Journalof Ethnopharmacology. 36 (1992) 127-132 Elsevier Scientific
Solanum
Publishers
Ireland
127
Ltd.
(Schldl.) a plant used in Mexico for the treatment of skin mycosis
chrysotrichum
X. Lozoya,
V. Navarro,
M. Garcia,
and M. Zurita
Truditionol Medicine und Drug Development Research Unit, Institute Meuicuno del Seguro Social. Xochitepec. Morelos (Mexico) (Accepted
December
9. 1991)
An ethnobotanical field study carried out in 200 rural communities determined that the leaves of So/unum chryx~trichum Schldl. are used in Mexican traditional medicine for the treatment of skin mycosis, being particularly recommended to cure Tinoe pedis. Clinical trials were performed using a cream containing 5% of a methanolic leaf extract of this plant. Results showed that 45% of the patients were completely cured after 4 weeks of topical treatment. The rest of the cases improved notably in comparison with the control group of patients using the habitual miconazole treatment. The same plant extract inhibited the growth in vitro of the dermatophytes Trychophyton mentagrophytes, T. rubrum and Microsporum gypseum at MICs under 15 mg/mI. Key words: Solarium; skin mycosis;
plant
fungicide
Introduction According to several ethnobotanical studies performed in Chiapas, Mexico, a number of plant remedies are used by local traditional medicine for the treatment of skin diseases (Miranda, 1975; Solis, 1980; Gallardo et al., 1983; Berlin et al., 1990). Among the plants reported to be used for the treatment of diverse skin infections, the genus Solanum shows a high index of citation and is represented by several species generally named under the Spanish term of ‘sosa’ (Lozoya and Zolla, 1984; Zurita and Zolla, 1986). Considering the epidemiological importance of skin infections in tropical countries like Mexico, and searching for new remedies for the treatment of this type of ailment, a specific study was performed to inquire deeper into the medical ethnobotany of ‘sosa’ and thus initiate its scientific evaluation. In Chiapas the botanical species Solanum chrysotrichum Schldl. is the most frequently used ‘sosa’ and particularly recommended for the treatment of the skin mycosis known as ‘Athlete’s foot’ (Tinae pedis). A clinical study was carried out to determine the efficacy of the methanolic extract Correspondence to: X. Lozoya. Unidad de Investigacibn en Medicina Traditional y Desarrollo de Medicamentos, I.M.S.S. Argentina 1. Xochitepec, Mor. CP 62790. Mexico.
037%8741/92/$05.00 0 1992 Elsevier Printed and Published in Ireland
Scientific
Publishers
Ireland
from S. chrysotrichum Schldl. leaves in curing this disease. Finally, in order to evaluate the possible bactericidal and/or fungicidal properties of this extract, a screening study was also performed using different bacteria and dermatophytes growing in vitro. The present paper reports the results obtained in these studies which confirm the antifungal properties of this popular plant remedy. Methodology Ethnobotanical study The study was performed in Chiapas, Mexico, with the aid of the communities of the 200 small Indian villages (circa 2500-3000 inhabitants each) where the Mexican Social Security Institute (IMSS) has built, during the past decade, clinics to provide primary medical attention to the local population. In all these places the physician in charge of the clinic has already established communication with the local healer, the midwife or with other traditional practitioners (TPs) of his community according to the strategy promoted by the ‘Programme of Interaction with Traditional Medicine’ implemented in the area since 1984 by the IMSS (Lozoya et al., 1988). A survey was applied by these doctors to the local TPs (250 persons of different ages and specialities). The Ltd.
128
questionnaire included the following aspects regarding the use of ‘sosa’: (a) other local names of the plant; (b) requirements for its collection and for the preparation of the remedy; (c) types of skin disease treated and diagnoses; (d) posology, method of administration of the drug and duration of treatment; (d) a characterization of its attributed properties and possible toxic effects. Plant specimens were collected in each case for further botanical classification and the appropriate vouchers were deposited in the IMSS Herbarium in Mexico City once the final taxonomical verification was established at the New York Botanical Garden. Clinical study
The study was performed at the IMSS General Hospital No. 1 of Cuernavaca city, in Morelos, Mexico. A group of 28, male and female, ambulatory adult patients with Tinae pedis, were selected considering the following exclusion criteria: onychomycosis; diabetes mellitus; peripheric neuropathy; renal or cardiac insufficiency; hemophilia; vasculitis and impetigo. Consent in writing from each patient was obtained to participate in the study after receiving a complete explanation about the purpose of the study and the type of products to be used in the treatments. Two groups were formed: group A (14 patients) used 2% miconazole nitrate cream, while group B, (14 patients) used a cream containing 5% of the methanolic extract of S. chrysotrichum Schldl. leaves (vide infra). Both products were prepared by Productos Medix, S.A., with the same vehicle (lanolin/Tween-SO/water/methylparaben) and having the same final presentation used by this company to produce Neomicol (R). The medicinal treatment described to the patients was the same for both groups: topical application of the respective cream, abundantly on the lesion, immediately after washing the skin area with soap and water, twice a day during 4 weeks. The evolution of each case was followed every week by the medical staff of the hospital recording changes in the signs and symptoms of both groups following a double-blind schema. The obtained final data (cure/no-cure) were statistically processed using the Fisher’s test. Plant extract
Leaves of S. chrysotrichum Schldl. (Solanaceae) were collected in Chiapas from uncultivated specimens, dried at room temperature and finely powdered. Plant material was macerated for 48 h with methanol at room temperature. The solvent
was distilled off under reduced pressure in a rotary evaporator until the extract became completely dry. The obtained product (yield 21.5% by weight) was used directly in the preparation of the cream for the clinical trials or redissolved in dimethylsulfoxide (DMSO) for the antimicrobial in vitro testing. Microbiological
