma bearing mice by 80% and 17% at 10 and 20 weeks of promotion, respectively. An 85% and 39% reduction in the number of papillomas per mouse were also observed during the same period. Previous studies on glycyrrhetic acid w12x showed 46 and 93% of mice with papillomas at 10 and 20 weeks of promotion with 2 and 5.6 papillomas per mouse, respectively while for euglobal G1 , 40% and 70% of mice bore papillomas with an average of 2 and 4.5 papillomas per mouse were observed. Comparatively, these results indicate that the inhibitory activity of niazimicin Ž3. for the two-stage carcinogenesis after 10 weeks of promotion is very much higher than that of glycyrrhetic acid and euglobal G1 , both previously reported potent antitumor promoters. However, after 20 weeks of promotion, the activity of niazimicin was about the same as that of glycyrrhetic acid but a little less potent than euglobal G1 . The results obtained in the present study, thus, indicate that 4Ž a-L-rhamnosyloxy.-benzyl isothio-
cyanate Ž2., niazimicin Ž3., 3-O-Ž6X-O-oleoyl-b-Dglucopyranosyl.-b-sitosterol Ž7., and b-sitosterol-3O-b-D-glucopyranoside Ž8. inhibit EBV-EA induction and could be potential antitumor promoters. Furthermore, our data strongly suggest that niazimicin Ž3) is a potent antitumor promoter in chemical carcinogenesis. Its inhibitory mechanism on tumor promotion will be studied upon isolation of more pure samples.
Acknowledgements The extraction procedures were conducted at the Institute of Chemistry, University of the Philippines Diliman ŽUPD. through the funding support of the Natural Science Research Institute, College of Science, UPD. The antitumor bioassays were conducted at the Kyoto Prefectural University of Medicine while the structure elucidation studies were conducted at the Kyoto Pharmaceutical University. The technical assistance of Miss Kayoko Oda, Kyoto Pharmaceutical University, for obtaining the mass spectra of the compounds is acknowledged. This study was also supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare for the Second-term Comprehensive Ten-year Strategy for Cancer Control, Japan. APG also acknowledges UP, the Japan Society for the Promotion of Science, and the Philip-
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pine Department of Science and Technology for the research visit to the Kyoto Pharmaceutical University. w14x
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An antitumor promoter from Moringa oleifera Lam. Amelia P. Guevara a,) , Carolyn Vargas a , Hiromu Sakurai b, Yasuhiro Fujiwara b, Keiji Hashimoto b, Takashi Maoka b, Mutzuo Kozuka c , Yoshohiro Ito c , Harukuni Tokuda d , Hoyoku Nishino d a
d
Institute of Chemistry, College of Science, UniÕersity of the Philippines, Diliman, Quezon City, Philippines b Kyoto Pharmaceutical UniÕersity, Misasagi, Yamashina-ku, Kyoto 607, Japan c Research Institute for Production DeÕelopment, Kyoto 606, Japan Department of Biochemistry, Kyoto Prefectural UniÕersity of Medicine, Kawaramachi-hirokoji, Kamigyo-ku, Kyoto 602, Japan Received 11 November 1998; received in revised form 25 January 1999; accepted 2 February 1999
Abstract In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-Ž a-L-rhamnosyloxy.benzyl carbamate Ž1. together with seven known compounds, 4Ž a-L-rhamnosyloxy.-benzyl isothiocyanate Ž2., niazimicin Ž3., niazirin Ž4., b-sitosterol Ž5., glycerol-1-Ž9-octadecanoate. Ž6., 3-O-Ž6X-O-oleoyl-b-D-glucopyranosyl.-b-sitosterol Ž7., and b-sitosterol-3O-b-D-glucopyranoside Ž8.. Four of the isolates Ž2, 3, 7, and 8., which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen ŽEBV-EA. activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate ŽTPA.. All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin Ž3. was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenzŽ a.anthracene ŽDMBA. as initiator and TPA as tumor promoter. From these results, niazimicin Ž3. is proposed to be a potent chemo-preventive agent in chemical carcinogenesis. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Antitumor promoter; Two-stage carcinogenesis; Epstein–Barr virus-early antigen activation; O-ethyl-4-Ž a-L-rhamnosyloxy.benzyl carbamate; Moringa oleifera Lam.
