Gastroprotective activity of polyphenolic-rich extract of Potentilla mooniana

Abstract

Context: Potentilla mooniana Wight. (Rosaceae) is a plant found in the Himalayan region where the root is traditionally used to treat stomach problems including gastric-ulcer.

Objective: To scientifically validate the gastro-protective effect and derive the possible mechanistic activity of the ethanol root extract from P. mooniana (EPM).

Materials and methods: The gastroprotective effect of EPM (100–400 mg/kg, p.o.) was evaluated on both the physical (Pyloric ligation, PL; Cold restrain stress, CRS) and chemical (absolute ethanol, EtOH; aspirin, ASP) ulcerogens induced ulceration in rats. The mechanistic activity of EPM was tested on various gastric-ulcer parameters, namely gastric pH, volume, acid–pepsin output, DNA content, histamine level, H⁺K⁺–ATPase activity, mucus content, microvascular permeability, antioxidant markers, and gastric-histopathological study.

Results: EPM significantly reduces the ulcer score against all the four tested gastric-ulcer models. In the PL model, EPM showed significant reduction (p < 0.05) in acid–pepsin output and cell shedding; however, no significant effect was observed on gastric volume, cell proliferation, stomach glandular weight, and histamine levels. EPM (400 mg/kg, p.o.) when compared with ulcer control showed significant increase in gastric pH by 41.6% and decrease in H⁺K⁺–ATPase activity by 47.73%. In addition, EPM showed significant increase in mucus content by 58.60% and a decrease in the microvascular permeability of Evans Blue by 85.00%, justifying its protective effects. Furthermore, EPM also showed significant antioxidant activity and histopathologically possessed excellent cytoprotective effect.

Conclusion: The gastro-protective effect of EPM is attributed mainly to the defensive mechanism owing to the presence of a good quantity of polyphenolic components.

• Antioxidant
• ethanol
• gastric-ulcer
• pyloric ligation
• mucus
• polysaccharides
• histopathology

Introduction

For more than a century, peptic ulcer was considered as the most frequent cause of surgery worldwide, with an increasing rate of morbidity and mortality (Yuan et al., 2006). Agents that may initiate the development of gastric ulcers are acid–pepsin secretion, stress, alcohol abuse, consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and infection with Helicobacter pylori (Laine et al., 2008; O’Malley, 2003). It is claimed that reactive oxygen species (ROS) also plays a significant role in the pathogenesis and development of gastric damage (Repetto & Llesuy, 2002). Until now, the current drug therapy given to the patients suffering from gastric ulcers is of synthetic nature such as antacids, sucralfate, proton pump inhibitors, prostaglandins, muscarinic, and histamine-(H₂) receptor antagonists. However, the long-term use of these drugs may cause several unwanted side effects. Hence, a demand for the use of an alternative medicine comes into play for the search of new, more effective and safer treatments, with lesser side effects. In this context, plant extracts stand out as the most promising substances in the search for new therapies for the treatment of gastric ulcer (Santin et al., 2010).

The genus Potentilla includes about 500 species that are of perennial, rarely biennial or annual, herbs and shrubs of the family Rosaceae. The genus name Potentilla is derived from the Latin diminutive of potens meaning “powerful” in reference to the medicinal properties of some species (Tomczyk & Latte, 2009). Potentilla mooniana Wight. (Syn. Potentilla polyphylla Wall. ex. Lehm.) is popularly known as many leaved Cinquefoil (English), Masi (Hindi), and Lynniang shynrang (Khasi tribes of Northeast India). The plant is commonly found in Asia at an altitude of 1500–3660 m (Anonymous, 1968). Traditionally, the root and sometimes the whole plant are used by the local healers (from Northeastern India) to treat gastric problems, mouth ulcers, and also act as stimulant and antiseptic. Sometimes the whole plant is ground and the juice extracted from it is taken for colic and spasmodic pain (Ahmed & Borthakur, 2005; Selvam, 2008). Since there is no evidence or detailed scientific investigation to define the gastroprotective activity of P. mooniana, the present work was aimed at studying the gastric-ulcer protective effects and mechanism of action of the ethanol root extract of P. mooniana (EPM) in various experimental models, namely pyloric ligation (PL), cold restrain stress (CRS), ethanol (EtOH), and aspirin (ASP)-induced gastric ulcer models.

Material and methods

Plant material, identification and extraction procedure

Fresh root of P. mooniana was collected and supplied by Mr. H. Carehome Pakyntein (Local healer and President of Jaintia Indigenous Medicine Association, Meghalaya, India) in the month of June–September (2010) from Jowai town region of the Jaintia Hills District, Meghalaya, North-East India. The botanical identification of the specimen was done by Dr. N. Odyuo (Scientist C in-charge), Botanical Survey of India, Shillong, Meghalaya (BSI/ERC/2010/Plant identification/281).

The fresh roots, after prewashing in running tap water, were dried under shade for 1 week. The roots were grounded into coarse powder using a mechanical grinder and passed through sieve no. 60. Extraction of coarse powdered drug (1 kg) was exhaustively extracted in a Soxhlet extractor using 95% ethanol (3.5 L) for 3 d. The extract was filtered and concentrated under reduced pressure to yield the crude ethanol extract of P. mooniana (EPM).

Phytochemical evaluation

The preliminary phytochemical screening of EPM was done to detect the presence of various phytochemical classes (Trease & Evans, 2002). Spectrophotometric quantification of phytochemical classes present in EPM was evaluated for the total content of phenolics and tannins (Makkar, 2000), flavonoids, and flavonols (Kumaran & Karunakaran, 2006), saponins (Hiai et al., 1976), sapogenin (Baccou et al., 1977), and total carbohydrate content (Yemm & Willis, 1954).

