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Antibacterial Activity of Sanguisorba officinalis against Helicobacter pylori

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In this study, a medicinal herbal plant, Sanguisorba officinalis, was examined and screened for anti-Helicobacter pylori (H. pylori) activity. Seventy percent ethanol was used for herbal extraction. For anti-H. pylori activity screening, inhibitory zone tests as an in vitro assay and in vivo study using a Mongolian gerbil (Meriones unguiculatus) model were performed. Also, the safety of herbal compounds was evaluated by animal study. As a result of inhibitory zone test, Sanguisorba officinalis extract demonstrated strong anti-H. pylori activities. Also, as results of in vivo animal studies, Sanguisorba officinalis extract demonstrated strong therapeutic effects against H. pylori infection according to the criteria of histological examination and rapid urease test. As results of the safety study, after 28 days treatment of the Sanguisorba officinalis extract, the animals were not detected any grossly and histological changes. These results demonstrate that it can be successfully cured against H. pylori infection and protected from H. pylori-induced pathology with Sanguisorba officinalis extract. It could be a promising candidate herb treatment for patients with gastric complaints including gastric ulcer caused by H. pylori.
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257
Antibacterial Activity of Sanguisorba officinalis against
Helicobacter pylori
Hyun-A Lee1, Sunhwa Hong1, Hong-Geun Oh2, Sang-Ho Park3, Youn-Chul Kim4,
Hyun Park5, Gil-Saeng Jeong5 and Okjin Kim1,6*
Center for Animal Resources Development, Wonkwang University, Iksan, Korea
Huvet Co. Ltd, Iksan, Korea
Korea DNA Valley Co. Ltd, Iksan, Korea
College of Pharmacy, Wonkwang University, Iksan, Korea
Zoonosis Research Center, Wonkwang University, Iksan, Korea
Institute of Animal Experiment & Efficacy Evaluattion, Wonkwang University, Iksan, Korea
In this study, a medicinal herbal plant, Sanguisorba officinalis, was examined and screened for anti-
Helicobacter pylori (H. pylori) activity. Seventy percent ethanol was used for herbal extraction. For anti-H.
pylori activity screening, inhibitory zone tests as an in vitro assay and in vivo study using a Mongolian
gerbil (Meriones unguiculatus) model were performed. Also, the safety of herbal compounds was
evaluated by animal study. As a result of inhibitory zone test, Sanguisorba officinalis extract demonstrated
strong anti-H. pylori activities. Also, as results of in vivo animal studies, Sanguisorba officinalis extract
demonstrated strong therapeutic effects against H. pylori infection according to the criteria of histological
examination and rapid urease test. As results of the safety study, after 28 days treatment of the
Sanguisorba officinalis extract, the animals were not detected any grossly and histological changes. These
results demonstrate that it can be successfully cured against H. pylori infection and protected from H.
pylori-induced pathology with Sanguisorba officinalis extract. It could be a promising candidate herb
treatment for patients with gastric complaints including gastric ulcer caused by H. pylori.
Key words: Sanguisorba officinalis, Helicobacter pylori, antibacterial activity, herb, therapy
Received 25 February 2010; Revised version received 21 July 2010; Accepted 2 September 2010
Helicobacter pylori (H. pylori) Marshall Warren
의하여 최초로 1984년에 만성 위염 환자의 위점막 생검
조직에서 나선형의 만곡형 그람음성 간균으로 분리 보고
되었고, 이후로 H. pylori 급성 만성위염, 소화불량,
흡수장애증, 저산증 , 위궤양, 십이지장궤양, 위암 , 위림프
종에 이르는 각종 소화기 질환의 주요한 원인 인자로
혀지고 있다(Marshall and Warren, 1984; Hansson et
al., 1996; Honda et al., 1998). Helicobacter (genus)
현재 24 (species) 보고되어 있으며, 사람과
물에서 위염 위궤양의 주요 인자로 보고되 있다 (Kim
and Kim, 2004). 또한 이들의 배설물 내에 Helicobacter
세균들이 배출될 있으며, 이들에 의한 음식물과 물의
오염 가능성이 보고되어 공중보건학적으로 중요성이
대두되고 있다 (Velazquez and Feirtag, 1999).
H. p y lori 치료방법으로 주로 사용되는 방법은 3제요법
(triple therapy)인데 이는 bismuth tetracycline
metronidazole 또는 amoxicillin 투여하는 방법이다 (Shim
et al., 2000). H. py l ori 박멸의 1 처방에는 bismuth
제를 포함하는 3제요법, omeprazole 같은 proton
pump inhibitors (PPI) 포함하는 2제요법, ranitidine,
bismuth citrate 제제를 포함하는 3제요법, bismuth
간으로 하는 3제요법에 PPI 추가하는 4제요법 등이
된다(Kim et al., 1999; Park et al., 2000). 오늘날
H. p y lori 박멸을 위하여 PPI 기본으로 항생제를 포함한
3제요법이 치료의 주를 이루고 있는데 이와 같은 항생제
사용은 필연적으로 이들 항생제에 대한 내성률의
가를 초래하며 우리나라는 서구보다도 높은 내성률을
이고 있다 (Han, 2007; Kim, 2007). 특히 clarithromycin
대한 내성률은 꾸준하게 증가하고 있는 실정이다(Kim
et al., 2004). H. pylori 감염률 아니라 항생제
*Corresponding author: Okjin Kim, Center for Animal Resources
Development, Wonkwang University, 344-2 Shinyoung-dong,
Iksan, Jeonbuk 570-749, Korea
Tel: +82-63-850-6668
Fax: +82-63-850-7308
E-mail: kimoj@wku.ac.kr
Lab. Anim. Res. 2010: 26(3), 257-263
258 Hyun-A Lee et al.
Lab. Anim. Res. | September, 2010 | Vol. 26, No. 3
성률이 다른 나라에 비해 상대적으로 높은 우리나라에서
H. p y lori 제균 치료율을 높이기 위해서는 지속적으로
항생제 내성을 관찰할 필요가 있다(Lee et al., 2005;
Kim, 2007).
현재 H. pylori 감염에 대한 대책은 H. pylori 감염
치료의 형태이며 사용되는 치료제는 항생제
metronidazole 주로 사용하고 있는데 이에 대한 내성과
항생제 부작용의 문제가 야기되고 있어 새로운 기전의
치료제의 개발이 필요한 실정이다(Kim et al.,
2004). 새로운 항생제의 개발 분야에서 크게 각광을
있는 것으로 기존 화합물에 기초한 항생제와 연관
계가 천연물 유래의 안전한 항생물질이 있으며 천연
물은 화합물보다 다양성이 크고 안전하여 천연물 유래
생물질의 사용은 높은 안전성과 항생제 내성 문제의
측면에서 장점을 가지고 있다 (Koehn and Carter,
2005).