Studies
screening
were
performed with cultures of (ATCC8937) Pseudomonas aeruginosa (ATCC9027) Staphylococcus aureus (ATCC6538), Bacillus subtilis (ATCC6633) and Candida albicans (ATCC 1023 1). The dermatophytes tested were Trychophyton mentagrophytes, Trychophyton rubrum, and Microsporum gypseum cultivated in our laboratory from clinical isolates. The bacteria were maintained on Trypticase soya agar (TSA), yeast and dermatophytes on Sabourand’s dextrose agar (SDA). The screening method was based on the commonly described procedures reported for the study of crude natural products with antimicrobial activity (Lennatte et al., 1985; Rios et al., 1988). Briefly, plant extract free of organic solvent was suspended in lo”/;, DMSO and added at various concentrations to the melted agar in petri dishes. Inoculum for each organism was prepared from broth culture (10’ colony forming units (CFU)/ml, for S. aureus, B. subtilis, P. aeruginosa, E. coli and C. albicans). The inoculum was applied as a spot with a loop calibrated to deliver 0.002 ml, resulting in a spot inoculum covering a circle of 5-8 mm diameter and containing lo4 CFU. The plates were incubated for 24 h at 37°C. Dermatophytes were grown at 26°C for 1 week on slants of SDA. The agar plates were inoculated by dropping approx. 0.05 ml of the spore and mycelial suspension in a concentration adjusted spectrophotometrically to a transmission of 8590% at 530 nm. The plates were incubated at 26°C until macroscopically visible growth appeared in the controls. Gentamicin, nystatin and amphotericin B were used as reference standards. Observations were performed in duplicate and results expressed as the lowest concentration of plant extract resulting in a complete suppression of microorganism growth, i.e. the minimal inhibitory concentrations (MICs). Escherichia
coli
Results Ethnobotanical
The collected
information
specimens
of ‘sosa’ were authen-
129
ticated at the New York Botanical Garden as Solanum chrysotrichum Schldl. and Solanum lanceolatum Cav. (Solanaceae). In the studied area both species are recognized by other names derived from the Maya language characteristic for the local cultures (in Tzotzil: K’ux peul; in Tzeltal: K’uxbal ch’ix; in chol: pajutiek). Inquiring on the popular medicinal use of these species it soon became clear that Solanum chrysotrichum Schldl. was the variety most frequently used and, according to the experiences described by the TPs, the only difference in using them was that S. lanceolatum Cav. was considered to be less active. A study of the cases in which Solanum chrysotrichum Schldl. was used by the TPs showed that the leaves of this plant are utilized in the following cases: (a) For the treatment of resistant and long lasting skin mycosis, among which the plant remedy was considered specifically useful in curing Tinae pedis (the ‘Athlete’s foot’ clearly recognized by traditional medicine as a fungal disease and referred to in Spanish with the local term of ‘mazamorra’) and also to heal other infectious dermatitis popularly named with the ambiguous term of ‘disipela’ (erysipelas). (b) For situations where a clean and fast process of cicatrization is desired, as in the treatment of abscess, sores, wounds, burns and other similar lesions. The leaves are considered the unique medicinal part of the plant and are used as a poultice since the use of the product is predominantly external (reported in 65.7% of the prescriptions obtained). However, the leaf is also used to prepare a water extract for oral administration (33.8% of the prescriptions); in such cases its use is for extensive skin diseases which have been unsuccessfully treated by topical means. There were few reports about the use of the water extract for rectal or vaginal administration to cure local infections (0.5%). For topical application both Solanum species are employed as a poultice, plaster or stupe, although in such cases an initial wash with the decoction is frequently recommended. Equally, the dried leaf in powdered form is sprinkled on the affected surface during several days. The decoction used for oral and topical administration is prepared with lo-20 g of leaves, boiled in 1 1water for 5-10 min. Adult oral doses vary between 250-400 ml every 8 h and the treatment lasts for 7-8 days. Topical treatment with the decoction consists in washing of the affected skin area 2 or 3 times daily until recovered. The leaves can be used dried or fresh. The plant is considered nontoxic by TP’s and in the cases of oral administra-
tions, treatments last for several weeks apparently without side effects. Clinical trials Results obtained in the clinical study of the antifungal properties of S. chrysotrichum Schldl. cream compared to miconazole nitrate are shown in Table 1. After 4 weeks of treatment, no patients receiving miconazole nitrate were cured although some signs and symptoms had disappeared. Patients treated with S. chrysotrichum Schldl. cream had a significantly better outcome: 42.8% were completely cured at the end of the 4 weeks treatment and for the remainder of the group the symptoms improved notably. Habitually, a complete cure from Tinae pedis is a difficult goal that takes several weeks of perseverance and discipline in the use of the fungal remedy, a situation that explains its high degree of recidivism. However, using the Solanum cream reduced pruritus, pain, edema and vesicles characteristic of the mentioned mycosis during the first week of treatment. The cases considered not cured at the end of the study presented only some degree of hyperkeratosis and fisures of the skin. Antimicrobial screening Results obtained during the screening of the antibacterial and antifungal properties of S. TABLE
I
(A) COMPARATIVE EFFECT BETWEEN MICONAZOLE AND SOLANLIM CREAMS AFTER 4 WEEKS OF TREATMENT IN PATIENTS WITH TINAE PEDIS AND (B) CHANGES IN SIGNS AND SYMPTOMS DURING THE SAME PERIOD OF TREATMENT
(4
Product Miconazole
Cured Number % Non-cured Number %
Solanum
0
6
0
42.8
14 100.0
a 57.1
@I Signs and symptoms Erythema Vesicles Hyperkeratosis Fisures Edema Pain Pruritus
Before
After
Before
After
9 2
2 0
7 4
0 0
a 6 2 5 14
a 5 0 0 7
7 4 2 6 12
6 2 0 0 0
I 30 TABLE
2
MIC VALUES
CUIIWP Ext. MeOH
OF S. CHRYSOTRICHUM
S. chrysotr.
SCHLDL.
