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Chinese medicinal herbs against antibiotic-resistant bacterial pathogens

Ben Chung-Lap Chan1,4

, Clara Bik-San Lau1,4

, Claude Jolivalt5,6

, Sau-Lai Lui1,2,4

, Carine Ganem-

Elbaz5,6

, Jean-Marc Paris5,6

, Marc Litaudon7, Kwok-Pui Fung

1,3,4, Ping-Chung Leung

1,4and Margaret

Ip2

*1Institute of Chinese Medicine; 2Department of Microbiology; 3School of Biomedical Sciences; 4State Key Laboratory of

Phytochemistry & Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories,Hong Kong, 5ENSCP ChimieParisTech, Laboratoire Charles Friedel and 6CNRS, UMR 7223, 75005 Paris, France,7Institut de Chimie des Substances Naturelles, France. *Corresponding author

Bacterial resistance to antibiotics has become a serious problem of public health that concerns almost all antibacterialagents and that manifests in all fields of their application. Novel antimicrobial compounds against new bacterial targetsand drug resistance mechanisms are urgently needed. Plant-derived antibacterials are always a source of novel therapeutics.

This article summarizes our studies in identifying the extracts and molecules which exhibit either direct growth inhibitionor resistance mechanisms against bacteria from 33 Chinese medicinal herbs which are commonly used and are claimed tohave antibacterial properties. A panel of Gram-positive, Gram-negative and drug resistant bacteria include Staphylococcus

aureus,Enterococcus faecalis,Pseudomonas aeruginosa,Escherichia coli andAcinetobacter baumannii were used in thestudy. In addition, evidence from published data of compounds from these Chinese herbs with promising antibacterial

effects will be reviewed.

Keywords Medicinal herbs; bacterial infection; drug resistant bacteria; MRSA; Chinese medicinal herbs

1. Introduction

Resistance to antimicrobials is a significant and growing problem, limiting treatment options, especially for seriousGram-positive infections [1]. Among them, methicillin resistant Staphylococcus aureus (MRSA) is the major cause ofworldwide outbreaks of both hospitals and the community infections [2-5]. At present, the pharmaceutical arsenalavailable to control MRSA is limited. Glycopeptide antibiotics, such as vancomycin, have traditionally been the

mainstay of treatment of MRSA but overuse has led to the emergence of vancomycin-resistant strains [6]. Concertedefforts are to be made to identify anti-MRSA materials from natural products [7-9] and the long history of Chinese

herbal medicine demonstrates the potential of plants as sources of lead compounds. More than 140 Chinese herbs areused in the treatment of bacterial infection [10] and some of the herbs have been shown to possess anti-MRSA activity[11-13]. In order to further explore the potential of Chinese medicinal herbs for the treatment of drug resistant bacterialinfections with particular reference to MRSA, we have chosen 33 commonly used Chinese herbs that are claimed topossess antibacterial properties for a systematic screening of their growth inhibition with a panel of bacteria strains. Inaddition, evidence from published data of compounds from these Chinese herbs with promising antibacterial effects willbe reviewed.

2. Materials and methods

2.1 Plant materials and preparation of plant extracts

Thirty-three Chinese medicinal herbs (Table 1) were included in the study. The herbs were purchased from the herbal

store in Hong Kong and the voucher specimens were deposited at the Institute of Chinese Medicine, the ChineseUniversity of Hong Kong and were extracted by standardized methods [14]. In brief, the herbal materials were soaked

for 1 h and then boiled twice with distilled water, 50% or 95% ethanol for 2 h under reflux. The aqueous or ethanolicextracts were individually collected and filtered. The filtrates were then concentrated under reduced pressure at 50 Cand lyophilized into powder.

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Table 1 List of Chinese Medicinal plants used in current study

Name of Chinese medicine Family Species

1 Caulis Spatholobi Leguminosae Spatholobus suberectus Dunn.2 Cortex Dictamni Rutaceae Dictamnus dasycarpus Turcz.

3 Cortex Ilicis Aquifoliaceae Ilex rotunda Thunb.4 Cortex Lycii Solanaceae Lycium chinense Mill.5 Cortex Moutan Ranunculaceae Paeonia suffruticosa Andr.6 Cortex Phellodendri Chinensis Rutaceae Phellodendron amurense Rup.

7 Flos Chrysanthemi Indici Compositae Chrysanthemum indicum L.8 Flos Lonicerae Japonicae Capri-foliaceae Lonicera japonica Thunb.

