16

Herbtaxis of Helicobacter pylori and eﬀects of herbs on its behavior

原著

Helicobacter pylori のハーブ走性及びハーブ抽出液による行動への影響
Herbtaxis of Helicobacter pylori and eﬀects of herbs on its behavior.
溝手朝子1）＊、石川直洋1）、人見英里1）、高野済2）、山本展久3）、望月聡4）

Tomoko Mizote1）＊, Naohiro Ishikawa1）, Eri Hitomi1）, Naru Takano2）, Nobuhisa Yamamoto3）,
and Satoshi Mochizuki4）【Abstract】
Herbs have been used as conservative substances, appetite stimulating agents or relaxing materials in the history of food culture as aright herb in the right country. The eﬀect of herbs on chemotactic activity of Helicobacter pylori (H.pylori) has not been investigatedpreviously. We performed in vitro inﬂuence of herb extracts on growth, motility and chemotaxis of H. pylori. Species of herb speciﬁc
inhibition on growth of H. pylori was observed. Lavender, Rosemary Stevia, Bergamot and Sage showed strong swarm inhibition.
However, chemotaxis to herb extracts (herbtaxis) was observed on Yellow Flower Lavender, Stevia and Cherry Sage. Inhibition of
growth and swarming ability of H. pylori by herb extracts might work as protective materials against H. pylori-associatedgastroduodenal disease in vivo. Further investigation will be needed to ensure that this eﬀect would be observed in animal model.

List of abbreviations: H. pylori : Helicobacter pylori, DMSO: dimethyl sulfoxide,
Keywords: H. pylori, herb extracts, chemotaxis, motility inhibition, swarming

Escherichia coli, and Helicobacter species, Helicobacter

Introduction
Helicobacter pylori is a well known bacterium which

species exhibits much higher motile7). Therefore, motility

induces a chronic inflammatory response in the stomach

in viscous environment seems necessary for bothand causes gastritis, peptic ulcer disease, gastriccolonization and persistence of the bacteria in the gastricadenocarcinoma

1,2), and gastriclymphoma

3,4). Thismucosa.

microorganism has a spiral body with unipolar sheathed

On the other hands, herbs are used in many ways all

flagella, which is well adapted for its motility in theover the countries, in many cases, as traditionally

5)viscous environment .

observed custom. It might be a possibility that difference

Motility in the viscous environment is one of theof the incidence ratio of gastric diseases by H. pylori

essential factors for H. pylori to colonize in the mucus

infection between other advanced countries and Japanlayer of the gastric epithelium, as it has been reported

would be ascribed by the difference of the food custom.

that high motility by flagella was a significant factor for

Several studies on the growth inhibition of H. pylori by

6)colonizing ability of H. pylori in the piglets model . By

herbs and capsaicin were reported8,9).

the comparison of the motile ability in viscous

We investigate here focusing on that how herb extracts

environment between enteric bacterium, Salmonella oraffect on the motile ability in viscous environment and1）Department of Nutrition, Yamaguchi Prefectural University, Yamaguchi, 753-8502, Japan
2）Find News Co.Lad., Oita 870-0954, Japan
3）Material Technology Division, Oita Industrial Research Institute, Takae-nishi, Oita, 870-1117, Japan
4）Faculty of Education and Welfare Science, Oita University, Dannoharu, Oita, 870-1192, Japan

