Research ArticleGastroprotective Activity of the Total Flavones fromAbelmoschusmanihot (L) Medic Flowers

Jun Zhang 12 Zai-Lin Fu 3 Zhao-Xing Chu4 and Bi-Wei Song2

1Department of Pharmacy e Second Affiliated Hospital of Zhejiang Chinese Medicine University HangzhouZhejiang 310005 China2Institute of Pharmacology Zhejiang University of Technology Hangzhou Zhejiang 310014 China3Department of Pharmacy First Peoplersquos Hospital of Yuhang District Hangzhou Zhejiang 311100 China4China Pharmaceutical University Nanjing Jiangsu 210009 China

Correspondence should be addressed to Zai-Lin Fu fuzailin163com

Received 4 July 2019 Accepted 4 January 2020 Published 25 February 2020

Academic Editor George B Lenon

Copyright copy 2020 Jun Zhang et al -is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Abelmoschus manihot (L) Medic flower is a medicinal plant for the treatment of diseases in China-e present studywas carried out to scientifically validate the gastroprotective activity and clarify the possible mechanism of the total flavones fromAbelmoschus manihot (L) Medic flowers (TFA)Methods Gastric ulcer was induced inmice by oral administration of ethanol-egastroprotective activity of TFA was evaluated by the gastric ulcer index and histological examinations -e gastric tissue wascollected in the form of homogenate -e level of malondialdehyde (MDA) and glutathione (GSH) the activity of superoxidedismutase (SOD) and protein content were measured Western blotting for the expression of Bax Bcl-2 TNF-α and NF-κB(p65)was also carried out -e effect of TFA was compared with that of standard antiulcer drug omeprazole (100mgkg) Results -isgastroprotective effect of TFA could be attributed to the increase in the activity of SOD andGSH and decrease in the levels ofMDAand also decrease in the levels of Bax TNF-α and NF-κB(p65) expressions and increase in the Bcl-2 expression level Conclusion-e findings of this study demonstrated that TFA could significantly attenuate ethanol-induced gastric injury via antioxidativeanti-inflammatory and antiapoptotic effects

1 Background

Gastric ulcer is a common digestive disease with an in-creasing incidence and prevalence attributed to the loss ofbalance between aggressive and protective factors [1] -ereis about 10 of the worldrsquos population suffering from thegastric ulcer [2] -e most common presenting symptom ofpatients with the gastric ulcer is often characterized withburning or gnawing pain in the center of the abdomen [3]

-e etiology of the gastric ulcer is multifactorial includingalcohol abuse stress smoking nonsteroidal anti-inflamma-tory drugs overuse and Helicobacter pylori infection [4 5]Excessive alcohol consumption is a major reported causesrcause for gastric ulcer [6] Alcohol as an ulcerogenic sub-stance can perturb the gastric mucosa and induce metabolicdisorders leading to inflammation hyperemia edema

hemorrhage and erosive lesions [7] Alcohol also markedlyincreases the accumulation of free radical oxygen and lipidperoxidation and synchronously suppresses the activity ofantioxidant enzymes such as superoxide dismutase andglutathione peroxidase in the gastric tissues which promotesthe necrosis and apoptosis of gastric epithelial cells [8] -usalcohol is commonly used to induce experimental gastriculcer models in animals [9] Most of the existing antiulcerdrugs mainly work via the manner of acid suppression andindirectly promote the self-healing of gastric mucosa butshow limited efficacy [10] Lots of clinical and experimentalstudies have demonstrated that utilization of herbal medicinescould be a valuable alternative to cure the gastric ulcer withfew adverse effects [11]

Abelmoschus manihot (L) Medic is a medicinal plantwhich belongs to the family of Malvaceae It is widely

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2020 Article ID 6584945 9 pageshttpsdoiorg10115520206584945

distributed in Papua New Guinea New Caledonia andChina [12] As a folk medicine in Eastern Europe and Asiaits flower has been used for a variety of purposes forhundreds of years [13] Pharmacological researches havedemonstrated that the extractive of its flowers has a widerange of pharmacological activities including analgesicanti-inflammory antioxidant hepatoprotective renal pro-tection and cardiovascular protection [14] -e extensivephytochemical and pharmacological studies have indicatedthat total flavonoids extracted from flowers of Abelmoschusmanihot (L) Medic (TFA) were the major bioactive com-pounds in the extractive [15 16] However no systematicstudy has been carried out on TFA to verify for the gas-troprotective activity yet -us this work was undertaken toinvestigate and validate the protection effect of TFA ongastric ulcer in mice and its possible mechanism of action

2 Materials and Methods

21 Drugs and Reagents Omeprazole was purchased fromAstraZeneca Pharmaceutical Co Ltd (London UK) GSHMDA and SOD kits were provided by Jiancheng Bioengi-neering Institute (Nanjing China) GAPDH antibody Bcl-2Bax NF-κB p65 and HRP-conjugated secondary antibodywere purchased from Boster (Wuhan China) TNF-α anti-body was provided by Proteintech Group Inc (ChicagoUSA) Hyperoside standard was provided by Anhui Instituteof Medical Science All other chemicals were of analytical-reagent grade with the highest quality commercially available

22 Plant Material and TFA Preparation -e dried flowersofAbelmoschus manihot (L) Medic were purchased from thewholesale market of Chinese Herbals in Bozhou AnhuiProvince -e flowers were identified by Prof Song Biwei(College of Pharmaceutical Sciences Zhejiang University ofTechnology) A voucher specimen was deposited in theHerbarium of the Department of Pharmacology of theCollege of Pharmaceutical Sciences Zhejiang University ofTechnology

-e TFA was extracted from the flowers of AbelmoschusManihot (L) Medic by the Department of PharamcologyZhejiang University of Technology Zhejiang China -edried flowers were shredded in an electric mill PowderedAbelmoschus Manihot (L) Medic flowers were immersed in70 ethanol (vv) -e mixture was extracted 3 times underreflux for 05 h -en the decoction was filtered through theanalytical filter paper and evaporated by rotary evaporationunder vacuum at 40degC -e ldquodrug extractrdquo ratio of Abel-moschus manihot ethanolic extract was 1 1 (1 gmL) -emain active components of TFA were hyperoside iso-quercitrin hibifolin quercetin-30-O-glycoside quercetinmyricetin and rutin [13]

23 Total Flavonoids Content Determination Total flavo-noids content of the decoction was determined by the AceticAcid-Sodium Acetate-Aluminum Chloride method withsome modifications [17] Briefly the decoction was dilutedto 25mL using 70 ethanol 1ml of the diluted ethanolic

extract was transferred to a 25mL volumetric flask 5mL ofAcetic Acid-Sodium Acetate buffer (2M Acetic Acid 2MSodium Acetate buffer 3 1) was added to the mixture fol-lowed by adding 3mL of 01M Aluminum Chloride solution-e final volume was adjusted to 25ml by ultrapure water-e mixture was incubated at room temperature for 40minand the absorbances of the mixture were measured in 401 nm-e standard hyperoside curve was used to calculate totalflavonoid content -e analysis was performed in triplicate

24 Hyperoside Content Determination by HPLC Samplesfor HPLC were prepared from the decoction 05mL of theTFA was transferred to a 25mL volumetric flask -e solu-tions were diluted to 25mL by HPLC grade methanol -eywere filtered through a 02 μm syringe filter Referencestandard hyperoside was prepared by weighing 01 mgmlof hyperoside A Hitach L2000 series HPLC system(Hitach Tokyo Japan) was used to analyze hyperoside inthis experiment -e analytical column used was a250 mm times 46 mm id HITACHI LaChrom C18 (5 μm)maintained at 30degC -e flow rate was kept at 1 mlminand the injection volume was 10 μL Two solvents A(acetonitrile) and B (01 phosphoric acid) were used forelution of constituents -e elution of the mobile phasecan be described as follows (A)(B) 1090 (0 min)⟶2080 (20sim30min)⟶ 2575 (30sim40min)⟶ 3070 (40sim45min)⟶ 1585 (45sim50min)⟶ 1090 (50sim60min)-e chromatogram peaks were detected at 360 nm [18]

25 Experimental Animals -e experiments were carriedout on healthy ICR mice (18ndash22 g 6ndash8 weeks) of either sex-ey were obtained from Zhejiang Academy of MedicalSciences (License no SCXK 2014-0001) All mice were keptin standardized animal house at 25plusmn 2degC light and darkcycles of 1212 hours respectively A balanced diet and freeaccess of water were provided -ey were starved for24 hours before use though water was allowed ad libitum-e mice were cared for in accordance with the ldquoPrinciplesof laboratory animal carerdquo (NIH publication no 82ndash23revised 1996) guidelines -e experiments were designedaccording to the Institutional Animal Ethical Committee

26 Ethanol-Induced Gastric Ulcer Model -e experimentwas carried out according to the method of Hollander et alwith a few modifications [19] -e mice were randomlydivided into 6 groups of 10 mice each control group modelgroup omeprazole group (100mgkg) and TFA group (300600 and 1200mgkg) -ey were all gavaged with 04mLabsolute alcohol except the control group-e same volumesof sterile water were gavaged to the mice of the controlgroup 1 hour later sterile water omeprazole or TFA wasadministrated to each group After 4 hours they were sac-rificed by cervical dislocation

27 Determination of Ulcer Index and Percentage of Inhibition-e isolated stomachs were immediately fixed with 4paraformaldehyde for 20 minutes -en they were opened

2 Evidence-Based Complementary and Alternative Medicine

along the greater curvature and washed with saline solution(09 NaCl) -e inner surface of the stomachs was ex-amined for ulceration with the help of a dissecting micro-scope -e ulcer index (UI) was calculated as described bythe method of Guth [20] -e percentage of inhibition wascalculated by the following formula [21]

(UIcontrol minus UItreated)

UIcontrol1113890 1113891 times 100 (1)

28 Biochemical Estimation -e tissues from stomachswere cut into pieces -e weight of each one was thenrecorded -e samples were homogenized in 9 volumes ofcold saline solution -e homogenates were centrifuged at10000 g for 20min at 4degC -e supernatants were used todetermine the biochemical parameters -e concentrationof total protein in the supernatants was measured by theBCA protein assay kit (Boster Wuhan Hubei China)Levels of GSH MDA and SOD were determined by thecommercial assay kits according to the manufacturerrsquosinstructions (Jiancheng Bioengineering Institute NanjingChina) for users

29 Histopathological Evaluation One part of the stomachswas fixed in 4 paraformaldehyde solution for 24 hours-en they were embedded in paraffin wax -e 5 μmthickness tissue sections were repaired and stained withhematoxylin and eosin (HE) -e slides were examinedunder light microscope to observe the morphologicalchanges and recorded

210 Western Blotting Gastric tissues were cut into piecesand homogenized with ice-cold cell lysis buffer for westernblotting and an IP kit (Beyotime shanghai China) for30min at 4degC -e homogenates were centrifuged at 14000 gfor 10min at 4degC -en the supernatants were collected andthe concentrations of total protein were detected by a BCAprotein assay kit (Boster Wuhan Hubei China) Equivalentamounts of proteins were separated by electrophoresis on 12and 5 sodium dodecylsulfate (SDS) polyacrylamide gels(SDS-PAGE) and transferred to nitrocellulose membranes(Beyotime shanghai China) -e membranes were blockedwith 5 BSA at room temperature for 2hours -e blots wereincubated overnight at 4degC with primary antibodies againstTNF-α (1 1000 Proteintech Group Inc Chicago USA) BaxBcl-2 GAPDH and NF-κB p65 (1 200 Boster WuhanChina) After washing themembranes were incubated with theappropriate HRP-conjugated secondary antibodies (BosterWuhan Hubei China) for 2 hours at room temperaturePhotodensity analysis was utilized with the chemiluminescencedetection system (Bio-Rad Hercules CA USA) after visual-izing by HRP substrate ECL solution (Boster Wuhan China)

211 Statistic Analysis -e results of the experiments werepresented as meanplusmn SD and one-way analysis of variance(ANOVA) was used to analyze comparison in different

groups plt 005 was considered to be significant Dataanalysis was achieved using the software progam GraphPadPrism 5

3 Results

31 Total Flavonoids Content After making a standardcalibration curve by hyperoside (y 00246x minus 00023r2 09999) the total flavonoids content of the TFA wasfound to be (468plusmn 007) (ww) respectively

32 Hyperoside Content -e standard calibration curve ofhyperoside was used to evaluate the content of flavonoid inthe extract -e hyperoside content of the TFA was(114plusmn 027) (ww) respectively (Figure 1)

33 Effect of TFA on the Ulcer Index and Percentage ofInhibition Ethanol is regarded as one of the major riskfactors for the pathogenesis of gastric ulcer A representativestomach of each group is shown in Figure 2 -e modelgroup presented severe mucosal injury (ulcer index4110 + 1828) TFA could significantly increase the inhibi-tion rate of ethanol-induced gastric ulcer at the dose of 600and 1200mgkg (Table 1) Compared with the positivecontrol group TFA showed better results than omeprazole-e effect of TFA was in a dose-dependent manner of whichthe high-dose group reached the maximum 6691 inhi-bition rate (plt 0001)

34 Effect of TFA on Biochemical Parameters All the resultsare shown in Figures 3(a)ndash3(c)) Ethanol caused a markedsignificant increase in the levels of MDA (plt 0001) andreduction in the activities of SOD (plt 0001) and GSH levels(plt 0001) in ulcerated mice as compared to the controlgroup Contrarily TFA (600mgkg) and TFA (1200mgkg)treatments dramatically decreased MDA levels in gastrictissue with a concomitant increase in the activities of SODand GSH levels as compared with the model group espe-cially the high-dose group

35 Histologic Evaluations of Gastric Tissue -e gastrictissue specimens of different groups were observed under amicroscope and the pathological changes are shown inFigures 4 and 5 In the control group the structure of thegastric tissue was intact and the epithelial cells were tightlyarranged (Figures 4(a) 5(a)) However there were varioushistopathological alterations including congestion cell-contained hyperaemia and edema gastric epithelial cellsnecrosis inflammatory changes and erosions in the gastrictissue of model group mice (Figures 4(b) 5(b)) Comparedwith the model group this poor sanitation was restrained byTFA or Omeprazole and the structure was tended to benormal -e results suggested that TFA could reduce gastricmucosa injury induced by ethanol

Evidence-Based Complementary and Alternative Medicine 3

36 e Expression of Proteins in the Gastric Tissue

361 Effect of TFA on the Expression of Bax and Bcl-2-e results of Bax and Bcl-2 protein expression were shownin Figures 6 and 7 -e expression of the proapoptotic factorBax in the model group was significantly higher than thecontrol group (plt 0001) 187 folds higher than the controlgroup and the expression of the antiapoptotic factor Bcl-2was significantly lower (plt 0001) 62 of the control group-e ratio of BaxBcl-2 in the each treatment group wassignificantly lower than that in themodel group especially inthe TFA high-dose group -is results demonstrated that

TFA can downregulate the expression of Bax upregulate theexpression of Bcl-2 and thus decrease the ratio of BaxBcl-2to inhibit the activation of the endogenous apoptoticpathway

362 Effect of TFA on the Expression of TNF-α NF-κB p65-e results of the expression of TNF-α and NF-κB p65 ingastric tissue are shown in Figures 6 and 8 -e expressionof TNF-α and NF-κB p65 in the TFA group was signifi-cantly lower than that in the model group especially in thehigh-dose group (plt 0001) -e results suggested that

