Gastroprotective and Antioxidant mechanism of Litchi chinensis Leaf extract

 

Vivek Srivastava^1,2*, Babita Viswakarma¹, Neha Mathur¹, Santosh Kumar Verma²

¹Amity Institute of Pharmacy, Amity University, Lucknow.

²Department of Pharmaceutical Sciences, Motherhood University, Roorkee.

 

ABSTRACT:

Litchi chinensis has been used to treat gastric ulcers in ayurvedic medicine but its efficacy was not validated yet. The dried leaves of Litchi chinensis were powdered and subjected to extraction with 90% ethanol using soxhlet extraction method. The extract was subjected to preliminary phytochemical analysis and was estimated for antiulcer activity against Ethanol induced ulcer model and cold stress-induced ulcer. The activity was assessed in Wistar rats at a dose of 200mg/kg orally and compared with ranitidine (45mg/kg). The extract showed significant lower the ulcer index in ethanol-induced ulcers as well as showed a significant reduction in cold stress-induced ulcers. Besides, it also shows concomitant attenuation of the level of antioxidant enzyme catalase and SOD. These findings indicate the ethanolic extract of Litchi chinensis exhibits convincing antiulcer action.

 

 

 

INTRODUCTION:

Peptic ulcer are one of the major ailments affecting about 60% of human adults and 80% of the pediatric population.¹ Gastric ulcers emerge because of variance in mucosal invading and protecting aspects.² Ulcer treatment are now primarily concentrated on reducing the harmful properties of invading secretion of acid, but novel search, safer medicine renewed the curiosity in medicine which defend the gastric mucosa from harmful agents without effecting secretin of acid or deactivating intragastric acidity. It is fully established that gastric mucosa could resist auto digestion though it is open to various toxic stimuli such as aggressive hydrochloric acid secretion, H. pylori infections, spicy food, and reflex of bile, pepsin, stress, reactive oxygen species, alcohol, 5-hydroxy tryptamine, substance P (SP), slow-releasing substance, platelet-activating factor and irritant receptors.^3-6

 

The difference between offensive features and defensive features might contribute to the development of gastric ulceration. Medicine increase the defensive features or

 

inhibit the offensive features which is the actions for antiulcerogenic effects.⁷ Litchi (Litchi chinensis Sonn.) is an evergreen plant and a non-climatic subtropical fruit arises from Southeast Asia, belonging to the family – Sapindaceae. Ayurveda has acknowledged the opportunity of the medicinal plants to heal different diseases and to foster immunity. Litchi belongs to the Soapberry genus which incorporates 150 subfamily and 2000 species. There is a need to evaluated herbal drugs technically for their mechanism of action, therapeutic efficacy and side effects. Litchi is one of the principal fruit plants which hold various bioactive compounds. Litchi fruits have a sweet taste and rich nutritious value. Litchi fruit is widely recognized by consumers over the world as other subtropical fruit.⁸ In conventional Chinese medicine litchi plant leaf has been used for the therapy of heart stroke, flatulence, and detoxification. Litchi pericarp tissue contains a notable quantity of flavonoids.⁹ Flavonoids engaged in a vital pharmacological role in treating diseases, such as cardiovascular disease, malignancy, inflammation, hypersensitivity, analgesic and anti-pyretic activities, petroleum extract of litchi leaf could inhibit the cyclo-oxygenase pathway of arachidonic acid metabolism rather than inhibiting arachidonic acid include inflammation.¹⁰ The principal source of toxic hepatitis is oxidative stress which may produce chronic liver disease. Polyphenolic compounds, like anthocyanin, tannins and numerous other ortho‑diphenolic compounds, have been recognized recently in Litchi.¹¹

 

MATERIALS AND METHOD:

Plant material:

The leaves part of the plant (Litchi chinensis) was collected from the Dehradun Uttarakhand, India. The herbarium was prepared and submitted to the National Botanical Research Institute (NBRI) Lucknow, for authentication. The Authentication reference number was NBRI/CIF/586/2018.