METHANOLIC
EXTRACT
ON MlCROORGANISMS
IN VITRO
S.a.
B.S.
E.c.
P.a.
C.a.
T.m.
T.r.
M.g.
N
N
N
N
10
5
5
I5
0.02
0.02
0.02
0.02
~
_
0.05
0.40
(mgiml) C0nrrol.v Gentamicin (mg/ml) Nistatin (mg/ml) Amphotericin
0.02
0.05
(mg/ml) %a.. Sluphylococcus aureus; B.S.. Bwillus suhtilis; Es.. Escherichia coli; P.a.. Pseudomonas aeruginosu; C.a., Candida ulhicans; T.m., Trychophyton meniagrofires; T.r.. Trychophyron ruhrum; M.g.. Microsporum gypscwm; (N) Negative, dose required up to 30 mgiml. (-) Not peformed.
chrysotrichum Schldl. extract are shown in Table 2. The plant product did not show antibacterial effects against selected gram-negative and grampositive microorganisms, at 30 mg/ml concentrations generally considered as the limit accepted for an active crude extract in this mode1 (Rios et al., 1988). Although inhibition of growth of the yeast Cundida albicans was obtained with the extract at concentrations of 20 mg/ml, the same extract possessed strong antifungal properties against T. mentugrofites, T. rubrum and M. gypseum at concentrations of 15.0 mg/ml or less.
Discussion According to the bibliographical sources there are no previous reports on the medicinal properties of S. chrysotrichum Schldl. or S. lanceolatum Cav. The ethnobotanical bibliography on Solunum species used by traditional medicines of other cultures can be summarized as follows: the leaves of S. verbascifolium L. are used in Indonesia as an abortifacient and to facilitate menstruation when orally administered as water extracts (Quisumbing, 1951; Couvee, 1952; Petelot, 1954; Burkill, 1966; Adam, 1980). S. hispidum Pers. leaves extracted in water are used in India for external treatment of skin tumors and to relieve inflammatory muscular processes (Bhakuni, 1969). The leaves of S. torvum Swartz. are reported to cure dysentery and malaria in Puerto Rico and rheumatism in New Guinea, when orally administered as water extracts (Mahmood, 1983; Holdsworth, 1975; Stimson, 197 1). In Central America, where traditional medicines
have a common origin and populations are closely related culturally, the medicinal use of some species of Solanum is similar. A water extract from leaves of S. nigrescens Mart. and Gal. has been reported in Guatemala as a useful remedy for the treatment of skin infections when topically administered (Caceres et al., 1987). The crushed fruit and juice of S. torvum Swartz and S. mammosum L. are used to cure Athlete’s foot in Belize (Arnason et al., 1980). Other experimental and clinical studies on species of Solanum that can be related to our results report a weak antimicrobial activity against Staphylococcus albus produced with a methanolic leaf extract of S. verbascifolium although the same product was inactive against Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis (Ogunlana,
Escherichiu
coli
1975). The product obtained by ethanolic maceration of leaves of S. nigrescens showed moderate anti-bacteria1 effect on Bacillus subtilis and Staphylococcus aureus and was inactive against S. pyogenes, Shigellaflexneri, Proteus vulgaris, Salmonella typhi and Pseudomonas aeruginosa (Caceres et al., 1987). However, a methanolic extract from leaves of the same plant inhibited the in vitro growth of Candida albicans and satisfactory results were obtained in the treatment of vaginal candidiasis in a group of patients using intravaginal suppositories containing the extract (Giron et al., 1988). According to the chemical literature several Solanum species are frequently used as a source of steroidal alkaloids because some yield solasodine, the nitrogen analogue of diosgenin. A compound that can easily be converted to 16-dehydropreg-
131
nenolone,
the basic intermediate
in the synthesis
of
many steroid drugs used for diverse purposes in modern medicine. Solasodine has been isolated mainly from S. torvum and S. verbascifolium, but a group of other steroidal saponins such as hispigenin, hispinin, chlorogenin, neochlorogenin, solaspigenin and neoslaspigenin can be found in S. hispidum, and S. nigrescens together with the alkaloids solasonine, solaverbascine, tomatidine and solamargine (Dopke, 1975; Verbist, 1977; Chakvarty, 1978, 1979, 1980, 1981, 1982; Adam, 1980). It is interesting to note that the antifungal activity of none of the mentioned compounds has been reported, although according to recent studies some steroidal glycosides such as pennogenin (a spirastanol saponin) show antifungal properties (Okunji et al., 1990). The antiinflammatory property recognized for some of the classical steroidal compounds of the Solanum type cannot explain the effects observed in the clinical trials or in the in vitro tests reported here for the S. chrysotrichum leaf extract. Studies in progress will determine the identity of the antifungal compounds present in the leaf of the popular ‘sosa’ remedy. Acknowledgments This work would not have developed without the assistance of the medical participants from traditional and institutional medicines. To all of them our recognition. We thank Michael Nee, Assistant Curator of the New York Botanical Garden for assistance in the identification of botanical specimens. We are also indebted for bibliographical support to Norman Farnsworth and the NAPRALERT team at the College of Pharmacy, University of Illinois at Chicago. We thank Mrs. Hilda Rodriguez from Productos Medix, S.A. for the preparation of the drugs used in the clinical study. References Adam, G., alkaloids:
Huong, H. and Khoi, N.H. (1980) Solarturn solaverbascine, a new 22,26-epidemionocholest-
ane alkaloid from Solanum verbasctfolium. Phytorhemistry 19, 1002-1003. Amason, T., Uck, F., Lambert, J. and Hebda, R. (1980) Maya medicinal plants of San Jose Succotz, Belize. Journal of Ethnopharmacology 2, 345-364. Berlin, B., Berlin, E.A., Breedlove, D.E., Duncan, T., Jara, V.M., Laughlin, R.M. and Velasco, T. (1990) La herbolaria medica T:eltal-Tsot:il en 10s Altos de Chiapas. Gobierno del Estado, Chiapas, Mexico. p. 154.
