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Anti-secretory and cyto-protective effects of peganine hydrochloride isolated from the seeds of Peganum harmala on gastric ulcers.

ARTICLE INFO

Keywords:

Peganum harmala

Peganine hydrochloride

Proton pump

Gastric ulcer

Gastric acid secretion

ABSTRACT

Gastroprotective mechanism of peganine hydrochloride isolated from Peganum harmala seeds was investigated. Peganine hydrochloride was evaluated against cold restraint (CRU), aspirin (AS), alcohol (AL) and pyloric ligation (PL) induced gastric ulcer models in rats. Potential anti-ulcer activity of peganine was observed against CRU (50.0%), AS (58.5%), AL (89.41%) and PL (62.50%) induced ulcer models. The reference drug omeprazole (10 mg/kg, p.o.) showed 77.45% protection against CRU, 49.97% against AS and 69.42% against PL model. Sucralfate. another reference drug (500 mg/kg, p.o.) showed 62.50% protection in AL induced ulcer model. Peganine significantly reduced free acidity (33.38%), total acidity (38.09%) and upregulated mucin secretion by 67.91%, respectively. Further, peagnine significantly inhibited H. IC-ATPase activity in vitro with [IC.sub.50] of 73.4714/mlas compared to the [IC.sub.50] value of omeprazole (30.24 p,g/ml) confirming its anti-secretory activity.

[C] 2013 Elsevier GmbH. All rights reserved.

Introduction

Plant derived metabolites have provided important basis for discovery and development of modern therapeutics. Peganum harmala Linn (Zygophyllaceae), commonly known as 'harmal' is one among the most important medicinal plants of India (Chopra et al. 1956). Its different parts are used in traditional systems of medicine for the treatment of variety of human ailments such as lumbago, asthma, colic, jaundice and as a stimulant emmenagogue (Bukhari et al. 2008). From current pharmaceutical studies, several pharmacological activities have been reported for P. harm ala such as anti-tumor, insecticidal and antimalarial effects (God et al. 2009), antileishmanial (Mirzaie et al. 2007; Khaliq et al. 2009; Pragya et al. 2008), anti-spasmodic, anti-histaminic, vasorelaxant effects (Asghari and Lockwood 2002), wound healing, anti-oxidant activity, immunomodulator properties leukemia healing (Zaker et al. 2007), hypoglycemic effects (Singh et al. 2008), analgesic and anti-inflammatory properties, antinociceptive effects (Monsef et al. 2004), antitumor activity (Madadkar et al. 2002), hepatoprotective effect (Khaled et al. 2008) and cytotoxic activity among others. Also, it has been reported that this plant had antibacterial, anti fungal and antiviral effects (Darabpour et al. 2011). Gastric or peptic ulcer constitutes major ailment that affects human gastrointestinal tract and presents major global health problem both in terms of morbidity and mortality (Laine 2004). Consumption of alcohol and analgesics has been identified as major risk factors responsible for acute gastric mucosal injury in humans and presents life-threatening hemorrhages that require surgical intervention (Salim 1987). The inhibition of gastric acid secretion has been proven to be a powerful therapeutic principle in the treatment of gastric and duodenal ulcer disease (Prous 1988).

Though various biological activities of the plant P. harmala has been reported earlier, anti-ulcer activity of this plant's active constituents has not been reported till date. In our literature survey, we found very little information available regarding its pharmacological effect on the gastrointestinal system. Keeping these facts in considerations, we have assessed the anti-ulcer activity of pega-nine hydrochloride also known as vasicine, (Joshi et al.1994, 1996) isolated from the seeds of P. harmala (Fig. 1).

Materials and methods

Plant material

The uncrushed seeds of P. harmala were collected from Kashmir, J & K, India, and authenticated and identified by the botany division of CDRI Lucknow (Plant code No. 4666).

Extraction and fractionation

The dried and powdered plant material (seeds) was extracted with 95% ethanol. The ethanolic extract was further fractionated into chloroform and aqueous fractions (Fig. 2).

