Chiang Mai J. Sci. 2014; 41(5.1) 1121

Chiang Mai J. Sci. 2014; 41(5.1) : 1121-1131http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Gastroprotective Effects and Antioxidant Activities ofPaederia pilifera Hook.f. Root ExtractKanokporn Saenphet, Supap Saenphet and Kantarat Jirakittirat*Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.*Author for correspondence; e-mail: stit.lilo123@gmail.com

Received: 2 May 2013Accepted: 11 October 2013

ABSTRACTGastric ulcer is a type of gastrointestinal diseases, which affects people all over the

world. Modern gastric ulcer drugs have various severe side effects and show less efficacy.Thus, more effective and safer anti-ulcer agents are needed. Reactive oxygen species haveimportant roles in the pathogenesis of various diseases, including gastric ulcers.The gastroprotective effects and the antioxidant activities of Paederia pilifera Hook.f.(Rubiaceae) root extract were examined on ethanol-induced gastric ulcers in rats. The effectsof the plant extract and omeprazole on ulcer index, percentage inhibition, antioxidant activitiesand lipid peroxidation were evaluated. Antioxidant properties of the plant in vitro were alsodetermined. Root extract of P. pilifera exhibited high total phenolic and ascorbic acid contentsand showed free radical scavenging activities. Pretreatment with P. pilifera extract at all doses(250, 500, 750 mg/kg BW) and omeprazole exhibited a significant reduction in gastric mucosallesions. The percentage inhibition in the rats treated with the extract was dose dependent.Pretreatment with P. pilifera extract and omeprazole showed a significant decrease in lipidperoxidation and an increase in the activities of catalase (CAT) and superoxide dismutase(SOD) enzymes and glutathione (GSH) levels. The cyto-protective and antioxidant propertiesof the extract found in this study could be due to the presence of the bioactive compounds inextract.

Keywords: gastroprotective effect, antioxidant activities, Paederia pilifera extract, gastric ulcers,free radical

1. INTRODUCTIONGastric ulcer is a major gastrointestinal

disorder, which affects many people in theworld. This disease causes deep lesionspenetrating through the muscularis mucosa.It is well known that the development ofgastric ulcers occurs due to an imbalancebetween various aggressive factors (ethanol,NSAIDS, smoking, stress, leukotrienes,

reactive oxygen species) and endogenousdefensive factors (mucus-bicarbonate barrier,mucosal blood flow, prostaglandins,enzymatic and non-enzymatic antioxidants).Gastric damage caused by ethanol andNSAIDS leads to an overproduction of thereactive oxygen species (ROS), such assuperoxide anions and hydroxyl radicals

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[5,13]. There is evidence of the participationof reactive oxygen species in the etiologyand pathophysiology of human diseases,such as gastrointestinal disorders andgastric ulcers. ROS attacks essential cellconstituents, such as proteins, lipids, andDNA, and induces lipid peroxidation inthe cell membranes [24]. Enzymatic andnonenzymatic antioxidants, includingvitamin C, catalase (CAT), superoxidedismutase (SOD) and glutathione (GSH)play an important role in the prevention ofthe toxicity of ROS and in gastric damage.Although many modern drugs on the markethave been widely applied in the preventionand treatment of gastric ulcers, most of thesedrugs have various severe side effects and havelow efficacy. It is believed that herbal drugshave few side effects, are effective and lessexpensive than synthetic drugs. Many medicinalplants listed in ancient pharmacopoeias andin older herbal books have a reputation fortreating gastric disorders, including gastriculcers. In certain scientific literature, a largenumber of medicinal plants with gastricantiulcer potential have been reported. It isalso known that medicinal plants contain awide variety of antioxidants, such as phenoliccompounds and vitamin C, which minimizegastric injuries in experimental animals[1, 2, 6, 27].

Paederia pilifera Hook.f. (Rubiaceae),known in the Thai language as Kra-pang-hohm,is a medicinal plant distributed throughoutThailand. It is a vine climbing plant withleaves that are 6-15 cm long and 2-6 cm wide.The opposite, green leaves may release a strongfetid odor when bruised. This plant can befound in the deciduous forests and tropicalrainforests throughout India and SoutheastAsia. In Thai herbal pharmacopoeias, thisplant has been used in the treatment ofgastrointestinal disorders such as diarrhea,gastiritis, food poisoning, dyspepsia, jaundice

and hyperbilirubinemia [29]. AlthoughP. pilifera has been used to treat numerousaliments, its gastroprotective effects andantioxidant activities based upon scientificstudies, have not yet been published.The aim of our work is to determine thegastroprotective effects and antioxidantactivities of Paederia pilifera root extract onthe ethanol-induced gastric mucosal damagein rats.