지유(Sanguisorbae officinalis) 장미과에 속하는 다년생
초본으로 중국 , 일본 한국에 자생하며, 방종형으로
근의 울곡이 불규칙하고, 다수의 분지가 있는데 길이가
10~20 cm, 직경이 0.5~1.5 cm 이르며, 근도에는 개개
아경이 있고, 이면은 암갈색을 띄고 세로주름이 현저하다
(An et al., 2004). 지유의 뿌리는 지혈제 , 진통제, 수렴효
과를 가지며 , 화상, 내인성 뇌출혈 치료제로 전통 중국
약제로 이용되어져 왔다 (An et al., 2004). 지유에서 분리
dimeric ellagitanin sanguiin H-6 DNA topoisomerases
저해와 세포독성활성과 관련되어진 것으로 알려져 있으
(Bastow et al., 1993), sanguiin H-6 H-11 질소
산화물을 억제하는 것으로 알려져 있다(Yokozawa et al.,
2000; An et al., 2004). 또한 지유에는 사포닌배당체,
pomolic acid, vitamin A, tritepenoides (Reher et al.,
1992), 탄닌과 관련 화합물(Tanaka, 1983; An et al.,
2004) phenolic acid, sanguisorbic acid dilactone
3가지 ellagitanins sanguiins H-1, H-2, H-3 (An et
al., 2004) 등을 17% 정도 함유하며, 탄닌 성분은
생리활성 기능 고혈압 억제, 통풍예방, 항산화 효과 ,
항암 다양한 약리효과가 검증 있으나 , 현재까지
지유의 H. pylori 항균 효과에 관한 연구는 없는 실정 이다 .
연구에서는 사람에 위암 위궤양을 유발하여
각한 임상결과를 초래하는 H. pyl ori 효과적인 천연물
유래 항균물질을 개발하고자 지유의 H. pylori 항균효과
탐색하였다.
재료 방법
지유 추출물 조제
시험에 사용된 지유는 익산시 대학한약국으로부터
국산으로 판매되고 있는 건조된 지유를 구입하여 대성아
트론 분쇄기 DA700 입자 크기가 30 mesh 이하가
도록 분쇄하여 천연물 분말을 수득한 , 상기에서 수득
건조된 천연물 분말 (1 kg) 질량의 3(v/w) 해당하는
증류수를 포함하는 70% 에탄올 수용액을 가하여 100 C
에서 3시간 동안 환류 냉각 추출하고 여과 감압
축한 , 동결 건조하여 분말상태의 지유 추출물 600 g
수득하여 실험의 시료로 사용하였다.
H. pylori 배양
H. pylori (ATCC 43504, American Tissue Culture
Collection, Rockville, MD, USA) 균주를 10% calf serum
첨가된 브루셀라 한천배지에 접종 , 10% CO
100% 습도가 유지되는 37 C incubator에서 3일간 배양
하였다. 배양된 H. pylori 멸균된 phosphate-buffered
saline (PBS, pH 7.2) 들어 있는 튜브에 , 1 mL
2.0×10 colony-forming unit (CFU) 균수를 포함하
준비하여 실험에 사용하였다.
실험동물
의약안전 실험동물자원과에서 specific-pathogen
free 상태로 계통이 유지되고 있는 Mongolian gerbil
(Meriones unguiculatus) 공급받아 원광대학 동물자원
개발연구 실험동물사실에서 1주일 동안 화사
실험에 사용하였다 . Mongolian gerbil H. pylori
사람의 태생리와 유사하게 만성으로 되어 위염,
궤양과 위암을 유발하는 것으로 알려져 있다 (Honda et
al., 1998). 연구자들은 사람 H. pylori 감염 동물
Mongolian gerbil 이용하여 H. pylori 감염
추출물 적용에 의한 치료효과를 인하고자 하였다.
기간 도는 23±1C, 습도 50±5%, 소음 60
phone 이하, 시간 08:00~20:00 (1 12시간), 조도
150~300 Lux, 환기는 시간당 10~12 환경에서
되었으며, 사료는 실험동물 전용사료 (Samtaco, Osan,
Gyeonggi, Korea) 2.0 Mrad 방사선으로 멸균시
유급식 으며 , 음수는 filter 여과 멸균수를
자유 급수하였다 . 연구에 사용된 동물실험에 관련된
모든 실험과정과 차는 원광대학 동물실험리위원
사전심의와 규정을 준수하여 되었다.
In vitro 헬리코박터 항균 효능 실험
H. p y lori 균주를 10% calf serum 첨가된 브루셀라
한천 배지 플레이트에 도말 (streak)하고 대조를 위한 항생
디스 Gentamicin (바이동물약, Suweon, Korea),
Kanamycin (바이동물약) Enrofloxacin (바이동물
) 적용용량으로는 각각 0.25, 0.5, 0.25 mg 적용
하였다. 천연물 추출물 30 mg dimethyl sulfoxide
(DMSO; Sigma, St. Louis, MO, USA) 100 µL
Anti-Helicobacter activity of Sanguisorba officinalis 259
Lab. Anim. Res. | September, 2010 | Vol. 26, No. 3
300 mg/mL 농도로 디스 여과지에 5µL 10 (1.5 mg),
10 (0.15 mg) 용매대조를 위한 DMSO 각각 적용하
였다. 실험에서는 균이 접종된 배지에 멸균된 디스
(지름 0.7 mm) 배치하고, 이후 10% CO , 100% 습도
유지되는 기에서 배양하고 4 가하였다 .
접종한 6, 12, 24, 48 60시간 각각 준자
이용하여 clear zone 지름으로 현되는 억제
(the zone of inhibition) 측정하여 균이 자라지
clear zone 관찰하였다 . 디스크는 균주 6
사하여 억제 위의 하였다. 결과의 가는
항생제 디스 각각의 clear zone 형성을 관찰하고
형성된 clear zone 지름을 측정하여 지름이 수록
효과가 높은 것으로 판정하였다.
In vivo 헬리코박터 항균 효능 실험
1주일간 7 Mongolian
gerbil 40수를 실험에 사용하였다. 10수의 동물
들을 사용하여 H. pylori 감염 지유 추출물 적용(I
), H. pylori 감염 지유 추출물 처치 적용(II)
H. pylor i 감염 없이 지유 추출물 적용(III), H.
pylori 감염 없이 지유 추출물 처치 적용(IV) 4
으로 실험을 하였다 . 실험에 사용된 7
Mongolian gerbil 12시간 식시 , I II
물들은 1 mL 2.0×10 CFU H. pylori 균수가 포함
되게 준비된 배양액을 두당 0.5 mL 존대를
이용하여 경구로 투여하여 H. pyl ori 감염시. III
IV 동물들은 12시간 멸균 PBS (pH 7.2)
0.5 mL 경구로 투여하였다. 투여 동물
들은 12시간의 사료를 급여하였. 동안에
음수는 자유급이할 있도록 하였다 . H. p ylori 또는 PBS
투여한 I III 동물들은 2주일 후인 9부터
지유 추출물을 체중 기준으로 400 mg/kg 용량으로 경구
투여를 4주간, 13까지 연일 투여하였다. 기간
동안 II IV 동물들은 다른 처치 없이 사료와
수를 자유 급여하였다.
9에서 13까지, H. pylori 감염이 유지되는
시점으로부터 4주간의 시험물질 투여 기간 , 시험
동물들은 1주에 1 체중을 측정하고 실험 종료일인 13
개체를 12시간 식한 ether 마취 하에
사시 위를 적출하여 병변 점수를 구하였다.
또한 요소분해효소 검사를 통하여 감염 여부를
인하였으며, 추가적인 리조직학적 검사를 통하여
조직 점수를 구하여 지유 추출물 투여 박터
감염에 미치는 영향 가하였다 .
점막의 병변 점수(gross lesion score) 산정은
Yam et al. (2009) 방법에 하였다. 시험방법을
하면 다음과 같다. 시험 군별 시험
료일에 적출된 (stomach) (great curvature)
개하여 펼친 후에 실체현미경(×10) 사용하여
관찰하고, 병변 점수를 산정하였다.
