Hepatoprotective, Anti-Inflammatory and Antioxidant Activities Of

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provided by Elsevier - Publisher Connector Bulletin of Faculty of Pharmacy, Cairo University (2016) 54, 209–217

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ORIGINAL ARTICLE Hepatoprotective, anti-inﬂammatory and antioxidant activities of Flacourtia montana J. Grah leaf extract in male Wistar rats

Chinchu Joshy, P.A. Thahimon, R. Arun Kumar, Betty Carla, Christudas Sunil *

St. Joseph’s College of Pharmacy, Cherthala, Kerala, India

Received 16 February 2016; revised 6 May 2016; accepted 5 June 2016 Available online 21 June 2016

KEYWORDS Abstract Flacourtia montana and its related species have been used traditionally for the treatment Hepatoprotective; of various diseases. The present study evaluated the hepatoprotective, anti-inflammatory and Anti-inflammatory; antioxidant activities of F. montana methanolic extract. The hepatoprotective effect of F. montana Antioxidant; was evaluated against paracetamol induced hepatotoxicity in Wistar rats. Administration of parac- Flacourtia montana; etamol (2 g/kg) showed a significant biochemical and histological deterioration in the liver of exper- Carrageenan imental animals. Pretreatment with F. montana (200 and 400 mg/kg b.wt. p.o) significantly (P 6 0.001) reduced the elevated levels of serum enzymes like serum glutamic-oxaloacetic transaminase (AST), serum glutamic-pyruvic transaminase (ALT), alkaline phosphatase (ALP) and reversed the hepatic damage in the liver which evidenced the hepatoprotective activity. The anti- inflammatory activity of F. montana was evaluated by carrageenan-induced paw edema and cotton pellet-induced granuloma models. F. montana (200 and 400 mg/kg) showed a significant (P 6 0.001) reduction in rat paw edema with 76.39% and 80.32%, respectively induced by carrageenan against the reference anti-inflammatory drug ibuprofen (10 mg/kg) (83.10%). Oral administration of F. montana (200 and 400 mg/kg) also significantly (P 6 0.001) reduced the granuloma mass formation in cotton pellet granuloma method. The reducing power and hydrogen peroxide radical scavenging were increased at increasing doses of F. montana. The results of the present study demonstrate that the methanolic extract of F. montana possess hepatoprotective, anti-inflammatory and antioxidant activities. Ó 2016 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc- nd/4.0/).

