Gastric Antisecretory and Antiulcer Activities of Decalepis arayalpathra.

Abstract

Decalepis arayalpathra. Joseph & Chandrasekharan (Periplocaceae) is locally called “amrithapala.” The Kani tribe of the Trivandrum district of Kerala uses the tuberous roots of this plant to treat peptic ulcers. In the current study, the ethanol extract of D. arayalpathra. roots significantly decreased the pepsin secretion at a dose of 250 mg/kg and the gastric juice volume and acid output at a dose of 500 mg/kg in pylorus ligated rats. Pretreatment with D. arayalpathra. extract (500 mg/kg, p.o.) provided significant protection against the peptic ulcerogenic effect of ethanol administered individually or in combination with indomethacin or hydrochloric acid. Our studies also revealed that preitreatment with D. arayalpathra. significantly decreased malondialdehyde levels and increased gastric wall mucus production and the protein concentration of the stomach wall of ethanol-treated rats at a dose of 500 mg/kg. The gastroprotective effect of D. arayalpathra. observed in the current study may be attributed to its effect on stimulating protein concentration and mucus production of the stomach wall. In addition to its gastric antisecretory activity, D. arayalpathra. exerts a cytoprotective effect, which could be partly due to the presence of antioxidant phytocompounds like flavonoids.

Keywords:

• Antisecretory
• antiulcer
• cytoprotection
• Decalepis arayalpathra
• Periplocaceae

Introduction

Decalepis arayalpathra. Joseph & Chandrasekharan (syn: Janakia arayalpathra. Joseph & Chandrasekharan), a rare and endemic species of the family Periplocaceae, is found in the southern part of the Western Ghats of Kerala. The local name of this plant is “Amrithapala.” The plant has milky latex and aromatic tuberous roots. The tuberous roots are used by the Kani tribe of the Trivandrum district of Kerala as an effective remedy for peptic ulcer, cancer-like afflictions, and also as a rejuvenating tonic (Pushpangadan et al., 1990). Phytochemical investigations at the Tropical Botanic Garden and Research Institute showed that D. arayalpathra. contained α.-amyrin acetate, 4-methoxy-salicyl aldehyde, magnificol, β.-sitosterol, naringenin, kaempferol, and aromadendrin (Chacko et al., 2000). In the current study, the gastric antisecretory effect of the ethanol extract of D. arayalpathra. and its antiulcer activities on ethanol-induced, ethanol- and indomethacin-induced, and ethanol- and hydrochloric acid-induced acute gastric lesions in rats is reported.

Materials and Methods

Plant material and preparation of the extract

D. arayalpathra. roots were collected from Njaraneeli in the Trivandrum district of Kerala during November 2003 by V.J Shine, Tropical Botanic Garden and Research Institute, and authenticated by Dr. Mathew Dan, plant taxonomist at the Tropical Botanic Garden and Research Institute. A voucher specimen has been deposited at the Tropical Botanic Garden and Research Institute Herbarium (TBGT 57005, dated December 30, 2003). The roots were washed thoroughly in tap water, shade-dried, and powdered. The powder (100 g) was extracted with 1000 mL 95% ethanol overnight using a Soxhlet apparatus. The extract was then filtered, and the filtrate was concentrated under reduced pressure to yield 20 g of the crude extract (5% w/w, with respect to the dried plant material). This crude extract was referred to as D. arayalpathra.. It was suspended in 2% gum acacia solution to required concentrations and used for the experiments.

Animals

Male Wistar albino rats (200–250 g) and male Swiss albino mice, (20–25 g), obtained from the institute's animal house, were used for the current study. They were maintained under standard laboratory conditions. All experiments involving animals were done according to NIH guidelines, after getting the approval of the institute's animal ethics committee. Twenty-four to 48 h before the start of the experiments, the animals was deprived of solid diet but received boiled water ad libitum..