0 5 10 15 20 25 30 35 40 45 50 55 60Retention time (min)

000

005

010

015

020

025

030

307

316

391 218

9

266

3

298

831

39

358

3

444

9

Abs

orba

nce (

AU

)

OH

OH

OH

O

OO

O

OHHO

HO

OH

HO

Hyperoside

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60Retention time (min)

000

002

004

006

008

010

248

265

303

327

353

385

449

479

509 539

563

584 6

507

01 738

776 817 867

911 9

4110

12

105

910

89

112

111

81

124

412

77

132

813

79

142

614

76

151

715

63

161

916

49 17

03

180

718

49

189

919

86

207

3 211

121

75 222

322

57

236

424

31

251

425

47

265

027

14

282

0

300

131

27

321

032

45

331

734

06 352

335

69

381

539

29

421

442

83 44

91

Abs

orba

nce (

AU

)

TFA

(b)

Figure 1 HPLC profile of hyperoside and TFA

4 Evidence-Based Complementary and Alternative Medicine

TFA could significantly downregulate the expression ofinflammation-related proteins in the gastric tissue ofgastric ulcer mice

4 Discussion

In the present study the model of ethanol-induced gastriculcer was used to evaluate the gastroprotective activity ofTFA Ethanol is widely used in experimental gastric ulcermodels because of its disadvantages-emodel is simple andrepeatable -e morphology histological features healingand recurrence processes of ulcer are similar to human [22]-e severity of gastric injury was determined by ulcer indexand we found that TFA could inhibit the occurrence ofulcers and reduce the area of ulceration It was shown by HEstaining that TFA could relieve the pathological changes ofgastric ulcer tissue in mice

Oxidative stress is an important etiological factor in thedamaged gastric mucosa [3] GSH is a major antioxidant byinteraction with lipid hydroperoxides -e ulcerogenic ac-tivity is related to the tissue levels of GSH Increase in the

levels of GSH can inhibit gastric ulceration [23] MDA is theend-product of lipid peroxidation and it is also an im-portant marker of oxidative damage -e increase in MDAcontent is associated with the necrosis and apoptosis of cells[24] SOD can exert its effects by removing oxygen freeradicals from the body [25] -erefore GSH MDA andSOD were measured in this study to evaluate oxidative stresslevels -rough the detection of the abovementioned oxi-dative damage indicators we found TFA can increase theactivity of SOD the concentration of GSH and at the sametime decrease the level of MDA in gastric tissue

Bcl-2 family proteins is a family of evolutionarily relatedproteins which derives its name from B-cell lymphoma 2[26] -ere are a total of 25 genes in the Bcl-2 family knownto date Bcl-2 family proteins are pivotal regulators of ap-optosis by controlling mitochondrial outer membranepermeabilization (MOMP) [27] When the cell is stimulatedby apoptotic signals the structure of Bax changes shiftingfrom the cytoplasm to the mitochondrial membrane andrecruiting an amount of Bax proteins to form a polymer-en the polymer is inserted into the outer mitochondrialmembrane causing formation of mitochondrial apoptosis-induced channel (MAC) CytC AIF and other proapoptoticproteins release into the cytoplasm via MAC resulting inapoptotic cell death [27 28] A heterodimer may be formedby the proapoptotic protein and the antiapoptotic proteinand the ratio of BaxBcl-2 in the dimer is directly related tothe regulation of apoptosis [29] TFA can significantlyupregulate the expression of antiapoptosis protien Bcl-2 anddownregulate the expression of proapoptosis factor Bax-us the apoptosis of cell was prevented through down-regulation of BaxBcl-2 ratio

Increasing evidence suggests that inflammatory media-tors may play a role in the development and progression ofgastric ulcer A lot of literature had reported that TNF-α the

(a) (b) (c) (d)

(e) (f )

Figure 2 -e gastric mucosal injury in mice (n 10) (a) Control group (b) model group (c) omeprazole group (d) high-dose group(e) middle-dose group (f ) low-dose group

Table 1 Ulcer index and inhibition rate of ulceration (Meanplusmn SDn 10)

Groups Dose(mgkg) Ulcer index Inhibition

ration ()Control mdash mdash mdashModel mdash 4110 + 1828 mdashOmeprazole 100 2830 + 583 3114High-dose 1200 1340 + 860lowastlowastlowast 6691Midium-dose 600 2410 + 1210lowast 4209Low-dose 300 4030 + 1784 195Note lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Evidence-Based Complementary and Alternative Medicine 5

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

20

15

10

5

0

MD

A (n

mol

mgp

rot)

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

60

45

20

0

SOD

(Um

gpro

t)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

1500

1000

500

0

GSH

(μm

olg

prot

)

lowastlowastlowast

lowast

(c)

Figure 3 Effect of TFA on biochemical parameters of gastric tissue inmice with GU (a)MDA levels of mice gastric tissue (b) SOD activitiesof mice gastric tissue and (c) GSH levels of mice gastric tissue (Meanplusmn SD n 6) plt 0001 vs control lowastplt 005 lowastlowastplt 001lowastlowastlowastplt 0001 vs model

(a) (b) (c) (d)

(e) (f )

Figure 4 Histologic evaluations of gastric tissue (HE staining times100) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c) (d)

(e) (f )

Figure 5 Histologic evaluations of gastric tissue (HE staining times200) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

TFA (mgkg)Control Model Ome 300 600 1200

TNF-α (17kd)

Bax (17kd)

Bcl-2 (26kd)

NF-κB p65 (65kd)

GAPHD (37kd)

Figure 6 Expression of the proteins with western blot

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of Bax

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

200

150

100

50

0

o

f con

trol

Expression of Bcl-2

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 7

main proinflammatory cytokine was the important con-tributing factor in intestinal mucosal injury [9 30] TNF-α isthe initiator of the exogenous death pathway It can combinewith autologous proximate or distant cell membrane deathreceptor (TNFR) -e initiation of the death receptorpathway leads to apoptosis (activated caspase-8) pro-grammed necrosis (via RIP1 and RIP3) or inflammation(via NF-κB) responses NF-κB a classic proinflammatorytranscription factor is kept inactive in resting cells bybinding with a member of the IKB α inhibitor protein family[31] NF-κB is an ideal target for the mediation of proin-flammatory factor expression in gastric ulcer [32] Manynatural products that have been promoted to have anti-inflammatory activity have also been shown to inhibit NF-κB In the current study western blot analysis indicated thesignificant downregulation of TNF-α and NF-κB p65

expressions in the gastric tissue which evidenced the anti-inflammatory effect of TFA

In summary the present study suggested that TFA couldsignificantly attenuate ethanol-induced gastric injury viaantioxidative anti-inflammatory and antiapoptotic effectsOur data reinforced the therapeutic potential of TFA in themanagement of gastric ulcer

Data Availability

-e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this article

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of TNF-α

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

150

100

50

0

o

f con

trol

Expression of NF-κB p65

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 8 Analysis of TNF-α and NF-κB p65 protein expression with Western blot (Meanplusmn SD) (a) expression of TNF-α (b) expression ofNF-κB p65 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

0

1

2

3

4

Bax

Bcl-2

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 7 Analysis of Bax and Bcl-2 protein expression with western blot (Meanplusmn SD) (a) Expression of Bax (b) expression of Bcl-2 and (c)BaxBcl-2 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

8 Evidence-Based Complementary and Alternative Medicine

References

[1] A S Tarnawski ldquoIncreased susceptibility of aging gastricmucosa to injury the mechanisms and clinical implicationsrdquoWorld Journal of Gastroenterology vol 20 no 16 p 4467 2014

[2] A M S Gomaa N A Abd El-Mottaleb and H A AamerldquoAntioxidant and anti-inflammatory activities of alpha lipoicacid protect against indomethacin-induced gastric ulcer in ratsrdquoBiomedicine amp Pharmacotherapy vol 101 pp 188ndash194 2018

[3] S Palle A Kanakalatha and C N Kavitha ldquoGastroprotectiveand antiulcer effects of Celastrus paniculatus seed oil againstseveral gastric ulcer models in ratsrdquo Journal of DietarySupplements vol 15 no 4 pp 373ndash385 2017

[4] Y Yang B Yin L Lv et al ldquoGastroprotective effect of aucubinagainst ethanol-induced gastric mucosal injury in micerdquo LifeSciences vol 189 pp 44ndash51 2017

[5] R H Hunt M Camilleri S E Crowe et al ldquo-e stomach inhealth and diseaserdquo Gut vol 64 no 10 pp 1650ndash1668 2015

[6] M Akanda I-S Kim D Ahn et al ldquoAnti-inflammatory andgastroprotective roles of rabdosia inflexa through down-regulation of pro-inflammatory cytokines and MAPKNF-κBsignaling pathwaysrdquo International Journal of Molecular Sci-ences vol 19 no 2 p 584 2018

[7] J Yoo J Lee Y Lee et al ldquoProtective effect of bovine milkagainst HCl and ethanolndashinduced gastric ulcer in micerdquoJournal of Dairy Science vol 101 no 5 pp 3758ndash3770 2018

[8] H Bernstein C Bernstein C Payne K Dvorakova andH Garewal ldquoBile acids as carcinogens in human gastroin-testinal cancersrdquo Mutation ResearchReviews in MutationResearch vol 589 no 1 pp 47ndash65 2005

[9] W Li X Wang W Zhi et al ldquo-e gastroprotective effect ofnobiletin against ethanol-induced acute gastric lesions inmice impact on oxidative stress and inflammationrdquo Immu-nopharmacology and Immunotoxicology vol 39 no 6pp 354ndash363 2017

[10] E Mohamed I Abu Hashim R Yusif et al ldquoPolymericmicelles for potentiated antiulcer and anticancer activities ofnaringinrdquo International Journal of Nanomedicine vol 13pp 1009ndash1027 2018

[11] W-P Bi H Man and M Man ldquoEfficacy and safety of herbalmedicines in treating gastric ulcer a reviewrdquoWorld Journal ofGastroenterology vol 20 no 45 pp 17020ndash17028 2014

[12] X-X Pan J-H Tao S Jiang Y Zhu D-W Qian andJ-A Duan ldquoCharacterization and immunomodulatory ac-tivity of polysaccharides from the stems and leaves of Abel-moschus manihot and a sulfated derivativerdquo InternationalJournal of Biological Macromolecules vol 107 pp 9ndash16 2018

[13] G Ai Q Liu W Hua Z Huang and D Wang ldquoHep-atoprotective evaluation of the total flavonoids extracted fromflowers of Abelmoschus manihot (L) Medic in vitro and invivo studiesrdquo Journal of Ethnopharmacology vol 146 no 3pp 794ndash802 2013

[14] D Lv X Cheng L Tang and M Jiang ldquo-e cardioprotectiveeffect of total flavonoids on myocardial ischemiareperfusion inratsrdquoBiomedicineampPharmacotherapy vol 88 pp 277ndash284 2017

[15] C Xue S Jiang J Guo D Qian J-a Duan and E ShangldquoScreening for in vitro metabolites of Abelmoschus manihotextract in intestinal bacteria by ultra-performance liquidchromatographyquadrupole time-of-flight mass spectrome-tryrdquo Journal of Chromatography B vol 879 no 32pp 3901ndash3908 2011

[16] C Xue J Guo D Qian et al ldquoIdentification of the potentialactive components of Abelmoschus manihot in rat blood andkidney tissue by microdialysis combined with ultra-

performance liquid chromatographyquadrupole time-of-flight mass spectrometryrdquo Journal of Chromatography Bvol 879 no 5-6 pp 317ndash325 2011

[17] J Li J Gong L Zhao H Zou and Y Yan ldquoExtraction process oftotal flavonoids of Abelmoschus manihotrdquo Journal of Interna-tional Pharmaceutical Research vol 43 no 2 pp 370ndash373 2016

[18] X-x Lv and Z-l Chen ldquoImprovement of determinationmethod of flower of sunset Abelmoschus in Chinese phar-macopoeia (2010 edition)rdquo Strait Pharmaceutical vol 27no 10 pp 67ndash69 2015

[19] D Hollander A Tarnawski W J Krause and H GergelyldquoProtective effect of sucralfate against alcohol-induced gastricmucosal injury in the ratrdquo Gastroenterology vol 88 no 1pp 366ndash374 1985

[20] P H Guth D Aures and G Paulsen ldquoTopical aspirin plusHCl gastric lesions in the ratrdquo Gastroenterology vol 76 no 1pp 88ndash93 1979

[21] I Szelenyi and K -iemer ldquoDistention ulcer as a model fortesting of drugs for ulcerogenic side effectsrdquo Archives ofToxicology vol 41 no 1 pp 99ndash105 1978

[22] W XIAO A XU and J Hui ldquoAdvances in models of gastriculcerationrdquo Pharmaceutical and Clinical Research vol 24no 2 pp 145ndash150 2016

[23] A P Jayaraj K R Rees F I Tovey et al ldquoAmolecular basis ofpeptic ulceration due to dietrdquo British Journal of ExperimentalPathology vol 67 no 1 pp 149ndash155 1986

[24] M Xie H Chen S Nie W Tong J Yin and M XieldquoGastroprotective effect of gamma-aminobutyric acid againstethanol-induced gastric mucosal injuryrdquo Chemico-BiologicalInteractions vol 272 pp 125ndash134 2017

[25] J-W Song C-S Seo T-I Kim et al ldquoProtective effects ofmanassantin a against ethanol-induced gastric injury in ratsrdquoBiological amp Pharmaceutical Bulletin vol 39 no 2 pp 221ndash229 2016

[26] A R D Delbridge S Grabow A Strasser and D L Vauxldquo-irty years of BCL-2 translating cell death discoveries intonovel cancer therapiesrdquo Nature Reviews Cancer vol 16 no 2pp 99ndash109 2016

[27] V Andreu-Fernandez M Sancho A Genoves et al ldquoBaxtransmembrane domain interacts with prosurvival Bcl-2proteins in biological membranesrdquo Proceedings of the Na-tional Academy of Sciences vol 114 no 2 pp 310ndash315 2017

[28] A Ashkenazi W J Fairbrother J D Leverson andA J Souers ldquoFrom basic apoptosis discoveries to advancedselective Bcl-2 family inhibitorsrdquo Nature Reviews Drug Dis-covery vol 16 no 4 pp 273ndash284 2017

[29] S -angarajan A Vedagiri S SomasundaramR Sakthimanogaran and M Murugesan ldquoNeuroprotectiveeffect of morin on lead acetate- induced apoptosis by pre-venting cytochrome c translocation via regulation of BaxBcl-2 ratiordquo Neurotoxicology and Teratology vol 66 pp 35ndash452018

[30] R Kamel E M El Morsy and A S Awad ldquoImmunomod-ulatory effect of candesartan on indomethacin-induced gastriculcer in ratsrdquo Immunopharmacology and Immunotoxicologyvol 34 no 6 pp 956ndash961 2012

[31] B Sun Q Xia and Z Gao ldquoTotal flavones of choerospondiasaxillaris attenuate cardiac dysfunction and myocardial in-terstitial fibrosis by modulating NF-κB signaling pathwayrdquoCardiovascular Toxicology vol 15 no 3 pp 283ndash289 2015

[32] X Chang F Luo W Jiang et al ldquoProtective activity ofsalidroside against ethanol-induced gastric ulcer via theMAPKNF-κB pathway in vivo and in vitrordquo InternationalImmunopharmacology vol 28 no 1 pp 604ndash615 2015