1. Introduction

* Corresponding author at: Department of Pharmacology, St. Joseph’s College of Pharmacy, Cherthala, Kerala, India. Tel.: +91 Hepatotoxicity refers to liver dysfunction or liver damage that 9789688425. is associated with an overload of drugs or xenobiotics such as E-mail address: [email protected] (C. Sunil). acetaminophen, cadmium chloride, ethanol, carbon tetrachlo- 1,2 Peer review under responsibility of Faculty of Pharmacy, Cairo ride (CCl4) and allyl alcohols. The chemicals that cause liver University. injury are called hepatotoxins or hepatotoxicants. Hepatotox- http://dx.doi.org/10.1016/j.bfopcu.2016.06.001 1110-0931 Ó 2016 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 210 C. Joshy et al. icants are exogenous compounds of clinical relevance and may has not been studied extensively both for its pharmacological include overdoses of certain medicinal drugs, industrial chem- as well as phytochemical properties. But related species of F. icals, and natural chemicals like microcystins, herbal remedies montana, Flacourtia jangomas, Flacourtia sepiaria, Flacourtia and dietary supplements.3,4 Certain drugs may cause liver inermis and Flacourtia ramontchi have been reported with var- injury when introduced even within the therapeutic ranges. ious pharmacological activities like antibacterial, antidiabetic, Hepatotoxicity may result not only from direct toxicity of anti-inflammatory and hepatoprotective.19–21 Hence this study the primary compound but also from a reactive metabolite was designed to evaluate the hepatoprotective, anti- or from an immunologically-mediated response affecting hep- inflammatory and antioxidant activities of F. montana leaves atocytes, biliary epithelial cells and/or liver vasculature.5,6 methanolic extract. Hepatotoxic response is expressed in the form of characteristic patterns of cytolethality in specific zones of the acinus. Its 2. Materials and methods related symptoms may include jaundice or icterus appearance causing yellowing of the skin, eyes and mucous membranes 2.1. Plant material due to high level of bilirubin in the extracellular fluid, pruritus, severe abdominal pain, nausea or vomiting, weakness, severe fatigue, continuous bleeding, skin rashes, generalized itching, The plant material was collected during the month of January, swelling of the feet and or legs, abnormal and rapid weight 2015, from Alappuzha district in Kerala, India. The plant was gain in a short period of time, dark urine and light color stool. taxonomically identified and authenticated by Dr. Kiranraj M. The liver disorders are one of the serious health problems, S. Assistant Professor of Botany S. N College, Cherthala. A throughout the world. Hepatoprotection or antihepatotoxicity voucher specimen was deposited at the Herbarium of Depart- is the ability to prevent damage to the liver.7,8 Hepatoprotec- ment of Botany with accession No. SNCCH089 for future tive effect was studied against chemicals and drug induced hep- reference. atotoxicity in rats. Alcohol, CCl4, galactosamine, paracetamol, isoniazid rifampicin, antibiotics, peroxidised oil, aflatoxin have 2.2. Preparation of methanolic extract of F. montana leaves been used as hepatic agents.9 Inflammation is a part of the complex biological response Leaves were washed thoroughly with water to remove the soil of body tissues to harmful stimuli, such as pathogens, dam- particles, shade dried and grounded. 1 kg of powder was aged cells, or irritants.10 It is a protective response that soaked in methanol sequentially for 72 h at room temperature. involves immune cells, blood vessels, and molecular mediators. Plant debris were removed from the extract by filtration and The purpose of inflammation is to eliminate the initial cause of defatted with petroleum ether and the filtrate was concentrated cell injury, clear out necrotic cells and tissues damaged from under reduced pressure using a rotary evaporator at 40 °C the original insult and the inflammatory process, and to initi- until extraction solvent was completely dried. The yield of ate tissue repair.11 Inflammation is beneficial when needed, methanol extract was 6.1 w/v. The extract was stored in the but it is disastrous when chronic. Chronic inflammation gener- refrigerator at 4 °C for further use. The extract was dissolved ates a constant supply of free radicals that overwhelm our in normal saline and used for animal studies. antioxidant defenses and damage DNA, aging us and causing disease of every description. Inflammation can also be defined 2.3. Experimental animals as body reaction against injury or irritant.12 Microorganisms, physical agents, chemicals, inappropriate immunological Healthy male Wistar rats (150–180 g) were obtained from the responses, and tissue death are the factors that can stimulate Animal house of Gov. Veterinary College, Mannuthy. They inflammation. Infectious agents such as viruses and bacteria were housed in polypropylene cages in a conducive environ- are some of the most common stimuli of inflammation.13 mental situation i.e. temperature (22 ± 2 °C), humidity (45 Since the beginning of human civilization, medicinal plants ±5°C) and 12 h of dark and light cycle. The animals were have been used by mankind for its therapeutic value. Nature fed with normal laboratory chow standard pellet diet. The ani- has been a source of medicinal agents for thousands of years mals were allowed to acclimatize for 7 days before commenc- and an impressive number of modern drugs have been isolated ing the experiments. All the studies were conducted in from natural sources.14 The plant-based, traditional medicine accordance with the animal ethics committee of the institute systems continue to play an essential role in health care, with (IAEC, proposal number SJCP/IAEC/21/03/15/01) St. about 80% of the world’s inhabitants relying mainly on tradi- Joseph’s college of pharmacy, Cherthala. tional medicines for their primary health care.15 The medicinal properties of plants could be based on the antioxidant, antimi- crobial antipyretic effects of the phytochemicals in them.16 2.4. Acute toxicity study According to World Health Organization, medicinal plants would be the best source to obtain a variety of drugs. There- To evaluate the toxicity of F. montana extract the acute toxic- fore, such plants should be investigated to better understand ity study was performed based on Irwin test.22 Four groups of their properties, safety and efficacy.17 fasted healthy rats (six per group) were orally administered the Flacourtia montana J. Grah is a thorny tree that belongs to extract at a dose of 1–4 g/kg; the control group was given dis- the family flacourtiaceae. They are found in the evergreen and tilled water. The rats were observed for 1 h continuously and semi-evergreen forests and endemic to the Western Ghats- then hourly for 4 h and finally after every 24 h up to 14 days common trees in South, Central and South Maharashtra for any physical signs of toxicity, such as writhing, gasping, Sahyadris.18 F. montana and its related species have been used palpitation and decreased respiratory rate or mortality. No traditionally for the treatment of various diseases. F. montana animals died. The animals showed some toxic behavior above Hepatoprotective and antioxidant activities of Flacourtia montana J. Grah leaf extract 211

