|Year : 2017 | Volume
| Issue : 1 | Page : 37-42
Investigation of mast cell stabilization and antiulcer activity of protein extract of Perna viridis
K Venkateskumar1, S Parasuraman2, PV Christopher2, Syed Ali3, Leow Yu Chuen2, Winnie Tang1, Siaw Yee Sii1, Tan Chun Yin1
1 Unit of Pharmaceutical Technology, AIMST University, Bedong, Malaysia
2 Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong, Malaysia
3 Advanced Medical and Dental Institute, Kepala Batas, Penang, Malaysia
|Date of Web Publication||21-Apr-2017|
Unit of Pharmacology, Faculty of Pharmacy, AIMST University, Bedong, Kedah
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: To study the mast cell stabilization and antiucler activity of protein extract of Perna viridis using rodent models. Materials and Methods: The total protein from P. viridis was extracted, purified, and screened for mast cell stabilization and antiulcer activity. Intestinal mesentery of male rats was used to study the peritoneal mast cell stabilization of protein extract of P. viridis. The rat intestinal mesentery was exposed to 10, 30, 100, 300, and 600 μg/mL of protein extract of P. viridis and the peritoneal mast cell stabilization was compared with that of standard (ketotifen) at a dose of 20 μg/mL. Antiulcer activity of protein extract of P. viridis was studied at the dose levels of 100, 200, and 400 mg/kg and the effect was compared with the omeprazole (20 mg/kg) using ethanol-induced ulcer model. At the end of the study, ulcer index and percentage of ulcer inhibition was calculated. Results: The total protein content in extract was found to be 32 μg/mL. The protein extracts of P. viridis showed significant mast cell stabilization only at high dose (600 μg/mL) and did not show any significant antiulcer activity for doses of 100 and 200 mg/kg in administered animals, but the significant antiulcer activity was observed at a dose of 400 mg/kg. Conclusion: The present study findings suggests that the protein extract of P. viridis did not exhibited significant mast cell stabilization and antiulcer activity at optimal doses.
Keywords: Antiucler activity, mast cell stabilization, Perna viridis, total protein
|How to cite this article:|
Venkateskumar K, Parasuraman S, Christopher P V, Ali S, Chuen LY, Tang W, Sii SY, Yin TC. Investigation of mast cell stabilization and antiulcer activity of protein extract of Perna viridis. J Pharm Negative Results 2017;8:37-42
|How to cite this URL:|
Venkateskumar K, Parasuraman S, Christopher P V, Ali S, Chuen LY, Tang W, Sii SY, Yin TC. Investigation of mast cell stabilization and antiulcer activity of protein extract of Perna viridis. J Pharm Negative Results [serial online] 2017 [cited 2017 Sep 25];8:37-42. Available from: http://www.pnrjournal.com/text.asp?2017/8/1/37/204912
| Introduction|| |
The enormous growth of world population has overburdened the existing resources for the drugs. Emergence of new diseases is at an alarming pace due to changing environments and lifestyle, and due to that ailments are changing their normal patterns. Marine environment is one of the promising and the viable resources that could be explored for more potential drugs to manage various diseases. Ocean offers a large biodiversity of flora and fauna which is estimated to be over 5,000,000 species more than double of the species in land. Since 1960s, nearly 6500 bioactive compounds have been isolated from marine organisms and nearly 300 bioactive marine natural products were filed for patent., These classes of bioactive compounds that have been isolated from marine organisms have been reported to possess a broad spectrum of biological activity, such as antibacterial, antiviral, anti-inflammatory, and anticancer activity. Molluscs are widely distributed throughout the world and they represent good candidates for discovery and development of drugs. It is also emphasized that molluscs have been studied as major sources for biologically active compounds and certain species have been screened for their pharmacological activity or other characteristics that are beneficial to biomedical arena.
Green mussel (Perna viridis) is one of the important mollusc category bivalves which are abundantly present in Southeast Asia and have been widely consumed for their nutritional values. This species has been widely reported for their biological potential and its use to assess marine pollution. Few studies have also reported their anti-inflammatory activity, bioadhesive property, inhibition of osteoclasts formation, inhibition of replication of Plasmodium falciparum, and inhibition of HIV virus replication. It was also reported that the mussels biological activity vary with their geographical locations and the environmental conditions in which they grow. It was also suggested that the full biopotential of green mussels has not been extensively reported. Hence, the present study is aimed to screen the protein extracts of P. viridis for mast cell stabilization and antiulcer activity using animal models.
| Material and Methods|| |
Green mussel belong to Kingdom Animalia, phylum of Mollusca, and class of Bivalvia, and it is one of the largest, oldest, and majority diverse phyla in nature consisting more than 110,000 species. The phylum Mollusca has a characteristic of '‘soft-bodies'’ and the class Bivalvia means it contains “two shells.” These groups have a shell in common with other edible clams. However, they vary in possessing a very elongated and asymmetrical outline as compared with other edible clams, which are usually more or less rounded or oval. The edible bivalve, P. viridis, was collected from local fishing outlets and in local markets of Semeling, Kedah, Malaysia.
