chinese medicinal herbs against antibiotic-resistant...

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 Fung1,3,4, Ping-Chung Leung1,4 and 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 antibacterial agents and that manifests in all fields of their application. Novel antimicrobial compounds against new bacterial targets and 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 inhibition or resistance mechanisms against bacteria from 33 Chinese medicinal herbs which are commonly used and are claimed to have antibacterial properties. A panel of Gram-positive, Gram-negative and drug resistant bacteria include Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumannii were used in the study. 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 serious Gram-positive infections [1]. Among them, methicillin resistant Staphylococcus aureus (MRSA) is the major cause of worldwide outbreaks of both hospitals and the community infections [2-5]. At present, the pharmaceutical arsenal available 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]. Concerted efforts 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 are used 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 bacterial infections with particular reference to MRSA, we have chosen 33 commonly used Chinese herbs that are claimed to possess antibacterial properties for a systematic screening of their growth inhibition with a panel of bacteria strains. In addition, evidence from published data of compounds from these Chinese herbs with promising antibacterial effects will be 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 Chinese University 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 ethanolic extracts were individually collected and filtered. The filtrates were then concentrated under reduced pressure at 50 °C and lyophilized into powder.

773©FORMATEX 2011

Science against microbial pathogens: communicating current research and technological advances A. Méndez-Vilas (Ed.)_______________________________________________________________________________

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 Liliaceae Paris 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 of Staphylococcus aureus (ATCC 25923) were used in preliminary screening. Five laboratory MRSA strains with known resistance mechanisms were used for further screening. A fluoroquinolone resistant strain (SA-1199B) of S. aureus harbouring the NorA gene that encodes 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 is resistant to gentamicin,, SA-APH3’ [18] (aminoglycoside- 3’-O-phosphoryltransferase) which is resistant to kanamycin and SA-ANT4’ [19] (aminoglycoside-4’-O- nucleotidyl transferase) which is resistant to tobramycin. Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606 and Acinetobacter 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 was diluted to obtain initial inoculums of 106 conlony forming unit (CFU)/ml. The actual CFU counts of initial inoculums were enumerated by the standard plate counting method: broth aliquot was serially diluted, plated in duplicate onto MH agar plates, incubated at 37oC for 18–24 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 µl aliquot of the extract solution was added to each well of the plate containing MH broth and bacteria (106 CFU/ml) to yield a final volume in each well of 200 l.

774 ©FORMATEX 2011

Science against microbial pathogens: communicating current research and technological advances A. Méndez-Vilas (Ed.)______________________________________________________________________________

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 a positive control and 1% of DMSO was used as a reagent control. Plates were incubated at 37◦C, and bacterial growth was 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 were calculated as one minus the percentage of growth in each well. The percentage of growth was calculated as the OD of each 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 bacterial growth 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 the growth 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 on E. coli ATCC25922 and S. aureus ATCC25923

Bacteria strains E.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/ml Type 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, <90% growth inhibition

3. Results

3.1 Antibacterial activities of the herbal extracts against E. coli and S. aureus

In the initial screening using E. coli (ATCC 25922) and a susceptible strain of S .aureus (ATCC 25923), most of the herbal extracts exhibited mild inhibitory effects on the growth of S. aureus. The extracts from 7 herbs possessed strong antibacterial activities against the 2 tested strains and the results were summarized in Table 2. Strongest antibacterial activities were observed in the ethanol extracts of Radix Sophorae Flavescentis and both 50 and 95% ethanolic extracts could completely inhibit the growth of S. aureus at 0.1 mg/ml. Complete growth inhibition of S. aureus were also observed in the ethanolic extracts of Radix Salviae Miltiorrhizae, Radix Scutellariae, the 95% ethanolic extract of Cortex Lycii and the aqueous extracts of Carpophorum Calvatiae and Rhizoma Coptidis at 1mg/ml. The ethanolic extract of Cortex Moutan was also active in suppressing the growth of S. aureus. However, none of the extracts from 33 herbs was active against the growth of the Gram-negative bacteria E. coli. The extracts from these 7 Chinese medicinal herbs were chosen for further screening.