2. MATERIALS AND METHODS2.1 Animals

Male and female albino Wistar rats(Rattus norvegicus) were purchased from theNational Laboratory Animal Center,Mahidol University, Salaya Campus,Thailand. Animals were housed in atemperature controlled (25±1°C) room witha 12-h dark and 12-h light cycle. The ratswere fed ad libitum with a standard pellet dietand had free access to water. All proceduresinvolving the animals were conducted withstrict adherence to the guidelines andprocedures reviewed and approved by theInstitutional Animal Care and Use Committeeof the Biology Department, Faculty ofScience, Chiang Mai University (protocol no.Re 002/12).

2.2 Plant PreparationThe roots of the Paederia pilifera

Hook.f. were collected from Chiang MaiProvince, Thailand in April 2012. The plantmaterial was identified by a botanist from theDepartment of Biology, Faculty of Science,Chiang Mai University. A voucher specimen(WP 3137) was deposited at Queen SirikitBotanic Garden, Mae Rim, Chiang Mai,Thailand. The root was washed with water,cut into small pieces, dried in an oven at60°C and then ground into a fine powder.One hundred grams of the root powder wassubjected to continuous hot extraction in a

Chiang Mai J. Sci. 2014; 41(5.1) 1123

soxhlet apparatus with 50% ethanol (700 ml)as a solvent. The extract was evaporated underreduced pressure using a rotary evaporatorand then freeze-dried respectively.

The extract was subjected to qualitativephytochemical screening according to themethod of Rasool et al [22].

Determination of total phenolic and,ascorbic contents and antioxidant activityof the extract

Total phenolic content was quantitativelydetermined by the procedure described bySingleton and Rossi [28]. Ascorbic acid contentof the root extract was determined accordingto the method of Schlessier et al [25].The scavenging effect of the extract on theDPPH radical was examined based on themethod of Brand-William et al [9]. The ABTsassay was measured by the method of Reet al [23].

2.3 Acute Toxicity TestFemale albino rats weighing 100 to

120 g were used for the acute toxicity study.The study was carried out as per OECDguideline 423[20]. No adverse effects ormortality were detected in the rats at up to2 g/kg, p.o., during the 24-hour observationperiod. Based on the results obtained fromthis study, and on the recommended dailyintake in humans, the determined dose forgastroprotective activity was fixed to be 250,500 and 750 mg/kgBW.

2.4 Study of The Gastroprotective Effectand Antioxidant Activity of Paederiapilifera Using an Ethanol Induced GastricUlcer Method2.4.1 Gastroprotective study

The effects of Paederia pilifera root extractand omeprazole on the ethanol-inducedgastric damage were determined. Thirty maleWistar rats weighing between 180-200 gwere divided into 5 groups with 6 rats in

each group. Group 1 rats were a negativecontrol group that received 1 ml of distilledwater. Group 2 rats received 8 mg/kg bodyweight of omeprazole and served as a positivecontrol group. Groups 3, 4 and 5 rats receivedroot extract at doses of 250, 500 and750 mg/kg body weight, respectively.The animals were given the root extract orallyfor a period of 15 days. On the 15th day,thirty minutes after pretreatment with theextract and drug, gastric ulcers were inducedwith absolute ethanol (5ml/kg body weight)in all rats, which had been fasted overnight.The animals were later sacrificed byan overdose of ether and their stomachswere immediately excised. The stomachswere opened along the greater curvature andwashed with normal saline solution.