Isolation of peganine hydrochloride

Aqueous fraction on column chromatographic purification yielded compound 1 (Fig. 2). It was UV-fluorescent and conformed well to Dragendorff's test (Munier and Macheboeuf 1951) indicating its alkaloid nature. Structure elucidation was performed by spectroscopic techniques (see [.sup.1]H, [.sup.13]H NMR spectra and Mass spectra of compound in supplementary information). On the basis of these data, this compound was identified as peganine hydrochloride which was confirmed by comparison of its physicochemical data with that reported in the literature (Sen and Ghose 1924).

All experimental protocols were approved by our Institutional Ethical Committee following the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) which complies with International norms of INSA (Indian National Science Academy). Adult Sprague Dawley rats of either sex, weighing 180-200g were housed in raised bottom mesh cages to prevent coprophagy and were kept in environmentally controlled rooms (25 [+ or -] 2 [degrees]C, 12h light and dark cycle). Animals were fed with reference laboratory food pellets and water was provided ad libitum.

Materials

Omeprazole and other chemicals were obtained from M/s. Sigma Chemicals, St. Louis, MO, USA whereas sucralfate was obtained from Meranani Pharmaceuticals, India.

Dose selection and drug preparation: In a pilot investigation, the dose dependent effect of pega nine (1) (10, 20 and 40 mg/kg, p.o.), reference drug omeprazole (OMZ) (10 mg/kg, p.o.) and sucralfate (SUC) (500 mg/kg, p.o.) were freshly prepared in 1% carboxymethyl cellulose (CMC) as suspension and administered orally 45 min before to exposure of ulcerogens to the animals at a volume of 1 m1/200g of body weight.

All animals were deprived of food for 16h before ulcerogens exposure and were divided into three groups (n = 6).

Group I (Control group): Control group of animals were treated with 1% CMC, 45 min prior to the induction of gastric ulcer in all ulceration models.

Group II (peganine treated): Rats were treated with peganine (1) hydrochloride (20 mg/kg, p.o.), 45 min prior to the induction of gastric ulcer in all ulceration models.

Group III (reference drug treated): Third group was sub categorizes into further sub-groups based on selection of models.

Sub-group A for CRU model: Rats were administered with reference drug omeprazole (10 mg/kg), 45 min prior to the induction of gastric ulcer.

Sub-group B for PL model: In case of PL model rats were administered with omeprazole (10 mg/kg), 45 min prior to the induction of gastric ulcer.

Sub-group C for AS model: When we were using AS models rats administered with reference drug omeprazole (10 mg/kg), 45 min prior to the induction of gastric ulcer.

Sub-group D for AL model: Sucralfate (SUC; 500 mg/kg) used as reference in AL model were administered to rats, 45 min prior to the induction of gastric ulcer.

Anti-ulcer studies

Cold restraint induced gastric ulcer (CRU)

The method described by (Levine 1971) was used in this assay. Animals were subjected to cold restraint stress after 45 min of treatment with peganine (1) and reference drug omeprazole (OMZ). All the animals were immobilized in restraint cage and kept at 4[degrees]C in an environmental chamber. Two hours later the animals were sacrificed and stomachs were observed and scored under Magnascope for ulcers.

Alcohol induced gastric ulcer model (AL)

Gastric ulcer was induced in rats by administering chilled absolute alcohol (1 m1/200g, body weight of animals) (Robert 1979). The peganine (1) and reference drug sucralfate were administered

Experimental animals

Aspirin induced gastric ulcer model (AS) The experiment was carried out according to the method of (Djahanguiri 1969). Gastric lesions were induced with Aspirin (150 mg/kg) administered to rats after 45 min of treatment of pega-nine (1) and reference drug omeprazole (OMZ). The animals were sacrificed after 5 h of aspirin treatment and the stomach was dissected out, incised along the lesser curvature and the lesion was scored.

