CHEMICAL COMPOSITION AND ANTI-INFLAMMATORY ACTIVITY OF ESSENTIAL OILS FROM THE LEAVES OF OCIMUM BASILICUM L. AND OCIMUM GRATISSIMUM L. (LAMIACEAE)

CHEMICAL COMPOSITION AND ANTI-INFLAMMATORY ACTIVITY OF ESSENTIAL OILS FROM THE LEAVES OF OCIMUM BASILICUM L. AND OCIMUM GRATISSIMUM L. (LAMIACEAE)

B. C. Okoye-Festus ^{* 1}, O. Obonga Willfred ², A. Onyegbule Felix ¹, I. Okoro Ogheneogaga ², Okokpedi Peace ², I. Nwosu Ngozi ², Ajibo Jane ² and O. Ndu Okechukwu ³

Department of Pharmaceutical and Medicinal Chemistry ¹, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.

Department of Pharmaceutical and Medicinal Chemistry ², Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, 410001, Enugu state, Nigeria.

Department of Pharmacology and Toxicology ³, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, 410001, Enugu state, Nigeria.

ABSTRACT: Ocimum basilicum L. and Ocimum gratissimum L. (Lamiaceae) are widely distributed aromatic herbs used in ethnomedicinal management of a range of inflammatory disorders. In the present work, we evaluated the topical anti-inflammatory effects of the volatile constituents extracted from the fresh leaves of these plant species. Fresh leaves of the plants were subjected to hydro distillation to obtain the volatile oils OBV and OGV from O. basilicum and O. gratissimum respectively.  The fresh leaves were also extracted with n-hexane to obtain OBHE and OGHE respectively. OBV, OGV, OBHE, and OGHE were screened for anti-inflammatory effect using xylene-induced ear edema as a model of inflammation. Their chemical constituents were also analyzed using GC/MS apparatus. At 50 µg/ear OBV, OGV, OBHE and OGHE exhibited significant (P<0.05) topical anti-inflammatory effect with edema inhibitions of 50.0, 63.3, 62.7 and 80 % respectively. The effects were comparable (P<0.05) with that of 100 µg/ear hydrocortisone (% edema inhibition of 54.8). 11 of the compounds from OGV are monoterpenes while 4 are sesquiterpenes. 8 of the compounds identified in OGHE are oxygenated monoterpene derivatives, 5 are sesquiterpenes, and the others are long chain carboxylic acid, eugenol and phthalate derivatives. OGV and OGHE contain linalool, 1-terpinen-4-ol, alpha-caryophylene and trans-longipinocarveol in common. Compounds identified in OBV include 2 monoterpenes, 7 oxygenated monoterpene derivatives, 2 sesquiterpenes, a long chain monocarboxylic acid and a triterpene, alpha-amyrin. OBV and OBHE contain eugenol acetate in common. Some of these identified volatile constituents may be contributing to the observed anti-inflammatory effects.

Ocimum basilicum, Ocimum gratissimum, Essential oils, Topical anti-inflammatory, Xylene-induced ear edema, GC-MS

INTRODUCTION: Ocimum basilicum L. and Ocimum gratissimum L. (Lamiaceae) are widely distributed aromatic herbs ¹.

Ocimum basilicum (Sweet basil) is an erect herb that grows to a height of 90 cm. The stem is purplish, and the leaves are lanceolate, glossy, and fragrant. The flowers are tubular, bilabiate, purplish, and packed in whorled racemes. Ocimum gratissimum (Wild basil) is also a perennial herb with erect stem and can grow up to 1-3 m tall. The stem is round glandular, much branched, glabrous or pubescent and woody at the base. The leaves are opposite, slender and the left blade elliptical to ovate ². The leaves of Ocimum basilicum have been used in ethnomedicine for a variety of ailments ranging from respiratory disorders ³, fever ⁴, as a remedy for gonorrhea, catarrh conditions, cough, constipation, dysentery, ringworm, carminative and hypertension ^{4, 5}. The whole plant and the essential oil of Ocimum gratissimum have many applications in traditional medicine, especially in Africa and India. Preparations from the whole plant are used as stomachic and in treating sunstroke, headache, and influenza. The seeds have laxative properties and are prescribed against gonorrhea. The essential oil is applied against fever, inflammations of the throat, ears or eyes, stomach pain, diarrhea and skin diseases ^{1, 2, 6, 7} and previous studies have confirmed its hypoglycaemic effect ⁸.

