Study on Anti-Ulcer Activity of Daucus carota Juice

 

G Chandra Mohan Rao^1*, D Sujatha¹, VLS Rupa¹, ES Sindhu Priya¹, K Mallikarjuna Rao² and M Venkateswarlu¹

¹Dept. of Pharmacology, Sri Padmavathi School of Pharmacy, Thiruchanoor, Tirupathi-517503, A.P, India.

²Department of Pharmaceutics, Annamacharya College of Pharmacy, Rajampeta, Kadapa(dist), A.P, India.

*Corresponding Author E-mail: chanduganjikunta@gmail.com

ABSTRACT:

A number of drugs are now available for treatment of peptic ulcer disease. These drugs are mainly considered the counter acid secretion and cytoprotection by virtue of their effects on mucosal defensive mechanisms. Although these drugs have brought remarkable changes in the ulcer therapy, the efficacy of these drugs is still debatable. Reports on clinical evaluation of these drugs show that there are incidences of relapse, adverse effects and danger to drug interaction during ulcer therapy. The search to find a suitable palliative or curative agent for the treatment of peptic ulcer disease has been extended to dietary fibre. The Main aim of the present study was to verify the claim of Daucus carota as an antiulcer agent, mentioned in traditional system of medicine. Hence, the objective of the present study was to investigate the antiulcer activity of juice of Daucus carota by employing the pylorus ligation method in rats. The group of animals treated with Ranitidine HCl at a dose of 26.57 mg/ kg b.wt, a significant decrease in the acid volume (13.5 ml) and increase in the gastric pH ⁽6) was noted. Group receiving D. carota   at a dose of 1 g/ kg. b. wt. a significant decrease in the acid volume (5.96 ml) and increase in gastric pH ⁽6) was observed when compared with the control animals. The anti-ulcer activity was observed by using the ulcer index values such as control (14.9), standard (5.15) and test samples (3.833). By on the observation of ulcer index values the prepared Daucus carota juice having the best anti-ulcer activity compared with standard sample.

 

 

INTRODUCTION:

A natural form of medicine, i.e., herbal medicine is the most ancient form of medicine. Today, many plants are synthesized for pharmaceutical use, stemming from ancient, healing traditions of herbal medicine. In India, Peptic ulcer disease is common. In the Indian pharmaceutical industry, antacids and antiulcer drugs share 6.2 billion rupees and occupy 4.3% of the market share. Today, there are two main approaches for treating peptic ulcer. The first deals with reducing the production of gastric acid and the second with re-enforcing gastric mucosal protection¹.

 

Researchers have long suspected that diet is associated with peptic ulcer, and dietary differences have been cited to explain the geographic variation in this disease. Dietary factors that have been investigated include dietary fibre; fatty acids, particularly linoleic acid; vitamins C, E, and A; alcohol; and caffeine².

 

Aldoori and colleagues found an inverse association between vitamin A and risk of duodenal ulcer. But review of literature has shown no scientific support for the antiulcer activity of Daucus carota Linn, a rich source of vitamin ‘A’. Hence, the present study aims for the systematic evaluation of the antiulcer activity of juice of D. carota

 

The search to find a suitable palliative and/or curative agent for the treatment of peptic ulcer disease has also been extended to dietary fibre. Although a great deal of research in dietary fibre was stimulated throughout the world, it is still early to assign clear health claims to dietary fibre. This difficulty is due in great part to the fact that dietary fibre includes many complex substances, each having unique chemical structure and physical properties. Nevertheless, numerous prospective and well designed experimental studies have highlighted several physiological and metabolic effects of dietary fibre The present study is also an attempt towards this direction.  The present study aims to verify the claim of D. carota as antiulcer agent as mentioned in traditional system of medicine. Hence, the objective of the present study  is to investigate the antiulcer activity of juice of D. carota by employing the pylorus ligation in rats³.

MATERIALS AND METHODS:

Fresh carrots were procured from Reliance mart super market, Thirupati, Andhra Pradesh, India. Ranitidine hydrochloride was obtained from the Micro labs as a gift sample.  Carboxy methyl cellulose, Phenolphthalein solution,  Methyl orange solution, Sodium hydroxide was procured from Sd. Fine Chemicals Ltd., India. Plant profile was mentioned in the table No:- 01 and the plant image also mentioned in the figure No:- 01^4,5.

 

Table No:- 01,  Plant profile

Scientific classification

Kingdom Plantae Division Angiosperms Class Eudicots Order Apiales Family Apiaceae Genus Daucus Species D. carota

Binomial name

Daucus carota synonym:-  Wild Carrot, Bishops Lace,  Queenanne’s  Lace.

 

Figure No:- 01, Image of the plant Daucus carrota

 

Preparation of the extract:

The collected fresh carrots were washed under tap water and cut into small pieces using a sharp knife. These pieces were crushed in a mortar into a soft paste. The paste thus obtained was squeezed using a muslin cloth to get the juice. The volume of the juice was measured and the weight per ml calculated.