Pyloric ligation induced ulcer model (PL) After 45 min of administration of peganine (1) and reference drug omeprazole, ulcer was induced in rats by pyloric ligation. Under chloral hydrate anesthesia (300 mg/kg, i.p.), the abdomen was opened and the pyloric end of the stomach was ligated avoiding any damage to the adjacent blood vessels (Shay et al. 1945). Stomach was replaced carefully and the animals were allowed to recover with free access to water. After 4 h the animals were sacrificed and the stomach was dissected out. Lesions were scored and gastric fluid was collected and centrifuged at 2000 rpm for 10 min. The collected supernatant was used for the estimation of gastric secretion studies. 45 min before alcohol treatment. After 1 h of alcohol administration, the animals were sacrificed and stomach was cut open along the greater curvature to observe the gastric lesions which appear as hemorrhagic bands along the mucosal ridges of the stomach. The lengths of the lesions were measured using Biovis image analyzer software and summated to give a total lesion score.

Gastric secretion study

Free and total acidity was measured from the collected gastric juice by titrating against 0.01 N NaOH, using phenolphthalein as an indicator and expressed in terms of [micro]equiv./m1 (Anoop and Jegacleesan 2003). Mucin level in gastric juice was quantified by Crowther and Wetmore (1987).

Direct fluorometric assay

In gastric juice, mucin was quantified with a fluorometric assay as described by Crowther and Wetmore (1987). Before the fluorometric assay, gastric juice was delipidate using the method of Wessel and Flugge (1984). Briefly describe, 50 [micro] of gastric juice was diluted 1:1(v/v) in PBS buffer and 400 [micro]l methanol was added. After a short centrifugation (9000 x g for I min), 200 [micro]l of chloroform and 300 [micro]l of distilled water were added, thoroughly mixed, and centrifuged once more. The upper phase was discarded and 300 [micro].1 of methanol was added. After a further centrifugation (9000g for 2 min), lipid-free proteins were recovered in the pellet. For the fluorometric mucin determination, the pellet was resuspended in 200 [micro]l of PBS, 250 [micro]l of alkaline reagent (1 ml 0.15 N NaOH and 200 [micro]l of 0.6 M 2-cyano-acetamide) was added and the mixture was incubated at 100 [degrees]C for 30 min. Subsequently, 2 ml of 0.6 M borate buffer (pH 8) was added and the fluorescence was measured by varion fluorimeter at 383 nm (excitation 336 nm). Antioxidant assays

Scavenging/inhibitory activity coefficient: The scavenging or percentage inhibitory activity of peganine (PG) in each assay was calculated from:

%Inhibition = (AO--Al)/AOx100

AO= absorbance of the control (without peganine), and

Al =absorbance of the treated (with peganine). The [IC.sup.50] value of the peganine (PG) was extrapolated from the reference linear regression curve.

Free radical scavenging activity

DPPH method: The antioxidant activity of the peganine (1) was assessed on the basis of the radical scavenging effect of the stable DPPH free radical (Blois 1958). Peganine (1) (10-100 [micro]g/ml) was added to 200 [micro]l of DPPH in methanol solution (100 M) in a 96-well microtitre plate (Tarsons Product (P) Ltd., India). After incubation at 37 [degrees]C for 30 min, the absorbance of each solution was determined at 490 nm using ELISA micro plate reader (Bio Rad Laboratories Inc., California, USA, Model 550). The corresponding blank readings were also taken and the remaining DPPH was calculated [IC.sub.50] value is the concentration of the sample required to scavenge 50% DPPH free radical.

Superoxide anion (SOD) scavenging activity assay: superoxide dismutase activity was measured based on its ability to inhibit the autoxidation of epinephrine to adrenochrome at alkaline pH (Misra and Fridovich 1972). The absorbance of reaction mixture was followed for 4 min at 480 nm in a spectrophotometer (Model 1201, Shimadzu). Enzymatic activity was expressed as U/mg protein at 30 [degrees]C. The amount of enzyme that caused 50 percent inhibition of epinephrine autoxidation was defined as one unit (U).

