38 M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44 ´also used as an anti-inﬂammatory agent, while secticidal) performed with crude extracts fromV. gaumeri is used to treat colds and coughing different plant parts of V. trifolia.spells (Ekundayo et al., 1990; Chawla et al.,1992; Argueta et al., 1994; Damayanti et al.,1996). It is well known that a considerable num- 2. Materials and methodsber of plant species, besides their popular use asmedicine in many countries, possess insecticidal 2.1. Plant materialactivities. The genus Vitex sp. is not an excep-tion. V. negundo has larvicidal activity against Leaves and stems of V. trifolia L. (Verbenaceae)the mosquito species Culex quinquefasciatus and were collected at Poblado ‘El Cinco’ (El Carrizo Valley, Sinaloa, Mexico). A specimen wasAnopheles stephensi (Pushpalatha and Muthukr- deposited at the IMSSM Herbarium, Mexico City,ishnan, 1995), and acts as a deterrent to the voucher number 11878. Collected material wasmosquito Aedes aegypti (Hebbalkar et al., 1992). dried in the dark at room conditions. Later, bothV. rotundifolia also shows deterrent properties leaves and stems were extracted by maceration intowards A. aegypti (Watanabe et al., 1995). Sev- hexane during 72 h in darkness. Residuals wereeral other Vitex species are currently being in- further extracted with dichoromethane (DCM)vestigated in speciﬁc programs of pest control following the same procedure. Final extraction was(Rahman and Bhattacharya, 1982; Epila and performed with methanol. The plant extracts wereRuyooka, 1988; Sudarsanam et al., 1995). then evaporated under reduced pressure and V. trifolia has been reported to have both prepared to perform the assays.medicinal and insecticidal properties. It alleviatespain derived from rheumatism and sprained 2.2. Cytotoxicity assaysjoints when applied topically. Also, the leavesare used to treat intermittent fever, while the All cell lines representing cervix carcinomatiny ﬂowers are administered as an infusion to (SQC-1 UISO), ovarian cancer (OVCAR-5), colontreat fever accompanied by vomiting and severe carcinoma (HCT-15 COLADCAR) and humanthirst (Ramesh et al., 1986; Thein et al., 1995). nasopharyngeal carcinoma (KB) were maintainedAn insect antifeeding activity has been recorded on RPMI medium supplemented with 10% fetalfor seeds (Hosozawa et al., 1974). There have bovine serum (FBS), and incubated at 37°C in anbeen various studies on the chemical structures atmosphere of 5% CO2 in air (95% humidity). Theof compounds isolated from both V. trifolia cells at a log phase of their growth cycle wereleaves and fruit extracts. The isolation of treated in triplicate with various concentrations ofﬂavonoids: casticin, 3,6,7-trimethyl quercetagetin the extracts (1, 10, 100 mg/ml) dissolved in 20%(Zeng et al., 1996), vitexin, artemetin, 5-methyl dimethylsulfoxide (DMSO) in water. Initial cell suspensions which contained 25 000 cells/mlartemetin, 7-desmethyl artemetin, luteolin (Nair (counted on a hemocytometer), were incubatedet al., 1975), luteolin-7-O-b-D-glucuronide, lute- with the corresponding extract as previouslyolin-3-O-b-D-glucuronide and isoorientin described during 72 h. Controls were test tubes(Ramesh et al., 1986), have been reported. Also, containing the cells alone, free of plant extracts.the triterpenoid friedelin and other steroids, b- The ﬁnal cell concentrations were determined bysitosterol and b-sitosterol-b-D-glucoside (Vedan- protein analysis to obtain the effective dose thattham and Subramanian, 1976; Zeng et al., inhibits 50% growth after the incubation period1996), have been described, and the chemical (ED50). The values were estimated by means of aconstituents of essential oils obtained from semilog curve derived from extract concentrationsleaves have been analyzed (Pan et al., 1989). (1 mg/ml) plotted against a percentage of viable In this paper we report several biological ac- cells. Extracts having an ED50 5 20 mg/ml weretivities (fungicidal, bactericidal, cytotoxic and in- considered active (Villarreal et al., 1992).