Evidence-Based Complementary and Alternative Medicine 9

along the greater curvature and washed with saline solution(09 NaCl) -e inner surface of the stomachs was ex-amined for ulceration with the help of a dissecting micro-scope -e ulcer index (UI) was calculated as described bythe method of Guth [20] -e percentage of inhibition wascalculated by the following formula [21]

(UIcontrol minus UItreated)

UIcontrol1113890 1113891 times 100 (1)

28 Biochemical Estimation -e tissues from stomachswere cut into pieces -e weight of each one was thenrecorded -e samples were homogenized in 9 volumes ofcold saline solution -e homogenates were centrifuged at10000 g for 20min at 4degC -e supernatants were used todetermine the biochemical parameters -e concentrationof total protein in the supernatants was measured by theBCA protein assay kit (Boster Wuhan Hubei China)Levels of GSH MDA and SOD were determined by thecommercial assay kits according to the manufacturerrsquosinstructions (Jiancheng Bioengineering Institute NanjingChina) for users

29 Histopathological Evaluation One part of the stomachswas fixed in 4 paraformaldehyde solution for 24 hours-en they were embedded in paraffin wax -e 5 μmthickness tissue sections were repaired and stained withhematoxylin and eosin (HE) -e slides were examinedunder light microscope to observe the morphologicalchanges and recorded

210 Western Blotting Gastric tissues were cut into piecesand homogenized with ice-cold cell lysis buffer for westernblotting and an IP kit (Beyotime shanghai China) for30min at 4degC -e homogenates were centrifuged at 14000 gfor 10min at 4degC -en the supernatants were collected andthe concentrations of total protein were detected by a BCAprotein assay kit (Boster Wuhan Hubei China) Equivalentamounts of proteins were separated by electrophoresis on 12and 5 sodium dodecylsulfate (SDS) polyacrylamide gels(SDS-PAGE) and transferred to nitrocellulose membranes(Beyotime shanghai China) -e membranes were blockedwith 5 BSA at room temperature for 2hours -e blots wereincubated overnight at 4degC with primary antibodies againstTNF-α (1 1000 Proteintech Group Inc Chicago USA) BaxBcl-2 GAPDH and NF-κB p65 (1 200 Boster WuhanChina) After washing themembranes were incubated with theappropriate HRP-conjugated secondary antibodies (BosterWuhan Hubei China) for 2 hours at room temperaturePhotodensity analysis was utilized with the chemiluminescencedetection system (Bio-Rad Hercules CA USA) after visual-izing by HRP substrate ECL solution (Boster Wuhan China)

211 Statistic Analysis -e results of the experiments werepresented as meanplusmn SD and one-way analysis of variance(ANOVA) was used to analyze comparison in different

groups plt 005 was considered to be significant Dataanalysis was achieved using the software progam GraphPadPrism 5

3 Results

31 Total Flavonoids Content After making a standardcalibration curve by hyperoside (y 00246x minus 00023r2 09999) the total flavonoids content of the TFA wasfound to be (468plusmn 007) (ww) respectively

32 Hyperoside Content -e standard calibration curve ofhyperoside was used to evaluate the content of flavonoid inthe extract -e hyperoside content of the TFA was(114plusmn 027) (ww) respectively (Figure 1)

33 Effect of TFA on the Ulcer Index and Percentage ofInhibition Ethanol is regarded as one of the major riskfactors for the pathogenesis of gastric ulcer A representativestomach of each group is shown in Figure 2 -e modelgroup presented severe mucosal injury (ulcer index4110 + 1828) TFA could significantly increase the inhibi-tion rate of ethanol-induced gastric ulcer at the dose of 600and 1200mgkg (Table 1) Compared with the positivecontrol group TFA showed better results than omeprazole-e effect of TFA was in a dose-dependent manner of whichthe high-dose group reached the maximum 6691 inhi-bition rate (plt 0001)

34 Effect of TFA on Biochemical Parameters All the resultsare shown in Figures 3(a)ndash3(c)) Ethanol caused a markedsignificant increase in the levels of MDA (plt 0001) andreduction in the activities of SOD (plt 0001) and GSH levels(plt 0001) in ulcerated mice as compared to the controlgroup Contrarily TFA (600mgkg) and TFA (1200mgkg)treatments dramatically decreased MDA levels in gastrictissue with a concomitant increase in the activities of SODand GSH levels as compared with the model group espe-cially the high-dose group

35 Histologic Evaluations of Gastric Tissue -e gastrictissue specimens of different groups were observed under amicroscope and the pathological changes are shown inFigures 4 and 5 In the control group the structure of thegastric tissue was intact and the epithelial cells were tightlyarranged (Figures 4(a) 5(a)) However there were varioushistopathological alterations including congestion cell-contained hyperaemia and edema gastric epithelial cellsnecrosis inflammatory changes and erosions in the gastrictissue of model group mice (Figures 4(b) 5(b)) Comparedwith the model group this poor sanitation was restrained byTFA or Omeprazole and the structure was tended to benormal -e results suggested that TFA could reduce gastricmucosa injury induced by ethanol

Evidence-Based Complementary and Alternative Medicine 3

36 e Expression of Proteins in the Gastric Tissue

361 Effect of TFA on the Expression of Bax and Bcl-2-e results of Bax and Bcl-2 protein expression were shownin Figures 6 and 7 -e expression of the proapoptotic factorBax in the model group was significantly higher than thecontrol group (plt 0001) 187 folds higher than the controlgroup and the expression of the antiapoptotic factor Bcl-2was significantly lower (plt 0001) 62 of the control group-e ratio of BaxBcl-2 in the each treatment group wassignificantly lower than that in themodel group especially inthe TFA high-dose group -is results demonstrated that

TFA can downregulate the expression of Bax upregulate theexpression of Bcl-2 and thus decrease the ratio of BaxBcl-2to inhibit the activation of the endogenous apoptoticpathway

362 Effect of TFA on the Expression of TNF-α NF-κB p65-e results of the expression of TNF-α and NF-κB p65 ingastric tissue are shown in Figures 6 and 8 -e expressionof TNF-α and NF-κB p65 in the TFA group was signifi-cantly lower than that in the model group especially in thehigh-dose group (plt 0001) -e results suggested that

0 5 10 15 20 25 30 35 40 45 50 55 60Retention time (min)

000

005

010

015

020

025

030

307

316

391 218

9

266

3

298

831

39

358

3

444

9

Abs

orba

nce (

AU

)

OH

OH

OH

O

OO

O

OHHO

HO

OH

HO

Hyperoside

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60Retention time (min)

000

002

004

006

008

010

248

265

303

327

353

385

449

479

509 539

563

584 6

507

01 738

776 817 867

911 9

4110

12

105

910

89

112

111

81

124

412

77

132

813

79

142

614

76

151

715

63

161

916

49 17

03

180

718

49

189

919

86

207

3 211

121

75 222

322

57

236

424

31

251

425

47

265

027

14

282

0

300

131

27

321

032

45

331

734

06 352

335

69

381

539

29

421

442

83 44

91

Abs

orba

nce (

AU

)

TFA

(b)

Figure 1 HPLC profile of hyperoside and TFA

4 Evidence-Based Complementary and Alternative Medicine

TFA could significantly downregulate the expression ofinflammation-related proteins in the gastric tissue ofgastric ulcer mice

4 Discussion

In the present study the model of ethanol-induced gastriculcer was used to evaluate the gastroprotective activity ofTFA Ethanol is widely used in experimental gastric ulcermodels because of its disadvantages-emodel is simple andrepeatable -e morphology histological features healingand recurrence processes of ulcer are similar to human [22]-e severity of gastric injury was determined by ulcer indexand we found that TFA could inhibit the occurrence ofulcers and reduce the area of ulceration It was shown by HEstaining that TFA could relieve the pathological changes ofgastric ulcer tissue in mice

Oxidative stress is an important etiological factor in thedamaged gastric mucosa [3] GSH is a major antioxidant byinteraction with lipid hydroperoxides -e ulcerogenic ac-tivity is related to the tissue levels of GSH Increase in the

levels of GSH can inhibit gastric ulceration [23] MDA is theend-product of lipid peroxidation and it is also an im-portant marker of oxidative damage -e increase in MDAcontent is associated with the necrosis and apoptosis of cells[24] SOD can exert its effects by removing oxygen freeradicals from the body [25] -erefore GSH MDA andSOD were measured in this study to evaluate oxidative stresslevels -rough the detection of the abovementioned oxi-dative damage indicators we found TFA can increase theactivity of SOD the concentration of GSH and at the sametime decrease the level of MDA in gastric tissue

Bcl-2 family proteins is a family of evolutionarily relatedproteins which derives its name from B-cell lymphoma 2[26] -ere are a total of 25 genes in the Bcl-2 family knownto date Bcl-2 family proteins are pivotal regulators of ap-optosis by controlling mitochondrial outer membranepermeabilization (MOMP) [27] When the cell is stimulatedby apoptotic signals the structure of Bax changes shiftingfrom the cytoplasm to the mitochondrial membrane andrecruiting an amount of Bax proteins to form a polymer-en the polymer is inserted into the outer mitochondrialmembrane causing formation of mitochondrial apoptosis-induced channel (MAC) CytC AIF and other proapoptoticproteins release into the cytoplasm via MAC resulting inapoptotic cell death [27 28] A heterodimer may be formedby the proapoptotic protein and the antiapoptotic proteinand the ratio of BaxBcl-2 in the dimer is directly related tothe regulation of apoptosis [29] TFA can significantlyupregulate the expression of antiapoptosis protien Bcl-2 anddownregulate the expression of proapoptosis factor Bax-us the apoptosis of cell was prevented through down-regulation of BaxBcl-2 ratio

Increasing evidence suggests that inflammatory media-tors may play a role in the development and progression ofgastric ulcer A lot of literature had reported that TNF-α the

(a) (b) (c) (d)

(e) (f )

Figure 2 -e gastric mucosal injury in mice (n 10) (a) Control group (b) model group (c) omeprazole group (d) high-dose group(e) middle-dose group (f ) low-dose group

Table 1 Ulcer index and inhibition rate of ulceration (Meanplusmn SDn 10)

Groups Dose(mgkg) Ulcer index Inhibition

ration ()Control mdash mdash mdashModel mdash 4110 + 1828 mdashOmeprazole 100 2830 + 583 3114High-dose 1200 1340 + 860lowastlowastlowast 6691Midium-dose 600 2410 + 1210lowast 4209Low-dose 300 4030 + 1784 195Note lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Evidence-Based Complementary and Alternative Medicine 5

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

20

15

10

5

0

MD

A (n

mol

mgp

rot)

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

60

45

20

0

SOD

(Um

gpro

t)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

1500

1000

500

0

GSH

(μm

olg

prot

)

lowastlowastlowast

lowast

(c)

Figure 3 Effect of TFA on biochemical parameters of gastric tissue inmice with GU (a)MDA levels of mice gastric tissue (b) SOD activitiesof mice gastric tissue and (c) GSH levels of mice gastric tissue (Meanplusmn SD n 6) plt 0001 vs control lowastplt 005 lowastlowastplt 001lowastlowastlowastplt 0001 vs model

(a) (b) (c) (d)

(e) (f )

Figure 4 Histologic evaluations of gastric tissue (HE staining times100) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c) (d)

(e) (f )

Figure 5 Histologic evaluations of gastric tissue (HE staining times200) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

TFA (mgkg)Control Model Ome 300 600 1200

TNF-α (17kd)

Bax (17kd)

Bcl-2 (26kd)

NF-κB p65 (65kd)

GAPHD (37kd)

Figure 6 Expression of the proteins with western blot

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of Bax

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

200

150

100

50

0

o

f con

trol

Expression of Bcl-2

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 7

main proinflammatory cytokine was the important con-tributing factor in intestinal mucosal injury [9 30] TNF-α isthe initiator of the exogenous death pathway It can combinewith autologous proximate or distant cell membrane deathreceptor (TNFR) -e initiation of the death receptorpathway leads to apoptosis (activated caspase-8) pro-grammed necrosis (via RIP1 and RIP3) or inflammation(via NF-κB) responses NF-κB a classic proinflammatorytranscription factor is kept inactive in resting cells bybinding with a member of the IKB α inhibitor protein family[31] NF-κB is an ideal target for the mediation of proin-flammatory factor expression in gastric ulcer [32] Manynatural products that have been promoted to have anti-inflammatory activity have also been shown to inhibit NF-κB In the current study western blot analysis indicated thesignificant downregulation of TNF-α and NF-κB p65

expressions in the gastric tissue which evidenced the anti-inflammatory effect of TFA

In summary the present study suggested that TFA couldsignificantly attenuate ethanol-induced gastric injury viaantioxidative anti-inflammatory and antiapoptotic effectsOur data reinforced the therapeutic potential of TFA in themanagement of gastric ulcer

Data Availability

-e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this article

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of TNF-α

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

150

100

50

0

o

f con

trol

Expression of NF-κB p65

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 8 Analysis of TNF-α and NF-κB p65 protein expression with Western blot (Meanplusmn SD) (a) expression of TNF-α (b) expression ofNF-κB p65 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

0

1

2

3

4

Bax

Bcl-2

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 7 Analysis of Bax and Bcl-2 protein expression with western blot (Meanplusmn SD) (a) Expression of Bax (b) expression of Bcl-2 and (c)BaxBcl-2 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

8 Evidence-Based Complementary and Alternative Medicine

References

[1] A S Tarnawski ldquoIncreased susceptibility of aging gastricmucosa to injury the mechanisms and clinical implicationsrdquoWorld Journal of Gastroenterology vol 20 no 16 p 4467 2014

[2] A M S Gomaa N A Abd El-Mottaleb and H A AamerldquoAntioxidant and anti-inflammatory activities of alpha lipoicacid protect against indomethacin-induced gastric ulcer in ratsrdquoBiomedicine amp Pharmacotherapy vol 101 pp 188ndash194 2018

[3] S Palle A Kanakalatha and C N Kavitha ldquoGastroprotectiveand antiulcer effects of Celastrus paniculatus seed oil againstseveral gastric ulcer models in ratsrdquo Journal of DietarySupplements vol 15 no 4 pp 373ndash385 2017

[4] Y Yang B Yin L Lv et al ldquoGastroprotective effect of aucubinagainst ethanol-induced gastric mucosal injury in micerdquo LifeSciences vol 189 pp 44ndash51 2017

[5] R H Hunt M Camilleri S E Crowe et al ldquo-e stomach inhealth and diseaserdquo Gut vol 64 no 10 pp 1650ndash1668 2015

[6] M Akanda I-S Kim D Ahn et al ldquoAnti-inflammatory andgastroprotective roles of rabdosia inflexa through down-regulation of pro-inflammatory cytokines and MAPKNF-κBsignaling pathwaysrdquo International Journal of Molecular Sci-ences vol 19 no 2 p 584 2018

[7] J Yoo J Lee Y Lee et al ldquoProtective effect of bovine milkagainst HCl and ethanolndashinduced gastric ulcer in micerdquoJournal of Dairy Science vol 101 no 5 pp 3758ndash3770 2018

[8] H Bernstein C Bernstein C Payne K Dvorakova andH Garewal ldquoBile acids as carcinogens in human gastroin-testinal cancersrdquo Mutation ResearchReviews in MutationResearch vol 589 no 1 pp 47ndash65 2005

[9] W Li X Wang W Zhi et al ldquo-e gastroprotective effect ofnobiletin against ethanol-induced acute gastric lesions inmice impact on oxidative stress and inflammationrdquo Immu-nopharmacology and Immunotoxicology vol 39 no 6pp 354ndash363 2017