Measurement of ulcer

Ulcers were observed under Magnascope (5x magnification). Scored according to the arbitrary scoring system described by Srivastava et al. 1991. The severity and intensity of the lesions were graded as following: (i) shedding of epithelium =10; (ii) petechial and frank hemorrhages= 20; (iii) one or two ulcers= 30; (iv) more than two ulcers= 40; and (v) perforated ulcers= 50.

Estimation of [PGE.sub.2]

Gastric tissue obtained from control and treatment groups used for [PGE.sub.2] quantification. Briefly, mucosa was scrapped and rapidly rinsed with ice-cold saline. The tissue was weighed and 10 volumes of phosphate buffer (0.1 M, pH-7.4) used for homogenization. Contain 1 mM EDTA and 10M indomethacin. The homogenate was centrifuged (10,000 rpm, 10 min, 4 [desrees]C), and the supernatant was processed for [PGE.sub.2] estimation using the Biotrak enzyme immunosorbent assay kit (Cayman), following the manufacturer's instructions. Results were expressed as pg [PGE.sub.2]/mg protein.

In vitro assay of [H.sup.+] [K.sup.+]-ATPase activity

According to Berglindh 1990 gastric microsomes was isolated from normal fasted rat stomach for [H.sup.+] [K.sup.+]-ATPase enzymatic activity. For the enzyme assay, gastric microsomes incubated with different concentrations of peganine (10-100) as well as reference drug omeprazole for 10 min at 37 [degrees]C, were added to an assay buffer containing (in mM) 150 KC1, 10 PIPES, 1 Mg[SO.sub.4], 5 Mg ATP, 1 EGTA and 0.1 ouabain, at pH 7.2 and 10[micro],g/ml valinomycin, 2.5 [micro]g/ml oligomycin. The reaction was carried out at 37 [degrees]C for 20 min and was stopped by adding 10% ice-cold trichloroacetic acid. After centrifugation (2000g for 1 min), inorganic phosphate release was determined from the resulting supernatant spectrophotometrically at 310 nm wavelength (Sanui 1974) and expressed as [micro]M/h/mg protein.

Statistical analysis

All values shown in the figures and tables represent the means [+ or -]- S.E.M. [IC.sub.50] values with 95% confidence limits were estimated using Maximum Likelihood Iterative Procedure (Finney 1952). Statistical analysis was performed with Prism version 3.0 software using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. p < 0.05 was considered to be statistically significant.

Results and discussion

Anti-ulcer effect of peganine against cold restraint induced ulcer in rats

Peganine (1) doses at (10, 20 and 40 mg/kg, p.o.) showed percentage protection of 37.5, 50.0 (p < 0.05) and 58.5 (p < 0.05) respectively. From this observation we found that 20 mg/kg dose of peganine (1) was effective and selected for further studies. Reference drug omeprazole (OMZ) found 77.4% (p < 0.01) protection in comparison to control (10 mg/kg, p.o.). CRU is a well-accepted model for the induction of gastric ulcers, in which peripheral sympathetic activation and increased acid secretion play important roles (Djahanguiri et al. 1973). The results are graphically represented in Fig. 3.

Anti-ulcer effect of synthetic compounds against cold restraint induced ulcer in rats

We synthesized two closely related analogs of peganine (1) (Vasicine) i.e. Vasicinone (2) and Acetyl vasicinone hydrochloride (3) (Fig. 1) in our laboratory using previously reported procedure (Tetsuji et al. 1977; Ahmed et al. 2001) (please see supporting information) and tested them at dose of 20 mg/kg, p.o. They exhibited poor protection (37.5%) in CRU ulceration model (Fig. 4) in comparison to peganine (1), which might be varied pharmacokinetic properties.

Effect of peganine against alcohol induced ulcer

Peganine (1) showed significant anti-ulcer activity against ethanol induced ulcer having 89.41% protection (p<0.001), whereas the reference drug, sucralfate (SUC), showed 62.5% protection (p < 0.01)as depicted in Fig. 5. Peganine (1) exerted a protective effect against ethanol in contrast to reference drug. Ethanol damages the superficial epithelial layers and inhibit the release of mucosal prostaglandins (Miller and Henagan 1984) and depresses the gastric defensive mechanisms, these agents appear to augment the gastric mucosal defense (Kinoshita et al. 1995) indicating the cytoprotective potentials of peganine (1).

