Hepatoprotective activity of the hydro-alcoholic extract of the

Gracilaria edulis (Gmelin)

 

Pranabesh Sikdar*, M. Venkata Ramana

¹Department of Pharmaceutical Chemistry, Seven Hills College of Pharmacy, Ramachandra Puram Mandal, Tirupati-517561, Andhra Pradesh, India.

²Gurram Balanarasaiah Institute of Pharmacy, Edulabad (V), Ghatkesar (M), Ranga Reddy– 501 301, Andhra Pradesh, India

 

ABSTRACT:

In recent years seaweeds have increasingly attracted interest in the search for new drugs and have been shown to be a primary source of bioactive natural compounds and biomaterials. In the present investigation, the biochemical composition of the red seaweed Gracilaria edulis, collected seasonally in the tuticorin (South India), was assayed by means of advanced analytical techniques, in the present study the hydro-alcoholic extract of the  Edulis was taken for Hepatoprotective activity using paracetamol model. The Phenolic content of the extract was also determined and it can be expressed as gallic acid equivalent i.e 49.33±1.23 mg/g of the extract. The extract have found to show significant decrease in the level of biomarker release at 300mg/kg and 400mg/kg. But the bodyweight have not shown the significant increase.

 

 

 

 

INTRODUCTION:

In ancient Indian literature, it is mentioned that every plant on this earth is useful for human beings, animals and other plants. The liver is the key organ regulating homeostasis in the body. It is involved with almost all the biochemical pathways related to growth, fight against diseases, nutrient supply, energy provision and reproduction The liver is expected not only to perform physiological functions but also to protect the hazards of harmful drugs and chemicals^1,2. In spite of tremendous scientific advancement in the field of hematology in recent years, liver problems are on the rise. Jaundice and hepatitis are two major hepatic disorders that account for a high death rate. Presently only a few hepatoprotective drugs and those from natural sources are available for the treatment of liver disorders.

 

The disorders associated with the liver are also numerous and varied^3,4,5. More than 900 drugs have been implicated in causing liver injury and it is the most common reason for a drug to be withdrawn from the market. ancient Indian literature, it is mentioned that every plant on this earth is useful for human beings, animals and other plants. The liver is the key organ regulating homeostasis in the body. It is involved with almost all the biochemical pathways related to growth, fight against diseases, nutrient supply, energy provision and reproduction The liver is expected not only to perform physiological functions but also to protect the hazards of harmful drugs and chemicals. In spite of tremendous scientific advancement in the field of hematology in recent years, liver problems are on the rise. Jaundice and hepatitis are two major hepatic disorders that account for a high death rate. Presently only a few hepatoprotective drugs and those from natural sources are available for the treatment of liver disorders. The disorders associated with the liver are also numerous and More than 900 drugs have been implicated in causing liver injury and it is the most common reason for a drug to be withdrawn from the market. Seaweeds also assumed greater significance as they are rich sources of various types of antioxidant compounds.  species were found to have Hepatoprotective activity (G.crassa). Based on the previous studies we planned to carry out the Hepatoprotective activity of the G.edulis by CCl₄ induced activity^6,7.

 

MATERIALS AND METHODS:

Plant material:

G. edulis (Gmelin) were collected from Tuticorin island of Tamil Nadu, India.

 

Extraction of plant material:

The algae were shade dried and were coarse powdered. About 400 gm of dry powder was taken in a Stoppered conical flask and it was defatted with Petroleum ether. The deffating was continued for 3-4 days with occasional shaking. The Petroleum ether extract was filtered. The marc left after deffating was taken out and dried under shade to get a dry mass, then extracted with ethanol and water (hydroalcoholic) by using cold maceration extraction. The extraction was continued for 6-8 days with occasional shaking. The hydroalcoholic extract was filtered, concentrated under reduced pressure to a semisolid mass and was made free from solvent. The final obtained extract was weighed; percentage yield was calculated and stored in a cool place^8-10.

 

Total Phenolic content:

Procedure

The test (hydroalcoholic extract) and standard (gallic acid) various concentrations were separately mixed with distill water (5ml), methanol (1ml) F-c reagent(0.5ml) and sodium carbonate (1ml) and the reaction mixture was mixed thoroughly and 200µl each of test sample and standard(diff. con) was kept in 96 well plate and incubated for 2 h in microplate reader(tecan i-control) and the absorbance values measured at 750nm. Using the gallic acid standard curve the total phenolic content of the sample was calculated^11-13.