山口県立大学

Table 1 List of herb extracts used in this work.
No.
Herb
1
Sweet Basil
2
Darkopal Basil3
Lettuce Basil
4
Cinnamon Basil5
Licorice Basil
6
Purpule Russell Basil
7
Bush Basil8
Holy Basil
9
Ruby Basil10
Lemon Basil11
Sweet Basil Seed
12
Yellow Flower Lavender13
Summer Lavender14
Hidcote Lavender
15
French Lavender16
French Stoechas Lavender17
Spike Lavender18
English Lavender
19
Apple Mint
20
Water Mint
21
eau de cologne Mint
22
Orange Mint
23
Cool Mint
24
Spear Mint
25
Pineapple Mint
26
Bush Mint
27
Pepper Mint
28
Rosemary Tuscanablue
29
Rosemary Wood30
Rosemary Dreamyblue
31
Apple Eucalyptus
32
Lemon Eucalyptus
33
Pineapple Sage
34
Cherry Sage
35
Amethyst Sage
36
Clary Sage
37
Lemongrass38
Lemon Thyme
39
Lemon Scented Verbena

看護栄養学部紀要

No.
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77

創刊号

2008年３月

17

Herb
Lemon Balm
Dill
Fennel(Leaf)
Wild Strawberry(leaf)
Sweet Herb MEXICAN
HopSteviaNasturtium(Leaf)
OreganoSweet Marjoram
Common Mallow
Lamb’s Ears
Echinacea
ChivesChicory
ChivesCommon Soapwort)
Black Mallow
Roman Chamomile
Chervil
Wild Rocket
Rose Geranium
Common Comfrey
Russian Trragon
Rhubarb
Salad Burnet
Common Yarrow
GarlicEchinacea (Flowers)
Echinacea (Stem)
Echinacea (Leaf)
Echinacea (Root)Commom Mullein (Leaf)
Commom Mullein (Fruit)
Bergamot (Flower)
Bergamot (Leaf)
Hyssop (Leaf)
Hyssop (Flower)

attract H. pylori in vitro, in addition of dose-dependent

broth) or on agar plates with brucella-serum brothgrowth inhibition by herb extracts, which are very

solidifi ed with 1.5 % agar (brucella-serum agar) andcommon in western advanced countries.

incubated under microaerobic conditions (N2, 85%; O2,
5%; CO2, 10%) at 37 ℃ .

Materials and methods
Bacteria strains and growth conditions.

Herb extracts preparation.
Herbs used in this study were listed in Table 1. 5.0 g ofHelicobacter pylori ATCC43504 was used in thisrow leaves of each herb was homogenized in ethanol ands t u d y. B a c t e r i a w a s g r ow n o n b r u c e l l a b r o t hfiltered. The filtrates were adjusted to 50 ml. 10ml of

supplemented with 3 % horse serum (brucella-serum

ethanol extraction was evaporated at 40 ℃ under reduced

18

Herbtaxis of Helicobacter pylori and eﬀects of herbs on its behavior

Table 2 Dose dependent growth inhibition of H. pylori by herb extracts.
amount of row
AVR (cm)leaf (mg)French Lavender
control
1.38
Cherry Sage
40
2.80
4
1.72
0.8
1.43
0.4
1.44
French Stoechas Lavender
control
1.28
Amethyst Sage
40
3.42
4
2.17
0.8
1.61
0.4
1.55
eau de cologne Mint
control
ND
Bergamot (Flower)
40
2.82
4
1.81
0.8
1.26
0.4
0.98
Rosemary Tuscanablue
control
1.28
Bergamot (Leaf)
40
3.35
4
2.07
0.8
1.63
0.4
1.52
Rosemary Dreamyblue
control
1.20
Hyssop (Flower)
40
3.07
4
2.26
0.8
1.81
0.4
1.67
Lemon Thyme
control
1.03
Common Soapwort)
40
3.13
4
1.64
0.8
1.58
0.4
1.19
Steviacontrol
1.29
Roman Chamomile
40
3.12
4
2.40
0.8
1.91
0.4
1.53
Pineapple Sage
control
1.15
Common Yarrow
40
3.65
4
1.55
0.8
1.16
0.4
1.06

amount of row
leaf (mg)control
40
4
0.8
0.4
control
40
4
0.8
0.4
control
40
4
0.8
0.4
control
40
4
0.8
0.4
control
40
4
0.8
0.4
control
40
4
0.8
0.4
control
40
4
0.8
0.4
control
40
4
0.8
0.4

