Antimicrobial Activity and Pharmaceutical Development of Essential Oil from Hyptis suaveolens

Transcription

1 Antimicrobial Activity and Parmaceutical Development of Essential Oil from Hyptis suaveolens S. Okonogi, S. Cansakaow, S. Vejabikul and V. Cuamanocan Faculty of Parmacy Ciang Mai University Ciang Mai, Tailand J. Lerpokanont Payao Hospital Ampur Muang Payao, Tailand P. Taravicitkul Faculty of Medicine Ciang Mai University Ciang Mai, Tailand A. Nakano and F. Ikegami Graduate Scool of Parmaceutical Sciences Ciba University Ciba Japan Keywords: antibacterial, antifungal, dermatological, volatile oil, formulation, stability, skin, irritation, imum inibitory concentration, MIC Abstract Volatile oil distilled from te overground part of Hyptis suaveolens was preliary quality controlled by GC. Te antibacterial activity of tis volatile oil was tested against various kinds of bacteria and fungi tat caused dermatological diseases by agar dilution metod. It was found tat te volatile oil from H. suaveolens inibited certain bacteria and fungi. A number of cream bases were prepared and evaluated for te satisfactory pysical properties and stability. Te creams containing 30% w/w of oil in four selected bases were tested for teir stability, antimicrobial activity and primary irritation test using Garamycin cream (0.1% gentamicin) as a positive control. It was found tat all formulations sowed a moderate antibacterial activity. INTRODUCTION Hyptis suaveolens (Labiatae) is a medium aromatic srub found in te tropics and subtropics, and distributed as te aggressive annual weedy species in te nortern part of Tailand. Te genus Hyptis is known to be used for traditional medicine as an anticancer (Mabberley, 1990), and antifertility agent in females (Oliver-Bever, 1986) and for te treatment of gastrointestinal infections, cramp, and pain, as well as in te treatment of skin infection (Correa, 1931). Some previous works regarding te compositions and antifungal (Pandey, 1982; Sing et al., 1992; Zollo-Amvan et al., 1998), antibacterial (Asekum and Ekundaya, 2000; Iwu et al., 1990) and anticonvulsant activities (Aka et al., 1993) of H. suaveolens leaf oil. It was reported tat te major compositions were 1,8- cineole, sabinene, β-caryopyllene, limonene and β-pellandrene depending on te geograpic factors of plant collection. Te present study is a part of our ongoing works on te researc and development of te Tai medicinal plants. We now report te antimicrobial activity of te volatile oil from H. suaveolens growing in te nortern part of Tailand and te development of a topical cream of H. suaveolens tat could serve as an antiseptic agent for te treatment of or skin infection. MATERIALS AND METHODS Plant Material and Preparation of H. suaveolens Oil H. suaveolens plants were collected at te flowering stage from Ciang Mai province, Tailand in September to December Te plant samples were identified and te voucer specimens were deposited in te Herbarium of Faculty of Parmacy, Ciang Mai University, Tailand. Proc. WOCMAP III. Vol. 4: Targeted Screening of MAPs, Economics & Law Eds. C. Franz, Á. Máté, L.E. Craker and Z.E. Gardner Acta Hort. 678, ISHS

