APPLIED AND ENVIRONMENTAL Mickobiotocy, May 1986, p. 1085-1088 0095-224086!051085-04802.000 Copyright © 1986, American Society for Microbiology Schinus moll: Val. 51, No. 5 a New Source of Natural Fungitoxicantt ANUPAM DIKSHIT,* ALI A. NAQVI, AND AKHTAR HUSAIN Central Institute of Medicinal and Aromatic Plants, Lucknow-226016, India Received 12 February 1986/Accepted 21 Febnuary 1986 ‘The oil of Schinus molle exhibited the maximum fungitoxic activity during the sereening of some essential oils agninst some common storage and animal pathogenic fungi. It showed absolute toxicity against animal pathogens and mild activity against storage fungi. The effective concentrations ofthe oll varied from 200 to 900 pm. The toxkity of the oll persisted up to 80°C and 90 days of storage but declined when autoclaved. It withstood heavy inoculum density. The oil exhibited a narrow range of activity and was found to be more ‘effective than Multfungin, an antifungal drug. The oll i was characterized by its various physicochemical Droperties. It was found to comprise 0 constituents. It appeared that some changes in the oll constituents during storage affected is fungitoxie potency. ‘Naturally occurring fungitoxicants described to date are mostly biodegradable (3) and are devoid of side effects (9) compared with commerical available fungitoxicants. ‘We report here resulis of our investigation of Schinus ‘molle L.,a member of the Anacardiaceae, as anew source of, fungitoxicant. Essential oils extracted from higher plants were investigated for their fungitoxic activity: test fungi used were those Which caused dermatomycoses in animals and fungal deterioration of foodstuffs during storage. MATERIALS AND METHODS Essential oils were extracted from different parts of 10 taxa of phanerogams by hydrodistillation with a Clevenger apparatus (4) and were evaluated at various concentrations TABLE 1. Inhibition of animal pathogenic and st ‘and storage fungi such as Alternaria alternata (Fries) Keissler, Aspergillus flavus Link, and Penicillium italicum jehmer. ‘The evaluations were performed by using the ‘poisoned food" technique (7) with Sabouraud dextrose agar ‘medium, Minimum fungistatic and fungicidal concentrations of the oils were evaluated by the method of Garber and Houston (00). The influence of temperature, storage, and inoculum density on the mycelial growth inhibition (MGD of the oils at ‘500 ppm were observed as previously described by Dikshit and Dinit (6). ‘A otal of 19 animal or phytopathogenic fungi were used to test the MGT of §. mole oil (500 ppm) by the conventionally used poisoned food technique. The fungistatic fungicidal torage fungi by essential ols from higher plants MGT aan secs iy nsouce Ania og ar Sona a ee OTT “pins aravecens 1. (ee) Tiscne Seed i666 sa Gfimanlomioncetdodapine Mein, hmctae Frit Sa S30 a MS ee ‘Surandh Kok) loa poltachys Benth Lamicese Leaves 3 656 0560 AMO ‘Bhangar) Junipers communis L.Gunipes)—Cpresaceae Leaves 4 a1 3 Bo wr mo ‘vende oficalic hatx (avers Laminceae Leaves Pee ee) ap Mentha arvensis L. apanese mint) Lamiaceae «= Actipars = 642 S387 O53 Plirorpermum ongcicoder(DC) paces” Prat Ss oma kB KI toon ‘Sava sean. (crying Lanigcene —Aealpans «S80 OA O23 Sehims made (eaorta pepper Araardns eaves wm ae SS re) eae Zanthoryum aatum Roxb. Tomes) Ratacexe Sood ys eS Oma {MB M:zppseu: Tn, T, mentagropiote: Te. Tribu: xed ot 40 pps SARA oleate: Atv A. flausB, . lalicum: tested a 500 Pp for fungitoxic activity against common animal pathogenic fungi, namely, Microsporum gypseum (Bodin) Guiart and Grigorakis, ‘Trichophyton mentagrophytes (Robin) Blanchard, Trichophyton rubrum (Castellani) Sabouraud, * Corresponding author. 