ABSTRACT
In South Africa, Centella asiatica. (L.) Urb is used traditionally for the treatment of various diseases. Analyses of the essential oil of this medicinal plant revealed 11 monoterpenoid hydrocarbons (20.20%), nine oxygenated monoterpenoids (5.46%), 14 sesquiterpenoid hydrocarbons (68.80%), five oxygenated sesquiterpenoids (3.90%), and one sulfide sesquiterpenoid (0.76%). α.-Humulene (21.06%), β.-caryophyllene (19.08%), bicyclogermacrene (11.22%), germacrene B (6.29%), and myrcene (6.55%) were the predominant constitutes. The essential oil extract exhibited a broad spectrum of antibacterial activities against Gram-positive (Bacillus subtilis, Staphylococcus aureus.) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei.) organisms.
Introduction
Centella asiatica. (L.) Urb (Apiaceae), commonly called pennywort or gotu kola., is a perennial creeping weed commonly found in moist places (Fosberg et al., Citation1979; Whistler, Citation1988; Van Wky et al., Citation1997). In South Africa, the plant is used for the treatment of leprosy, wounds, cancer, fever, and syphilis. It is also used for the treatment of acnes and allergy (Van Wky et al., Citation1997).
A number of phytochemical and biological studies have been reported on the plant. In Europe, an extract from C. asiatica. was used for many years for the treatment of wounds (Maquart et al., Citation1999). It was believed that the extract contains three active triterpenes, namely, asiatic acid, madecassic acid, and asiaticoside, which have healing properties (Boiteau & Chanez, Citation1967; Van Wky et al., Citation1997; Maquart et al., Citation1999). Recent studies have led to the isolation of other triterpenes with healing potential, namely, terminolic acid, madecassoside, and asiaticosdie-B (Schaneberg et al., Citation2003). Sceffoleoside A and saponins (centellasaponins B, C, and D) with four ursane- and oleanane-type triterpene oligoglycosides were isolated from C. asiatica. growing in Sir Lanka by Matsuda et al. (2001). Extensive clinical investigation on C. asiatica. has led to the use of the plant in managing diabetics (Cesarone et al., Citation2001), hypertension, and edema (De Sanctis et al., Citation2001; Incandela et al., Citation2001aCitationb) in addition to its evaluations in wound treatment. The antitumor and cytotoxic properties of the crude extract and partially purified fractions were reported by Babu et al. (Citation1995). According to the authors, the partially purified extract was more effective on tumor cells than the crude extracts. Dermatologically, extracts of C. asiatica. has been used in scar management and in cosmetic formulation (Martelli et al., Citation2000; Widgerow et al., Citation2000).
The essential oil composition of C. asiatica. from Tokushima, Japan, was reported by Yoshinori et al. (Citation1982). The major constituent of the oil (36.4%) was an unidentified terpenic acetate, while other prominent constituents were β.-caryophyllene, trans.-β.-farnesene, and germacrene D.
There are no literature reports on the chemical constituents of C. asiatica. growing in South Africa, despite its extensive usage in folk medicine. This paper reports the chemical composition of the essential oil of C. asiatica., as compared to other studies, and its antimicrobial activity.
Materials and Methods
Plant samples
Fresh mature plants were collected along Tyumie river in Alice, South Africa, and voucher specimen (OYA 03/02, authenticated by Dr. DS. Grierson) was deposited at the university herbarium. The fresh plants was air-dried.
Isolation of the volatile oils
Essential oil was hydrodistillated from dried plant of C. asiatica. (500 g) using a Clevenger apparatus in accordance with the British Pharmacopoeia. (1980) method for 3 h.
Gas chromatography–mass spectrometry (GC-MS) analyses
GC-MS analysis of the oil was carried out on a Hewlett Packard gas chromatography HP 5973 (USA) interfaced with a VG analytical 70-280s double-focusing mass spectrometer. Electron ionization was at 70 eV with ion source temperature at 240°C. HP-5 column was used (30 m × 0.25 mm i.d.), which was similar to DB 5, film thickness was 0.25 µm, while helium was used as the carrier gas. The oven temperature was 70–240°C at 5°/min. The oil (0.2 µl) was injected into the GC-MS. n.-Alkanes were run at the same condition for the determination of Kováts indices.
Identification of constituents
Constituents of the oil were identified by comparison of their mass spectral pattern and retention (Kovát) indices with those of standard samples and literature data (Adams, Citation1989; Joulain & Koenig, Citation1998; ESO Citation1999).