Gastric secretion in pylorus-ligated rats

For the estimation of gastric secretion, the pylorus-ligated model described by Shay et al. (1945) was used. Animals fasted for 24 h were anesthetized using pentothal sodium. A small abdominal incision was made, and the pylorus was ligated. Immediately after ligation, D. arayalpathra. (250, 500, and 1000 mg/kg) or ranitidine (50 mg/kg) or vehicle (2%gum acacia) was administered intraduodenally to the rats. Then the incision was closed and the animals were sacrificed 4 h later. The stomach was excised, opened along the greatest curvature, and the lumenal contents were collected and centrifuged for 15 min at 4500 rpm to remove the residual debris. The gastric juice volume was measured, and the total acid output was determined by titration against 0.01 N NaOH using phenolphthalein as indicator as reported by Yesilada et al. (1997). Pepsin concentration was determined by modification of the colorimetric method of Anson (1983) involving digestion of 2% hemoglobin in 0.02 N HCl (pH 2.0, 37°C, 15 min) followed by alkaline condensation with Folin-Ciocalteu reagent and spectrophotometric measurement at 578 nm. Pepsin output was expressed as milligrams of pepsin in 4 h.

Gastric lesions induced by ethanol

Lesions were induced according to the method of Al-Bekairi et al. (1992). Rats fasted for 24 h were used. Thirty minutes after the treatments (D. arayalpathra., 250, 500, 1000 mg/kg; or ranitidine, 50 mg/kg; or omeprazole, 5 mg/kg; or vehicle, 2% gum acacia), 1 mL of 80% ethanol was administered orally. The animals were sacrificed under anesthesia using ether 1 h after ethanol treatment. The stomach of each animal was excised and opened along the greater curvature. The gastric ulcers were scored according to the method of Valcavi et al. (1982).

Histopathologic studies

The stomach samples from the above experiments were preserved in 10% buffered formalin and processed for routine paraffin block preparation. Using a rotary microtome (American Optical Co., USA), sections of thickness about 5 µm were cut and stained with hematoxylin and eosin. These were examined under the microscope for histopathologic changes such as congestion, edema, ulceration, and necrosis by an observer who was blinded with respect to the treatment groups.

Malondialdehyde estimation

The method of Fong et al. (1973) was followed. The glandular part of the stomach tissue was homogenized in trichloroacetic acid (TCA) and the homogenate was suspended in thiobarbituric acid. After centrifugation, the optical density of the clear pink solution was read at 532 nm.

Estimation of gastric protein content

The level of protein in the stomach samples was determined according the following procedure: the stomachs were rapidly dissected from the animals, frozen in liquid nitrogen, and stored at − 20°C until analysis. Total protein was determined by the method of Lowry et al. (1951).

Gastric wall mucus determination

The modified procedure of Come et al. (1974) was used to determine gastric wall mucus. The glandular segment from the stomach was removed and weighed. Each segment was transferred immediately to 10% Alcian blue solution (in sucrose solution, buffered with sodium acetate, pH 5), and the excess dye was removed by rinsing in sucrose solution. The dye complexed with the gastric wall mucus was extracted with 0.5% magnesium chloride. A 4-mL sample of the extract was then shaken with an equal volume of diethyl ether. The resulting emulsion was centrifuged, and the absorbance of the aqueous layer was recorded at 580 nm. The quantity of Alcian blue extracted pergram of glandular tissue was then calculated.

Gastric lesions induced by ethanol and indomethacin

Indomethacin was used to inhibit endogenous formation of prostaglandin through enhancement of prostaglandin cycloxygenase according to the method described by Robert et al. (1983). The rats were fasted for 48 h with access to water ad libitum.. The animals were treated with 2.5 mg/kg (s.c.) indomethacin 1 h before oral administration of D. arayalpathra.. Twenty minutes later, these rats were given orally 1 mL of 80% ethanol. The animals were sacrificed 1 h after ethanol administration. The stomachs were excised and examined for lesions. The lesions were quantified.