M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44 ´ 392.3. Fungicidal assays inhibition; + + , growth was inhibited above 50%; + , growth was inhibited less than 50%; − , Five species of fungi were assayed to determine no inhibition occurred with respect to the control.the fungicidal activity of the plant extracts: Peni-cillium sp., Aspergillus ﬂa6us, A. parasiticus, Tri- 2.5. Insecticidal assayschoderma sp. and Fusarium sp. All strains of fungiwere grown in potato dextrose agar (PDA). Plant Insecticidal properties of V. trifolia were testedextracts dissolved in cyclohexane, acetone, water in the lepidopteran Spodoptera frugiperda (Noctu-or a mixture of them were added to 5 ml of sterile idae), the most important pest of corn in Mexico,PDA in a concentration of 500 mg/ml, while con- and also a pest of other crops, such as wheat, soytrols only contained the solvent where the sample and sorghum, as well as ornamentals (Brown andwas applied. Control and experimental assays Dewhurst, 1975). Larvae of S. frugiperda werewere done by triplicate. Petri dishes were inocu- reared on artiﬁcial diet as described (Bell andlated with 7–10-day-old fungi cultures, and incu- Joachim, 1976). The diet included 0.044% forma-bated at 28°C during 6 days. Radial mycelial lin, 0.03% acetic acid, and 0.11% choline chloridegrowth was monitored every 2 days. Data are (ﬁnal concentrations) to restrict the growth ofgiven as percent inhibition as compared to the undesirable microorganisms (Aranda et al., 1996).controls that reached 100% growth (March et al., Third instar larvae were fed with the plant ex-1991). tracts contaminating the diet, either mixing the extracts with the nutrients or spreading a thin2.4. Bacterial growth inhibition assays layer on top of the congealed diet. Plant extracts were dissolved in cyclohexane, acetone, water or a The bacterial growth inhibition assays were per- mixture of them at a concentrations of 10, 100formed using cultures of Pseudomonas aeruginosa and 1000 mg/ml (mixed with the diet) or 10, 100(ATCC 9027), Staphylococcus aureus (ATCC and 1000 mg/cm2 (over the surface of the diet),6538), Shigella sonei (ATCC 11060), Proteus and poured into 24-well polystyrene ELISAmirabilis, Salmonella typhi (ATCC-CDC-99), as plates. One plate was used per sample dilutionwell as the yeast Candida albicans (ATCC 10231). plus one control with the solvents used to dissolveBacteria strains were maintained on trypticase soy the extracts. The plates were incubated at 28°C,agar (TSA) and the yeast on Sabourand’s dex- 60% relative humidity and 16:8 h light:dark pho-trose agar (SDA). The method was based on toperiod during 7 days. After 7 days, all larvaeconventional disk assays. Plant extracts at 10, 5, were weighed including controls (Kubo, 1991).2.5, and 1.25 mg/ml were dissolved in 20% DMSO The data were statistically analyzed for a studentin water. However, in order to dissolve the most t-test (Dowdy and Wearden, 1983).non-polar samples, 20% Tween-20 was added.The inoculum for each microorganism was pre-pared from broth culture (108 colony forming 3. Resultsunits per milliliter, CFU/ml). For testing, 104CFU were placed by means of a tiny droplet Yields of extracts were as follows (w/w): leaf-ranging from 5 to 8 mm in diameter, using a hexane, 0.97%; stem-hexane, 0.42%; leaf-DCM,micropipette calibrated to 2 ml. Controls were 1.95%; stem-DCM, 0.47%; leaf-methanol, 5.61%;prepared using the same solvents employed to stem-methanol, 2.88%.dissolve the plant extracts, and as positive controlgentamicin (Pharmacia) was used as reference 3.1. Cytotoxic acti6itystandard. Each assay was done in duplicate. Theplates were incubated 24 h at 37°C (Villarreal et Table 1 shows the ED50 values for positiveal., 1994). The observed effects were recorded extracts of V. trifolia assayed on four lines ofusing pluses as follows: + + +, 100% growth human tumor cells. Hexanic and DCM extracts
