Antimicrobial activity and cytotoxicity of extracts of Suea Khao capsule formulation and its herbal components
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Abstract
Introduction and Objectives: The Sua Khaw Capsule is a formula developed from the original Chinese medicine formula, Yu Ping Feng San. The recent COVID–19 outbreak has significantly impacted the health and lives of the global population. The Faculty of Chinese Medicine, Huachiew Chalermprakiet University therefore developed and improved this herbal medicine formula which has the properties of increasing vital energy, strengthening the lungs and spleen, and nourishing yin. However, it still lacks research support in various fields. Hence, this research aims to study antimicrobial activity and cytotoxicity of different extracts of Sua Khaw capsule formula and its herbal components, and conduct quality assessment of the finished product to prepare data for future herbal medicine registration.
Methods: Sua Khaw Capsules and its 12 herbal components; namely, Huang Qi (Astragalus membranaceus), Dang Shen (Codonopsis pilosula), Tai Zi Shen (Pseudostellaria heterophylla), Bai Zhu (Atractylodes macrocephala), Fang Feng (Saposhnikovia divaricate), Nan Sha Shen (Adenophora tetraphylla), Jin Yin Hua (Lonicera japonica), Lian Qiao (Forsythia suspensa), Zi Su Ye (Perilla frutescens), Huo Xiang (Pogostemon cablin), Shen Qu (Medicated Leaven), and Gan Cao (Glycyrrhiza uralensis) were extracted with 3 types of solvents, i.e., ethyl acetate, ethanol, and water. The crude extracts were then tested for their antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans using agar well diffusion method; and extract concentrations that inhibited and killed such microorganisms were also determined. Regarding the safety of this herbal formula, these extracts were tested for cytotoxicity against Vero cells; and microbial and heavy metal contaminations of Sua Khaw capsules were also determined using methods described in the Thai Herbal Pharmacopoeia (THP).
Results: Ethyl acetate (EA) extracts of 12 Chinese herbs and Sua Khaw capsule formula demonstrated inhibitory activity against S. aureus with inhibition zone diameters (IZD) ranging from 13.3 – 28.0 mm, with MICs in the range of 0.39 – 12.5 mg/ml. EA extract from Gan Cao showed highest inhibitory activity, with IZD of 28.0 mm and MIC of 0.39 mg/ml. Gan Cao and Bai Zhu EA extracts also showed inhibitory activity against C. albicans, while EA extracts of Tai Zi Shen and Jin Yin Hua also inhibited E.coli. However, such inhibitory activity was weaker than that against S. aureus. Ten ethanol (ET) extracts (except those of Dang Shen and Nan Sha Shen) exhibited inhibitory activity against S. aureus with IZDs ranging from 14.0 – 28.3 mm, and MICs of 0.097 – 12.5 mg/ml. Gan Cao ET extract showed the best inhibitory activity, with IZD of 28.3 mm and MIC of 0.097 mg/ml. For Sua Khaw Capsule formula, both EA and ET extracts, demonstrated inhibitory activity against S. aureus with IZDs of 16.5 and 17.5 mm, respectively, and equal MICs of 3.13 mg/ml. For the water extracts, only those of Lian Qiao, Su Ye and Jin Yin Hua showed inhibitory activity against S. aureus. All extracts showed cytotoxicity against Vero cell cultures with the IC50 range of 60.70 µg/ml to >1,000 µg/ml. The results of heavy metal contaminations in Sua Khaw capsules showed that the contents of arsenic, cadmium, and lead passed the acceptance levels of THP, while the results of microbiological quality assessment of Sua Khaw capsules passed the acceptance levels of THP after subjected to gamma irradiation.
Discussion: EA extracts of all herbal components and ET extracts of 10 out of 12 herbs exhibited inhibitory activity against S. aureus, and the EA and ET extracts of Sua Khaw capsule had equal MICs against S. aureus of 3.13 mg/ml, indicating that the two solvents had similar extractive activity efficiency to extract antibacterial compounds. EA and ET extracts of Gan Cao showed the highest inhibitory activity with MIC values of 0.39 and 0.097 mg/ml, respectively. This may be due to a high content of isoflavonoids present in the roots of Gan Cao, which can be well extracted by ET and EA. It was found that the Tai Zi Shen EA extract showed a relatively high toxicity to Vero cell cultures, with IC50 of 60.70 µg/ml, while EA extract of Sua Khaw capsule showed a much lower cytotoxicity (IC50 314.6 µg/ml). This was partly due to a small proportion of such cytotoxic herb in the herbal formula.
Conclusions and Recommendation: Of all the extracts, EA extracts showed highest inhibitory activity against S. aureus, followed by ET extracts; however, all extracts tested showed lower antimicrobial activities than the positive controls (levofloxacin and clotrimazole). Water appeared to be an inappropriate solvent for the extraction of antimicrobial compounds from this herbal formula. Only EA or ET extracts of Tai Zi Shen, Gan Cao, Bai Zhu and Jin Yin Hua also showed inhibitory activity against E. coli or C. albicans as well, but with lower activity. Although Sua Khaw capsule formula showed low cytotoxicity to Vero cells, more toxicity studies as well as pharmacological and clinical studies should be conducted to support its safe use in humans and therapeutic efficacy.
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