Gastric lesions induced by ethanol and hydrochloric acid

Following the method of Hara and Okabe (1985), ulceration was induced in the stomachs of 24 h fasted rats. The ulcer-inducing agent, 150 mM HCl in 60% (v/v) ethanol, was administered orally via an intragastric tube 1 h after the test groups and the control group of rats had received orally D. arayalpathra. (250, 500, and 1000 mg⁻¹ mL) and 2% gum acacia (1 mL), respectively. The stomachs were removed from the anesthetized and exsanguinated rats 1 h after ethanol/HCl dosing.

The esophageal orifice of the stomach was tied with a piece of thread, and 10 mL of 2% v/v formaldehyde solution was introduced through the pyloric orifice into the stomach. The pyloric orifice was then closed with a clip. Ten minutes later, the stomach was cut open along the greater curvature. The diameter or lengths of the lesions of the glandular portion of the stomach were summed up to obtain lesion indices for both the D. arayalpathra. treated and control rat stomachs, according to the method of Noamesi et al. (1994).

Behavioral and toxic effects

Five groups of 10 mice were administered orally 250, 500, 750, 1000, and 1250 mg/kg of the D. arayalpathra. extract. They were observed continuously for 1 h for any gross behavioral changes, like drowsiness, restlessness, writhing, convulsion, piloerection, symptoms of toxicity and mortality, if any, and intermittently for the next 6 h and then again 24 h after dosing with D. arayalpathra. extract.

Statistical analysis

Data were expressed as mean±standard deviation of the mean (SD), and statistical comparisons were performed using Student's t.-test (Snedecor & Cochran, 1980).

Results

Gastric secretion measurements of pylorus-ligated rats showed that D. arayalpathra. significantly decreased the pepsin secretion (250 mg/kg), the gastric juice volume, and HCl output at 500 mg/kg dose. Ranitidine (50 mg/kg), also caused a marked decrease in all these secretory parameters (Table 1). The highest dose used in the study (1000 mg/kg) was not as effective as the two lower doses used in eliciting these three responses.

Table 1. Effect of Decalepis arayalpathra. root ethanol extract on basal gastric secretion in pylorus-ligated rats.

	Gastric juice volume	Acid output	Pepsin output
Groups	(mL/4 h)	(µEq H^+/4 h)	(mg/mL)
Control (2% gum acacia)	4.50±0.42	282.39±56.42	5.50±0.99
D. arayalpathra. (250 mg/kg)	3.90±0.14	141.20±40.11	3.14±0.54
D. arayalpathra. (500 mg/kg)	2.62±0.12	56.35±20.12	3.30±0.63
D. arayalpathra. (1000 mg/kg)	3.97±0.15	59.41±15.32	3.61±0.91
Ranitidine (50 mg/kg)	1.70±0.21	50.39±20.21	2.35±0.77

Values are the mean±SD for 6 animals in each group.

*p ≤ 0.01 compared with control.

Oral administration of 80% ethanol produced severe hemorrhagic lesions in the glandular mucosa of the stomach. Administration of indomethacin or HCl along with ethanol was found to potentiate the gastric damage in terms of the lesion scores (Table 2). D. arayalpathra. was found to offer the gastric mucosa a statistically significant protection against ulceration caused by ethanol or a combination of ethanol and indomethacin or ethanol and HCl, even better than the omeprazole-treated groups. There was no significant reduction of gastric lesions in any of the ranitidine-treated groups. The maximum protection to the gastric mucosa was observed at the dose of 500 mg/kg D. arayalpathra. in the case of the ethanol group and the ethanol + indomethacin group, but in the case of the ethanol + HCl group, the maximum protection was observed at 1000 mg/kg D. arayalpathra..

Table 2. Effect of Decalepis arayalpathra. root ethanol extract on gastric lesions induced by ethanol or ethanol and indomethacin or ethanol and hydrochloric acid (HCl) in rats.