[10] E Mohamed I Abu Hashim R Yusif et al ldquoPolymericmicelles for potentiated antiulcer and anticancer activities ofnaringinrdquo International Journal of Nanomedicine vol 13pp 1009ndash1027 2018

[11] W-P Bi H Man and M Man ldquoEfficacy and safety of herbalmedicines in treating gastric ulcer a reviewrdquoWorld Journal ofGastroenterology vol 20 no 45 pp 17020ndash17028 2014

[12] X-X Pan J-H Tao S Jiang Y Zhu D-W Qian andJ-A Duan ldquoCharacterization and immunomodulatory ac-tivity of polysaccharides from the stems and leaves of Abel-moschus manihot and a sulfated derivativerdquo InternationalJournal of Biological Macromolecules vol 107 pp 9ndash16 2018

[13] G Ai Q Liu W Hua Z Huang and D Wang ldquoHep-atoprotective evaluation of the total flavonoids extracted fromflowers of Abelmoschus manihot (L) Medic in vitro and invivo studiesrdquo Journal of Ethnopharmacology vol 146 no 3pp 794ndash802 2013

[14] D Lv X Cheng L Tang and M Jiang ldquo-e cardioprotectiveeffect of total flavonoids on myocardial ischemiareperfusion inratsrdquoBiomedicineampPharmacotherapy vol 88 pp 277ndash284 2017

[15] C Xue S Jiang J Guo D Qian J-a Duan and E ShangldquoScreening for in vitro metabolites of Abelmoschus manihotextract in intestinal bacteria by ultra-performance liquidchromatographyquadrupole time-of-flight mass spectrome-tryrdquo Journal of Chromatography B vol 879 no 32pp 3901ndash3908 2011

[16] C Xue J Guo D Qian et al ldquoIdentification of the potentialactive components of Abelmoschus manihot in rat blood andkidney tissue by microdialysis combined with ultra-

performance liquid chromatographyquadrupole time-of-flight mass spectrometryrdquo Journal of Chromatography Bvol 879 no 5-6 pp 317ndash325 2011

[17] J Li J Gong L Zhao H Zou and Y Yan ldquoExtraction process oftotal flavonoids of Abelmoschus manihotrdquo Journal of Interna-tional Pharmaceutical Research vol 43 no 2 pp 370ndash373 2016

[18] X-x Lv and Z-l Chen ldquoImprovement of determinationmethod of flower of sunset Abelmoschus in Chinese phar-macopoeia (2010 edition)rdquo Strait Pharmaceutical vol 27no 10 pp 67ndash69 2015

[19] D Hollander A Tarnawski W J Krause and H GergelyldquoProtective effect of sucralfate against alcohol-induced gastricmucosal injury in the ratrdquo Gastroenterology vol 88 no 1pp 366ndash374 1985

[20] P H Guth D Aures and G Paulsen ldquoTopical aspirin plusHCl gastric lesions in the ratrdquo Gastroenterology vol 76 no 1pp 88ndash93 1979

[21] I Szelenyi and K -iemer ldquoDistention ulcer as a model fortesting of drugs for ulcerogenic side effectsrdquo Archives ofToxicology vol 41 no 1 pp 99ndash105 1978

[22] W XIAO A XU and J Hui ldquoAdvances in models of gastriculcerationrdquo Pharmaceutical and Clinical Research vol 24no 2 pp 145ndash150 2016

[23] A P Jayaraj K R Rees F I Tovey et al ldquoAmolecular basis ofpeptic ulceration due to dietrdquo British Journal of ExperimentalPathology vol 67 no 1 pp 149ndash155 1986

[24] M Xie H Chen S Nie W Tong J Yin and M XieldquoGastroprotective effect of gamma-aminobutyric acid againstethanol-induced gastric mucosal injuryrdquo Chemico-BiologicalInteractions vol 272 pp 125ndash134 2017

[25] J-W Song C-S Seo T-I Kim et al ldquoProtective effects ofmanassantin a against ethanol-induced gastric injury in ratsrdquoBiological amp Pharmaceutical Bulletin vol 39 no 2 pp 221ndash229 2016

[26] A R D Delbridge S Grabow A Strasser and D L Vauxldquo-irty years of BCL-2 translating cell death discoveries intonovel cancer therapiesrdquo Nature Reviews Cancer vol 16 no 2pp 99ndash109 2016

[27] V Andreu-Fernandez M Sancho A Genoves et al ldquoBaxtransmembrane domain interacts with prosurvival Bcl-2proteins in biological membranesrdquo Proceedings of the Na-tional Academy of Sciences vol 114 no 2 pp 310ndash315 2017

[28] A Ashkenazi W J Fairbrother J D Leverson andA J Souers ldquoFrom basic apoptosis discoveries to advancedselective Bcl-2 family inhibitorsrdquo Nature Reviews Drug Dis-covery vol 16 no 4 pp 273ndash284 2017

[29] S -angarajan A Vedagiri S SomasundaramR Sakthimanogaran and M Murugesan ldquoNeuroprotectiveeffect of morin on lead acetate- induced apoptosis by pre-venting cytochrome c translocation via regulation of BaxBcl-2 ratiordquo Neurotoxicology and Teratology vol 66 pp 35ndash452018

[30] R Kamel E M El Morsy and A S Awad ldquoImmunomod-ulatory effect of candesartan on indomethacin-induced gastriculcer in ratsrdquo Immunopharmacology and Immunotoxicologyvol 34 no 6 pp 956ndash961 2012

[31] B Sun Q Xia and Z Gao ldquoTotal flavones of choerospondiasaxillaris attenuate cardiac dysfunction and myocardial in-terstitial fibrosis by modulating NF-κB signaling pathwayrdquoCardiovascular Toxicology vol 15 no 3 pp 283ndash289 2015

[32] X Chang F Luo W Jiang et al ldquoProtective activity ofsalidroside against ethanol-induced gastric ulcer via theMAPKNF-κB pathway in vivo and in vitrordquo InternationalImmunopharmacology vol 28 no 1 pp 604ndash615 2015

Evidence-Based Complementary and Alternative Medicine 9

36 e Expression of Proteins in the Gastric Tissue

361 Effect of TFA on the Expression of Bax and Bcl-2-e results of Bax and Bcl-2 protein expression were shownin Figures 6 and 7 -e expression of the proapoptotic factorBax in the model group was significantly higher than thecontrol group (plt 0001) 187 folds higher than the controlgroup and the expression of the antiapoptotic factor Bcl-2was significantly lower (plt 0001) 62 of the control group-e ratio of BaxBcl-2 in the each treatment group wassignificantly lower than that in themodel group especially inthe TFA high-dose group -is results demonstrated that

TFA can downregulate the expression of Bax upregulate theexpression of Bcl-2 and thus decrease the ratio of BaxBcl-2to inhibit the activation of the endogenous apoptoticpathway

362 Effect of TFA on the Expression of TNF-α NF-κB p65-e results of the expression of TNF-α and NF-κB p65 ingastric tissue are shown in Figures 6 and 8 -e expressionof TNF-α and NF-κB p65 in the TFA group was signifi-cantly lower than that in the model group especially in thehigh-dose group (plt 0001) -e results suggested that

0 5 10 15 20 25 30 35 40 45 50 55 60Retention time (min)

000

005

010

015

020

025

030

307

316

391 218

9

266

3

298

831

39

358

3

444

9

Abs

orba

nce (

AU

)

OH

OH

OH

O

OO

O

OHHO

HO

OH

HO

Hyperoside

(a)

0 5 10 15 20 25 30 35 40 45 50 55 60Retention time (min)

000

002

004

006

008

010

248

265

303

327

353

385

449

479

509 539

563

584 6

507

01 738

776 817 867

911 9

4110

12

105

910

89

112

111

81

124

412

77

132

813

79

142

614

76

151

715

63

161

916

49 17

03

180

718

49

189

919

86

207

3 211

121

75 222

322

57

236

424

31

251

425

47

265

027

14

282

0

300

131

27

321

032

45

331

734

06 352

335

69

381

539

29

421

442

83 44

91

Abs

orba

nce (

AU

)

TFA

(b)

Figure 1 HPLC profile of hyperoside and TFA

4 Evidence-Based Complementary and Alternative Medicine

TFA could significantly downregulate the expression ofinflammation-related proteins in the gastric tissue ofgastric ulcer mice

4 Discussion

In the present study the model of ethanol-induced gastriculcer was used to evaluate the gastroprotective activity ofTFA Ethanol is widely used in experimental gastric ulcermodels because of its disadvantages-emodel is simple andrepeatable -e morphology histological features healingand recurrence processes of ulcer are similar to human [22]-e severity of gastric injury was determined by ulcer indexand we found that TFA could inhibit the occurrence ofulcers and reduce the area of ulceration It was shown by HEstaining that TFA could relieve the pathological changes ofgastric ulcer tissue in mice

Oxidative stress is an important etiological factor in thedamaged gastric mucosa [3] GSH is a major antioxidant byinteraction with lipid hydroperoxides -e ulcerogenic ac-tivity is related to the tissue levels of GSH Increase in the

levels of GSH can inhibit gastric ulceration [23] MDA is theend-product of lipid peroxidation and it is also an im-portant marker of oxidative damage -e increase in MDAcontent is associated with the necrosis and apoptosis of cells[24] SOD can exert its effects by removing oxygen freeradicals from the body [25] -erefore GSH MDA andSOD were measured in this study to evaluate oxidative stresslevels -rough the detection of the abovementioned oxi-dative damage indicators we found TFA can increase theactivity of SOD the concentration of GSH and at the sametime decrease the level of MDA in gastric tissue

Bcl-2 family proteins is a family of evolutionarily relatedproteins which derives its name from B-cell lymphoma 2[26] -ere are a total of 25 genes in the Bcl-2 family knownto date Bcl-2 family proteins are pivotal regulators of ap-optosis by controlling mitochondrial outer membranepermeabilization (MOMP) [27] When the cell is stimulatedby apoptotic signals the structure of Bax changes shiftingfrom the cytoplasm to the mitochondrial membrane andrecruiting an amount of Bax proteins to form a polymer-en the polymer is inserted into the outer mitochondrialmembrane causing formation of mitochondrial apoptosis-induced channel (MAC) CytC AIF and other proapoptoticproteins release into the cytoplasm via MAC resulting inapoptotic cell death [27 28] A heterodimer may be formedby the proapoptotic protein and the antiapoptotic proteinand the ratio of BaxBcl-2 in the dimer is directly related tothe regulation of apoptosis [29] TFA can significantlyupregulate the expression of antiapoptosis protien Bcl-2 anddownregulate the expression of proapoptosis factor Bax-us the apoptosis of cell was prevented through down-regulation of BaxBcl-2 ratio

Increasing evidence suggests that inflammatory media-tors may play a role in the development and progression ofgastric ulcer A lot of literature had reported that TNF-α the

(a) (b) (c) (d)

(e) (f )

Figure 2 -e gastric mucosal injury in mice (n 10) (a) Control group (b) model group (c) omeprazole group (d) high-dose group(e) middle-dose group (f ) low-dose group

Table 1 Ulcer index and inhibition rate of ulceration (Meanplusmn SDn 10)

Groups Dose(mgkg) Ulcer index Inhibition

ration ()Control mdash mdash mdashModel mdash 4110 + 1828 mdashOmeprazole 100 2830 + 583 3114High-dose 1200 1340 + 860lowastlowastlowast 6691Midium-dose 600 2410 + 1210lowast 4209Low-dose 300 4030 + 1784 195Note lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Evidence-Based Complementary and Alternative Medicine 5

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

20

15

10

5

0

MD

A (n

mol

mgp

rot)

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

60

45

20

0

SOD

(Um

gpro

t)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

1500

1000

500

0

GSH

(μm

olg

prot

)

lowastlowastlowast

lowast

(c)

Figure 3 Effect of TFA on biochemical parameters of gastric tissue inmice with GU (a)MDA levels of mice gastric tissue (b) SOD activitiesof mice gastric tissue and (c) GSH levels of mice gastric tissue (Meanplusmn SD n 6) plt 0001 vs control lowastplt 005 lowastlowastplt 001lowastlowastlowastplt 0001 vs model

(a) (b) (c) (d)

(e) (f )

Figure 4 Histologic evaluations of gastric tissue (HE staining times100) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c) (d)

(e) (f )

Figure 5 Histologic evaluations of gastric tissue (HE staining times200) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

TFA (mgkg)Control Model Ome 300 600 1200

TNF-α (17kd)

Bax (17kd)

Bcl-2 (26kd)

NF-κB p65 (65kd)

GAPHD (37kd)

Figure 6 Expression of the proteins with western blot

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of Bax

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

200

150

100

50

0

o

f con

trol

Expression of Bcl-2

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 7

main proinflammatory cytokine was the important con-tributing factor in intestinal mucosal injury [9 30] TNF-α isthe initiator of the exogenous death pathway It can combinewith autologous proximate or distant cell membrane deathreceptor (TNFR) -e initiation of the death receptorpathway leads to apoptosis (activated caspase-8) pro-grammed necrosis (via RIP1 and RIP3) or inflammation(via NF-κB) responses NF-κB a classic proinflammatorytranscription factor is kept inactive in resting cells bybinding with a member of the IKB α inhibitor protein family[31] NF-κB is an ideal target for the mediation of proin-flammatory factor expression in gastric ulcer [32] Manynatural products that have been promoted to have anti-inflammatory activity have also been shown to inhibit NF-κB In the current study western blot analysis indicated thesignificant downregulation of TNF-α and NF-κB p65

expressions in the gastric tissue which evidenced the anti-inflammatory effect of TFA

In summary the present study suggested that TFA couldsignificantly attenuate ethanol-induced gastric injury viaantioxidative anti-inflammatory and antiapoptotic effectsOur data reinforced the therapeutic potential of TFA in themanagement of gastric ulcer

Data Availability

-e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this article

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of TNF-α

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

150

100

50

0

o

f con

trol

Expression of NF-κB p65

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 8 Analysis of TNF-α and NF-κB p65 protein expression with Western blot (Meanplusmn SD) (a) expression of TNF-α (b) expression ofNF-κB p65 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

0

1

2

3

4

Bax

Bcl-2

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 7 Analysis of Bax and Bcl-2 protein expression with western blot (Meanplusmn SD) (a) Expression of Bax (b) expression of Bcl-2 and (c)BaxBcl-2 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

8 Evidence-Based Complementary and Alternative Medicine

References

[1] A S Tarnawski ldquoIncreased susceptibility of aging gastricmucosa to injury the mechanisms and clinical implicationsrdquoWorld Journal of Gastroenterology vol 20 no 16 p 4467 2014

[2] A M S Gomaa N A Abd El-Mottaleb and H A AamerldquoAntioxidant and anti-inflammatory activities of alpha lipoicacid protect against indomethacin-induced gastric ulcer in ratsrdquoBiomedicine amp Pharmacotherapy vol 101 pp 188ndash194 2018

[3] S Palle A Kanakalatha and C N Kavitha ldquoGastroprotectiveand antiulcer effects of Celastrus paniculatus seed oil againstseveral gastric ulcer models in ratsrdquo Journal of DietarySupplements vol 15 no 4 pp 373ndash385 2017

[4] Y Yang B Yin L Lv et al ldquoGastroprotective effect of aucubinagainst ethanol-induced gastric mucosal injury in micerdquo LifeSciences vol 189 pp 44ndash51 2017