Experimental setup and drug treatment protocol

The gastric ulcer study was carried out on inbred Charles–Foster albino rats of either sex (150–180 g). The pharmacological experiments were performed after getting necessary approval from the Institutional Animal Ethical Committee, Institute of Medical Sciences, Banaras Hindu University (Approval no.: Dean/10-11/59). The rats were housed under standard laboratory conditions and were fed with standard commercial rat feed (Amrut Pvt. Ltd., Chembur East, Mumbai, India) and water ad libitum. In all the experimental models, the animals were divided into seven groups, each group containing six animals (n = 6). All the standard and tested drugs were suspended in 0.5% carboxymethyl cellulose (CMC) and administered orally (by gavage) for a period of 7 d.

• Group I rats (normal control; NC) received distilled water as vehicle.

• Group II rats (ulcer control; UC) received 0.5% carboxymethyl cellulose (CMC) in distilled water.

• Groups III and IV rats received standard drugs omeprazole (20 mg/kg, p.o.; OMZ 20) and ranitidine HCl (50 mg/kg, p.o.; RTD 50), respectively.

• Groups V, VI, and VII rats received EPM at dose levels of 100, 200, and 400 mg/kg, p.o., respectively.

Fasting of the animals (with free access to water ad libitum) in all the gastric ulcer models was done for 18 h prior to all the experiments conducted.

Acute oral toxicity study

The acute oral toxicity study of EPM was performed according to the up and down methods for dose selection as proposed in Organization for Economic Co-operation and Development Guidelines no. 425 (OECD, 2008). Inbred Charles–Foster albino rats of either sex (150–180 g) were used for the study. EPM was administered to the overnight fasted rats at two dose levels: 2000 and 5000 mg/kg, b.w (p.o). The toxicity signs and symptoms or any abnormalities associated with the EPM administration were observed within 24 h and then daily up to 14 d. The number of rats that survived was recorded at the end of the study period.

Gastroprotective activity studies

On the seventh day and after the last dose of drug administration, physical and chemical ulcerogens were used to induce ulcer to the overnight fasted rats, described as below.

Gastroprotection against pylorus ligation-induced ulcer (PL)

Pylorus ligation (PL) on the fasted rat stomach was done under anaesthetized conditions using pentobarbitone (35 mg/kg, i.p.). The abdomen was opened and the ligation was made at the pyloric end without causing any damage to the blood supply. The stomach was carefully replaced and the abdomen was closed with interrupted sutures. During this post-operative period, the rats were deprived of water up to 4 h (Oliveira et al., 2004). After 4 h, stomachs were finally dissected out and cut open along the greater curvature and ulcers were scored by a person unaware of the experimental protocol in the glandular portion of the stomach (Sanyal et al., 1983). The ulcer index was monitored and calculated by adding the total number of ulcers per stomach and the total severity of ulcers per stomach. The total severity of the ulcers was determined by recording the severity of each ulcer after histological confirmation as shown below:

• [0]: no ulcer.

• [+]: pin point ulcer and histological changes limited to superficial layers of mucosa and no congestion.

• [++]: ulcer size less than 1 mm and half of the mucosal thickness showed necrotic changes.

• [+++]: ulcer size 1–2 mm with more than two-thirds of the mucosal thickness destroyed with marked necrosis and congestion, muscle is remaining unaffected.

• [++++]: ulcer either more than 2 mm in size or perforated with complete destruction of the mucosa with necrosis and hemorrhage, muscle is still remaining unaffected.

The pooled group ulcer score was then calculated according to the method of Sanyal et al. (1983); and the % protection was calculated by using the following formulae.

Gastroprotection against ethanol-induced ulcer (EtOH)

In the EtOH-induced ulcer model, gastric ulcers were induced by administering absolute ethanol (1 mL/200 g, p.o., 1 h). The animals were killed by decapitation and their stomachs were incised along the greater curvature and finally examined for ulcers. Ulcer index (in mm²) was scored following the standard method of Hollander et al. (1985).

Gastroprotection against cold restrain stress-induced ulcer (CRS)

Cold restrain stress was induced by strapping the animal’s fore and hind limbs on a wooden plank and kept for 2 h, at a temperature of 4–6 °C (Sairam et al., 2003). Later, the animals were killed by decapitation and ulcers were examined on the dissected stomachs. Ulcer indices were calculated and scored as per method described under PL-induced ulcer model.

Gastroprotection against aspirin-induced ulcer (ASP)

On the day of the experiment, aspirin at a dose of 200 mg/kg p.o. was administered to the overnight fasted animals (Rao et al., 2004). The animals were sacrificed after 4 h of aspirin administration and the ulcers were scored as per the method described in the PL-induced ulcer model.

Biochemical parameters

Determination of gastric volume, pH, acid–pepsin output, and mucin activity

The determination of gastric volume, pH, acid–pepsin output, and mucin activity was evaluated after the gastric juice was collected from 4 h PL rats, which was centrifuged at 3000 rpm for 10 min at 25 °C. The gastric juice was determined for the total volume content (mL/100 g body weight), gastric pH (using pH meter), acid output by titrating with 0.1 N NaOH using Topfer’s reagent (free acid) and phenolphthalein as indicators (expressed as μEq/4 h acid output), and total pepsin output using hemoglobin as a substrate (expressed as μmol tyrosine/4 h) (Sairam et al., 2003). The dissolved mucosubstances (mucoprotein) in the gastric juice (precipitated by adding 90% alcohol) was evaluated for total carbohydrate (hexose, hexosamine, fucose, and sialic acid) (Sanyal et al., 1983) and protein content (Lowry et al., 1951). The results were expressed as μg/mL gastric juice and the ratio of total carbohydrate to protein (TC: P) was taken as an index of mucin activity. Total DNA content in the gastric juice (cell shedding) was estimated and expressed as μg DNA/mL gastric juice (Mukhopadhyay et al., 1987).