3.2 Antibacterial activities of the herbal extracts against A. baumannii, E. faecalis and P. aeruginosa

Those active extracts were also tested with 4 other bacterial strains and the results were summarized in Table 3(a). The ethanolic extracts of Radix Sophorae Flavescentis could inhibit the growth of Gram-positive strain E. faecalis completely but they were inactive in inhibiting the growth of the other 3 Gram-negative strains P. aeruginosa ATCC 27853, A. baumannii ATCC 19606 and A. baumannii 4405, suggesting that the antibacterial action of Radix Sophorae Flavescentis is likely to be restricted to Gram-positive strains. On the other hand, the 95% ethanolic extracts of Cortex Moutan and Cortex Lycii were active against both E. faecalis and the A. baumannii strains at 1mg/ml. The ethanolic extracts of Rhizoma Coptidis and the 95% ethanolic extract of Radix Scutellariae were active against Gram-positive strain E. faecalis. Interestingly, they were active against the efflux pump overexpressed A. baumannii 4405, but not to

775©FORMATEX 2011


its parent strain A. baumannii ATCC 19606. Finally, the aqueous extracts of Carpophorum Calvatiae and the ethanolic extracts 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 the ethanol 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 of S. aureus RN4220. These results suggested that 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 and ethanolic extracts of Rhizoma Coptidis at 1mg/ml were also active in inhibiting the MRSA strains. A weaker antibacterial activity was observed in Cortex Moutan when compared other tested extracts. For the aqueous extracts of Carpophorum Calvatiae, the antibacterial actions on the tested MRSA strains were inactive.

776 ©FORMATEX 2011


Tab

le 3

Ant

ibac

teri

al a

ctiv

ity o

f th

e he

rbal

ext

ract

s on

(a)

oth

er b

acte

rial

str

ains

; and

(b)

met

hici

llin

resi

stan

t S. a

ureu

s st

rain

s

(a)

Bac

teri

al s

trai

ns

E.

faec

alis

AT

CC

292

12

P. a

erug

inos

a A

TC

C 2

7853

A

. bau

man

nii A

TC

C 1

9606

A

. bau

man

nii 4

405

Nam

e of

Chi

nese

med

icin

e C

once

ntra

tion

1

mg/

ml

0.1

mg/

ml

1 m

g/m

l0.

1 m

g/m

l 1

mg/

ml

0.1

mg/

ml

1 m

g/m

l0.

1 m

g/m

l

Typ

e of

ext

ract

C

arpo

phor

um C

alva

tiae

W

ater

-

- -

- -

- -

- C

orte

x L

ycii

95

% e

than

ol

++

-

- -

++

-

++

-

Cor

tex

Mou

tan

95%

eth

anol

+

+

- -

- +

-

+

- R

adix

Sal

viae

Mil

tior

rhiz

ae

95%

eth

anol

-

- -

- -

- -

-

50%

eth

anol

-

- -

- -

- -

- R

adix

Scu

tell

aria

e 95

% e

than

ol

++

-

- -

- -

+

-

50%

eth

anol

-

- -

- -

- -

- R

adix

Sop

hora

e F

lave

scen

tis

95

% e

than

ol

++

+

+

- -

- -

- -

50

% e

than

ol

++

+

+

- -

- -

- -

Rhi

zom

a C

opti

dis

Wat

er

- -

- -

- -

++

-

95

% e

than

ol

++

-

- -

- -

++

-

50

% e

than

ol

++

-

- -

- -

++

-

(++

) ve

ry a

ctiv

e, c

ompl

ete

grow

th in

hibi

tion

), (

+)

acti

ve , 9

0% g

row

th in

hibi

tion

and

(–

) no

t act

ive,

<90

% g

row

th in

hibi

tion

(b)

Bac

teri

al s

trai

ns

S.au

reus

119

9B

S.au

reus

RN

4220

S.

aure

us A

PH

2”-A

AC

6’

S.au

reus

AP

H3’

S.

aure

us A

NT

4’

Nam

e of

Chi

nese

med

icin

e C

once

ntra

tion

0.

1 m

g/m

l 0.

1 m

g/m

l 1

mg/

ml

0.1

mg/

ml

1 m

g/m

l0.

1 m

g/m

l 1

mg/

ml

0.1

mg/

ml

1 m

g/m

l0.