[5] R H Hunt M Camilleri S E Crowe et al ldquo-e stomach inhealth and diseaserdquo Gut vol 64 no 10 pp 1650ndash1668 2015

[6] M Akanda I-S Kim D Ahn et al ldquoAnti-inflammatory andgastroprotective roles of rabdosia inflexa through down-regulation of pro-inflammatory cytokines and MAPKNF-κBsignaling pathwaysrdquo International Journal of Molecular Sci-ences vol 19 no 2 p 584 2018

[7] J Yoo J Lee Y Lee et al ldquoProtective effect of bovine milkagainst HCl and ethanolndashinduced gastric ulcer in micerdquoJournal of Dairy Science vol 101 no 5 pp 3758ndash3770 2018

[8] H Bernstein C Bernstein C Payne K Dvorakova andH Garewal ldquoBile acids as carcinogens in human gastroin-testinal cancersrdquo Mutation ResearchReviews in MutationResearch vol 589 no 1 pp 47ndash65 2005

[9] W Li X Wang W Zhi et al ldquo-e gastroprotective effect ofnobiletin against ethanol-induced acute gastric lesions inmice impact on oxidative stress and inflammationrdquo Immu-nopharmacology and Immunotoxicology vol 39 no 6pp 354ndash363 2017

[10] E Mohamed I Abu Hashim R Yusif et al ldquoPolymericmicelles for potentiated antiulcer and anticancer activities ofnaringinrdquo International Journal of Nanomedicine vol 13pp 1009ndash1027 2018

[11] W-P Bi H Man and M Man ldquoEfficacy and safety of herbalmedicines in treating gastric ulcer a reviewrdquoWorld Journal ofGastroenterology vol 20 no 45 pp 17020ndash17028 2014

[12] X-X Pan J-H Tao S Jiang Y Zhu D-W Qian andJ-A Duan ldquoCharacterization and immunomodulatory ac-tivity of polysaccharides from the stems and leaves of Abel-moschus manihot and a sulfated derivativerdquo InternationalJournal of Biological Macromolecules vol 107 pp 9ndash16 2018

[13] G Ai Q Liu W Hua Z Huang and D Wang ldquoHep-atoprotective evaluation of the total flavonoids extracted fromflowers of Abelmoschus manihot (L) Medic in vitro and invivo studiesrdquo Journal of Ethnopharmacology vol 146 no 3pp 794ndash802 2013

[14] D Lv X Cheng L Tang and M Jiang ldquo-e cardioprotectiveeffect of total flavonoids on myocardial ischemiareperfusion inratsrdquoBiomedicineampPharmacotherapy vol 88 pp 277ndash284 2017

[15] C Xue S Jiang J Guo D Qian J-a Duan and E ShangldquoScreening for in vitro metabolites of Abelmoschus manihotextract in intestinal bacteria by ultra-performance liquidchromatographyquadrupole time-of-flight mass spectrome-tryrdquo Journal of Chromatography B vol 879 no 32pp 3901ndash3908 2011

[16] C Xue J Guo D Qian et al ldquoIdentification of the potentialactive components of Abelmoschus manihot in rat blood andkidney tissue by microdialysis combined with ultra-

performance liquid chromatographyquadrupole time-of-flight mass spectrometryrdquo Journal of Chromatography Bvol 879 no 5-6 pp 317ndash325 2011

[17] J Li J Gong L Zhao H Zou and Y Yan ldquoExtraction process oftotal flavonoids of Abelmoschus manihotrdquo Journal of Interna-tional Pharmaceutical Research vol 43 no 2 pp 370ndash373 2016

[18] X-x Lv and Z-l Chen ldquoImprovement of determinationmethod of flower of sunset Abelmoschus in Chinese phar-macopoeia (2010 edition)rdquo Strait Pharmaceutical vol 27no 10 pp 67ndash69 2015

[19] D Hollander A Tarnawski W J Krause and H GergelyldquoProtective effect of sucralfate against alcohol-induced gastricmucosal injury in the ratrdquo Gastroenterology vol 88 no 1pp 366ndash374 1985

[20] P H Guth D Aures and G Paulsen ldquoTopical aspirin plusHCl gastric lesions in the ratrdquo Gastroenterology vol 76 no 1pp 88ndash93 1979

[21] I Szelenyi and K -iemer ldquoDistention ulcer as a model fortesting of drugs for ulcerogenic side effectsrdquo Archives ofToxicology vol 41 no 1 pp 99ndash105 1978

[22] W XIAO A XU and J Hui ldquoAdvances in models of gastriculcerationrdquo Pharmaceutical and Clinical Research vol 24no 2 pp 145ndash150 2016

[23] A P Jayaraj K R Rees F I Tovey et al ldquoAmolecular basis ofpeptic ulceration due to dietrdquo British Journal of ExperimentalPathology vol 67 no 1 pp 149ndash155 1986

[24] M Xie H Chen S Nie W Tong J Yin and M XieldquoGastroprotective effect of gamma-aminobutyric acid againstethanol-induced gastric mucosal injuryrdquo Chemico-BiologicalInteractions vol 272 pp 125ndash134 2017

[25] J-W Song C-S Seo T-I Kim et al ldquoProtective effects ofmanassantin a against ethanol-induced gastric injury in ratsrdquoBiological amp Pharmaceutical Bulletin vol 39 no 2 pp 221ndash229 2016

[26] A R D Delbridge S Grabow A Strasser and D L Vauxldquo-irty years of BCL-2 translating cell death discoveries intonovel cancer therapiesrdquo Nature Reviews Cancer vol 16 no 2pp 99ndash109 2016

[27] V Andreu-Fernandez M Sancho A Genoves et al ldquoBaxtransmembrane domain interacts with prosurvival Bcl-2proteins in biological membranesrdquo Proceedings of the Na-tional Academy of Sciences vol 114 no 2 pp 310ndash315 2017

[28] A Ashkenazi W J Fairbrother J D Leverson andA J Souers ldquoFrom basic apoptosis discoveries to advancedselective Bcl-2 family inhibitorsrdquo Nature Reviews Drug Dis-covery vol 16 no 4 pp 273ndash284 2017

[29] S -angarajan A Vedagiri S SomasundaramR Sakthimanogaran and M Murugesan ldquoNeuroprotectiveeffect of morin on lead acetate- induced apoptosis by pre-venting cytochrome c translocation via regulation of BaxBcl-2 ratiordquo Neurotoxicology and Teratology vol 66 pp 35ndash452018

[30] R Kamel E M El Morsy and A S Awad ldquoImmunomod-ulatory effect of candesartan on indomethacin-induced gastriculcer in ratsrdquo Immunopharmacology and Immunotoxicologyvol 34 no 6 pp 956ndash961 2012

[31] B Sun Q Xia and Z Gao ldquoTotal flavones of choerospondiasaxillaris attenuate cardiac dysfunction and myocardial in-terstitial fibrosis by modulating NF-κB signaling pathwayrdquoCardiovascular Toxicology vol 15 no 3 pp 283ndash289 2015

[32] X Chang F Luo W Jiang et al ldquoProtective activity ofsalidroside against ethanol-induced gastric ulcer via theMAPKNF-κB pathway in vivo and in vitrordquo InternationalImmunopharmacology vol 28 no 1 pp 604ndash615 2015

Evidence-Based Complementary and Alternative Medicine 9

TFA could significantly downregulate the expression ofinflammation-related proteins in the gastric tissue ofgastric ulcer mice

4 Discussion

In the present study the model of ethanol-induced gastriculcer was used to evaluate the gastroprotective activity ofTFA Ethanol is widely used in experimental gastric ulcermodels because of its disadvantages-emodel is simple andrepeatable -e morphology histological features healingand recurrence processes of ulcer are similar to human [22]-e severity of gastric injury was determined by ulcer indexand we found that TFA could inhibit the occurrence ofulcers and reduce the area of ulceration It was shown by HEstaining that TFA could relieve the pathological changes ofgastric ulcer tissue in mice

Oxidative stress is an important etiological factor in thedamaged gastric mucosa [3] GSH is a major antioxidant byinteraction with lipid hydroperoxides -e ulcerogenic ac-tivity is related to the tissue levels of GSH Increase in the

levels of GSH can inhibit gastric ulceration [23] MDA is theend-product of lipid peroxidation and it is also an im-portant marker of oxidative damage -e increase in MDAcontent is associated with the necrosis and apoptosis of cells[24] SOD can exert its effects by removing oxygen freeradicals from the body [25] -erefore GSH MDA andSOD were measured in this study to evaluate oxidative stresslevels -rough the detection of the abovementioned oxi-dative damage indicators we found TFA can increase theactivity of SOD the concentration of GSH and at the sametime decrease the level of MDA in gastric tissue

Bcl-2 family proteins is a family of evolutionarily relatedproteins which derives its name from B-cell lymphoma 2[26] -ere are a total of 25 genes in the Bcl-2 family knownto date Bcl-2 family proteins are pivotal regulators of ap-optosis by controlling mitochondrial outer membranepermeabilization (MOMP) [27] When the cell is stimulatedby apoptotic signals the structure of Bax changes shiftingfrom the cytoplasm to the mitochondrial membrane andrecruiting an amount of Bax proteins to form a polymer-en the polymer is inserted into the outer mitochondrialmembrane causing formation of mitochondrial apoptosis-induced channel (MAC) CytC AIF and other proapoptoticproteins release into the cytoplasm via MAC resulting inapoptotic cell death [27 28] A heterodimer may be formedby the proapoptotic protein and the antiapoptotic proteinand the ratio of BaxBcl-2 in the dimer is directly related tothe regulation of apoptosis [29] TFA can significantlyupregulate the expression of antiapoptosis protien Bcl-2 anddownregulate the expression of proapoptosis factor Bax-us the apoptosis of cell was prevented through down-regulation of BaxBcl-2 ratio

Increasing evidence suggests that inflammatory media-tors may play a role in the development and progression ofgastric ulcer A lot of literature had reported that TNF-α the

(a) (b) (c) (d)

(e) (f )

Figure 2 -e gastric mucosal injury in mice (n 10) (a) Control group (b) model group (c) omeprazole group (d) high-dose group(e) middle-dose group (f ) low-dose group

Table 1 Ulcer index and inhibition rate of ulceration (Meanplusmn SDn 10)

Groups Dose(mgkg) Ulcer index Inhibition

ration ()Control mdash mdash mdashModel mdash 4110 + 1828 mdashOmeprazole 100 2830 + 583 3114High-dose 1200 1340 + 860lowastlowastlowast 6691Midium-dose 600 2410 + 1210lowast 4209Low-dose 300 4030 + 1784 195Note lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Evidence-Based Complementary and Alternative Medicine 5

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

20

15

10

5

0

MD

A (n

mol

mgp

rot)

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

60

45

20

0

SOD

(Um

gpro

t)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

1500

1000

500

0

GSH

(μm

olg

prot

)

lowastlowastlowast

lowast

(c)

Figure 3 Effect of TFA on biochemical parameters of gastric tissue inmice with GU (a)MDA levels of mice gastric tissue (b) SOD activitiesof mice gastric tissue and (c) GSH levels of mice gastric tissue (Meanplusmn SD n 6) plt 0001 vs control lowastplt 005 lowastlowastplt 001lowastlowastlowastplt 0001 vs model

(a) (b) (c) (d)

(e) (f )

Figure 4 Histologic evaluations of gastric tissue (HE staining times100) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c) (d)

(e) (f )

Figure 5 Histologic evaluations of gastric tissue (HE staining times200) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

TFA (mgkg)Control Model Ome 300 600 1200

TNF-α (17kd)

Bax (17kd)

Bcl-2 (26kd)

NF-κB p65 (65kd)

GAPHD (37kd)

Figure 6 Expression of the proteins with western blot

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of Bax

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

200

150

100

50

0

o

f con

trol

Expression of Bcl-2

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 7

main proinflammatory cytokine was the important con-tributing factor in intestinal mucosal injury [9 30] TNF-α isthe initiator of the exogenous death pathway It can combinewith autologous proximate or distant cell membrane deathreceptor (TNFR) -e initiation of the death receptorpathway leads to apoptosis (activated caspase-8) pro-grammed necrosis (via RIP1 and RIP3) or inflammation(via NF-κB) responses NF-κB a classic proinflammatorytranscription factor is kept inactive in resting cells bybinding with a member of the IKB α inhibitor protein family[31] NF-κB is an ideal target for the mediation of proin-flammatory factor expression in gastric ulcer [32] Manynatural products that have been promoted to have anti-inflammatory activity have also been shown to inhibit NF-κB In the current study western blot analysis indicated thesignificant downregulation of TNF-α and NF-κB p65

expressions in the gastric tissue which evidenced the anti-inflammatory effect of TFA

In summary the present study suggested that TFA couldsignificantly attenuate ethanol-induced gastric injury viaantioxidative anti-inflammatory and antiapoptotic effectsOur data reinforced the therapeutic potential of TFA in themanagement of gastric ulcer

Data Availability

-e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this article

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of TNF-α

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

150

100

50

0

o

f con

trol

Expression of NF-κB p65

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 8 Analysis of TNF-α and NF-κB p65 protein expression with Western blot (Meanplusmn SD) (a) expression of TNF-α (b) expression ofNF-κB p65 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

0

1

2

3

4

Bax

Bcl-2

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 7 Analysis of Bax and Bcl-2 protein expression with western blot (Meanplusmn SD) (a) Expression of Bax (b) expression of Bcl-2 and (c)BaxBcl-2 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

8 Evidence-Based Complementary and Alternative Medicine

References

[1] A S Tarnawski ldquoIncreased susceptibility of aging gastricmucosa to injury the mechanisms and clinical implicationsrdquoWorld Journal of Gastroenterology vol 20 no 16 p 4467 2014

[2] A M S Gomaa N A Abd El-Mottaleb and H A AamerldquoAntioxidant and anti-inflammatory activities of alpha lipoicacid protect against indomethacin-induced gastric ulcer in ratsrdquoBiomedicine amp Pharmacotherapy vol 101 pp 188ndash194 2018

[3] S Palle A Kanakalatha and C N Kavitha ldquoGastroprotectiveand antiulcer effects of Celastrus paniculatus seed oil againstseveral gastric ulcer models in ratsrdquo Journal of DietarySupplements vol 15 no 4 pp 373ndash385 2017

[4] Y Yang B Yin L Lv et al ldquoGastroprotective effect of aucubinagainst ethanol-induced gastric mucosal injury in micerdquo LifeSciences vol 189 pp 44ndash51 2017

[5] R H Hunt M Camilleri S E Crowe et al ldquo-e stomach inhealth and diseaserdquo Gut vol 64 no 10 pp 1650ndash1668 2015

[6] M Akanda I-S Kim D Ahn et al ldquoAnti-inflammatory andgastroprotective roles of rabdosia inflexa through down-regulation of pro-inflammatory cytokines and MAPKNF-κBsignaling pathwaysrdquo International Journal of Molecular Sci-ences vol 19 no 2 p 584 2018

[7] J Yoo J Lee Y Lee et al ldquoProtective effect of bovine milkagainst HCl and ethanolndashinduced gastric ulcer in micerdquoJournal of Dairy Science vol 101 no 5 pp 3758ndash3770 2018

[8] H Bernstein C Bernstein C Payne K Dvorakova andH Garewal ldquoBile acids as carcinogens in human gastroin-testinal cancersrdquo Mutation ResearchReviews in MutationResearch vol 589 no 1 pp 47ndash65 2005