AVR (cm)
1.09
3.89
2.30
1.42
1.26
1.21
3.18
1.27
1.18
1.12
ND
4.02
2.64
1.56
1.16
1.46
4.78
2.12
1.44
1.39
ND
5.43
2.26
1.25
1.17
ND
3.58
1.45
1.14
1.06
ND
4.95
2.32
1.51
1.35
ND
3.32
1.83
1.31
1.13

pressure. The residues were dissolved in 500 µl of

Roshi, Tokyo Japan) exudated 20 µl of herb extracts were

DMSO and used as herb extracts for further experiments.

placed onto the brucella soft agar. Herb extracts wereBacterial growth inhibition studies.

diluted with DMSO for the dose dependent inhibition.

0.5 ml of 2 days culture of H. pylori cells were added

Plates were incubated under microaerobic condition forto the 2.0 ml of brucella-serum broth containing 0.5 %3days at 37 ℃. The diameter of clear zone was measured.

refined agar (brucella soft agar) and poured onto brucella-

Data are expressed as the means and standard errors forserum agar. The 8mm-diameter filter paper disk (Toyotriplicate experiments.

山口県立大学

看護栄養学部紀要

創刊号

2008年３月

19

Motility assay.
H. pylori (2 X 106 cells) were stabbed with toothpick
onto a motility agar containing 0.3 % refined agar in
brucella-serum broth. 10 µl of herb extract (equivalent
with 20 mg of row leaves) was premixed to the motility
agar. Plates were incubated for 5 days in microaerobic
condition at 37 ℃. Data were expressed as the means andstandard errors for triplicate experiments.
Chemotactic response to herb extracts.
Cells grown in brucella-serum broth were collected
and washed twice with 50 mM potassium phosphatebuffer, pH7.0 (chemotaxis buffer) and suspended inchemotaxis buffer. 10-3 diluted herb extracts were filledin 1 µl capillaries and performed to the chemotaxis assay
10)

to herb extract (herbtaxis) described previously . Afterincubation for 5, 10 and 30 min, bacteria incorporated
into the capillary tube were spread over a 0.125 cm2 area

Fig.1. Motility testing of H. pylori. Single colony of H. pylori
ATCC43504 was stabbed onto motility agar plate with DMSO(A), French Stoechas Lavender extract (B) and Bergamot
extract (C). The sizes of haloe were measured after 5 days
incubation.

on a slide glass, Gram stained and counted. Data wasexpressed as the means and standard errors for three
determinations.
Results
Growth inhibition of H. pylori by herb extracts.
First screenings of the growth inhibition of H. pylori
by 77 kinds of herb extracts were tested (data not shown).

Table 3 Effect of herb extracts to swarming ability of H.
pylori16 herb extracts were selected to examine a dosed e p e n d e n t m a n n e r o f g r ow t h i n h i b i t i o n a t acorresponding concentration of 0.4 mg raw leaf ml -1(Table 2). The inhibitory effect was observed in several
herbs associated with the biological classifi cation as
overall view. For example, Basil spp. and Mint spp. has
weak inhibition by the comparison with Lavender,
Rosemary and Sage. Remarkable dose-dependent growth
inhibition was observed in16 herb extracts as shown in
Table 3. Interestingly though DMSO did not affectgrowth of the bacteria, clear growth inhibition circle was

AVR(cm)
0.61
1.38
1.46
1.24
1.37
0.57
0.65
0.12
0.98

±SD
0.17
0.42
0.30
0.10
0.26
0.49
0.40
0.05
0.10

Roman Chamomile
Common Yarrow

1.67
1.39

0.51
0.29

Lemon Thyme
Stevia

1.32
0.12

0.35
0.16

Bergamot (Flower)
Bergamot (Leaf)