4 Central Institute of Medicinal and Aromatic Plants publication no. 568. ros nature ofthe oil against the pathogens that did not proliferate (100% MGI was examined by using a conventional tech- rnigue (10), The efficacy of the oil was also compared with that of Multifungin (synthetic lotion for the control of ringworm infections; Bochringer Knoll Ltd.) in Sabouraud ‘dextrose agar by using the poisoned food technique. ‘The physicochemical characterization of $. moile oil was performed by the method of Langenau (13). Gas-liquid1086 DIKSHIT ET AL TABLE 2. Influence of temperature, storage, and inoculum density on MGI by 5. molle a MGT aint Contos a 7 Lepr i : ‘Temperature we 1010100 ore 10 = 10100 wre 10 10100 Autoclaving (5 loin’. 20 min) «75:8 OO ‘Storage at #C (days) ° 101000 0 10 = 100 o 100 100 0 10 100100 10 7% OBS 150 m3 SLB 180 0 gl 83 20 a9 3188 20 sk MS 8D Inoculum density (90, of fungal isk) 2 100 100 4 100 100 ® 100 100 16 100 10) 2 100 10. Conceiaton, 30 pe chromatographic analysis of the oil was done by using a Perkin-Elmer GC model 3920 equipped with TCD and an SS column (2 m by 0.125 in. [.3175 em)) packed with 10% Carbowax 20 M on 80/100 Chromosorb WAW. The oven temperature was programmed from 60°C, with an inital hold (of § min, to 180°C at a rate of min with a final hold time of 20 min. A hydrogen flow of 30 ml/min was maintained through the column. The relative retention and coinjection techniques were used to identify the constituents. A Hew- lett-Packard integrator (HP 3390 A) attached to the detector output was used to calculate percent area. RESULTS ‘The efficacy of the essential oils against animal pathogens and storage fungi varied. The oil of S. molle was the most effective, inhibiting the animal pathogens completely and exhibiting moderate activity against the storage fungi. All other essential oils showed either partial or poor activity Table 0. The minimum fungistatic concentrations of S. molle oil were 300, 200, and 200 ppm against M. gypseum, T. ‘mentagrophytes, and T. rubrum, respectively. The minimum fungicidal concentrations were 900 ppm against T. ‘mentagrophytes and 400 ppm against 7. rubrum. M. _gypseum was completely resistant 0 the fungicidal action of Sumolle oil, even at concentrations of 900 ppm. The toxicity of the oil persisted at temperatures as high as 80°C and for up to 90 days of storage but declined when autoclaved. It withstood heavy inoculum densities (Table 2). The oil exhibited a narrow range of activity as i¢fungistati= cally checked the mycelial growth of animal pathogens, namely. Epidermophyton floccosum, Histoplasma capsula ‘um, Microsporum canis, Microsporum ferrugineum, Tricho- APPL. ENVIRON, MicRouioL. TABLE 3. MGI of fungi by 5. male oil Fangs Bien Cladosporium eladospor Leaf spot a9 fodder (Fresenius) de Cladosporium richoides Cerebral chromomycosis 60.0 ‘Emmons Gurvatara tunata (Wakker) Leaf spot 764 Boedija Epidermophyton floccosum —Dermatomycosis 10° (Harz) Langeroa and Mi Tochevitch Fonsecaca pedrosoi Chromomycosis as “Bromp0) Negroni Fusarium moniiforme Shel. Fruit rot 4S ‘don Georichum candidum Link Geotrichosis uaa Histoplasma capsulatum Histoplasmosis or Daring Microsporum canis Bodin Dermatomycosis 10" Microsporum ferrupineum — Dermatomycosis 108" ‘Oe Nocardia asteroides (Ep Nocardiosis oo pinger) Blanchard Nocardia bresiienss (Lin- Nocardiosis 250 ‘denterg) Casteliani and Chalmers Phialophora jeanselmel Mycetoma 0 (Langeron) Emmons Sporotrichum schenckii_Sporotrchosis 40 Hektoen and Perkins Trichophyton equinum (Ma-Detmatomycosis 200" ‘ruchot and Dassanvile) Gedoelst, Trichophyton simi (Pinoy) Dermatomycosis woo Stockdale, Mackenzie and Austwick Trichophyton terrestre Durie. Dermatomycosis 66 and Frey Trichophyton tonsurans —Dermatomycosis 100° Mamsten Trichophyton violaceum Sa- Dermatomycosis 653 bouraud phyton eguinum, and Trichophyton ronsurans, from the ‘group of 19 fungi tested (Table 3). The minimum fungistatic concentrations of $. molle oil were 60, 75, and $5 times more active against M. gypseum, T. mentagrophytes, and T. rubrum, respectively, when compared with Mulifungin. In terms of minimum fungicidal concentrations, $. mole oil was 125 times more effective than Multifungin against 7, rubrum and 58.5 times more effective against 7. mentagrophytes (Table 4). Physi- ‘cochernical properties of the oil were determined (Table 5). (Of 50 components resolved by gas-liquid chromatography, 14 were found above 1% (vol/vol); however, only 10 com: ponents could be identified (Table 6) DISCUSSION Pharmacological, physical, and chemical properties of 5. ‘molle oil have already been reported by various workers (1, 2, 5, 8 11, 12, 14-19). To our knowledge, its fungitoxic action against tested pathogenic organisms is being reported for the first time, "The fungitoxie character ofthe ol declined after 90 days of storage (Table 2), suggesting that some effective chemical ‘changes in ils composition had occurred (Fable 6). Out of ldVou. 51, 1986 5. MOLLE—A NEW SOURCE OF NATURAL FUNGITOXICANT 1087 TABLE 4. Efficacy of S, molle oil compared with Multiungin ‘Aatimycate ‘Minimum fangstateconen (ppm rma nga cance PO Antica Active inzedient, Me Te Tr ¥i Te c S mole oi = 300, 200) 200 300 300 Mulitungin’ 2% s-Bromosalicy-4-chlorani- 18.00 15,000 11.000 50,000 50,000 $0,000 (standard) lide-19e N-phenyl-N-benzyl4- amino-I-methyl piperidine salicylate 7 Ma. M-sypreum: Ti, T, mentagrophytes Te. rar + See Tait « Remained state, * Active ipedints are daslved in polythene aco TABLE 5. Physicochemical properties of S. molle antifungal activity might have been caused by the relative Property Vale _ changes in these four constituents, Teaves Because of its potent and narrow spectrum of fungitoxicity Moisture (9) and its superior effectiveness over Multifungin (standard), ‘Summer 62.76 the oll from leaves of $. mole is an effective addition to the Winter. 6745 ‘armory of antifungal agents. This effectiveness is even Dry matter (%) ‘greater against animal pathogenic fungi and should be further ‘Summer 3728 studied ia animals, Winter. 3257 ACKNOWLEDGMENTS "i ote tbs) ‘Thanks are due to L-N_ Mohapatra, Division of Microbiology All ‘Summer ue 0 India Institut of Medical Sciences, New Delhi, for providing animal Winer O74 pathogenic fungal cultures. Yield) (dry wi basis) ‘Wealso thank the Council of Scent and Industral Research, cae Ar) New Delhi, for nancial assistance Winer 277 Spegitc gravity 3 30°C. (0.9004395, LITERATURE CITED fae. 37 4 1, Bernhard, R. A.» T. Sehibamoto, K. Yamaguchl, and E. White. Refractive index at 37°C. 1.504 1983. The volatile constituents of Schinut molle L. J. Agric. pH 40 ood Chem. 31:463-466 Cola Light yellow 2. Bernhard, R. A., and R. Wrolitad. 