1 m

g/m

l

Typ

e of

ext

ract

C

arpo

phor

um C

alva

tiae

W

ater

-

- -

- -

- -

- -

- C

orte

x L

ycii

95

% e

than

ol

- +

+

+

- +

-

++

-

++

+

C

orte

x M

outa

n

95%

eth

anol

-

- +

-

+

- +

+

+

-

Rad

ix S

alvi

ae M

ilti

orrh

izae

95

% e

than

ol

- +

+

+

- +

-

++

-

++

-

50

% e

than

ol

- +

+

+

- -

- +

-

++

-

Rad

ix S

cute

llar

iae

95%

eth

anol

-

++

+

+

- +

+

- +

+

+

++

-

50

% e

than

ol

- +

+

- -

++

-

++

-

++

-

Rad

ix S

opho

rae

Fla

vesc

enti

s

95%

eth

anol

+

+

++

+

-

++

+

+

++

+

+

++

+

+

50

% e

than

ol

++

+

+

+

- +

+

++

+

+

++

+

+

++

R

hizo

ma

Cop

tidi

s W

ater

-

++

-

- -

- +

+

- +

+

-

95%

eth

anol

-

+

+

- -

- +

+

- +

+

-

50%

eth

anol

-

++

+

-

- -

++

-

++

-

(++

) ve

ry a

ctiv

e, c

ompl

ete

grow

th in

hibi

tion

), (

+)

acti

ve , 9

0% g

row

th in

hibi

tion

and

(–

) no

t act

ive,

<90

% g

row

th in

hibi

tion

777©FORMATEX 2011


Tab

le 4

Sum

mar

y of

the

anti

-mic

robi

al a

ctio

n of

the

activ

e in

gred

ient

s fr

om th

e C

hine

se M

edic

ine

used

in c

urre

nt s

tudy

Nam

e of

Chi

nese

med

icin

e A

nti-

mic

robi

al a

ctiv

ities

A

ctiv

e in

gred

ient

s re

fere

nces

C

orte

x L

ycii

A

nti-

MR

SA a

ctiv

ity

Ant

i-fu

ngal

act

ivit

ies

(+)-

Lyo

nire

sino

l-3

alph

a-O

-bet

a-D

-glu

copy

rano

side

Dih

ydro

-N-c

affe

oylt

yram

ine,

tran

s-N

-fer

uloy

loct

opam

ine,

tran

s-N

-ca

ffeo

ylty

ram

ine

and

cis-

N-c

affe

oylt

yram

ine

[46]

[47]

Cor

tex

Mou

tan

Ant

imic

robi

al a

ctiv

ity a

gain

st S

. aur

eus,

S. E

pide

rmid

is, C

. A

lbic

ans,

C. T

ropi

cali

s an

d C

. Gla

brat

a

Pae

onol

fro

m th

e vo

lati

le f

ract

ions

of

the

herb

al e

xtra

cts

[48]

Rad

ix S

alvi

ae M

ilti

orrh

izae

A

ntib

acte

rial

act

ivity

aga

inst

B. s

ubti

lis,

E. c

asse

lifla

vus,

M.

lure

us, P

. aci

dila

ctic

i, S.

aur

eus

and

S. E

pide

rmid

is

Ant

ibac

teri

al a

ctiv

ity a

gain

st 2

1 S.

aur

eus

stra

ins

Cry

ptot

ansh

inon

e an

d di

hydr

otan

shin

one

I

C

rypt

otan

shin

one

[44]

[45]

Rad

ix S

cute

llar

iae

Syne

rgis

tic

effe

ct w

ith -

lact

am-r

esis

tant

str

ains

of

S. a

ureu

s A

nti-

Hel

icob

acte

r py

lori

act

ivity

Sy

nerg

ies

betw

een

baic

alei

n, te

trac

ycli

ne,

-lac

tam

s an

d ci

prof

loxa

cin

agai

nst M

RS

A a

nd in

hibi

t MR

SA

PK

In

duce

apo

ptos

is C

andi

da a

lbic

ans

and

Inhi

bit b

iofi

lm f

orm

atio

n

Bai

cali

n

Bai

cali

n

Bai

cale

in

Bai

cale

in

[29]

[34]

[30,

31]