점수는 병변 가로길이 ×세로길이(mm ) 합으
계산하였다. 개체의 점수를 구한 군별
점수를 구하였다.
점막의 조직병변 점수 (histological lesion score) 산정
Yam et al. (2009) 방법에 하였. 시험방
법을 하면 다음과 같다 . 리조직학적 검사를
위하여 적출된 위의 일부를 10% 중성 포르말 고정
하고, 선위(glandular stomach) 부위를 가로 으로 5
mm 으로 , 개체 3 부위를 선정하였고,
부위들은 다른 개체들 간에 서로 동일한 부위
되도록 주의하여 선정하였다 . 선정된 부위들의 조직들
리조직학적 검사를 위한 통상적인 방법을 사용하여
포매한 , 4 µm 절편하여 hematoxylin
and eosin (H&E) 염색 리조직학적인 검사를
였다. 조직 병변 대한 가를 하여 0:
no lesion, 1: mild, 2: moderate, 3: severe 등급을
점수로 환산하여 기록하고, 개체당 3 부위의 점수의
구하여 개체의 조직병변 점수를 구한 ,
점수를 구하였다.
요소분해효소 검사(rapid urease test) CLO 검사
시약인 ASAN Helicobacter Test (Asan Pharmaceutical
Co., Seoul, Korea) 이용하여 되었다. 균적으로
출한 날문부위(pyloric region) 점막의 조직을 채취하여
ASAN Helicobacter Test 각각을 제조사의
서에 배양하여 노란 배지가 적색으로 하는
우를 양성으로 판정하였다. 검사과정을 하면
미리 방치한 시약을 노란 한천 보이도록
스티커 벗긴 바늘을 사용하여 위점막 조직을
속에 스티커 다시 동물번호
시간을 기입하고 incubator 이용하여 37 C 12시간
후에 판독하였다. 판독의 결과 노란색에서 적색으로
배지의 경우를 양성으로 판정하였다. 실험
종료 양성 판정된 개체의 수를 분율로 구하여 양성
(positive percent) 구하였고 음성 개체수를 전체수
분율을 구하여 치료율(therapeutic percent)
구하였다.
Polymerase chain reaction (PCR) 검사는 실험 종료
공시동물들의 pyloric region 점막 일부를 채취하여
DNA 추출한 H. pylori 존재 인하기 위한
PCR 하였다. DNA 추출은 bead beater-phenol
extraction method 사용하였다(Kim and Kim, 2004).
검체를 1 mL 멸균 증류수가 들어있는 Mini-Bead Beater
(Biospec product) 전용 2 mL tube 채취하여
멸균 3 증류수에 부유시 glass bead (0.1 mm
260 Hyun-A Lee et al.
Lab. Anim. Res. | September, 2010 | Vol. 26, No. 3
size, Biospec product) 200 µL phenol-chlorform-
isoamyl alcohol 용액 (50:49:1) 200 µL Mini-
Bead Beater 30초간 5,000 rpm으로 하였다.
4C에서 12,000 rpm으로 15분간 원심분리한
액을 멸균 2 mL tube 옮겼. 3 M sodium acetate 10
µL ice-cold ethanol 250 µL 20 C에서 10분간
정체시 , 15,000 rpm으로 15분간 원심분리하였다.
전물은 70% alcohol 하여 에서 건조시 Tr i s
EDTA (pH 8.0) 60 µL 용해시 실험에 사용하였다.
Kim et al. (2004) 방법에 genus Helicobacter
rpoB DNA 있는 primer 사용하
PCR 하였다. Forward primer (HF; 5' ACTTT
AACGCATGAAGATAT 3') Reverse primer (HR; 5' ATA
TTTTGACCTTCTGGGGT 3') 사용하여 PCR Taq
DNA polymerase 1 U, dNTP 250 µM, 50 mM Tris-
HCl (pH 8.3), 40 mM KCl, 1.5 mM MgCl 포함하는
AccuPower PCR Premix (Bioneer, Daejeon, Korea)
용하였다. 추출된 DNA template 50 ng, primer
20 pmol AccuPower PCR Premix tube 멸균
증류수로 최종 20 µL 맞춘 , 94 C에서 30,
52에서 30, 72 C에서 45초의 30 cycles PCR
, 1.2% agarose gel에서 458 bp 특이 밴드
인하였다.
지유추출물의 독성 평가
지유 추출물을 기간 섭취함으로 인해 독성발현으
의심되는 세포 상이 있는지 알아보기 위한 독성
실험을 하였다. 실험 9 Mongolian
gerbil 사용하여 되었으며 , 28 동안 매일 400 mg/
kg 용량으로 시험물질을 경구투여하였다. 실험 종료일
ether 마취 하에 사하여 부검을 실시하고 실질
기의 병변 관찰한 , 대뇌(cerebrum), 소뇌
(cerebellum), 연수(pons), 고환(testis), 심장(heart), (liver),
(lung), (kidney), (muscle), 비장 (spleen),
(prostate), (pancreas), (thymus), (adrenal
gland), 소장(small intestine), 대장(large intestine),
(bone marrow), 상선(thyroid gland) (seminal
vesicle) 적출하여 10% 중성완충 포르말용액(neutral
buffered formalin) 고정하였다. 고정이 료된 조직들은
리조직학적 검사를 위한 통상적인 방법을 사용하여
포매한 , 4 µL 절편하여 H&E 염색
리조직학적인 검사를 하였다.
통계학적 분석
시험 양성률에 대한 통계는 Repeated Measured
ANOVA SPSS v.12 USA (Duncans 사후검정) 실시하였
으며 P<0.05 이하일 통계적으로 유의한 것으 판단
되었다. Rapid urease 검사인 CLO test 시험
양성율과 치료율에 대한 95% confidential interval (CI)
MINITAB software (Minitab Inc., State College, PA,
USA) 사용하여 구해. 양성률에 대한 CI
차이가 있으면 통계적으로 유의한 것으로 판단되었다 .
결과
In vitro 헬리코박터 항균 효능
H. p y lori 세균 배양 플레이트에 적용한 대조 항생제
크들과 지유 추출물을 적용한 37 C incubator에서
추가 배양하면서 적용 디스 clear zone 지름
기를 측정한 결과 각각의 디스 H. pyl o ri 세균
증식하지 하는 clear zone 관찰할 있었다.
험결과 Ta bl e 1 같이 적용 12시간 가장 clear
zone 지름을 관찰할 있었는데 , DMSO 경우 clear
zone크기를 관찰할 없었고 대조으로 사용된
Gentamicin 경우 9 mm, Kanamycin 8 mm, Enrofloxacin
13 mm이었으며 , 지유 추출물 1.5 mg 적용시 13 mm이었다.
In vivo 헬리코박터 항균 효능
지유 추출물을 H. pylori 세균 감염 비감염 동물에
투여한 , 체중화를 인한 결과 Ta b l e 2 같은 결과
었다. H. p ylori 세균 감염 지유 추출물 투여
비투여 II 하여 유의한 체중 증가 보였
(P<0.05).