Antibacterial test
A collection of five laboratory bacteria which included two Gram-positive and three Gram-negative strains, was obtained from this Microbiology Department, Rhodes University. These are Bacillus subtilis, Staphylococcus aureus., Escherichia coli., Pseudomonas aeruginosa., and Shigella sonnei.. The minimum inhibitory concentration (MIC) values of the essential oils were determined with a microplate dilution method against the bacteria using 96-well microtiter plates. Essential oil in 40 µl of hexane was dissolved in 40 µl of acetone. Each test organism was prepared by diluting 24-h-old broth culture with sterile nutrient broth. The culture was then diluted 100-fold to give approximately 106 bacteria ml−1. The microtiter plates were prepared using serial dilutions (Afolayan & Meyer, Citation1997; Amvam Zollo et al., Citation1998; Eloff, Citation1998) and incubated for 24–48 h at 37°C. As an indicator of bacterial growth, 40 µl of 0.2 mg/ml p.-iodonitrotetrazolium (INT) solution was added to each well and incubated at 37°C for 30 min. The colorless tetrazolium salt was reduced to a red-colored product by biological activity of the organisms, thereby making the inhibition of bacterial growth visible as clear wells. MIC values were recorded as the lowest concentration resulting in complete inhibition of bacterial growth. Each treatment was replicated three times. Streptomycin, chloramphenicol, solvents, and sample-free solutions were used as standard and blank controls.
Results and Discussion
A colorless mild scented oil of 0.06% (w/w) yield was obtained from the hydrodistillation of C. asiatica.. Forty constituents were identified from the GC-MS spectra accounting for 99.12% of the oil composition. The oil was characterized by a high concentration of sesquiterpenes (68.80%). α.-Humulene (21.06%), β.-caryophyllene (19.08%), bicyclogermacrene (11.22%), germacrene B (6.29%), and germacrene D (4.01%) were the dominant sesquiterpene constituents. The percentage composition of monoterpenes was 20.20% with myrcene (6.55%), γ.-terpinene (5.77%), and α.-pinene (3.49%) as the main constituents. The oxygenated mono- and sesquiterpene content were negligible (5.45% and 3.90%, respectively). A small amount of mintsulfide was also detected in the oil (). The unidentified constituents occurred only in small or trace amounts. In general, the germacrane group of compounds was conspicuous in the compositional profile of the essential oil (21.78%).
Table 1. Chemical composition of the essential oil of C. asiatica..
Comparing this study with that of Yoshinori et al. (Citation1982), both oils are dominated by sesquiterpenes, but compositional constituents differs. Also, only five monoterpenoids (1.6%) were present in the essential oil of the C. asiatica. growing in Japan, whereas 20 monoterpenoids are being reported in the South African plant.
The antibacterial tests also revealed that the essential oil of C. asiatica. had a broad-spectrum antimicrobial activity against all the tested organisms with MIC values ranging from 1.25 to 0.039 mg/ml (). The oil was, however, more active against the Gram-positive bacteria than the Gram-negative ones. This observation is particularly noteworthy because plants extracts are known to be more active against Gram-positive than Gram-negative bacteria (Rabe & Van Staden, Citation1997; Grierson & Afolayan, Citation1999; Afolayan, Citation2003).
Table 2. Antibacterial activity of the essential oil of C. asiatica..
In conclusion, the chemical profile of the essential oil of C. asciatica. has revealed high contents of germacrene compounds that are of economical significance. These compounds are known to be strong antimicrobial and antitumor agents, hence, their presence could have been the rationale behind the strong inhibitory effects on Gram-positive bacteria.
Acknowledgments
The authors are grateful to the NRF, South Africa, and the Lagos State University, Nigeria, for financial support.
References
- Adams RP ((1989):): Identification of Essential Oils by Ion Trap Mass Spectroscopy. New York, Academic Press.
- Afolayan AJ (2003): Extracts from the shoot of Arctotis arctotoides. inhibit the growth of bacteria and fungi. Pharm Biol 41: 22–25.
- Afolayan AJ, Meyer JM (1997): The antimcrobial activity of 3,5,7-trihydroxyflavone isolated from the shoot of Helichrysum aureonites.. J Ethnopharmacol 57: 177–181. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Amvam Zollo PH, Biyiti L, Tchoumbougnang F, Menut C, Lamaty G, Bessiere JM (1998): Aromatic plants of tropical Central Africa. XXXII. Chemical composition and antifungal activity of thirteen essential oils from aromatic plants of Cameroon. Flav Fragr J 13: 107–114. [CROSSREF]
- Babu TD, Kuttan G, Padikkala J (1995): Cytotoxic and anti-tumor properties of certain taxa of Umbelliferae with special reference to Centella asiatica. (L.) Urban. J Ethnopharmacol 48: 53–57. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Boiteau P, Chanez M (1967): Isolation of a new triterpenic acid from Centella asiatica. (L.) Urb. of Madagascar: Madecassic acid. CR Acad Sci Hebd Seances Acad Scid 9: 407–410. [CSA]
- British Pharmacopoeia. (1980): Her Majesty's Stationary Office: London, 11: A109.