Bhakuni, O., Dhar, M.L., Dhar, M.M.. Dhawan, B.N. and Mehrotra, B.N. (1969) Screening of Indian plants for biological activity. Part Indian Journal of Experimental Biology 7, 250-262. Burkill, I.H. (1935) Dictionary of the Economic Products of the Malay Peninsula. Vol. II, 1966 reprint. Ministry of Agriculture and Cooperatives, Kuala Lumpur, Malaysia, p. 10. Chakravarty, A.K., Dhar, T.K. and Pakrashi, SC. (1978) Hispigenin a novel 22-beta ortho-spirostane from Solanum hispidum. Tetrahedron letters 3875-3878. Chakravarty, A.K., Saha, C.R. and Pakrashi, S.C. (1979). New spirostane saponins and sapogenins from Solanum hispidum seeds. Phytochemistry 18, 902-904. Chakravarty, A.K., Dhar, T.K., and Pakrashi, S.C. (1980) Studies on Indian medicinal plants 55: solaspigenin and neosolaspigenin, two new spirostane sapogenins from Solanum hispidum. Phytochemistry 19, 1249-1251. Chakravarty, A.K., Pakrashi, SC. and Uzawa. J. (1981) Studies on Indian medicinal plants Part 62: 13C Nuclear Magnetic Resonance spectra of 23-hydroxy spirostane sapogenins of Solanum hispidum. Canadian Journal of Chemistry 59. 1328- 1330. Chakravarty, A.K., Das, B., and Pakrashi, S.C. (1982) Studies on Indian medicinal plants Part 65: solanolide, a steroid lactone sapogenin from Solanum hispidum. Phytochemistry 21, 2033-2035. Caceres, A., Giron, L., Alvarado, S. and Torres M. (1987) Screening of antimicrobial activity of plants popularly used in Guatemala for the treatment of dermatomucosal diseases. Journal of Ethnopharmacology 20, 223-237. Couvee, L. (1952) Compilation of herbs, plants and crops supposed to be effective in various complaints and illnesses. Journal of Scienttjk Research 18, I-10. Dopke, W., Neguerias, C., Hegs, U. (1975) On the steroid alkaloi- and saponin contents of Solanum torvum. Pharmazie 30. 755-756. Gallardo, M., Tellez, M., Vargas, M. and Vazquez, L. (1983) Aspectos etnobotanicos y bacterioldgicos en la medicina Traditional en 10s Altos de Chiapas. Universidad National Autonomade Mexico. Escuela National de Estudios Profesionales Iztacala, Mexico, p. 35. Giron, L.M., Aguilar, G.A., Cdceres, A. and Arroyo, L.G. (1988) Anticandidal activity of plants used for the treatment of vaginitis in Guatemala and Clinical Trial of a Solanum nigrescens preparation. Journal of Ethnopharmacology 22. 307-313. Holdsworth, D. (1975) Traditional medicinal plants used in the treatment of Malaria and fevers in Papua New Guinea. Papua New Guinea Medical Journal 18, 142-148. Lennette, E.H., Balows, A., Hansler, Jr. W.J., and Shadomy, H.J. (1985) (Eds.) Manual of Clinical Microbiology. Washington, D.C.. American Society of Microbiology. pp. 143-153. Lozoya, X., and Zolla, C. (1984) Medicina Traditional en MPxice. B-letin Oticina Sanitaria Panamericana 96, 360-364. Lozoya, X., Vehizquez, G., and Flares, A. (1988) La Medicina Traditional en Mexico: Experiencia del Programa IhISSCOPLAMAR 1982-1987. Instituto Mexican0 de1 Seguro Social, Mexico, p. 13. Mahmood, U., Thakun, R., and Blunden, G. (1983) Neochlorogenin, neosolaspigenin and sclaspigenin from Solanum torvum leaves. Journal of Natural Products 46. 427-428.
132 Miranda, (1976) La Vegetacidn de Chiapus. Vol. 2. Gobierno de1 Estado, Chiapas, Mexico p. 22. Ogunlana, E.D., and Ramstad, E. (1975) Investigations into the antibacterial activities of local plants. Planta Medica 27. 354-356.
Research
127-150.
Stimson,
Okunji, C.O., Okeke. C.N.. Gugnani, H.C. and Iwu, M.M. (1990) An antifungal spirostanol saponin from fruit pulp of Dracaena
Rios, J.L.. Recio, M.C. and Villar, A. (1988) Screening methods for natural products with antimicrobial activity: a review of the literature. Journal of Ethnopharmacology 23.
mannii.
international
Journal
qf
Crude
Drug
28, 193- 199.
A. (1954) Les plantes medicinales du Camgodge, da Laos et du Vietnam. Vol. 2, Archives des researches Agronomiques et Pastorales au Vietnam, Vietnam, p. 195.
Petelot,
Quisumbing, E. (1951) Medicinal Plants of the Philippines. Technical Bulletins 16, Department of Agriculture and Natural Resources, Manila, Philippines, p. 126.
W.R. (1971) Ethnobotanical
Lloydia
notes from Puerto
Rico.
34, 165- 170.
Solis, G. (1980) Recopilacidn de Plantas Medicinales de 10s Altos de Chiapas. lnstituto National Indigenista, Chiapas, Mexico, p. 15. Verbist, J.F.. Monnet, R. and Dobremez. J.R. (1977) Identification and quantification of steroid alkaloids from Nepalese Solanum species. Planta Medica II, 40-42. Zurita, en
M., and Zolla, C. (1986) Enfermedades la medicina
Sanitaria
traditional
Panamericana
de
MPxico.
