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FEMS Immunology and Medical Microbiology 43 (2005) 295–300

Screening of anti-Helicobacter pylori herbs derivingfrom Taiwanese folk medicinal plants

Yuan-Chuen Wang *, Tung-Liang Huang

Department of Food Science, National Chung-Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, 402, ROC

Received 5 May 2004; received in revised form 25 August 2004; accepted 20 September 2004

First published online 30 October 2004

Abstract

In this study, extracts from 50 Taiwanese folk medicinal plants were examined and screened for anti-Helicobacter pylori activity.

Ninety-five percent ethanol was used for herbal extraction. Paederia scandens (Lour.) Merr. (PSM), Plumbago zeylanica L. (PZL),

Anisomeles indica (L.) O. Kuntze (AIOK), Bombax malabaricum DC. (BMDC) and Alpinia speciosa (J. C. Wendl.) K. Schum.

(ASKS) and Bombax malabaricum DC. (BMDC) all demonstrated strong anti-H. pylori activities. The minimum inhibitory concen-

tration values of the anti-H. pylori activity given by the five ethanol herb extracts ranged from 0.64 to 10.24 mg ml�1. Twenty-six

herbs, including Artemisia argvi Levl. et Vant (AALEV), Phyla nodiflora (Linn.) Greene (PNG) and others, showed moderate anti-

H. pylori activity. The additional 19 herbs, including Areca catechu Linn. (ACL), Euphorbia hirta Linn. (EHL) and Gnaphalium adn-

atum Wall. ex DC. (GAWEDC), possessed lower anti-H. pylori effects. About half of the Taiwanese folk medicinal plants tested,

demonstrated to possess higher anti-H. pylori activity.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Anti-Helicobacter pylori activity; Taiwanese folk medicinal plants; Ethanol extract

1. Introduction

Infection with Helicobacter pylori is strongly associ-

ated with duodenal and gastric ulcers [1]. Substantial epi-

demiological data has revealed that high rates of

H. pylori infection might be related to high rates of gas-

tric cancer and gastric adenocarcinoma [2]. Various phar-macological regimens have been studied in the treatment

of H. pylori infection. Antibiotics [3,4], proton–pump

inhibitors [5,6], H2-blockers [7,8], and bismuth salts [9]

are suggested standard treatment modalities, which are

typically combined in dual, triple and quadruple therapy

regimens in order to eradicate H. pylori infection [10,3].

Some problems may arise upon administration of these

0928-8244/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsim.2004.09.008

* Corresponding author. Tel.: +886 4 22840385 7x17; fax: +886 4

22854053.

E-mail address: ycwang@dragon.nchu.edu.tw (Y.-C. Wang).

eradication regimens, i.e. the cost [10], the efficacy of anti-

biotics regarding the pH (for instance, amoxicillin is most

active at a neutral pH and tetracycline has greater activity

at a low pH) [10] and resistance to the antibiotics [11].

However, above 15% of the patients undergoing such

drug regimens experienced therapeutic failure [10].

Hence, numerous studies have concentrated on theeradication of H. pylori infection using traditional her-

bal medicines. Garlic and Pteleopsis extracts exhibited

weak and modest, respectively, anti-H. pylori activity

[12,13]. Fifty-four Chinese herbs were screened for

anti-H. pylori activity, exhibiting Rheum palmatum,

Rhus javanica, Coptis japonica and Eugenia caryophyl-

lata strong anti-H. pylori activity [14]. Cranberry juice

possesses modest anti-H. pylori activity [15]. The anti-H. pylori activities of Aristolochia paucinervis, black

myrobalan and cinnamon were also examined [16–18].

Anti-H. pylori compounds from the Brazilian medicinal

. Published by Elsevier B.V. All rights reserved.

296 Y.-C. Wang, T.-L. Huang / FEMS Immunology and Medical Microbiology 43 (2005) 295–300

plant Myroxylon peruiferum have successfully isolated

[19]. Extracts and fractions from seven Turkish plants

were also demonstrated to elicit anti-H. pylori activity

[20]. The leaves, roots and stems of Korean and Japa-

nese wasabi exhibited bactericidal activities against

H. pylori, having the leaves the highest bactericidalactivity [21]. In addition, some flavonoids and isoflavo-

noids isolated from licorice such as licochalcone A, licoi-

soflavone B, and gancaonols have been reported to

exhibit inhibitory activities against H. pylori [22].

In this study, 50 Taiwanese folk medicinal plants

were examined and screened for anti-H. pylori activity.