1. Introduction Moringa oleifera Lam., a member of family Moringacae, is commonly known as horse radish. The tree was introduced to the Philippines from tropical Asia or Malaya in the prehistoric period w1x. )
Corresponding author.
The young leaves, flowers, and green pods are common vegetables in the Filipino diet. The medicinal value of the seeds and the different parts of the plant have long been recognized in folklore medicine w1–7x. Because of the many traditional uses of M. oleifera, it is not surprising that several investigations have been conducted to isolate bioactive compounds from various parts of the plant. Nitrile, mus-
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 0 2 5 - X
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tard oil glycosides and thiocarbamate glycosides have been isolated and found to be responsible for the hypotensive principles of the leaves w3–5x while 4-Ž a-L-rhamnosyloxy.benzyl thiocyanate Ž2. had been found to be responsible for the antimicrobial activity of the roots w6x and seeds w7x. A mutagen, 4-Ž a-L-rhamnosyloxy.benzyl acetonitrile Žniazirin 4., was isolated from the roasted seeds w8x. As part of our research program on the isolation of bioactive compounds from Philippine plants, the seeds of M. oleifera Lam. were examined for antigenotoxic and anti-inflammatory activities. In this
paper, we report the results of our investigations on the effect of our isolates on the short-term in vitro EBV-EA activation assay for antitumor promoters and present evidence on the role of niazimicin as inhibitor against the two-stage mouse tumorigenesis. Although of already known structure, it is the first time that the antitumor promoting activity of niazimicin is reported. In our study, we isolated a new carbamate rhamnoside Ž1. together with seven known compounds, 4Ž a-L-rhamnosyloxy.benzyl isothiocyanate Ž2. w6x, niazimicin Ž3. w3x, niazirin Ž4. w3,4x, b-sitosterol Ž5.,
Scheme 1.
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glycerol-1-Ž9-octadecanoate. Ž6. w9x, 3-O-Ž6X-Ooleoyl-b-D-glucopyranosyl.-b-sitosterol Ž7. w10x, and b-sitosterol-3-O-b-D-glucopyranoside Ž8. w11x. The structure of the new compound was determined from spectral data to be O-ethyl-4-Ž a-L-rhamnosyloxy. benzyl carbamate Ž1.. A primary screening for antitumor promoting activity was carried out on the ethanol extract and four of the isolates Ž2, 3, 7, and 8., which were obtained in relatively good yields. The screening process employed the in vitro synergistic assay, the Epstein–Barr virus-early antigen ŽEBV-EA. activation method w12–14x wherein the inhibitory effect of the isolates on the EBV-EA activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoylphorbol-13acetate ŽTPA., was observed. Since many compounds that inhibit in vitro EBV-EA activation have been found to be antitumor promoters in vivo w12x, we eventually tested the in vivo antitumor promoting activity of isolate 3 Žniazimicin. with the two-stage chemical carcinogenesis on mice skin. Niazimicin Ž3. was chosen for the in vivo assay because the structural type of this compound is not yet known as an antitumor promoter ŽScheme 1..