101. 339-345.
dermatolbgicas
Boletin
Oficina
Solanum
Publishers
Ireland
127
Ltd.
(Schldl.) a plant used in Mexico for the treatment of skin mycosis
chrysotrichum
X. Lozoya,
V. Navarro,
M. Garcia,
and M. Zurita
Truditionol Medicine und Drug Development Research Unit, Institute Meuicuno del Seguro Social. Xochitepec. Morelos (Mexico) (Accepted
December
9. 1991)
An ethnobotanical field study carried out in 200 rural communities determined that the leaves of So/unum chryx~trichum Schldl. are used in Mexican traditional medicine for the treatment of skin mycosis, being particularly recommended to cure Tinoe pedis. Clinical trials were performed using a cream containing 5% of a methanolic leaf extract of this plant. Results showed that 45% of the patients were completely cured after 4 weeks of topical treatment. The rest of the cases improved notably in comparison with the control group of patients using the habitual miconazole treatment. The same plant extract inhibited the growth in vitro of the dermatophytes Trychophyton mentagrophytes, T. rubrum and Microsporum gypseum at MICs under 15 mg/mI. Key words: Solarium; skin mycosis;
plant
fungicide
Introduction According to several ethnobotanical studies performed in Chiapas, Mexico, a number of plant remedies are used by local traditional medicine for the treatment of skin diseases (Miranda, 1975; Solis, 1980; Gallardo et al., 1983; Berlin et al., 1990). Among the plants reported to be used for the treatment of diverse skin infections, the genus Solanum shows a high index of citation and is represented by several species generally named under the Spanish term of ‘sosa’ (Lozoya and Zolla, 1984; Zurita and Zolla, 1986). Considering the epidemiological importance of skin infections in tropical countries like Mexico, and searching for new remedies for the treatment of this type of ailment, a specific study was performed to inquire deeper into the medical ethnobotany of ‘sosa’ and thus initiate its scientific evaluation. In Chiapas the botanical species Solanum chrysotrichum Schldl. is the most frequently used ‘sosa’ and particularly recommended for the treatment of the skin mycosis known as ‘Athlete’s foot’ (Tinae pedis). A clinical study was carried out to determine the efficacy of the methanolic extract Correspondence to: X. Lozoya. Unidad de Investigacibn en Medicina Traditional y Desarrollo de Medicamentos, I.M.S.S. Argentina 1. Xochitepec, Mor. CP 62790. Mexico.
037%8741/92/$05.00 0 1992 Elsevier Printed and Published in Ireland
Scientific
Publishers
Ireland
from S. chrysotrichum Schldl. leaves in curing this disease. Finally, in order to evaluate the possible bactericidal and/or fungicidal properties of this extract, a screening study was also performed using different bacteria and dermatophytes growing in vitro. The present paper reports the results obtained in these studies which confirm the antifungal properties of this popular plant remedy. Methodology Ethnobotanical study The study was performed in Chiapas, Mexico, with the aid of the communities of the 200 small Indian villages (circa 2500-3000 inhabitants each) where the Mexican Social Security Institute (IMSS) has built, during the past decade, clinics to provide primary medical attention to the local population. In all these places the physician in charge of the clinic has already established communication with the local healer, the midwife or with other traditional practitioners (TPs) of his community according to the strategy promoted by the ‘Programme of Interaction with Traditional Medicine’ implemented in the area since 1984 by the IMSS (Lozoya et al., 1988). A survey was applied by these doctors to the local TPs (250 persons of different ages and specialities). The Ltd.
128
questionnaire included the following aspects regarding the use of ‘sosa’: (a) other local names of the plant; (b) requirements for its collection and for the preparation of the remedy; (c) types of skin disease treated and diagnoses; (d) posology, method of administration of the drug and duration of treatment; (d) a characterization of its attributed properties and possible toxic effects. Plant specimens were collected in each case for further botanical classification and the appropriate vouchers were deposited in the IMSS Herbarium in Mexico City once the final taxonomical verification was established at the New York Botanical Garden. Clinical study
The study was performed at the IMSS General Hospital No. 1 of Cuernavaca city, in Morelos, Mexico. A group of 28, male and female, ambulatory adult patients with Tinae pedis, were selected considering the following exclusion criteria: onychomycosis; diabetes mellitus; peripheric neuropathy; renal or cardiac insufficiency; hemophilia; vasculitis and impetigo. Consent in writing from each patient was obtained to participate in the study after receiving a complete explanation about the purpose of the study and the type of products to be used in the treatments. Two groups were formed: group A (14 patients) used 2% miconazole nitrate cream, while group B, (14 patients) used a cream containing 5% of the methanolic extract of S. chrysotrichum Schldl. leaves (vide infra). Both products were prepared by Productos Medix, S.A., with the same vehicle (lanolin/Tween-SO/water/methylparaben) and having the same final presentation used by this company to produce Neomicol (R). The medicinal treatment described to the patients was the same for both groups: topical application of the respective cream, abundantly on the lesion, immediately after washing the skin area with soap and water, twice a day during 4 weeks. The evolution of each case was followed every week by the medical staff of the hospital recording changes in the signs and symptoms of both groups following a double-blind schema. The obtained final data (cure/no-cure) were statistically processed using the Fisher’s test. Plant extract
Leaves of S. chrysotrichum Schldl. (Solanaceae) were collected in Chiapas from uncultivated specimens, dried at room temperature and finely powdered. Plant material was macerated for 48 h with methanol at room temperature. The solvent
was distilled off under reduced pressure in a rotary evaporator until the extract became completely dry. The obtained product (yield 21.5% by weight) was used directly in the preparation of the cream for the clinical trials or redissolved in dimethylsulfoxide (DMSO) for the antimicrobial in vitro testing. Microbiological