2 Te volatile oil was obtained by te ydrodistillation (3) from fres aerial plants. Te oil was separated from te aqueous solution, dried by treating wit anydrous Na 2 SO 4, cooled into glass vials and stored in refrigerator at 2-8 C until used. Oil samples were preliary quality controlled by GC Simadzu-GC 14B instrument employing te following conditions: column: DB-1 (J and W Scientific), carrier gas: He (40 ml/), detector: FID, temp. program: initially at 60 C wit an increase of 7 C/ until 250 C and time: 25. Antimicrobial Activity Test Te antibacterial activity of volatile oil was tested by te well diffusion metod against S. aureus (ATCC 29213) and E. coli (ATCC 25922). Te well diameter was 12 mm. Te diameter of zones of inibition was measured after - incubation. Te imum inibitory concentration (MIC) of te oil was detered by using te agar dilution metod against S. aureus (ATCC 29213); 2 isolates of S. aureus from patients, S. aureus (NP3HC) and S. aureus (PCD); and 2 isolates of S. pyogenes from patients, S. pyogenes (P31) and S. pyogenes (P183). Te temperature of incubation was 37 C. Using 5-mm diameter well diffusion metod did te antifungal activity of te essential oil. Te test organisms were Tricopyton mentagropytes and T. rubrum isolated from dermatological patients. Te diameter of zones of inibition was measured after 5-7 days incubation at 25 C. Te experiment of all tests was done in tree replications. Formulation and Stability Test of Cream Bases Several cream bases containing different kinds of ingredients were prepared using two-pase eat system. Te oil soluble ingredients and aqueous soluble ingredients were weiged and placed into two separated containers. Te oil pase was melted to about 78 C. At te same time, te aqueous pase was eated to 75 C. Te melted oil pase was added into te ot aqueous pase wit constant stirring until forg good emulsion. Te bases were slowly cool down to room temperature. All cream bases were tested for teir pysical stability by using 6 cycles of eating and cooling acceleration. One cycle is defined as alteration of storage condition from 45 C for to 4 C for. Using ph paper strip and Brookfield viscometer detered te pysical parameters of cream bases suc as ph and average viscosity, respectively. Formulation and Biological Activity of H. suaveolens Creams Formulations containing 10, 20, and 30% w/w of H. suaveolens oil in te selected cream bases were prepared. Te antibacterial activity of tose formulations was tested against S. aureus (ATCC 29213) by a well diffusion metod. Te commercial product, Garamycin cream was used as a positive control and te cream bases were also used as negative controls. Stability Test of H. suaveolens Creams Te fresly prepared H. suaveolens creams wit good appearance and biological activities were selected. Eac cream, after fresly prepared, was divided and put into tree containers. Te stability test was carried out wit tree conditions; 4 C (in a refrigerator) for 3 monts, 30 C (at room temperature) for 3 monts, and 6 cycles of eating (45 C for ) and cooling (4 C for ) accelerated condition. Te cange in pysical properties of te creams suc as color, creag, and cracking was observed. Te release beavior wit antibacterial activity of te creams after storage in eac condition was also investigated using S. aureus (ATCC 29213) as a test organism. Primary Skin Irritation Test Te primary skin irritation test was performed in te ealty albino rabbits (Mc Gees and Steinberg, 1987) wit an average body weigt of 3.5 kg. Te patces containing H. suaveolens cream were applied to te air-saven dorsal site of te rabbit for 4. Te signs of irritaion suc as erytema, papules and edema were observed

3 ,, and after patc removal. Te cream bases were used as negative controls. Te solution containing 10, 30, 50, 70, 80% v/v of H. suaveolens oil in olive oil were also tested for skin irritation, in te same manner as creams, using an olive oil as a negative control. RESULTS AND DISCUSSION Te yield of H. suaveolens oil obtained from ydrodistillation was about 0.21%. Te pysical appearance of te oil was pale yellow clear liquid and acid to litmus paper. It demonstrated density and viscosity of about 0.88 g/ml and 2.54 centipoise, respectively. It was previously reported tat te essential oil of H. suaveolens exibited several compositions caracterized by te occurrence of β-caryopyllene (Nigeria), 1,8-cineol and sabinene (India and Aruba), 1,8-cineol and β-pinene (Brazilian Amazonian and Brazilian Norteast) (Luz et al., 1984; Fun and Svendsen, 1990; Mallavarapu et al., 1993; Amed et al., 1994). H. suaveolens oil obtained in tis study sowed several components (Fig. 1). Te major components identified by autentic samples were β-caryopyllene, 1,8-cineol, and pellandrene at te retention time of , 6.916, and 5.840, respectively. Te differences in te oil composition could be due to te geograpic factors as mentioned above and te collection time (Azevedo et al., 2001). In our study, te essential oil was obtained from te plant collected at flowering time in te nortern part of Tailand. Te essential oil of H. suaveolens displayed significantly inibitory activity against all bacteria and fungi used in te study (Table 1). Te inibition was stronger to gram positive tan gram negative bacteria. Altoug te smaller well was used, te diameter of inibition zones against fungi, particularly T. rubrum was almost as wide as tat of bacteria in te bigger wells. Tese results sowed tat H. suaveolens oil was very active against tose dermatopytes. Te imal inibitory concentration (MIC) for H. suaveolens oil was detered for four bacteria. Te MIC values were between mg/ml based on te species and variety of tested organisms (Table 2). Factors tat affect te release of an active principle from a base include its affinity for te base and te viscosity of te preparation (Florence and Attwood, 1990; Oyedele et al., 2000). Moreover, te good appearance wit ph similarly to te skin and igly stable sould be considered for te suitable base. Te color of all cream bases fresly prepared in tis study was wite. Te ph and average viscosity of te bases varied between 6-7 and ,000 centipoise depending on te amount and kinds of ingredients in eac base. Altoug 17 bases were prepared, we found tat only four formulations (Table 3) were stable after stability test. Terefore, tose four formulations were selected for furter studies. Creams containing 10, 20, and 30% H. suaveolens oil in eac selected base were prepared. Te ph of all formulations sowed an optimum value, similar to te ealty skin ph, of 5-6, lower tan tat of te bases. Tis could be due to te low ph of te oil. Te fresly prepared creams exibited te antimicrobial activity wereas te bases sowed no activity. Te antibacterial effect of te creams increased wit increase in H. suaveolens oil content (Table 4). Te nature of base in wic a drug is formulated as considerable effect on its efficacy (Omotoso et al., 1986; Florence and Attwood, 1990). Te sligtly wider inibition zone of formulation 1 can be attributed to te low viscosity of te cream base. Te oter cream bases of iger viscosity would retard diffusion of te active ingredient into te agar. Garamycin cream sowed markedly iger activity tan H. suaveolens creams containing 10 or 20% oil but sligtly similar to te cream wit 30% oil. Te results from rabbit skin irritation test sowed a well-defined erytema, a significant sign of irritation, (Mazulli and Maiback, 1987) from H. suaveolens oil at a concentration iger tan 70% (Table 5). Te above-mentioned results of antibacterial activity of creams demonstrated tat te iger content of te oil, te iger activity of te cream. Terefore, te concentration of 30% oil in cream base was considered to be suitable for cream formulations for furter studies. After storage in refrigerator of 4 C for 3 monts, H. suaveolens formulations 165