	Lesion score (mean±SD)
Treatments	Ethanol	Ethanol + Indomethacin	Ethanol + HCl
Control (2% gum acacia)	18.14±1.94	23.14±1.94	94.20±6.20
D. arayalpathra. (250 mg/kg )	4.53±1.72 (23%)	10.00±1.72 (75%)	72.50±5.10 (43%)
D. arayalpathra. (500 mg/kg)	0.90±0.21 (95%)	2.30±0.21 (90%)	36.15±7.10 (61%)
D. arayalpathra. (1000 mg/kg)	4.88±1.21 (73%)	6.40±2.13 (72%)	16.14±5.10 (82%)
Ranitidine (50 mg/kg)	14.50±1.20 (20%)	18.14±1.17 (21%)	72.14±5.30 (23%)
Omeprazole (5 mg/kg)	9.60±1.21 (47%)	12.21±1.46 (47%)	50.32±2.1 (46%)

Values are the mean±SD for 6 animals in each group.

*p ≤ 0.01 compared with control. Values in parentheses indicate the percentage of inhibition of the lesions.

In the current study, ethanol treatment significantly increased the malondialdehye levels of the stomach. D. arayalpathra. pretreatment significantly reduced the malondialdehyde concentration in a dose-dependent manner up to 500 mg/kg (Table 3).

Table 3. Effect of Decalepis arayalpathra. root ethanol extraction the gastric wall mucus, protein concentration, and malondialdehyde levels in gastric ulcers induced by 80% ethanol in rats.

	Gastric wall mucus	Protein	Malondialdehyde
Treatments	(Alcian blue µg/g wet glandular tissue)	(mg/100 mg)	(nmol/g wet tissue)
Control (2% gum acacia) + ethanol	290.39±11.20	8.34±0.24	72.58±1.84
D. arayalpathra. (250 mg/kg) + ethanol	315.31±9.61	13.24±0.13	63.56±1.67
D. arayalpathra. (500 mg/kg) + ethanol	387.31±11.56	14.62±0.17	50.31±1.85
D. arayalpathra. (1000 mg/kg) + ethanol	385.22±7.89	12.93±0.24	53.52±1.17
Ranitidine (50 mg/kg) + ethanol	300.11±8.17	8.96±0.14	68.12±1.72
Omeprazole (5 mg/kg) + ethanol	350.11±7.12	15.13±0.17	46.13±1.27

Values are the mean±SD for 6 animals in each group.

*p ≤ 0.01 compared with control.

Gastric protein concentration of the ethanol-treated rats was also significantly increased by the administration of D. arayalpathra. at 500 mg/kg dose, beyond which it declined (Table 3).

D. arayalpathra. was also found to significantly inhibit the depletion of stomach wall mucus, caused by ethanol treatment (Table 3) at 500 mg/kg dose, even to a greater extent than in the omeprazole reference group. Increasing the dose further did not increase the production of mucus. Ranitidine did not overcome the depletion of mucus induced by ethanol.

Ethanol treatment caused necrosis, congestion, and edema in the gastric mucosa of rat compared with normal control, which showed well defined gastric mucosa (Figs. 1 and 2). Pretreatment with D. arayalpathra. (500 mg/kg) was found to significantly inhibit the ethanol-induced gastric histopathologic changes. The stomachs of the animals pretreated with D. arayalpathra. before ethanol administration showed only mild congestion; otherwise, the stomach appearance was normal (Fig. 3), almost comparable with that of the omeprazole-treated group (Fig. 4).

Figure 1 The stomach wall of control rat showing its normal appearance, with the well-defined gastric mucosa.

Figure 2 The stomach wall of rat after treatment with 80% ethanol (1 mL) showing congestion, edema, and necrosis of the gastric mucosa.

Figure 3 The stomach wall of the rat treated with D. arayalpathra. (500 mg/kg) and 80% ethanol (1 mL) showing almost normal histologic architecture.

Figure 4 The stomach wall of rat treated with omeprazole (5 mg/kg) and 80% ethanol (1 mL). The necrotic changes are minimal compared with that in Figure 2.

In the toxicity study, no mortality occurred within 24 h with the four doses of D. arayalpathra. tested. The LD₅₀ was therefore greater than 1250 mg/kg p.o. in mice (data not shown).

