INTRODUCTION:
Ulcers are an open sore of the skin or mucus membrane characterized by sloughing of inflamed dead tissue, most common on the skin of the lower extremities and in the gastrointestinal tract.[1] The etiology of peptic ulcer is not clearly known, probably due to disproportion between aggressive (acid, pepsin, bile, and H.pylori) and defensive (gastric mucus, bicarbonate secretion, prostaglandins, nitric oxide, innate resistance of mucosal cells) factors.[2] Damage of the mucosal layer is an initial step in generation of ulcer, known to be primarily due to hyper secretion of HCl from parietal cells of gastric mucosa through proton pump and oxidative stress.
Free radicals are detrimental to the integrity of mucosal layer and mediate their injury.[3] Hence there is a need for agents to minimize and repair free-radical induced damage. Due to lesser side effects compared to synthetic drugs, presently 80% of the world population depends on natural products for the first line of primary health care.[4] The plant Ficus racemosa L (Moraceae) is used in traditional system of medicine for a long time, for the treatment of various disorders like diabetes, liver disorders, diarrhoea, inflammatory conditions, haemorrhoids, respiratory and urinary diseases. Latex is found in specialized secretor cells of plants known as laticifers, which is an aqueous suspension of complex mixture of molecule. Latex is used topically on chronic infected wounds to alleviate edema, pain and to promote the healing.[5] Ficus racemosa latex is used as folklore medicine for ulcer treatment in Vandavasi region, Tamil Nadu. In this preliminary study, an attempt has been made to demonstrate the in-vitro antiulcer and antioxidant activities of ethanolic extract of Ficus racemosa latex.
MATERIAL AND METHODS:
Collection and Extraction of latex:
Fresh latex was collected from the Ficus racemosa trees (15-20 years old), locally grown in Vandavasi region, in the month of May, 2016. Single incision at the bark was made using stainless steel knife and the exuded latex was collected drop by drop, treated with twice the volume of ethanol, filtered immediately and kept at -20°C.[6] The fresh latex was identified and authenticated by Dr. P. Sathiyarajeswaran, Siddha Central Research Institute, Chennai- 106. The Ficus racemosa latex was extracted using ethyl alcohol as a solvent and was dried by using rotator evaporator under reduced pressure.[7] The ethanolic extract of Ficus racemosa L. latex (EEFRL) was stored and utilized for further studies.
In vitro Antiulcer activity:
H+K+ATPase Assay:
The
mucosal layer was scrapped from freshly slaughtered goat, suspended in saline
and homogenized in ice-cold phosphate buffer, pH 7.4. The homogenate was
centrifuged for 20 min at 5000 rpm and the supernatant obtained was centrifuged
for 60 min at 14,000 rpm. The pellet was re-suspended in phosphate buffer.[8]
Protein was determined by the method of Lowry et al.[9] EEFRL
(10-50 µg/ml) were incubated in the reaction mixture (40mM Tris-Hcl buffer, pH
7.4, containing 2 mM MgC
and 20 µl membrane protein) to make a volume of 1 ml.
Then, 2 mM ATP disodium salt was added to initiate the reaction; this
preparation was incubated for 20 min at 37°C. The reaction was terminated by
adding 1 ml of ice-cold trichloroacetic acid (10% v/v). The H+K+ATPase
activity was assayed in the presence of different concentration of extract and
Omeprazole. The amount of inorganic phosphate released from ATP was determined
spectrophotometrically at 400 nm.[8]
Antacid activity:
The antacid activity of EEFRL was studied by assessing the pH value, neutralization capacity and duration of antacid effect on modified Vatier’s artificial stomach model[10]. Artificial gastric acid was prepared by 2.0 g of NaCl and 3.2 g of pepsin dissolved in 7.0 ml of hydrochloric acid and distilled water up to 1000 ml to adjust pH 1.20. The freshly prepared EEFRL 500 mg, 1000 mg (90 ml) and control Sodium bicarbonate (90 ml) were added separately to the artificial gastric acid (100 ml) at pH 1.2 and continuously stirred (30 rpm) with magnetic stirrer. Artificial gastric acid at pH 1.2 was pumped at 3 ml/min into the container of artificial stomach, and pumped out at 3 ml/min at the same time. The change of pH in the container of the artificial stomach was continuously monitored. The duration of the antacid effect was calculated when the pH value returned to its initial value (1.20).