[35-

37]

Rad

ix S

opho

rae

Fla

vesc

enti

s

Ant

ibac

teri

al a

ctiv

itie

s ag

ains

t the

Gra

m-p

osit

ive

bact

eria

: S.

aure

us, B

. sub

tili

s, S

. epi

derm

idis

, and

P. a

cnes

A

ntif

unga

l and

ant

ibac

teri

al a

ctiv

ity

Ant

ibac

teri

al a

ctiv

ity a

gain

st 1

0 is

olat

es o

f M

RSA

A

nti-

MR

SA a

nd V

RE

(V

anco

myc

in r

esis

tant

ent

eroc

occi

)

Kur

arid

in, S

opho

rafl

avan

one

G, k

urar

inon

e an

d 5-

met

hyls

opho

rafl

avan

one

B

Pr

enyl

Fla

vone

s

Sop

hora

flav

anon

e G

7,9,

2',4

'-tet

rahy

drox

y-

8-is

open

teny

l-5-

met

hoxy

chal

cone

(T

HIP

MC

)

[22]

[2

3]

[2

5]

[2

6]

Rhi

zom

a C

opti

dis

Ant

i-he

rpes

sim

plex

vir

us e

ffec

ts

Inhi

bit S

. epi

derm

idis

bio

film

for

mat

ion

Ant

imic

robi

al a

ctio

n of

ber

beri

ne p

oten

tiat

ed b

y 5’

-m

etho

xyhy

dnoc

arpi

n an

d in

com

bina

tion

wit

h am

pici

llin

or

oxac

illi

n

Ber

beri

ne

B

erbe

rine

Ber

beri

ne

[39]

[40]

[41,

43]