2000 mg/kg, therefore the LD50 is greater than 2000 mg/kg. before injection of carrageenan. Paw volume was measured Pre-screening investigation with 1/20th, 1/10th and 1/5th of with plethysmometer at 1, 2, 3 and 4 h after injection. The inhi- 2000 mg/kg, i.e. 100, 200 and 400 mg was done. The doses bitory activity was calculated using the following formula.25 200 and 400 mg/kg were found to be effective against hepato- ð Þ¼ð Þ protective and anti-inﬂammatory activities. Percentage inhibition PI Vt Vo control ðVt VoÞtreated=

2.5. Evaluation of hepatoprotective activity ðVt VoÞcontrol 100

2.5.1. Induction of Paracetamol induced hepatotoxicity Vo = Mean paw volume at 0 h, Vt = Mean paw volume at a particular time interval Male Wistar rats weighing 150–180 g were used for the study. Animals were divided into 5 groups of 6 animals each. 2.6.2. Cotton pellet-induced granuloma in rats Group I: (normal control) received normal saline (1 ml/kg b.wt. p.o.) for 5 days. Male Wistar rats weighing 150–180 g were used for the study. Group II: (toxic group) received saline (1 ml/kg b.wt. p.o.) Group I: (normal control) rats received normal saline. Group for 5 days and paracetamol (2 g/kg b.wt. p.o) on the 2nd and II: (standard group) received Ibuprofen (10 mg/kg b.wt.) 3rd day. Group III: (test group) received methanolic extract of F. mon- Group III: (standard group) received Silymarin (100 mg/kg tana (200 mg/kg b.wt. p.o). Group IV: (test group) received b.wt. p. o.) for 5 days and paracetamol (2 g/kg b.wt. p.o.) on methanolic extract of F. montana (400 mg/kg b.wt. p.o). the 2nd and 3rd day, 30 min after Silymarin administration. A sterilized cotton pellet weighing 10 ± 1 mg was implanted Group IV: (test group), received methanolic extract of subcutaneously into the groin region of rats after which four F. montana (200 mg/kg b. wt. p.o) for 5 days and were admin- groups were treated (once daily) with 200 and 400 mg/kg of F. istered paracetamol (2 g/kg b.wt. p.o.) on the 2nd and 3rd day, montana for seven consecutive days. Animals in control and 30 min after methanolic extract administration. standard groups received saline and Ibuprofen (10 mg/kg) Group V: (test group), received methanolic extract of respectively. The animals were sacriﬁced on the 8th day. There- F. montana (400 mg/kg, b.wt. p.o) for 5 days and were admin- after, the pellets surrounded by granuloma tissue were dissected istered paracetamol (2 g/kg b.wt. p.o.) on the 2nd and 3rd day out carefully and the weights of wet cotton pellets were noted 30 min after methanolic extract administration.23 and thereafter the cotton pellets were dried in an oven at 60 ° Animals were sacriﬁced and blood was collected directly C for 24 h to obtain a dry cotton pellet weight and the mean 26 through retro-orbital plexus. Serum was separated after coag- weight of granuloma tissue. The percentage inhibition (PI) ulating at 37 °C for 30 min and centrifuged at 3000 rpm for of granuloma tissue development was calculated: 15–20 min. Serum was used for the estimation of biochemical PI ¼ Weight of pellet ðcontrolÞ parameters like serum glutamate pyruvate transaminase (AST), serum glutamate oxaloacetate transaminase (ALT) weight of pelletðtestÞ=Weight of pelletðcontrolÞ100 and alkaline phosphatase (ALP) and the liver tissues collected were subjected to histopathology. AST, ALT and ALP were measured by using diagnostic kits. 2.7. Antioxidant activity

