ANTI-ULCER ACTIVITY OF BAICALEIN-7-O-GLUCURONIDE ISOLATED FROM PERSICARIA GLABRA FLOWERS AGAINST PYLORIC LIGATION INDUCED ULCER IN ALBINO RATS

Vijayaraj Chellaiyan * and Senguttuvan Swaminathan Department of Chemistry, Thiru.Vi.Ka. Government Arts College, Thiruvarur, Tamilnadu, India 610 003 ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

To isolate a flavonoid glycoside from flowers of Persicaria glabra using chromatography separation techniques, spectral characterization and its anti-ulcer activity. The isolated compound was purified, analyzed and characterized by chemical tests, HPLC and spectroscopic methods such as UV, IR, 1 H-NMR and 13 C-NMR. Anti-ulcer activity of Baicalein-7-O-glucuronide (250 mg) and methanolic extract (500 mg) of Persicaria glabra was examined on pyloric ligated induced ulcer model in rats. Omeprazole was used as standard. The anti-ulcer activity of flower extract of Persicaria glabra was estimated with the help of pH, gastric volume, free acidity, total acidity and ulcer index. The extract showed substantial reduction in pH, gastric volume and ulcer index in dose dependent manner as compared to control. The extract did not produce any toxic effects even at high doses.

…………………………………………………………………………………………………….... Introduction:-
Nature always attitudes as a golden mark to illustrate the outstanding phenomenon of symbiosis. The plant Persicaria glabra belongs to the family Polygonaceae. The family Polygonaceae consists of several important medicinal plants with wide range of biological activities and interesting phyto chemical constituents. The plant mostly found in river banks, stream sides and marshy areas. The plant Persicaria glabra is claimed to have medicinal uses such as astringent, diuretic, treatment of pneumonia, piles, jaundice, rheumatism, relieves pain, and in some areas used as remedy for fever. The root stocks are reported to be used in piles, jaundice, debility and consumption. The herb possess anti-bacterial activity against Micrococcus pyrogens and Diplococcus pyrogens (Sivakumar et al., 2011).
Ulcer is defined as the erosion in the lining of the stomach and is caused by the distractions of the gastric mucosal defense and repair organisms (Anoop and Jegadeesan, 2003). Peptic ulcer disease (PUD) is a disease that affects a considerable number of people worldwide. It progresses when there is an inequality between the aggressive and protective aspects at the luminal surface of the epithelial cells of stomach (Harold et al., 2007).
In recent years, there is a dynamic search to discover novel and alternative agents useful to contest gastric dyspepsia and peptic ulcer disease. Pyloric ligation induced ulcer signifies a unique ulcer model in examining the cause, course, consequence and treatment of peptic ulcer. Pylorus ligation induced ulcer is results of auto digestion of the gastric mucosal barrier possibly due to excess production and accumulation of HCl in the stomach (Sairam et al., 2003).
The residue from ethyl acetate segment was taken up in acetone and left in an ice-chest for two days when a yellow solid separated. It eminent a greenish brown colour with alc. Fe 3+ , formed yellow precipitate with basic lead acetate solution and reduced ammoniacal AgNO 3 but not Fehling's solution. It contributed yellow colour with aqueous NaOH intense yellow with Con.H 2 SO 4 and magenta colour with Mg/HCl. It seemed deep purple under UV which revolved yellow and disclosure to NH 3 . It retorted to Wilson's Boric Acid, Gibbs and Molisch's tests but not answer to Horhammer-Hansel tests.
Supporting intention for the structure of the flavone glycoside is provided by the HPLC (Shimadzu, Columbia), UV (Perkin Elmer Spectrophotometer), IR (Perkin -Elmer spectrometer) and NMR (125 MHz and 500 MHz, DMSO-d 6 ) spectral data were recorded on a Bruker AMX NMR spectrometer. Chemical shifts were reference to the corresponding residual solvent peaks and the values were recorded in δ.

Hydrolysis of the glycoside:-
The glycoside dissolved in hot aqueous methanol was hydrolyzed with H 2 SO 4 (5%) at 100˚C for around 2 hrs. The excess of alcohol was distilled off in vacuo and the succeeding aqueous solution was extracted with ether. The residue from ether fraction was isolated as designated below. The glycoside was subjected to partial hydrolysis by treatment with 10% formic acid in cyclohexane and the resultant solution extracted with ethyl acetate.

Phytochemical screening of plant extract:-
A small amount of the dry extract was used for the phytochemical tests (Harborne, 1994) for compounds which contain alkaloids, flavonoids, saponins and glycosides while steroids and tannins are absent in entirely the crude extracts.
Animals:-Male albino rats of Wistar strain almost weighing 160-180g were used in this study. They were healthy animals bought from the Indian Institute of Science, Bangalore. The animals were kept in spacious polypropylene cages bedded with rice husk. The animal room was well ventilated and preserved under standard experimental conditions (Temperature 27 ±2˚C and 12 hour light/dark cycle) through the experimental period. All the animals were fed with standard pellet diet and water were provided and libitum. They were familiarized to the environment for one week prior to experimental use (Alaghawani and Naser, 2013).