Studies
screening
were
performed with cultures of (ATCC8937) Pseudomonas aeruginosa (ATCC9027) Staphylococcus aureus (ATCC6538), Bacillus subtilis (ATCC6633) and Candida albicans (ATCC 1023 1). The dermatophytes tested were Trychophyton mentagrophytes, Trychophyton rubrum, and Microsporum gypseum cultivated in our laboratory from clinical isolates. The bacteria were maintained on Trypticase soya agar (TSA), yeast and dermatophytes on Sabourand’s dextrose agar (SDA). The screening method was based on the commonly described procedures reported for the study of crude natural products with antimicrobial activity (Lennatte et al., 1985; Rios et al., 1988). Briefly, plant extract free of organic solvent was suspended in lo”/;, DMSO and added at various concentrations to the melted agar in petri dishes. Inoculum for each organism was prepared from broth culture (10’ colony forming units (CFU)/ml, for S. aureus, B. subtilis, P. aeruginosa, E. coli and C. albicans). The inoculum was applied as a spot with a loop calibrated to deliver 0.002 ml, resulting in a spot inoculum covering a circle of 5-8 mm diameter and containing lo4 CFU. The plates were incubated for 24 h at 37°C. Dermatophytes were grown at 26°C for 1 week on slants of SDA. The agar plates were inoculated by dropping approx. 0.05 ml of the spore and mycelial suspension in a concentration adjusted spectrophotometrically to a transmission of 8590% at 530 nm. The plates were incubated at 26°C until macroscopically visible growth appeared in the controls. Gentamicin, nystatin and amphotericin B were used as reference standards. Observations were performed in duplicate and results expressed as the lowest concentration of plant extract resulting in a complete suppression of microorganism growth, i.e. the minimal inhibitory concentrations (MICs). Escherichia
coli
Results Ethnobotanical
The collected
information
specimens
of ‘sosa’ were authen-
129
ticated at the New York Botanical Garden as Solanum chrysotrichum Schldl. and Solanum lanceolatum Cav. (Solanaceae). In the studied area both species are recognized by other names derived from the Maya language characteristic for the local cultures (in Tzotzil: K’ux peul; in Tzeltal: K’uxbal ch’ix; in chol: pajutiek). Inquiring on the popular medicinal use of these species it soon became clear that Solanum chrysotrichum Schldl. was the variety most frequently used and, according to the experiences described by the TPs, the only difference in using them was that S. lanceolatum Cav. was considered to be less active. A study of the cases in which Solanum chrysotrichum Schldl. was used by the TPs showed that the leaves of this plant are utilized in the following cases: (a) For the treatment of resistant and long lasting skin mycosis, among which the plant remedy was considered specifically useful in curing Tinae pedis (the ‘Athlete’s foot’ clearly recognized by traditional medicine as a fungal disease and referred to in Spanish with the local term of ‘mazamorra’) and also to heal other infectious dermatitis popularly named with the ambiguous term of ‘disipela’ (erysipelas). (b) For situations where a clean and fast process of cicatrization is desired, as in the treatment of abscess, sores, wounds, burns and other similar lesions. The leaves are considered the unique medicinal part of the plant and are used as a poultice since the use of the product is predominantly external (reported in 65.7% of the prescriptions obtained). However, the leaf is also used to prepare a water extract for oral administration (33.8% of the prescriptions); in such cases its use is for extensive skin diseases which have been unsuccessfully treated by topical means. There were few reports about the use of the water extract for rectal or vaginal administration to cure local infections (0.5%). For topical application both Solanum species are employed as a poultice, plaster or stupe, although in such cases an initial wash with the decoction is frequently recommended. Equally, the dried leaf in powdered form is sprinkled on the affected surface during several days. The decoction used for oral and topical administration is prepared with lo-20 g of leaves, boiled in 1 1water for 5-10 min. Adult oral doses vary between 250-400 ml every 8 h and the treatment lasts for 7-8 days. Topical treatment with the decoction consists in washing of the affected skin area 2 or 3 times daily until recovered. The leaves can be used dried or fresh. The plant is considered nontoxic by TP’s and in the cases of oral administra-
tions, treatments last for several weeks apparently without side effects. Clinical trials Results obtained in the clinical study of the antifungal properties of S. chrysotrichum Schldl. cream compared to miconazole nitrate are shown in Table 1. After 4 weeks of treatment, no patients receiving miconazole nitrate were cured although some signs and symptoms had disappeared. Patients treated with S. chrysotrichum Schldl. cream had a significantly better outcome: 42.8% were completely cured at the end of the 4 weeks treatment and for the remainder of the group the symptoms improved notably. Habitually, a complete cure from Tinae pedis is a difficult goal that takes several weeks of perseverance and discipline in the use of the fungal remedy, a situation that explains its high degree of recidivism. However, using the Solanum cream reduced pruritus, pain, edema and vesicles characteristic of the mentioned mycosis during the first week of treatment. The cases considered not cured at the end of the study presented only some degree of hyperkeratosis and fisures of the skin. Antimicrobial screening Results obtained during the screening of the antibacterial and antifungal properties of S. TABLE
I
(A) COMPARATIVE EFFECT BETWEEN MICONAZOLE AND SOLANLIM CREAMS AFTER 4 WEEKS OF TREATMENT IN PATIENTS WITH TINAE PEDIS AND (B) CHANGES IN SIGNS AND SYMPTOMS DURING THE SAME PERIOD OF TREATMENT
(4
Product Miconazole
Cured Number % Non-cured Number %
Solanum
0
6
0
42.8
14 100.0
a 57.1
@I Signs and symptoms Erythema Vesicles Hyperkeratosis Fisures Edema Pain Pruritus
Before
After
Before
After
9 2
2 0
7 4
0 0
a 6 2 5 14
a 5 0 0 7
7 4 2 6 12
6 2 0 0 0
I 30 TABLE
2
MIC VALUES
CUIIWP Ext. MeOH
OF S. CHRYSOTRICHUM
S. chrysotr.
SCHLDL.