지유 추출물을 H. pyl o ri 세균 감염과 비감염 동물에
여한 , 병변 점수 조직병변 점수를 인한
결과 Table 3 같은 결과를 었다. H. pylori 감염 없이
Table 1. Anti-H. pylori activities of Sanguisorba officinalis extract using the paper disk diffusion bioassay
Treatment Clear zone (mm)
6 hrs 12 hrs 24 hrs 48 hrs 60 hrs
Dimethyl sulfoxide 00000
Gentamicin 69777
Kanamycin 58877
Enrofloxacin 91313129
Sanguisorba officinalis 10 (1.5 mg) 8 13 12 12 10
10 (0.15 mg)46655
Anti-Helicobacter activity of Sanguisorba officinalis 261
Lab. Anim. Res. | September, 2010 | Vol. 26, No. 3
지유 추출물 적용(III), H. pylori 감염 없이 지유 추출
처치 적용(IV) 경우에 병변 관찰되지 않았
. , H. p ylori 감염 지유 추출물 처치 적용
(II) 지유 추출물 적용(I) 보다 병변 점수
조직병변 점수가 유의하게 높은 점수를 보였다 (P<0.05)
요소분해효소 검사는 지유 추출물을 H. p ylori
감염과 비감염 동물에 투여하고 H. pylori 세균의
료에 의한 효과를 인하기 위하여 되었으며,
실험결과는 Table 4 . 요소효소분해 검사인
CLO test 결과는 검체에 감염된 박터 세균 성장
요소효소분해와 제공된 배지의 요소와의 반응 결과로
유도되는 화로 감염 여부를 손쉽 있는
방법이다(Lee et al., 2006). 실험결과, H. pylo ri 감염
지유 추출물 적용(III), H. p ylori 감염 없이 지유
추출물 처치 적용(IV) 경우에 요소분해효소
검사에 의하여 H. pylo r i 세균에 음성 결과 보였다 .
, H. pyl o ri 감염 지유 추출물 처치 적용(II)
10 개체 두에서 적색의 H. pylor i 세균 양성
반응 검출되었다. H. p ylori 감염 지유 추출물 적용
(I) 10 두에서 음성의 결과를 보여 H. pyl o ri
양성대조 II 유의한 화를 보였다 (P<0.05).
PCR 검사를 통하여 H. pylo r i 특이적인 밴드
함으로 검사된 위조직 내에 H. py l ori 감염 여부를
정할 있었다(Table 4). H. pylori 양성 검체는 특이
인할 있었다(Figure 1). 실험결과, H.
pylori 감염 지유 추출물 처치 적용(II) 10
개체 두에서 양성반응 검출되었다(Table 4). , H.
pylori 감염 지유 추출물 적용(I) 10 두에서
음성의 결과를 보여 H. pylori 감염 양성대조 II
의한 화를 보였다(P<0.05).
지유 추출물의 독성
지유 추출물을 기간 섭취함으로 인해 독성발현으
의심되는 세포 상이 있는지 알아보기 위해 9
Table 2. Change in body weights (g) of mice treated with H. pylori and/or Sanguisorba officinalis extract in vivo study
Group Treatment Week after inoculation
01234
IH. pylori+Sanguisorba 132.5± 2.0 136.0±2.1 140.0±2.1* 142.5±1.6* 144.0±1.1*
II H. pylori+PBS 132.5±1.5 135.0±2.0 135.0±2.1 135.5±2.0 136.5±1.6
III PBS+Sanguisorba 133.0±2.1 138.0±2.1 142.0± 2.0* 144.5±2.0* 145.0±2.5*
IV PBS+PBS 133.5±2.1 138.5±1.1 143.0±1.5* 145.0±2.0* 146.0±2.5*
Sanguisorba officinalis treatment was contuinued with 400 mg/kg dose during 4 weeks after Helicobacter pylori infection. PBS,
phosphate-buffered saline. Significantly different from the positive control group II (P<0.05).
Table 3. Gross and histopathological lesion scores of mice treated with H. pylori and/or Sanguisorba officinalis extract in vivo study
Group Treatment Gross lesion scores Histopathological lesion scores
IH. pylori+Sanguisorba 12.0±3.5* 2.0±0.50*
II H. pylori+PBS 95.0±5.5 8.0±1.50
III PBS+Sanguisorba 0* 0*
IV PBS+PBS 0* 0*
PBS, phosphate-buffered saline. *Significantly different from the positive control group II (P<0.05).
Table 4. Results of rapid urease test (CLO) and polymerase chain reaction (PCR) with gastric mucosal tissues after the study on
therapeutic effects of H. pylori infection with Sanguisorba officinalis extract
Group Inoculation nPositive reaction (positive percent)
H. pylori Treatment CLO PCR
I Yes Sanguisorba 10 0*
( 0 %, CI 0-25.9)
0*
( 0 %, CI 0-25.9)
II Yes PBS 10 10
( 100 %, CI 74.1-100)
10
( 100 %, CI 74.1-100)
III No Sanguisorba 10 0*
(0 %, CI 0-25.9)
0*
(0 %, CI 0-25.9)
IV No PBS 10 0*
(0 %, CI 0-25.9)
0*
(0 %, CI 0-25.9)
Treatment was conducted daily during 2 weeks after H. pylori inoculation. The positive percent revealed H. pylori colonization, which
was observed as red color change from yellow color medium. Incidence percentage (95% confidential interval; CI) was calculated with
MiniTab statistic software program. *Significantly different from the positive control group II (P<0.05).
262 Hyun-A Lee et al.
Lab. Anim. Res. | September, 2010 | Vol. 26, No. 3
Mongolian gerbil 용하여 28 동안 매일 400
mg/kg 용량으로 시험물질을 경구투여하고 지유의 독성발
가한 결과 대조 지유 추출물 투여
모든 개체에서 또는 특이 임상증상은 관찰되지
. 실험 종료일 ether 마취 하에 사하여 부검을
실시하고 실질 장기의 병변 관찰한 결과 유의
병변 관찰할 없었다. 또한 , 실질 장기들의
조직학적 검사에서도 다른 세포상은 관찰되지 않았.
고찰
H. pylori 감염은 만성 위염, 장상화생(intestinal
metaplasia) 소화성 궤양의 중요한 원인이며 , 위암발생
기여한다(Ahn et al., 1991). 한국에서 위암은 암으로
인한 주요 원인을 차지하고 있다(Ahn et al.,
1991). 한국인의 연간 위암 발생율은 10
57.9 여성 25.1 보고되고 있으며, 이는 다른 나라의
위암 발생율에 하여 매우 높은 수치임을
. 위암은 임상적 결과가 심각하여 한국 성인
률의 주요 원인중의 하나로 보고되고 있다(Ahn et al.,
1991). H. p yori 율은 한국인에서 8 80%, 20
이상에서는 90% 보이는 것으로 보고되고 있다(Jung
et al., 2000). 자료들에 의하면 대부분의 한국인
들은 Helicobacter 염되어 있으며, 40-45
상의 생애 동안 H. py lori 감염된 상태로 다는
있다. 또한 국내 높은 위암 발생율은 H.
pylori 높은 율과 연관된 결과임을 있다(Jung
et al., 2000). 연구 결과들에 의하면 H. pyl o ri
위암의 유발 인자로서 H. pylori 치료 감염대책
시급한 것을 있다.