 

Animals:

^{The preferred rodent species is the rat, although} other rodent species may be used. Male rats are used as the literature suggests higher metabolic rate in males when compared to female rats. Healthy young adult animals of Wistar strain were employed for the study^14,15.

 

DOSE SELECTION:

During the study period for all the animals treated with Test item at a dose of 2000 mg/kg. The oral LD50 of these formulations were therefore, considered greater than 2000 mg/kg, b.w.t. Therefore 1/10th, 1/15th, and 1/20th  of the LD50  were selected as test doses¹⁶.

PROCEDURE:

Animal used – winstar rats

Animals were divided into 6 groups of 6 each

GP 1:	Normal (0.5% Tween 80,10ml/kg, p.o.)
GP 2:	Control (0.5% Tween 80, 10ml/kg, p.o.)
GP 3:	Test dose 1 (200 mg/kg,p.o.)
GP 4:	Test dose 2 (300 mg/kg,p.o.)
GP 5:	Test dose 3 (400 mg/kg,p.o.)
GP 6:	Silymarin (50mg/kg, p.o)

 

Cirrhosis was induced in Wistar rats by chronic exposure to CCl₄ vapours in an inhalation chamber and phenobarbitone in drinking water (500mg/l) for a period of 4 weeks(20min/ twice weekly) for as described by Mclean et.al., After 4 weeks of CCl₄ treatment animals were divided in to 6 groups of 6 animals each. All the animals were given the above assigned treatment for a period of 30 days. After 30 days of treatment the animals were anesthetized using diethyl ether, and the blood was collected from retro-orbital plexus for biochemical estimations. The animals were then sacrificed by cervical dislocation, and the liver was removed for histopathological and antioxidant status estimation.

 

BIOCHEMICAL STUDIES:

Estimation of serum aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (ALP) and Total Proteins (TP) levels.

The blood sample collected was allowed to clot for 45 min at room temperature. The serum was separated by centrifugation at 3000 rpm at 30°C for 15 min and used for assaying ASAT, ALAT, ALP and total protein using Precision Biomed assay kits.

 

^{Estimation of liver Glutathione (GSH), Superoxide dismutase (SOD), Catalase and} glutathione peroxidase (GPx) levels:

GSH (Cayman-703102), SOD (Cayman-706002), catalase (Cayman-707002) and GPx (Cayman-703102) activities/levels were estimated using Cayman assay kits (cayman chemical company, Ann Arbor, USA)

 

Estimation of liver peroxide levels:

Liver tissue pieces were weighted and immediately minced and homogenized in 10% (w/v) ice-cold 100 mM phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 × g for 20 min and were kept at −70°C for estimation of levels of malondialdehyde (MDA). The protein concentrations of liver tissue were determined by the Lowry method. The above homogenate (0.25 ml) was mixed with 0.25 ml Tris–HCl, 0.05 ml of 4 mM FeCl₂, and 0.05 ml of 0.2 mM ascorbate. The mixture was incubated at 37 ◦C for 1 h in a capped tube, then 0.5 ml of 0.1 N HCl, 0.2 ml of 10% SDS, 0.9 ml distilled water, and 2 ml 0.6% thiobarbituric acid were added to each tube and vigorously shaken. The tubes were placed in 95˚C for 30 min. After cooling, tubes were added 5 ml n-butanol and centrifuged at 1000 rpm for 25 min, and the supernatant was subsequently measured with a spectrophotometer at 532 nm. The results were expressed as nmol MDA per milligram of protein¹⁷.

 

Histopathological studies:

The liver tissue was fixed in the 10% formalin, dehydrated in gradual ethanol (50–100%), cleared in xylene, and embedded in paraffin wax. The sections, which are 5-6 μm thick, was then prepared using rotary microtome and stained with hematoxylin and eosin dye for microscopic observation of histopathological changes in the liver.

 

STATISTICAL ANALYSIS:

For determination of significant inter-group differences of each parameter one-way analysis of variance (ANOVA) was carried out. Dunnet’s test was used for individual comparisons after significant ANOVA results. The differences with p < 0.05 was considered statistically significant. Graphpad prism 6 software (Graphpad software, Inc. California, was used for the statistical analysis¹⁷.