Discussion

Gastric ulcer is caused by the imbalance between acid and pepsin and the weakness of the mucosal barrier (Mitra et al., 1996). In the current study, on pylorus-ligated rats, D. arayalpathra. at doses of 250 mg/kg and 500 mg/kg decreased the pepsin secretion and gastric acid output, respectively. The cytoprotective role played by D. arayalpathra. on the gastric mucosa may be due to its inhibitory effect on gastric acid and pepsin output.

The protective effects of D. arayalpathra. on various ulceration models were evaluated. In ethanol-induced ulceration, pretreatment with D. arayalpathra. was found to inhibit necrosis, congestion, and edema in the gastric mucosa of the treated rats. These results were substantiated by the histopathologic observations. Ethanol-induced gastric lesions are caused by the direct action of ethanol. The resulting gastric mucosal damage was due to the stimulation of leucotriene-C₄ (LTC₄), mast cell secretory products (Oates & Hakkinen, 1988), and free radical formation. Ethanol is metabolized in the body to cause increased production of O₂⁻ within the tissues, and simultaneously this increased the cellular free radical concentration. These free radicals caused breaking of DNA strands and protein denaturation (Halliwell & Gutteridge, 1987). They also induced lipid peroxidation of the cell membrane significantly by increasing the malondialdehye levels and reducing protein and nucleic acid concentration of the stomach (Al-Bekairi et al., 1992). In the current study, D. arayalpathra. was found to be effective in decreasing malondialdehyde concentration of the glandular stomach, which had possibly accumulated due to the effect of toxic free radicals in the mucosal cells (Al-Bekairi et al., 1992), indicating the presence in the D. arayalpathra. extract of some phytoconstituents with an antioxidative nature. Phytochemical investigations of D. arayalpathra. at our laboratory showed that it contained flavonoids (naringenin, kaempferol, aromadendrin,), besides triterpenes (α-amyrin acetate), sterols, and so forth. (Chacko et al., 2000). It has been already reported that flavonoids have the potential to reduce free radical activity, thus acting as antioxidants (Sharda, 1999), which play an important role in cytoprotection (Szabo & Hollander, 1989). Flavonoids are known to have antiulcer properties (Yamahara et al., 1990). α-Amyrin is reported to have an effective gastroprotective role (Oliveira et al., 2004).

D. arayalpathra. significantly elevated the gastric mucus in ethanol-treated rats. Gastric mucus serves as the first line of defense against ulcerogens. It is secreted by the neck cells of the gastric mucosa, and it prevents physical damage of the gastric mucosa and also supports diffusion of hydrogen ions (Williams & Turnberg 1980). It is considered important in the mucosal defense against endogenous aggressions such as acid and pepsin and also as an agent in facilitating its repair (Robert et al., 1985; Matuz, 1992). Thus, D. arayalpathra. possesses antioxidant and gastroprotective properties, which may be responsible for its antiulcer effects. Ethanol-induced gastric mucosal lesions are caused by the direct action of ethanol, and therefore ethanol-induced gastric lesions are not inhibited by antisecretory agents like ranitidine but are inhibited by agents that enhance the mucosal defensive mechanisms (Morimoto et al., 1991) like omeprazole. Also, in the ethanol-induced ulceration of the current study, ranitidine had no gastroprotective effect. This finding is supported by the work of other authors (Hakkinen et al., 1991; Palacios et al., 1998).

In conclusion, D. arayalpathra. contains antiulcerogenic compounds, which have gastroprotective effects on ulceration induced by ethanol alone or in combination with indomethacin or HCl. This could be through its involvement in scavenging of free radicals, reduction of HCl output, and enhancement of protein synthesis and mucus secretion of the gastric mucosa. Further studies are required to isolate the antiulcer compounds and to elucidate their mechanism of action.

Acknowledgment

The authors wish to thank Dr. G.M. Nair, the director of the institute, for facilities; Mr. K.P. Pradeep Kumar for photographic assistance; and Mr. S. Radhakrishna Pillai for technical assistance. Funding from the Kerala State Council for Science, Technology and Environment is gratefully acknowledged.