Although there are reported studies on the anti-inflammatory effect of some species of Ocimum ^9-11, there is to date no known scientific study validating the ethnomedicinal use of Ocimum basilicum and Ocimum gratissimum in inflammatory conditions. Inflammatory mycoses, a clinical condition associated with inflammation, fungal and bacterial infection is commonly treated with a combination of the anti-inflammatory, antifungal and antibacterial agent in one formulation ^12-15.

There are several reported studies on the antimicrobial potentials of Ocimum species ^16-27. Our aim in the present study is thus to evaluate the topical anti-inflammatory potentials of the two Ocimum spp with the view of ascertaining their suitability for use in topical herbal formulations for inflammatory mycoses. We have also adopted two methods of essential oil extraction namely hydrodistillation and solvent extraction.

MATERIALS AND METHODS

Plant Material: Fresh leaves of Ocimum basilicum and Ocimum gratissimum were purchased from a local market in Nsukka, Enugu State Nigeria. The leaves were chopped into smaller pieces and used immediately for essential oil extraction.

Extraction of the Essential Oils: About 600 g fresh leaves of Ocimum basilicum and Ocimum gratissimum were coarsely milled and the volatile constituents isolated by hydro-distillation for 4 h using Clevenger apparatus. The volatile oil fractions OBV and OGV respectively were recovered. About 200 g each of coarsely milled fresh leaves of Ocimum basilicum and Ocimum gratissimum were extracted in 500 mL hexane for 48 h with constant checking at room temperature. The hexane extracts were concentrated in vacuo to yield the volatile oil fractions OBHE and OGHE respectively. OBV, OGV, OBHE, and OGHE were stored in the refrigerator (0 ºC) before topical anti-inflammatory tests and analysis of the constituents with GC/MS apparatus.

Gas Chromatography-Mass Spectrometry: OBV, OGV, OBHE, and OGHE were analyzed by GC/MS (GCMS-QP2010 PLUS, Shimadzu Japan). Operating conditions were as follows: carrier gas, helium with a flow rate of 2 ml/min; column temperature, 60-280 ºC at 5 ºC/min; injector and detector temperatures, 250 ºC; injected volume 2 μl; split ratio, 1:50. The MS operating parameters were as follows: ionization potential, 70 eV; ionization current, 1A; ion source temperature, 200 ºC and resolution of 1000.

Identification of Compounds: Identification of components in OBV, OGV, OBHE, and OGHE were based on a comparison of the retention indices and computer matching of MS fragments with the NISTO5.LIB.

Topical Anti-inflammatory Tests: The effect of OBV, OGV, OBHE, and OGHE on acute topical edema was evaluated by a modification of previously reported methods ^{28, 29}. Adult albino mice (20 ± 5g) of either sex were divided into groups of 5 animals. The treatment groups received the extracts dissolved in xylene at doses of 50, 100, 200 and 400 μg/ear applied on the anterior surface of the right ear. Control animals received either equivalent volume of the phlogistic agent (xylene) or hydrocortisone dissolved in xylene (100 μg/ear). Two hours after application, the mice were sacrificed and both ears removed.

Circular sections (5mm) of both the right (treated) and left (untreated) ears were punched out using a cork borer and weighed. Edema was quantified as weight differences between the two earplugs. The anti-inflammatory activity was evaluated as percent edema inhibition in the treated animals relative to the control animals ^{28, 29} using the relation:

Where Rt = mean weight of right earplug of treated animals; Lt = mean weight of left earplug of treated animals; Rc = mean weight of the right earplug of control (vehicle treated) animals; Lc = mean weight of the left earplug of control (vehicle treated) animals.

Statistical Analysis: Results of the anti-inflammatory effect obtained were analyzed by SPSS version 11 using one way ANOVA and subjected to Fischer LSD post hoc tests and expressed as mean ± SEM. Differences between means were considered significant at P<0.05.