2.5.2. Histopathological studies 2.7.1. Reducing power assay Livers of different groups were fixed in 10% buffered neutral Reducing power was determined according to the method of formalin for 48 h and then with bovine solution for 6 h. Paraf- Oyaizu.27 10 mg of methanol extract and ascorbic acid, the fin sections were taken at 5 mm thickness, processed in standard were dissolved in 10 ml of methanol. From the above, alcohol-xylene series and were stained with alum hematoxylin different concentrations (100, 200, 300, 400 and 500 lg/ml) and eosin. The sections were examined microscopically for were pipetted out and made up to 1 ml with distilled water. histopathological changes.23 2.5 ml of phosphate buffer (pH 6.6) and 2.5 ml of potassium ferricyanide were added to each of the test tubes and incubated 2.6. Evaluation of anti-inflammatory activity of F. montana at 40 ⁰C for 20 min. After incubation, 2.5 ml of trichloroacetic acid was added and centrifuged for 5 min. To 2.5 ml of this 2.6.1. Carrageenan induced paw edema reaction mixture, 0.5 ml ferric chloride (0.1% w/v) and 2.5 ml of water were added. The absorbance was measured In this study the animals were divided into 4 groups of 4 rats at 700 nm using UV spectrophotometer. each. Group I: (normal control) rats received normal saline. Group II: (standard group) received Ibuprofen (10 mg/kg b. wt. p.o). Group III: (test group), received methanolic extract 2.7.2. Hydrogen peroxide scavenging assay of F. montana (200 mg/kg b.wt. p.o). Group IV: (test group), A solution of hydrogen peroxide (40 mM) was prepared in received methanolic extract of F. montana (400 mg/kg b.wt. phosphate buffer (50 mM, pH 7.4). 10 mg of F. montana and p.o.). ascorbic acid standard was dissolved in 10 ml of methanol. The acute anti-inflammatory effect was evaluated by Different concentrations of F. montana and ascorbic acid carrageenan induced rat paw edema according to the method (100, 200, 300, 400 and 500 lg/ml) was added to 0.6 ml hydro- of Winter et al.24 Edema was induced by injection of 1% sus- gen peroxide and absorbance at 230 nm was determined after pension of carrageenan in 0.9% sterile saline solution into the 10 min against a blank solution containing phosphate buffer right plantar region of the rat. F. montana (200 and 400 mg/kg) without hydrogen peroxide.28 The percentage of hydrogen per- and Ibuprofen (10 mg/kg b.wt.) were administrated orally 1 h oxide scavenging was calculated as: 212 C. Joshy et al.