Ninety-five percent ethanol was used for herbal extrac-

tion. Inhibitory zone and minimum inhibitory concen-tration tests (MIC) were utilized for screening.

2. Materials and methods

2.1. Herbal plants and herbal extracts preparation

Fifty Taiwanese medicinal plants were purchasedfrom local herbal markets. These herbs and its parts

were used for drug showed in Table 1.

Ninety-five percent ethanol was used for the extrac-

tion of all herbs. Two hundred millilitres of 95% ethanol

was added to 30 g of herb powder followed by the stir-

ring of the mix at room temperature for 1 h. The mixture

was then centrifuged at 9000 rpm for 15 min at 4 �C. Theresidue was extracted twice with 2 · 200 ml of 95% eth-anol. The supernatants were mixed and concentrated to

dryness in a rotary vacuum evaporator below 40 �C, andthe concentrate was then weighed. Extraction yield (%,

w/w) was calculated as the ratio of the weight of the con-

centrate to the weight of the herb powder.

2.2. Bacterial strains and cultivation

H. pylori BCRC 17021, BCRC 17023, BCRC 17026,

BCRC 17027 and BCRC 15415 were obtained from the

Bioresources Collection and Research Center (BCRC),

Hsinchu, Taiwan, ROC. Five clinical isolates of H. py-

lori QU 108, QU 141, QU 144, QU 150 and QU 181

were isolated from the stomach of patients from Kao-

hsiung Chang-Gung Memorial Hospital (Kaohsiung,

Taiwan, ROC). These isolates were identified usingstandard diagnostic procedures [23].

All H. pylori strains were cultured in 5-ml tryptic soy

broth (TSB, Difco, USA; each liter contained: a pan-

creatic digest of casein [17 g], an enzymatic digest of

soybean meal [3 g], dextrose [2.5 g], sodium chloride

[5 g], dipotassium phosphate [2.5 g], pH 7.3,) with a

Columbia agar (bioMerieux, France; each liter con-

tained: bio-polyone [10 g], bio-lysat [10 g], bio-myotone[3 g], corn starch [1 g], sodium chloride [5 g], agar [13.5

g], pH 7.3) slant containing 5% (v/v) of defibrinated

sheep blood formed at the bottom of the test tube.

The broth incubated in a microaerophilic jar system

(BBL), featuring a gas composition of 5% O2 and

10% CO2-in-air (an OXOID BR 056A gas-generating

kit was used for this purpose), at 37 �C for 72 h. The

cell suspension was then diluted with 0.1% peptone toprovide a cell concentration of 0.5–1.0 · 106 cfu ml�1

for antimicrobial testing.

2.3. Inhibitory-zone testing

Different strains of anti-H. pylori inhibitory zone test-

ing for plant extracts were performed according to the

method of Johnson and Christine [24]. A volume of

0.1 ml for each of the bacterial suspensions tested

(0.5–1.0 · 106 cfu ml�1) was spread onto a Columbia

agar plate containing 5% (v/v) defibrinated sheep blood.

Wells sized 7-mm in diameter were punched on theplates with 30 ll of the herbal extract [0.2 g ml�1; di-

methyl sulfoxide (DMSO) as solvent] to be individually

incorporated into the wells. DMSO was used as control.

The plates were diffused at 4 �C for 2 h, and incubated in

a microaerophilic jar system (BBL), featuring a gas com-

position of 5% O2 and 10% CO2-in-air, at 37 �C for 72 h.

The clear zone around each well was observed and its

diameter was examined.

2.4. Minimum inhibitory concentration testing

Abroth-dilutionmethod [25]wasused forMIC testing.

A volume of 0.1-ml of cell suspensions (initial bacterial

count 0.5–1.0 · 106 cfu ml�1) was spread onto Columbia

agar plates containing 5% (v/v) defibrinated sheep blood.

DMSO was used as control. Following incubation in amicroaerophilic jar system, featuring a gas composition

of 5% O2 and 10% CO2-in-air, at 37 �C for 72 h, the colo-

nies that had formed on the plates were enumerated. The

MIC was defined as the minimum concentration of the

test sample (antibacterial agent) in a given culturemedium

above which bacteria are not able to form colonies.

3. Results and discussion

3.1. Extraction yield

Ethanol-extraction yields of product deriving from

the 50 herbs tested, exhibited large differences from

each other, and the yields ranged from 1.91% to

23.58% (w/w) (Table 1). Product-extraction yields great-er than 10% (w/w) were found for a total of 11 herbs,

which included PGL, PSM, CAU, CJG, etc. (see Table

1). While a yield of less than 5% (w/w) was observed for

20 herbs, being among them CAR, XSL, SRL, HML,

etc. (see Table 1).