2. Materials and methods 2.1. General experimental procedures 1
H-NMR and 13 C spectra were recorded on a Varian XL-300 spectrometer in CDCl 3 using TMS as an internal standard. EI and FAB mass spectra were measured on a JEOL JMS-SX 102 A QQ mass spectrometer. Silica gel ŽMerck G60, 230 mesh. was used for rapid column flash chromatography w15x. Columns of 2–3 in. in height and of varying diameters depending on the amounts of extracts Žranging from a 2-in. diameter for about 12 g crude extracts to 1 in. diameter for a few grams of column fractions. were used. Preparative high-performance liquid chromatography ŽPrep. HPLC. was performed using Shimadzu LC 6AD liquid chromatograph and Shimadzu SPD-10A UV–VIS detector Ž250 nm. with a Shimpak PreSil column ŽShimadzu. Ž250 mm = 20 mm, i.d. 5 mm. and hexaner2-propanol Ž6:4. as mobile phase. Pre-coated silica gel plates ŽMerck, 0.25 mm. were used for thin layer chromatography ŽTLC..
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Compounds were visualized by spraying with vanillin-concentrated H 2 SO4 solution followed by heating. 2.2. Plant material, extraction and isolation of compounds 1–8 The seeds of M. oleifera Lam. were collected from Abucay, Bataan, Philippines in April and May. Decoated seeds were ground and soaked in distilled ethanol for a few days with occasional stirring. The ethanol extract was partitioned between CCl 4 and water and thence, the CCl 4 fraction was subjected to repeated and sequential flash column chromatography on silica gel using vacuum elution. A total of 12 g of the extract were loaded into 2–3 in. height= 2 in. diameter silica gel columns and eluted with hexane-ethyl acetate as the solvent system. A combination of gradient and isocratic elution was employed, starting with hexane and increasing the polarity of the eluting solvent with ethyl acetate by 10% increment until pure isolates were obtained ŽFig. 1.. The progress of separation was monitored by TLC. Successive purification of fractions D 21 and D 22 by high-performance liquid chromatography on silica gel afforded four isolates 1 Ž5 mg., 2 Ž15 mg., 3 Ž20 mg. and 4 Ž10 mg.. Isolates 1, 2, 3, and 4 were identified as O-ethyl-4-w a-L-rhamnosyloxy.benzylxcarbamate, 4-Ž a-L-rhamnosyloxy.benzyl isothiocyanate, niazimicin, and niazirin, respectively, by IR, NMR and MS data. Isolates 5 Ž50 mg., 6 Ž65 mg., 7 Ž30 mg., and 8 Ž10 mg. were also obtained from fractions B, C, D 1 and E, respectively ŽFig. 1.. These isolates were identified as b-sitosterol Ž5., glycerol-1-Ž9-octadecanoate. Ž6. w9x, 3-O-Ž6X-O-oleoyl-b-D-glucopyranosyl.-b-sitosterol Ž7. w10x, and b-sitosterol-3-O-glucopyranoside Ž8. w11x by NMR and MS data. Isolates 5, 7, and 8 each contained about 10% of the stigmasterol analog. 2.3. In Õitro bioassay for antitumor-promoting actiÕity: the Epstein–Barr Virus-Early Antigen (EBV-EA) actiÕation 2.3.1. Chemicals The cell culture reagents, n-butyric acid and Triton X100 were purchased from Nacalai Tesque, ŽKyoto, Japan.. TPA, 7,12-dimethylbenzw axanthra-
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Fig. 1.