2.4.2 Assessment of ulcer index andpercentage inhibition

The ulcer lesions were examined andscored (mm2) under light microscopes.The opened stomachs were cut in halfand the right part was scraped with a glassslide to obtain samples of gastric mucosa.The samples were stored at 4°C forsubsequent antioxidant activity assay. Ulcerindex and percentage inhibition werecalculated using the following formulas:

Ulcer index (UI) =

Percentage Inhibition =

2.4.3 Histological evaluationThe left part of each stomach was fixed

in 10% buffered formalin. Tissues weredehydrated and processed for histologicalexamination using standard techniques.Sections were cut into 6 μm thickslicesand stained with hematoxylin and eosin. Thesections were mounted and histopathological

Number of animals ulcerated

total ulcer score

UI of ethanol treated group

UI of ethanol treated group-UIof pretreated groups × 100

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changes were observed under a lightmicroscope.

2.4.4 Estimation of antioxidant activitiesThe mucosal scrapings were weighed

and gastric mucosa homogenates wereprepared in 20 mM Tris HCl buffer(100g/L). The homogenate was centrifugedat 10,000 g for 20 minutes using a coldcentrifuge. The supernatant was used tomeasure MDA, GSH, CAT and SOD.

Lipid peroxidation in gastric mucosawas estimated by determination ofmalondialdehyde (MDA) using thethiobarbituric acid reaction substances(TBARS) test according to the method ofBuege and Aust [10]. Superoxide dismutase(SOD) activity was measured according to themethod of Xin et al [30]. The amount ofglutathione (GSH) was estimated accordingto the method of Sedlak and Lindsey [26].Catalase activity was determined accordingto the Goldblith and Proctor method [12].The protein content was assayed by themethod used by Bradford [8].

2.5 Statistical AnalysisResults were expressed in terms of mean

± SD. The statistical significance of differencesbetween groups was assessed using one-wayANOVA followed by a least significantdifference test (LSD). A value of p< 0.05 wasconsidered significant.

3. RESULTS AND DISCUSSIONThe phytochemical analysis confirmed

the presence of various components, such asalkaloids, glycones, terpenoids, saponins,phenols and anthraquinones in the rootextract of P. pilifera. The yield of the extractwas 6.71% w/w.

3.1 Determination of Total Phenolic andAscorbic Contents and AntioxidantActivity of P. pilifera Extract

The root extract of P. pilifera exhibiteda high total phenolic content (58±0.01 mggallic/g extract). The ascorbic acid contentin the extract was found to be 35±0.05 mg/gextract. The free radical scavenging activitiesof the extract were measured by DPPHand ABTS assays. The extract scavengedthe DPPH and ABTS radicals with an IC50

value of 17.02 mg/ml and 57.57 mg/ml,respectively.

3.2 Study of Gastroprotective EffectsThe oral administration of absolute

ethanol induced elongated reddish bands ofhemorrhagic erosions in the gastric mucosa(Figure 1b). Mild hemorrhagic erosionsdeveloped in the gastric mucosa ofthe omeprazole treated group (Figure 1c).P. pilifera treated groups showed a dose-dependent decrease in hemorrhagingwhere compared with the ethanol group(Figure 1d,e,f).

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Figure 1. Gross appearance of gastric mucosa in rats: (a) gastric mucosa of a normal controlrat (b) gastric ulcerated rat group (c) omeprazole treated group, and (d)(e)(f) pretreatmentwith P. pilifera extract at the doses of 250, 500 and 750 mg/kg BW, respectively.

a b

c d

e f

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The gastric ulcerated group revealed thehighest ulcer index rating (Table 1). As shownin Table 1, pretreatment with P. pilifera extractand omeprazole exhibited a significantreduction in the ulcer index, when comparedwith the gastric ulcerated rat group (p < 0.05).The percentage inhibitions were 75.62%,

81.17% and, 91.03% in rats treated with 250,500 and 750 mg/kg BW of the extract,respectively, while the rats treated withomeprazole showed a lower percentage ofinhibition. Ulcer index and percentageinhibition in the rat treated with the extractwere determined to be dose dependent.

Table 1. Gastroprotective effect of P. pilifera extract on ethanol-induced gastric lesion in rat.Experimental Groups Ulcer index % inhibition

Normal control - -Gastric ulcerated group 84.09±16.4a -P. pilifera 250 mg/kg 20.50±6.40b 75.62P. pilifera 500 mg/kg 15.83±1.00b 81.17P. pilifera 750 mg/kg 7.54±1.40c 91.03Omeprazole 8 mg/kg 31.64±0.50d 62.37

Data are presented as mean ± standard deviation. Differences in superscript letters are considered significantlydifferent (p < 0.05).