항균제내성은 H. pylori 제균 주요 원인이다
(Harris, 1997). 적으로 1 제균 치료제로는 PPI,
amoxicillin, clarithromycin 또는 metronidazole 3제요법이
권장되지만, 국내의 metronidazole 내성률이 25.8-66.2%
정도이며(Kim et al., 2004; Lee et al., 2005),
clarithromycin 내성률은 2.5-13.8% 보고되어 있다(Kim
et al., 2004). 해당 에서 또는 특정 환자에서
clarithromycin 대한 원발성 내성률이 15% 으면
험적인 1 치료제로 clarithromycin 사용이 (Lee et
al., 2002; Kim et al., 2004). 제균율 감소에
영향 미치는 요인은 항생 내성으로서 국내
경우 amoxicillin clarithromycin 1987년에는 0%
내성율에서 2003년에는 각각 18.5% 13.8%으로 내성
율이 증가하였으며 metronidazole 52.9%에서 66.2%
내성율이 증가음을 있다(Wouden et al., 1999).
항생제 내성의 대안으로서 천연물 유래 항균효능
물질의 개발이 새로운 대안으로 제시되고 있다(Lim et al.,
2002).
연구에서 항균효능이 기대되는 지유 추출물의 anti-
H. p ylori 효능을 in vitro 실험에서 인하였다. 또한 지유
추출물을 이용하여 실험동물을 이용한 in vivo H. pylori
감염치료 효능 연구를 결과 지유 추출물은 H.
pylori 감염을 억제하고 감염에 의한 병변 화시주는
것을 있었다. 지유 추출물을 적용한 H. py lori
염에 대한 in vivo 치료효과 실험결과 점막의 H. pylori
효과는 rapid urease CLO test PCR 결과로
있었다. 또한, 우수한 anti-H. pylori 효능을 인한
지유 추출물에 대한 28 투여 안전 가를
, 지유 추출물의 400 mg/kg 연일 투여에 의하여 유의
독성 화는 관찰되지 않았. 라서 지유 추출물은
안전성이 높은 것으로 판단되었다.
지유는 중국, 일본 한국에 분포하는 장미과에
속하는 다년생 식물로서 동속식물의 뿌리를 생약에서
지유라 부르며 , 산비 습기 에서 자라는
것으로 알려져 있다. 지유의 약리성분으로 뿌리에 tannin,
triterpenoid saponin 함유되어 있고 가지에는 qurcetin
kaempferol 배당체인 ursol triterpenoid saponin
함유되어 있으며, 에는 vitamin C, 에는
chrysanthemin, cyanin 함유되어 있다(An et al., 2004).
현재 지유에 대한 연구는 성분 (An et al., 2004)
경세포보작용(Nguyen et al., 2008), 억제효과
(Park et al., 2004) 대한 연구는 있으나 현재까지 지유
anti-H. pylori 효능에 대한 보고는 없었다. 연구
지유는 in vitro in vivo 실험 다에서 우수한
anti-H. pylori 효능이 있음이 인되었다. 라서 연구
결과는 지유 anti-H. pylori 효능에 대한 최초의 보고이
. 지유의 성분으로 현재 보고된 tannin 관련 화합물
phenolic acid, sanguisorbic acid dilactone 3가지
ellagitanins sanguiins H-1, H-2, H-3 (Tanaka, 1983;
An et al., 2004) 다른 천연물 추출물 성분에서 활성을
가지고 있는 성분으로 알려져 있어 이들 성분이 연구
Figure 1. Results of polymerase chin reaction in the study o
f
anti-H. pylori activity with Sanguisorba officinalis extract. M: 100
bp marker, P: DNA from H. pylori ATCC 43504, N: negative
control, 1~2: Group I (H. pylori+Sanguisorba officinalis), 3~6:
Group II (H. pylori + phosphate-buffered saline).
Anti-Helicobacter activity of Sanguisorba officinalis 263
Lab. Anim. Res. | September, 2010 | Vol. 26, No. 3
에서 인한 지유 추출물의 H. pylori 항균효과에 관여하
생리활성물질이 추정해 있으며,
유의 anti-H. pyl o ri 효능에 관여하는 성분에 대한 추가
구가 되어야 것으로 생각된다.
연구의 결과를 종합한다면 , 항생제 내성문제가 없으
섭취 안전성이 높은 천연물 유래 H. py lori 항균물
질로서 지유 추출물은 in vitro in vivo 실험에서
우수한 anti-H. p ylori 효능이 인되었기 때문에 H.
pylori 치료제 개발의 우수한 후보물질로서 이용
것으로 판단되었다.
감사의
연구는 2010 원광대학 연구비를 지원
되었으며, 이에 감사 드립니다 .
참고문헌
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and Son, J. H. (2004) Antioxidant effects and application as
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... It has been widely used in the treatment of colitis, ulcerative colitis, pyelonephritis, hemostasis, and burns [3]. Besides, it was also reported to possess the following activities: antioxidant [4], anti-inflammatory [5], antiviral [6], antimicrobial [7,8], and antitumor [9]. Recently, more attention has been paid on its anti-cancer activities, such as antiproliferative effects against breast cancer [10], oral cancer [11], prostate cancer [12], and colorectal cancer [13]. ...
... The chemical constituents isolated from S. officinalis are mainly triterpene glycosides [14], phenolics, and flavonoids [15,16]. The polyphenols ( Figure 1) include gallic acid (1), sanguiin H-4 (2), catechin (3), methyl-6-O-galloyl-β-D-glucopyranoside (4), caffeic acid (5), syringic acid (6), ethyl gallate (7), 3,4-dihydroxy-5-methoxybenzyl ester (8), ferulic acid (9), ellagic acid (10), and 3,3-dihydroxy ellagic acid (11). The triterpenes ( Figure 1) include 2-oxo-3β,19α-dihydroxy-olean-12-en-28-oic acid β-D-glucopyranosyl ester (12), rosamultin (13), ziyuglycoside I (14), 3β-[(α-L-arabinopyranosyl)oxy]-urs-12,18 (19)-dien-28oic acid β-D-glucopyranosyl ester (15), 3β-[(α-L-arabinopyranosyl)oxy]-urs-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester (16), 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-Dglucopyranosyl ester (17), pomonic acid (18), euscaphic acid (19), 2α, 3α-duhydroxyursa-12,19(29)-dien-28-oic acid β-D-glucopyranosyl ester (20), 1β-hydroxyeuscaphic acid (21), 3β,19α-dihydroxy-12-ursen-28-acid (22), and 2α-hydroxyoleanolic acid (23). ...
... for syringic acid (6), m/z 197.0→124.1 for ethyl gallate (7), m/z 197.1→182.2 for 3, 4-dihydroxy-5-methoxybenzyl ester (8), m/z 193.1→134 for ferulic acid (9), m/z 301→145 for ellagic acid (10), m/z 328.7→314 for 3, 3-dihydroxy ellagic acid (11), m/z 673.4→511.4 for rosamultin (13), m/z 784.5→437.4 ...
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A novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for simultaneous determination of 11 phenolic acids and 12 triterpenes in Sanguisorba officinalis L. Chromatographic separation was conducted with gradient elution mode by using a DiamonsilTM C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase of 0.1% acetic acid water (A) and methanol (B). The drift tube temperature of ELSD was set at 70 °C and the nitrogen cumulative flow rate was 1.6 L/min. The method was fully validated to be linear over a wide concentration range (R2 ≥ 0.9991). The precisions (RSD) were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L. and could also be successfully applied to study the influence of processing method on those functional components in Sanguisorba officinalis L.