RESULTS:

Anti-inflammatory Activity: The result of the topical anti-inflammatory screening of the volatile oils is shown in Table 1 and 2. At the dose of 50 μg/ear, all the tested oils exhibited significant (p<0.05) inhibition of edema induced by topical application of hexane. OGHE showed better anti-inflammatory effect than OGV at the tested doses, while OBV showed better anti-inflammatory effect than OBHE. When compared with hydrocortisone, OGHE and OBV exhibited the better anti-inflammatory effect. The anti-inflammatory effect exhibited by the oils is however not dose-dependent.

TABLE 1: EFFECT OF THE VOLATILE OILS FROM OCIMUM GRATISSIMIUM LEAVES ON XYLENE-INDUCED EAR EDEMA IN MICE

OGV = Volatile oil from Ocimum gratissimium leaves obtained by hydrodistillation

OGHE = Volatile oil from Ocimum gratissimium leaves obtained by solvent (hexane) extraction.

The test materials were applied topically at the stated doses. Treatment animals were compared to control animals which had received vehicle only or prednisolone;

*P<0.05, **P<0.01, n = 5.

TABLE 2: EFFECT OF THE VOLATILE OILS FROM OCIMUM BASILICUM LEAVES ON XYLENE-INDUCED EAR EDEMA IN MICE

OBV = Volatile oil from Ocimum basilicum leaves obtained by hydrodistillation

OBHE = Volatile oil from Ocimum basilicum leaves obtained by solvent (hexane) extraction

The test materials were applied topically at the stated doses. Treatment animals were compared to control animals which

had received vehicle only or prednisolone.

*P<0.05, **P<0.01, n = 5

Volatile Oil Compositions of the Leaves: The results of the chemical composition of the volatile oils extracted from the two species of Ocimum by solvent extraction and hydrodistillation are shown in Table 3 and 4. A total of 15 compounds were identified in OGV as against 18 compounds identified in OGHE. 11 of the compounds from OGV are monoterpenes while 4 are sesquiterpenes. 8 of the compounds isolated from OGHE are oxygenated monoterpene derivatives, 5 are sesquiterpenes, and the others are long chain carboxylic acid, eugenol and phthalate derivatives. OGV and OGHE contain linalool, 1-Terpinen-4-ol, alpha-caryophyllene and trans longipinocarveol in common. A total of 14 and 11 compounds were identified in OBV and OBHE respectively. Compounds identified in OBV include 2 monoterpenes, 7 oxygenated monoterpene derivatives, 2 sesquiterpenes, a long chain monocarboxylic acid and a triterpene, alpha-amyrin. OBV and OBHE contain eugenol acetate in common.

TABLE 3: CHEMICAL COMPOSITION OF THE VOLATILE OILS EXTRACTED FROM OCIMUM GRATISSIMIUM AND LEAVES

OGV = Volatile oil from Ocimum gratissimium leaves obtained by hydrodistillation

OGHE = Volatile oil from Ocimum gratissimium leaves obtained by solvent (hexane) extraction

TABLE 4: CHEMICAL COMPOSITION OF THE ESSENTIAL OIL ISOLATED FROM OCIMUM BASILICUM LEAVES

OBV = Volatile oil from Ocimum basilicum leaves obtained by hydrodistillation

OBHE = Volatile oil from Ocimum basilicum leaves obtained by solvent (hexane) extraction

DISCUSSION: The whole leaves and essential oil of various species of Ocimum have several applications in traditional medicines, especially in Africa and India. Previous studies have validated the hypoglycaemic effect ⁸, antibacterial ^21-27, antinociceptive and anti-inflammatory ^{10, 11} properties of some Ocimum species. Our present studies have shown that the essential oils from Ocimum gratissimum and Ocimum basilicum possess significant topical anti-inflammatory effect in xylene-induced mouse ear edema model. The anti-inflammatory activity was found to vary based on the method of essential oil extraction, which has a direct bearing on the chemical compositions. In general, the activity increases with increase in the proportion of some essential oils like eugenol, linalool, D-fenchone, 1-terpene-4-ol, thymol, alpha-caryophyllene and the presence of diterpenes and triterpenes, which have earlier been reported to possess anti-inflammatory activity ^30-35.

