Evaluation of Traditional Chinese Medicinal Plants for Anti-MRSA Activity with Reference to the Treatment Record of Infectious Diseases

The in vitro antimicrobial activities of 30 Chinese medicinal plants were evaluated with reference to the treatment record of infectious diseases in the Traditional Chinese Medicine (TCM) literature. The plant materials were extracted with 80% ethanol and the extracts were primarily screened against conventional clinical pathogens like Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans by the agar diffusion method. Their inhibition zone diameters (IZDs, mm, 50 mg/mL) ranged from <8 to 24. The 21 extracts which showed IZDs ≥10 mm against MSSA were also active against methicillin-resistant S. aureus (MRSA) with lower IZDs of 9.0–18.8 mm. They were further subjected to minimal inhibitory concentration and minimal bactericidal concentration (MIC/MBC, μG/mL) assays, which were 8-2,048/32->2,048 by the standard broth microdilution method. The seven extracts from M. yunnanensis, S. sinensis, G. morella, E. daneillii, M. squamulata, S. arborescens and B. hancei were determined as the most active extracts, with MICs of 8–64 μg/mL. The results were in good agreement with their traditional applications in skin and other infections.


Introduction
Staphylococcus aureus infections range from common skin infections, such as furunculosis and impetigo, to severe deep-seated infections. S. aureus ranks first or second among bacterial pathogens causing bloodstream infections. It is the leading cause of nosocomial pneumonia and it also causes infections of surgical wounds and prosthetic implants. Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) have become the most common cause of infections among pathogenic bacteria around the Globe, and many life-threatening diseases such as endocarditis, pneumonia and toxin shock syndrome are ascribed to them. Contrary to methicillin-susceptible S. aureus (MSSA), MRSA tend to be multi-drug resistant (MDR), that is, resistant not only to β-lactam antibiotics but also to a wide range of different antibiotic classes, such as fluoroquinolones, tetracyclines, macrolides, lincosamides and aminoglycosides, and even strains of vancomycin intermediate susceptible or full resistant (VISA and VRSA, respectively) have emerged [1]. Therefore, the search for novel anti-MRSA agents is urgently needed. Meanwhile, great emphasis has been placed on the value of plants used in ethnomedicine and traditional medicine for drug discovery has currently been laid greater stress worldwide [2][3][4][5].
Traditional Chinese medicines (TCM), like Ayurveda, Unani and Kampo, have flourished as systems of medicine in use for thousands of years [2]. With more than 5,000 years of Chinese history and as a part of Chinese culture, TCM mainly used plant materials in organized traditional medical systems which have been playing a critical role in fighting various diseases and maintaing human health. Thousands of Chinese herbal medicines have been recorded in a great variety of national and local literatures such as Shen Nong's Materia Medica [6], Compendium of Materia Medica [7], Dictionary of Chinese Materia Medica [8], Chinese Materia Medica [9], Compilation of Chinese Herbal Medicine [10] and Selected Yunnan Traditional Chinese Herbs [11] from ancient to modern times. They have been widely used throughout the country or in local areas by the Chinese people of all nationalities.
In TCM clinical practice, the Chinese disease name "Chuang-Yang" (pyogenic infection and ulceration of skin) is a general terminology for the surgical and skin disease visible over the body surface and dermatosis, including "Yong Ju, Ding-Chuang, Jie-Zhong, Liu-Zhu, Liu-Tan, etc." (carbuncle, deep-seated sore, furuncle, multiple abscess, tuberculosis of bone and joint, scrofula, etc.). The diseases are commonly seen in clinic, due to stagnation of qi and blood stasized from the attack of evils and consumption of blood by heat-evil [12].
Many Chinese herbal medicines (or medicinal plants) have been documented as treatment of these diseases, which prompted us to investigate their inhibitory activity against MRSA. We herein report the in vitro anti-MRSA effects of a collection of 30 Chinese medicinal plants with reference to the treatment record of skin and other kinds of infections (Table 1).  Table 1 shows the recorded TCM indications and phytohemical constitution of the 30 Chinese medicinal plants [6][7][8][9][10][11]. The antibacterial susceptible spectrum of the tested seven MRSA strains is listed in Table 2. The initial screening results of the 30 plant extracts against MSSA and other conventional standard strains of Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Candida albicans (ATCC Y0109) are expressed as inhibition zone diameters (IZDs) in Table 3, while anti-MRSA activities of the selected 21 extracts which were active with IZDs ≥10 mm against MSSA were shown in Table 4. Their corresponding MICs/MBCs (µg/mL) are shown in Table 5.