9 Folium Isatidis Cruciferae Isatis indigotica Fort.10 Fructus Forsythiae Oleaceae Forsythia suspensa (Thunb.) Vahl.11 Fructus Gardeniae Rubiaceae Gardenia jasminoides Ellis.

12 Herba Andrographis Acanthaceae Andrographis paniculata (Burm. F.) Nees.13 Herba Centellae Umbelliferae Centella asiatica (L.) Urb.14 Herba Patriniae Valerianaceae Patrinia villosa Juss.

15 Herba Portulacae Portulacaeae Portulaca oleracea L.16 Herba seu Rhizoma Nerviliae Fordii Orchidaceae Nervilia fordii (Hanee) Schltr.17 Herba Taraxaci Compositae Taraxacum mongolicum Hand. -Mazz.18 Herba Violae Violaceae Viola yedoensis Makino.19 Carpophorum Calvatiae Lycoperdaceae Calvatia lilacina (Mont.et Berk.) Lloyd.

20 Radix Gentianae Gentianaceae Gentiana manshurica Kitag.21 Radix Isatidis Cruciferae Isatis indigotica Fort.22 Radix Pulsatillae Ranunculaceae Pulsatilla chinensis (Bge.) Regel.

23 Radix Salviae Miltiorrhizae Labiatae Salvia miltiorrhiza Bge.24 Radix Scutellariae Labiatae Scutellaria baicalensis Georgi.25 Radix Sophorae Flavescentis Leguminosae Sophora flavescens Ait.

26 Radix Trichosanthis Cucurbitaceae Trichosanthes kirilowii Maxim.

27 Rhizoma Anemarrhenae Liliaceae Anemarrhena asphodeloides Bge.28 Rhizoma Belamcandae Iridaceae Belamcanda chinensis (L.) DC.

29 Rhizoma Coptidis Ranunculaceae Coptidis chinensis Franch.

30 Rhizoma Paridis LiliaceaeParis polyphylla Smith var.yunnanensis (Franch.)Hand.-Mazz.

31 Rhizoma Picrorhizae Scrophulariaceae Picrorhiza scrophulariiflora Pennell.32 Semen Vignae Radiatae Fabaceae Vigna radiata (L.) R.Wilczek.33 Spica Prunellae Labiatae Prunella vulgaris L.

2.2 Bacterial strains

Initially, an Escherichia coli (strain ATCC 25922) and a susceptible strain ofStaphylococcus aureus (ATCC 25923)

were used in preliminary screening. Five laboratory MRSA strains with known resistance mechanisms wereused for further screening. A fluoroquinolone resistant strain (SA-1199B) ofS. aureus harbouring the NorA gene thatencodes a membrane-associated protein mediating active efflux of fluoroquinolones [15]. SA-RN4220-pUL5054,which is resistant to 14- and 15-membered macrolides including erythromycin and contains the multicopies plasmid

pUL5054 coding for MsrA, an efflux pump [16]. The other three strains were experimentally induced aminoglycosides

resistance: SA-APH2-AAC6 [17] (aminoglycoside- 6-N-acetyltransferase/ 2-O- phosphoryltransferase) which isresistant to gentamicin,, SA-APH3 [18](aminoglycoside- 3-O-phosphoryltransferase) which is resistant to kanamycinand SA-ANT4 [19] (aminoglycoside-4-O- nucleotidyl transferase) which is resistant to tobramycin. Enterococcusfaecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606 andAcinetobacter baumannii 4405 (Efflux pump overexpressed strain) were also included in the study.

2.3 Determination of antibacterial susceptibilities

All strains were cultured on Mueller-Hinton (MH) broth (Oxoid) at 37oC. An overnight culture broth of each strain wasdiluted to obtain initial inoculums of 106 conlony forming unit (CFU)/ml. The actual CFU counts of initial inoculumswere enumerated by the standard plate counting method: broth aliquot was serially diluted, plated in duplicate onto MHagar plates, incubated at 37oC for 1824 h and the CFU/ml determined from counting the numbers of single colonies.The antibacterial activity screening of extracts was carried out in MH broth medium using a Biomek model 3000 liquid-

handling robot (Beckman-Coulter) and 96 well microtiter plates. A 5 l aliquot of the extract solution was added toeach well of the plate containing MH broth and bacteria (10 6 CFU/ml) to yield a final volume in each well of 200 l.

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The final concentration of sample was either 1 mg/ml or 0.1 mg/ml in 1% DMSO. Ampicillin (16 mg/L) was used as apositive control and 1% of DMSO was used as a reagent control. Plates were incubated at 37C, and bacterial growthwas monitored by measuring the optical density (OD) of the broth at 620 nm following 24 h of incubation.