1963. The essential oil of TABLE 6. Chemical constituents (above 1% (volvo) detected in ‘Smo oil a intervals by gas-liquid chromatography ‘Consent tention tine % Composition at day Ta ore a-Pinene 230) 196 Ra 4 Myrcene”,a-phellandrene 29 Sz 29.06 639) Limonene*, B-phellandrene? 15.46 18.03. 24.13 55) p-Cymene (10.48) aa oa 6.90 6-Caryopbyllene (33.87) 18 oa ost CCryptone (37°56) 293 3931.66 ‘aTerpineol (39.25) 5396 am io Unidentied $4.7) sal 43500 Ler ‘Unidentified (56.6) 437 eas ‘Unidentified (58.3), 2.30 2 Oss Carvacrol 61.) 132 re 2a Unidentified (67.42) 1039 jas 27 ed ad confrmed on a8 $5 oluna @ m by TR in, (3175 ew tacked with 10 OVeIDI 09 8/100 Chromonore WHD. The temperature ‘Ebanges andthe flow of yarouen were the same as those used To the Carbowax 20 M cola, ‘constituents only 10 could be identified. However, percep tible changes during storage occurred in «-pinene, limonene, and B-phellandrene in a defined pattera of gradual increase, ‘but there was a decrease in the four unidentified constituents (Table 6). Therefore, it could be inferred that deterioration in Schinus mole, the tenpene hydrocarbon fraction. J. Food Sei DBS9-68. 3 Beye, F. 1978 Insecticides from the vegetable kingdom, Plant Rew: Dev. 7213-31, 4. Clevenger, J. F. 1928. Apparatus for the determination of ‘Volatile oil J. Am. Pharm, Assoc. 17:36, 5S. Ceemonini, A. 1928. lavestigation on fruits on Schinus molle. ‘Ann. Chim. Applicata 18:361-365, 6. Dikshit, A. and S..N. Dist. 1982. Cedrus olla promising antifungal agent. Indian Perfum. 26:216-297 7. Dikhit, A., and A. Hasain. 1984. Antifungal action of some essential ils against animal pathogens. Fitoterapia S8:171-17. Dominguez, A..J- F. Carmona, and . B. De Venegas. 1971 Lignocenc’acid and other compounds of Schinus molle Phytochemistry 10:1687 9, Fawcett, C. H., and D. M. Spencer, 1970. Plant chemotherapy ‘with natural products, Annu. Rev. Phytopathol. 403-418, 30. Garber, HR. H., and B. R. Houston. 1959. An inhibitor of Vertcltwm -albo trum in colton’ seed. Phytopathology 9449-450, 1, Hashim, F. M., G. A. ELHossary, and F. S. ELSakhuwy. 1978 Preliminary phytochemical stay of Schinus molleL. growing in Egypt. J. Pharm. Sci. 19:235-246, 12, Jennings, W. Gu, and R. A. Bernhard. 1975. Identification of ‘components ofthe essential ol fom the California pepper tree (Schinus mole L-). Chem. Mikrobiol. Technol. Lebensm 495-96, 13, Langenaa, E.E. 1948. The examination and analysis ofessentiat ells syathetics, and solates, p. 227-348, In E. Guenther (ed), ‘The ersential gil. Van Nostrand Reinhold Co., Inc, New York 14, Montes, A. L., C0. Bandinel, and E. Davidson. 1961. Schinus 4"Mtudy of the composition of the essential oils matte ‘obtained fom the leaves and from oleoresin extracted from the1088 16. n, DIKSHIT ET AL bers. An, Soc. Cient, Argent. 173:3-16, Ottling, G. 1948. Schinus mole esseti ‘Accad. Pugliese Sc. 649-54 Pabliction and Information Directorate. 1972, Schinus, 9, 247-289. In The wealth of India: raw material, vol. 8. Coun (of Scientific and Industrial Research, New Delhi, India Terhune, 5. J, J. W. Hogg, and’ B.M. Lawrence. ll Ani Retuz 1914, APrL. ENVIRON. Mickontot B.Spathulene: a new sesquitepene in Schinus molle ol Phytochemistry 13:868-866, van dee Lingen, G. W. B. 1930. South Aftiean pepper tee oi Pertum. Essent. Oi Rec. 21:1 Zakdi,S. M. Aw. A. Hanan, and 8. Babar, 1970. Some studies of the pharmacological activities of Schinus mole. Pak. Sei nd Res, 13:53-58