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

Zuo, Guo-Ying; Zhang, Xin-Juan; Yang, Cui-Xian; Han, Jun; Wang, Gen-Chun; Bian, Zhong-Qi

doi:10.3390/molecules17032955

Open AccessArticle

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

¹

Research Center for Natural Medicines, Kunming General Hospital, PLA, Kunming 650032, China

²

Kunming Medical College, Kunming 650032, China

³

Yunnan Traditional Chinese Medical College, Kunming 650500, China

⁴

Center for Infectious Diseases, Kunming General Hospital, PLA, Kunming 650032, China

^*

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Molecules 2012, 17(3), 2955-2967; https://doi.org/10.3390/molecules17032955

Submission received: 8 February 2012 / Revised: 27 February 2012 / Accepted: 29 February 2012 / Published: 9 March 2012

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Abstract

:

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.

Keywords:

Chinese medicinal plants; traditional Chinese medicine (TCM); methicillin-resistant Staphylococcus aureus (MRSA); inhibition zone diameters (IZD); minimal bactericidal concentrations (MIC)

1. 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. Traditional indications and phytohemical constitution of the 30 Chinese plants.

**Table 1.** Traditional indications and phytohemical constitution of the 30 Chinese plants.
No.	Species	Traditional Indication	Phytohemical Constitution
1	A. nepalensis	bleeding of the nose, enteritis and dysentery	tannins, triterpenoids, flavonoids, phenols
2	B. balsamifer	anti-rheumatism, ringworm and sores, dysentery, detoxification and snake bite	flavonoids, simple terpenoids
3	B. hancei	heat clearing and detoxicating, dysentery, jaundice, boils, swelling, tuberculosis injury hematemesis, osteomyelitis, periostitis, rheumatism and pain	hydroxytyrosol derivatives and glycosides
4	C. austroglauca	astringing sores, carbuncles, dysentery, hemostasis and vaginal discharge	none
5	C. japonica	carbuncles, boils, mumps, erysipelas, rheumatism, jaundice, dysentery, hematuria and gonorrhea	flavonoids (delphinidin glycosides)
6	C. orbiculatus	dysentery, multiple abscess, Herpes zoster, detoxification, inflammatory, cellulites and snake bite	sesquiterpene, flavonoids
7	C. orchioides	diarrhea, ulcer, pus and muscles atrophy	triterpenoids, lignans, flavonoids, alkaloids, stereoids
8	C. prainii	antipyresis, diuretic and chyluria	alkaloids, polyphenols, flavonoids
9	E. Burm	heat clearing and detoxicating, pharyngitis, dysentery, diarrhea, furuncle ulcer, skin itching, swelling and pain of hemorrhoids, throat red and swollen, bleeding gums and traumatic injury.	quinones, triterpenoids, flavonoids
10	E. daneillii	diarrhea, abdominal pain and vomiting	alkaloids, flavonoid glycosides, flavaprin, limonoids
11	E. fortunei	chronic diarrhea, dysentery, dispersing blood stasis and traumatic bleeding	alkaloids, triterpenoids, flavonoids
12	E. laxiflorus	pesticide, rheumatism, bone fractures and hemoptysis	alkaloids, triterpenoids, flavonoids, sesquiterpenes (agarofurans)
13	G. morella	wound rot, carbuncle, tinea, ulcer and sore, anthelminthic and containing toxic substances	phenols (gambogic acid), flavonoids (xanthones), triterpenoids
14	I. simonsii	scabies, bladder hernias, mixed cropping of edible spices and containing toxic substances	terpenoids, lignans, flavonoids, phenols
15	K. angustifolia	anti-infection, swelling and pain, ulcer and enteritis and heat stroke	lignans, triterpenoids
16	L. lancifolia	stomach pain, vomiting and swelling	alkaloids, terpenoids, flavonoids, essential oils
17	M. hongheensis	vomiting, diarrhea, dysentery, constipation and geriatric hacking cough	alkaloids
18	M. salicina	carbuncle, furunculosis and sore pain	alkaloids, lignans
19	M. squamulata	scabies, carbuncle boils swollen poison, hemorrhoids, enterobiasis, beriberi, rheumatoid and snake bite	triterpenoids
20	M. yunnanensis	hepatitis, sore, otitis media, stomach and duodenal ulcer, enlarged spleen and boils swelling, hematuria leucorrhea and traumatic bleeding	Polyphenols,tannins，flavonoids,coumarins, various terpenoids
21	O. javanica	lump in the abdomen, boils and swelling of throat and containing toxic substances	falcarindiol, carotatoxin, 5-allylpyrogallol
22	P. edulis	(no record)	unsaturated organic acids
23	P. molle	carbuncle, swollen abscess, fistula and scrofula	tannins, flavonoids, alkaloids
24	R. japonicus	subsiding swelling, jaundice, malaria lymph node tuberculosis and pterygium	lactones (anemonin, protoanemonin), flavonoids
25	S. arborescens	HBV (skimmianine), rheumatoid, paralysisa, beriberi, and containing toxic substances	alkaloids, coumarins, triterpenoids, phenols
26	S. davidii	antipyretic, detoxicate, subsiding swelling, laryngitis, pneumonia, dysentery, cystitis and edema	polyphenols
27	S. parasitica	anti-rheumatism and anticancer	flavonoids
28	S. sinensis	furuncle and swelling	benzoquinone, tannins, phenols, lignans, flavonoids, triterpenoids
29	S. tamariscina	inflammation, pharyngolaryngitis and bacteriostasis	flavonoids, phenol glycosides, trehalose
30	S. viridis	urticaria, herpes zoster, rheumatism and analgesia	lignans, triterpenoids, organic acids