1.05
0.63

0.30
0.09

Brucella agar
DMSO 10µℓ

1.86
1.59

0.34
0.32

Yellow Flower Lavender
Hidcote LavenderFrench LavenderFrench Stoechas Lavender
Rosemary Tuscanablue
Rosemary Wood
Rosemary Dreamyblue
Cherry Sage
Amethyst Sageobserved around the disk of DMSO which was placed asa control disk of each herb extract on the several agar
plates, such as French Lavender, French Stoechas
Lavender, Rosemary Tuscanable, Rosemary Dreamyblue,
Lemon Thyme, Stevia, Pineapple Sage, Amethyst Sage,
Cherry Sage, Roman Chamomile and Bergamot.
Swarming inhibition of H. pylori by herb extracts.
15 herbs were selected to test the swarming inhibition

20

Herbtaxis of Helicobacter pylori and eﬀects of herbs on its behavior

Fig.2. Herbtaxis of H. pylori. (A)Effect of Basil and Mint extracts (□ : Lemon Basil, ■ :Spear Mint), (B) effect of Lavender spp(□:Yellow Flower Lavender, ■:Hidcote Lavender, ○:French
Lavender, Δ:French Stoechas Lavender), (C) effect of Rosemary
spp. (□:Rosemary Tuscanablue, ■:Rosemary Wood,○:Rosemary Dreamyblue), (D) effect of Sage spp. (□:Cherry
Sage, ■:Amethyst Sage), (E) effect of Stevia, Roman
Chamomile and Bergamot (□:Stevia, ■:Roman Chamomile ,○: Bergamot ). Symbols with dotted line showed ●: 50 mM
potassium phosphate buffer (pH7.0) and ▲: DMSO. The
number of cells attracted by herb extract in the capillary wascounted. Error bars show standard deviations.

efficacy from the growth inhibition data. The concentric

Chemotaxis to herb extracts.

circle of the swarm zone shown in Fig. 1 for the example

Herbs which did not show growth inhibition of H.

was observed on the motility plate. The motility plate

pylori, Basil spp. and Mint spp., acted as attractant to H.

containing 10 µl of DMSO did not affect any swarm

pylori (Fig. 2 (A)). As shown in Fig.2 (B), mostinhibition of H. pylori. However, Yellow Flower

herbtaxis to Lavender spp.of H. pylori was low, however,Lavender, Rosemary Wood, Rosemary Dreamyblue,

Yellow Flower Lavender showed high activity as an

Cherry Sage, Stevia and Bergamot (leaf) showed strong

attractant. Rosemary Dreamyblue highly attracted H.

swarm inhibition (Table 3).

pylori among Rosemary spp.(Fig.2 (C)). Amethyst Sage

山口県立大学

看護栄養学部紀要

創刊号

2008年３月

21

showed the highest activity as an attractant among Sage

Bergamot (leaf) and Cherry Sage all displayed strongspp(Fig. 2 (D)). Stevia, Roman Chamomile and Bergamot

inhibition of growth so in these cases the results may

showed positive activity to attract H. pylori though thereflect lack of growth rather than inhibition of motility.

number of cells accumulated in the capillary was reduced

The motility inhibition observed using Yellow Flower

after 10 min incubation in case of Stevia (Fig. 2 (E)).

Lavender and Rosemary Wood suggests that these herb
extracts might inhibit H. pylori movement in the mucin
layer in the stomach.

Discussion
11)

H. pylori has a small genome of only 1.7 Mb

in size.