Determination of mucin activity on gastric juice and mucosal scrapings

The mucosal scrap obtained from the 4 h PL rat stomach was dissolved in 90% alcohol and was estimated for total mucosal glycoproteins (total carbohydrate to protein ratio) as described above for gastric juice content (Sairam et al., 2003). Stomach glandular weight and DNA content (cell proliferation) of mucosal scrap (homogenized in 2.5 mL ice cooled 0.6 N perchloric acid) were estimated. Results for cell proliferation were expressed as μg DNA/100 mg tissue (Mukhopadhyay et al., 1987).

Estimation of mucus content

The mucus content obtained from the 1 h absolute EtOH-induced ulcer rats was estimated by reacting with Alcian blue dye solution (Corne et al., 1974). The concentration of the dye was determined spectrophotometrically at 580 nm from the Alcian blue standard calibration curve. The mucus content was calculated and expressed in terms of μg Alcian blue/g wet tissue.

Determination of gastric mucosal microvascular permeability

The microvascular permeability test was studied on 1 h absolute EtOH-induced gastric ulcer rats using the Evans blue dye (EBD) as an indicator for increased capillary permeability. On the day of the experiment and after the drug administration, the rats were gavaged with absolute EtOH (1 mL/200 g) for 1 h. Thirty minutes before the animals were sacrificed; EBD (10 mg/kg; i.v.) was administered. The stomach was removed and the scrapped mucosal tissue obtained was soaked overnight in a stoppered glass tube containing 1 mL 1N KOH at 37 °C. Then, 9 mL of a mixed solution of 0.6 N phosphoric acid and acetone (5:13) were added to the tube, shaken vigorously for a few seconds, and centrifuged at 3000 rpm for 15 min. Absorbance of supernatant was measured at 620 nm and the result was expressed as μg Evans blue dye/g of tissue (Katayama et al., 1978).

Determination of histamine content

The histamine content was estimated in the stomach tissue of the animals following the spectroflourimetric methods of determination at 450 nm emission and 360 nm activation (Shore et al., 1959). The results were expressed as μg histamine liberated/g tissue.

Determination of H⁺K⁺–ATPase inhibitory activity

The evaluation of H⁺K⁺–ATPase enzymatic activity was done in gastric microsomes of 4 h PL rats (Dorababu et al., 2006). The stomach mucosal scrapings bearing the parietal cells were isolated, dissolved, and suspended in EGTA–sucrose buffer solution (250 mM sucrose, 2 mM MgCl₂, 1 mM EGTA, and 2 mM Tris maintained at pH 7.4). The gastric microsomes were prepared by successive centrifugation at 3000 rpm for 10 min and then at 20 000 rpm for 30 min twice (4 °C). The resultant supernatant was discarded and the microsome pellet obtained was dissolved and homogenized in mannitol buffer solution (250 mM mannitol, 2 mM magnesium chloride, and 2 mM Tris maintained at pH 7.4). The total protein was estimated as per the methods of Lowry et al. (1951), whereas the H⁺K⁺–ATPase inhibitory activity was assayed by the methods of Reyes-Chilpa et al. (2006). About 0.1 mL homogenate containing the enzymes was mixed with 0.2 mL Tris-HCl (20 mM, pH 7.4), 0.2 mL 2 mM MgCl₂, and 0.2 mL 2 mM KCl. The reaction mixture was initiated by adding 0.2 mL 2 mM ATP, incubated at 37 °C for 30 min and later terminated by adding 10% tricholoroacetic acid followed by centrifugation at 2000 × g for 10 min. The amount of inorganic phosphate (P_i) liberated from the ATP was measured spectrophotometrically at 640 nm following the methods of Griswold et al. (1951). Results of the final solution were expressed as μM P_i liberated/mg protein/h.

Antioxidant and free radical determination

Tissue mucosal scrap obtained from the rat stomach of 1 h absolute EtOH-induced gastric ulcers was used for the determination of antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and lipid peroxides (LPO). The mucosal scrap was homogenized for 30 s in 0.9% ice cold saline solution, which was centrifuged initially at 800 × g for 10 min and later at 12 000 × g for 15 min. The clear supernatant containing the mitochondrial fraction was used for the following estimations (Goel et al., 2001). The LPO level was estimated in terms of malondialdehyde (MDA) which was determined as per the methods of Ohkawa et al. (1979). SOD was determined as per the methods of Kakkar et al. (1984). The results were expressed as units (U) of SOD activity/g tissue. The decomposition of hydrogen peroxide (H₂O₂) in the presence of catalase enzyme was estimated as per the methods of Beers and Sizer (1952). Results obtained are expressed as units (U) of CAT activity/g of tissue. GSH level was estimated following the method of Sedlak and Linsday (1968). Reduced glutathione was used for generating the standard calibration curve. Absorbance of all the solutions was measured spectrophotometrically at 412 nm and the results were expressed as μg of GSH/g of tissue.

Histopathological studies

Stomach sample (glandular portion) of all the untreated and treated rats obtained from 1 h absolute EtOH-induced ulcerated rats were fixed in 10% formalin which was embedded in paraffin blocks for microtome sectioning. The obtained sections (1–3 μm) were stained with hematoxylin and eosin dye and finally monitored and photographed using a Nikon digital microscope (Eclipse 200, Nikon Instruments Inc., Melville, NY) at 10 × magnifications.

Statistical analysis

All the results were expressed as mean ± S.E.M. (n = 6). The analysis of variance was performed with Graphpad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA) using a one-way ANOVA followed by Tukey’s post test for multiple group comparison. The difference was considered to be significant when p < 0.05.

Results

Phytochemical evaluation of EPM

The ethanol extract of EPM obtained from Soxhlet extraction was found to produce a percentage yield of 20.8% w/w air dried plant material. The qualitative phytochemical screening of EPM showed the presence of phenolics, tannins, flavonoids, carbohydrates, saponins, steroids, amino acids, coumarins, and anthraquinone glycosides. Quantification of total phenolic and tannin content in EPM was found to be 84.15 and 65.31 mg/g (tannic acid equivalent per gram plant material), respectively. Total flavonoids and flavonols content was found to be 9.53 and 2.01 mg/g (rutin equivalent per gram plant material), respectively. Total saponins and sapogenin content was found to be 20.75 and 15.4 mg/g, respectively (diosgenin equivalent per gram plant material), whereas total carbohydrate content was found to be 56.8 mg/g (d-fructose equivalent per gram plant material).

