Screening of antibacterial activity of 20 Chinese herbal medicines in Yunnan

The 80% ethanol extracts from 20 Chinese herbal medicines collected in Yunnan Province, China, were screened for antibacterial activity against Staphylococcus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (EC) and Pseudomonas aeruginosa (PA), including antifugal activity against Candida albicans (CA) by the agar hole diffusion test. The extracts were prepared through macerating at room temperature and hot refluxing procedures. 19 of the 20 extracts showed effects with different potency and their inhibition zone diameters (IZD) ranged from 9 to 27 mm. The extracts from Psychotria henryi Levl and Marsdenia tinctoria R. Br. (maceration and reflux extraction) were the most active against both SA and MRSA, with mean IZDs = 19.7 to 22.0 mm. The anti-SA and MRSA active extracts were further determined for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by serial dilution with a standard broth microdilution method. 12 extracts showed effects on both SA and MRSA strains with MICs/MBCs ranges among 32 to ≥ 1024 / 64 to ≥ 1024 mg/L, respectively. P. henryi, M. tinctoria and Sapium baccatum Roxb also showed inhibition against EC, PA and CA strains, and displayed a broad spectrum of antimicrobial activities.


Since
methicillin-resistant Staphylococcus aureus (MRSA) occurred in early 1961 (Jevons, 1961), it has become a troublesome pathogen responsible for serious infections owing to its resistance to a large group of antibacterial agents, resulting in high morbidity and mortality.It is resistant not only to β-lactam antibiotics, but also to other antibacterial groups, such as fluoroquinolones, tetracyclines, macrolides, lincosamides and aminoglycosides.Some strains that are partially or fully resistant to vancomycin have been found (Pantosti and Venditti, 2009).The search for novel drugs to combat MRSA infection is urgently needed.
Traditional Chinese medicine (TCM) has accounted for a wealth of health needs over thousands of years in China (The Compiler Group, 2009).A great number of plant species have been used as healing herbs in TCM for the treatment of infectious diseases, such as skin ulcers, cold, diarrhea, pneumonitis and tuberculosis, etc (Jiangsu New Medical College, 1977;Sima et al., 2012;Jinous and Fereshteh, 2012;Muhammad et al., 2012).There is increasin interest in researching Chinese herbal drugs for application as antibacterial agents (Zuo et al., *Corresponding author.E-mail: zuoguoying@263.net.Tel: +86-871-477-4941.The tested MRSA strains were multi-drug resistant to β-lactams, aminoglycosides, fluoroquinolones and macrolides, and they were susceptible to vancomycin (Table 2).

Plants
The selected plants were collected in Xishuangbanna of Yunnan Province in August, 2011.They were identified at the Botany Department, Kunming Institute of Botany (KIB), the Chinese Academy of Sciences.The voucher specimens were preserved at the herbarium of KIB (Table 2).

Media used
Standard Mueller-Hinton agar and broth (MH-A and MH-B), and Sabouraud agar and broth (S-A and S-B) (Tianhe Microbial Agents Co., Hangzhou, China) were used as the bacterial and fungal culture media, respectively.

Extract preparation
Two types of 80% ethanol extracts were prepared from the 20 Southern Yunnan plants by the macerating extraction at room temperature (25°C) and the hot reflux extraction (85°C), respectively as the follows: An amount (50 g, each) of powdered air-dried aerial parts of the plant material was macerated with 80% ethanol (150 ml) for one week, filtered and the residue was further macerated twice with the same amount of solvent overnight and filtered after sonication for 30 min.The filtrates were combined and the solvent was evaporated at 40°C under reduced pressure to afford each of the plant extracts.Another amount (50 g, each) of the same plant material was refluxed with 80% ethanol for two hours, filtered and the residue was further refluxed twice with the same amount of solvent and filtered.The filtrates were combined and the solvent was evaporated at 40°C in vacuum to afford each of the plant extracts (Table 3).