... Biochemical analysis also confirmed that the indicated dosages of SRE and anti-PD-1 antibodies have no significant toxic effect on the liver or kidneys in humanized PD-1 mice with humanized PD-L1 MC38 tumors ( Table 1). In a Mongolian gerbil, daily oral administration of SRE at a dose of 400 mg/kg for 28 days, no gross histological changes were found (33). Moreover, the no changes were found in the relative weights of liver and kidney in the gerbil, or in AST and ALT in high-fat-diet-induced obese C57BL/6J mice after 8 weeks' treatment with 200 mg/kg/day SRE extracted with 50% ethanol (34). ...
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Immune checkpoints such as programmed death-1 (PD-1) have been proven as antitumor targets by enhancing cytotoxic T cell activity. All immune checkpoint blockades are antibody therapeutics that have large size and high affinity, as well as known immune-related side effects and low responses. To overcome the limitation of antibody therapeutics, we have explored PD-1/PD-L1 (programmed death-ligand 1) blockades in traditional oriental medicine, which has a long history but has not yet studied PD-1/PD-L1 blockades. Sanguisorbae Radix extract (SRE) blocked PD-1 and PD-L1 binding in competitive ELISA. SRE effectively inhibited the PD-1/PD-L1 interaction, thereby improving T cell receptor (TCR) signaling and the NFAT-mediated luciferase activity of T cells. SRE treatment reduced tumor growth in the humanized PD-L1 MC38 cell allograft humanized PD-1 mouse model. Additionally, the combination of SRE and pembrolizumab (anti-PD-1 antibody) suppressed tumor growth and increased infiltrated cytotoxic T cells to a greater extent did either agent alone. This study showed that SRE alone has anticancer effects via PD-1/PD-L1 blockade and that the combination therapy of SRE and pembrolizumab has enhanced immuno-oncologic effects.
... Sanguisorba officinalis L. is a perennial plant widely distributed throughout Eastern Asia, Northern Africa, Southern Europe, and other parts of the world. Several studies (Do et al., 2005;Janovska, Kubikova, & Kokoska, 2003;Lee et al., 2010) have reported that ethanolic extracts of S. officinalis L. had broad-spectrum antibacterial properties. Previous work demonstrated that polyphenolic extracts of S. officinalis L. exerted remarkable antibacterial activity against five pathogenic bacteria (gram-negative Escherichia coli and Salmonella Typhimurium, gram-positive S. aureus, Listeria monocytogenes, and Bacillus subtilis) and tentatively identified 44 polyphenolic compounds, most of which are recognized as active antibacterial compounds . ...
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Sanguisorba officinalis L. (family Rosaceae, subfamily Rosoideae) is a plant found throughout Southern Europe, Northern Africa, and Eastern Asia. This study demonstrated the antibacterial activity of a purified polyphenolic extract (PPE) from S. officinalis L. against Bacillus subtilis using growth inhibitory and apoptosis assays, and investigated the antibacterial mechanism responsible for changes in cell membrane properties. Fourier transform infrared spectroscopy suggested that PPE altered the cell wall and membrane properties of B. subtilis . Further determination of cell membrane integrity and permeability revealed that B. subtilis membrane integrity was more severely damaged by PPE at the minimum inhibitory concentration (MIC) than at the minimum bactericidal concentrati on (MBC). Instead, PPE at the MBC reduced cell membrane fluidity by significantly decreasing the proportion of anteiso‐ and iso‐branched phospholipid fatty acids (PLFAs) from 64.17 ± 0.28% and 27.23 ± 0.03% in the control to 5.57 ± 1.06% and 6.00 ± 1.40%, respectively (P < 0.001). Scanning electron microscopy revealed different effects of PPE on cell morphology, demonstrating that, at the MIC and MBC, PPE exerted antibacterial activity by disrupting the cell membrane and reducing cell membrane fluidity, respectively. Consequently, this study elucidated changes in the bacterial membrane due to exposure to PPE and its potential use as an antimicrobial agent. Practical Application The abuse of synthetic chemical preservatives raises food safety concerns; however, plant‐derived polyphenolic compounds may be a safe and effective alternative. This study demonstrated the strong antibacterial activity of a purified polyphenolic extract (PPE) of Sanguisorba officinalis L. and revealed its antibacterial mechanism against Bacillus subtilis , suggesting that it may provide a useful antimicrobial agent in food industry applications.
... Based on the highly positive relationships between antioxidant activity and antibacterial activity proved by Shan et al. [20], it was believed that the polyphenolic extract of S. officinalis L. had the potential of inhibiting bacteria. So far, there have been several studies reporting the antibacterial activities of ethanol extract of S. officinalis L. against Helicobacter pylori, Pseudomonas aeruginosa, Lactobacillus plantarum, and so on, suggesting that this extract had broad-spectrum antibacterial property [26][27][28]. However, these studies did not explore the relationship between the polyphenolic extract of S. officinalis L. and its antibacterial activity. ...
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Sanguisorba officinalis L., one kind of perennial plants, has been widely distributed in southern Europe, northern Africa and China. The objective of the present work was to evaluate the antibacterial captivities of polyphenolic extract (PE) of S. officinalis L. on five pathogenic bacteria, including Gram-negative bacteria (Escherichia coli and Salmonella typhimurium) and Gram-positive bacteria (Staphylococcus aureus, Listeria monocytogenes and Bacillus subtilis). The antibacterial activities were determined by the diameter of inhibition zone, minimum inhibitory concentration and minimum bactericidal concentration tests. The results showed that purified PE had significantly better performance in inhibiting bacteria than crude PE (P < 0.05), and PE had better inhibition effect on Gram-positive bacteria than Gram-negative bacteria. Using LC–ESI–QTOF–MS/MS technology, a total of 44 polyphenolic compounds were tentatively identified, 26 of which have been discussed for the first time in S. officinalis L. Gallic acid, ellagic acid, catechin and their derivatives, which have been identified as antibacterial bioactivities previously, were the major constituents with the amounts of 10, 8 and 11, respectively. Besides, compared with potassium sorbate and sodium benzoate, purified PE with a low concentration had significantly stronger antibacterial ability against all the tested bacteria (P < 0.05), suggesting that purified PE of S. officinalis L. could be a promising source of food preservatives.
... The stomach was opened along the greater curvature and washed with saline, and half of the glandular mucosa was scraped off for detection of colonizing H. pylori, while the residual portion was formalin-fixed and embedded in paraffin for histological analysis. H. pylori colonization was confirmed by the rapid urease test CLO as previously described [23]. Mucosal damage was evaluated according to established criteria [24]. ...
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The efficacy of standard therapeutic strategies for Helicobacter pylori (H. pylori) infection is decreasing over time due to the emergence of drug-resistant strains. As an alternative, the present study investigated the capacity of Lactobacilllus paracasei (L. paracasei) HP7, isolated from kimchi, to inhibit H. pylori growth. The effects of L. paracasei HP7 on H. pylori adhesion and H. pylori-induced inflammation were examined in AGS human gastric adenocarcinoma epithelial cells and a mouse model of H. pylori SS1 infection. L. paracasei HP7 reduced H. pylori adhesion to AGS cells and suppressed the inflammatory response in infected cells by downregulating interleukin-8. H. pylori colonization in the stomach of C57BL/6 mice was demonstrated by rapid urease test, and results showed significant decrease in mice post-treated with L. paracasei HP7. Additionally, L. paracasei HP7 decreased gastric inflammation and epithelial lesions in the stomach of H. pylori-infected mice. These results demonstrate that L. paracasei HP7 treatment can inhibit H. pylori growth and is thus a promising treatment for patients with gastric symptoms such as gastritis that are caused by H. pylori infection.