Acute oral toxicity studies

From the acute oral toxicity study, it was observed that EPM at tested doses 2000 and 5000 mg/kg, p.o., produce no behavioral sign, symptoms of toxicity or mortality on the experimental animals. Hence, the drug was found to be safe up to 5000 mg/kg, p.o.

Gastroprotective activity studies

Oral administration of EPM graded doses for 7 d showed a protective activity that is significantly effective against all the physical (PL and CRS) and chemical (EtOH and ASP) ulcerogens, when compared with the ulcer control group. However, the maximum gastroprotective effect of EPM was observed in EtOH-induced ulceration with a percentage protection of up to 92.14% which is significantly more effective as compared with the standard omeprazole (83.33%) and ranitidine (69.18%), respectively. The scoring of the ulcer index and percentage inhibition is shown in Table 1.

Table 1. Effect of oral administration of EPM, ranitidine and omeprazole on physical (pylorus ligation and cold restrain stress) and chemical (ethanol and aspirin) induced ulcerogens in experimental rats.

		Physical induced ulcerogens				Chemical induced ulcerogens
		Pylorus ligation		Cold restrain stress		Ethanol		Aspirin
Groups/treatment	Dose (mg/kg, p.o.)	Ulcer Index (mm^2)	% Protection	Ulcer Index	% Protection	Ulcer Index	% Protection	Ulcer Index	% Protection
Ulcer control	0.5% CMC	18.0 ± 2.9	–	16.8 ± 1.8	–	31.8 ± 1.8	–	27.3 ± 3.7	–
Ranitidine	50	4.80 ± 1.1^a	73.33	4.80 ± 0.9^a	71.43	9.8 ± 1.3^a	69.18	9.2 ± 1.8^a	58.74
Omeprazole	20	6.00 ± 0.7^a	66.66	7.70 ± 1.1^a	54.16	5.3 ± 1.0^a	83.33	6.8 ± 1.7^a	69.51
EPM	100	13.3 ± 2.2^b	26.11	11.5 ± 1.1^b	11.90	5.0 ± 1.2^a	84.27	16.8 ± 1.9^c	24.66
EPM	200	6.80 ± 1.7^a	62.22	9.80 ± 1.2^a	30.95	2.8 ± 0.7^ab	91.20	14.2 ± 2.2	36.32
EPM	400	7.00 ± 1.1^a	61.11	8.80 ± 1.4^a	47.61	2.5 ± 0.6^ab	92.14	13.8 ± 2.5^a	38.11

Values are expressed as mean ± S.E.M (n = 6 in each group). Statistical comparison was analyzed by a one-way ANOVA followed by Tukey’s multiple comparison tests. ^ap < 0.05, statistically significant as compared with the ulcer control; ^bp < 0.05, statistically significant as compared with the ranitidine; ^cp < 0.05, statistically significant as compared with the omeprazole.

Effect of EPM on gastric volume, pH, acid-pepsin output and mucin activity

The effect of EPM (200–400 mg/kg, p.o.), ranitidine, and omeprazole on gastric secretion measurements in 4 h PL-rats showed significant decrease in the volume of gastric acid accompanied by a fall in total acid–pepsin output (Table 2). However, the effect on gastric pH was observed only at higher doses when the rats were pre-treated with EPM at 400 mg/kg (p.o.) dose. EPM (200–400 mg/kg, p.o.) also showed significant effects on the mucin activity (TC: P) in both gastric juice mucoproteins and mucosal scrap glycoproteins whereas no effect was seen with that of the standard drug omeprazole and ranitidine (Table 3). The increase in mucin activity was observed specifically from the increased level of hexosamine and sialic acid content.

Table 2. Effect of oral administration of EPM graded doses on gastric juice parameters in 4 h PL rats for 7 d.

			Acidity (µEq/mL)			Pepsin
Groups/treatment	Volume (mL/100 g)	pH	Free acid	Total acid	Total acid output (µEq/4 h)	Conc. (µmol tyrosine/mL)	Output (µmol tyrosine/4 h)
			41.00 ± 6.9	103.3 ± 7.1	209.8 ± 31.61	403.0 ± 38.7	834.8 ±118.8
Ranitidine 50	0.51 ± 0.08^a	4.33 ± 0.44	26.70 ± 4.6	75.33 ± 8.0	86.26 ± 19.6^a	282.7 ± 32.9^a	248.7 ± 28.9^a
Omeprazole 20	0.69 ± 0.11	6.27 ± 0.41^ab	16.60 ± 2.8^a	64.00 ± 8.8^a	58.63 ± 14.9^a	201.5 ± 19.6^a	225.6 ± 36.4^a
EPM 100	0.94 ± 0.16	3.42 ± 0.28^c	29.50 ± 5.6	95.00 ± 10.7	107.7 ± 25.20	309.1 ± 36.2^c	519.9 ± 114.7
EPM 200	0.57 ± 0.10	4.00 ± 0.15^c	26.00 ± 7.5	68.67 ± 7.6^ad	70.40 ± 22.2^a	262.7 ± 16.4^a	276.7 ± 65.3^a
EPM 400	0.59 ± 0.09	5.62 ± 0.21^ad	17.66 ± 2.7	30.67 ± 4.8^abe	47.63 ± 14.9^a	199.4 ± 14.3^ad	223.9 ± 51.3^a

Values are expressed as mean ± S.E.M (n = 6 in each group). Statistical comparison was analyzed by a one-way ANOVA followed by Tukey’s multiple comparison tests. ^ap < 0.05, statistically significant as compared with the ulcer control; ^bp < 0.05, statistically significant as compared with the ranitidine; ^cp < 0.05, statistically significant as compared with the omeprazole; ^dp < 0.05, statistically significant as compared to the EPM 100; ^ep < 0.05, statistically significant as compared with the EPM 200.