Gastric ulcers occur due to an imbalancebetween aggressive factors and endogenousdefensive factors. Gastric mucus andbicarbonate secretion, maintenance ofmucosal blood flow and prostaglandinproduction are known to protect thegastric mucosa against damage. It is knownthat increases of gastric acid secretion,reduced mucosal blood flow, inhibition ofprostaglandin production and a decrease ofgastric mucus enhance the susceptibility ofthe mucosa to injuries induced by theaggressive factors [11]. Ethanol is an aggressivefactor which induces gastric ulceration.It rapidly penetrates the gastric mucosa,promotes membrane damage and ulcerformation through a destruction of the mucusbarrier, increases vascular permeability anddamages the capillary endothelium [3, 21].Ethanol also has the ability to induce theformation of leucotriene, a lipoxygenasederived metabolite of arachidonic acid, andinhibits the agents that enhance the mucosaldefensive factors, such as prostaglandin [17].We showed that the administration of ethanolproduced significant gastric damage in rats,

while the damage was reduced in the extractpre-treated groups. As shown in Table 1,pretreatment with P. Pilifera extract at the dosesof 250, 500, 750 mg/kg BW revealed aprotective action against ethanol-inducedgastric mucosa damage as demonstrated bythe reduction of the gastric ulcer area.

This result was confirmed by thehistological examination showing a preventionof mucosal hyperemia, edema and epitheliumdisruption found in gastric ulcer induced rats.Histological observations of ethanol inducedgastric damage showed severe disruption ofthe surface epithelium, hemorrhaging in themucosa and deep mucosa, edema andneutrophills infiltration of the submucosallayer (Figure 2b). Mild epithelium disruptionsand edema of the submucosa developed inthe gastric mucosa of the omeprazole treatedgroup (Figure 2c). Pretreatment withP. pilifera extract provided better protectionof the gastric mucosa than did the drug.This was indicated by a reduction of damagedmucosal epithelium, reduced or absent edemaand neutrophills infiltration in the submucosa(Figuresure 2d,e,f ).

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Figure 2. Histological study of gastric mucosa in rats: (a) Normal control rat; (b) gastriculcerated rat damage showing severe disruption in the surface epithelium (1), hemorrhage inmucosa and deep mucosa (2), edema and neutrophills infiltration of the submucosal layer (3);(c) omeprazole treated rat showing mild epithelium disruptions (1) and edema of submucosa(2); (d) pretreatmant with P. pilifera extract at a dose of 250 mg/kg BW showing mild epitheliumdisruptions (1) and submucosa edema (2); (e) pretreatmant with P. pilifera extract at a dose of500 mg/kg BW; (f) pretreatmant with P. pilifera extract at a dose of 750 mg/kg BW.

a b

c d

e f

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It is clear that P. pilifera extracteffectively prevents mucosal damage.The gastroprotective effect of P. piliferaextract may be attributed to variouscompounds in the extract, includingphenolic compounds, vitamin C, alkaloids,flavonoids, saponins and glycosides.These compounds have been found topossess antiulcer activities. Flavonoids andphenolic compounds increase mucosalprostaglandin production. Prostaglandinsare found to produce a gastroprotectiveeffect by decreasing acid secretion and alsoincreasing gastric mucus and bicarbonatesecretion, promoting angiogenesis andtriggering mucosal cell proliferation [5].It is possible that P. pilifera extract mightbe rich in flavonoids and phenoliccompounds, and could enhance the functionof prostaglandins. Flavonoids have beenshown to increase the formation of newcapillaries, decrease histamine secretion bythe inhibition of histidine decarboxylaseand chelate free radicals and reactive oxygen

species, while inhibiting leucotriene [16].The protective activities of saponins areprobably due to the activation of the mucusmembrane protection factors and theimprovement of blood flow [4, 7]. Alkaloidshave been reported to inhibit gastric acidsecretion in rats [16].

3.3 Estimation of Antioxidant ActivitiesThe effects of the pretreatment with

P. pilifera extract on enzymatic andnonenzymatic antioxidants in gastricmucosa are presented in Table 2. Ascompared to normal control rats, ulceratedrats showed significant an increase in thelevel of MDA in gastric mucosa and adecrease in SOD, CAT activities and GSHlevels (p < 0.05). Rats pretreated withP. pilifera extract at doses of 250, 500 and750 mg/kg BW and omeprazole showed asignificant decrease in lipid peroxidationand an increase in the activities of theantioxidant enzymes, as well as glutathionelevels.

