1. INTRODUCTION:

Coptidis Rhizoma refers to a rhizome nearly without root of Coptis japonica Makino (in Japan), Coptis chinensis Franch (in China), or other similar buttercup family (Ranunculaceae)^1,2. Coptidis Rhizoma is a little smelly. Its taste is very bitter and its residual makes saliva tinged with yellow color. For its pharmacological actions, it has efficacy of strong stomach, soothing, anti-inflammation, and antibacterial etc., which is effective foremesis due to diarrhea or gastric fever generated as a result of heat accumulation over intestine or stomach^3,4.

It is also known to be excellent in antipyretic action and deintoxication. The main ingredient of Coptidis Rhizoma is berberine, which has been reported to have a strong antimicrobial activity. The dosage conversion indicates that the dried herb medicine contains more than 4.2% of berberine chloride (C20H18ClNO4: 371.81). It includes other ingredients such as coptisine, epiberberine, berberrubine, palmatine, columbamine, jaterrhizine, worenine, magnoflorine, ferulic acid, obakunone, and obakulacton^5,6. Candida albicans is a fungus that causes the largest problems among pathogenic fungi^7,8. According to the statistics published in the National Institutes of Health in the USA, Candida albicans is ranked the fourth infection bacillus in hospitals and the first among fungi^9,10. It can be fatal to patients with wasting diseases such as diabetes, tuberculosis, cancer, and acquired immuno-deficiency^11,12. It can also cause many problems including colpitis even in healthy women whose immune function is normal. For treatment drugs, polyene-based amphotericinB and azole-based fluconazole and ketoconazole are employed¹³. However, due to the characteristics of low full-recovery rate, high drug tolerance, high relapse rate after treatment, and side effects such as nephrotoxicity, a new discovery of drugs and treatment method are demanded¹^4,15. In this regard, the present study selected Coptidis Rhizoma among many herbal drug ingredients and investigated the antifungal effect from the fraction of the herbal medicine, focusing on discovery of new substance or ingredients with antifungal effects. In recent years, a large number of studies have been conducted to identify the effect of natural substances against various diseases around the world^16,17. A variety of studies have also been conducted to determine the drug effect of natural substance in Korea at a national level. With this purpose in mind, our laboratory selected some natural substances through literature reviews and searched for antifungal effect of Candida albicans as a pathogenic fungus. The present study tested and investigated antifungal effect of Candida albicans by using fractions extracted from Coptidis Rhizoma, which has been most widely used in herbal medicines. Thus, the present study searched the antifungal effect on Candida albicans by extracting Coptidis Rhizoma into Hexane, Chloroform, n-butanol (n-BuOH), and ethyl acetate (EtOAc).

2. MATERIALS AND METHODS:

2.1. Preparation of Coptidis Rhizoma and fraction:

Dried Coptidis Rhizoma (600g) was pulverized to powder, and maceration extraction of three times for five hours was conducted at 50⁰ while performing decoction using methanol. The extracted solution was decompressed and concentrated at a water bath to obtain around 4g of methanol extr) act^18,19. This methanol extract was suspended with distilled water and then each of the fractions was obtained in the following order by performing solvent fraction: n-hexane, methylene chloride, ethyl acetate, and n-butanol of the same quantity according to the phase method.

2.2 Experimental strain and culture:

The Korean Culture Center of Microorganisms (KCCM) purchased Candida albicans KCCM 50235, which was then inoculated in yeast malt (YM) (Difco, USA) broth and cultured for 24 hours at 37⁰ in the shaking incubator. The cultured fungus was verified using gram straining, which was one of the bacterial staining methods. Prior to the experiment, the cultured fungus was activated by a number of subcultures^20,21.

2.3Paper disc diffusion method:

In order to measure the antifungal effect of the extract from Coptidis Rhizoma on Candida albicans, agar diffusion assay was conducted. 100 ul of Candidaalbicans, which was diluted with 10⁷CFU/mland placed on the YM liquid broth, was put on the YM solid broth and spread using a sterilized glass spreader. 50 ul of 70% methanol and Fluconazole (50mg/ml) (positive control group) and Coptidis Rhizoma extract (experimental group) were dropped on the paper disc (8 mm, Whatman, USA) and dried, and then placed on the YM solid broth where experimental fungus was spread, respectively. They were cultured in the incubator at 37°C for 24 hours and transparent circle diameter around the paper disc was measured. In addition, the minimum concentration where a significant difference was revealed and transparent circle was created was set to the minimum inhibitory concentration (MIC) of the Coptidis Rhizoma extract against Candidaalbicans.

2.4 Statistical Analysis:

The experimental results were calculated using mean± standard error, and significance verification between groups was done using a Student's t-test. When P value was less than 5%, it was determined as significant.

3. RESULTS AND DISCUSSION:

The inhibitory effect of Coptidis Rhizoma extract on the growth of Candidaalbicans was determined by using agar diffusion assay method, which was the most widely-used method in antifungal susceptibility test, and the minimum inhibitory concentration, was measured. The results showed the following inhibitory effects in figure 1: 10mm in 70% ethanol, 29mm in Fluconazole (50mg/ml) as positive control group.