 

Phytochemical screening:

Preliminary phytochemical screening of the extract was performed for the presence of flavonoids, tannins.

 

a) Test for  Tannins:⁶

The extract was treated with a few drops of neutral ferric chloride solution. Tannins form an in sky blue precipitate.

The extract was treated with lead acetate solution. A white precipitate developed shows the presence of tannins.

 

b) Test for  Flavonoid⁶

Shinoda test: The extract was treated with dilute HCl, in presence of small piece of magnesium ribbon. Development of pink, orange or red color shows the presence of flavonoids.

 

Table:2 : Treatment schedule for evaluation of anti ulcer activity

Group	Drug	Dose	Purpose
I	Saline	2 ml	To serve as control
II	Ranitidine	26.57 mg/kg	To serve as Standard
III	D. carota Juice	1 g/kg	To assess the antiulcer Activity

 

 

 

 

 

Acute toxicity studies:⁷

Healthy adult male albino rats were fasted overnight with free access to drinking water. They were divided into five groups each consisting of six animals. Group-1 animals were treated with distilled water (2ml/kg/p.o) and Group-2 to Group-5 animals received 500 mg, 1 g, 2 g, 4 g/kg/p.o of D.carota juice respectively. The animals were observed continuously for 2 hours, then intermittently and at the end of 24 hours. The numbers of the deaths were noted to calculate LD_{50 .}

 

Gross behavioral changes:⁷

The animals were observed for behavioral, neurological and autonomic profiles during acute toxicity studies.

 

PHARMACOLOGICAL EVALUATION:

ANTIULCER ACTIVITY:^{8, 9}

In the present study, antiulcer activity was assessed by employing pylorus ligation model. The animals were divided into 3 groups of 6 animals each and treated as shown in the table.02¹⁰. A simple and reliable method for production of gastric ulceration in the rat was based on ligature of the pylorus. The ulceration was caused by accumulation of acidic gastric juice in the stomach. Ulcer index and acidity of the gastric content of treated animals are compared with controls. The rats were fasted for 48 hours before the experiment but with free access to water and just two hours before starting the experiment the water also were removed. One hour after drug or saline administration, under light ether anesthesia the abdomen was opened by small midline incision below the xiphoid process, pyloric portion of the stomach was slightly lifted out and ligated avoiding traction to the pylorus or damage to its blood supply the stomach was replaced carefully and the abdominal wall closed by interrupted sutures. Nineteen hours later, the pylorus ligated rats were sacrificed by ether over dosing and their stomachs were dissected out after ligating the esophagus at cardiac end. Each stomach was cut opened along the greater curvature and the contents were collected into a centrifuge tube, then the mucosa was washed under slow running tap water and the number and size of ulceration was scored as per the method of Rao et al.   The gastric juice collected from each stomach was centrifuged and its volume was measured. Free and total acidity were estimated titrimetrically with 0.1NaOH using methyl orange and phenolphthalein as indicators. The following parameters were measured

 

Table:3: Effect Of D. carota Juice On Acid Volume And pH

S. NO:	GROUP	TREATMENT	DOSE	ACID VOLUME(ml)	PH
1	Control	Saline	2 ml/animal	20.26 ± 0.5944	1 ± 0
2	Standard	Ranitidine	26.57 mg/kg b. wt.	13.5 ± 2.1984*	6 ± 0
3	Treatment	D.carota juice	1 g/kg b. wt.	5.96 ± 4.7120*	6 ± 0

* p< 0.05 when compared with the control group

 

Table: 4:Effect Of D. carota Juice On Free Acidity And Total acidity:

S. NO:	GROUP	TREATMENT	DOSE	FREE ACIDITY	TOTAL ACIDITY
1	Control	Saline	2ml/animal	36.41 ±  0.5322	226.41 ± 2.0920
2	Standard	Ranitidine	26.57 mg/kg b. wt.	39 ± 1.9356	66 ± 1.4719*
3	Treatment	D.carota juice	1 g/kg b. w.t	39.25  ± 0.4193	35  ± 0.9092*

*p< 0.05 when compared with the control group.

 

Table :5:Effect Of D. carota Juice On Ulcer Score

S. NO:	GROUP	TREATMENT	DOSE	MUN	MUS	MUP	UI
1	Control	Saline	2ml/animal	46 ± 17.1658	3 ± 1.8257	100%	14.9
2	Standard	Ranitidine	26.57 mg/kg b. wt.	1±0.8164*	0.5	50%	5.15
3	Treatment	D.carota juice	1 g/kg b.wt.	5±6.6332*	0	33.33%	3.833

*p< 0.05 when compared with the control group.

 

 

Acid volume and pH:

The stomach was removed and the contents were drained into a graduated centrifuge tube through a small nick along the greater curvature adjacent to pyloric ligation. The volume of the juice was measured (acid volume)¹¹. The contents were drained into a graduated centrifuge tube, which was centrifuged at 3000 rpm for 10 minutes and the centrifuged samples were decanted and analyzed for pH (using broad range pH paper)¹².