[9] W Li X Wang W Zhi et al ldquo-e gastroprotective effect ofnobiletin against ethanol-induced acute gastric lesions inmice impact on oxidative stress and inflammationrdquo Immu-nopharmacology and Immunotoxicology vol 39 no 6pp 354ndash363 2017

[10] E Mohamed I Abu Hashim R Yusif et al ldquoPolymericmicelles for potentiated antiulcer and anticancer activities ofnaringinrdquo International Journal of Nanomedicine vol 13pp 1009ndash1027 2018

[11] W-P Bi H Man and M Man ldquoEfficacy and safety of herbalmedicines in treating gastric ulcer a reviewrdquoWorld Journal ofGastroenterology vol 20 no 45 pp 17020ndash17028 2014

[12] X-X Pan J-H Tao S Jiang Y Zhu D-W Qian andJ-A Duan ldquoCharacterization and immunomodulatory ac-tivity of polysaccharides from the stems and leaves of Abel-moschus manihot and a sulfated derivativerdquo InternationalJournal of Biological Macromolecules vol 107 pp 9ndash16 2018

[13] G Ai Q Liu W Hua Z Huang and D Wang ldquoHep-atoprotective evaluation of the total flavonoids extracted fromflowers of Abelmoschus manihot (L) Medic in vitro and invivo studiesrdquo Journal of Ethnopharmacology vol 146 no 3pp 794ndash802 2013

[14] D Lv X Cheng L Tang and M Jiang ldquo-e cardioprotectiveeffect of total flavonoids on myocardial ischemiareperfusion inratsrdquoBiomedicineampPharmacotherapy vol 88 pp 277ndash284 2017

[15] C Xue S Jiang J Guo D Qian J-a Duan and E ShangldquoScreening for in vitro metabolites of Abelmoschus manihotextract in intestinal bacteria by ultra-performance liquidchromatographyquadrupole time-of-flight mass spectrome-tryrdquo Journal of Chromatography B vol 879 no 32pp 3901ndash3908 2011

[16] C Xue J Guo D Qian et al ldquoIdentification of the potentialactive components of Abelmoschus manihot in rat blood andkidney tissue by microdialysis combined with ultra-

performance liquid chromatographyquadrupole time-of-flight mass spectrometryrdquo Journal of Chromatography Bvol 879 no 5-6 pp 317ndash325 2011

[17] J Li J Gong L Zhao H Zou and Y Yan ldquoExtraction process oftotal flavonoids of Abelmoschus manihotrdquo Journal of Interna-tional Pharmaceutical Research vol 43 no 2 pp 370ndash373 2016

[18] X-x Lv and Z-l Chen ldquoImprovement of determinationmethod of flower of sunset Abelmoschus in Chinese phar-macopoeia (2010 edition)rdquo Strait Pharmaceutical vol 27no 10 pp 67ndash69 2015

[19] D Hollander A Tarnawski W J Krause and H GergelyldquoProtective effect of sucralfate against alcohol-induced gastricmucosal injury in the ratrdquo Gastroenterology vol 88 no 1pp 366ndash374 1985

[20] P H Guth D Aures and G Paulsen ldquoTopical aspirin plusHCl gastric lesions in the ratrdquo Gastroenterology vol 76 no 1pp 88ndash93 1979

[21] I Szelenyi and K -iemer ldquoDistention ulcer as a model fortesting of drugs for ulcerogenic side effectsrdquo Archives ofToxicology vol 41 no 1 pp 99ndash105 1978

[22] W XIAO A XU and J Hui ldquoAdvances in models of gastriculcerationrdquo Pharmaceutical and Clinical Research vol 24no 2 pp 145ndash150 2016

[23] A P Jayaraj K R Rees F I Tovey et al ldquoAmolecular basis ofpeptic ulceration due to dietrdquo British Journal of ExperimentalPathology vol 67 no 1 pp 149ndash155 1986

[24] M Xie H Chen S Nie W Tong J Yin and M XieldquoGastroprotective effect of gamma-aminobutyric acid againstethanol-induced gastric mucosal injuryrdquo Chemico-BiologicalInteractions vol 272 pp 125ndash134 2017

[25] J-W Song C-S Seo T-I Kim et al ldquoProtective effects ofmanassantin a against ethanol-induced gastric injury in ratsrdquoBiological amp Pharmaceutical Bulletin vol 39 no 2 pp 221ndash229 2016

[26] A R D Delbridge S Grabow A Strasser and D L Vauxldquo-irty years of BCL-2 translating cell death discoveries intonovel cancer therapiesrdquo Nature Reviews Cancer vol 16 no 2pp 99ndash109 2016

[27] V Andreu-Fernandez M Sancho A Genoves et al ldquoBaxtransmembrane domain interacts with prosurvival Bcl-2proteins in biological membranesrdquo Proceedings of the Na-tional Academy of Sciences vol 114 no 2 pp 310ndash315 2017

[28] A Ashkenazi W J Fairbrother J D Leverson andA J Souers ldquoFrom basic apoptosis discoveries to advancedselective Bcl-2 family inhibitorsrdquo Nature Reviews Drug Dis-covery vol 16 no 4 pp 273ndash284 2017

[29] S -angarajan A Vedagiri S SomasundaramR Sakthimanogaran and M Murugesan ldquoNeuroprotectiveeffect of morin on lead acetate- induced apoptosis by pre-venting cytochrome c translocation via regulation of BaxBcl-2 ratiordquo Neurotoxicology and Teratology vol 66 pp 35ndash452018

[30] R Kamel E M El Morsy and A S Awad ldquoImmunomod-ulatory effect of candesartan on indomethacin-induced gastriculcer in ratsrdquo Immunopharmacology and Immunotoxicologyvol 34 no 6 pp 956ndash961 2012

[31] B Sun Q Xia and Z Gao ldquoTotal flavones of choerospondiasaxillaris attenuate cardiac dysfunction and myocardial in-terstitial fibrosis by modulating NF-κB signaling pathwayrdquoCardiovascular Toxicology vol 15 no 3 pp 283ndash289 2015

[32] X Chang F Luo W Jiang et al ldquoProtective activity ofsalidroside against ethanol-induced gastric ulcer via theMAPKNF-κB pathway in vivo and in vitrordquo InternationalImmunopharmacology vol 28 no 1 pp 604ndash615 2015

Evidence-Based Complementary and Alternative Medicine 9

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

20

15

10

5

0

MD

A (n

mol

mgp

rot)

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

60

45

20

0

SOD

(Um

gpro

t)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Con

trol

Mod

el

Om

epra

zole

1200 60

0

300

TFA (mgkg)

1500

1000

500

0

GSH

(μm

olg

prot

)

lowastlowastlowast

lowast

(c)

Figure 3 Effect of TFA on biochemical parameters of gastric tissue inmice with GU (a)MDA levels of mice gastric tissue (b) SOD activitiesof mice gastric tissue and (c) GSH levels of mice gastric tissue (Meanplusmn SD n 6) plt 0001 vs control lowastplt 005 lowastlowastplt 001lowastlowastlowastplt 0001 vs model

(a) (b) (c) (d)

(e) (f )

Figure 4 Histologic evaluations of gastric tissue (HE staining times100) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c) (d)

(e) (f )

Figure 5 Histologic evaluations of gastric tissue (HE staining times200) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

TFA (mgkg)Control Model Ome 300 600 1200

TNF-α (17kd)

Bax (17kd)

Bcl-2 (26kd)

NF-κB p65 (65kd)

GAPHD (37kd)

Figure 6 Expression of the proteins with western blot

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of Bax

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

200

150

100

50

0

o

f con

trol

Expression of Bcl-2

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 7

main proinflammatory cytokine was the important con-tributing factor in intestinal mucosal injury [9 30] TNF-α isthe initiator of the exogenous death pathway It can combinewith autologous proximate or distant cell membrane deathreceptor (TNFR) -e initiation of the death receptorpathway leads to apoptosis (activated caspase-8) pro-grammed necrosis (via RIP1 and RIP3) or inflammation(via NF-κB) responses NF-κB a classic proinflammatorytranscription factor is kept inactive in resting cells bybinding with a member of the IKB α inhibitor protein family[31] NF-κB is an ideal target for the mediation of proin-flammatory factor expression in gastric ulcer [32] Manynatural products that have been promoted to have anti-inflammatory activity have also been shown to inhibit NF-κB In the current study western blot analysis indicated thesignificant downregulation of TNF-α and NF-κB p65

expressions in the gastric tissue which evidenced the anti-inflammatory effect of TFA

In summary the present study suggested that TFA couldsignificantly attenuate ethanol-induced gastric injury viaantioxidative anti-inflammatory and antiapoptotic effectsOur data reinforced the therapeutic potential of TFA in themanagement of gastric ulcer

Data Availability

-e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this article

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of TNF-α

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

150

100

50

0

o

f con

trol

Expression of NF-κB p65

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 8 Analysis of TNF-α and NF-κB p65 protein expression with Western blot (Meanplusmn SD) (a) expression of TNF-α (b) expression ofNF-κB p65 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

0

1

2

3

4

Bax

Bcl-2

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 7 Analysis of Bax and Bcl-2 protein expression with western blot (Meanplusmn SD) (a) Expression of Bax (b) expression of Bcl-2 and (c)BaxBcl-2 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

8 Evidence-Based Complementary and Alternative Medicine

References

[1] A S Tarnawski ldquoIncreased susceptibility of aging gastricmucosa to injury the mechanisms and clinical implicationsrdquoWorld Journal of Gastroenterology vol 20 no 16 p 4467 2014

[2] A M S Gomaa N A Abd El-Mottaleb and H A AamerldquoAntioxidant and anti-inflammatory activities of alpha lipoicacid protect against indomethacin-induced gastric ulcer in ratsrdquoBiomedicine amp Pharmacotherapy vol 101 pp 188ndash194 2018

[3] S Palle A Kanakalatha and C N Kavitha ldquoGastroprotectiveand antiulcer effects of Celastrus paniculatus seed oil againstseveral gastric ulcer models in ratsrdquo Journal of DietarySupplements vol 15 no 4 pp 373ndash385 2017

[4] Y Yang B Yin L Lv et al ldquoGastroprotective effect of aucubinagainst ethanol-induced gastric mucosal injury in micerdquo LifeSciences vol 189 pp 44ndash51 2017

[5] R H Hunt M Camilleri S E Crowe et al ldquo-e stomach inhealth and diseaserdquo Gut vol 64 no 10 pp 1650ndash1668 2015

[6] M Akanda I-S Kim D Ahn et al ldquoAnti-inflammatory andgastroprotective roles of rabdosia inflexa through down-regulation of pro-inflammatory cytokines and MAPKNF-κBsignaling pathwaysrdquo International Journal of Molecular Sci-ences vol 19 no 2 p 584 2018

[7] J Yoo J Lee Y Lee et al ldquoProtective effect of bovine milkagainst HCl and ethanolndashinduced gastric ulcer in micerdquoJournal of Dairy Science vol 101 no 5 pp 3758ndash3770 2018

[8] H Bernstein C Bernstein C Payne K Dvorakova andH Garewal ldquoBile acids as carcinogens in human gastroin-testinal cancersrdquo Mutation ResearchReviews in MutationResearch vol 589 no 1 pp 47ndash65 2005

[9] W Li X Wang W Zhi et al ldquo-e gastroprotective effect ofnobiletin against ethanol-induced acute gastric lesions inmice impact on oxidative stress and inflammationrdquo Immu-nopharmacology and Immunotoxicology vol 39 no 6pp 354ndash363 2017

[10] E Mohamed I Abu Hashim R Yusif et al ldquoPolymericmicelles for potentiated antiulcer and anticancer activities ofnaringinrdquo International Journal of Nanomedicine vol 13pp 1009ndash1027 2018

[11] W-P Bi H Man and M Man ldquoEfficacy and safety of herbalmedicines in treating gastric ulcer a reviewrdquoWorld Journal ofGastroenterology vol 20 no 45 pp 17020ndash17028 2014

[12] X-X Pan J-H Tao S Jiang Y Zhu D-W Qian andJ-A Duan ldquoCharacterization and immunomodulatory ac-tivity of polysaccharides from the stems and leaves of Abel-moschus manihot and a sulfated derivativerdquo InternationalJournal of Biological Macromolecules vol 107 pp 9ndash16 2018

[13] G Ai Q Liu W Hua Z Huang and D Wang ldquoHep-atoprotective evaluation of the total flavonoids extracted fromflowers of Abelmoschus manihot (L) Medic in vitro and invivo studiesrdquo Journal of Ethnopharmacology vol 146 no 3pp 794ndash802 2013

[14] D Lv X Cheng L Tang and M Jiang ldquo-e cardioprotectiveeffect of total flavonoids on myocardial ischemiareperfusion inratsrdquoBiomedicineampPharmacotherapy vol 88 pp 277ndash284 2017

[15] C Xue S Jiang J Guo D Qian J-a Duan and E ShangldquoScreening for in vitro metabolites of Abelmoschus manihotextract in intestinal bacteria by ultra-performance liquidchromatographyquadrupole time-of-flight mass spectrome-tryrdquo Journal of Chromatography B vol 879 no 32pp 3901ndash3908 2011

[16] C Xue J Guo D Qian et al ldquoIdentification of the potentialactive components of Abelmoschus manihot in rat blood andkidney tissue by microdialysis combined with ultra-

performance liquid chromatographyquadrupole time-of-flight mass spectrometryrdquo Journal of Chromatography Bvol 879 no 5-6 pp 317ndash325 2011

[17] J Li J Gong L Zhao H Zou and Y Yan ldquoExtraction process oftotal flavonoids of Abelmoschus manihotrdquo Journal of Interna-tional Pharmaceutical Research vol 43 no 2 pp 370ndash373 2016

[18] X-x Lv and Z-l Chen ldquoImprovement of determinationmethod of flower of sunset Abelmoschus in Chinese phar-macopoeia (2010 edition)rdquo Strait Pharmaceutical vol 27no 10 pp 67ndash69 2015

[19] D Hollander A Tarnawski W J Krause and H GergelyldquoProtective effect of sucralfate against alcohol-induced gastricmucosal injury in the ratrdquo Gastroenterology vol 88 no 1pp 366ndash374 1985

[20] P H Guth D Aures and G Paulsen ldquoTopical aspirin plusHCl gastric lesions in the ratrdquo Gastroenterology vol 76 no 1pp 88ndash93 1979

[21] I Szelenyi and K -iemer ldquoDistention ulcer as a model fortesting of drugs for ulcerogenic side effectsrdquo Archives ofToxicology vol 41 no 1 pp 99ndash105 1978

[22] W XIAO A XU and J Hui ldquoAdvances in models of gastriculcerationrdquo Pharmaceutical and Clinical Research vol 24no 2 pp 145ndash150 2016

[23] A P Jayaraj K R Rees F I Tovey et al ldquoAmolecular basis ofpeptic ulceration due to dietrdquo British Journal of ExperimentalPathology vol 67 no 1 pp 149ndash155 1986

[24] M Xie H Chen S Nie W Tong J Yin and M XieldquoGastroprotective effect of gamma-aminobutyric acid againstethanol-induced gastric mucosal injuryrdquo Chemico-BiologicalInteractions vol 272 pp 125ndash134 2017

[25] J-W Song C-S Seo T-I Kim et al ldquoProtective effects ofmanassantin a against ethanol-induced gastric injury in ratsrdquoBiological amp Pharmaceutical Bulletin vol 39 no 2 pp 221ndash229 2016