2. Results and Discussion

2.1. Results

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.

Table 2. The antibacterial agent-susceptibility test results of MRSA strains.

**Table 2.** The antibacterial agent-susceptibility test results of MRSA strains.
MRSA Strains	Resistant (R)	Intermediate (I)	Susceptible (S)
MRSA 008	PEN, OXS, AMP, LEV, CTX, PIP/S, ERY, CLI, AZM, RIF	NONE	VAN, TEC, LNZ, FOS,
MRSA 082	AMP, CLI, RIF, CTX V, CIP, LEV, CAZ	FOS	LNZ, VAN
MRSA 092	AZM, OXS, AMP, FOX, CLI, CLR, LEV	NONE	COT, RIF, PIP/S, VAN, GAT, FOS
MRSA 123	PEN, OXS, FOX, IPM CTS	NONE	LNZ, VAN, MXF, FOS
MRSA 144	PEN, AMP, OXS, FOX, FUR, CFZ, AZM, RIF, CLI, CLR	NONE	VAN, FOS
MRSA 189	PEN, OXS, AMP, FOX, FUR, CTX, COT, CIP, LEV	FOS	VAN, LNZ, MXF
MRSA 321	CLR, CLI, AZM, PEN, OXS, AMP, ERY. CTX, FUR, LEV	CAT	VAN, GAT, PIP/T

AMP: ampicillin; AZM: azithromycin; CAT: cefathiamidine; CAZ: ceftazidime; CFZ: cefazolin; CIP: ciprofloxacin; CLI: clindamycin; CLR: clarithromycin; COT: Cotrimoxazole; CTS: cilastatin sodium; CTX: Cefotaxime; ERY: erythromycin; FOS: fosfomycin; FOX: cefoxitin; FUR: cefuroxime; GAT: gatifloxacin; IPM: imipenem; LEV: levofloxacin; LNZ: linezolid; MXF: moxifloxacin; OXS: oxacillin; PEN: Penicillin; PIP: piperacillin; PIP/S: piperacillin/sulbactam; PIP/T: piperacillin/tazobactam; RIF: rifampin; TEC: teicoplanin; VAN: Vancomycin.

Table 3. Screening results of the antimicrobial activities of the extracts from 30 Chinese medicinal plants (IZD: mm) ^a.

**Table 3.** Screening results of the antimicrobial activities of the extracts from 30 Chinese medicinal plants (IZD: mm) ^a.
No.	Species	Part ^b	Weight (g) ^c	Ratio (%) ^d	SA ^e	EC ^f	PA ^g	CA ^h
1	A. nepalensis	TBL	3.85	7.7	14	<8	12	9
2	B. balsamifer	WP	0.61	1.2	12	9	<8	5
3	B. hancei	WP	4.96	9.9	19	10	13	11
4	C. austroglauca	TBL	1.50	3.0	23	14	15	9
5	C. japonica	V	1.57	3.1	9	11	13	8
6	C. orbiculatus	V	2.06	4.1	11	10	<8	10
7	C. orchioides	WP	2.28	4.6	16	13	14	8
8	C. prainii	WP	1.96	3.9	10	10	<8	9
9	E. Burm	L	3.03	6.1	10	10	10	<8
10	E. daneillii	TBL	2.52	5.0	18	12	10	9
11	E. fortunei	V	4.42	8.8	15	13	12	11
12	E. laxiflorus	TBL	2.68	5.4	<8	<8	<8	10
13	G. morella	WP	7.38	14.8	17	12	<8	<8
14	I. simonsii	TBL	3.48	7.0	13	11	11	8
15	K. angustifolia	TBL	0.99	2.0	<8	8	<8	8
16	L. lancifolia	TBL	2.28	4.6	<8	<8	<8	<8
17	M. hongheensis	TBL	3.09	6.2	21	11	13	<8
18	M. salicina	TBL	2.91	5.8	12	9	8	10
19	M. squamulata	TBL	1.48	2.9	17	12	12	11
20	M. yunnanensis	TBL	2.91	5.8	24	12	<8	9
21	O. javanica	WP	2.65	5.3	<8	8	<8	<8
22	P. edulis	TBL	4.49	9.0	<8	<8	<8	<8
23	P. molle	WP	2.99	6.0	11	10	16	12
24	R. japonicus	WP	5.24	10.5	<8	9	8	<8
25	S. arborescens	TBL	1.86	3.7	24	13	14	11
26	S. davidii	TBL	2.54	5.1	<8	8	9	10
27	S. parasitica	TBL	1.04	2.1	<8	<8	<8	<8
28	S. sinensis	TBL	6.49	13.0	18	8	13	21
29	S. tamariscina	WP	4.44	8.9	12	9	11	9
30	S. viridis	V	2.63	5.3	12	<8	<8	9

