Biological Activities of an Ethnomedicine: Alternanthera sessilis

INTRODUCTION:

Alternanthera sessilis R.Br. ex D.C. (Amaranthaceae) is a very common weed found throughout India¹. It has been used in Indian traditional system of medicine since a long time, for promoting memory and intelligence and externally for complexion; Bhavapraakasha attributed blood-purifying properties to this herb. During the 16^th century, it was used in diseases due to vitiated blood, skin diseases, ulcers and wounds^{2, 3}. The plant is a good adjuvant with sex tonics and for females a natural galactagogue¹; entire plant and its parts, alone or in combination with other plants are used traditionally throughout the world and in India for intestinal cramps, diarrhea and dysentery (intestinal disorder), as a diuretic, as a cooling agent to treat fever, hepatitis, tight chest, bronchitis, asthma and lung troubles, to stop bleeding, as a hair tonic, for itch, common skin problems such as cuts, burns, eczema, boils, and leucoderma; poultice of pounded fresh material is used in sprains, carbuncle, erysipelas and acute conjunctivitis; the plant is also used for hazy vision and night blindness^1-10.

A review of the literature revealed that anti-ulcer activity of this plant has not been subjected to scientific evaluation, therefore this study was undertaken to investigate the anti-ulcer potential of chloroform insoluble fraction of alcoholic extract (CIAE) using rats.

MATERIAL AND METHODS:

Plant material

Whole plants were collected from the fields around the campus of Indira Gandhi Krishi Vishvavidyalaya (Agriculture University), Raipur (Chhattisgrah), India, during the months of December and January 2004-05, authentication was made by Prof. P. Jayaraman, Director Plant Anatomy Research Centre; Chennai- India. The specimen was voucher, and deposited at Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur; Chhattisgrah, India.

Extraction

One kg of powdered drug was packed in soxhlet apparatus and successively extracted with petroleum ether (60-80âC) and ethanol (95%). The alcoholic extract was fractioned with chloroform. The chloroform insoluble fraction of alcoholic extract (CIAE), 5.9 % w/w of powdered drug, was used for further experimental work.

Experimental animal

Inbred, male, Wister Albino rats (100 – 130 grams) were selected for these studies. Six rats were taken for each group. The rats were used after an acclimatization period of seven days to the laboratory environment. They were provided with food and water adlibitum. All animal experiments were carried out at Periyar College of Pharmaceutical Sciences for Girls, Trichy, Tamilnadu- India, according to the guidelines and approval of the Animal Ethics Committee (Registration Number 265/CPCSEA).

Evaluation of in vitro antioxidant activity

Diphenylpicrylhydrazyl (DPPH) radical scavenging activity, Hydrogen peroxide (H₂O₂) radical scavenging activity and reducing power of CIAE was determined by procedures mentioned earlier^11-15.

DPPH radical scavenging activity: 0.1 mM solution of DPPH in ethanol was prepared and 1.0 ml of this solution was added to 3.0 ml of extract solution in water at different concentrations (10-100 µg/ml). Thirty minutes later, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The capability to scavenge the DPPH radical and the standard (BHT) was calculated using the following equation:

Where A_cont is the absorbance of the control reaction and A_test is the absorbance in the presence of the sample of the extracts.

H₂O₂ radical scavenging activity: A solution of H₂O₂was prepared in phosphate buffer (pH 7.4). H₂O₂ concentration was determined spectroscopically measuring absorption with extinct coefficient for H₂O₂. Different concentrations of extracts in distilled water were added to a H₂O₂ solution (0.6 ml, 40 mM). Absorbance of H₂O₂ at 230 nm was determined 10 min later against a blank solution containing the phosphate buffer without H₂O₂. a-tocopherol was used as the standard. The % of H₂O₂ scavenging was calculated by the equation:

Where A_cont is the absorbance of the control reaction and A_test is the absorbance in the presence of the sample of the extracts.

Reducing power: Various concentrations of the extracts in 1.0 ml of deionized water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 1% potassium ferricyanide (2.5 ml). The mixture was incubated at 50°C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of

the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml. 0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Butylated hydroxy toluene (BHT) was used as a standard.

Evaluation of anti-ulcer activity

In present study the effect of CIAE on pylorus ligature, aspirin induced and cold-restraint stress-induced gastric lesions in rats was evaluated according to methods described earlier^16-18.