H. pylori showed a chemotactic attraction toward

However, detailed investigation of the genome structure

extracts of Yellow Flower Lavender, Stevia, Rosemaryof different isolates showed that this organism has aDreamyblue, Bergamot and Amethyst Sage suggesting12)

diverse genome , which was explained by the highthese herbs may be useful in eliciting H. pylori from the13)

occurrence of natural transformation in this organism .

basement toward the surface of the gastric mucosa. In

Investigation of factors influencing eradication of H.

the cases of Bergamot and Stevia the number of cells

pylori with triple therapy

14)

showed that it is subject to15)

accumulated in capillaries was decreased after ten

failure related to bacterial resistance to the antibiotics .

minutes incubation, suggesting the extracts containOur results demonstrate that growth inhibition of H.

compounds with a toxic effect on H. pylori, consistentpylori in vitro inclined towards genus specific efficacy.

with the strong growth inhibition observed with theseFor example, in the same family of Labiatae, Basil andextracts. Herb extracts which work both as growth

Mint groups did not show strong growth inhibition ininhibitors and as attractants, such as rosemarycomparison with that shown by Lavenders and Sages.

Dreamyblue, Amethyst Sage and Bergamot, may

The major compounds which are contained in the former

effectively elicit H. pylori.

groups are methyl carbitol (Basils) and menthol

From the genome annotation data, H. pylori has only 4derivatives (Mints). Major components in the lattertransducer-like protein coding regions 11), 12), thoughgroups are limonene, linalyl acetate, cineol and pinene.

substrates for their proteins are still unknown. However,

Linalool is contained in both groups. Each compound

the active compounds in herb extracts which attracted H.

was tested for the growth inhibition. α-pinene, βpylori might have a similar three-dimensional molecular-pinene and 1.8-cineol showed quite strong growth

structure to the substrates for the sensors. Thei n h i b i t i o n ove r 2 . 5 µ m o l , 5 µ m o l a n d 5 µ m o linvestigation of the response of H. pylori, and of H.

respectively. However, other compounds but linalool did

pylori mutants in which transducer-coding genes havenot show typical growth inhibition. Minimum molecule

been disrupted, to concentration gradients of herbof linalool for growth inhibition of H. pylori was 25 µextracts will be needed to obtain further information.

mol. However, the active compounds which were

The data presented here suggests that the proper usage

contained in these herb extracts have not yet beenof herbs may help to decrease gastric disease caused by H.

determined. The growth inhibition observed on control

pylori infection. Appropriate use of herbs in daily foods

disks in experiments using extracts of Lavenders andmight be preferable to medical treatment with the risks ofSages indicated that these herb extracts containedside effects or causing the appearance of antibiotic

volatile, DMSO-soluble compounds which acted as

resistant strains of H. pylori. This effect deserves further

inhibitory factors, though the identity of these compounds

study in animal models.

is not known.
Motility assays in soft agar using extracts of Yellow

Acknowledgements

F l ow e r L ave n d e r, R o s e m a r y Wo o d , R o s e m a r yThis study was supported in part by a Grant-in-Aid

Dreamyblue, Stevia, Bergamot (leaf) and Cherry Sage allscientific research for Food and Food culture of Uragami

showed reduction in motility relative to control

fi nancial group. We also thank Oita prefecture forexperiments. However, Rosemary Dreamyblue, Stevia,

supplying many kinds of herb extracts to support this22

Herbtaxis of Helicobacter pylori and eﬀects of herbs on its behavior

study.

Sutton G. G., Fleischmann R. D., Ketchum K. A.,
Klenk H. P., Gill S., Dougherty B. A., Nelson K.,

1) Blaser, M. J., Helicobacter pylori: microbiology of

Quackenbush J., Zhou L., Kirkness E. F., Peterson S.,‘slow’ bacterial infection. Trends Microbiol. , 1,

Loftus B., Fitzegerald L. M., Lee N., Adams M. D.,

255-260 (1993).

Hickey E. K., Berg D. E., Gocayne J. D., Utterback T.

2) The EUROGAST Study Group., An international

B., Peterson J. D., Kelley J. M., Cotton M. D.,

association between Helicobacter pylori infection andWeidman J. M., Fujll C., Bowman C., Watthey L.,

gastric cancer. Lancet., 341, 1359-1362 (1993).