METHANOLIC
EXTRACT
ON MlCROORGANISMS
IN VITRO
S.a.
B.S.
E.c.
P.a.
C.a.
T.m.
T.r.
M.g.
N
N
N
N
10
5
5
I5
0.02
0.02
0.02
0.02
~
_
0.05
0.40
(mgiml) C0nrrol.v Gentamicin (mg/ml) Nistatin (mg/ml) Amphotericin
0.02
0.05
(mg/ml) %a.. Sluphylococcus aureus; B.S.. Bwillus suhtilis; Es.. Escherichia coli; P.a.. Pseudomonas aeruginosu; C.a., Candida ulhicans; T.m., Trychophyton meniagrofires; T.r.. Trychophyron ruhrum; M.g.. Microsporum gypscwm; (N) Negative, dose required up to 30 mgiml. (-) Not peformed.
chrysotrichum Schldl. extract are shown in Table 2. The plant product did not show antibacterial effects against selected gram-negative and grampositive microorganisms, at 30 mg/ml concentrations generally considered as the limit accepted for an active crude extract in this mode1 (Rios et al., 1988). Although inhibition of growth of the yeast Cundida albicans was obtained with the extract at concentrations of 20 mg/ml, the same extract possessed strong antifungal properties against T. mentugrofites, T. rubrum and M. gypseum at concentrations of 15.0 mg/ml or less.
Discussion According to the bibliographical sources there are no previous reports on the medicinal properties of S. chrysotrichum Schldl. or S. lanceolatum Cav. The ethnobotanical bibliography on Solunum species used by traditional medicines of other cultures can be summarized as follows: the leaves of S. verbascifolium L. are used in Indonesia as an abortifacient and to facilitate menstruation when orally administered as water extracts (Quisumbing, 1951; Couvee, 1952; Petelot, 1954; Burkill, 1966; Adam, 1980). S. hispidum Pers. leaves extracted in water are used in India for external treatment of skin tumors and to relieve inflammatory muscular processes (Bhakuni, 1969). The leaves of S. torvum Swartz. are reported to cure dysentery and malaria in Puerto Rico and rheumatism in New Guinea, when orally administered as water extracts (Mahmood, 1983; Holdsworth, 1975; Stimson, 197 1). In Central America, where traditional medicines
have a common origin and populations are closely related culturally, the medicinal use of some species of Solanum is similar. A water extract from leaves of S. nigrescens Mart. and Gal. has been reported in Guatemala as a useful remedy for the treatment of skin infections when topically administered (Caceres et al., 1987). The crushed fruit and juice of S. torvum Swartz and S. mammosum L. are used to cure Athlete’s foot in Belize (Arnason et al., 1980). Other experimental and clinical studies on species of Solanum that can be related to our results report a weak antimicrobial activity against Staphylococcus albus produced with a methanolic leaf extract of S. verbascifolium although the same product was inactive against Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis (Ogunlana,
Escherichiu
coli
1975). The product obtained by ethanolic maceration of leaves of S. nigrescens showed moderate anti-bacteria1 effect on Bacillus subtilis and Staphylococcus aureus and was inactive against S. pyogenes, Shigellaflexneri, Proteus vulgaris, Salmonella typhi and Pseudomonas aeruginosa (Caceres et al., 1987). However, a methanolic extract from leaves of the same plant inhibited the in vitro growth of Candida albicans and satisfactory results were obtained in the treatment of vaginal candidiasis in a group of patients using intravaginal suppositories containing the extract (Giron et al., 1988). According to the chemical literature several Solanum species are frequently used as a source of steroidal alkaloids because some yield solasodine, the nitrogen analogue of diosgenin. A compound that can easily be converted to 16-dehydropreg-
131
nenolone,
the basic intermediate
in the synthesis
of
many steroid drugs used for diverse purposes in modern medicine. Solasodine has been isolated mainly from S. torvum and S. verbascifolium, but a group of other steroidal saponins such as hispigenin, hispinin, chlorogenin, neochlorogenin, solaspigenin and neoslaspigenin can be found in S. hispidum, and S. nigrescens together with the alkaloids solasonine, solaverbascine, tomatidine and solamargine (Dopke, 1975; Verbist, 1977; Chakvarty, 1978, 1979, 1980, 1981, 1982; Adam, 1980). It is interesting to note that the antifungal activity of none of the mentioned compounds has been reported, although according to recent studies some steroidal glycosides such as pennogenin (a spirastanol saponin) show antifungal properties (Okunji et al., 1990). The antiinflammatory property recognized for some of the classical steroidal compounds of the Solanum type cannot explain the effects observed in the clinical trials or in the in vitro tests reported here for the S. chrysotrichum leaf extract. Studies in progress will determine the identity of the antifungal compounds present in the leaf of the popular ‘sosa’ remedy. Acknowledgments This work would not have developed without the assistance of the medical participants from traditional and institutional medicines. To all of them our recognition. We thank Michael Nee, Assistant Curator of the New York Botanical Garden for assistance in the identification of botanical specimens. We are also indebted for bibliographical support to Norman Farnsworth and the NAPRALERT team at the College of Pharmacy, University of Illinois at Chicago. We thank Mrs. Hilda Rodriguez from Productos Medix, S.A. for the preparation of the drugs used in the clinical study. References Adam, G., alkaloids:
Huong, H. and Khoi, N.H. (1980) Solarturn solaverbascine, a new 22,26-epidemionocholest-
ane alkaloid from Solanum verbasctfolium. Phytorhemistry 19, 1002-1003. Amason, T., Uck, F., Lambert, J. and Hebda, R. (1980) Maya medicinal plants of San Jose Succotz, Belize. Journal of Ethnopharmacology 2, 345-364. Berlin, B., Berlin, E.A., Breedlove, D.E., Duncan, T., Jara, V.M., Laughlin, R.M. and Velasco, T. (1990) La herbolaria medica T:eltal-Tsot:il en 10s Altos de Chiapas. Gobierno del Estado, Chiapas, Mexico. p. 154.