Susceptibility test
The ethanol extracts of the 20 plants were initially subjected to susceptibility test according to the agar diffusion method on MH-A (for the bacteria) or S-A (for C. albicans) plates (Zuo et al., 2008).Briefly, crude extracts were prepared at concentrations of 50 mg/ml in dimethylsulfoxide (DMSO).The microbial suspension was firstly plated onto the agar plates with inoculums of 1.5 × 10 8 CFU/ml for the bacteria and 5 × 10 5 CFU/ml for C. albicans.Then, holes of 6.0 mm diameter each in the agar plates were punched with a sterilized hole-puncher.The samples were pipetted into the holes with no overflowing.The plates were incubated at 35°C for 24 h and measured and the inhibition zone diameters (IZDs) recorded.For every experiment, a sterility check (DMSO and medium), negative control (DMSO, medium and inoculums) and positive antibacterial control (vancomycin) were included (Table 3).All experiments were performed in triplicate.
The samples of IZDs > 12 mm against standard S. aureus (SA) were subjected to assay of their IZDs against MRSA (Table 4).The subsequent samples with IZDs > 12 mm against one of the MRSA strain (MRSA111) were further subjected to assay of their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for MRSA by standardized broth microdilution techniques (Zuo et al., 2008).Briefly, the starting inoculums were 5 × 10 5 CFU/ml.The 96-well plates were incubated at 35°C for 24 h according to CLSI guidelines (CLSI, 2006(CLSI, , 2007)).All experiments were determined in duplicate, with concentrations ranging up to 2048 mg/L.For the MBC assays, 0.1 ml aliquots from drug dilution wells with no visual growth inhibition were plated onto MH-A.The lowest drug concentration that yielded three or fewer microorganism colonies was recorded as the MBC NCCLS, 1999).Vancomycin (Eli Lilly Japan K.K., Seishin Laboratories) was also used as a positive control anti-MRSA agent (Table 5).The anti-MRSA activities of the selected 17 extracts which were active with IZDs ≥ 10 mm against MSSA were shown in Table 4.The extracts from Psychotria henryi Levl and Marsdenia tinctoria R. Br. (maceration and reflux extraction) were the most active against both SA and MRSA, with mean IZDs = 19.7 to 22.0 mm (Table 4).The anti-SA and MRSA active extracts were further determined for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by serial dilution with a standard broth microdilution method.12 extracts showed effects on both SA and MRSA strains with MICs/MBCs ranges between among 32 to ≥ 1024 / 64 to ≥ 1024 mg/L, respectively.P. henryi, M. tinctoria and Sapium baccatum Roxb also showed inhibition against EC, PA and CA strains, and displayed a broad spectrum of antimicrobial activities.

DISCUSSION
The screening results revealed that most of the plant extracts were much more active against Gram positive pathogen (SA) than those of Gram negative pathogens (EC and PA), which is a common phenomenon of plant antimicrobial property (Tegos et al., 2002).None of the plants showed strong inhibition against PA (Table 3).The 4 plant extracts from D. repens (reflux extraction), M. tinctoria (macerating extraction), S. baccatum (reflux extraction) and P. henryi (reflux extraction) showed IZDs of 9 to 12 mm against standard EC.There were 15 plant extracts active against standard CA and the IZDs were in the ranges of 10 to 20 mm.Some plants showed a wide spectrum of antimicrobial activity and were in agreement with their folk uses (Table 3).However, several extracts showed no antimicrobial activity at 50 mg/ml which were not consistent with their folk uses for infectious diseases.It might be due to the antimicrobial potency which was much lower in this study and the different criterion for the antimicrobial judgment.We also noted the inconsistent values between IZD and MIC of an extract; this might be due to the different diffusion capacity of the active constituents in the culture media of agar and broth.
It was found that different extraction methods produced different yields of active extracts.Meanwhile, the higher temperature of extraction may cause the active ingredient change, hence the antimicrobial activity decreases (Tables 3 to 5).
In this study, the 80% ethanol extracts of 20 plant materials were screened for their antimicrobial activities for the first time.We found that P. henryi was active against not only Gram-positive bacteria but also Gram-negative bacteria.The chemical composition and pharmacological effects of P. henryi has not been reported yet, but there were reports (Muhammad et al., 2003;Kerber et al., 2001) about the same genus, that is, P. sychotria brachyceras and P. sychotria klugii which contained indole alkaloids.So, it is worthy to further investigate the anti-MRSA components.Our study suggested that M. tinctoria showed promising activity against MSSA and MRSA.The plant contained steroidal components such as tinctoralactone and marsdenone (Chowdhury et al., 1993) and showed antifertility activity (Chowdhury et al., 1994).But its anti-MRSA effect has not been noted previously.Further research for the corresponding active components from these plants and their systematic anti-MRSA properties are been carried out.

Table 1 .
The antibacterial agent-susceptibility testing results of MRSA strains.
MATERIALS AND METHODSBacterial strainsStrains of S. aureus (SA, ATCC25923), E. coli (EC, ATCC25922), P. aeruginosa (PA, ATCC27853), C. albicans (CA, ATCCY0109) and antibiotic susceptibility disks were provided by the National Institute of Control of Pharmaceutical and Biological products (NICPBP, Beijing, China).MRSA strains were clinical isolates from infectious samples of critically ill patients in Kunming General Hospital (KGH) (Table1

Table 2 .
Species of the 20 medicinal plants.
RESULTSThe antibacterial agent susceptibility spectrum of the tested three MRSA strains is listed in Table1.They were used for anti-MRSA evaluation.The 20 Yunnan medicinal plants are shown in Table2, together with their plant families, specimen numbers and folk uses.The initial antimicrobial screening results (IZDs) of 50 mg/ml ethanol extracts of the 20 plant extracts against standard SA, that is, methicillin-susceptible Staphylococcus aureus (MSSA, ATCC25923) and other standard strains of EC, PA and CA are shown in Table3.19 of the 20 extracts showed effects with different potency and their IZDs ranged from 9 to 27 mm.
Table 4．The testing results of the 17 extracts with IZD ≥ 10mm against SA and MRSA strains (unit: mm).