Pylorus ligature induced gastric ulcer: The animals were deprived of food for 24 hours before the commencement of the experiment, but water was allowed adlibitum. Care was being taken to avoid coprophagy. The drugs were given orally 2 hours prior to pylorus ligation. Two hour before the experiment, the pylorus of each rat was tied under light ether anesthesia and the abdominal incision was closed. Six hours after the ligation, the animals were sacrificed using overdose of ether and the stomach removed. A small nick was made at the junction of pylorus ligation and greater curvature. The contents of the stomach were collected in centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10 min. The volume of supernatant fluid was measured and used for pH analysis. The stomach was then excised and cut along the greater curvature. The gastric juice was collected, centrifuged and its pH and volume were measured. The stomachs were washed carefully with 5.0 ml of 0.9% NaCl, rinsed under a slow stream of water and pinned flat on a corkboard. A person scored ulcers unaware of the experimental protocol in the glandular portion of the stomach. The stomachs were coded to avoid observer bias and examined with a hand lens (x10). Erosions formed on the glandular portion of the stomachs were counted and each one given a severity rating. Ulcer index was calculated by adding the total number of ulcers per stomach and the total severity of ulcers per stomach. The ulcer score was obtained by measuring the dimensions of the duodenal ulcer(s) in square millimeters and ulcer index was determined as follows¹⁹.

Loss of normal morphology 1 point

Discoloration of mucosa 1 point

Mucosal edema 1 point

Hemorrhages 1 point

Petechial points (until 9) 2 points

Petechial points (>10) 3 points

Ulcers up to 1mm n ´ 2 points*

Ulcers>1mm n ´ 3 points*

Perforated ulcers n ´ 4 points*

*Number of ulcers found

Aspirin (ASP)-induced ulcers: The animals were treated with control or drug samples for seven days. At the end of 7^th day, rats were fasted for 24 hours. ASP in dose of 200 mg/kg (20 mg/ml) was administered to the animals on the day of the experiment. After 4 h of aspirin induction animals were sacrificed and the stomach was then excised

and cut along the greater curvature. The ulcer index was then determined as described above.

Cold-restraint stress (CRS)-induced ulcers: CIAE (100 and 200 mg/kg) and test drug (Famotidine 20 mg/kg) was administered to rats for 7 days. The control group was fed normal saline solution. Thereafter the rats were deprived of food, but not water, for about 18 h before the experiment. On day eight, the experimental rats were immobilized by strapping the fore and hind limbs on a wooden plank and kept for 2 h, at temperature of 4-6âC. Two hours later, the animals were sacrificed by cervical dislocation and ulcers were examined on the dissected stomachs as described above.

Statistical analysis

All the data were subjected to statistical analysis using SPSS 14.0 for Windows. The results of the experiments are expressed as mean ± SEM. After confirming the variances homogeneity of results by Bartlett’s test, the differences were estimated by one-way ANOVA followed by Tukey’s test for the single dose studies, or by means of Dunnet’s test for individual comparison of groups with control. When the probability (p) was <0.05, the results were considered to be significant.

RESULTS:

Antioxidant activity: The extract exhibited scavenging potential with IC50 value of 33.26 µg/ml and 219.83µg/ml for DPPH and H₂O₂ radicals respectively. The value was found to be lesser than that of BHT (18.92µg/ml) and a-tocopherol (129.84µg/ml), used as standards in respective assays (Table 1-2). The extract showed dose dependant increase in reducing power that was comparable to the standard BHT (Table 3).

Anti-ulcer activity: CIAE exhibited a significant protective effect on ulceration induced by pylorus ligation, aspirin induced and cold restraint stress in rats (Tables 4-7). The control animals had ulcers and hemorrhagic streaks, whereas in animals administered with the extract there was reduction in ulcer index in all the models in a dose dependant manner. The ulcer index significantly (p<0.001) decreased after treatment with CIAE, at both the tested doses (100 and 200 mg/kg) and was comparable to the standard drug famotidine (20 mg/kg). The percent inhibitions of the gastric lesions were recorded more than 50 % in all the models. The inhibition was 64.57 % and 68.88 % respectively in pylorus ligation induced ulcer (Table 4); in ASP model the inhibition was 55.49 % and 66.21% respectively (Table 6), while in case of cold restraint stress induced ulcers, the inhibition was marginally higher (72.71 %) with CIAE at dose of 200 mg/kg body weight compared to famotidine at 20 mg/kg dose (71.80 %) (Table 7). Significant changes in the total gastric acid levels were also found after intraduodenal administration of the extract in the legated pylorus model. The results indicate that the extract not only significantly (p <

0.001) decreases the volume of gastric acid secretion but it also reduced free acid and total acid with respect to control and comparable to the standard drug (Table 5).