778 ©FORMATEX 2011


4. Discussion

In this study, we have identified strong antibacterial properties in the extracts from 7 Chinese herbs and the anti-microbial activities of the active components from those Chinese herbs from literature were summarized in Table 4. Gram positive bacteria were more sensitive to the tested Chinese medicinal extracts than Gram negative organisms and this phenomenon is consistent to previous findings [20, 21]. Those plant extracts demonstrating the highest levels of antibacterial activity were from Radix Sophorae Flavescentis. Its potent antibacterial activities are mainly contributed by a group of chemicals called flavonoids. Kuroyanagi et al [22] have isolated a panel of flavonoids which included kushenol, kushecarpins, norkurarinone, kurarinone and l-maackiain and they showed that these compounds exhibited significant antibacterial activities against the Gram-positive bacteria S. aureus, Bacillus subtilis, S. epidermidis and Propionibacterium acnes with the MIC ranged from 2.5-25 g/ml. Direct anti-microbial effects were also observed in four prenylated flavonoids: kuraridin, kurarinone, 5-methylsophoraflavanone B, and sophoraflavanone G were isolated from Radix Sophorae Flavescentis against 2 fungal strains Candida albicans ATCC 10231, Saccharomyces cerevisiae, and 5 bacterial strains E. coli KCTC 1924, Salmonella typhimurium KCTC 1926, S. epidermis ATCC 12228, and S. aureus KCTC 1621 [23, 24]. For anti-MRSA activities, direct antibacterial effects against MRSA by two Radix Sophorae Flavescentis isolated compounds sophoraflavanone G and prenylated Chalcone 7,9,2',4'- tetrahydroxyl-8- isopentenyl-5-methoxychalcone (THIPMC) were reported by Cha et al. [25] and Lee et al. [26] respectively. They also showed that these compounds could synergistically inhibit the growth of bacteria with ampicillin. Recently, flavonoids isolated from Radix Sophorae Flavescentis were shown to inhibit an enzyme called sortase A in vitro and kurarinol was the most potent inhibitor of sortase A, with an IC50 value of 107.7 ± 6.6 μM [21]. Sortase A plays a critical role in the pathological effects of Gram-positive bacteria by modulating the ability of the bacterium to adhere to host tissue via the covalent anchoring of adhesion molecules and other virulence associated proteins to cell wall peptidoglycans and S. aureus mutants lacking sortase fail to display surface proteins and are defective in the establishment of infections but microbial viability is not affected [27]. The inhibition of sortase A by the flavonoids might contribute to the antibacterial actions of Radix Sophorae Flavescentis. Besides direct antibacterial action, drug combinations offer a promising strategy to overcome bacterial resistance mechanisms and restore the effectiveness of antibiotics. Some flavones had a weak antibacterial effect on MRSA, but that at sub-MIC concentrations they greatly increased the susceptibility of these strains to -lactam antibiotics [28]. One example is Radix Scutellariae and its active components, baicalin and baicalein. Both flavones have been shown to restore the effectiveness of -lactam antibiotics against MRSA and other strains of beta-lactam-resistant S. aureus [29, 30]. In Gram-negative bacteria, baicalein was shown to reverse the resistance in TetK overexpressing E. coli by inhibiting tetracyclin efflux pump TetK [30]. Furthermore, two novel mechanisms of actions of baicalein against MRSA were reported recently. Firstly, by using MRSA 1199B and a panel of ciprofloxacin, it was showed that baicalein could significantly reverse the ciprofloxacin resistance of possibly by inhibiting the NorA efflux pump in vitro [31]. Secondly, a MRSA pyruvate kinase (PK), which is an enzyme essential for S. aureus growth and survival has been identified [32], and it was suggested to be as a potential S. aureus drug target [33]. Baicalein was shown to inhibit the MRSA PK and this inhibition could lead to a deficiency of ATP which might further contribute to the antibacterial actions of baicalein against MRSA [31]. Anti-Helicobacter pylori were also observed in baicalein [34]. For anti-fungal activities, baicalein was shown to dose dependently inhibit the biofilm formation of C. albicans from 4 to 32 g/ml [35], and in vitro synergism of fluconazole and baicalein against clinical isolates of fluconazole resistant C. albicans were also observed [36]. Recently, baicalein alone or in combination with amphotericin was shown to induce programmed cell death in C. albicans [37, 38]. The plant alkaloid berberine is an active ingredient of Rhizoma Coptidis. It has been shown to possess different antimicrobial activities. Anti-herpes simplex virus effects were observed in berberine and the possible antiviral mechanism was demonstrated to be inhibition of viral DNA synthesis [39]. In modest concentrations of 30-45 g/ml, berberine were sufficient to exhibit an antibacterial effect and to inhibit biofilm formation significantly [40]. Moreover, the antimicrobial action of berberine was potentiated by a multidrug pump inhibitor, 5’-methoxyhydnocarpin, from 32 to 2 g/ml against NorA overexpressed S. aureus [41]. To further enhance the efficacy of berberine, Samosorn et al. [42] conjugated the berberine with the NorA inhibitor 5-nitro-2-phenyl-1H-indole via a methylene ether linking group and they found that this hybrid showed excellent antibacterial activity against S. aureus 1199B (MIC: 1.7 μM), which was over 382-fold more active than the parent antibacterial berberine. Synergistic effects of berberine and -lactam antibiotics against MRSA were observed [43]. Tanshinone is an active ingredient of Radix Salviae Miltiorrhizae which contributes to the anti-microbial activities. Cryptotanshinone and dihydrotanshinone I were found to act against a broad range of Gram-positive bacteria included Bacillus subtilis, Bacillus thuringiensis, Micrococcus luteus, S. aureus and S. epidermidis with MIC ranged from 3.1 to 25 g/ml, and the mechanism of action may be related to the non-selective inhibiting properties of DNA, RNA and protein syntheses [44]. Recently, transcriptional profiling revealed that the action mechanism of cryptotanshinone on S.

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aureus is correlated to its action as active oxygen radical generator and S. aureus might undergo an oxygen-limiting state upon exposure to cryptotanshinone [45]. For Cortex Lycii, an isolated compound (+)-Lyoniresinol-3 alpha-O-beta-D-glucopyranoside exhibited potent antimicrobial activity against MRSA isolated from patients and human pathogenic fungi without having any hemolytic effect on human erythrocytes [46]. Antifungal effects against Candida albicans were also observed in 4 phenolic amides, dihydro-N-caffeoyltyramine, trans-N-feruloyloctopamine, trans-N–caffeoyltyramine and cis-N- caffeoyltyramine isolated from an ethyl acetate extract of Cortex Lycii [47]. Literature concerning the antibacterial effects of Cortex Moutan is very limited and one of active ingredients paeonol may contribute to the anti-microbial activities of this herb [48].