cene ŽDMBA., and ribonuclease ŽRNase. were obtained from Sigma ŽSt. Louis, MO, USA.. EBV-EA positive serum from a patient with nasopharyngeal carcinoma ŽNPC. was a gift from Prof. H. Hattori, Department of Otorhinolaryngology, Kobe University and used for the immunofluorescence test. 2.3.2. Cells The EBV genome carrying lymphoblastoid cells ŽRaji cells derived from Burkitt’s lymphoma. were cultured in RPMI-1640 medium ŽNissui, Tokyo,
Japan. under the conditions as described previously w12–14x. Spontaneous activation of EBV-EA in the subline Raji cells was less than 0.1%. 2.3.3. Experimental procedure The indicator cells ŽRaji, 1 = 10 6rml. were incubated at 378C for 48 h in 1 ml of medium containing 4 mM n-butyric acid Žinducer., TPA Ž32 pmol. solution in 2 ml dimethylsulfoxide ŽDMSO.x, and various amounts of the test compounds dissolved in 5 ml of DMSO w12x. Smears were made from the cell
A.P. GueÕara et al.r Mutation Research 440 (1999) 181–188
suspension. The EBV-EA inducing cells were stained with high titer EBV-EA positive sera from NPC patients and detected by an indirect immunofluorescence technique w16x. In each assay, at least 500 cells were counted and the number of stained cells Žpositive cells. among them was recorded. Triplicate assays were performed for each data point. The EBV-EA inhibitory activity of the test compound was expressed by making a comparison with that of the positive control experiment which was carried out with n-butyric acid Ž4 mM. plus TPA Ž32 nM.. In the experiments, the EBV-EA induction was ordinarily at around 35% and these values were taken as the positive control Ž100%.. Inhibition of EBV-EA activation Ž Percentage inhibition s 100% y percentage to positive control. was taken as an indication for potential antitumor promoting activity. An amount of 4 mM n-butyric acid alone induced 0.1% EA-positive cells. Less than 20 ml of DMSO per milliliter of medium did not show toxicity against our assay system. The viability of treated Raji cells was assayed by the trypan-blue staining method. A high viability of the Raji cells is necessary for the in vitro assay. 2.4. In ÕiÕo bioassay for antitumor-promoting actiÕity: two-stage carcinogenesis test on mouse skin papillomas 2.4.1. Animals Specific pathogen-free female ICR mice Ž6 weeks old. were obtained from Nippon SLC ŽHamamatsu, Japan.. 2.4.2. Experimental procedure Specific pathogen-free female ICR mice Ž6 weeks old. were housed, five per polycarbonate cage, in a temperature-controlled room at 24 " 28C and given food and water ad libitum. The backs of mice were shaved with surgical clippers and the mice were topically treated with DMBA Ž100 mg, 390 nmol. in acetone Ž0.1 ml.. One week after the initiation, carcinogenic growth was promoted twice a week by the application of TPA Ž1 mg, 1.7 nmol. in acetone Ž0.1 ml. on the skin. The animals were then divided into two experimental groups, of 15 mice per group. Group I received TPA treatment alone while group II received topical applications of compound 3
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Žniazimicin. Ž85 nmol. in acetone Ž0.1 ml. at 1 h before each TPA treatment. The incidence and number of papillomas were observed weekly for 20 weeks, as described previously w17,18x.
3. Results and discussion 3.1. In Õitro screening for potential antitumor promoting actiÕity Various assay methods are currently being used to detect tumor promoters as defined by the two-step hypothesis of skin carcinogenesis. Such methods include the classical protocol of skin painting in mice, activation of protein kinase C, induction of ornithine decarboxylase w22x and others w23x. From our previous studies on the induction of EBV-genome-carrying human lymphoblastoid cells, it was reported that TPA and other well-known tumor promoters are all efficient EBV-inducers w24x. This led to our assumption that the EBV-inducing system could be utilized as an in vitro assay for the detection of such tumor promoter substances. Thus, the ethanol extract and isolates 2, 3, 7, and 8 were tested for their potential antitumor promoting activity by observing their inhibitory effect on the induction of EBV-EA activation. Their inhibitory effects at various concentrations, the corresponding viability of the Raji cells and the calculated IC 50 for the pure isolates are summarized in Tables 1 and 2. At a concentration of 100 mgrml, the ethanol extract inhibited the induction of EBV-EA activation by 73%. However, upon dilution by 100-fold, such inhibitory effect was reduced to 11%. No activity was observed at 0.1 mgrml. These inhibitory effects
Table 1 Inhibitory effect of ethanol extract of seeds from M. oleifera Lam. on the induction of EBV-EA activation Concentration Žmgrml.
Percentage inhibition of induction of EBV-EA activation Ž% cell viability a .