Table 3. Effect of oral administration of EPM graded doses on content of gastric juice mucoprotein and mucosal scrap glycoprotein in gastric ulcer induced by 4 h PL.

Groups/treatment	Total hexose (TH)	Total hexosamine (THA)	Total fucose (TF)	Total sialic acid (TSA)	Total carbohydrates TC (TH + THA + TF + TSA)	Total proteins (TP)	TC:TP
Gastric juice mucoprotein estimation (µg/mL)
Ulcer control	110.1 ± 11.2	209.0 ± 22.1	28.12 ± 4.6	27.5 ± 3.1	389.6 ± 22.6	250.2 ± 19.6	1.5 ± 0.40
Ranitidine 50	165.3 ± 12.5	288.2 ± 18.3	30.15 ± 2.0	45.4 ± 5.1	519.3 ± 31.1^a	157.5 ± 24.2	3.2 ± 0.29
Omeprazole 20	130.0 ± 16.7	270.2 ± 20.7	41.19 ± 5.8	39.2 ± 3.8	490.5 ± 26.3	200.6 ± 25.6	2.5 ± 0.25
EPM 100	142.7 ± 10.9	249.7 ± 16.4	31.47 ± 2.5	40.1 ± 4.2	489.7 ± 18.5	183.4 ± 20.2	2.5 ± 0.41
EPM 200	166.1 ± 18.0	312.1 ± 26.1^a	35.41 ± 1.2	44.8 ± 5.0	566.8 ± 28.4^a	161.2 ± 22.9	3.5 ± 0.33^a
EPM 400	182.4 ± 28.1	358.2 ± 23.5^ad	41.83 ± 2.8	60.2 ± 4.9^ad	635.4 ± 31.2^abcd	145.4 ± 15.2^a	4.2 ± 0.65^a
Mucosal scrap glycoprotein estimation (µg/100 mg tissue)
Ulcer control	772.4 ± 87.2	1599.4 ± 334.1	131.2 ± 15.0	132.2 ± 20.1	2710 ± 181.2	2131.2 ± 150	1.3 ± 0.22
Ranitidine 50	892.9 ± 77.5	1887.2 ± 271.2	150.5 ± 24.9	206.3 ± 26.7	3150 ± 202.8	1812.4 ± 131	1.7 ± 0.17
Omeprazole 20	911.2 ± 65.4	1789.1 ± 301.4	180.2 ± 25.3	216.2 ± 27.2	3162 ± 298.3	1745.9 ± 103	1.8 ± 0.33
EPM 100	975.2 ± 89.9	2001.2 ± 342.1	140.3 ± 22.5	129.0 ± 20.7	3111 ± 321.5	1952.1 ± 191	1.6 ± 0.30
EPM 200	1034.8 ± 71.2	2411.2 ± 381.2	150.3 ± 18.2	189.2 ± 17.2	3780 ± 271.2^a	1701.3 ± 221	2.2 ± 0.46
EPM 400	1112.2 ± 102	3012.1 ± 166.6^a	201.2 ± 23.5	239.6 ± 16.2^ad	4319 ± 403.1^a	1241.2 ± 120^ad	3.2 ± 0.81^a

All results are expressed as mean ± S.E.M (n = 6 in each group). Statistical comparison was analyzed by a one-way ANOVA followed by Tukey’s multiple comparison tests. ^ap < 0.05, statistically significant as compared with the ulcer control; ^bp < 0.05, statistically significant as compared with the ranitidine; ^cp < 0.05, statistically significant as compared with the omeprazole; ^dp < 0.05, statistically significant as compared with the EPM 100.

Effect of EPM on stomach glandular weight and DNA content (cell shedding and proliferation)

Administration of EPM (200–400 mg/kg, p.o.), omeprazole, and ranitidine showed a significant decrease in the DNA content of the gastric juice (cell shedding) when compared with the ulcerated control group. However, effects on mucosal cell proliferation and stomach glandular weight were found to be insignificant (Table 4).

Table 4. Effect of oral administration of EPM graded doses on gastric juice cell shedding, mucosal cell proliferation and on stomach glandular weight in 4 h PL rats for 7 d.

Groups/treatment	Gastric juice cell shedding (μg DNA/mL)	Mucosal cell proliferation (μg DNA/100 mg wet tissue)	Weight of the glandular portion of stomach (mg/100 g, BW)
Ulcer control	61.1 ± 8.6	121.6 ± 15.5	468.9 ± 29.1
Ranitidine 50	21.4 ± 7.9^a	176.1 ± 16.4	501.5 ± 25.2
Omeprazole 20	27.9 ± 4.2^a	152.8 ± 14.1	518.8 ± 34.6
EPM 100	34.4 ± 6.1	155.8 ± 21.5	565.0 ± 38.2
EPM 200	29.1 ± 5.6^a	187.1 ± 15.2	579.9 ± 44.3
EPM 400	17.6 ± 5.3^a	195.5 ± 23.7	610.8 ± 83.2

All results are expressed as mean ± S.E.M (n = 6 in each group). Statistical comparison was analyzed by a one-way ANOVA followed by Tukey’s multiple comparison tests. ^ap < 0.05, statistically significant as compared with the ulcer control.

Effect of EPM on mucus content

In EtOH-induced gastric ulcer model, oral administration of absolute ethanol (p.o.) to the untreated rats (ulcer control group) when compared with the normal control group showed a significant decrease of 55.2% in the level of the gastric mucus content, signifying a marked depletion of mucus in the stomach. Rats pre-treated with EPM (100–400 mg/kg, p.o.) when compared with the ulcer control group produce a significant effect in increasing the mucus secretion as represented in Figure 1. However, no significant effect on mucus content was observed with that of the standard drug ranitidine and omeprazole.