The antimicrobial effects of the extracts were expressed as the percent of reduction after 24 h of incubation and werecalculated as one minus the percentage of growth in each well. The percentage of growth was calculated as the OD ofeach well divided by the OD of the drug-free well after subtracting the background OD obtained from microorganism-

free microtiter plates. The antibacterial activities of the extracts were classified into 3 different classes: if no bacterialgrowth was detected after 24 h of incubation, the extracts were classified as very active (indicated by ++ in Table 2 and

3), and as active (+) if the percent reduction of the bacterial growth is greater than or equal to 90% compared to thegrowth control (with no extracts), and as not active (-) if the reduction of bacterial growth was less than 90% [9]. All

experiments were performed in duplicates.

Table 2 Antibacterial activity of the herbal extracts onE. coli ATCC25922 and S. aureus ATCC25923

Bacteria strainsE.coli ATCC 25922 S.aureus ATCC 25923

Name of Chinese medicine Concentration 0.1 mg/ml 1 mg/ml 0.1 mg/ml 1 mg/mlType of extract

Carpophorum Calvatiae Water - - - ++Cortex Lycii 95% ethanol - - - -

Cortex Moutan 95% ethanol - - - +Radix Salviae Miltiorrhizae 95% ethanol - - - ++

50% ethanol - - - ++Radix Scutellariae 95% ethanol - - - ++

50% ethanol - - - -Radix Sophorae Flavescentis 95% ethanol - - ++ ++

50% ethanol - - ++ -Rhizoma Coptidis Water - - - ++

95% ethanol - - - ++50% ethanol - - - ++

(++) very active, complete growth inhibition, (+) active, 90% growth inhibition and () not active,

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its parent strainA. baumannii ATCC 19606. Finally, the aqueous extracts of Carpophorum Calvatiae and the ethanolicextracts of Radix Salviae Miltiorrhizae were inactive in all 4 tested bacteria strains.

3.3 Antibacterial activities of the herbal extracts against MRSA with known resistant mechanism

The active extracts identified from the initial screening were tested with 5 MRSA strains with known resistant

mechanisms and the results were summarized in Table 3(b). Again, strongest antibacterial activities were found in theethanol extracts of Radix Sophorae Flavescentis and complete growth inhibitions were observed at 0.1mg/ml against 4

out of 5 MRSA strains and it was less effective in inhibiting the growth ofS. aureus RN4220. These results suggestedthat Radix Sophorae Flavescentis were effective in inhibiting the growth of MRSA with different resistant mechanisms.

The ethanolic extracts of Cortex Lycii, Radix Salviae Miltiorrhizae, Radix Scutellariae and both the aqueous andethanolic extracts of Rhizoma Coptidis at 1mg/ml were also active in inhibiting the MRSA strains. A weakerantibacterial activity was observed in Cortex Moutan when compared other tested extracts. For the aqueous extracts ofCarpophorum Calvatiae, the antibacterial actions on the tested MRSA strains were inactive.

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Table3Antibacterialactivityofthehe

rbalextractson(a)otherbacterialstrains

;and(b)methicillinresistantS.aureusstrains

(a)

Bacterialstrains

E.

faecalisATCC29212

P.aeruginosaATCC27853

A.baumanniiATCC19606

A.bauman

nii4405

N

ameofChinesemedicine

Concentra

tion

1mg/ml

0.1mg/ml

1mg/ml

0.1mg/ml

1mg/ml

0.1mg/ml

1mg/ml

0.1mg/ml

Typeofextract

Ca

rpophorumCalvatiae

Water

-

-

-

-

-

-

-

-

Co

rtexLycii

95%eth

anol

++

-

-

-

+

+

-

++

-

Co

rtexMoutan

95%eth

anol

++

-

-

-

+

-

+

-

Ra

dixSalviaeMiltiorrhizae

95%eth

anol

-

-

-

-

-

-

-

-

50%eth

anol

-

-

-

-

-

-

-

-

Ra

dixScutellariae

95%eth

anol

++

-

-

-

-

-

+

-

50%eth

anol

-

-

-

-

-

-

-

-

Ra

dixSophoraeFlavescentis

95%eth

anol

++

++

-

-

-

-

-

-

50%eth

anol

++

++

-

-

-

-

-

-

Rh

izomaCoptidis

Water

-

-

-

-

-

-

++

-

95%eth

anol

++

-

-

-

-

-

++

-

50%eth

anol

++

-

-

-

-

-

++

-

(++)ver

yactive,completegrowthinhibition),(+)active,

90%growthinhibitionand()

notactive,