... acnes compounds are rich in the roots but not in the aerial parts of S. officinalis L. Among the extracts and their respective EA fractions, the SOR ME and ME-EA showed the lowest MIC and MBC values against P. acnes. Furthermore, the SOR ME and ME-EA showed antimicrobial activity against numerous Gram-negative and -positive bacteria, which corresponds to earlier works [31,37,38]. It suggested that the SOR ME displayed a broad spectrum of antibacterial activity, whereas both the CWE and HWE displayed a narrow spectrum of antibacterial activity. ...
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Identification of medicinal plants and naturally derived compounds as new natural antioxidant and antibacterial sources for topical acne treatment has long been important. To determine anti-Propionibacterium acnes activity and in vitro antioxidant activities, Sanguisorba officinalis L. root (SOR) was extracted with cold water (CWE), hot water (HWE), and methanol (ME), and each extract was fractionated successively with hexane, ethyl acetate (EA), and butanol to determine whether the activities could be attributed to the total phenolic, flavonoid, terpenoid, and condensed tannin contents. Pearson’s correlation coefficients were analyzed between the respective variables. The SOR CWE, HWE, ME, and their respective EA fractions showed anti-P. acnes activity based on the paper disc diffusion method on agar plates, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC). The MIC against P. acnes had a moderate (+) correlation with the total phenolic content, but not with the other measures. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity (SC) had a strong (–) correlation with the total phenolic content and a moderate (–) correlation with the total flavonoid content. The total antioxidant capacity had a strong (+) correlation with the condensed tannin content. Linoleic acid peroxidation inhibition had a strong (–) correlation with the total phenolic content. To elucidate the major active phytochemicals in the CWE-EA, HWE-EA, and ME-EA fractions, high performance liquid chromatography-ultraviolet (HPLC-UV) and ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) were performed. The HPLC-UV analysis showed the presence of nine compounds in common (arjunic acid and/or euscaphic acid, gallic acid, kaempferol, caffeic acid, ferulic acid, tannic acid, and coumarin, quercetin). The UHPLC-QTOF-MS analysis showed the presence of nine compounds in common (gallic acid; caffeic acid; umbelliferone; arjunic acid, euscaphic acid, and/or tormentic acid; pomolic acid; rosamultic acid; and benzoic acid). When standards of the identified phytochemicals were tested against the same bacterium, quercetin, coumarin, and euscaphic acid showed antibacterial activity against P. acnes.
... The precursor ion was [M + H] + at m/z 418.9 Da for VII, and the product ion peak at m/z 342. 8 Da was attributable to one molecule of CH3 (15 Da) and two molecules of OCH3 loss (62 Da). The mass parameters for six analytes and IS are summarized in Table 1. ...
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A selective and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of ziyuglycoside I (I), 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester (II), 3β-[(α-l-arabinopyranosyl) oxy]-urs-12,18(19)-dien-28-oic acid β-d-glucopyranosyl ester (III), rosamultin (IV), 1β-hydroxyeuscaphic acid (V) and alpinoside (VI) in rats after oral administration of Sanguisorba officinalis L. (S. officinalis) extract. The 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester, 3β-[(α-l-arabinopyranosyl) oxy]-urs-12,18(19)-dien-28-oic acid β-d-glucopyranosyl ester, rosamultin, 1β-hydroxyeuscaphic acid and alpinoside in rat plasma were the first report in the pharmacokinetics study in the present study. The analytes were quantified using the multiple reaction monitoring (MRM) mode with the electrospray ion source in positive electrospray ionization. Plasma was extracted with ethyl acetate via liquid–liquid extraction. Bifendate was used as internal standard (IS). The current method was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery, matrix effect and stability. The lower limits of quantification of ziyuglycoside I, 3β,19α-dihydroxyurs-12-en-28-oic-acid 28-β-d-glucopyranosyl ester, 3β-[(α-l-arabinopyranosyl) oxy]-urs-12,18(19)-dien-28-oic acid β-d-glucopyranosyl ester, rosamultin, 1β-hydroxyeuscaphic acid and alpinoside were 6.1, 4.9, 1.3, 3.8, 1.5 and 5.7 ng/mL, respectively. Intra-day and inter-day precision and the accuracy of the assay were in range from −9.48 to 12.74%. The extraction recoveries of analytes and bifendate (IS) from rat plasma ranged from 77.17% to 92.48%. Six compounds could be rapidly absorbed into blood (Tmax, 0.58–1.58 h), and then eliminated relatively slowly (t1/2, 6.86–11.63 h). The pharmacokinetic results might contribute to further guide the clinical application of S. officinalis.
... H pylori infection is the main cause of chronic gastritis, which is the most frequent chronic bacterial disease [1,27]. H. pylori induces an inflammatory immune response in the gastric mucosa, and many studies have demonstrated a significant correlation between H. pylori density and acute and chronic inflammatory grade. ...
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Among several diagnostic tests, a Helicobacter pylori stool antigen (HpSA) test may offer a useful noninvasive method for diagnosing infection without sacrificing animals. In this study, male C57BL/6 mice (n=6) were infected with H. pylori ATCC 49503 (1×10(8) CFU/mouse) by intragastric inoculation three times at 2-day intervals, and H. pylori infected stool specimens were collected 1, 3, 5, 7, 14, 21 days after infection to assess reliability of the HpSA test. Five of six specimens were positive at 5-21 days after infection, and the sensitivity of the HpSA test was 83.33%. The presence of H. pylori infection was confirmed by the rapid urease test and genomic DNA polymerase chain reaction (PCR), and showed the same results as the HpSA. However, the rapid urease test and genomic DNA PCR are invasive tests and require animal sacrifice to detect H. pylori in gastric biopsy samples. We suggest that an HpSA test kit would be useful and effective for monitoring H. pylori in various laboratory animals, as H. pylori can be easily monitored without sacrificing animals.
Article
A selective, accurate and efficient LC-MS/MS method was developed for the simultaneous determination of 13 phenolic acids. Additionally, for more comprehensively determining the chemical constituents in Sanguisorba officinalis L. extract, a previously developed method was employed for the simultaneous determination of six triterpenes. Thus, two methods were used to ensure the comprehensiveness and reliability of this study. Based on these methods, the pharmacokinetic profiles of the 13 phenolic acids and 6 triterpenes in normal and leukopenia rats after oral administration of Sanguisorba officinalis L. extract were compared for the first time in present study. Quantitative detection of the 13 phenolic acids and 6 triterpenes was performed using the multiple reaction monitoring mode with the electrospray ion source in negative and positive electrospray ionization, respectively. Chromatographic separation was performed on an Agilent Eclipse Plus C18 RRHD column (50 × 2.1 mm, 1.8μm), using gradient elution with a mobile phase composed of methanol-0.1% aqueous formic acid. The pharmacokinetic results demonstrated that the pharmacokinetic characteristics of the 19 analytes in leukopenia rats differed significantly from those determined in normal rats, which could provide a helpful reference for the clinical application of Sanguisorba officinalis L. in the prevention and treatment of leucopenia. This article is protected by copyright. All rights reserved.