Wallin E., Hayes W. S., Borodovsky M., Karp P. D.,

3) Parsonnet, J., Hansen S., Rodrigoez L., Gells A. B.,

Smith H. O., Fraser C. M., and Venter J. C., The

Warnke R. A., Jellum E., Orentreich N., Vogelman J.

complete genome sequence of the gastric pathogen

H., and Freidman G. D., Helicobacter pylori infection

Helicobacter pylori. Nature, 388, 539-547 (1997).

and gastric lymphoma. N. Engl J. Med., 330,

12) Alm R.A., Ling L.S., Moir D.T., King B.L., Brown

1267-1271 (1994).

E.D., Doig P.C., Smith D.R., Noonan B., Guild B.C.,

4) Wotherspoon, A.C., Orth-Hidalgo C., Falzon M. R.,

deJonge B.L., Carmel G., Tummino P.J., Caruso A.,

and Isaacson P. G., Helicobacter pylori-asocciatedUria-Nickelsen M., Mills D.M., Ives C., Gibson R.,

gastritis and primary B-cell gastric lymphoma. Lancet,

Merberg D., Mills S.D., Jiang Q., Taylor D.E., Vovis

338, 1175-1176 (1991).

G.F., Trust T.J., Genomic-sequence comparison of two5) Hazel, S. L., Lee A., Brady L., and Hennessy W.,

unrelated isolates of the human gastric pathogen

Campylobacter pyloridis and gastritis association with

Helicobacter pylori. Nature, 397, 176-180 (1999).

intercellular spaces and adaptation to an environment13) Tsuda M., Karita M., Nakazawa T.: Geneticof mucus as important factors in colonization of thetransformation in Helicobacter pylori. Microbiol.

gastric epithelium. J. Infect. Dis., 153, 658-663 (1986).

Immun.,37, 85-89, (1993).

6) Krakowku, M., Morgan D. R., Kraft W. G., and Leunk

14) (Graham, D. Y., Lew, G. M. Malaty, H. M., Evans, D.

R. D., Establishment of gastric Campylobacter pylori

G., Evans, Jr., D. J., Klein, P. D., Alpen, L. C., andinfection is the neonatal gnotobiotic piglet. Infect.

Genta R. M.: Factors influencing the eradication of

Immun., 55, 2789-2796 (1987).

H e l i c o b a c t e r p y l o r i w i t h t r i p l e t h e r a p y.

7) Hazell, S. L., Lee A., Brady L., and Heunessy W..

Gastroenterology, 102, 493-496 (1992).

Campylobacter pyloridis and gastric association with

15) Banatvaia N., Davis, G.R., Abdi Y., Clements L.,

intercellular spaces and adaptation to an environment

Rampton D.S., Hardie J.M., and Feidman, R.A., Highof mucus as important factors in colonization of theprevalence of Helicobacter pylori metronidazole

gastric epithelium. J. Infect. Dis., 153, 658-663 (1986).

resistance in mignants to east London: relation to8) Imamura, L., Tsuchiya M., Inada A., Nakanishi T., andprevious nitromidazole exposure and gastroduodenal

Kobashi K., Inhibition of urease and growth of
Helicobacter pylori by herb extracts. J. Traditional
Medicines, 12, 129-136 (1995).
9) Jones, N. L., Shabib S., and Sherman P. M.. Capsaicin
as an inhibitor of the gastric pathogen Helicobacter
pylori. FEMS Microbiology Letters, 146, 223-227(1997).
10) Mizote, T., Yoshiyama H., and Nakazawa T.. Ureaseindependent chemotactic response of Helicobacter
pylori to urea, urease inhibitors, and sodium
bicarbonate. Infect. Immun., 65, 1519-1521 (1997).
11) Tomb J.F. White O., Kerlavage A. R., Clayton R. A.,

disease. Gut 35, 1562-1566 (1994).