5. Conclusion

In conclusion, we have identified some herbal extracts with promising direct antibacterial activities or synergistic with antibiotic activity. Evidence from published data showed that compounds isolated from these Chinese herbal medicines are effective against a wide range of pathogenic microorganisms as well as drug resistant MRSA.

Acknowledgements We acknowledge the technical support of Mr C.P. Lau and the support from the BIO-Asia program from the French Ministry of Foreign and European Affairs, and CUHK Scheme D grant for 2010/2. We would also like to thank the Ming Lai Foundation and the International Association of Lions Clubs District 303- Hong Kong and Macau Tam Wah Ching Chinese Medicine Resource Centre.

References

[1] Chambers HF. Ceftobiprole: in-vivo profile of a bactericidal cephalosporin. Clinical Microbiology and Infection 2006;12 Suppl 2:17-22.

[2] Hilliard JJ, Fernandez J, Melton J, Macielag MJ, Goldschmidt R, Bush K, et al. In vivo activity of the Pyrrolopyrazolyl-Substituted Oxazolidinone RWJ-416457. Antimicrobial agents and chemotherapy 2009.

[3] Clark NM, Patterson J, Lynch JP, 3rd. Antimicrobial resistance among gram-negative organisms in the intensive care unit. Current Opinion in Critical Care 2003;9:413-23.

[4] Bell JM, Turnidge JD. High prevalence of oxacillin-resistant Staphylococcus aureus isolates from hospitalized patients in Asia-Pacific and South Africa: results from SENTRY antimicrobial surveillance program, 1998-1999. Antimicrobial agents and chemotherapy 2002;46:879-81.

[5] Rubinstein E, Kollef MH, Nathwani D. Pneumonia caused by methicillin-resistant Staphylococcus aureus. Clinical Infectious Diseases 2008;46 Suppl 5:S378-85.

[6] Sakoulas G, Moellering RC, Jr. Increasing antibiotic resistance among methicillin-resistant Staphylococcus aureus strains. Clinical Infectious Diseases 2008;46 Suppl 5:S360-7.

[7] Quave CL, Plano LR, Pantuso T, Bennett BC. Effects of extracts from Italian medicinal plants on planktonic growth, biofilm formation and adherence of methicillin-resistant Staphylococcus aureus. Journal of ethnopharmacology 2008;118:418-28.

[8] Pesewu GA, Cutler RR, Humber DP. Antibacterial activity of plants used in traditional medicines of Ghana with particular reference to MRSA. Journal of ethnopharmacology 2008;116:102-11.

[9] de Lima MR, de Souza Luna J, dos Santos AF, de Andrade MC, Sant'Ana AE, Genet JP, et al. Anti-bacterial activity of some Brazilian medicinal plants. Journal of ethnopharmacology 2006;105:137-47.

[10] Xu QQ, Li YR. Discussion on sensitivity of 18 Chinese Herbage Medicines against MRSA. China Pharmaceuticals 2006;15:49. [11] Zuo GY, Wang GC, Zhao YB, Xu GL, Hao XY, Han J, et al. Screening of Chinese medicinal plants for inhibition against

clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Journal of ethnopharmacology 2008;120:287-90. [12] Kim KJ, Yu HH, Cha JD, Seo SJ, Choi NY, You YO. Antibacterial activity of Curcuma longa L. against methicillin-resistant

Staphylococcus aureus. Phytotherapy Research 2005;19:599-604. [13] Lechner D, Stavri M, Oluwatuyi M, Pereda-Miranda R, Gibbons S. The anti-staphylococcal activity of Angelica dahurica (Bai

Zhi). Phytochemistry 2004;65:331-5. [14] Chan BC, Hon KL, Leung PC, Sam SW, Fung KP, Lee MY, et al. Traditional Chinese medicine for atopic eczema: PentaHerbs

formula suppresses inflammatory mediators release from mast cells. Journal of ethnopharmacology 2008;120:85-91. [15] Kerns RJ, Rybak MJ, Kaatz GW, Vaka F, Cha R, Grucz RG, et al. Structural features of piperazinyl-linked ciprofloxacin

dimers required for activity against drug-resistant strains of Staphylococcus aureus. Bioorganic & medicinal chemistry letters 2003;13:2109-12.