 

RESULT AND DISCUSSIONS:

The extracted was obtained by the hot soxhlet method. It was dark green in color and semisolid in nature which was stored in a dry cool place.

 

Total phenolic content can be expressed as gallic acid equivalent i.e 49.33±1.23 mg/g of the extract

 

Fig-1 Total Phenolic content of the HAE of G.edulis

 

Body weights:

There were no significant changes in the body weight in all the treatment groups when compared to control (p<0.05) (Table 1)

 

Effect on serum ASAT, ALAT, ALP and TP:

All the treatment groups showed a significant dose dependent protection against CCl4 induced increase in the serum ASAT, ALAT and ALP (P<0.05), In the case of total proteins, all the treated groups significantly prevented the CCl₄ induced decrease in the serum total protein levels however significant protection was observed at a dose of 400mg/kg (P<0.05) (Table 1, Appendix-I).

 

Effect on liver GSH, SOD, Catalase GPx and and lipid peroxide levels

All the treatment groups showed a significant dose dependent protection against CCl₄ induced decrease in the liver GSH and SOD (P<0.05), catalase enzyme In the case of GPx, MDA all the treatment groups showed a significant dose dependent protection (P<0.05), (Table1).

 

Effect on liver histopathology

The Section of liver from normal rats (Group 01) showed normal appearance of the Hepatic parenchyma including the portal areas. The liver sections of the control rats treated with CCl₄ showed severe degeneration and necrosis of hepatocytes. In addition, there was a significant portal cirrhosis was observed. The liver sections of treatment groups (Group 3-5) showed a dose dependent protection against CCl4 induced changes. However, Group 6 treated with Silymarin (50mg/kg) showed highest protection with near normal appearance of liver sections, indicating a significant hepatoprotection.

 

In the present study, the HAE was obtained and the Phenolic content was determined with the help of gallic acid as standard. Total phenolic content can be expressed as gallic acid equivalent i.e 49.33±1.23 mg/g of the extract The hepatoprotective activity of test item algae extract (200, 300 and 400mg/kg b.wt.,) were studied for their hepatoprotective activity against CCl4 induced cirrhosis in rats. The serum biochemistry (ASAT, ALAT, ALP and TP), liver antioxidant status (SOD, Catalase, GSH, GPx, and lipid peroxide levels) and liver histopathology analysis revealed a varying degree of hepatoprotection by the test item. The test dose 200mg/kg and 300mg/kg body wt have shown increased level of biochemical markers. The total protein content have shown significant increase in G3,G4, G5 cases. In G5 the ASAT and SOD level have increased significantly. Among these, Group 4 and 5 animals treated with test item at an oral dose 300 and 400 mg/kg, respectively, showed highest degree of hepatoprotection as evident from the biochemical and histopathological studies. The above protection was near to the hepatoprotective activity exhibited by Group-6 silymarim (50mg/kg). Hence, the above results clearly demonstrate that the test item has significant hepatoprotective activity.

Table 1: Effect of Test item on body weight, serum biochemistry and liver antioxidant parameters in paracetamol induced hepatotoxicity in rats

Note: Data expressed as mean±SD: n=6 per group. * indicate p<0.05,** indicate p<0.01,*** indicate p<0.001

 

Group 1: Normal

                      

                             Treatment: 0.5% Tween 80                                                                                     Dose : 10 ml/kg, p.o.

Section of liver from normal rats (Group 1) showing the normal appearance of the hepatic parenchyma including the portal areas. (H and E 10x and 40x).

 

Group 2: Control

                       

                                  Treatment: 0.5%Tween 80                                                                              Dose : 10 ml/kg,

 

Sections of the control rats (Group 2) treated with ccl₄ inhalation for 4 weeks showed severe degeneration changes in the hepatic parenchyma with loss of liver architecture and portal cirrhosis. (H and E 10x and 40x).

 

 

 

Group 3:

                            

                                Treatment: Test dose 1                                                                                   Dose : 200 mg/kg, p.o

 

Sections of the treated rats (Group 3) treated with low dose 200mg/kg extract showed mild hepatoprotection with minimal degeneration and necrosis of hepatocytes and minimal degree of neutrophil infiltration and inflammation. (H and E: Χ 10x and 40x).

 

Group 4:

                            

                                 Treatment: Test dose 2                                                                                     Dose : 300 mg/kg, p.o.