Figure 1. Quantification of adherent mucus in gastric mucosa of rats treated with EPM (100–400 mg/kg, p.o.), ranitidine and omeprazole in 1 h absolute EtOH induced gastric ulcers. Statistical comparison was analyzed by a one-way ANOVA followed by Tukey’s multiple comparison tests. ^ap < 0.05, statistically significant as compared with the normal control (NC); ^bp < 0.05, statistically significant as compared with the ulcer control (UC); ^cp < 0.05, statistically significant as compared with the ranitidine 50 mg/kg, p.o. (RTD 50); ^cp < 0.05, statistically significant as compared with the omeprazole 20 mg/kg, p.o. (OMZ 20).

Effect of EPM on mucosal microvascular permeability

Standard drugs ranitidine and omeprazole were observed to have a significant activity in decreasing the amount of EBD in the gastric mucosa by 51.73% and 46.00%, respectively. EPM 100 mg/kg, p.o. which was found to be the minimal effective dose, also produced marked decrease in the extravagated amount of EBD by 85.00%, showing significant difference when compared with the ulcerated control group (Figure 2). A significant difference was also observed when EPM (100 mg/kg, p.o.) was compared with that of OMZ (20 mg/kg, p.o.), signifying that the extract of P. mooniana was more effective in decreasing the microvascular permeability as compared with the standard omeprazole.

Figure 2. Effect of minimal effective dose of EPM (100 mg/kg, p.o.) on microvascular permeability (extravagation of Evans blue) in gastric mucosa induced by 1 h absolute EtOH (data are mean ± S.E.M., n = 6 in each group). Statistical comparison was analyzed by a one-way ANOVA followed by Tukey’s multiple comparison tests. ^ap < 0.05, statistically significant as compared with the ulcer control, ^cp < 0.05, statistically significant as compared with the OMZ 20.

Effect of EPM on histamine level

In 4 h PL-model, the ulcer control rats produced a significant increase in the level of histamine by 55.72%, when compared with the normal control group. However, rats treated with standard antihistaminic drug ranitidine (50 mg/kg, p.o.) when compared with the ulcer control group produce significant effect in decreasing the histamine levels by 45.00%. Meanwhile, there was no significant effect observed in all the EPM treated groups, signifying that the extract might not act namely inhibiting the histamine (H₂) receptor (Figure 3A).

Figure 3. Effect of EPM (100, 200, and 400 mg/kg) on histamine content and H⁺ K⁺–ATPase activity in 4 h pylorus ligated rats for 7 d (data are mean ± S.E.M., n = 6 in each group). Statistical comparison was analyzed by a one-way ANOVA followed by Tukey’s multiple comparison tests. ^ap < 0.05, statistically significant as compared with the normal control (NC); ^bp < 0.05, statistically significant as compared with the ulcer control (UC); ^cp < 0.05, statistically significant as compared with the RTD 50 (for histamine estimation)/OMZ 20 (for H⁺K⁺–ATPase activity).

Effect of EPM on H⁺K⁺–ATPase inhibitory activity

In 4 h PL-model, there was a three-fold (74.00%) increase in the level of inorganic phosphate ions (P_i) in the ulcerated control group when compared with the normal control group, indicating the activation of the H⁺K⁺–ATPase pump. Rats treated with a standard proton pump inhibitor (omeprazole 20 mg/kg, p.o.) and higher dose of EPM (400 mg/kg, p.o.) showed a significant decrease in the level of phosphate ions (P_i) when compared with the ulcerated control group, indicating the inhibitory effect of both the drugs on the gastric H⁺K⁺–ATPase enzyme (Figure 3B). However, a lower dose of EPM (100 and 200 mg/kg, p.o.) showed no significant effect.

Effect of EPM on antioxidant enzymes

Administration of absolute ethanol in the ulcerated control showed an increased level of LPO by 62.46% and a decreased level of CAT (67.33%), SOD (59.09%) and GSH (58.40%) when compared with the normal control group significantly. Pre-treatment with EPM graded doses and standard ranitidine significantly maintains the antioxidant enzyme activities leading to a decrease in the LPO levels (Table 5).

Table 5. Effects of oral administration of EPM graded doses on the quantity of LPO, SOD, CAT, and glutathione (GSH) levels in rats with gastric ulcers induced by absolute EtOH.

		Antioxidant enzymes
Groups/treatment	LPO (MDA, nmol/g tissue)	SOD (units/g tissue)	CAT (units/g tissue)	GSH (μg GSH/g tissue)
Normal control	25.9 ± 3.1	37.4 ± 0.5	34.9 ± 1.3	278.1 ± 27.0
Ulcer control	69.0 ± 8.1^a	15.3 ± 3.3^a	11.4 ± 1.1^a	115.7 ± 29.6^a
Ranitidine 50	28.6 ± 7.2^b	28.0 ± 2.3^ab	22.7 ± 2.2^ab	285.3 ± 20.0^b
Omeprazole 20	37.2 ± 4.5^b	30.5 ± 1.2^b	28.9 ± 1.9^b	274.1 ± 25.3^b
EPM 100	41.5 ± 3.7^b	27.9 ± 1.6^ab	25.2 ± 2.6^ab	275.9 ± 25.1^b
EPM 200	38.6 ± 4.7^b	32.9 ± 2.1^b	28.3 ± 1.5^b	325.3 ± 57.9^b
EPM 400	27.5 ± 4.6^b	36.0 ± 1.1^b	31.2 ± 2.1^bc	344.1 ± 36.2^b

All results are expressed as mean ± S.E.M (n = 6 in each group). Statistical comparison was analyzed by a one-way ANOVA followed by Tukey’s multiple comparison tests. ^ap < 0.05, statistically significant as compared with the normal control; ^bp < 0.05, statistically significant as compared with the ulcer control; ^cp < 0.05, statistically significant as compared with the ranitidine.

Histopathological studies

Oral administration of absolute ethanol produces lesions on the gastric mucosa leading to various histopathological changes such as congestion, erosion, necrosis, and hemorrhage, thus, altering the normal architecture of the stomach glandular portion. Pre-treatment with EPM graded doses, ranitidine and omeprazole offered significant protection to the gastric mucosa against all such damages causes by ethanol (Figure 4).