Effect of peganine against aspirin induced ulcer in rats

Peganine (1) exhibited 58.50% protection against aspirin induced ulcer model (Fig. 5). Omeprazole (OMZ) showed 49.97% protection in comparison to control. To confirm the cytoprotective potency of peganine (1) NSAIDs induced ulcer model was investigated. Peganine reduced ulcer incidence, which further supports cytoprotective effect, which may be mediated by prostaglandins.

Anti-ulcer effect of peganine against pyloric ligation induced ulcer in rats

Anti-ulcer activity of peganine (1) was also studied against pyloric ligation induced ulcer in rats where it showed protection of 62.50% (p < 0.01) and reference drug omeprazole (OMZ) showed 69.42% (p <0.01) protection (Fig. 5). In this model, auto-digestion of mucosa by gastric acid results in the development of ulcers (Godl and Bhattacharya 1991). Peganine (1) significantly reduced free and total acidity in this model, which suggests its anti-secretory potency.

Effect of peganine on gastric secretion

The antisecretory effect of peganine (1) was evaluated by estimating free and total acidity of gastric juice and by estimating the activity of mucin as shown in Table 1. Gastric acid is an important factor for the genesis of ulceration in pyloric-ligated model vessels (Shay et al. 1945). Peganine (1) has reduced free acidity (33.38%, p <0.01), total acidity (38.09%) which was comparable with reference drug omeprazole (OMZ) (50.56%, p <0.01) and (54.68%, p <0.05) respectively. Mucin secretion was upregulated by 67.91% (p <0.01), whereas omeprazole (OMZ) increased mucin secretion by 42.25% (p <0.05) in comparison to control.

Table 1 Effect of peganine (PG) and reference drug omeprazole (OMZ)
on free acidity. total acidity and mucin contents in pyloric
ligation model (n =6 in each group).

Treatment              Free acid          Total acid            Mucin
             ([micro].equiv./ml)  ([micro]equiv,/ml)    ([micro]g/ml)

Control     56.50 [+ or -] 3.592      94.23 [+ or -]  561.06 [+ or -]
                                               3.480            53.76

PC (l) (2a        37.64 [+ or -]      58.33 [+ or -]    1748.70 [+ or
mg/kg)                    5.634'              4.500'       -] 189.20"

OMZ               27.93 [+ or -]      42.70 [+ or -]  972.41 [+ or -]
(10mg/kg)                 1.305"              1.309"           9.918'

* Statistically significant at p< 0.05 in comparison to control.

**Statistically significant at p <0.01 in comparison to control.


Effect of peganine on [H.sup.+] [K.sup.+] -ATPase activity

The antisecretory mechanism of action of peganine (1) (10-100 [micro]g/ml), has been confirmed through the inhibition of gastric H (+) K (+) -ATPase activity in comparison with control with an [IC.sub.50] value of 73.47 [micro]g/ml. Omeprazole (10-50 [micro]g/ml) reduced the enzyme activity with an [IC.sub.50] value of 30.24 [micro]g/ml (Fig. 6). Proton pump is a membrane bound enzyme that catalyses H (+) transport at the expense of ATP hydrolysis. Thus suggesting that peganine might be imparting anti-ulcer activity through decrease in acid secretion via proton pump inhibition.

Effect of peganine and omeprazole on [PGE.sub.2] level

Estimation of prostaglandin generation is of immense importance in context to NSAIDs induced gastropathy. The PGE2 generation in the ulcer control group was 2131.0 [+ or -] 163.4 pg/mg tissue protein. The [PGE.sub.2] value of peganine (1) and reference drug omeprazole (OMZ) treated group was found to be 4893.0 [+ or -]472.7 (Pb < 0.01), 4341.0 [+ or -] 449.7 (Pb <0.01) respectively (Fig. 7). Improvement of prostaglandin generation in peganine (1) treated groups indicates its cytoprotective activity.