 

Sections of the treated rats (Group 4) treated with 300mg/kg of test item showed a moderate hepatoprotection with minimal degeneration and necrosis of hepatocytes and minimal degree of neutrophil infiltration and inflammation. (H and E: 10x and 40x).

 

Group 5

                              

                                       Treatment: Test dose 3                                                                               Dose : 400 mg/kg, P.o.

 

Sections of the treated rats (Group 5) treated with high dose of plant extract 400mg/kg showed a significant hepatoprotection with near normal appearance of the hepatic parenchyma. (H and E: 10x and 40x)

 

 

Group 6

                           

                                    Treatment: Silymarin                                                                                 Dose : 50 mg/kg, p.o.

 

Sections of the treated rats (Group 6) treated silymarin (50mg/kg) showed a significant hepatoprotection with near normal appearance of the hepatic parenchyma (H and E: 10x and 40x

 

 

REFERENCES

1.       Faulkner DJ. Marine natural products. Nat. Prod. Rep. 2001, 18, 1–49.

2.       Bold HC, Wynne MJ. Introduction to the algae and reproduction, Englewood Cliffs, NJ, 1985; 1–33.

3.       Davis AR, Targett NM, Mc Connell OJ and Young CME pibiosis of marine algae and benthic invertebrates: natural products chemistry and other mechanisms inhibiting settlement and overgrowth. Bioorg. Mar. Chem, 1989; 3: 85-114.

4.       Dennis J, Mc Hugh. Prospects for seaweed production in developing countries. Food Agric. Org. 2002; 120-122.

5.       Blunt JW, Copp BR, Munro, MHG North cote and Prinsep MR. Marine natural products. Nat. Prod. Rep., 2005; 22: 15-61.

6.       Smith AJ. (2004). Medicinal and pharmaceutical uses of seaweed natural products: A review. J. Appl. Phycol, 2004; 16: 245-262.

7.       Hosokawa M, Bhaskar N, Sashima T, and Miyashita K. Fucoxanthin as a bioactive and nutritionally beneficial marine carotenoid: a review. Carotenoid Sci.. 2006; 10:15-28.

8.       Davis AR, Targett NM, McConnell OJ, and Young CM.. Epibiosis of marine algae and benthic invertebrates: natural products chemistry and other mechanisms inhibiting settlement and overgrowth. Bioorg. Mar. Chem.1989; 3: 85-114.

9.       Davit D, Fernandez R, Mariezcurrena R, Mombru AW, Saldaña J, Dominguez L, Coll J, and Manta E. A New indole derivative from the red alga Chondriaatro purpurea. Isolation, Structure determination, and anthelmintic activity. J. Nat. Prod. 1998; 61: 1560-1563.

10.     Connan S, Delisle F, Deslandes E, and Ar Gall E. Intra-thallusphlorotannin content and antioxidant activity in Phaeophyceae of temperate waters. Bot. 2006; 49: 34-46.

11.     Jha M, Youssef A, et al. Comparative Phycochemical investication of Hydrocarbons content on some marine Seaweeds Algae. Res. J .Phytochem., 2008; 2(1): 10-17.

12.     Zeliha Demire. Antimicrobial and antioxidant activity of brown algae from the Aegean Sea. Chem. Soc. 2009; 619-628.

13.     Aswathi EM, Peres Ciênc, Lavras V. Evaluation of antifungal activity of seaweed extracts. 2012; 294-299.

14.     Jamila P. (2012). The Antiviral Activities and Antifungal activities and Mechanisms of Marine Polysaccharides: An Overview. Mar. Drugs. 2795-2816.

15.     Mourelle, M., et al. "Prevention Of CCl₄‐Induced Liver Cirrhosis By Silymarin." Fundamental and Clinical Pharmacology. 1989; 3 (3): 183-191.

16.     Pavanato, Amalia, et al. "Effects of quercetin on liver damage in rats with carbon tetrachloride-induced cirrhosis." Digestive Diseases and Sciences. 2003; 48(4): 824-829.

17.     Bosek, P., and M. Nakano. "Hepatoprotective effect of rooibos tea (Aspalathus linearis) on CCl₄-induced liver damage in rats." Physiological Research. 2003; 52 (4): 461-466.

 

 

 

 

Accepted on 24.04.2017        © RJPT All right reserved