Screening of anti-Helicobacter pylori herbs deriving from Taiwanese folk medicinal plants

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 concentration 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 adnatum 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.

Anti-Helicobacter pylori activity, Taiwanese folk medicinal plants, Ethanol extract

1 Introduction

Infection with Helicobacter pylori is strongly associated with duodenal and gastric ulcers [1]. Substantial epidemiological data has revealed that high rates of H. pylori infection might be related to high rates of gastric cancer and gastric adenocarcinoma [2]. Various pharmacological 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 eradication regimens, i.e. the cost [10], the efficacy of antibiotics 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 the eradication of H. pylori infection using traditional herbal 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 caryophyllata 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 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 Japanese wasabi exhibited bactericidal activities against H. pylori, having the leaves the highest bactericidal activity [21]. In addition, some flavonoids and isoflavonoids isolated from licorice such as licochalcone A, licoisoflavone 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 extraction. Inhibitory zone and minimum inhibitory concentration tests (MIC) were utilized for screening.

2 Materials and methods

2.1 Herbal plants and herbal extracts preparation

Fifty Taiwanese medicinal plants were purchased from local herbal markets. These herbs and its parts were used for drug showed in 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

Ninety-five percent ethanol was used for the extraction of all herbs. Two hundred millilitres of 95% ethanol was added to 30 g of herb powder followed by the stirring of the mix at room temperature for 1 h. The mixture was then centrifuged at 9000 rpm for 15 min at 4 °C. The residue was extracted twice with 2 × 200 ml of 95% ethanol. The supernatants were mixed and concentrated to dryness in a rotary vacuum evaporator below 40 °C, and the concentrate was then weighed. Extraction yield (%, w/w) was calculated as the ratio of the weight of the concentrate 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. pylori QU 108, QU 141, QU 144, QU 150 and QU 181 were isolated from the stomach of patients from Kaohsiung Chang-Gung Memorial Hospital (Kaohsiung, Taiwan, ROC). These isolates were identified using standard diagnostic procedures [23].

All H. pylori strains were cultured in 5-ml tryptic soy broth (TSB, Difco, USA; each liter contained: a pancreatic 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 (bioMérieux, France; each liter contained: 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 to provide 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 testing 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 the plates with 30 µl of the herbal extract [0.2 g ml⁻¹; dimethyl 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 composition 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

A broth-dilution method [25] was used for MIC 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 a microaerophilic jar system, featuring a gas composition of 5% O₂ and 10% CO₂-in-air, at 37 °C for 72 h, the colonies 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 culture medium 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 greater 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).

3.2 Inhibitory-zone testing

The inhibitory-zone testing results for the ethanol extracts of 50 herbs are given in Table 2. Fifty herbs were divided into three classes based on the relative effectiveness of anti-H. pylori actions. Strong anti-H. pylori-activity herbs, included PZL, PSM, AIOK, BMDC and ASKS, for which 9–10 H. pylori strains were inhibited from the ten strains tested by the ethanol extracts of herbs, with either large diameter of inhibitory zones or lower values of hazy zones.

Table 2

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

Herb extract	Strain
	BCRC 17021	BCRC 17023	BCRC 17026	BCRC 17027	BCRC 15415	Qu 108	Qu 141	Qu 144	Qu 150	Qu 181
AALEV	++	+	++(H)	++(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	++	+(H)	+	−	−	+++(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.

Concentration of all extracts was 0.2 g ml⁻¹ and 30 µl of extract was incorporated into each well.

++++, >20 mm (dia); +++, 16–20 mm (dia); ++, 11–15 mm (dia); +, 8–10 mm (dia);: ≤7 mm (dia).

Hazy zone.

Twenty-six herbs including AALEV, BBL, BFS, CAL, CAR, CAU, EARB, HCT, LCP, LOR, MBOKEJKM, MCDD, PCL, PGL, PNG, PSFES, RSMVRDC, SAL, SCC, SCL, SPS, SSBH, VCL, WICAM, XSL, and ZNDC were classified as moderate anti-H. pylori-activity herbs, being 6–9 H. pylori strains inhibited by their herbal extracts.

The additional 19 herbs were classified as lower-activity 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.

Results shown in Table 2 indicate that approximately half of the Taiwanese folk medicinal plants tested were demonstrated to possess higher anti-H. pylori activity. These herbs exhibit potentially a high therapeutic interest 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 PZM extracts. 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 haemorrhages [26a]. Moreover, the roots of PZL have been used for the treatment of rheumatic pain, dysmenorrhea, carbuncles, ulcers and killing intestinal parasites [26b].

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.

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 decoction and methanol eluted fractions of Pteleopsis suberosa 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. solstitialis, 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 µg ml⁻¹[20]. The Greek herbal medicine extracts of Anthemis melanolepis, Cerastium candidissimum, Chamomilla recutita, Conyza albida, Dittrichia 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 active ingredient in chilli, inhibited H. pylori growth in a dose-dependent concentration, exhibiting a concentration greater than 10 µg ml⁻¹ and a MIC₉₀ of 0.5 mg 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 obtained 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.