Table 1

The reference table of scientific name, abbreviation, parts were used for drug and extraction yield of 50 folk medicinal plants in Taiwan

Scientific name Abbreviation Part using for drug Extraction yield (%, w/w)

Artemisia argvi Levl. et Vant AALEV Leaf and stem 6.19

Areca catechu Linn. ACL Seed 4.03

Anisomeles indica (L.) O. Kuntze AIOK Leaf and stem 4.52

Agrimonia pilosa Ledeb. APL Whole plant 3.23

Alpinia speciosa (Wendl.) K. Schum. ASKS Root 7.03

Amaranthus spinosus L. ASL Root 7.85

Amaranthus virdis L. AVL Stem 13.36

Bidens bipinnata L. BBL Whole plant 4.90

Bletilla formosana (Hayata) Schltr BFS Stem 4.15

Bischofia javanica Blume BJB Stem 13.95

Bombax malabaricum DC. BMDC Root 10.04

Bidens pilosa L. var. minor (Blume) Sherff BPLVMS Whole plant 6.78

Chenopodium ambrosioides L. CAL Leaf and stem 11.20

Canarium album (Lour.) Raeuschel CAR Root 1.91

Centella asiatica (L.) Urban. CAU Whole plant 17.64

Cayratia japonica (Thunb.) Gagnep. CJG Whole plant 16.20

Catharanthus roseus (L.) G. Don. CRGD Whole plant 7.06

Cycas revoluta Thunb. CRT Leaf 14.50

Ehretia acuminata R. Br. EARB Root 6.50

Euphorbia hirta Linn. EHL Whole plant 4.53

Flemingia philippinensis Merr. & Rolfe FPMR Stem 7.86

Gnaphalium adnatum Wall. ex DC. GAWEDC Whole plant 3.72

Houttuynia cordata Thunb. HCT Whole plant 6.56

Hibiscus muthtabilis Linn. HML Whole plant 2.72

Litsea cubeba (Lour.) Persoon LCP Stem 3.65

Ludwigia octovalvis (Jacq.) Raven. LOR Stem 5.03

Murdannia bracteata (C. B. Clarke) O. Kuntze ex J. K. Morton MBOKEJKM Whole plant 4.56

Melastoma candidum D. Don MCDD Stem and root 7.50

Milletia reticulata Bentham MRB Whole plant 3.53

Polygonum chinense Linn. PCL Root 6.21

Psidium guajava L. PGL Leaf 23.58

Phyla nodiflora (Linn.) Greene PNG Whole plant 13.30

Polygonum senticosum (Meissn) Franch. et Sav. PSFES Whole plant 4.73

Paederia scandens (Lour.) Merr. PSM Whole plant 15.32

Phyllanthus urinaria Linn. PUL Whole plant 5.50

Plumbago zeylanica L. PZL Stem 6.28

Rhus semialata Merr. var. roxburghiana DC. RSMVRDC Stem 3.38

Sonchus arvensis Linn. SAL Whole plant 4.44

Sphenomeris chusana (L.) Copel SCC Whole plant 5.55

Sambucus chinensis Lindl. SCL Whole plant 3.25

Sophora flavescens Ait. SFA Root 11.15

Solanum nigrum Linn. SNL Whole plant 3.21

Setaria palmfolia Stapf. SPS Whole plant 5.71

Sida rhombifolia Linn. SRL Whole plant 2.05

Senecio scandens Buch-Ham. SSBH Whole plant 5.36

Vernonia cinerea (L.) Less. VCL Whole plant 7.75

Viola mandshurica VM Whole plant 8.06

Wikstroemia indica (L.) C. A. Mey. WICAM Stem 4.45

Xanthium strumarium Linn. XSL Whole plant 2.05

Zanthoxylum nitidum (Roxb.) DC. ZNDC Root 5.57

Y.-C. Wang, T.-L. Huang / FEMS Immunology and Medical Microbiology 43 (2005) 295–300 297

3.2. Inhibitory-zone testing

The inhibitory-zone testing results for the ethanol ex-

tracts of 50 herbs are given in Table 2. Fifty herbs were

divided into three classes based on the relative effective-

ness of anti-H. pylori actions. Strong anti-H. pylori-ac-

tivity herbs, included PZL, PSM, AIOK, BMDC and

ASKS, for which 9–10 H. pylori strains were inhibitedfrom the ten strains tested by the ethanol extracts of

herbs, with either large diameter of inhibitory zones or

lower values of hazy zones.