% ¼½ð Þ= scavenged Ai At Ai 100 3.2. Histopathological examination where Ai is the absorbance of control and At is the absorbance of test. Histopathological examination of liver sections of the control group showed normal cellular architecture with distinct hep- 2.8. Estimation of total phenolic content atic cells and central vein (Fig. 1–I). In the liver sections of paracetamol intoxicated rats (Fig. 1–II), there was disarrange- Total phenolic content was assessed according to the Folin– ment and degeneration of normal hepatic cells. The hepatic Ciocalteau method. A standard curve was prepared using dif- cells also showed a small nucleus with necrosis; vacuolization ferent concentrations of gallic acid (50, 100, 150, 200 and and inflammation. Treatment with F. montana extract (200 250 mg/ml) in methanol. To the above standard solutions of and 400 mg/kg) and silymarin, exhibited prominent protection gallic acid (1 ml each), 5 ml Folin–Ciocalteau reagent (1:10 against paracetamol intoxication, which was evidenced by less dilution in distilled water) was added and after 8 min, 4 ml vacuole formation, less disarrangement and degeneration of of 7.5% sodium carbonate was added. These solutions were hepatocytes (Fig. 1III–V). Table 2 summarizes the data on incubated at room temperature for 2 h and their absorbance the degree of liver damage induced by paracetamol for each at 765 nm was measured spectrophotometrically. For the test group. The histoscores for vacuolization, inflammation and solution, 1 ml of F. montana methanolic extract (1 g in 10 ml cellular necrosis of the livers were significantly higher after methanol) was added to 5 ml of Folin–Ciocalteau reagent. paracetamol treatment. In contrast, pretreatment with F. mon- 4 ml of 7.5% sodium carbonate was added after 8 min and this tana (400 mg/kg) and Silymarin (100 mg/kg) significantly was incubated at room temperature for 2 h and its absorbance reduced the injury histoscores of the livers from these groups. was measured at 765 nm. The samples were tested in triplicate and a calibration curve with five data points for gallic acid was 3.3. Effect of methanolic extract of F. montana on carrageenan obtained. The results were compared to a Gallic acid calibra- induced paw edema in rats tion curve and the total phenolic content of F. montana methanolic extract was expressed as mg of gallic acid equiva- The effect of methanolic extracts of F. montana (200 and lents per gram of extract.29 400 mg/kg) in carrageenan induced paw edema in rats is shown in Table 3. After carrageenan induction there was a significant 2.9. Statistical analysis increase in edema formation in normal rats. Methanolic extract at 200 mg/kg at 1, 2, 3 and 4 h prevented the car- Results were expressed as mean ± SEM. Statistical analyses of rageenan induced paw edema with a percentage inhibition of all the data obtained were evaluated using a one-way ANOVA 18.62 ± 1.52, 42.24 ± 0.67, 67.11 ± 1.48, 76.39 ± 1.01 at 1, followed by Dunnett’s post – hoc multiple comparison test 2, 3 and 4 h respectively, while 29.65 ± 0.70, 58.39 ± 0.62, with SPSS Program; Version 20. All the results were also 69.54 ± 0.972, 80.32 ± 0.833 at a dose of 400 mg/kg at 1, 2, expressed as graph by Graph Pad Prism software (v.5). P val- 3 and 4 h respectively. Ibuprofen decreased the carrageenan ues 6 0.05 were considered as statistically significant. induced paw edema with a percentage of 46.76 ± 1.12, 61.18 ± 0.88, 75.20 ± 1.15, 83.10 ± 1.21 at 1, 2, 3 and 4 h respectively. The results showed that methanolic extract with 3. Results a dose of 200 and 400 mg/kg showed maximum inhibition significantly (P 6 0.001) on carrageenan induced rat paw edema 3.1. Effect of F. montana on serum levels of AST, ALT and ALP at the end of 4 h compared to standard group rats. in hepatotoxic rats 3.4. Cotton pellet-induced granuloma in rats In paracetamol induced hepatoxic study, after paracetamol administration to the normal rats showed a significant increase The effect of methanolic extracts of F. montana (200 and of AST, ALT and ALP levels. But methanolic extract of F. 400 mg/kg) in cotton pellet induced granuloma in rats is shown montana (200 and 400 mg/kg) treatment brought the AST, in Table 4. The results revealed that the methanolic extract of ALT and ALP levels significantly (P 6 0.001) to near normal F. montana shows a dose dependant inhibition of weight of (Table 1). both wet and dry cotton pellets. The mean decrease in weight