Figure 4. Morphological and histological study of stomach tissues after ulcers induction by absolute ethanol and protection by standard drug omeprazole, ranitidine, and EPM (100, 200, and 400 mg/kg, p.o.). The histopathological sections were stained with hematoxylin and eosin dyes and the photographs were taken with a Nikon digital microscope (Eclipse 200) with 10 × magnification.

Discussion

It is well known that the degree of damage to the gastric mucosa results mainly by various noxious physical and chemical stimuli induced ulceration, which ultimately leads to the imbalance between the defensive and aggressive factors. In the present study, for the screening of gastroprotective activity of EPM, four gastric-ulcer models were selected (PL, CRS, EtOH, and ASP-induced ulceration). Ulcer induction in these models is claimed to be a multifactorial event, but each one has a principle mechanism that explains the etiology of the lesions (García-Barrantes & Badilla, 2011). Based on the results, it was observed that EPM at 400 mg/kg, p.o., showed a significant decrease in the ulcer score in all the tested gastric ulcer models. However, the maximum gastroprotective activity was observed in EtOH and PL-induced gastric ulceration; hence, both these models were selected for the evaluation of mechanism of action of EPM on various aggressive and defensive factors.

Gastric secretion studies play an important role in ulcer induction through pyloric ligations (Singh et al., 2010). Pylorus ligation is a physically induced gastric ulcer model that permits one to study more variables in detail which helps to elucidate the gastroprotective mechanisms of the tested drug(s) from natural or synthetic sources. In the present study, gastric parameters such as gastric volume, pH, acid–pepsin output, gastric mucosal barrier (mucin activity), gastric juice DNA (cell shedding), mucosal cell proliferation, histamine level, and H⁺K⁺–ATPase inhibitory activity were evaluated using the PL-induced gastric ulcer model. The gastric secretion study showed that the total acid–pepsin output decreases significantly when the rats were pre-treated with EPM (200–400 mg/kg, p.o.) and ranitidine (50 mg/kg, p.o.). However, the gastric juice volume secreted by the animals did not produce any significant difference between treatments except with ranitidine where the volume was found to decrease significantly. This phenomenon has also been reported in other studies, indicating that the gastric volume and acid-pepsin parameters are not always closely related and can be independent of each other (Calvo et al., 2007; Hiruma-Lima et al., 2006). Moreover, there is no significant effect produce by EPM on the histamine level when compared with the ulcer control group, thus ensuring that the EPM has no antihistaminic effect. However, the effect of omeprazole and the higher dose of EPM (400 mg/kg, p.o.) on gastric pH were found to increase significantly by 47.00% and 41.00%, respectively. This outcome was further supported by the effect of EPM (400 mg/kg, p.o.) on gastric H⁺K⁺–ATPase inhibitory activity which was found to be significant (p < 0.05) when compared with the ulcerated control group. This activity ensures that higher dose of EMP possesses proton pump inhibitory effect. Many authors also reported the positive relationship between the gastric pH and the H⁺K⁺–ATPase enzymatic activity (Armstrong, 2004; McCarty & Whitaker, 2010). Gastric pH signifies the amount of protons present in the stomach, whereas the H⁺K⁺–ATPase enzymatic activity is an integral membrane protein that actively transports protons (H⁺) and potassium (K⁺) ions across the plasma membrane and is important for acid–base balance and potassium homeostasis (Gumz et al., 2010). The effect of EPM (200–400 mg/kg, p.o.) on mucin activity (defensive factor) was also observed, resulting in a significantly increase in total carbohydrate to protein ratio (TC:P) in both gastric juice mucoproteins and mucosal scrap glycoproteins. The increase in mucin activity produce by EPM was observed from the significant increase in individual mucopolysaccharide (hexosamine and sialic acid) which finally lead to the elevated level of total carbohydrates in the stomach tissue. Hexosamine has been reported as the main biochemical marker responsible for the enrichment of gastric adherent mucus in the stomach glandular tissue (Saxena & Singh, 2011). Mucin is a viscous glycoprotein having physiochemical properties producing relatively resistant acid barrier and it constitutes the major part of the mucus, an important pre-epithelial factor acting as a first-line defense against destructive ulcerogens (Sairam et al., 2003). Apart from enhancing the mucin activity, the effect of EPM as a defensive substance was further exemplified by a decrease in the cell exfoliation (cell shedding) as seen from a decrease in the DNA content of gastric juice when compared with the ulcer control group. Cell shedding is an indication of integrity of the gastric mucosa. The enhanced level of cell shedding from the inner lining of the gastric mucosa denotes the loss of integrity and decrease life span of cell, whereas decreased shedding could indicate enhanced life span of cells and promotion of defensive mechanism (Megala & Geetha, 2012). In the present study, EPM was observed to increase the mucin activity and, at the same time, act as a reliable index for cell exfoliation, thereby possesses a defensive mechanism.

The mechanistic activity of EPM was also ascertained using chemical-induced gastric ulcer models with absolute ethanol as a chemical ulcerogen. Various parameters such as mucus content, microvascular permeability test, antioxidant activity, and study of histopathological effects of EPM were examined in the stomach samples of both treated and untreated rats. Ethanol has been reported by many authors to produce gastric damage to the mucosa. The severity of gastric ulcer could be mainly due to the depletion of gastric mucus, disruption of the vascular endothelium resulting in increased vascular permeability, and also generates free radicals, thus leading to the imbalance in the antioxidant enzymes (Mitsuyama et al., 2005). Several studies have recently shown the importance of gastric mucus in gastroprotection (Zanatta et al., 2009). The results from the present study showed that EPM (100–400 mg/kg, p.o.) has excellent protective activity against the destructive nature of absolute ethanol; thereby, significantly producing an increasing amount of gastric mucus content when compared with the ulcer control group. This outcome was further supported by the positive effect of EPM on mucin activity as previously observed in the pyloric ligation model, which further signifies the protective effects namely strengthening of the gastric mucosal barrier. Moreover, the effect of EPM (100 mg/kg, p.o.) was also observed to significantly decrease the mucosal microvascular permeability of the Evans blue dye produced by ethanol. In this context, it can be predicted that the gastroprotective effect of EPM might be due to the protective nature of the mucus which forms a defensive layer on the mucosal endothelium.