 

Preparation of ethanol extracts:

Fresh leaves of Litchi chinensis were collected and 100gm of the air-dried powdered drug was taken and extracted with 90% ethanol in a soxhlet extractor for 72h. The extract was concentrated in water-bath at temperature 50-55°C and dried in a desiccator.^12-15 Ethanolic extracts of Litchi chinensis leaves extracts (EELC) were subjected for preliminary phytochemical analysis.^16-18

 

Experimental animal:

Healthy albino Wistar rats (150-200g) were purchased from the Laboratory Animal Services Division of Central Drug Research Institute, Lucknow. A total of 48 animals were used for this study. The animals used during the study were treated humanely throughout the study period under international guidelines OECD. The Animals were kept in polyacrylic cages and maintained under standard housing conditions (room temperature 24-27^◦C) with a 12 h light and dark cycle. Food and water were available ad libitum. Food was not allowed from 1 h before and till completion of the behavioral study. All procedures described were reviewed and approval was obtained from the Institutional Animal Ethics Committee (IAEC) or Committee for control and supervision of Experiments on Animals (CPCSEA), Amity Institute of Pharmacy. Amity University Uttar Pradesh, Lucknow (1492/PO/Re/S/11/CPCSEA28/03/ 2018).

 

Evaluation of anti-ulcer activity in Wistar rats:

Treatment schedule:

For both models i.e. ethanol induced ulcer and cold stress-induced ulcer model the Wistar albino rats of either sex weighing between 150-250gram was used and divided into 4 groups. Each group contains four rats. Treatment given for 14 days in all animals. Group I: Normal Control (0.9% normal saline), p.o; Group II: Ulcer Control (Cold Restraint stress-induced), p.o; Group III: Standard (Ranitidine 45mg/kg b.w), p.o; and Group IV: Litchi chinensis leaves extract of 200mg/kb b.w p.o. was used.

 

 

1.     Cold Restrain stress induced gastric ulcer model:

Animal were getting rid of food for 24 hours with a free approach to the water before the experiments. The Litchi chinensis, Normal control (0.9% saline), Ulcer control (Cold Restraint stress-induced) and Standard drug (Ranitidine 24mg/kg b.w) were delivering 30 min before to restraining the animals, For stress-induced ulcers, 30 min post delivering of medicines, every rat was individually restrained in stress cage and kept at 4-6°C for three hour in refrigerator. By using cervical dislocation, the animals was sacrificed, stomach isolated and the stomach was opened along the greater curvature, rinsed under a stream of water and pinned flat on a corkboard. Formed Erosions on the glandular region of the stomach was counted and severity score on the 1-3 scale founded on ulcers diameter. The total ulcers diameter in 1 stomach divided by factor 10 were labelled as ulcer index (UI).^19-20

 

2.     Ethanol induced gastric ulcer model:

By using 1ml of 90% v/v Ethanol gastric ulcers induced in wistar rats, animal fasted for 24 hour with free access to water prior to the test Litchi chinensis leaves extract, ulcer control (0.9% normal saline) and standard drug (Ranitidine) given orally thirty minutes before administration of ethanol and animal was sacrificed the stomach was dissected out record the ulcer score, in glandular region linear heamorrhagic lesions was also recorded,²¹ picture of opened stomach was taken and percentage ulcer inhibition was calculated.

 

Method of ulcer rating:

Ulcer index calculated by counting the wounds with the help of hand lens (10X) and rated as follows:

 

Normal control stomach 0, Red coloration 0.5, Ulcer spot 0, Haemorrhagic streaks 0.5, Ulcers ˃ 3 but ˂ 5 2.0, Ulcers ˃ 5 3.0.

For each animal average score of ulcers was stated as ulcer index

 

Calculation of ulcer index:

The extent of ulceration in the excised stomach was matched to the control in all the models. The extent of ulceration was stated as an ulcer index obtained by the average of the scores in each treatment group.²²

Ulcer index = (UN + US + UP) × 1\10

Where, UN= Average number of ulcers per animal

US = Severity score average

UP = Percentage of animal with ulcers

 

The percentage of ulcer protection was calculated by using formula

% of ulcer Protection = Control mean ulcer index - test mean ulcer index / Control mean ulcer index × 100

 

Histopathological Evaluation of Ethanol induced Gastric Ulcers:

All group animals’ stomach was dip in Formalin (10%) to evaluate the possible modifications which are seen by Histopathology. For tissue staining Haematoxylin-eosin was used. By which we used to review some transformations like gastric epithelium removal, infiltration of neutrophils, gastric erosions, inflammation and edema.²³

 

Estimation of Lipid peroxidation (LPO):

Took 1.0ml sample added 2.0ml TCA-TBA-HCL reagent and heated for fifteen minutes in boiling water bath. Cooled the solution and centrifuged at 1,000g for ten minutes the resultant clear upper layer was useful and flocculent precipitate was discarded. Took reading at 535nm, Lipid peroxidation stated as nmoles of formed MDA/min/mg/protein.^24-26

 

Estimation of catalase:

Took 0.5ml mixture of reagent A and B added 1.0ml buffer, 0.4ml water and 0.2ml enzyme and incubate for 0, 30, 60, 90 seconds then added 2.0ml acetic acid and heated for ten minutes the developed color was read at 610nm. Catalase activity stated as moles of decomposed H₂O₂/min/mg protein.²⁷

 

RESULTS:

The antiulcer effect of ethanolic extract of leaves of Lichi chinensis was studied in ethanol induced ulcer and cold stress induced ulcers in Wistar rats. The extract at dose of 200mg/kg b.w produced significant inhibition of gastric lesion by ethanol and cold stress induced ulcer in rats using ranitidine as a standard drug. Selected animals were divided into four groups of 6 animals each.

 

Cold Restrain stress induced gastric ulcer model:

Stress is major factor for the production of ulcer. In this model, ulcer was produced by giving stress by cold. Animal were deprived of food for 24 hours with free access to water prior to tests. 30 minutes prior to the cold stress drugs or vehicle was given via oral route. The animals were kept in special cage and the cages were kept for a temp. of 3 to 4⁰C for 3 hours. Litchi chinensis has shown protection index of 70% with the dose of 200 mg/kg b.w respectively in the comparison the control, Ranitidine as reference standard drug and protection of 97%. (Result are tabulated in Table 1)

 

Table 1: Effect of Litchi chinensis on Ulcer Inhibition in Cold Restrain Stress Induced Gastric Ulceration in Rats

All values are Mean ± SEM, n= 4, *P < 0.0001 when compared to control group.

 

Normal control

 

 

Standard

 

 

Disease Control

 

 

Test

Figure 1: (a) Normal control (b) Disease Control (Ulcer induced by Cold Restrain stress), (c) Standard (treated with Ranitidine 45mg/kg b.w and shows protected mucosal layer), (d) Test (treated with ethanolic extract of Litchi chinensis 200mg/kg b.w and shows protected mucosal layer)

 

Table 2: Effect of Litchi chinensis on Ulcer Inhibition in Ethanol Induced Gastric Ulceration in Rats b.w and shows protected mucosal layer), (d) Test (treated with ethanolic extract of Litchi chinensis 200mg/kg b.w and shows protected mucosal layer)

All values are Mean ± SEM, n= 4, *P < 0.0010 when compared to control group.

Ethanol induced gastric ulcer model:

Oral administration of absolute (90% ethanol) ethanol produced distinguishing lesion in control group animal. Standard and extract treated groups. The glandular portion of rat stomach which appeared as shows the bands of thick, dark red and black lesions. Litchi chinensis has shown protection index of 45% with the dose of 200 mg/kg b.w respectively in the comparison the control, Ranitidine as reference standard drug and protection of 31%. (Result are tabulated in Table 2)

 

 

(a) Normal control

 

(b) Standard

 

c) Disease control

 

(d) Test

Figure 2: (a) Normal control (b) Disease Control (treated with 1m/kg b.w Ethanol), (c) Standard (treated with Ranitidine 45mg/kg, (d) Test

Macroscopical and histopathological evaluation:

Histopathological changes on Ethanol induced model showed the oedematous, inflammation, degeneration, haemorrhage, appearance of the gastric tissue, whereas Litchi chinensis (200 mg/kg b.w) treated group’s shows regeneration and prevents the formation of haemorrhage and edema and those are shown in figure.