40 M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44 ´Table 1 54% growth inhibition of Fusarium sp. within 4Cytotoxicity of crude extracts from Vitex trifolia (ED50 mg/ml) days of the experiment, then, after 6 days, theCancer cell linesa Extract inhibition percentage dropped 1.7 times. Growth inhibition was poor or negligible on the other Hexane Dichloromethane tested fungi for all extracts (Table 2). Leaf Stem Leaf Stem 3.3. Bacterial growth inhibitionSQC-1 UISO 15.5 38 2.2 37.1OVCAR-5 7.6 17.4 2.9 8.5 Table 3 shows the growth inhibition producedHCT-15 COLAD- 3.6 2.8 B1 1.9 by leaf extracts of V. trifolia toward six species of CARKB 6.0 30.2 1.9 4.1 bacteria (two Gram-positives and four Gram-neg- atives) and one species of yeast. All extracts com- a SQC-1 UISO, cervix carcinoma; OVCAR-5, ovarian can- pletely inhibited the growth of Gram-positivecer; HCT-15 COLADCAR, colon cancer; KB, nasopharyngeal species (except the methanolic extract assayed atcarcinoma. the lowest dose). Growth of Gram-negative bacte- ria were 100% inhibited at 10 mg/ml in all ex-have shown interesting ED50 values, the DCM tracts, except for S. typhi. No bacterial growthleaf extract being the most active, with an ED50 was observed when 5 mg/ml of DCM leaf extractless than 1 mg/ml towards HCT-15 COLADCAR, were assayed, except for S. typhi which was inhib-which proved to be most sensitive cell line. On the ited over 50% growth. Also, the methanolic leafother hand, the SQC-1 UISO cell line was the extract inhibited E. coli and P. mirabilis over 50%least sensitive. No effects were detected with the growth. At the following dose assayed (2.5 mg/methanolic extracts of either leaves or stems ml), it was only the DCM leaf extract that par-(ED50 20 mg/ml). tially inhibited the growth of S. sonei and S. typhi; the lowest dose of DCM leaf extract that3.2. Fungicidal acti6ity caused inhibition to C. albicans was 5 mg/ml (Table 3). Percentage of growth inhibition of ﬁve fungalspecies by V. trifolia leaf extracts are shown in 3.4. Insecticidal acti6ityTable 2. Hexanic leaf extract caused 100% inhibi-tion of Fusarium sp. within 2 days of growth, later Larvae of S. frugiperda were force-fed fromdropping to 47% at day 4 and to 15% at day 6. contaminated diet with V. trifolia extracts. After 7On the other hand, the DCM extract exhibited a days, larvae were weighed. A high antifeedingTable 2Antifungal activity of crude extracts from Vitex trifolia leaves (percentage of radial mycelial growth inhibition compared to thecontrol at 2, 4 and 6 days of growth)Microorganism Percentage inhibition of mycelial growth Hexane (days) Dichloromethane (days) Methane (days) 2 4 6 2 4 6 2 4 6Penicillium sp. 22 20 17 27 23 20 0 0 0Aspegillus ﬂa6us 28 19 15 23 33 29 21 4 4Aspergillus parasiticus 0 0 0 29 35 23 0 9 4Tricoderma sp. 20 18 0 21 27 5 0 0 0Fusarium sp. 100 47 15 54 54 38 31 18 0
42 M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44 ´ triterpenoid friedelin and sitosterol, did not show cytotoxic values against this same cell line (Zheng, 1994; Wu et al., 1995; Hirobe et al., 1997). None of the ﬂavonoids that have been already described in V. trifolia were investigated for their cytotoxic potential against the cell lines studied in this investigation, except for KB. There are several reports which suggest a correlation between the structures of methoxy ﬂavones as well as 5,7-dihy- droxy ﬂavones (with substitutions in the B ring), and with cytotoxic activities (Kupchan et al., 1971; Woerdenbag et al., 1994). However, other investigations describe inactive polymethoxy-Fig. 1. Insecticidal activity of crude dichloromethane (DCM) ﬂavonoids (Kingston et al., 1979), or no