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Acute toxicity studies:-Male albino rats were used to study the acute toxicity of methanolic extract and Baicalein-7-O-glucuronide extracted from Persicaria glabra flowers. Animals were fasted overnight and administered with methanolic extract. Changes in the animal behavior were prominent before and after administration for 24 hours. Treated animals continued further detected for up to 14 days for any toxic symptoms (Turner, 1965).
Pyloric ligation induced ulcer in rats:-Animals were divided into four groups, each consisting of six rats. Group-1 treated as control group, received saline orally for 7 days. Group-2 treated as standard group was received Omeprazole 30 mg/kg for 7 days. Group 3 and 4 treated as treatment groups were received Baicalein-7-O-glucuronide and methanolic extract from Persicaria glabra in a dose of 250 and 500 mg/kg for 7 days respectively. Animals in all groups were fasted for 18 hours after the respective assigned treatment and were anaesthetized with diethyl ether at the dose of 35 mg/kg. Ligation was done without causing any wound to the blood supply of the stomach. Animals were allowed to recover and steady in individual cages and were deprived of water during postoperative period. After 4 h of surgery, rats were surrendered and gastric contents were collected into the centrifuge tubes and centrifuged at 1000 rpm for 10 min and the pH, free acidity, total acidity of the gastric juice was determined (Nguelefack et al., 2005;Hirohashi et al., 1993). In addition the ulcer index was determined by opening the stomach on greater curvature and scores were given 0 to 3 depending upon the austerity of ulcers.

Statistical analysis:-
Values were expressed as mean  SD for six rats in the each group and statistical significant differences between mean values were determined by one way analysis of variance (ANOVA) followed by the Tukey's test for multiple comparisons. The results were statistically analyzed by Graphpad Instat Software (Graphpad Software, San Diego, CA, USA) version 3 was used and p< 0.01 was considered to be significant.     (Fig 1). 1 H-NMR spectrum ( Figure 2) showed multiplet signals at δ 8.93 -δ 7.14 ppm (m, 6H, Ar) assigned to aromatic protons and also showed signals at δ 9.10 ppm (bs, 1-OH in acid) assigned to -OH in carboxylic acid group. In addition, 1 H-NMR showed a signal at δ 5.13 (s, 1H, vinyl) assigned to vinyl proton. The 5-OH proton resonates at δ 12.36 ppm (bs, 1-OH in 5 th position). The multiplet signals at δ 3.88 -δ 3.36 ppm (m, unresolved, pyranose protons) assigned to the sugar protons. The 13 C-NMR data (Figure 3) shown that were 21 carbons in this structure, 15 of which were typical for a flavone skeleton and the others were consigned to glycoside. 13  By associating all the above exposed physical and chemical part of evidences the flavonoid found from Persicaria glabra flowers has been characterized as the Baicalein-7-O-glucuronide (Fig 4).

Pyloric Ligation induced ulcer in albino rats:-
The ulcer formation in each of these models occurs by different mechanisms pylorus ligation-induced ulcers are caused by enhanced acid pepsin secretion leading to auto-digestion of the gastric mucosa and break down of the gastric mucosal barrier, and the digestive effect of accumulated gastric juice and interference of gastric blood circulation are responsible for the induction of ulceration (Aditi Chaturvedi et al., 2007). The effect of orally administered four groups on gastric damage induced by pyloric ligation is shown in Table 1. It was detected that increase the ulcer lesion in ulcer control rats. Significant reduction in ulcer lesion was observed in treatment with four groups. It is significant to note that increased the volume, total acidity and free acidity and decreased pH of gastric juice were observed in ulcer control rats as compared to normal rats. Administration of Baicalein-7-Oglucuronide decreased the volume, total acidity and free acidity and increased pH of gastric juice were observed as compared to control rats. Animal groups treated with the Baicalein-7-O-glucuronide exhibited a reduction of gastric damage against pyloric ligation induced gastric ulceration. The percentage of ulcer protection was 94.80% for Group 2, 90.37% for Group 3, 89.63% for Group 4 were recorded. Omeperazole, the positive control included for the study also offered significant protection (94.80%) against pyloric ligation induced gastric ulcer. Figure 5 shows the photographic representation of control and experimentally induced ulcer model. The increase in volume in the ulcer control rats is undoubtedly due to increase protection of hydrochloric acid as evident from the total acidity and decrease pH value of gastric juice. In the present study, the decrease in volume of the gastric juice and concomitant decrease in the acidity and increase in pH proving the anti-ulcer activity of Baicalein-7-O-glucuronide. Further evidenced by the reduced edema formation and epithelial lifting were observed in morphometric study ( Fig  5). Therefore, it can be believed that Baicalein-7-O-glucuronide may stimulate the secretion of prostaglandin or possess prostaglandin like substances.

Conclusion:-
Gastro protective role for Baicalein-7-O-glucuronide against gastric mucosal damage induced by pyloric ligation were investigated in the present study. Pyloric ligation induced gastric ulcer rats show increased gastric volume, acidity and depleted pH. The observed gastro protection is possible mediated to a major extent by a gastric mucosal secretion mechanism as the isolated compound Baicalein-7-O-glucuronide are able to restore the increased volume, acidity and depleted pH by pyloric ligation almost towards normal levels seen in control. This is further evidenced by morphometric study.