[16] Ross JI, Eady EA, Cove JH, Cunliffe WJ, Baumberg S, Wootton JC. Inducible erythromycin resistance in staphylococci is encoded by a member of the ATP-binding transport super-gene family. Molecular Microbiology 1990;4:1207-14.

[17] el Solh N, Moreau N, Ehrlich SD. Molecular cloning and analysis of Staphylococcus aureus chromosomal aminoglycoside resistance genes. Plasmid 1986;15:104-18.

[18] Welch KT, Virga KG, Whittemore NA, Ozen C, Wright E, Brown CL, et al. Discovery of non-carbohydrate inhibitors of aminoglycoside-modifying enzymes. Bioorganic & medicinal chemistry 2005;13:6252-63.

[19] Lelievre H, Lina G, Jones ME, Olive C, Forey F, Roussel-Delvallez M, et al. Emergence and spread in French hospitals of methicillin-resistant Staphylococcus aureus with increasing susceptibility to gentamicin and other antibiotics. Journal of clinical microbiology 1999;37:3452-7.

780 ©FORMATEX 2011


[20] Palaniappan K, Holley RA. Use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria. International journal of food microbiology 2010;140:164-8.

[21] Oh I, Yang WY, Chung SC, Kim TY, Oh KB, Shin J. In vitro sortase a inhibitory and antimicrobial activity of flavonoids isolated from the roots of Sophora flavescens. Archives of pharmacal research 2011;34:217-22.

[22] Kuroyanagi M, Arakawa T, Hirayama Y, Hayashi T. Antibacterial and antiandrogen flavonoids from Sophora flavescens. Journal of natural products 1999;62:1595-9.

[23] Sohn HY, Son KH, Kwon CS, Kwon GS, Kang SS. Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Morus alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora koreensis Nakai. Phytomedicine 2004;11:666-72.

[24] Cha JD, Jeong MR, Jeong SI, Lee KY. Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens. Journal of microbiology and biotechnology 2007;17:858-64.

[25] Cha JD, Moon SE, Kim JY, Jung EK, Lee YS. Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens against methicillin-resistant Staphylococcus aureus. Phytotherapy Research 2009;23:1326-31.

[26] Lee GS, Kim ES, Cho SI, Kim JH, Cho G, Ju YS, et al. Antibacterial and Synergistic Activity of Prenylated Chalcone Isolated from the Roots of Sophora flavescens. Journal of Korean Society Applied Biology and Chemistry 2010;53:290-6.

[27] Mazmanian SK, Liu G, Jensen ER, Lenoy E, Schneewind O. Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proceedings of the National Academy of Sciences of the United States of America 2000;97:5510-5.

[28] Sato Y, Shibata H, Arai T, Yamamoto A, Okimura Y, Arakaki N, et al. Variation in synergistic activity by flavone and its related compounds on the increased susceptibility of various strains of methicillin-resistant Staphylococcus aureus to beta-lactam antibiotics. International journal of antimicrobial agents 2004;24:226-33.

[29] Liu IX, Durham DG, Richards RM. Baicalin synergy with beta-lactam antibiotics against methicillin-resistant Staphylococcus aureus and other beta-lactam-resistant strains of S. aureus. The Journal of pharmacy and pharmacology 2000;52:361-6.

[30] Fujita M, Shiota S, Kuroda T, Hatano T, Yoshida T, Mizushima T, et al. Remarkable synergies between baicalein and tetracycline, and baicalein and beta-lactams against methicillin-resistant Staphylococcus aureus. Microbiology and immunology 2005;49:391-6.

[31] Chan B, Ip M, Lau C, Lui S, Jolivalt C, Ganem-Elbaz C, et al. Synergistic effects of baicalein with ciprofloxacin against Nor A over-expressed methicillin-resistant Staphylococcus aureus (MRSA) and inhibition of MRSA pyruvate kinase. Journal of ethnopharmacology 2011: 137:767-73

[32] Cherkasov A, Hsing M, Zoraghi R, Foster LJ, See RH, Stoynov N, et al. Mapping the Protein Interaction Network in Methicillin-Resistant Staphylococcus aureus. Journal of proteome research 2011;10:1139-50.

