Evaluation of biological activities of thymus seravschanicus and its main compounds

suMMARY

Thymus seravschanicus Klokov (Lamiaceae) is an aromatic medicinal plant native for Uzbekistan. This plant used in Uzbek traditional medicine as antimicrobial and antiseptic in treatment of infected throat, bronchitis, toothache, radiculitis, neuritis in addition to its antispasmodic. This is the first study reporting the volatile compounds and in vitro biological investigation for T. seravschanicus. Phytochemical investigations on the aerial part of T. seravschanicus resulted in the isolation of flavonoids, phenolics and terpenoids. Our in vitro assay results demonstrated that plant of T. seravschanicus can be used as considered useful source of antioxidant and cytotoxic constituents.

Keywords: Thymus seravschanicus, GC-MS, HPLC, volatiles, flavonoids, terpenes

introduction. Thyme plants (Thymus) are aromatic perennial herbs and sub-shrubs, native to Europe, North Africa and Asia. Only two species from the genus Thymus (Thymus seravschanicus Klokov and T. incertus Klokov) have been found in the Uzbek Flora [1]. T. seravschanicus is a perennial plant and grows in Pamir-Alay and Tyan Shan mountains (Central Asia) where it is widely known as Tog’jambil. It is popularly used in Uzbek traditional medicine as antimicrobial and antiseptic in treatment of infected throat, bronchitis, toothache, radiculitis, neuritis in addition to its antispasmodic [2]. To our knowledge there is no report regarding the chemical constituents, or the biological activities of this plant. The present study is aimed to investigate the composition of the MeOH, CHCl3 and H2O extracts of the aerial parts T. seravschanicus using GC-MS. In addition, cytotoxic properties of the extracts and individual compounds identified by HPLC investigations and were tested on HeLa and HL60 cancer cell models; antioxidant activities of these samples were also evaluated.

Materials and Methods. The aerial parts of T. seravschanicus were collected from a wild population during the flowering season in July, 2010 in Tahtaqaracha pass of Samarqand region, Uzbekistan. The aerial part of the plant material (100 g) was extracted with 500 ml solvents (MeOH, CHCl₃ and H₂O, respectively). Chromatographic profiles of T. seravschanicus extracts were generated using a high performance liquid chromatograph LC-10ATvp connected to a UV-VIS detector SPD-10Avp (Shimadzu Co, Kyoto, Japan). For separation of these extracts, a Nucleosil 100-5 C18 column with a size 250 mm x 4 mm (Macherey-Nagel GmbH & Co, KG) was used. Elution was carried out by a mobile phase consisted of A (0.025M phosphoric acid in water) and solvent B (acetonitrile) in different gradient profiles. GC-MS was carried out in a

Hewlett-Packard 5890 gas chromatograph equipped with a DB-5ms fused silica column, interfaced with a Hewlett-Packard mass selective detector 5971 (Agilent Technologies) operated by HP Enhanced ChemStation software, version A.03.00.

Samples were screened for cytotoxic activity using HeLa (human epithelial cervical cancer, ATCC CCL-2) and HL-60 (human promyelocytic leukemia, ATCC CCL-240) cell lines. Cytotoxicity of extracts was determined in triplicate using the MTT cell viability assay [3]. DMSO was used as negative control, whereas doxorubicin was used as positive control.

The antioxidant and radical scavenging activities of samples were evaluated according to BrandWilliams et al. [4] using diphenyl picryl hydrazyl (DPPH). Quercetin was used as a positive control. Data represent mean ± standard error of the mean (SEM) of at least n = 3 replicates. The IC₅₀ was determined as the drug concentration which resulted in a 50% reduction in cell viability or inhibition of the biological activity. Statistical analysis was performed using Student’s t-test. The criterion for statistical significance was p<0.05.

Results and Discussion. The MeOH and CHCl3 extracts of T. seravschanicus were analyzed by GC- MS in order to identify and characterize the major components responsible for the biological activity and altogether 41 components were identified. The most abundant components were mainly hydrocarbons, terpenes, fatty acids, flavones and sterols. Thymol presented in the percentages of 31.13 and 38.23 %, for the MeOH and CHCl₃ extracts, respectively. The HPLC-UV/VIS investigation of T. seravschanicus allowed the identification and characterization of nine major components in either MeOH, CHCl₃ or H₂O extracts of the aerial parts. These compounds were identified by comparison with authentic samples. Apigenin-7-glucoside (1), thymol (5) and p-cymene (7) were the most abundant compounds in the extract; eriodictyol (2), naringenin (3), quercetrin (4), carvacrol (6), ellagic acid (8) and gallic acid (9) were also present (Fig. 1).

The antiproliferative activity of 3 different extracts of the aerial parts of T. seravschanicus along with 9 identified compounds were assessed on both HeLa and HL-60 cells using the MTT assay and the IC50 values are presented in Table 1. The cytotoxic activity of MeOH and CHCl3 extracts against HeLa cells may be attributed to their content of thymol and carvacrol. Possessing phenolic group, these compounds dissociate to negatively charged phenolate ions under physiological conditions thus interacting with transporters and enzymes by binding to them via hydrogen bridges and ion bonding. As a result, the conformations of proteins could be disturbed and their biological activity altered. Moreover, being monoterpene in nature, thymol can dissolve in biomembranes resulting in disturbance of the close interaction between membrane lipids and proteins and leading to a loss of function.

HL-60 cells result consistently affected by both MeOH and CHCl₃ extracts. In this case the antiproliferative activities of thymol and carvacrol are in the same range of the other identified compounds and the antiproliferative activity could be probably related/dependent to a synergic action of compounds consisting the extracts. Water extract does not seem to have cytotoxic activity on HeLa cells, although when tested as single compound ellagic acid shows a consistent level of affection. HL-60 resulted consistently affected by H2O extract and when tested as single compound gallic acid shows a high level of affection; in this view gallic acid results as the main candidate for cytotoxic activity of the H2O extract.

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Table 1 - Results of antiproliferative (MTT assay) and antioxidant (DPPH* radical scavenging) assays of the extracts and individual compounds isolated from T. seravschanicus

Both MeOH and H₂O extracts showed a promising antioxidant activity when it is compared to the control (Table 1). The mean radical scavenging activity of the extracts ranges from 15.87-16.72 pg/ml. respectively, which is much closer to the standard reference. Based on the identified compounds, the potent antioxidant activity. can be related to the phenolic content including flavonoids and phenolic acids. Ellagic and gallic acids showed the most potent activity with IC50 values of 0.79 and 0.96 pg/ml. respectively.

Conclusion.

Our in vitro assay results demonstrated that plant of T. seravschanicus can be used as considered useful source of antioxidant and cytotoxic constituents. Carvacrol. thymol. ellagic and gallic acids of T. seravschanicus are strongly and selectively able to induce cell death of studied cell lines. The present study therefore can provide new context for further researches.

Acknowledgement

Financial support by UNESCO-L’OREAL foundation for a research fellowship to N.Z. Mamadalieva is gratefully acknowledged.

REFERENCES

1. Vvedenskiy A. Flora of Uzbekistan. Tashkent: AS RUz Fan Publ; 1961.
2. Kholmatov K. K.. Qosimov A. Medicinal plants. Tashkent: Ibn-Sino Publ; 1994.
3. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays //Journal of Immunol Methods. -1983. -V. 65. P. 55-63.
4. Brandwilliams W.. Cuvelier M.. Berset C. Use of a free-radical method to evaluate antioxidant activity //LWT-Food Sci Technol. -1995. -V. 28. -P. 25-30.