Effect of peganine on anti-oxidant assays

Antioxidants can reduce the oxidative stress and consequently ameliorate the progress of stress related diseases. The peganine (1) exhibited strong antioxidant activity in the DPPH inhibition assay as evidenced by the low [IC.sub.50] values (Table 2). The [IC.sub.50] values obtained are 71.97 [+ or -] 3.79 [micro],g/ml, respectively in the DPPH inhibition assays. In vivo antioxidant studies of peganine (1) were also carried out to corroborate its antioxidant properties. Superoxide dismutase (SOD) was assayed according to Misra and Fridovich (1972) based on the inhibition of epinephrine auto-oxidation by the enzyme. In the control group the SOD activities were 0.079 [+ or -] 0.37 (U/mg protein) after the treatment with peganine it increased the SOD activity 0.275 [+ or -] 0.21 (U/mg protein) similar to reference antioxidant ascorbic acid treated group's SOD 0.284 [+ or -] 0.83 (p <0.01. when compared with control). The antioxidant activity of the peganine (1) has been suggested to play a role in the relief of long-term complications and the oxidative stress.

Table 2

In vitro antioxidant activity of peganine (1) on DPPH antioxidant
(% inhibition) (a)

Concentration ([micro]g/ml)  1.1-Diphenyl 1-2-picryl-hydrazyl
                                                       (DPPH)

10                                         16.97[+ or -] 1.24

20                                        29.43 [+ or -] 3.59

40                                         37.02 [+ or -]4.10

60                                         46.81 [+ or -]8.16

80                                         51.G5[+ or -]12.73

100                                      62.23 [+ or -] 10.08

[IC.sub.50]                                71.97 [+ or -]3.79

Reference (ascorbic acid)                  42.06 [+ or -]5.27
[IC.sub.50]

a Values are expressed as percentage mean of 3 replicates.


Conclusion

In conclusion our results revealed that, peganine (1) isolated from the seeds of P. harmala possess remarkable anti-ulcer activity in rats and our study is the first of its kind to show significant anti-secretory, antioxidant and cytoprotective effect of peganine. However two synthetic analogs vasicinone (2) and its acetate (3) prepared in our laboratory did not exhibit significant activity, which indicated that 1 might has better pharmacokinetic properties (ADME) than 2 and 3.

Acknowledgements

Authors are thankful to the Director, CDRI for constant encouragement for the program on natural products, SAIF Division for spectral data, Botany Division for identification of plant material. Vinay Kumar Singh to UGC, New Delhi, Vaibhav Mishra and Tanvir Khaliq gratefully acknowledge CSIR, New Delhi, for providing financial support. Mrs. Shibani Sen Gupta for her technical work.This is CDRI Communcation No 8486.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phyma1.2013.06.017.

* Corresponding authors. Tel.: +91 522 2612411: fax: +91 522 2623405.

E-mail addresses: gpalitcdri@gmail.com (G. Palit), t_narendra@cdri.res.in, tnarender@rediffmail.com (T. Narencler).

0944-7113/$--see front matter [c] 2013 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.phymed.2013.06.017

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Vinay Kumar Singh (a), Vaibhav Mishra (b), (c), Sriniwas Tiwari (a), Tanvir Khaliq (a), Manoj Kumar Barthwal (b), Haushila Prasad Pandey (c), Gautam Palit (b), *, Tadigoppula Narender (a), *

a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, UP., India

b Division of Pharmacology, CSIR-Central Drug Research Institute. Lucknow 226001, UP., India

c Department of Biochemistry (BHU), Varanasi 221005. UP., India
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Author:Singh, Vinay Kumar; Mishra, Vaibhav; Tiwari, Sriniwas; Khaliq, Tanvir; Barthwal, Manoj Kumar; Pandey
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Oct 15, 2013
Words:4746
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