Neutralization capacity:
Each freshly prepared test sample (90 ml) was placed in a 250 ml beaker and a magnetic stirrer was continuously run at 30 rpm to imitate the stomach movements. The test samples were titrated with artificial gastric acid to end point of pH 3. The consumed volume of the artificial gastric acid was measured and the experiment was repeated for the standard (Sodium bicarbonate) and water.
Antioxidant activity:
Free radical scavenging activity was evaluated using DPPH, nitric oxide radical and TBARS assays with different concentrations of the extract EEFRL and Ascorbic acid using standard methods.[11,12,13] The reaction mixture containing 1.9 ml of DPPH solution with different concentration of EEFRL (100, 200, 400, 800, 1000µg/ 0.1ml) was shaken and incubated in dark for 20min at room temperature. The resultant absorbance was recorded on 517 nm. For Nitric oxide radical assay 3ml of reaction mixture containing sodium nitroprusside (10mM in phosphate buffered saline) and various concentrations of the EEFRL (100, 200, 400, 800, 1000µg/ml) were incubated at 370C for 4 hours. To the incubation solution, 0.5ml of Griess reagent was added and absorbance was read at 546 nm. For TBARS assay a stock solution of EEFRL in DMSO (1 mg/ml) was prepared and different levels (100, 200, 400, 800 and 1000 μl) from each stock solution were transferred into different test tubes and volumes were adjusted to 1 ml with the same solvent. To each test tube 3.0 ml of 10% liver homogenate was added and incubated for 30 min. Lipid peroxidation was initiated by adding 40 μl of Ferric chloride (400 mM) and 40 μl L-ascorbic acid (200 mM) and incubated for 1 h at 37°C. After incubation, 3 ml of 0.25 N HCl containing 15% trichloroacetic acid and 0.375% thiobarbituric acid was added. The reaction mixture was boiled for 30 min, then cooled, and centrifuged at 5000rpm for 5 min. A blank was prepared with the same reagents without the sample, and using Ascorbic acid as a positive control (100 µg/ ml). The absorbance was measured at 532 nm. The percentage inhibition was calculated using the following formula:
Control-Sample
Percentage Inhibition= ---------------------------------×100
Control
Where Control and Samples are the absorbance of control and sample respectively.
Statistical Analysis:
The results of antacid activity are expressed as mean ± SEM. Results were statistically analyzed using one-way ANOVA, followed by the Tukey’s HSD test for individual comparison. P<0.01 was considered as significant.
RESULTS:
In H+K+ATPase assay the EEFRL showed comparable proton pump inhibitory effect with that of standard Omeprazole in a concentration dependent manner on goat gastric mucosal homogenate. EEFRL at 50µg/ml showed maximum proton pump inhibitory effect (1.72±0.01 μM of Pi/hr/mg protein) as like standard Omeprazole (1.88±0.04 μM of Pi/hr/mg protein) (Fig.1).
Fig 1: Effect of Ficus racemosa Latex on H+K+ATPase assay
From the determination of pH on modified Vatier’s artificial stomach model, it has been found that EEFRL has significant (P<0.01) antacid effect at 500 mg and 1000 mg concentrations when compared with water. The duration of antacid action of EEFRL at both concentrations were significantly (P<0.01) higher than that of water. EEFRL 500 mg and 1000 mg and Sodium bicarbonate were shown significant (P<0.01) neutralization capacity when compared with water (Table.1).