Essential oils obtained from Ocimum gatissimum by solvent (n-hexane) extraction (OGHE) showed better topical anti-inflammatory activity than the oils obtained from the same plant species by hydro-distillation (OGV). Conversely, the essential oils obtained from Ocimum basilicum by hydro-distillation (OBV) showed better anti-inflammatory activity than the oils obtained from the same plant species by solvent (n-hexane) extraction (OBHE). These observations can be correlated with the chemical compositions of the essential oils from both plant species isolated by the two reported methods Table 3 and 4. OBV and OBHE both contained eugenol derivatives previously reported to possess significant anti-inflammatory activity ^{36, 37}. OBV, however, also contained several other compounds like eucalyptol, linalool, borneol acetate, alpha-bergamot, germacrene and a triterpenoid alpha-amyrin Table 4. These compounds have been reported to exhibit anti-inflammatory activity ^38-40 or detected in essential oils reported exhibiting anti-inflammatory activity ^{41, 42}. This might explain the significantly higher anti-inflammatory activity of OBV compared to OBHE. OGV and OGHE both contained linalool, alpha-caryophyllene, (-) alpha Pangasinan and 1-terpinene-4-ol along with other volatile constituents like alpha-thujone, camphene, borneol, verbenone, D-Fenchone, alpha cardinal, etc. Table 3. These volatile constituents have been detected in some essential oil isolated from plants with anti-inflammatory activity ^{41, 42}.

The presence of a eugenol derivative (Dehydrodihydrodiisoeugenol), a diterpene (Eudesma-4(14), 11-diene) and Di-n-octyl phthalate in OGHE might be the plausible explanation for the observed better anti-inflammatory activity that OGV. It is also an interesting observation that OGHE and OBV (at 50 μg/ear) exhibited close to 2 times topical anti-inflammatory activity compared to indomethacin (100 μg/ear) Table 3 and 4. This observation thus supports the use of volatile oils obtained by solvent (n-hexane) extraction from Ocimum gratissimum and the oils obtained by hydro distillation from Ocimum basilicum for topical application in inflammatory conditions.

Previous studies have indicated the chemical variability in the composition of the essential oil extracted from Ocimum species. At least six chemotypes namely eugenol, thymol, citral, ethyl cinnamate, geraniol, and linalool have been identified, with eugenol being the most important economically ². In a recent study, phenylpropanoids were shown to be the major constituents of Ocimum gratissimum and Ocimum basilicum ⁴³.

Specifically, in the study, methyl chavicol and linalool were shown to be the major constituent of Ocimum basilicum along with bicyclogermacrane and alpha-terpineol. Also, in the study the major constituents in Ocimum gatissimum were eugenol, ^{1, 8} cineole, germacrene D and beta-caryophyllene. Our current results show somewhat a slightly different variability. Linalool, germacrene, alpha-terpineol were also shown present in Ocimum basilicum along with eugenol derivatives, while caryophyllene and ^{1, 8} cineole were shown present in Ocimum gratissimum along with linalool and alpha-terpineol.

Our results so far have shown that the volatile oils isolated from Ocimum basilicum and Ocimum gatissimum show varying degrees of topical anti-inflammatory activity. This supports the ethnomedicinal uses of these plant materials in the management of inflammatory disease states. These plant materials have also been reported to possess significant antifungal and antibacterial activities. Our work thus supports there use as topical agents in the management of inflammatory mycoses.

ACKNOWLEDGEMENT: The authors are grateful to National Research Institute for Chemical Technology, Zaria, Kaduna State, Nigeria for the measurement of GC/MS spectra.

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Festus-Okoye BC, Willfred OO, Felix A, Ogheneogaga IO, Peace O, Ngozi IN, Jane A and Ndu-Okechukwu O: Chemical composition and anti-inflammatory activity of essential oils from the leaves of ocimum basilicum l. and ocimum gratissimum l. (lamiaceae). Int J Pharmacognosy 2014; 1(1): 59-65. doi: 10.13040/IJPSR.0975-8232.1(1).59-65.

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