Twenty-six herbs including AALEV, BBL, BFS,

CAL, CAR, CAU, EARB, HCT, LCP, LOR, MBO-

KEJKM, MCDD, PCL, PGL, PNG, PSFES,

RSMVRDC, SAL, SCC, SCL, SPS, SSBH, VCL, WI-

CAM, XSL, and ZNDC were classified as moderate

anti-H. pylori-activity herbs, being 6–9 H. pylori strainsinhibited by their herbal extracts.

Table 2

Anti-H. pylori activity spectra of ethanol extracts of fifty folk medicinal plants

Herb extracta Strain

BCRC 17021 BCRC 17023 BCRC 17026 BCRC 17027 BCRC 15415 Qu 108 Qu 141 Qu 144 Qu 150 Qu 181

AALEV ++b + ++(H)c ++(H) ++ + � ++(H) ++ ++

ACL � + � + + � � � + �AIOK ++ +(H) ++ ++ ++(H) ++ +++ +++ +++(H) ++(H)

APL + + ++ + � � + � � �ASKS ++ ++ +++ ++ + � ++(H) + + ++

ASL � + � � � � +++(H) + � �AVL + + + � + � + � � �BBL + +(H) + ++ + +(H) � + + +

BFS + + + ++ +(H) +(H) +(H) + � �BJB + � � � � + � ++ ++ +

BMDC ++ ++ ++ ++ ++ ++(H) +++(H) ++ ++ +++

BPLVMS � � � � � +(H) � + ++(H) +(H)

CAL � ++(H) + ++ + + + ++(H) + �CAR ++ ++ ++ + ++(H) � � ++++(H) + ++

CAU + +(H) + + + ++ � + + ++

CJG � � � � � +++(H) � +++(H) +++ ++(H)

CRGD ++ � � � +(H) � � ++ + +

CRT � +(H) � � � � � � + �EARB � + + ++ + ++ � + + +

EHL � + + + + + � � � �FPMR � � � � � ++(H) � +(H) + +(H)

GAWEDC � � + + + + � � � �HCT + + +++ + + � ++(H) + � +(H)

HML � � � � � � � + + +

LCP � ++ � + � ++ + + ++ ++

LOR � + � ++ + + ++ � ++ �MBOKEJKM + + ++(H) + +(H) � � + + +

MCDD � ++ � ++ ++ ++ +(H) + + ++(H)

MRB � + � + + � � � � �PCL � + + + + + � + + +

PGL � + +++ ++ + ++ � + + ++

PNG +++ ++ � + ++ � � +(H) + +(H)

PSFES ++b +(H)c + � � +++(H) � + + ++

PSM ++ ++ +++ +++ ++ ++ � +++ ++ +++

PUL � � � + + � � � � +(H)

PZL +++ ++++ +++(H) ++++ ++++ ++ ++(H) ++++ +++ ++ +

RSMVRDC � ++(H) + ++ + +(H) ++ +(H) ++(H) ++(H)

SAL + + +++ + + � +++(H) + + +

SCC � + + + + + � � + �SCL +(H) +(H) � +(H) + � � + + +

SFA � + � � � +(H) � +(H) + +(H)

SNL � + � � � +(H) + � + +(H)

SPS + +(H) ++(H) + + � � +(H) + ++(H)

SRL � + � � � + +(H) � + +(H)

SSBH +++ ++ � +(H) +++ ++(H) � +++(H) ++ ++

VCL � + ++ + ++ ++ +++ � ++ �VM � +(H) � + + +(H) � � � +(H)

WICAM � + + ++ +++ + � + + �XSL � + + � + + � ++ + +(H)

ZNDC � + + � + +++(H) � +++ ++ ++

All the plates were incubated in a microaerophilic jar system at 37 �C for 72 h, in which the gas composition was 5% O2 and 10% CO2-in-air.a Concentration of all extracts was 0.2 g ml�1 and 30 ll of extract was incorporated into each well.b ++++, > 20 mm (dia); +++, 16–20 mm (dia); ++, 11–15 mm (dia); +, 8–10 mm (dia); : 67 mm (dia).c Hazy zone.

298 Y.-C. Wang, T.-L. Huang / FEMS Immunology and Medical Microbiology 43 (2005) 295–300

The additional 19 herbs were classified as lower-activ-

ity anti-H. pylori herbs. They included ACL, APL, ASL,

AVL, BJB, BPLVMS, CJG, CRGD, CRT, EHL,

FPMR, GAWEDC, HML, MRB, PUL, SFA, SNL,

SRL and VM, for which 2–5 H. pylori strains were

inhibited by the ethanol extracts of herbs. The inhibitory

zones of ethanol extracts given by these herbs either

exhibited small diameter or more haziness.