- Cesarone MR, Incandela L, De Sanctis MT, Belcaro G, Bavera P, Bucci M, Ippolito E. (2001): Evaluation of treatment of diabetic microangiopathy with total triterpenic fraction of Centella asiatica.: A clinical prospective randomized trial with a microcirculatory model. Angiology 52 (Suppl 2): S49–54.
- De Sanctis MT, Belcaro G, Incandela L, Cesarone MR, Griffin M, Ippolito E, Cacchio M (2001): Treatment of edema and increase capillary filtration in venous hypertension with total triterpenic fraction of Centella asiatica.: A clinical, prospective, randomized, dose-ranging trial. Angiology 52 (Suppl 2): S55–59. [PUBMED], [INFOTRIEVE]
- Eloff JN (1998): A sensitive and quick microplate method to determine the minimum inhibitory concentration of plant extracts for bacteria. Planta Med 64: 711–713. [PUBMED], [INFOTRIEVE], [CSA]
- ESO 2000 (1999): The complete database of essential oil: B.A.C.I.S. The Netherlands.
- Fosberg FR, Sachet MH, Oliver RL (1979): A geographical checklist of the Micronesian dictoledonae. Micronesia 15: 200. [CSA]
- Grierson DS, Afolayan AJ (1999): Antimcrobial activity of some indigenous plants used for the treatment of wounds in the Eastern Cape, South Africa. J Ethnopharmacol 66: 103–106. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Incandela L, Cesarone MR, Cacchio M, De Sanctis MT, Santavenere C, D'Auro MG, Bucci M, Belcaro G (2001a): Total triterpenic fraction of Centella asiatica. in chronic venous insufficiency and in high-perfusion microangiopathy. Angiology 52 (Suppl 2): S9–13. [PUBMED], [INFOTRIEVE]
- Incandela L, Belcaro G, De Sanctis MT, Cesarone MR, Griffin M, Ippolito E, Bucci M, Cacchio M (2001b): Total triterpenic fraction of Centella asiatica. in treatment of venous hypertension: A clinical, prospective, randomized trial using a combined microcirculatory model. Angiology 52 Suppl 2: S61–67. [PUBMED], [INFOTRIEVE]
- Joulain D, Koenig WA (1998): The Atlas of Spectra Data of Sesquiterpene Hydrocarbons. Hamburg, E.B.-Verlag.
- Martelli L, Berardesca E, Martelli J (2000): Topical formulation of a new plant extract complex with refiming properties: Clinical and non-invasive evaluation in a double-blind trial. Int J Cosmetics Sci 22: 201–206. [CROSSREF]
- Matsuda H, Morikawa T, Ueda H, Yoshikawa M (2001): Medicinal foodstuffs. XXVII. Saponin constituents of gotu kola (2): Structure of new ursane and oleanane-type triterpene oligoglycosides, centellasaponins B, C and D, from Centella asiatica. in Sri Lanka. Chem Pharm Bull (Tokyo) 49: 1368–1371. [CROSSREF]
- Maquart FX, Chastang F, Simeon A, Birembaut P, Gillery P, Wegrowski Y (1999): Triterpenes from Centella asiatica. stimulate extracellular matrix accumulation in rat experimental wounds. Eur J Dermatol 9: 289–296. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Rabe T, Van Staden J (1997): Antibacterial activity of South African plants used for medicinal purposes. J Ethnopharmacol 56: 81–87. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Schaneberg BT, Mikell JR, Bedir E, Khan IA (2003): An improved HPLC method for quantitative determination of six triterpenes in Centella asiatica. extracts and commercial products. Pharmazie 58: 381–384. [PUBMED], [INFOTRIEVE]
- Whistler WA (1988): Checklist of the weed flora of Western Polynesia. Technical paper 194. Noumea, New Caledonia, South Pacific Commission, p. 38.
- Widgerow AD, Chait LA, Stals R, Stals PJ (2000): New innovations in scar management. Aesthetic Plast Surg 24: 227–234. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- van Wyk BE, van Oudtshoorn B, Gericke N (1997): Medicinal Plants of South Africa. Pretoria, Briza Publications, pp. 78–79.
- Yoshinori A, Reiko M, Tsunematsu T (1982): Mono- and sesquiterpenoids from Hydrocotyle. and Centella. species. Phytochemistry 21: 2590–2592. [CROSSREF]