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Sanguisorba officinalis Linne (S. officinalis, Rosaceae) has been used as a medicinal plant for the treatment of burns, hematemesis, melena, intestinal infections, and dermatitis for a long time in China, Korea, and Japan. The therapeutic efficacy of this herb is intimately associated with its anti-oxidant, anti-inflammatory, antiviral, antifungal, hemostatic, and anticancer activities. Its root contains triterpenoid saponins (zigyuglycoside I: C[Formula: see text]H[Formula: see text]O[Formula: see text] and ziyuglycoside II: C[Formula: see text]H[Formula: see text]O8) and tannins (sanguiin H-6: C[Formula: see text]H[Formula: see text]O[Formula: see text]). It has been recently revealed that these active constituents of S. officinalis possess antiwrinkle properties without cytotoxicity. They also have anticancer effects by inducing apoptosis and cell cycle arrest. Moreover, they can inhibit proliferative tumorigenesis. The underlying mechanism involved in the pharmacological actions of these active constituents is mainly related to p38 MAPK signaling. Although various studies have reported its therapeutic activities and major chemical components, review articles that extensively organize various properties of S. officinalis and its major constituents are still scarce. Taken together, the objective of this paper is to provide overall pharmacological and phytochemical profiles of S. officinalis and its constituents (including ziyuglycoside I, ziyuglycoside II, and sanguiin H-6), and their potential roles in clinical applications for the treatment of inflammatory diseases, bleeding disorders, and cancer.
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Helicobacter pylori (H. pylori) causes chronic gastritis, peptic ulcer disease, primary B-cell gastric lymphoma, and adenocarcinoma of the stomach. The eradication of H. pylori infection requires combination of antibiotics including proton pump inhibitors. However, development of antibiotic resistance is a major cause of treatment failure. To select an appropriate regimen, systemic information on the antibiotic resistance is mandatory. H. pylori acquires resistance essentially via point mutations, and this phenomenon is found with most antibacterials. The prevalence of primary antibiotic resistance in H. pylori strains isolated from Korean patients has been increasing along with the shift to high minimum inhibitory concentrations from 1987 to 2009. Moreover, MIC values of secondary isolates were higher than those of primary isolates. In addition, there is an increasing tendency for the emergence of strains with multi-drug resistance. Resistance rates of H. pylori to amoxicillin, clarithromycin, metronidazole, tetracycline, ciprofloxacin, levofloxacin, and moxifloxacin have been reported up to 18.5%, 38.5%, 66.2%, 34.6%, 34.6%, 29.5%, and 23.2%, respectively. Especially, antibiotic resistance to metronidazole or clarithromycin affects undermining the efficacy of eradication treatment. Further nation-wide surveillance regarding the effect of antibiotic resistance on the eradication rate is necessary to establish the appropriate treatment for H. pylori infection.
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Orthosiphon stamineus Benth., which is used as a gastroprotective herbal remedy in Malaysia, was assessed for its anti-ulcerogenic activity against ethanol-induced ulcers in rats. Fifty percent methanol was used to extract the oven-dried O. stamineus leaves. The extract was then lyophilized with a rotary evaporator and freeze-dried. Oral administration of O. stamineus methanolic extract (OSME) (125, 250, 500, and 1,000 mg/kg) was found to significantly decrease the ulcer index (P < .01, P < .001, P < .001, and P < .001, respectively). Histological study of a section of the rat stomach also showed a marked improvement in the gastric mucosal damage in groups receiving OSME. In order to further investigate the gastroprotective mechanism of OSME, mucus secretion and lipid peroxidation level were estimated in vitro and ex vivo. OSME exhibited dose-dependent stimulation of mucus secretion (r = 0.718, P < .001) and inhibition of lipid peroxidation in rat gastric mucosal homogenates (both in vitro [r = 0.819, P < .05] and ex vivo [r = 0.981, P < .05]). It was concluded that the gastroprotective mechanism of OSME was partly due to its ability to inhibit lipid peroxidation and stimulate gastric mucus secretion.
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An actinomycetes strain which produced chitinase, urease, and antifungal substances to Monilinia fructicola was isolated from peaches mummified by Monilinia fructicola. The strain TH-04 was identified as Streptomyces sp. based on cultural and morphological characteristics, cell wall diaminopimelic acid, and sugar patterns of whole-cell extracts. Streptomyces sp. TH-04 showed antifungal activity to several fungi including Monilinia fructicola, Colletotrichum gloeosporioides. Magnaporthe grisea. Rhizoctonia solani, Phytophthora capsici. Alternaria kikuchiana, Fusarium solani, and Fusarium oxysporum. The optimum cultural conditions for the production of antifungal substances were 20°C, pH 7. and 7 days.
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Recently, the development of antibiotic resistance emerged as a significant clinical problem in the eradication of Helicobacter pylori. We investigated the MICs of antibiotics for 135 H. pylori isolates from adults in Seoul, South Korea, over the past 16 years. The MICs of amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, and ciprofloxacin increased from 1987 to 2003. Rates of primary resistance to clarithromycin increased from 2.8% in 1994 to 13.8% in 2003. The A2144G mutation was frequently observed in the 23S rRNA gene in clarithromycin-resistant isolates. The increase in resistance to clarithromycin seems to result in a decrease in eradication efficacy for H. pylori. These results suggest that the MICs of several antibiotics for H. pylori have increased over the past 16 years in Seoul.
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From the leaves of Bencomia caudata and Dendriopoterium menendezii a new triterpenoid glycoside has been isolated. Its structure was determined as 3-O-galloyl-23-hydroxytormentic acid 28-O-β-d-glucopyranoside mainly from the NMR spectra. In addition, several known triterpenoids have been isolated from six other species of the Sanguisorbeae. A comparative study of the triterpene occurrence pattern has been performed.
Article
Sanguisorbae radix (SR), the root of Sanguisorba officinalis L. (Rosaceae), has been traditionally used for its anti-inflammatory, anti-infectious and analgesic activities in Korea. Previous work has shown that SR prevents neuronal cell damage induced by Abeta (25--35) in cultured rat cortical neurons. The present study was carried out to further investigate the neuroprotective effect of SR on oxidative stress-induced toxicity in primary culture of rat cortical neurons, and on ischemia-induced brain damage in rats. SR, over a concentration range of 10--50 microg/ml, inhibited H2O2 (100 microM)-induced neuronal death, which was significantly inhibited by MK-801 (5 microM), an N-methyl-D-aspartate (NMDA) receptor antagonist, and verapamil (20 microM), an L-type Ca2+ channel blocker. Pretreatment of SR (10-50 microg/ml), MK-801 (5 microM), and verapamil (20 microM) inhibited H2O2-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) measured by a fluorescent dye, Fluo-4 AM. SR (10-50 microg/ml) inhibited H2O2-induced glutamate release into medium measured by HPLC, and generation of reactive oxygen species (ROS) measured by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In vivo, SR prevented cerebral ischemic injury induced by 2-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. The ischemic infarct and edema were significantly reduced in rats that received SR (10, 30 mg/kg, orally), with a corresponding improvement in neurological function. Catechin isolated from SR inhibited H2O2-induced neuronal death in cultures. Taken together, these results suggest that SR inhibits H2O2-induced neuronal death by interfering with the increase of [Ca2+]i, and inhibiting glutamate release and generation of ROS, and that the neuroprotective effect of SR against focal cerebral ischemic injury is due to its anti-oxidative effects. Thus SR might have therapeutic roles in neurodegenerative diseases such as stroke.