Table 3

Minimum inhibitory concentrations of ethanol extract of five folk medicinal plants against H. pylori.

Test strain MIC (mg ml�1)

AIOK ASKS BMDC PSM PZL

Helicobacter pylori BCRC 17021 5.12 >5.12 5.12 2.56 0.64

H. pylori BCRC 17023 >5.12 >5.12 >5.12 0.64 2.56

H. pylori BCRC 17026 5.12 5.12 >5.12 1.28 10.24

H. pylori BCRC 17027 2.56 >5.12 1.28 2.56 2.56

H. pylori BCRC 15415 >5.12 >5.12 >5.12 5.12 0.64

All the plates were incubated in a microaerophilic jar system at 37 �C for 72 h, in which the gas composition of 5% O2 and 10% CO2-in-air.

Y.-C. Wang, T.-L. Huang / FEMS Immunology and Medical Microbiology 43 (2005) 295–300 299

Results shown in Table 2 indicate that approximately

half of the Taiwanese folk medicinal plants tested weredemonstrated to possess higher anti-H. pylori activity.

These herbs exhibit potentially a high therapeutic inter-

est either to be used in food health or as novel drugs for

the eradication of H. pylori infection in the future.

3.3. Minimum inhibitory concentration testing

The MIC values for the five stronger anti-H. pylori

herbal extracts were determined. As shown in Table 3,

the ethanol extract of PSM exhibited the lowest MIC,

where values ranged from 0.64 to 5.12 mg ml�1. This

was followed by the extract of PZL with a MIC ranged

from 0.64 to 10.24 mg ml�1. The ethanol extracts of

AIOK, BMDC and ASKS featured MIC values ranging

from 1.28 to 5.12 mg ml�1 and gradually showed lower

anti-H. pylori activity as compared with PSL and PZMextracts. Results in Table 3 show that for the five extracts

having the stronger anti-H. pylori activity, PSM and PZL

were demonstrated to possess the greatest anti-H. pylori

activity, and consequently being the most commonly

used as folk medicinal plants in Taiwan. The entire plant

of PSM has been used for the treatment of rheumatic

affections, diarrhoea in children and internal haemor-

rhages [26a]. Moreover, the roots of PZL have been usedfor the treatment of rheumatic pain, dysmenorrhea, car-

buncles, ulcers and killing intestinal parasites [26b].

The literature has revealed that a large number of the

anti-H. pylori components abound in plants and exhibit

different degrees of anti-H. pylori activities. MICs for

the aqueous garlic extract against nineteen strains of

H. pylori ranged from 2 to 5 mg ml�1 [12]. Both decoc-

tion and methanol eluted fractions of Pteleopsis sube-

rosa have been shown to reveal anti-H. pylori activity

against one America Type Culture Collection (ATCC),

Rockville, MD, USA, strain and five clinical strains,

presenting MICs ranging from 0.0625 to 0.5 mg ml�1

[13]. Screening of Turkish anti-ulcerogenic folk remedies

for anti-H. pylori activity, revealed that flowers of Cistus

laurifolius and Spartium junceum, cones of Cedrus libani,

herbs and flowers of Centaurea solstitialis ssp. solsti-

tialis, fruits of Momordica charantia, herbaceous parts

of Sambucus ebulus, and flowering herbs of Hypericum

perforatum, showed anti-H. pylori activity, with MICs

in the range of 1.95–250 lg ml�1 [20]. The Greek herbal

medicine extracts of Anthemis melanolepis, Cerastium

candidissimum, Chamomilla recutita, Conyza albida, Dit-

trichia viscosa, Origanum vulgare and Stachys alopecuros

have been proved to be active against one standard

strain and 15 clinical isolates of H. pylori, with MICs

ranging from 0.625 to 5 mg ml�1 [27]. Capsicin, an ac-

tive ingredient in chilli, inhibited H. pylori growth in a

dose-dependent concentration, exhibiting a concentra-

tion greater than 10 lg ml�1 and a MIC90 of 0.5mg ml�1 [28]. The ethyl-acetate extract of Elephantopus

scaber Linn. presenting MIC values ranging from 2.56

to 10.24 mg ml�1 against 8 strains of H. pylori have been

reported in our previous study [29]. From the results ob-

tained in this study, PSM and PZL exhibited lower MIC

values when compared with other studies. Both herbs

were shown to have strong anti-H. pylori activity.

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