Administration of absolute ethanol to the rats results in a decreased level of antioxidant enzymes (SOD and CAT) and GSH, whereas the levels of LPO were observed to increase significantly (Laloo et al., 2013). Any disturbance in the levels of these endogenous enzymes can result in the accumulation of highly reactive free radical species (ROS), thereby producing deleterious effects on the cell membrane integrity and its function (Moraes et al., 2009). Superoxide dismutase catalyses the reactions of superoxide anion radicals and dismutation of hydrogen peroxide (H₂O₂), while catalase degrades H₂O₂ into a molecule of oxygen and water (Saravanan et al., 2003). GSH, in contrast, is an essential antioxidant parameter found in mammalian cells and is responsible for maintaining the gastric mucosal integrity from various noxious agents that generate harmful free radicals in the body (Devi et al., 2007). Treatment with EPM (100–400 mg/kg, p.o) restored the antioxidant levels to normal conditions, thereby reflecting the effect as antioxidant potential. The illustration in Figure 4 represents the histological alteration of the gastric mucosa in untreated and treated groups when the rats were administered with absolute ethanol. Administration of absolute ethanol in the ulcerated control group (Group II) when compared with the normal control group (Group I) produces various cytological destruction such as severe detachment of surface epithelium (blue arrow), cytological changes in the mucosal layers, formation of gastric pit lesions (red arrow), and hemorrhage (green arrow). Animals pre-treated with standard drug omeprazole (Group III) and EPM graded doses (100–400 mg/kg, p.o.) (Groups V, VI, and VII) showed protective effect in preserving the normal functional cytoarchitecture of the entire gastric mucosa, thus producing a characteristic appearance almost similar to that of the normal control (black arrow) (Group I). This finding confirmed the cytoprotective nature of EPM against gastric lesions produced by absolute ethanol in which the gastric glands were seen to be intact, normal and no congestion and hemorrhage was observed.

Many plant extracts and their isolated compounds have been scientifically proven to have good therapeutic source of gastroprotection against various chemical or physical ulcerogens induced gastric ulceration. The gastroprotective effects as reported by many authors were attributed mainly to the presence of various phytoconstituents like tannins, phenolics, flavonoids, polysaccharides, and many other bioactive components. These phytochemical classes either individually or in combined synergistic complex can offer protection to the gastric mucosa in different mechanistic ways. Polyphenolics component (tannins, phenolics acids, and flavonoids) has been reported to possessed gastroprotective effects namley (1) strengthening the defensive mucosal barrier by increasing the mucus secretion, (2) antisecretory effects by depleting the aggressive factors, and (3) potent antioxidants by scavenging the free radicals (Laloo et al., 2013; Sairam et al., 2003). In contrast, polysaccharides have gastroprotective effects namely the formation of a gelatinous layer on the gastric mucosa, increases mucus synthesis, averts the necrotic lesion, and prevents the gastric DNA fragmentation induced by absolute ethanol (Choi et al., 2009; Galati et al., 2001). Hence, from the quantification results of the present study, it was observed that EPM contains a good quantity of polyphenolics and polysaccharide classes which suggest that the gastroprotective activity acting on a defensive mucosal barrier might be due to the contribution of such components.

According to Tomczyk and Latte (2009), some Potentilla species, namely, P. reptans, P. speciosa, P. kleiniana, P. palustris, and P. erecta, have been used worldwide to treat various classes of ulcers (mouth, stomach, and colon ulcers). However, on the basis of a literature search and to the best of our knowledge, only P. reptans aqueous extract (870 mg/kg, p.o.) has been scientifically proven to possess antiulcerogenic activity in rat stomach induced by ethanol (96% v/v) (Gurbuz et al., 2005). Potentilla fulgens (200 and 400 mg/kg, p.o.) was another species recently reported by us to have potent gastroprotective effects against pyloric ligations and absolute ethanol induced gastric ulcer model with a validated mechanism of action in rats (Laloo et al., 2013). Comparatively, a mechanistic level, the results from present study signify that P. mooniana protect stomach ulceration by strengthening the mucosal defensive layer rather than decreasing the acid secretion through the aggressive factors. Nevertheless, P. fulgens was reported to act by decreasing aggressive factors, namely, anti-secretory effect on the gastric juice by decreasing the levels of histamine, acid–pepsin secretion, and by inhibiting the H⁺K⁺–ATPase pump. The reason behind such mechanistic activities can be predicted due to the present of major diversity of polyphenolics classes reported in the genus.

Conclusion

In conclusion, the data of the present study suggested that the ethanol root extract from P. mooniana (EPM) possesses significant gastroprotective activity, which is attributed to its defensive property, namely, strengthening the mucosal defensive barrier and also by acting as potent antioxidants. Such activity can be predicted by the presence of a good amount of polyphenolics and polysaccharide compounds present in the extract. Thus, the present finding scientifically confirms the traditional use of P. mooniana in the treatment of gastric ulcer.

Declaration of interest

All authors report no declaration of interest.

Acknowledgements

Financial assistance provided by Rajiv Gandhi National Fellowship Scheme (RGNFS) to Mr. Damiki Laloo is greatly acknowledged. Authors also wished to acknowledge The Botanical Survey of India, Shillong, Meghalaya, for plant identification. We are also thankful to Mr. H. C. Pakyntein (President and herbal practitioner: Jaintia Indigenous Medicine Association) for supplying and providing information regarding the medicinal uses of the plant as gastroprotective.