relationsextract from Vitex trifolia leaves. Doses were: 1, 0.1, and 0.01 may exist between structures and biological activi-mg/ml in diet and 1, 0.1 and 0.01 mg/cm2 on diet.Values are ties (Edwards et al., 1979; Mori et al., 1988).expressed as mean 9S.D. (n=24). *PB 0.05 and **PB 0.001signiﬁcantly different from the control. Moreover, most of these compounds have been described because of their potent activity against aactivity was recorded in the DCM leaf extract speciﬁc cell line, but they have not shown a wider(Fig. 1). While the larvae had a very low food range of action (Cushman and Nagarathnam,consumption at the lowest doses assayed, they 1991). It will be desirable to carry out a bioassaystop feeding at the highest dose employed (1000 guided fractionation of V. trifolia extracts usingmg/cm2), and were dead after a 7-day trial. This the tested cell lines to identify the responsiblebehaviour was common when the plant extract compounds for the observed cytotoxic activity,was layered on the surface of the diet, but when it and to establish whether or not the knownwas mixed with the components of the diet, the ﬂavones in this plant are contributing. Two im-antifeeding effect was recorded only for the portant reasons could support this approach, onehighest dose assayed. is that the most active ﬂavones described for this plant against KB cells (luteolin and its derivatives) were isolated from complete crude alcoholic ex- tracts in which we were unable to detect any4. Discussion activity, and the other is that important variations in the ﬂavonoid 6 pattern described for the Although all prepared extracts from V. trifolia same species have been related to different geo-showed interesting biological properties, it was graphical distribution of the plants (Nair et al.,the DCM leaf extract exhibiting the highest cyto- 1975).toxicity against SQC-1 UISO, OVCAR-5, HCT- It is also noteworthy that the important an-15 COLADCAR and KB cell lines. These tifeeding activity of the DCM leaves extractobserved actions could probably be attributed to against Spodoptera frugiperda that we observed incertain secondary compounds in the most apolar this investigation, has been also described withfraction of the DCM extract that could also be extracts from seeds against larvae of S. liturapresent in the hexanic one, in which speciﬁc toxic (Hosozawa et al., 1974).effects against the studied cell lines were alsodetected. Some ﬂavonoids already isolated fromthis species could have contributed to the general Acknowledgementstoxicity of the plant, i.e. luteolin has shown cyto-toxicity against KB (ED50 =0.3 1 mg/ml); how- This research was partially supported by Insti-ever, other ﬂavones, such as artemetin, the tuto Mexicano de Tecnologia del Agua (IMTA)
M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44 ´ 43and Programa de Naciones Unidas pare el De- controlling termite attacks on cassava (Manihot esculenta)sarrollo (PNUD). We thank Abigail Aguilar with Vitex doniana: a preliminary study (Isoptera). Sociobi- ology 14 (1), 291 – 297.(Herbarium of the Centro Medico Nacional, ´ Hebbalkar, D.S., Hebbalkar, G.D., Sharma, R.N., Joshi, V.S.,IMSS, Mexico City) for authentication of the Bhat, V.S., 1992. Mosquito repellent activity of oils fromplant specimen. We gratefully acknowledge Vitex negundo Linn. leaves. Indian J. Med. Res. 95, 200 –Daniel Alonso, Socorro Vallejo, Laura Lina and 203.Victor Navarro for their valuable help in the Hirobe, C., Qiao, Z.S., Takeya, K., Itokawa, H., 1997. Cyto-biological assays. We also thank the people from toxic ﬂavonoids from Vitex agnus-castus. Phytochemistry 46 (3), 521 – 524.El Carrizo for their help gathering the plant Hosozawa, S., Kato, N., Munakata, K., Chen, Y.L., 1974.material. 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