 

Determination of Total Acidity:¹³

The known amount of gastric residue was titrated with 0.1 N NaOH,  then add two drops of Methyl orange reagent which changes to a salmon color when total amount of free hydrochloric acid was neutralized. The total acidity was determined by titration using Phenolphthalein as indicator. Gastric juice specimen was transferred in a porcelain evaporating dish. 1-2 drops of Methyl orange reagent was added. A colour change was observed; a bright red color appears due to presence of free HCl.  Titrated with 0.1 NaOH from a burette, mixing was done after each addition until the last trace of red color disappeared and was replaced by   a canary yellow colour. The numbers of milliliters of NaOH used was read from the burette. This  represents the amount of free hydrochloric acid. 1-2 drops of phenolphthalein was added to the gastric juice . The titration was continued until the red color of phenolphthalein appeared (deep pink), titrated to the point at which the further addition of alkali did not deepen the color.  Reading was taken (ml NaOH) for total acidity.

Y    =    0.1 N NaOH (ml)   X   10

Where,

Y= Total acidity (m Eq/L)

 

Ulcer Index:

The stomach was removed and fixed on a cork plate and the number and severity of the ulcers was registered with a stereo-microscope using the following scores .

0             = Normal coloured stomach

0.5          = Red colouration

1             = Spot ulcer

1.5          = Hemorrhagic streaks

2             = Ulcers ≥ 3 but ≤5

3             = ulcers > 5

Calculation

Ulcer index was calculated as;

UI           = UN + US +   UP   X   10^-1

Where, UI             = ulcer index

UN         = average of number of ulcers per animal

US          = average of severity score

UP          = percentage of animals with ulcer

 

RESULTS AND DISCUSSION:

The roots of juice of  D.carota was found to be safe since no animal died at the dose of 4 g/kg b.w.  The root juice  of D. carota did not show any gross behavioural changes at the doses tested.

 

Preliminary phytochemical studies of the root juice of D. carota showed the presence of tannins, flavanoids and polyphenols.

 

In the presence study, after pylorus ligation a significant increase in the acid volume was observed in the control animals (20.26 ml). The group of animals treated with Ranitidine at a dose of 26.57 mg/ kg b.wt, a significant decrease in the acid volume (13.5 ml) and increase in the gastric pH (6) was noted. Group receiving D.carota at a dose of 1 g/ kg. b. wt. a significant decrease in the acid volume (5.96 ml) and increase in gastric pH (6) was observed when compared with the control animals. The results were shown in the table No:- 03.

 

Animals treated with Ranitidine at a dose of 26.57 mg/kg b.wt. showed a significant decrease in total acidity (66) when compared with the control group. The juice of D. carota at the dose of 1g/ kg b.wt. produced  a significant decrease in  total acidity (35) when compared to the control group of animals. The total acidity values were shown in the table No:- 04.

 

Figure No:-02, Images of the Rat stomach with different treatments.

 

Figure No:- 03, Graphical Reprasentation of the  anti-ulcer activity comparing with the different treatments.

 

In the standard group of animals which were treated with Ranitidine  the Mean ulcer number, Mean ulcer score, Mean ulcer percentage and Ulcer index was significantly less than that of control group.  The group of animals treated with the juice of D. carota at a dose of 1g/kg b.wt, showed a significant decrease in the mean ulcer number, mean ulcer score, mean ulcer percentage and ulcer index  when compared with the control group. The results were shown in the Table No:- 5.

 

The juice of D. carota has significantly increased the gastric pH and also exhibited a significant decrease in both total acidity and also volume of gastric content in pylorus ligation model. The reduction in acid output, peptic activity and increase in mucin secretion were the major mechanisms behind the protection shown in the pylorus ligation model by various plants like Rhamnus procumbens and Datura fastuosa, so the plant D. carota may also be exhibiting potential antisecretory effect by similar mechanisms.

 

CONCLUSION:

The various parameters governing the ulcer formation in the stomach were evaluated and it was observed that the juice of D. carota roots showed a significant decrease in the acid volume, free acidity, total acidity and an increase in the gastric pH. Moreover the mean ulcer number, mean ulcer score and ulcer index also decrease significantly. Thus, the roots of D carota were found to exhibit potential antisecretory effects by reducing the acid output.

 

ACKNOWLEDGEMENT:

The authors are thankful to Micro Labs Pvt. Ltd, for providing the gift sample of Ranitidine Hydrochloride.   The authors also thankful  to Sd. Fine Chemicals Ltd  for providing the chemicals such as Carboxy methyl cellulose, Phenolphthalein solution,  Methyl orange solution, Sodium hydroxide and also thankful to management of Sri Padmavathi School of Pharmacy, Thiruchanoor for providing the all facilities for carried out this research work.

 

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Received on 30.11.2009       Modified on 23.01.2010

Accepted on 27.02.2010      © RJPT All right reserved