100 10 1 0.1
72.9 Ž60. 40.4 Ž )80. 11.2 Ž )80. 0 Ž )80.
a
Values in parentheses are the percentage viability of Raji cells.
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Table 2 Inhibitory effects of isolates from M. oleifera Lam. on induction of EBV-EA activation Isolates
4-Ž a-L-rhamnosyloxy.benzyl isothiocyanate Ž2. Niazimicin Ž3. X 3-O-Ž6 -O-oleoyl-b-D-glucopyranosyl.-b-sitosterol Ž7. b-sitosterol-3-O-b-D-glucopyranoside Ž8. a b
Percentage inhibition Ž% cell viability. at different concentrationsa 1000 mol ratio
500 mol ratio
100 mol ratio
10 mol ratio
100 Ž60. b 100 Ž60. 100 Ž60. 100 Ž60.
83.1 Ž) 80. 79.3 Ž) 80. 65.3 Ž) 80. 93.5 Ž) 80.
52.6 Ž) 80. 47.2 Ž) 80. 15.2 Ž) 80. 62.7 Ž) 80.
20.3 Ž) 80. 18.1 Ž) 80. 0.0 Ž) 80. 11.4 Ž) 80.
IC 50 Žmgrml. 32.7 35.3 70.4 27.9
Mol RatiorTPA Ž20 ng s 32 pmolrml.. Values in parentheses are the percentage viability of Raji cells.
suggested that the ethanol extract had moderate antitumor promoting activity. All the isolates tested showed inhibitory effects on the induction of EBV-EA activation without significant cytotoxicity on Raji cells. Of the tested compounds, the isothiocyanate rhamnoside Ž2., niazimicin Ž3., and sitosterol glucoside Ž8. showed potent inhibitory activity in which about 50%, 80–90%, and 100% inhibitions of activation were observed at 100, 500, and 1000-fold mol ratio to 32 pmol of TPA, respectively, and preserved the high viability of Raji cells even at the highest concentration Ž1000 mol ratiorTPA.. On the other hand, steroid 7 was considered moderately active with about 15%, 65% and 100% inhibition at 100, 500, and 1000 mol ratiorTPA, respectively. These inhibitory activities for isolates 2, 3, 7, and 8 were higher than those of previously reported potent antitumor promoters like glycyrrhetic acid w12,19x, b-carotene w13x, and euglobals w12x. In our previous work, an intractable mixture of acylglucosylsterols, 3-O-w6X-O-palmitoyl-b-D-glucosylx-b stigmasta-5, 25-diene and its stearoyl derivative, isolated from the fruits of Momordica charantia Linn. w20x, was found to reduce the number of micronucleated polychromatic erythrocytes induced by mitomycin C, indicating the antimutagenic activity of the mixture of acylglucosylsterols. Considering the related structure of isolate 7, the antimutagenic activity of the acylglucosylsterol Ž7. isolated from M. oleifera may be worth testing, too. Also, in our previous work on the seeds of Leucaena leucocephala w21x, a mixture of 3-O-b-D-glucopyranosyl-b-sitosterol Ž8. and its stigmasterol analog was found to exhibit inhibitory activity on the EBV-EA activation as well as the antimutagenic
activity against mitomycin C on the micronucleus test. 3.2. In ÕiÕo two-stage carcinogenesis test on mouse skin Since the inhibitory effect on the induction of EBV-EA activation is well-correlated with antitumor-promoting activity in vivo w12x, the above results suggested that these test compounds, especially, compounds 2, 3, and 8 were potent antitumor promoters. Thus, on the basis of the results of the in vitro assay, the inhibitory effect of niazimicin Ž3. on the two-stage carcinogenesis on mouse skin using DMBA as an initiator and TPA as a promoter was examined. Although the EBV-EA inhibitory activity of 8 was higher than 2 and 3, and the activity of 2 was about the same as 3, niazimicin Ž3. was chosen for the in vivo assay because the structural type of this compound is not known as an antitumor promoter till now. Also, not enough of 2 and 8 were available for the in vivo assay at the time of investigation. Isolation of more samples of isolates 2 and 8 is underway. The inhibitory activities on tumor promotion were evaluated by the ratio Ž%. of papilloma-bearing mice ŽFig. 2A. and the average numbers of papillomas per mouse ŽFig. 2B.. In the positive control, papillomas started to appear on the 6th week of promotion with 20% of the mice already bearing papillomas. By the 10th week, all control mice already bore papillomas with an average of 6.2 papillomas per mouse. After 20 weeks of promotion, the number of papillomas increased to an average of 9.5 per mouse. On the other hand, when niazimicin was applied before each TPA treat-