Discussion
A collection of 30 Chinese medicinal plants were evaluated for their in vitro antimicrobial effects, especially their anti-MRSA potentials. Most of the plants with anti-infective effects related to skin infections have been indicated in various works of Chinese herbal medicines either by themselves or the different species belonging to the same botanical genus, such as the indication of carbuncle which means a severe abscess or multiple boil in the skin, typically infected with S. bacteria (Table 1) [6][7][8][9][10][11]. The antimicrobial screening of all the plant species is being reported for the first time to the best of our knowledge, especially the anti-MRSA activities.
The 80% ethanol extracts of the 30 Chinese medicinal plant extracts were initially subjected to the screening of their antimicrobial activities against MSSA and other conventional standard strains of Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Candida albicans (ATCC Y0109). Their activities as IZDs (mm) at a concentration of 50 mg/mL ranged from <8 to 24, of which 21 extracts showed IZDs ≥10 mm against MSSA ( Table 1). The results were in agreement with the literature record of traditional usages (Table 3) [6][7][8][9][10][11]. Judging from the IZD values, the extracts were generally more active against the Gram positive pathogen (MSSA) than against Gram negative (E. coli and P. aeruginosa) and fungal pathogens (C. albicans). The additional permeability barrier caused by the outer lipopolysaccharide layer in Gram negative bacteria makes them more resistant to plant natural products, as has been demonstrated previously [13,14]. Therefore, finding out anti-staphylococcal natural products from plant materials is much easier and is proved in the previous reports [15][16][17][18].
The tested MRSA strains were seven multi-drug resistant clinical isolates (Table 2). They were used for the further assay of their susceptibilities to the 21 extracts (Table 4). It is interesting that these extracts which were active against MSSA also showed more or less activity against MRSA with IZD values (mm) ranging between 9.0 and 18.8 and this has been noted previously [15][16][17][18][19]. The differences were from −3.0 to 6.5 mm in this study (Table 4).
Anti-MRSA potency of MICs/MBCs (μg/mL) of the 21 extracts was determined as 8-2,048/32->2,048 ( Table 5). Comparison of the values of IZD and MIC for a same extract in Tables 4 and 5, they were not always positively correlated. As different sample concentrations will produce different IZDs, the real inhibitory potency of a sample should be judged by its MIC. This should be noted for future studies.
The phytochemicals, including polyphenols and antimicrobial activity of Mallotus species have been reviewed [26,27]. Rottlerin from M. philippinensis exhibited potent bactericidal activity with an MBC value of 3.12-6.25 μg/mL against several clinical H. pylori isolates [28]. Garcinia genus is rich in caged xanthones [29]. Morellin isolated from gamboge (G. hanburyi, tenghuang in Chinese) was reported as an antibiotic principle in 1954 [30]. The anti-MRSA activity of morellin and other caged xanthones from Gamboge, G. morella and other Garcinia species have also reported [31,32]. Two antifungal compounds ulopterol (a coumarin) and a quinolone alkaloid 4-Methoxy-l-methyl-3-(2'-S-hydroxy-3'-ene-butyl)-2-quinolone were isolated from Skimmia laureola, a Pakistani medicinal plant [33], which were similar to our results of the inhibition of S. arborescens against C. albicans (Table 3). However, no active antimicrobial compounds from the other plant materials have been isolated up to now, especially the anti-MRSA compounds from the most active extracts of S. sinensis, E. daneillii, M. squamulata, and B. hancei, with MICs at 8-64 μg/mL. We are continuing our search for the corresponding phytochemicals from these plants and their further systematic anti-MRSA properties.
(Tianhe Microbial Agents Co., Hangzhou, China) were used as the bacterial and fungal culture media, respectively. The antibacterial agents-susceptibility testing results of MRSA strains were shown in Table 2.

Extract Preparation
The 30 samples of the air-dried and ground plant materials (50 g) were macerated with 80% ethanol (500 mL) for 7 days, filtered and the mare was further macerated twice with the same solvent overnight and filtered after being sonicated for 30 min. The filtrates were combined and the solvent was evaporated at 40 °C in vacuum to afford each of the plant extract (Table 3).

Antimicrobial Screening
The ethanol extracts of the 30 plants were primarily subjected to susceptibility screening against standard microbial strains according to the agar diffusion method on MHA (for the bacteria) or SA (for C. albicans) plates. The sample extracts (50 mg/mL in dimethyl sulfoxide) were pipetted into 6 mm (in diameter) holes punched in the agar of prepared agar plates, plating with inoculums of 1.5 × 10 8 CFU/mL for bacteria and 5 × 10 5 CFU/mL for C. albicans in advance, respectively and incubated at 35 °C (for C. albicans at 28 °C) for 24 h, measured and recorded the IZDs [15][16][17]37]. The solvent value was deducted accordingly to get final results of activity. All experiments were carried out in triplicate. The test results were interpreted based on IZD as <10 mm for the resistance, =10 mm for the mildly susceptible, 11-15 mm for moderately susceptible, ≥16 mm for the highly susceptible ( Table 2). The 21 extracts with IZDs ≥10 mm against MSSA were further subjected to the assay of their IZDs against MRSA strains (Table 3).

MICs and MBCs Assaying
The extracts with IZDs ≥10 mm against MSSA were further subjected to the assay of their IZDs and minimal inhibitory concentrations and minimal bactericidal concentrations (MICs/MBCs) against MRSA strains by serial dilution method according to the procedures reported previously [15][16][17]38,39]. Briefly, MICs/MBCs were determined by the standardized broth (using MHB) microdilution techniques with starting inoculums of 5 × 10 5 CFU/mL for the bacteria according to CLSI (formally NCCLS) guidelines and incubated at 35 °C for 24 h. For the MBCs assaying, 0.1 mL aliquots from drug dilution wells with visual growth inhibition were plated onto MHA media. The lowest drug concentration that yielded three or fewer microorganism colonies was recorded as the MBC. They were determined in triplicate, with concentrations ranging up to 2048 μg/mL for all the extracts (Table 5).

Conclusions
The screening of in vitro antimicrobial activity of the ethanol extracts from 30 Chinese medicinal plants led to the confirmation of 21 extracts displaying both anti-MSSA and MRSA effects with various levels of potency which were in good agreement with their TCM indications of skin infections and modern phytochemical constituents, with M. yunnanensis and S. arborescens extracts being the most active against MRSA. The combination of TCM indications and phytochemical profiles is a promising approach to the search of anti-MRSA plant natural products.