^a IZD: Inhibition zone diameter (the concentration of the extract at 50 mg/mL); ^b Part: The part of plant used for extraction (L: leaves; TBL: tender branches and leaves; WP: whole plant; V: vane); ^c Weight: The weight of extract; ^d Ratio: The ratio of extract; ^e SA: Staphylococcus aureus (ATCC 25923, MSSA); ^f EC: Escherichia coli (ATCC 25922); ^g PA: Pseudomonas aeruginosa (ATCC 27853); ^h CA: Candida albicans (ATCC Y0109).

Table 4. Comparison of IZDs of the 21 extracts against MRSA and MSSA strains (IZD: mm) ^a.

**Table 4.** Comparison of IZDs of the 21 extracts against MRSA and MSSA strains (IZD: mm) ^a.
No.	Extracts	MSSA	MRSA_ave ± SEM (n) ^b	△(MSSA-MRSA_ave)
1	M. yunnanensis	24	17.5 ± 1.04 (4)	6.5
2	S. arborescens	24	18.8 ± 2.06 (4)	5.2
3	C. austroglauca	23	18.5 ± 1.85 (4)	4.5
4	M. hongheensis	21	15.8 ± 1.65 (4)	5.2
5	B. hancei	19	16.0 ± 1.63 (4)	3.0
6	S. sinensis	18	14.3 ± 1.31 (4)	3.7
7	E. daneillii	18	13.8 ± 1.31 (4)	4.2
8	M. squamulata	17	14.5 ± 0.87 (4)	2.5
9	G. morella	17	15.7 ± 0.67 (3)	1.3
10	C. orchioides	16	17.3 ± 0.95 (4)	−1.3
11	E. fortunei	15	15.5 ± 1.55 (4)	−0.5
12	A. nepalensis	14	17.0 ± 1.41 (4)	−3.0
13	I. simonsii	13	12.8 ± 1.11 (4)	0.2
14	B. balsamifer	12	7.0 ± 1.22 (4)	5.0
15	S. viridis	12	11.0 ± 1.68 (4)	1.0
16	S. tamariscina	12	11.0 ± 1.00 (4)	1.0
17	M. salicina	12	9.7 ± 0.33 (3)	2.3
18	P. molle	11	15.8 ± 1.38 (4)	−4.8
19	C. orbiculatus	11	9.5 ± 0.50 (4)	1.5
20	E. Burm	10	13.0 ± 2.08 (3)	−3.0
21	C. prainii	10	9.0 ± 1.00 (2)	1.0

^a IZD: Inhibition zone diameter (the concentration of the extract at 50 mg/mL); ^b Number of MRSA isolates.

Table 5. Anti-MRSA activity of the extracts to the zone of inhibition against MSSA ≥10 mm (MIC/MBC: μg/mL).