 

 

Normal control

 

Disease control

 

Standard

 

Test

Figure 3: (a)Ethanol induced normal control shows normal mucosa (b) Ethanol induced ulcer method shows inflammation and mucosal ulceration control, (c) Standard drug Ranitidine (24mg/kg b.w) shows no significance change in histopathology almost normal appearance, (d) Litchi chinensis test (200mg/kg b.w) shows no significance change in histopathology almost normal appearance.

 

 

Effect of EELC on Catalase Activity:

The results revealed that at a dose of 200mg/kg EELC decreased the CAT activity significantly in the glandular tissue, when compared to the control group. The activity of the treated group 200mg/kg group was comparable and equipotent as that of animals treated with Ranitidine at 45mg/kg (p<0.01) (Table 3).

 

Effect of EELC on Lipid Peroxidation:

The results revealed that at a dose 200mg/kg, EELC decreased the LPO level significantly in the glandular tissue, in comparison to the control group. The activity of the group treated with 200mg/kg group was comparable to that of ranitidine at 45mg/kg treated group (p<0.001) (Table 3).

 

Table 3: Effect of EELC on In vivo Antioxidant Activity in Ethanol induced Ulcers in Rats

Values are expressed in terms of mean ± S.E.M, ** p<0.01, ***p<0.001 Vs

Ulcerated control group - One way ANOVA followed by Dunnett’s test

 

DISCUSSION:

The cause of gastric ulcers is variance in mucosal invading and protective factors. Stress ulcers are mediated by the brain-gut axis and complex neural mechanism.²⁸ Stress causes an ischemic condition in the gastric mucosa by activation of the parasympathetic and sympathetic nervous system resulting in vasoconstriction, which in turn causes free radical generation. Further stress has also been found to inactivate mucosal prostaglandin synthase by accumulating H₂O₂, which in turn inhibits the synthesis of prostaglandins known to favors the generation of oxygen species.²⁸ The results suggest that in the cold stress model pretreatment with EELC significantly suppressed the ulcer index as compared to control. This effect appears may be due to its Anticholinergics²⁹.

 

Ethanol serves as a most common ulcerogenic agent and when given intragastrically to rats it produces severe gastric hemorrhagic erosions. The genesis of ethanol-induced gastric lesions is multifactorial with the depletion of gastric walls mucus content as one of the involved factors and this damage induced by ethanol may be due to mucosal leukotrienes release. Ethanol-induced damage to the gastric mucosa is associated with significant production of oxygen free radicals by an effect of Xanthine oxidase,³⁰ Ca²⁺ -protease increased lipid peroxidation and damage to the cell and cell membrane. Accumulation of activated neutrophils in the gastric mucosa may be a source of free radicals.³¹

 

Ethanol treatment caused significant increases in the ulcer index whereas pretreatment with EELC shows significant inhibition in ethanol-induced gastric damage and exerts cytoprotection, by the reduction in lipid peroxidase level and increase in reduced Catalase level. The cytoprotective property of EELC observed in the present study is due to the strengthening of mucosa or by an increase of duodenal alkaline secretion or luminal prostaglandin secretion.³²

 

The histopathological observations showed that upon EELC pretreatment, the mucosal epithelium had almost normal architecture and it had less hemorrhage and less necrosis, as against the ethanol-induced damages in the mucosal epithelium. The prior administration of EELC protects the gastric cells against ethanol injury by decreasing the necrosis and hemorrhage.³³

 

CONCLUSION:

The present study concluded that the antiulcer potential of EELC may be attributed to cytoprotection and antioxidant. Certain phytoconstituents such as glycosides, tannins, saponins, phytosterols, carbohydrates, steroids, flavonoids, and phenolic compounds were detected during qualitative phytochemical screening of the successive leaf extractives of Litchi chinensis which may be responsible for the antiulcer activity and can be further fractionated and investigated for its role and utility in antiulcer mechanisms.

 

ACKNOWLEDGEMENTS:

The authors would like to thank to Amity University, Lucknow and Motherhood University, Roorkee for providing laboratory facility and encouraging our work.

 

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Accepted on 28.04.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(3):1705-1710.