Table 2. Effects of pretreatment with P. pilifera extract and omeprazole on lipid peroxidationand the antioxidant activities in the gastric mucosa of rats.

Treatment

Normal ControlEthanolP. pilifera250 mg/kgP. pilifera500 mg/kgP. pilifera750 mg/kgOmeprazole8 mg/kg

MDA(μmol/mg

protein)3.68 ± 0.21a

4.82 ± 0.00b

4.53 ± 0.04c

3.96 ± 0.57a

2.95 ± 1.26a

4.45 ± 0.35ac

CAT(U/mg protein)

0.0320 ± 0.004a

0.0043 ± 0.000b

0.0119 ± 0.0026c

0.0190 ± 0.0089c

0.0225 ± 0.0082ac

0.0111 ± 0.0010c

SOD(μmol/min/mg

protein)7.05 ± 0.00a

0.87 ± 0.12b

2.27 ± 0.24c

2.21 ± 0.15c

6.45 ± 0.14d

1.23 ± 0.15e

GSH(nmol/mg

protein)132.5 ± 3.54a

14.0 ± 0.00b

55.0 ± 12.73c

61.5 ± 7.78c

73.0 ± 2.83d

53.5 ± 4.90e

Data are presented as mean ± standard deviation. Differences in superscript letters are considered significantlydifferent (p < 0.05).

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Reactive oxygen species have animportant role in the pathogenesis ofvarious diseases, including gastric ulcers.The administration of ethanol results in anoverproduction of free radicals, such assuperoxide, hydroxyl and peroxyl radicals[18]. The production of free radicals leadsto increased lipid peroxidation resultingin an increased MDA level. Preventiveantioxidants, such as CAT, SOD and GSH,are the first line of defense against ROS.In the present study, the administration ofethanol significantly increased the levels ofMDA and decreased CAT, SOD activitiesand GSH levels in the gastric mucosa incomparison to the normal control group(Table 2). It is obvious that ethanol causedthe gastric injury by reducing the CAT andSOD activities, which were associated withincreased oxidative damage. Pretreatmentwith P. pilifera extract and omeprazole showedsignificant increases in SOD, CAT activitiesand GSH levels and a decrease in MDA levels.Plant chemical substances, such as phenoliccompounds and flavonoids which are presentin P. pilifera extract have been shown toscavenge free radicals. The root extract ofP. pilifera exhibited a high total phenoliccontent, which appears higher than thephenolic content that was detected in otherplant extracts that were reported to possessgastroprotective effects [2, 19]. Ascorbic acidis related to the positive effects on antioxidantsystems [15]. It acts as a chain breakingantioxidant, which impairs the formation offree radicals in the intracellular substancesthroughout the body. The qualitativedetermination of ascorbic acid in the extractof P. pilifera shows that it is a good source ofascorbic acid. This extract was found topossess antioxidant activity when tested inDPPH and ABTS assays. Thus, it is possiblethat the antioxidant activity of the root extractcontributes to its anti-gastric ulcer activity.

The extract components, known for theirantioxidant and antiulcer activities, have a highpotential for antioxidant activity and thegastroprotective effect.

4. CONCLUSIONThe results of our study provide

additional support for the traditional use ofthis plant as a gastric antiulcer drug.The root extract of P. pilifera hasantioxidant activity and gastroprotectiveeffects on ethanol-induced gastric ulcers.The cytoprotective and antioxidantproperties of the extract are due to thepresence of one, or a combination ofbioactive compounds in the root. This findingcould lead to the further isolation andpharmacological study of new therapeuticcompounds against ulcers. Therefore, furtherstudies are required to investigate the activecompounds and the mechanisms of actionof P. pilifera extract.

ACKNOWLEDGEMENTSWe wish to thank Doctor Pinkew

Ton-Nuan, a Lanna folk healer, forknowledge and advice about medicinal plants.Our sincere thanks are also extended to theGraduate School, Chiang Mai Universityand TRF-Master Research Grants for thefinancial support. Finally, we wish to thankMr. Russell Kirk Hollis for his review of thismanuscript.

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