Table1: In Vitro Antiulcer effect of F. racemosa Latex on artificial gastric juice
|
Treatment Group |
Antacid activity |
Neutralization Capacity (ml) |
|
|
pH value |
Duration (min) |
||
|
Water (90ml) |
1.1±0.078 |
0.013±0.003 |
0.063±0.007 |
|
NaHCO3(500mg) |
2.8±0.121* |
4.20±0.016* |
0.82±0.012* |
|
NaHCO3(1000mg) |
3.0±0.144* |
4.30±0.086* |
0.91±0.132* |
|
EEFRL (500mg) |
2.0±0.060* |
2.10±0.044* |
0.32±.0.028* |
|
EEFRL (1000mg) |
2.5±0.174* |
4.15±0.028* |
0.65±0.057* |
Values are expressed as mean ± SEM (n=3) * P< 0.01 when compared with water
The extract also showed concentration-dependent free radical scavenging activities in DPPH assay, nitric oxide radical assay and thiobarbituric acid reactive species (Table 2).
Table 2: Antioxidant activity of Ficus racemosa latex
|
Treatment |
Concentration (µg/ml) |
Percentage Inhibition (%) |
||
|
DPPH |
NO |
TBARS |
||
|
Ficus racemosa (EEFRL) |
100 |
46.42 |
61.36 |
54.29 |
|
200 |
74.42 |
74.23 |
57.31 |
|
|
400 |
83.00 |
74.43 |
57.94 |
|
|
800 |
83.57 |
74.50 |
66.17 |
|
|
1000 |
91.85 |
84.32 |
87.89 |
|
|
Vitamin C |
100 |
83.41 |
65.56 |
93.57 |
DISCUSSION:
The enzyme H+K+ATPase is exclusive to the parietal cells which transports the hydrogen ions against the concentration gradient. Mitochondria in the parietal cells, the richest source by consuming more ATP generates inorganic phosphate which may act as indirect measure of proton pump (H+K+ATPase) activity in gastric acid formation.[14] In this study, the ability of EEFRL to inhibit H+K+ATPase on isolated goat stomach was studied. Thus it has been indicated that EEFRL has potentially inhibited the proton pump responsible for the secretion of acid. From the in vitro evaluation of antacid activity using the modified Vatier’s artificial stomach model, it has been observed that the ethanolic extract of Ficus racemosa latex has acid neutralizing capacity with considerable duration which could be helpful in the treatment of hyper-secretor conditions of gastric ulcer. Further studies on identification and separation of phytochemical constituents from Ficus racemosa latex will suggest the actual compound responsible for its acid neutralizing capacity. Free radicals are highly reactive particles with an unpaired electron and are produced by radiation or as by-products of metabolic processes. They initiate chain reactions, which lead to disintegration of membranes and cellular components, including lipids, proteins, and nucleic acids. Lipid peroxidation may be pro inflammatory and can damage the tissues directly.[15] Protection against free radical lipid peroxidation by plant extract is of great significance for their traditional use against various disorders. In vitro studies of EEFRL revealed its antioxidant properties, scavenging the free radicals and reactive oxygen species in a concentration-dependent manner in DPPH, Nitric oxide and LPO assays which could be helpful in the process of ulcer healing and could be used as an alternative in the treatment of peptic ulcer due to the cyto-protective nature of its antioxidant constituents.
CONCLUSION:
From the In vitro assays carried out it is concluded that the ethanolic extract of Ficus racemosa latex may have beneficial effect in the treatment of gastric ulcer ascribed to the cytoprotective nature of antioxidant constituents, acid neutralization capacity and Proton pump inhibitory effect. However more work is required to explore the biological activity of the compound(s) present in the F.racemosa latex and to elucidate their mechanism of action.
ACKNOWLEDGEMENT:
The Authors are Thankful to Dr. S. Devaraj, Chairman and Dr. D. Dev Anand, Seceretary of Annai Veilankanni’s Pharmacy College, Chennai-15 for their valuable support.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 13.09.2018 Modified on 14.10.2018
Accepted on 21.10.2018 ©A&V Publications All right reserved
Res. J. Pharmacology & Pharmacodynamics.2018; 10(4): 159-162.
DOI: 10.5958/2321-5836.2018.00029.0