P. viridis is a type of Asian mussel, predominantly encompasses the Asia-Pacific and Indo-Pacific regions. Their habitats include mid-intertidal zones to subtidal zones. Size of the large mussel is up to 800-1000 mm in length and infrequently reaching 1650 mm. Outer edge of the P. viridis is vivid green to dark brownish-green in color, attachment points are in olive-green in color, juveniles are in bright and vivid green color, and the interior of the shell is pale bluish green and shiny. The outer shell has a smooth surface with noticeable concentric growth rings and tapers of a sharp down-turned beak and the beak has interlocking teeth in which two are inside the left and one is inside the right.,
Preparation of green mussel protein extracts
P. viridis was washed with distilled water and deshelled, and excess water was removed. The extracts were prepared from whole body tissue by cold phosphate buffer saline (PBS) at pH 7.4 with the protease inhibitor, phenylmethylsulfonyl fluoride (PMSF) by simple homogenization procedure. The homogenized mixtures were centrifuged at 10,000 RCF for 30 min at 4°C. The supernatant was obtained and freeze dried. The lyophilized crude extract was stored at-20°C till evaluation. The lyophilized crude extract of P. viridis was reconstituted in PBS and partially purified by 85% ammonium sulfate precipitation. The precipitate was centrifuged at 10,000 RCF for 1 hour at 4°C. Supernatant was carefully removed, and the pellet was collected and dissolved in sterile distilled water. Ammonium sulfate was removed by HiTrap desalting column. The desalted solution (protein extract) was then lyophilized and stored at-20°C.
Estimation of total protein concentration
The total protein in P. viridis extracts was estimated by method as described by Hartree (1972) using bovine serum albumin (BSA) as standard. The Hartree version is a modification of Lowry protein assay and this method is more suitable to compare the concentration of solutions of same protein than to absolute measurement, as the absorbance is dependent on protein composition. Protein samples and standards were processed in the same manner by mixing them with same solvent and assay reagents and absorbance was measured using a spectrophotometer. Hartree-Lowry reagent A (2 g of sodium potassium tartrate and 100 g sodium carbonate in 500 mL 1N NaOH and volume was made up to 1 L with distilled water), B (2 g of sodium potassium tartrate and 1 g copper sulfate dissolved in 90 mL distilled water and 10 mL 1N sodium hydroxide), and C (1 part of Folin-Ciocalteau reagent diluted with 15 parts of distilled water) were prepared by the method as described. A 50 mg of BSA was dissolved in distilled water in a 50 mL volumetric flask to give a clear solution of 1 mg/mL. From stock BSA solution, dilution series of calibration standard was prepared in distilled water in test tubes to give concentrations of 50-250 μg/mL. A 50 mg of lyophilized crude extracts of P. viridis was dissolved in distilled water in a 50 mL volumetric flask to get a clear solution of 1 mg/mL (stock). The stock solution was serially diluted to give a test solution with a concentration of 200 μg/mL.
In a test tube, 1 mL of distilled water/calibration standard or crude extracts of P. viridis was added to 0.9 mL of Hartree-Lowry reagent A, incubated at 50°C for 10 min, and cooled to room temperature. Later, 0.1 mL of Hartree-Lowry reagent B was added and the reaction mixture was incubated at room temperature for 10 min. In a reaction mixture, 3 mL of Hartree-Lowry reagent C was rapidly added to each test tube and mixed thoroughly, incubated at 50°C for 10 min, then cooled to room temperature. The final assay volume was 5 mL and the net absorbance of blank/calibration standards or crude extracts of P. viridis was measured spectrophotometrically at 650 nm. The calibration plot was prepared by graphing the net A650 values for the standard versus protein concentration. The protein concentration of crude extracts was determined by interpolation from the calibration curve. The protein content of the samples was analyzed in triplicate.
Healthy, adult, either gender of Sprague-Dawley (SD) rats, weighing 140-160 g, were obtained from Central Animal house, AIMST University, Malaysia. The animals were housed in large, spacious polyacrylic cages at an ambient room temperature with 12-h light/12-h dark cycle. The animals were fed with normal rodent pellet and water ad libitum. The study was approved by AIMST University Human and Animal Ethics Committee and the study was conducted according to Animal Research Review Panel guidelines.