Bhakuni, O., Dhar, M.L., Dhar, M.M.. Dhawan, B.N. and Mehrotra, B.N. (1969) Screening of Indian plants for biological activity. Part Indian Journal of Experimental Biology 7, 250-262. Burkill, I.H. (1935) Dictionary of the Economic Products of the Malay Peninsula. Vol. II, 1966 reprint. Ministry of Agriculture and Cooperatives, Kuala Lumpur, Malaysia, p. 10. Chakravarty, A.K., Dhar, T.K. and Pakrashi, SC. (1978) Hispigenin a novel 22-beta ortho-spirostane from Solanum hispidum. Tetrahedron letters 3875-3878. Chakravarty, A.K., Saha, C.R. and Pakrashi, S.C. (1979). New spirostane saponins and sapogenins from Solanum hispidum seeds. Phytochemistry 18, 902-904. Chakravarty, A.K., Dhar, T.K., and Pakrashi, S.C. (1980) Studies on Indian medicinal plants 55: solaspigenin and neosolaspigenin, two new spirostane sapogenins from Solanum hispidum. Phytochemistry 19, 1249-1251. Chakravarty, A.K., Pakrashi, SC. and Uzawa. J. (1981) Studies on Indian medicinal plants Part 62: 13C Nuclear Magnetic Resonance spectra of 23-hydroxy spirostane sapogenins of Solanum hispidum. Canadian Journal of Chemistry 59. 1328- 1330. Chakravarty, A.K., Das, B., and Pakrashi, S.C. (1982) Studies on Indian medicinal plants Part 65: solanolide, a steroid lactone sapogenin from Solanum hispidum. Phytochemistry 21, 2033-2035. Caceres, A., Giron, L., Alvarado, S. and Torres M. (1987) Screening of antimicrobial activity of plants popularly used in Guatemala for the treatment of dermatomucosal diseases. Journal of Ethnopharmacology 20, 223-237. Couvee, L. (1952) Compilation of herbs, plants and crops supposed to be effective in various complaints and illnesses. Journal of Scienttjk Research 18, I-10. Dopke, W., Neguerias, C., Hegs, U. (1975) On the steroid alkaloi- and saponin contents of Solanum torvum. Pharmazie 30. 755-756. Gallardo, M., Tellez, M., Vargas, M. and Vazquez, L. (1983) Aspectos etnobotanicos y bacterioldgicos en la medicina Traditional en 10s Altos de Chiapas. Universidad National Autonomade Mexico. Escuela National de Estudios Profesionales Iztacala, Mexico, p. 35. Giron, L.M., Aguilar, G.A., Cdceres, A. and Arroyo, L.G. (1988) Anticandidal activity of plants used for the treatment of vaginitis in Guatemala and Clinical Trial of a Solanum nigrescens preparation. Journal of Ethnopharmacology 22. 307-313. Holdsworth, D. (1975) Traditional medicinal plants used in the treatment of Malaria and fevers in Papua New Guinea. Papua New Guinea Medical Journal 18, 142-148. Lennette, E.H., Balows, A., Hansler, Jr. W.J., and Shadomy, H.J. (1985) (Eds.) Manual of Clinical Microbiology. Washington, D.C.. American Society of Microbiology. pp. 143-153. Lozoya, X., and Zolla, C. (1984) Medicina Traditional en MPxice. B-letin Oticina Sanitaria Panamericana 96, 360-364. Lozoya, X., Vehizquez, G., and Flares, A. (1988) La Medicina Traditional en Mexico: Experiencia del Programa IhISSCOPLAMAR 1982-1987. Instituto Mexican0 de1 Seguro Social, Mexico, p. 13. Mahmood, U., Thakun, R., and Blunden, G. (1983) Neochlorogenin, neosolaspigenin and sclaspigenin from Solanum torvum leaves. Journal of Natural Products 46. 427-428.
132 Miranda, (1976) La Vegetacidn de Chiapus. Vol. 2. Gobierno de1 Estado, Chiapas, Mexico p. 22. Ogunlana, E.D., and Ramstad, E. (1975) Investigations into the antibacterial activities of local plants. Planta Medica 27. 354-356.
Research
127-150.
Stimson,
Okunji, C.O., Okeke. C.N.. Gugnani, H.C. and Iwu, M.M. (1990) An antifungal spirostanol saponin from fruit pulp of Dracaena
Rios, J.L.. Recio, M.C. and Villar, A. (1988) Screening methods for natural products with antimicrobial activity: a review of the literature. Journal of Ethnopharmacology 23.
mannii.
international
Journal
qf
Crude
Drug
28, 193- 199.
A. (1954) Les plantes medicinales du Camgodge, da Laos et du Vietnam. Vol. 2, Archives des researches Agronomiques et Pastorales au Vietnam, Vietnam, p. 195.
Petelot,
Quisumbing, E. (1951) Medicinal Plants of the Philippines. Technical Bulletins 16, Department of Agriculture and Natural Resources, Manila, Philippines, p. 126.
W.R. (1971) Ethnobotanical
Lloydia
notes from Puerto
Rico.
34, 165- 170.
Solis, G. (1980) Recopilacidn de Plantas Medicinales de 10s Altos de Chiapas. lnstituto National Indigenista, Chiapas, Mexico, p. 15. Verbist, J.F.. Monnet, R. and Dobremez. J.R. (1977) Identification and quantification of steroid alkaloids from Nepalese Solanum species. Planta Medica II, 40-42. Zurita, en
M., and Zolla, C. (1986) Enfermedades la medicina
Sanitaria
traditional
Panamericana
de
MPxico.
101. 339-345.
dermatolbgicas
Boletin
Oficina