4 sowed no canges in pysical properties. A cange in color from wite to creamy-wite or pale yellow was observed in tose creams after storage in room temperature. Te color cange was muc obviously in creams after accelerated condition. Tis result suggested tat te iger temperature or stress condition could cause a degradation of some ingredients in te creams. Moreover, te stress condition could induce te agglomeration of te oil globules dispersed in te creams. Te antibacterial activity of te creams stored in eiter 4 C or 30 C demonstrated no difference from fres creams (Table 6). However sligtly decrease in activity was found in creams after an accelerated condition. Tis was considered tat te stress condition caused te evaporation of te separated volatile oil. Terefore, te oil content of eac cream was in lower concentration and producing te smaller diameter inibition zone. Te results from irritation test of H. suaveolens creams and bases sowed neiter erytema nor edema in dorsal skin of rabbits. As te concentration of H. suaveolens oil in te cream bases was 30%, tis result was in good agreement wit te result from irritation test of oil solution tat no reaction occurred wen te oil content was lower tan 80%. CONCLUSION Te essential oil of H. suaveolens growing in te nortern part of Tailand displayed significant inibitory activity against several type strain and clinical isolate bacteria and fungi tat caused dermatological diseases. Tree major components, β- caryopyllene, 1,8-cineol, and pellandrene were identified from te oil. Te solution containing oil content lower tan 80% exibited no sign of skin reaction. Semisolid formulations as o/w creams of te oil were designed in four selected suitable bases. Remarkable antibacterial effects, similarly to Garamycin te commercial product, were demonstrated at 30% H. suaveolens oil in some bases. It was found tat te properties of base into wic te oil was incorporated affected its activity. ACKNOWLEDGEMENTS Te autors would like to tank NRCT Tailand for financial support. Literature Cited Aka, P.A. and Nwambie, A.I Nigerian plants wit anti-convulsant property. Fitoterapia 64: Amed, M., Scora, R.W. and Ting, I.P Composition of leaf oil of Hyptis suaveolens (L.) Poit. J. Essent. Oil Res. 6: Asekum, O.T. and Ekundayo, O Essential oil constituents of Hyptis suaveolens (L.) Poit. (bus tea) leaves from Nigeria. J. Essent. Oil Res. 12: Azevedo, N.R., Campos, I.F.P., Ferreira, H.D., Portes, T.A., Santos, S.C., Serapin, J.C., Paula, J.R. and Ferri, P.H Cemical variability in te essential oil of Hyptis suaveolens. Pytocem. 57: Correa, M.P Discionario das Plantas Uteis e das Exoticas Cultivadas. Imprensa, Nacional, Rio de Janeiro. Din, L.B., Zakaria, Z., Sandudin, M.W., Bropy, J. and Toia, R.F Composition of te stem volatile oil from Hyptis suaveolens. Pertanika 11: Florence, A.T. and Attwood, D Pysicocemical Principles of Parmacy. 2 nd ed., Macmillan, Basingtoke. Fun, C.E. and Svendsen, A.B Te essential oil of Hyptis suaveolens Poit. grown in Aruba. Flav. Flag. J. 5: Iwu, M.M., Ezeugwu, C.O., Okunji, C.O., Sanson, D.R. and Tempesta, M.S Antimicrobial activity and terpenoids of te essential oil of Hyptis suaveolens. Int. J. Crude Drug Res. 28: Luz, A.I.R., Zogbi, M.G.B., Ramos, L.S., Maia, J.G.S. and Da Silva, M.L Essential oils of some Amazonian Labiatae, I. Genus Hyptis. J. Nat. Prod. 47: Mallavarapu, G.R., Rames, S., Kaul, P.N., Battacarya, A.K. and Rao, B.R.R Te essential oil of Hyptis suaveolens (L.) Poit. J. Essent. Oil Res. 5:

5 Marzulli, F.N. and Maiback, H.I Dermatotoxicology. 3 rd ed., Hemispere Publising Corporation, New York. p.758. Mc Gees, A.H. and Steinberg, M Skin irritation testing in animals. p In: F.N. Marzulli and H.I. Maiback (eds.), Dermatotoxicology, 3 rd ed., Hemispere Publising Corporation, New York. Mabberley, D.J Te Plant-Book. Cambridge University Press, London. Oliver-Bever, B Medicinal Plants in Tropical West Africa. Cambridge University Press, London. Omotoso, J.A., Wateley, T.L., Law, T.K. and Florence, A.T Te nature of te oil pase and te release of solutes from multiple w/o/w emulsion. J. Parm. Parmacol. Lett. 10:9-12. Oyedele, A.O., Orafidiya, L.O., Lamikanra, A. and Olaifa, J.I Volatility and mosquito repellancy of Hemizygia welwitscii oil and its formulations. Insect Sci. Applic. 20: Pandey, D.K., Tripati, N.N., Tripati, R.D. and Dixit, S.N Fungitoxic and Pytotoxic properties of te essential oil of Hyptis suaveolens. J. Plant Dis. Prot. 89: Sing, G., Upadyay, R.K. and Rao, G.P Fungitoxic activity of te volatile oil of Hyptis suaveolens. Fitoterapia 63: Zollo-Amvam, P.H., Biviti, L., Tcoumbougnang, F., Menut, C., Lamaty, G. and Boucet, P Aromatic plants of tropical Central Africa. Part XXXII. Cemical composition and antifungal activity of tirteen essential oils from aromatic plants of Cameroon. Flav. Flag. J. 13: Tables Table 1. Antimicrobial activity of H. suaveolens oil. Test organisms Inibition zone (mm) S. aureus (ATCC 29213) 27 a E. coli (ATCC 25922) 22 a T. mentagropytes 15 b T. rubrum b a well diameter was 12 mm, b well diameter was 5 mm Table 2. Te MIC of H. suaveolens oil. Test microorganism MIC (mg/ml) S. aureus (ATCC 29213) 8.82 S. aureus (NP3HC) 8.82 S. aureus (PCD) S. pyogenes (P31) 4.41 S. pyogenes (P183)

6 Table 3. Te appearance and some pysical parameters of te selected bases. Formulation Type of Viscosity Emulsifying agent Color ph cream (cp) 1 o/w Sodium lauryl sulfate wite o/w Sodium lauryl sulfate wite o/w Trietanolae stearate wite o/w Trietanolae stearate wite Table 4. Antibacterial activity of te selected creams containing various percentage of H. suavelens oil. Formulation Base a Inibition zones (mm) 10% 20% 30% 1 nz nz nz nz Garamycin cream b 27 a nz = no inibition zone b Garamycin cream contains 1% gentamicin Table 5. Te results of skin irritation test of solution containing various percentage of H. suaveolens oil. Response 30 0% a 10% 30% Erytema b Edema c % 70% 80% Response Erytema b Edema c a olive oil 100% b 0 = no erytema, 2 = well-defined erytema c 0 = no edema

7 Table 6. Antibacterial activity of H. suaveolens creams before and after stability test. Condition* Formulation Inibition zones (mm) Before After * 1 = 4 C, 2 = 30 C, 3 = eating and cooling accelerated condition Figures Fig. 1. GC cromatogram of H. suaveolens oil. 169