Mast cell stabilization activity of P. viridis extract
Mast cell stabilization activity of P. viridis extracts was studied using method as described elsewhere., Male SD rats were fasted overnight and sacrificed by cervical dislocation. The abdomen was cut open and mesentery was collected, washed with Ringer-Locke solution. Small pieces (2 cm in length) of mesentery was cut and placed in a beaker containing Ringer-Locke solution and they were used for further experiment. The processed tissues were divided into three sets and they were exposed with ketotifen 20 μg/mL, different concentrations of mussel extract (10, 30, 100, 300, and 600 μg/mL), and incubated for another 30 min. After incubation, the mesentery was fixed with 4% formaldehyde containing O-toluidine blue for ≈20 min on a clean glass slide. Then, the tissue preparation was washed with acetone and then with xylene. Later, the slide was examined under light microscope at 400× magnification. Randomly, 100 mast cells were counted (left to right, clockwise) and the number of intact and fragmented or disrupted mast cells were noted. Finally, the percentage of mast cells which were intact or fragmented or disrupted was calculated.
Acute toxicity testing
Healthy, adult, female genders of SD rats were used for acute toxicity testing. The fixed dose method (FDM) was used for acute toxicity testing. The acute toxicity testing of protein extracts of P. viridis was carried out at dose levels of 3, 30, 300, 600, and 2000 mg/kg body weight (BW), according to the Organization for Economic Cooperation Development (OECD) guidelines (new OECD FDM test guideline 420). Three female rats were sequentially dosed (single oral dose) at intervals of 24 h and observed for toxicity signs (autonomic profiles: defecation and urination; neurological activity: spontaneous activity, reactivity, touch response, pain response, and gait; and behavioral functions: alertness, restlessness, irritability, and fearfulness) for at least once daily for 14 days.
Antiulcer activity of P. viridis extract:
Healthy, adult, male genders of SD rats were used for the experiment. The rats were divided into six groups viz.,
Group I: Control
Group II: Ulcer control
Group III: Omeprazole 20 mg/kg
Group IV: P. viridis 100 mg/kg
Group V: P. viridis 200 mg/kg
Group VI: P. viridis 400 mg/kg
The rats were treated with respective treatment once daily per oral for 7 days and they were fed with normal rat pellet diet and normal tap water ad libitum. Later, the animals were fasted for 12 h (food but not water) and treated with respective drug or extract. After 30 min of treatment, the rats were administered with 2 mL/kg of 70% ethanol orally to induce gastric ulcer. The animals were sacrificed after 12 h of ethanol administration by cervical dislocation and their stomachs were immediately excised. The content of the stomach was rinsed with sodium chloride. The stomach was then dried slightly by dabbing on tissue paper and pinned on a board. The gastric mucosal lesions on the processed stomachs were examined under a stereomicroscope (3×).
Body weight was also monitored throughout the experiment. Degree of ulceration in alcohol-induced ulcer was quantified using the method as described by Szabo and Hollander. The harvested stomachs were pinned on a corkboard and the number of ulcers were determined using stereomicroscope with square-grid eyepiece based on grading on a 0-5 scale [0 = normal or almost normal mucosa; 1 = vascular congestions; 2 = one/ two lesions; 3 = severe lesions; 4 = very severe lesions; and 5 = mucosa full of lesions]. Total stomach area and ulceration area was measured to calculate ulcer index (UI). UI was calculated using formula [(ulcerated area/total stomach area) × 100] and percentage of ulcer inhibition was calculated using formula [(UI in control-UI in test) × 100/UI in control].
The mean ± SEM values were calculated for each group. Statistical differences among the groups were determined using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. BW variation before and after ulcer induction was analyzed using paired t-test. A P < 0.05 was considered to be significant.
| Results|| |
The yield of protein extracts was 6.89 g from 2 kg of live mussels. The total protein content in extracts was estimated by interpolation from a calibration curve prepared by using BSA [Figure 1]. The assay was performed in triplicates to obtain the average. Total protein content was measured using Hartree-Lowry method and the protein concentration was found to be 32 μg/mL [Table 1]. In this method, the absorbance values are dependent on concentration of protein.
It was observed that about 51.33 ± 10.26% of intact mass cells and 48.67 ±10.26% of degranulated mast cells in the mesentery tissue exposed with the protein extracts of P. viridis at a dose of 600 mg/Kg [Table 2]. These findings suggest that the protein extracts of P. viridis showed significant mast cell stabilization at a dose of 600 mg/kg and the effect was found to be very low in comparison with the standard ketotifen.