[26] A R D Delbridge S Grabow A Strasser and D L Vauxldquo-irty years of BCL-2 translating cell death discoveries intonovel cancer therapiesrdquo Nature Reviews Cancer vol 16 no 2pp 99ndash109 2016

[27] V Andreu-Fernandez M Sancho A Genoves et al ldquoBaxtransmembrane domain interacts with prosurvival Bcl-2proteins in biological membranesrdquo Proceedings of the Na-tional Academy of Sciences vol 114 no 2 pp 310ndash315 2017

[28] A Ashkenazi W J Fairbrother J D Leverson andA J Souers ldquoFrom basic apoptosis discoveries to advancedselective Bcl-2 family inhibitorsrdquo Nature Reviews Drug Dis-covery vol 16 no 4 pp 273ndash284 2017

[29] S -angarajan A Vedagiri S SomasundaramR Sakthimanogaran and M Murugesan ldquoNeuroprotectiveeffect of morin on lead acetate- induced apoptosis by pre-venting cytochrome c translocation via regulation of BaxBcl-2 ratiordquo Neurotoxicology and Teratology vol 66 pp 35ndash452018

[30] R Kamel E M El Morsy and A S Awad ldquoImmunomod-ulatory effect of candesartan on indomethacin-induced gastriculcer in ratsrdquo Immunopharmacology and Immunotoxicologyvol 34 no 6 pp 956ndash961 2012

[31] B Sun Q Xia and Z Gao ldquoTotal flavones of choerospondiasaxillaris attenuate cardiac dysfunction and myocardial in-terstitial fibrosis by modulating NF-κB signaling pathwayrdquoCardiovascular Toxicology vol 15 no 3 pp 283ndash289 2015

[32] X Chang F Luo W Jiang et al ldquoProtective activity ofsalidroside against ethanol-induced gastric ulcer via theMAPKNF-κB pathway in vivo and in vitrordquo InternationalImmunopharmacology vol 28 no 1 pp 604ndash615 2015

Evidence-Based Complementary and Alternative Medicine 9

(a) (b) (c) (d)

(e) (f )

Figure 5 Histologic evaluations of gastric tissue (HE staining times200) (a) Control group (b) model group (c) omeprazole group (d) low-dose group (e) middle-dose group and (f) high-dose group

TFA (mgkg)Control Model Ome 300 600 1200

TNF-α (17kd)

Bax (17kd)

Bcl-2 (26kd)

NF-κB p65 (65kd)

GAPHD (37kd)

Figure 6 Expression of the proteins with western blot

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of Bax

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

200

150

100

50

0

o

f con

trol

Expression of Bcl-2

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 7

main proinflammatory cytokine was the important con-tributing factor in intestinal mucosal injury [9 30] TNF-α isthe initiator of the exogenous death pathway It can combinewith autologous proximate or distant cell membrane deathreceptor (TNFR) -e initiation of the death receptorpathway leads to apoptosis (activated caspase-8) pro-grammed necrosis (via RIP1 and RIP3) or inflammation(via NF-κB) responses NF-κB a classic proinflammatorytranscription factor is kept inactive in resting cells bybinding with a member of the IKB α inhibitor protein family[31] NF-κB is an ideal target for the mediation of proin-flammatory factor expression in gastric ulcer [32] Manynatural products that have been promoted to have anti-inflammatory activity have also been shown to inhibit NF-κB In the current study western blot analysis indicated thesignificant downregulation of TNF-α and NF-κB p65

expressions in the gastric tissue which evidenced the anti-inflammatory effect of TFA

In summary the present study suggested that TFA couldsignificantly attenuate ethanol-induced gastric injury viaantioxidative anti-inflammatory and antiapoptotic effectsOur data reinforced the therapeutic potential of TFA in themanagement of gastric ulcer

Data Availability

-e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this article

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of TNF-α

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

150

100

50

0

o

f con

trol

Expression of NF-κB p65

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 8 Analysis of TNF-α and NF-κB p65 protein expression with Western blot (Meanplusmn SD) (a) expression of TNF-α (b) expression ofNF-κB p65 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

0

1

2

3

4

Bax

Bcl-2

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 7 Analysis of Bax and Bcl-2 protein expression with western blot (Meanplusmn SD) (a) Expression of Bax (b) expression of Bcl-2 and (c)BaxBcl-2 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

8 Evidence-Based Complementary and Alternative Medicine

References

[1] A S Tarnawski ldquoIncreased susceptibility of aging gastricmucosa to injury the mechanisms and clinical implicationsrdquoWorld Journal of Gastroenterology vol 20 no 16 p 4467 2014

[2] A M S Gomaa N A Abd El-Mottaleb and H A AamerldquoAntioxidant and anti-inflammatory activities of alpha lipoicacid protect against indomethacin-induced gastric ulcer in ratsrdquoBiomedicine amp Pharmacotherapy vol 101 pp 188ndash194 2018

[3] S Palle A Kanakalatha and C N Kavitha ldquoGastroprotectiveand antiulcer effects of Celastrus paniculatus seed oil againstseveral gastric ulcer models in ratsrdquo Journal of DietarySupplements vol 15 no 4 pp 373ndash385 2017

[4] Y Yang B Yin L Lv et al ldquoGastroprotective effect of aucubinagainst ethanol-induced gastric mucosal injury in micerdquo LifeSciences vol 189 pp 44ndash51 2017

[5] R H Hunt M Camilleri S E Crowe et al ldquo-e stomach inhealth and diseaserdquo Gut vol 64 no 10 pp 1650ndash1668 2015

[6] M Akanda I-S Kim D Ahn et al ldquoAnti-inflammatory andgastroprotective roles of rabdosia inflexa through down-regulation of pro-inflammatory cytokines and MAPKNF-κBsignaling pathwaysrdquo International Journal of Molecular Sci-ences vol 19 no 2 p 584 2018

[7] J Yoo J Lee Y Lee et al ldquoProtective effect of bovine milkagainst HCl and ethanolndashinduced gastric ulcer in micerdquoJournal of Dairy Science vol 101 no 5 pp 3758ndash3770 2018

[8] H Bernstein C Bernstein C Payne K Dvorakova andH Garewal ldquoBile acids as carcinogens in human gastroin-testinal cancersrdquo Mutation ResearchReviews in MutationResearch vol 589 no 1 pp 47ndash65 2005

[9] W Li X Wang W Zhi et al ldquo-e gastroprotective effect ofnobiletin against ethanol-induced acute gastric lesions inmice impact on oxidative stress and inflammationrdquo Immu-nopharmacology and Immunotoxicology vol 39 no 6pp 354ndash363 2017

[10] E Mohamed I Abu Hashim R Yusif et al ldquoPolymericmicelles for potentiated antiulcer and anticancer activities ofnaringinrdquo International Journal of Nanomedicine vol 13pp 1009ndash1027 2018

[11] W-P Bi H Man and M Man ldquoEfficacy and safety of herbalmedicines in treating gastric ulcer a reviewrdquoWorld Journal ofGastroenterology vol 20 no 45 pp 17020ndash17028 2014

[12] X-X Pan J-H Tao S Jiang Y Zhu D-W Qian andJ-A Duan ldquoCharacterization and immunomodulatory ac-tivity of polysaccharides from the stems and leaves of Abel-moschus manihot and a sulfated derivativerdquo InternationalJournal of Biological Macromolecules vol 107 pp 9ndash16 2018

[13] G Ai Q Liu W Hua Z Huang and D Wang ldquoHep-atoprotective evaluation of the total flavonoids extracted fromflowers of Abelmoschus manihot (L) Medic in vitro and invivo studiesrdquo Journal of Ethnopharmacology vol 146 no 3pp 794ndash802 2013

[14] D Lv X Cheng L Tang and M Jiang ldquo-e cardioprotectiveeffect of total flavonoids on myocardial ischemiareperfusion inratsrdquoBiomedicineampPharmacotherapy vol 88 pp 277ndash284 2017

[15] C Xue S Jiang J Guo D Qian J-a Duan and E ShangldquoScreening for in vitro metabolites of Abelmoschus manihotextract in intestinal bacteria by ultra-performance liquidchromatographyquadrupole time-of-flight mass spectrome-tryrdquo Journal of Chromatography B vol 879 no 32pp 3901ndash3908 2011

[16] C Xue J Guo D Qian et al ldquoIdentification of the potentialactive components of Abelmoschus manihot in rat blood andkidney tissue by microdialysis combined with ultra-

performance liquid chromatographyquadrupole time-of-flight mass spectrometryrdquo Journal of Chromatography Bvol 879 no 5-6 pp 317ndash325 2011

[17] J Li J Gong L Zhao H Zou and Y Yan ldquoExtraction process oftotal flavonoids of Abelmoschus manihotrdquo Journal of Interna-tional Pharmaceutical Research vol 43 no 2 pp 370ndash373 2016

[18] X-x Lv and Z-l Chen ldquoImprovement of determinationmethod of flower of sunset Abelmoschus in Chinese phar-macopoeia (2010 edition)rdquo Strait Pharmaceutical vol 27no 10 pp 67ndash69 2015

[19] D Hollander A Tarnawski W J Krause and H GergelyldquoProtective effect of sucralfate against alcohol-induced gastricmucosal injury in the ratrdquo Gastroenterology vol 88 no 1pp 366ndash374 1985

[20] P H Guth D Aures and G Paulsen ldquoTopical aspirin plusHCl gastric lesions in the ratrdquo Gastroenterology vol 76 no 1pp 88ndash93 1979

[21] I Szelenyi and K -iemer ldquoDistention ulcer as a model fortesting of drugs for ulcerogenic side effectsrdquo Archives ofToxicology vol 41 no 1 pp 99ndash105 1978

[22] W XIAO A XU and J Hui ldquoAdvances in models of gastriculcerationrdquo Pharmaceutical and Clinical Research vol 24no 2 pp 145ndash150 2016

[23] A P Jayaraj K R Rees F I Tovey et al ldquoAmolecular basis ofpeptic ulceration due to dietrdquo British Journal of ExperimentalPathology vol 67 no 1 pp 149ndash155 1986

[24] M Xie H Chen S Nie W Tong J Yin and M XieldquoGastroprotective effect of gamma-aminobutyric acid againstethanol-induced gastric mucosal injuryrdquo Chemico-BiologicalInteractions vol 272 pp 125ndash134 2017

[25] J-W Song C-S Seo T-I Kim et al ldquoProtective effects ofmanassantin a against ethanol-induced gastric injury in ratsrdquoBiological amp Pharmaceutical Bulletin vol 39 no 2 pp 221ndash229 2016

[26] A R D Delbridge S Grabow A Strasser and D L Vauxldquo-irty years of BCL-2 translating cell death discoveries intonovel cancer therapiesrdquo Nature Reviews Cancer vol 16 no 2pp 99ndash109 2016

[27] V Andreu-Fernandez M Sancho A Genoves et al ldquoBaxtransmembrane domain interacts with prosurvival Bcl-2proteins in biological membranesrdquo Proceedings of the Na-tional Academy of Sciences vol 114 no 2 pp 310ndash315 2017

[28] A Ashkenazi W J Fairbrother J D Leverson andA J Souers ldquoFrom basic apoptosis discoveries to advancedselective Bcl-2 family inhibitorsrdquo Nature Reviews Drug Dis-covery vol 16 no 4 pp 273ndash284 2017

[29] S -angarajan A Vedagiri S SomasundaramR Sakthimanogaran and M Murugesan ldquoNeuroprotectiveeffect of morin on lead acetate- induced apoptosis by pre-venting cytochrome c translocation via regulation of BaxBcl-2 ratiordquo Neurotoxicology and Teratology vol 66 pp 35ndash452018

[30] R Kamel E M El Morsy and A S Awad ldquoImmunomod-ulatory effect of candesartan on indomethacin-induced gastriculcer in ratsrdquo Immunopharmacology and Immunotoxicologyvol 34 no 6 pp 956ndash961 2012

[31] B Sun Q Xia and Z Gao ldquoTotal flavones of choerospondiasaxillaris attenuate cardiac dysfunction and myocardial in-terstitial fibrosis by modulating NF-κB signaling pathwayrdquoCardiovascular Toxicology vol 15 no 3 pp 283ndash289 2015

[32] X Chang F Luo W Jiang et al ldquoProtective activity ofsalidroside against ethanol-induced gastric ulcer via theMAPKNF-κB pathway in vivo and in vitrordquo InternationalImmunopharmacology vol 28 no 1 pp 604ndash615 2015

Evidence-Based Complementary and Alternative Medicine 9

main proinflammatory cytokine was the important con-tributing factor in intestinal mucosal injury [9 30] TNF-α isthe initiator of the exogenous death pathway It can combinewith autologous proximate or distant cell membrane deathreceptor (TNFR) -e initiation of the death receptorpathway leads to apoptosis (activated caspase-8) pro-grammed necrosis (via RIP1 and RIP3) or inflammation(via NF-κB) responses NF-κB a classic proinflammatorytranscription factor is kept inactive in resting cells bybinding with a member of the IKB α inhibitor protein family[31] NF-κB is an ideal target for the mediation of proin-flammatory factor expression in gastric ulcer [32] Manynatural products that have been promoted to have anti-inflammatory activity have also been shown to inhibit NF-κB In the current study western blot analysis indicated thesignificant downregulation of TNF-α and NF-κB p65

expressions in the gastric tissue which evidenced the anti-inflammatory effect of TFA

In summary the present study suggested that TFA couldsignificantly attenuate ethanol-induced gastric injury viaantioxidative anti-inflammatory and antiapoptotic effectsOur data reinforced the therapeutic potential of TFA in themanagement of gastric ulcer

Data Availability

-e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

-e authors declare that there are no conflicts of interestregarding the publication of this article

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

250

200

150

100

50

0

o

f con

trol

Expression of TNF-α

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

150

100

50

0

o

f con

trol

Expression of NF-κB p65

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 8 Analysis of TNF-α and NF-κB p65 protein expression with Western blot (Meanplusmn SD) (a) expression of TNF-α (b) expression ofNF-κB p65 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

Con

trol

Om

epra

zole

Mod

el

300

600

1200

TFA (mgkg)

0

1

2

3

4

Bax

Bcl-2

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(c)

Figure 7 Analysis of Bax and Bcl-2 protein expression with western blot (Meanplusmn SD) (a) Expression of Bax (b) expression of Bcl-2 and (c)BaxBcl-2 plt 0001 vs control group lowastplt 005 lowastlowastplt 001 lowastlowastlowastplt 0001 vs model group

8 Evidence-Based Complementary and Alternative Medicine

References

[1] A S Tarnawski ldquoIncreased susceptibility of aging gastricmucosa to injury the mechanisms and clinical implicationsrdquoWorld Journal of Gastroenterology vol 20 no 16 p 4467 2014

[2] A M S Gomaa N A Abd El-Mottaleb and H A AamerldquoAntioxidant and anti-inflammatory activities of alpha lipoicacid protect against indomethacin-induced gastric ulcer in ratsrdquoBiomedicine amp Pharmacotherapy vol 101 pp 188ndash194 2018

[3] S Palle A Kanakalatha and C N Kavitha ldquoGastroprotectiveand antiulcer effects of Celastrus paniculatus seed oil againstseveral gastric ulcer models in ratsrdquo Journal of DietarySupplements vol 15 no 4 pp 373ndash385 2017

[4] Y Yang B Yin L Lv et al ldquoGastroprotective effect of aucubinagainst ethanol-induced gastric mucosal injury in micerdquo LifeSciences vol 189 pp 44ndash51 2017