**Table 5.** Anti-MRSA activity of the extracts to the zone of inhibition against MSSA ≥10 mm (MIC/MBC: μg/mL).
No.	Extracts and Vancomycin	Activity	MSSA	MRSA 082	MRSA 092	MRSA 189	MRSA 144	MRSA 321
1	M. yunnanensis	MIC	32	8	32	64	16	32
1	M. yunnanensis	MBC	128	64	256	128	64	256
2	S. arborescens	MIC	64	64	64	64	16	32
2	S. arborescens	MBC	256	256	256	256	128	128
3	C. austroglauca	MIC	64	64	64	64	32	16
3	C. austroglauca	MBC	256	128	512	256	128	256
4	M. hongheensis	MIC	32	128	8	32	16	16
4	M. hongheensis	MBC	128	512	32	128	64	64
5	B. hancei	MIC	64	64	32	64	32	64
5	B. hancei	MBC	128	256	256	128	128	256
6	E. daneillii	MIC	32	32	32	32	32	64
6	E. daneillii	MBC	64	256	128	256	256	256
7	S. sinensis	MIC	32	64	32	32	32	16
7	S. sinensis	MBC	128	256	256	64	128	64
8	G. morella	MIC	32	32	64	32	16	32
8	G. morella	MBC	256	256	256	128	64	128
9	M. squamulata	MIC	64	32	64	32	64	32
9	M. squamulata	MBC	256	128	256	128	256	128
10	C. orchioides	MIC	512	256	512	512	512	256
10	C. orchioides	MBC	1,024	1,024	>2,048	>2,048	512	1024
11	E. fortunei	MIC	512	512	512	512	512	512
11	E. fortunei	MBC	>2,048	>2,048	1,024	>2,048	>2,048	>2,048
12	A. nepalensis	MIC	1024	512	256	512	512	256
12	A. nepalensis	MBC	>2,048	1,024	1,024	>2,048	>2,048	1,024
13	I. simonsii	MIC	512	512	512	1,024	1,024	1,024
13	I. simonsii	MBC	1,024	>2,048	>2,048	>2,048	>2,048	>2,048
14	B. balsamifer	MIC	256	256	256	64	256	128
14	B. balsamifer	MBC	1,024	256	1,024	256	1,024	256
15	M. salicina	MIC	512	512	1,024	512	512	512
15	M. salicina	MBC	1,024	>2,048	>2,048	>2,048	1,024	>2,048
16	S. viridis	MIC	256	128	64	128	128	128
16	S. viridis	MBC	1,024	512	256	512	512	512
17	S. tamariscina	MIC	512	1,024	1,024	1,024	1,024	1,024
17	S. tamariscina	MBC	1,024	>2,048	>2,048	2,048	>2,048	>2,048
18	C. orbiculatus	MIC	1,024	1,024	512	512	1,024	512
18	C. orbiculatus	MBC	>2,048	>2,048	1,024	1,024	>2,048	1,024
19	P. molle	MIC	512	256	256	256	256	256
19	P. molle	MBC	>2,048	1,024	1,024	1,024	1,024	1,024
20	C. prainii	MIC	1,024	1,024	1,024	1,024	1,024	2,048
20	C. prainii	MBC	>2,048	>2,048	2,048	>2,048	>2,048	>2,048
21	E. Burm	MIC	512	512	1,024	1,024	1,024	1,024
21	E. Burm	MBC	>2,048	1,024	>2,048	>2,048	>2,048	>2,048
/	Vancomycin	MIC	1	1	1	1	1	1
/	Vancomycin	MBC	2	2	2	2	2	2

2.2. 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 Table 4 and Table 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.

From the phytochemical constitution of the 30 plant materials listed in Table 1, it is found that most of the active extracts contained tannins, (poly)phenols (including flavonoids, lignans and coumarins), terpenoids or alkaloids which have been reported [18,20,21,22,23,24,25].

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.