Figure 1. Inhibitory zone of medicinal plant 70% ethanol extracts and fluconazole by disc diffusion method against Candida albicans after 24h

Inhibitory zone of 4 kinds fraction, Coptidis Rhizoma by disc diffusion method against Candida albicans after 24hshowed the following in figure 2: 10mm in 70% ethanol, 29mm in Fluconazole (50mg/ml) as positive control group. 23 mm in Hexane fraction extract, 30 mm in Chloroform fraction extract, 32 mm in n-butanol (n-BuOH) fraction extract, and 10 mm in ethyl acetate (EtOAc) fraction extract.

Figure 2. Inhibitory zone of 4 kinds fraction,Coptidis Rhizomaby disc diffusion method against Candidaalbicansafter 24h.

Inhibitory effect of positive control and 4 kind’s fraction against Candida albicans showed the following in figure 3.

Figure 3. Inhibitory zone of positive control and 4 kinds fraction,Coptidis Rhizomaby disc diffusion method against Candidaalbicansafter 24

In recent years, much attention has been paid to infectious pathogen due to the rapid increase in the number of cancer patients caused by various types of cancers along with immunodeficient patients^22,23. It is also highly critical problem caused by bacterial infection as well as mycosis. In particular, the types of antibiotic used in fungal infection treatment are not diverse compared with that of antibiotic for bacteria and have high side-effect occurrences, which is why a new development and alternative of antifungal agent is needed. In this context, it is highly important to discover antifungal effects from natural substances. Thus, the present study aimed to determine the inhibitory effect of Coptidis Rhizoma extracton the growth of Candida albicans, which was known as a causative agent of fungal infection inside the mouth. Coptidis Rhizoma is safe to eat and easy to buy. It has also been reported to have various efficacies as well as antimicrobial effect. The inhibitory effect of Coptidis Rhizoma extract on the growth of Candida albicans was determined by using agar diffusion assay method, which was the most widely-used method in antifungal susceptibility test and the minimum inhibitory concentration, was measured. The results showed the following inhibitory effects in figure 1: 10mm in 70% ethanol, 29mm in Fluconazole (50mg/ml) as positive control group, 23 mm in Hexane fraction extract, 30 mm in Chloroform fraction extract, 32 mm in n-butanol (n-BuOH) fraction extract, and 10 mm in ethyl acetate (EtOAc) fraction extract in figure 2. In particular, the inhibitory effect on Candida albicans was best revealed in n-BuOH as 32 mm compared with 29 mm in Fluconazole (50mg/ml) as positive control group, indicating much better antifungal effect. When the ingredients extracted from Rhizoma Coptidis was refined using the CPC and HPLC, the optimum solvent condition in the CPC was n-butanol: acetic acid: water(4:1:5). Berberine (16.8 mg) has been reported to be separated effectively by the CPC and HPLC method. Since existing antifungal agents have many side-effects, it would be highly encouraging that a dosage of existing drugs can be reduced through combined modality therapy using n-BuOH fraction or similar natural substances while similar or improved effect can be obtained. As discussed in the study result in this paper, the discovery of medicinal ingredient with antifungal effects as combined modality therapy can be regarded highly important. In summary, the present study searched antifungal agents of new substance and identified the antifungal effect of n-BuOH fraction extracted from Coptidis Rhizoma on Candida albicans infection.

4. CONCLUSION:

The present study aimed to determine the inhibitory effect of Coptidis Rhizoma extracton the growth of Candida albicans, which was known as a causative agent of fungal infection inside the mouth. Coptidis Rhizoma is safe to eat and easy to buy. It has also been reported to have various efficacies as well as antimicrobial effect. The inhibitory effect of Coptidis Rhizoma extract on the growth of Candida albicans was determined by using agar diffusion assay method, which was the most widely-used method in antifungal susceptibility test, and the minimum inhibitory concentration, was measured. The results showed the following inhibitory effects in figure 1: 10mm in 70% ethanol, 29mm in Fluconazole (50mg/ml) as positive control group, 23 mm in Hexane fraction extract, 30 mm in Chloroform fraction extract, 32 mm in n-butanol (n-BuOH) fraction extract, and 10 mm in ethyl acetate (EtOAc) fraction extract in figure 2. In particular, the inhibitory effect on Candida albicans was best revealed in n-BuOH as 32 mm compared with 29 mm in Fluconazole (50mg/ml) as positive control group, indicating much better antifungal effect.

5. ACKNOWLEDGMENT:

This study was supported by research funds from Nambu University, 2017

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Received on 23.06.2017 Modified on 24.07.2017

Research J. Pharm. and Tech. 2017; 10(7):2387-2390.

DOI: 10.5958/0974-360X.2017.00422.X

Mechanism in Antibacterial Activity on Fraction of Coptidis Rhizoma against Candida Albicans