Acute toxicity testing of single oral administration of protein extracts of P. viridis in rats did not show any mortality, behavioral changes up to 2000 mg/kg. Hence, further pharmacological studies were carried out at the dose levels of 100, 200, and 400 mg/kg dose levels.
Changes in BW were compared before and after ulcer induction. It was noticed that there were no significant changes in BW in control and treatment groups. Ulcer induction was evident in groups II-VI after the administration of 70% ethanol.
The absolute and relative stomach weights also did not show any significant variations [Table 3] between the control and the test group. Effect of P. viridis on UI of alcohol ulcerated rats is depicted in [Figure 3]. Ethanol-induced ulcer were characterized by presence of number of hemorrhagic red bands of different size [Figure 2]. A significant increased UI was observed in control group. The ulcer grading showed significant increase in ulcer control group (P < 0.001), P. viridis 100 mg/kg (P < 0.05), P. viridis 200 mg/kg (P < 0.01), and the animals treated with omeprazole 20 mg/kg (P < 0.001), P. viridis 400 mg/kg (P < 0.01) showed significant decrease in ulcer score ulcer score [Table 4]. A significant increase in UI was observed in all groups other than control group. The standard drug omeprazole exhibited reduction in UI when compared with ulcer control. The percentage of ulcer inhibition of extracts compared with ulcer control is depicted in [Figure 4].
|Figure 3: Effect of P. viridis on UI of alcohol-ulcerated rats. The values are mean ± SEM (n = 6). aP < 0.05 and bP < 0.001 compared with control, cP < 0.001 compared with ulcer control, one-way ANOVA followed by Tukey's post hoc test|
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|Figure 2: Morphological appearance of ethanol-induced hemorrhagic lesions in the stomach of rats. (a) Control, (b) ulcer control, (c) Omeprazole 20 mg/kg, (d) P. viridis 100 mg/kg, (e) P. viridis 200 mg/kg, and (f) P. viridis 400 mg/kg|
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|Figure 4: Effect of P. viridis on degree of protection (% ulcer inhibition) against ulceration in alcohol-ulcerated rats. The values are mean ± SEM (n = 6)|
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| Discussion|| |
Hartree-Lowry method is a colorimetric assay based on reaction between cupric ions and Folin-Ciocalteau reagent. This protein assay is relatively sensitive and is widely employed, but it is susceptible to many interfering compounds which are commonly used in buffers for preparing proteins. This factor could be suggested as one of the limitations of such assay. Therefore, distilled water was used to prepare the samples for assay. In addition, although crude samples are taken through the same procedures with pure protein, BSA, it does not accurately quantitates total protein in crude samples due to tangled mixtures of carbohydrates, lipids, nucleic acids with proteins and glycoproteins in real samples. The absorbance can be read in the region of 500-750 nm, with 650 nm being the most commonly used. The proposed method obeys Beer-Lambert law of absorption in the concentration range of 50-250 μg/mL, where the fraction of light absorbed is proportional to the concentration of absorbing species.
In the present study, antiulcer potential of protein extracts of P. viridis was studied against ethanol-induced ulcer in SD rats. Protein extracts of P. viridis did not show considerable antiulcer activity till 200 mg/kg. But at higher doses, significant antiulcer activity was evident and this effect was comparable with that of standard omeprazole 20 mg/kg treated group. Ethanol is frequently used to induce ulcers in experimental rats that lead to intense gastric mucosal damage. Studies suggest that ethanol damage to the gastrointestinal mucosa starts with microvascular injury, which is the disruption of the vascular endothelium triggered by increased in vascular permeability, edema formation, and epithelial lifting. Administered ethanol produces necrotic lesions in gastric mucosa by its direct toxic effect, reduces the secretion of bicarbonates and production of mucus, and increases the extension of cellular damage which is dose-dependent.,
P. viridis is widely used as a food substance due to their high nutritional values. Protein extracts or whole P. viridis was expected to have mucoprotective or antiulcer effect. But the present study findings revealed that the extracts did not show any significant antiulcer activity in 100 and 200 mg/kg treated animals and showed significant showed antiulcer activity in 400 mg/kg treated animals. It was suggested that, further studies are required to explore the antiulcer activity of protein extract of P. viridis in different animal models.
| Conclusion|| |
Antiulcer activity and mast cell stabilizing capacity of protein extract of P. viridis was found to be insignificant at lower doses. Although these properties were evident in higher doses, they were not comparable with that of the standards. Further, studies are required to establish the antiulcer and mast cell stabilization properties of protein extract of P. viridis.
The authors are grateful to Ministry of Higher Education, Malaysia for financial support through Fundamental Research Grant Scheme (FRGS) (Project Code: FRGS/1/2015/WAB09/AIMST/02/1) to carry out the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]