A.P. GueÕara et al.r Mutation Research 440 (1999) 181–188
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Fig. 2. All mice were initiated with DMBA Ž390 nmol. and promoted with 1.7 nmol of TPA given twice weekly starting 1 week after initiation. ŽA. Percentage of mice bearing papillomas. ŽB. Average number of papillomas per mouse. v, control TPA alone; `, TPA q 85 nmol of niazimicin. Each symbol represents the mean for 15 animals.
ment Žgroup II., the formation of papillomas in mouse skin was delayed from the 6th week to the 9th week and the incidence and the mean numbers of papillomas per mouse were reduced. Only 20% and 93.3% of mice bore papillomas at 10 to 20 weeks of promotion with 0.9 and 5.8 papillomas per mouse detected, respectively. Thus, niazimicin Ž3. exhibited 50% delay in the promotion of tumors and decreased the incidence of papilloma bearing mice by 80% and 17% at 10 and 20 weeks of promotion, respectively. An 85% and 39% reduction in the number of papillomas per mouse were also observed during the same period. Previous studies on glycyrrhetic acid w12x showed 46 and 93% of mice with papillomas at 10 and 20 weeks of promotion with 2 and 5.6 papillomas per mouse, respectively while for euglobal G1 , 40% and 70% of mice bore papillomas with an average of 2 and 4.5 papillomas per mouse were observed. Comparatively, these results indicate that the inhibitory activity of niazimicin Ž3. for the two-stage carcinogenesis after 10 weeks of promotion is very much higher than that of glycyrrhetic acid and euglobal G1 , both previously reported potent antitumor promoters. However, after 20 weeks of promotion, the activity of niazimicin was about the same as that of glycyrrhetic acid but a little less potent than euglobal G1 . The results obtained in the present study, thus, indicate that 4Ž a-L-rhamnosyloxy.-benzyl isothio-
cyanate Ž2., niazimicin Ž3., 3-O-Ž6X-O-oleoyl-b-Dglucopyranosyl.-b-sitosterol Ž7., and b-sitosterol-3O-b-D-glucopyranoside Ž8. inhibit EBV-EA induction and could be potential antitumor promoters. Furthermore, our data strongly suggest that niazimicin Ž3) is a potent antitumor promoter in chemical carcinogenesis. Its inhibitory mechanism on tumor promotion will be studied upon isolation of more pure samples.
Acknowledgements The extraction procedures were conducted at the Institute of Chemistry, University of the Philippines Diliman ŽUPD. through the funding support of the Natural Science Research Institute, College of Science, UPD. The antitumor bioassays were conducted at the Kyoto Prefectural University of Medicine while the structure elucidation studies were conducted at the Kyoto Pharmaceutical University. The technical assistance of Miss Kayoko Oda, Kyoto Pharmaceutical University, for obtaining the mass spectra of the compounds is acknowledged. This study was also supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare for the Second-term Comprehensive Ten-year Strategy for Cancer Control, Japan. APG also acknowledges UP, the Japan Society for the Promotion of Science, and the Philip-
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pine Department of Science and Technology for the research visit to the Kyoto Pharmaceutical University. w14x
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