[5] R H Hunt M Camilleri S E Crowe et al ldquo-e stomach inhealth and diseaserdquo Gut vol 64 no 10 pp 1650ndash1668 2015

[6] M Akanda I-S Kim D Ahn et al ldquoAnti-inflammatory andgastroprotective roles of rabdosia inflexa through down-regulation of pro-inflammatory cytokines and MAPKNF-κBsignaling pathwaysrdquo International Journal of Molecular Sci-ences vol 19 no 2 p 584 2018

[7] J Yoo J Lee Y Lee et al ldquoProtective effect of bovine milkagainst HCl and ethanolndashinduced gastric ulcer in micerdquoJournal of Dairy Science vol 101 no 5 pp 3758ndash3770 2018

[8] H Bernstein C Bernstein C Payne K Dvorakova andH Garewal ldquoBile acids as carcinogens in human gastroin-testinal cancersrdquo Mutation ResearchReviews in MutationResearch vol 589 no 1 pp 47ndash65 2005

[9] W Li X Wang W Zhi et al ldquo-e gastroprotective effect ofnobiletin against ethanol-induced acute gastric lesions inmice impact on oxidative stress and inflammationrdquo Immu-nopharmacology and Immunotoxicology vol 39 no 6pp 354ndash363 2017

[10] E Mohamed I Abu Hashim R Yusif et al ldquoPolymericmicelles for potentiated antiulcer and anticancer activities ofnaringinrdquo International Journal of Nanomedicine vol 13pp 1009ndash1027 2018

[11] W-P Bi H Man and M Man ldquoEfficacy and safety of herbalmedicines in treating gastric ulcer a reviewrdquoWorld Journal ofGastroenterology vol 20 no 45 pp 17020ndash17028 2014

[12] X-X Pan J-H Tao S Jiang Y Zhu D-W Qian andJ-A Duan ldquoCharacterization and immunomodulatory ac-tivity of polysaccharides from the stems and leaves of Abel-moschus manihot and a sulfated derivativerdquo InternationalJournal of Biological Macromolecules vol 107 pp 9ndash16 2018

[13] G Ai Q Liu W Hua Z Huang and D Wang ldquoHep-atoprotective evaluation of the total flavonoids extracted fromflowers of Abelmoschus manihot (L) Medic in vitro and invivo studiesrdquo Journal of Ethnopharmacology vol 146 no 3pp 794ndash802 2013

[14] D Lv X Cheng L Tang and M Jiang ldquo-e cardioprotectiveeffect of total flavonoids on myocardial ischemiareperfusion inratsrdquoBiomedicineampPharmacotherapy vol 88 pp 277ndash284 2017

[15] C Xue S Jiang J Guo D Qian J-a Duan and E ShangldquoScreening for in vitro metabolites of Abelmoschus manihotextract in intestinal bacteria by ultra-performance liquidchromatographyquadrupole time-of-flight mass spectrome-tryrdquo Journal of Chromatography B vol 879 no 32pp 3901ndash3908 2011

[16] C Xue J Guo D Qian et al ldquoIdentification of the potentialactive components of Abelmoschus manihot in rat blood andkidney tissue by microdialysis combined with ultra-

performance liquid chromatographyquadrupole time-of-flight mass spectrometryrdquo Journal of Chromatography Bvol 879 no 5-6 pp 317ndash325 2011

[17] J Li J Gong L Zhao H Zou and Y Yan ldquoExtraction process oftotal flavonoids of Abelmoschus manihotrdquo Journal of Interna-tional Pharmaceutical Research vol 43 no 2 pp 370ndash373 2016

[18] X-x Lv and Z-l Chen ldquoImprovement of determinationmethod of flower of sunset Abelmoschus in Chinese phar-macopoeia (2010 edition)rdquo Strait Pharmaceutical vol 27no 10 pp 67ndash69 2015

[19] D Hollander A Tarnawski W J Krause and H GergelyldquoProtective effect of sucralfate against alcohol-induced gastricmucosal injury in the ratrdquo Gastroenterology vol 88 no 1pp 366ndash374 1985

[20] P H Guth D Aures and G Paulsen ldquoTopical aspirin plusHCl gastric lesions in the ratrdquo Gastroenterology vol 76 no 1pp 88ndash93 1979

[21] I Szelenyi and K -iemer ldquoDistention ulcer as a model fortesting of drugs for ulcerogenic side effectsrdquo Archives ofToxicology vol 41 no 1 pp 99ndash105 1978

[22] W XIAO A XU and J Hui ldquoAdvances in models of gastriculcerationrdquo Pharmaceutical and Clinical Research vol 24no 2 pp 145ndash150 2016

[23] A P Jayaraj K R Rees F I Tovey et al ldquoAmolecular basis ofpeptic ulceration due to dietrdquo British Journal of ExperimentalPathology vol 67 no 1 pp 149ndash155 1986

[24] M Xie H Chen S Nie W Tong J Yin and M XieldquoGastroprotective effect of gamma-aminobutyric acid againstethanol-induced gastric mucosal injuryrdquo Chemico-BiologicalInteractions vol 272 pp 125ndash134 2017

[25] J-W Song C-S Seo T-I Kim et al ldquoProtective effects ofmanassantin a against ethanol-induced gastric injury in ratsrdquoBiological amp Pharmaceutical Bulletin vol 39 no 2 pp 221ndash229 2016

[26] A R D Delbridge S Grabow A Strasser and D L Vauxldquo-irty years of BCL-2 translating cell death discoveries intonovel cancer therapiesrdquo Nature Reviews Cancer vol 16 no 2pp 99ndash109 2016

[27] V Andreu-Fernandez M Sancho A Genoves et al ldquoBaxtransmembrane domain interacts with prosurvival Bcl-2proteins in biological membranesrdquo Proceedings of the Na-tional Academy of Sciences vol 114 no 2 pp 310ndash315 2017

[28] A Ashkenazi W J Fairbrother J D Leverson andA J Souers ldquoFrom basic apoptosis discoveries to advancedselective Bcl-2 family inhibitorsrdquo Nature Reviews Drug Dis-covery vol 16 no 4 pp 273ndash284 2017

[29] S -angarajan A Vedagiri S SomasundaramR Sakthimanogaran and M Murugesan ldquoNeuroprotectiveeffect of morin on lead acetate- induced apoptosis by pre-venting cytochrome c translocation via regulation of BaxBcl-2 ratiordquo Neurotoxicology and Teratology vol 66 pp 35ndash452018

[30] R Kamel E M El Morsy and A S Awad ldquoImmunomod-ulatory effect of candesartan on indomethacin-induced gastriculcer in ratsrdquo Immunopharmacology and Immunotoxicologyvol 34 no 6 pp 956ndash961 2012

[31] B Sun Q Xia and Z Gao ldquoTotal flavones of choerospondiasaxillaris attenuate cardiac dysfunction and myocardial in-terstitial fibrosis by modulating NF-κB signaling pathwayrdquoCardiovascular Toxicology vol 15 no 3 pp 283ndash289 2015

[32] X Chang F Luo W Jiang et al ldquoProtective activity ofsalidroside against ethanol-induced gastric ulcer via theMAPKNF-κB pathway in vivo and in vitrordquo InternationalImmunopharmacology vol 28 no 1 pp 604ndash615 2015

Evidence-Based Complementary and Alternative Medicine 9

References

[1] A S Tarnawski ldquoIncreased susceptibility of aging gastricmucosa to injury the mechanisms and clinical implicationsrdquoWorld Journal of Gastroenterology vol 20 no 16 p 4467 2014

[2] A M S Gomaa N A Abd El-Mottaleb and H A AamerldquoAntioxidant and anti-inflammatory activities of alpha lipoicacid protect against indomethacin-induced gastric ulcer in ratsrdquoBiomedicine amp Pharmacotherapy vol 101 pp 188ndash194 2018

[3] S Palle A Kanakalatha and C N Kavitha ldquoGastroprotectiveand antiulcer effects of Celastrus paniculatus seed oil againstseveral gastric ulcer models in ratsrdquo Journal of DietarySupplements vol 15 no 4 pp 373ndash385 2017

[4] Y Yang B Yin L Lv et al ldquoGastroprotective effect of aucubinagainst ethanol-induced gastric mucosal injury in micerdquo LifeSciences vol 189 pp 44ndash51 2017

[5] R H Hunt M Camilleri S E Crowe et al ldquo-e stomach inhealth and diseaserdquo Gut vol 64 no 10 pp 1650ndash1668 2015

[6] M Akanda I-S Kim D Ahn et al ldquoAnti-inflammatory andgastroprotective roles of rabdosia inflexa through down-regulation of pro-inflammatory cytokines and MAPKNF-κBsignaling pathwaysrdquo International Journal of Molecular Sci-ences vol 19 no 2 p 584 2018

[7] J Yoo J Lee Y Lee et al ldquoProtective effect of bovine milkagainst HCl and ethanolndashinduced gastric ulcer in micerdquoJournal of Dairy Science vol 101 no 5 pp 3758ndash3770 2018

[8] H Bernstein C Bernstein C Payne K Dvorakova andH Garewal ldquoBile acids as carcinogens in human gastroin-testinal cancersrdquo Mutation ResearchReviews in MutationResearch vol 589 no 1 pp 47ndash65 2005

[9] W Li X Wang W Zhi et al ldquo-e gastroprotective effect ofnobiletin against ethanol-induced acute gastric lesions inmice impact on oxidative stress and inflammationrdquo Immu-nopharmacology and Immunotoxicology vol 39 no 6pp 354ndash363 2017

[10] E Mohamed I Abu Hashim R Yusif et al ldquoPolymericmicelles for potentiated antiulcer and anticancer activities ofnaringinrdquo International Journal of Nanomedicine vol 13pp 1009ndash1027 2018

[11] W-P Bi H Man and M Man ldquoEfficacy and safety of herbalmedicines in treating gastric ulcer a reviewrdquoWorld Journal ofGastroenterology vol 20 no 45 pp 17020ndash17028 2014

[12] X-X Pan J-H Tao S Jiang Y Zhu D-W Qian andJ-A Duan ldquoCharacterization and immunomodulatory ac-tivity of polysaccharides from the stems and leaves of Abel-moschus manihot and a sulfated derivativerdquo InternationalJournal of Biological Macromolecules vol 107 pp 9ndash16 2018

[13] G Ai Q Liu W Hua Z Huang and D Wang ldquoHep-atoprotective evaluation of the total flavonoids extracted fromflowers of Abelmoschus manihot (L) Medic in vitro and invivo studiesrdquo Journal of Ethnopharmacology vol 146 no 3pp 794ndash802 2013

[14] D Lv X Cheng L Tang and M Jiang ldquo-e cardioprotectiveeffect of total flavonoids on myocardial ischemiareperfusion inratsrdquoBiomedicineampPharmacotherapy vol 88 pp 277ndash284 2017

[15] C Xue S Jiang J Guo D Qian J-a Duan and E ShangldquoScreening for in vitro metabolites of Abelmoschus manihotextract in intestinal bacteria by ultra-performance liquidchromatographyquadrupole time-of-flight mass spectrome-tryrdquo Journal of Chromatography B vol 879 no 32pp 3901ndash3908 2011

[16] C Xue J Guo D Qian et al ldquoIdentification of the potentialactive components of Abelmoschus manihot in rat blood andkidney tissue by microdialysis combined with ultra-

performance liquid chromatographyquadrupole time-of-flight mass spectrometryrdquo Journal of Chromatography Bvol 879 no 5-6 pp 317ndash325 2011

[17] J Li J Gong L Zhao H Zou and Y Yan ldquoExtraction process oftotal flavonoids of Abelmoschus manihotrdquo Journal of Interna-tional Pharmaceutical Research vol 43 no 2 pp 370ndash373 2016

[18] X-x Lv and Z-l Chen ldquoImprovement of determinationmethod of flower of sunset Abelmoschus in Chinese phar-macopoeia (2010 edition)rdquo Strait Pharmaceutical vol 27no 10 pp 67ndash69 2015

[19] D Hollander A Tarnawski W J Krause and H GergelyldquoProtective effect of sucralfate against alcohol-induced gastricmucosal injury in the ratrdquo Gastroenterology vol 88 no 1pp 366ndash374 1985

[20] P H Guth D Aures and G Paulsen ldquoTopical aspirin plusHCl gastric lesions in the ratrdquo Gastroenterology vol 76 no 1pp 88ndash93 1979

[21] I Szelenyi and K -iemer ldquoDistention ulcer as a model fortesting of drugs for ulcerogenic side effectsrdquo Archives ofToxicology vol 41 no 1 pp 99ndash105 1978

[22] W XIAO A XU and J Hui ldquoAdvances in models of gastriculcerationrdquo Pharmaceutical and Clinical Research vol 24no 2 pp 145ndash150 2016

[23] A P Jayaraj K R Rees F I Tovey et al ldquoAmolecular basis ofpeptic ulceration due to dietrdquo British Journal of ExperimentalPathology vol 67 no 1 pp 149ndash155 1986

[24] M Xie H Chen S Nie W Tong J Yin and M XieldquoGastroprotective effect of gamma-aminobutyric acid againstethanol-induced gastric mucosal injuryrdquo Chemico-BiologicalInteractions vol 272 pp 125ndash134 2017

[25] J-W Song C-S Seo T-I Kim et al ldquoProtective effects ofmanassantin a against ethanol-induced gastric injury in ratsrdquoBiological amp Pharmaceutical Bulletin vol 39 no 2 pp 221ndash229 2016

[26] A R D Delbridge S Grabow A Strasser and D L Vauxldquo-irty years of BCL-2 translating cell death discoveries intonovel cancer therapiesrdquo Nature Reviews Cancer vol 16 no 2pp 99ndash109 2016

[27] V Andreu-Fernandez M Sancho A Genoves et al ldquoBaxtransmembrane domain interacts with prosurvival Bcl-2proteins in biological membranesrdquo Proceedings of the Na-tional Academy of Sciences vol 114 no 2 pp 310ndash315 2017

[28] A Ashkenazi W J Fairbrother J D Leverson andA J Souers ldquoFrom basic apoptosis discoveries to advancedselective Bcl-2 family inhibitorsrdquo Nature Reviews Drug Dis-covery vol 16 no 4 pp 273ndash284 2017

[29] S -angarajan A Vedagiri S SomasundaramR Sakthimanogaran and M Murugesan ldquoNeuroprotectiveeffect of morin on lead acetate- induced apoptosis by pre-venting cytochrome c translocation via regulation of BaxBcl-2 ratiordquo Neurotoxicology and Teratology vol 66 pp 35ndash452018

[30] R Kamel E M El Morsy and A S Awad ldquoImmunomod-ulatory effect of candesartan on indomethacin-induced gastriculcer in ratsrdquo Immunopharmacology and Immunotoxicologyvol 34 no 6 pp 956ndash961 2012

[31] B Sun Q Xia and Z Gao ldquoTotal flavones of choerospondiasaxillaris attenuate cardiac dysfunction and myocardial in-terstitial fibrosis by modulating NF-κB signaling pathwayrdquoCardiovascular Toxicology vol 15 no 3 pp 283ndash289 2015

[32] X Chang F Luo W Jiang et al ldquoProtective activity ofsalidroside against ethanol-induced gastric ulcer via theMAPKNF-κB pathway in vivo and in vitrordquo InternationalImmunopharmacology vol 28 no 1 pp 604ndash615 2015

Evidence-Based Complementary and Alternative Medicine 9