3. Experimental

3.1. Plant Materials

The selected 30 Chinese medicinal plant samples were collected from the tropical mountain forests of southeastern Yunnan Province of China, at altitudes of 1,500–3,074 m in June 2010. They were identiﬁed by Y.M. Shui at the Kunming Institute of Botany (KIB); the Chinese Academy of Sciences. Voucher specimens are preserved at the herbarium of KIB [34]. The names of species/family (specimen No.) were as the following: Alnus nepalensis D. Don./Betulaceae (KUN 35); Blumea balsamifer (Linn.) D.C./Asteraceae (KUN 21); Brandisia hancei Hook. f./Scrophulariaceae (YFS 6); Carex prainii C.B. Clarke/Cyperaceae (KUN 11133); Cayratiy japonica (Thunb) Gngacp.var.japonica/Vitaceae (YFS 531); Celastrus orbiculatus Thunb./Celastraceae (KUN 110); Curculigo orchioides Gaertn./Hypoxidaceae (KUN 1151); Cyclobalanopsis austroglauca Y.T. Chang/Fagaceae (SWFC 851222); Embelia Burm f./Myrsinaceae (KUN355); Euonymusfortunei (Turcz.); Hand. Mazz./Celastraceae (YUKU (s.n)); Euonymus laxiflorus Charmp.ex.Benth./Celastraceae (YCP 851027); Evodia daneillii (Benn) Hemsl./Rutaceae (YFS 663); Garcinia morella Desr./Clusiaceae (YCP 851757); Illicium simonsii Maxim./Il1iciaceae (IBSC 125); Kadsura angustifolia A.C. Smith/Schisandraceae (KUN 4480); Litsea lancifolia (Roxb. ex. Nees); Benth. et. Hook. f. ex. F./Lauraceae (KUN 23); Machilus salicina Hance./Lauraceae (YUKU 1535); Mallotusyunnanensis Pax et. Hoffm./Euphorbiaceae (YFS 1144); Manglietia hongheensis Y.m Shui et. W.H. Chen./Magnoliaceae (KUN 262); Meliosma squamulata Hance./Lauraceae (KUN 2411); Oenanthe javanica (Bl.)DC./Umbellifera (KUN 214); Polygonummolle D. Don./Polygonaceae (KUN 367); Pyrularia edulis (Wall.) A.D.C./Santalaceae (KUN 60002100); Ranunculus japonicus Thumb./Ranunculaceae (KUN 2398); Schima sinensis (Hemsl. et. Wils) Airy-shaw./Theaceae (KUN158); Schisandra viridis A.c.Smith./Schisandraceae (YFS); Scurrula parasitica Linn.var. parasitica./Loranthaceae (YFS 327); Selaginella tamariscina (Seauv.) Spring./Selaginellaceae (YCP 85937); Skimmia arborescens Anders./Rutaceae (PE 100260); Sophora davidii (Franch.) Skeels./Leguminosae (YCP 851049). The traditional indications and phytochemical constitutions were listed in Table 1.

3.2. Microbial Strains and Culture Media

Standard bacterial and fungal strains, i.e., Staphylococcus aureus (ATCC 25923, MSSA), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Candida albicans (ATCC Y0109) were provided by the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, China). Clinical MDR MRSA strains of MRSA 8, MRSA 82, MRSA 92, MRSA 123, MRSA 144, MRSA 189 and MRSA 321 were clinical isolates from infectious samples of critically ill patients in Kunming General Hospital (KGH). Pathogen puriﬁcation and identiﬁcation (including colonial morphology, Gram staining and coagulase testing) were conducted in our clinical microbiology laboratory and further conﬁrmed by standard cefoxitin disk diffusion test following the Clinical and Laboratory Standards Institute (CLSI) guidelines [15,35,36]. ATCC 25923 was used as the control strain. Vancomycin (Eli Lilly Japan K.K., Seishin Laboratories) was used as a control anti-MRSA agent. Standard Mueller-Hinton agar and broth (MHA and MHB) and Sabouraud agar (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.

3.3. 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).

3.4. 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 × 10⁸ CFU/mL for bacteria and 5 × 10⁵ 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).

3.5. 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 × 10⁵ 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).

4. 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.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (NSFC 81073126, 81173504) and the supporting funds of the Department of Health, Chengdu Military Region, PLA of China (MB09030) and Yunnan Province of China (2008PY001).

Sample Availability: Not available.

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Zuo, G.-Y.; Zhang, X.-J.; Yang, C.-X.; Han, J.; Wang, G.-C.; Bian, Z.-Q. Evaluation of Traditional Chinese Medicinal Plants for Anti-MRSA Activity with Reference to the Treatment Record of Infectious Diseases. Molecules 2012, 17, 2955-2967. https://doi.org/10.3390/molecules17032955

AMA Style

Zuo G-Y, Zhang X-J, Yang C-X, Han J, Wang G-C, Bian Z-Q. Evaluation of Traditional Chinese Medicinal Plants for Anti-MRSA Activity with Reference to the Treatment Record of Infectious Diseases. Molecules. 2012; 17(3):2955-2967. https://doi.org/10.3390/molecules17032955

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Zuo, Guo-Ying, Xin-Juan Zhang, Cui-Xian Yang, Jun Han, Gen-Chun Wang, and Zhong-Qi Bian. 2012. "Evaluation of Traditional Chinese Medicinal Plants for Anti-MRSA Activity with Reference to the Treatment Record of Infectious Diseases" Molecules 17, no. 3: 2955-2967. https://doi.org/10.3390/molecules17032955

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

Abstract

1. Introduction

2. Results and Discussion

2.1. Results

2.2. Discussion

3. Experimental

3.1. Plant Materials

3.2. Microbial Strains and Culture Media

3.3. Extract Preparation

3.4. Antimicrobial Screening

3.5. MICs and MBCs Assaying

4. Conclusions

Acknowledgements

References and Notes

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