[33] Zoraghi R, See RH, Axerio-Cilies P, Kumar NS, Gong H, Moreau A, et al. Identification of Methicillin Resistant Staphylococcus aureus Pyruvate Kinase as a Novel Antimicrobial Drug Target. Antimicrobial agents and chemotherapy 2011: 55:2042-53.

[34] Wu J, Hu D, Wang KX. Study of Scutellaria baicalensis and Baicalin against antimicrobial susceptibility of Helicobacter pylori strains in vitro. Journal of Chinese medicinal materials 2008;31:707-10.

[35] Cao Y, Dai B, Wang Y, Huang S, Xu Y, Cao Y, et al. In vitro activity of baicalein against Candida albicans biofilms. International journal of antimicrobial agents 2008;32:73-7.

[36] Huang S, Cao YY, Dai BD, Sun XR, Zhu ZY, Cao YB, et al. In vitro synergism of fluconazole and baicalein against clinical isolates of Candida albicans resistant to fluconazole. Biological & Pharmaceutical Bulletin 2008;31:2234-6.

[37] Dai BD, Cao YY, Huang S, Xu YG, Gao PH, Wang Y, et al. Baicalein induces programmed cell death in Candida albicans. Journal of microbiology and biotechnology 2009;19:803-9.

[38] Fu Z, Lu H, Zhu Z, Yan L, Jiang Y, Cao Y. Combination of Baicalein and Amphotericin B Accelerates Candida albicans Apoptosis. Biological & Pharmaceutical Bulletin 2010;34:214-8.

[39] Chin LW, Cheng YW, Lin SS, Lai YY, Lin LY, Chou MY, et al. Anti-herpes simplex virus effects of berberine from Coptidis rhizoma, a major component of a Chinese herbal medicine, Ching-Wei-San. Archives of virology 2010;155:1933-41.

[40] Wang X, Yao X, Zhu Z, Tang T, Dai K, Sadovskaya I, et al. Effect of berberine on Staphylococcus epidermidis biofilm formation. International journal of antimicrobial agents 2009;34:60-6.

[41] Stermitz FR, Lorenz P, Tawara JN, Zenewicz LA, Lewis K. Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor. Proceedings of the National Academy of Sciences of the United States of America 2000;97:1433-7.

[42] Samosorn S, Tanwirat B, Muhamad N, Casadei G, Tomkiewicz D, Lewis K, et al. Antibacterial activity of berberine-NorA pump inhibitor hybrids with a methylene ether linking group. Bioorganic & medicinal chemistry 2009;17:3866-72.

[43] Yu HH, Kim KJ, Cha JD, Kim HK, Lee YE, Choi NY, et al. Antimicrobial activity of berberine alone and in combination with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus. Journal of medicinal food 2005;8:454-61.

[44] Lee DS, Lee SH, Noh JG, Hong SD. Antibacterial activities of cryptotanshinone and dihydrotanshinone I from a medicinal herb, Salvia miltiorrhiza Bunge. Bioscience, biotechnology, and biochemistry 1999;63:2236-9.

[45] Feng H, Xiang H, Zhang J, Liu G, Guo N, Wang X, et al. Genome-wide transcriptional profiling of the response of Staphylococcus aureus to cryptotanshinone. Journal of biomedicine & biotechnology 2009;2009:617509.

[46] Lee DG, Jung HJ, Woo ER. Antimicrobial property of (+)-lyoniresinol-3alpha-O-beta-D-glucopyranoside isolated from the root bark of Lycium chinense Miller against human pathogenic microorganisms. Archives of pharmacal research 2005;28:1031-6.

[47] Lee DG, Park Y, Kim MR, Jung HJ, Seu YB, Hahm KS, et al. Anti-fungal effects of phenolic amides isolated from the root bark of Lycium chinense. Biotechnology letters 2004;26:1125-30.

[48] Papandreou V, Magiatis P, Chinou I, Kalpoutzakis E, Skaltsounis AL, Tsarbopoulos A. Volatiles with antimicrobial activity from the roots of Greek Paeonia taxa. Journal of ethnopharmacology 2002;81:101-4.

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