DISCUSSION:

Oxidation is essential to many living organisms’ production of energy-to-fuel biological processes. However, oxygen-centered free radicals and other reactive oxygen species, which are continuously, produced in vivo, result in cell death and tissue damage. Therefore the role of reactive oxygen species and free radical reactions in biology has become an area of intense interest^{20, 21}. Antioxidants have been found to be important in the prevention of human diseases by functioning as free radical scavengers, complexers of pro-oxidant metals, reducing agents and quenchers of singlet oxygen formation. There are some synthetic antioxidant compounds, such as butylated hydroxytoluene, and butylated hydroxyanisole, which are commonly used in processed foods, it has been reported that these compounds may have side effects. In addition, it has been suggested that there is an inverse relationship between dietary intake of antioxidant-rich foods and the incidence of a number of human diseases^20-22. Therefore, research into the determination of natural antioxidant sources is important, but due to its increasing significance, there is a parallel increase in the use of methods for estimating the efficiency of such substances as antioxidants^20-25. In our work we have performed some more popular methods that were easy to perform in our laboratory conditions.

Table 1: DPPH radical scavenging activity

Drug Concentration	DPPH Scavenged %
Drug Concentration	BHT	A .sessilis
10 µg/ml	28.8±0.22	20.6± 0.19***
20µg/ml	61.8± 0.70	51.7±0.72***
50 µg/ml	79.8± 1.14	69.8± 1.01***
100 µg/ml	95.17± 1.45	88.7± 1.36**
IC₅₀	18.92µg/ml	33.26µg/ml

Values are mean ± S.E., n = 6; data analyzed by one way ANOVA and student’s “t” test; ** p< 0.001, *** p< 0.0001 vs control.

The DPPH method can be used for solid or liquid samples and is not specific to any particular antioxidant component, but applies to the overall antioxidant capacity of the sample²⁵. H₂O₂ is highly important because of its ability to penetrate biological membranes. H₂O₂ is not very reactive, but it can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cells. Thus, removing of H₂O₂ is important for protection of food systems²⁶. Antioxidant activity has been reported to be concomitant with the development of reducing power. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity²⁷. In our present study it was found that A. sessilis had mild to moderate scavenging as well as reducing capacity.

Peptic ulcer is a lesion of gastric or duodenal mucosa occurring at a site where the mucosal epithelium is exposed

to aggressive factors. Although in most of the cases the etiology of the ulcers is unknown, it is generally accepted that they result from an imbalance between aggressive factors and the maintenance of mucosal integrity through endogenous defense mechanisms²⁸.

Table 2: H₂O₂ radical scavenging activity

Drug Concentration	H₂O₂Scavenged %
Drug Concentration	a-Tocopherol	A.sessilis
50 µg/ml	31.17± 0.48	15.17± 0.31***
100 µg/ml	46.33± 0.84	30.75± 0.75**
200 µg/ml	67.67± 1.23	53.42± 1.16***
300 µg/ml	79.83± 1.58	62.48± 1.38**
350 µg/ml	88.67± 2.04	70.65± 1.67***
IC₅₀	129.84µg/ml	219.83µg/ml

Values are mean ± S.E., n = 6; data analyzed by one way ANOVA and student’s “t” test; ** p< 0.001, *** p< 0.0001 VS control.

Also it has been reported that non-steroidal anti-inflammatory drugs (NSAIDs) associated gastric ulceration occurs in 30 per cent of users that leads to hospitalization and is also associated with high mortality. Aspirin (ASP) is one of the most widely used NSAIDs, which damages gastrointestinal mucosa by irritant action, causing alterations in mucosal permeability and/or suppression of prostaglandin synthesis²⁹. Most of the commonly used drugs such as H2- blockers (ranitidine, famotidine etc), M1- blockers (pirenzepine, telenzepine etc), proton pump inhibitors (omeprazole, lansaprazole etc), decrease secretion of acid while, drugs like sucralfate and carbenoxolone promote mucosal defenses. Not long, the role of these drugs on the defensive factors is gaining importance. It is now assumed that these drugs ultimately balance the aggressive factors (acid, pepsin, H. pylori, bile salts) and defensive factors (mucin secretion, cellular mucus, bicarbonate secretion, mucosal blood flow and cell turnover). Although these drugs have brought about remarkable changes in ulcer therapy, the efficacy of these drugs is still debatable. Reports on clinical evaluation of these drugs show that there are incidences of relapses and adverse effects and danger of drug interactions during ulcer therapy. The search for an ideal anti-ulcer drug continues and has also been extended to herbal drugs in search for new and novel molecules, which afford better protection and decrease the incidence of relapse ³⁰.

Table 3: Reducing power

Drug Concentration	BHT	A.sessilis
100µg	0.987± 0.02	0.87± 0.02**
200µg	1.25±0.02	1.18± 0.02***
500µg	1.61±0.02	1.53± 0.05
600µg	1.8±0.05	1.66± 0.01*
1000µg	2.28±0.06	2.1± 0.05*

Values are mean ± S.E., n = 6; data analyzed by one way ANOVA and student’s “t” test; *p<0.01, ** p< 0.001, *** p< 0.0001 vs control.

Table 4: Effect on pylorus ligature induced gastric lesions

Treatment	Ulcer Index (Sq mm)	Inhibition (%)
Control (5% w/v Acacia)	34.8 ± 2.1	---
A.sessilis (100 mg/kg)	12.3** ± 0.9	64.57
A.sessilis (200 mg/kg)	10.8** ± 0.4	68.88
Famotidine (20mg/kg)	10.5** ± 0.8	69.74

Values are mean ± S.E, n=6; ** p< 0.001 vs control by students ‘t’ test.

Experimental studies to determine the role of plant extracts for their gastric ulcer activity needs to evaluate acid output and status of the mucosal barrier in gastric ulcer^{16-18, 28-30}.

The present study was therefore undertaken to describe the effect of plant extracts on pylorus ligature, aspirin induced and cold-restraint stress-induced gastric lesions in rats. The parameters taken to assess anti-ulcer activity were volume of gastric secretion, pH, free acidity, total acidity, ulcer index and % inhibition of gastric ulcers in pylorus ligature model. While in aspirin induced and cold resistant stress induced models, ulcer index and % inhibition of gastric ulcers was determined. Pylorus ligature induced gastric ulcer model was used to investigate the neutralizing properties and/or the antisecretory effects of extracts¹⁶.

Table 5: Effects on gastric juice parameters in pylorus ligature induced gastric lesions

Treatment	Volume of gastric juice (ml)	pH	Total acidity (mEq/l)	Free acidity (mEq/l)
Control (5% w/v Acacia)	2.1 ± 0.05	1.2 ± 0.06	98 ± 4.7	79 ± 3.8
A.sessilis (100 mg/kg)	0.73* ± 0.03	3.6** ± 0.06	43** ± 3.3	37** ± 2.1
A.sessilis (200 mg/kg)	0.53** ± 0.02	4.4** ± 0.08	31** ± 0.18	18** ± 0.5
Famotidine (20mg/kg)	0.50** ± 0.03	4.5** ± 0.38	28** ± 1.2	16** ± 0.9

Values are mean ± S.E, n=6; * p< 0.01 vs control by students ‘t’ test.

The other two methods were performed to evaluate the protective potential of the drug on gastric mucosal cells^{17, 18}. Additionally it has been reported that the reactive oxygen species generated by the metabolism of arachidonic acid, platelets, macrophages, smooth muscle cells may contribute to gastric mucosal damage.

Table 6: Effect on aspirin induced gastric lesions

Treatment	Ulcer Index (mm²)	Inhibition (%)
Control (saline solution)	39.5 ± 0.99	---
A.sessilis (100 mg/kg)	17.58 ± 0.2**	55.49
A.sessilis (200 mg/kg)	13.33 ± 0.66**	66.21
Famotidine (20mg/kg)	13.83 ± 0.31**	64.94

Values are mean ± S.E, n=6; * p< 0.01 vs control by students ‘t’ test.

Table 7: Effects on cold restraint stress induced ulcers

Groups	Ulcer Index (mm²)	Inhibition (%)
Control (saline solution)	28.69 ! 0.70	---
A.sessilis (100 mg/kg)	9.57 ! 0.02**	66.64
A.sessilis (200 mg/kg)	7.83 ! 0.09**	72.71
Famotidine (20mg/kg)	8.09 !0.05***	71.80

Values are mean ± S.E, n=6; **p< 0.001 and ***p< 0.0001 vs control by students ‘t’ test.

Therefore, by scavenging free radicals, the reactive oxygen metabolites might be useful by protecting the gastric mucosa from oxidative damage or by accelerating healing of gastric ulcers³¹.

Our results imply that A. sessilis possesses significant antiulcer property either due to cytoprotective action of the drug or by strengthening of gastric and duodenal mucosa or due to its antioxidant activity. There is also a possibility that all of these mechanisms may be acting simultaneously and thus enhancing mucosal defense. These activities may also be due to the presence of various phytochemicals present in it and detected during phytochemical analysis. However, further studies are required to establish its exact mode of action and the active principles involved in its anti-ulcer effect.

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Received on 08.02.2008 Modified on 20.02.2008

Research J. Pharm. and Tech. 1(2): April-June. 2008;Page 75-79

Table 4: Effect on pylorus ligature induced gastric lesions

Treatment

Treatment

Treatment