Table 1 Effect of F. montana on AST, ALT and ALP levels. Groups AST (IU/L) ALT (IU/L) ALP (IU/L) Normal control 32.70 ± 1.22 69.98 ± 3.4 37.53 ± 1.62 Paracetamol (2 g/kg b.wt.) 115.74 ± 3.4### 138.94 ± 1.4### 120.44 ± 2.72### Paracetamol (2 g/kg b.wt.) + Silymarin (100 mg/kg) 48.41 ± 1.3***,# 93.55 ± 1.0***,# 52.06 ± 1.14***,# Paracetamol (2 g/kg b.wt.) + F. montana extract (200 mg/kg b. wt.) 57.32 ± 0.58***,## 105.01 ± 0.44***,## 59.78 ± 0.52***,## Paracetamol (2 g/kg b.wt.) + F. montana extract (400 mg/kg b. wt.) 53.07 ± 0.67***,# 97.22 ± 0.62***,# 54.60 ± 0.29***,# Results are expressed as mean ± SEM. The results were considered statistically signiﬁcant when *P 6 0.05, **P 6 0.01, ***P 6 0.001 compared with paracetamol, #P 6 0.05, ##P 6 0.01, ###P 6 0.001 compared with normal control. Hepatoprotective and antioxidant activities of Flacourtia montana J. Grah leaf extract 213

Figure 1 Histological section of Group I – Liver section of normal rats showing normal central vein (A) and radially arranged hepatocytes (B). Group II – Liver section of hepatotoxic rats showing hepatocellular changes with small nucleus and necrosis (A); vacuolization and inﬂammation (C). Group III – Liver section of standard treated group with normal central vein (A) and normal hepatocytes (B). Group IV and Group V – Liver section of F. montana showed normal central vein (A) and radially arranged hepatocytes with less vacuole formation.

Table 2 Histoscores of rat livers treated with F. montana and Silymarin in paracetamol-induced hepatotoxicity assay. Histo-grade Groups Control Paracetamol F. montana Silymarin 100 mg/kg 200 mg/kg 400 mg/kg Vacuolization 0 3.3 ± 0.1## 2.4 ± 0.5* 1.44 ± 0.2** 1.5 ± 0.1** Inflammation 0 2.8 ± 0.3## 1.93 ± 0.2** 1.58 ± 0.08** 1.6 ± 0.3** Cellular necrosis 0 3.7 ± 0.3## 2.9 ± 0.3* 1.63 ± 0.4** 1.4 ± 0.09** To quantify the histological indices for vacuolization and hepatocellular necrosis of liver, the slides were graded 0–4 according to the method of Knodell et al. The liver damage was graded 0–4 as follows: 0 = no visible cell damage; 1 = slight (1–25%); 2 = moderate (26–50%); 3 = moderate/severe (51–75%); 4 = severe/high (76–100%). #Significantly different from the control group (##P 6 0.01); *Significantly different from the paracetamol group (*P 6 0.05, **P 6 0.01); Values are mean ± SEM (n = 6). of both wet and dry cotton pellets for rats, which received 200 was found to be more effective at a dose of 400 mg/kg body and 400 mg/ kg body weight of the extract was significantly weight. The extract at the dose of 400 mg/kg had shown (P 6 0.001) lower than those in the control rats. The extract 61% inhibition in weight of wet cotton pellets and 67% 214 C. Joshy et al.

Table 3 Effect of F. montana on Carrageenan induced rat paw edema volume. Groups Paw volume (cm) 1h 2h 3h 4h Normal saline 0.43 ± 0.47 0.53 ± 0.33 0.61 ± 0.30 0.72 ± 0.45 Ibuprofen (10 mg/kg) 0.23 ± 0.49** 0.20 ± 0.47** 0.15 ± 0.71*** 0.12 ± 0.87*** F. montana extract (200 mg/kg) 0.35 ± 0.66* 0.31 ± 0.36* 0.20 ± 0.91** 0.17 ± 0.73** F. montana extract (400 mg/kg) 0.31 ± 0.7* 0.22 ± 0.33** 0.18 ± 0.60*** 0.14 ± 0.60*** Results are expressed as mean ± SEM. The results were considered statistically signiﬁcant when *P 6 0.05, **P 6 0.01, ***P 6 0.001 compared with normal control.

Table 4 Effect of F. montana on cotton pellet granuloma. Treatment group Mean of wet cotton pellets % Mean of dry cotton pellets % Mean of transudative (mg) Inhibition (mg) Inhibition weight Normal saline 190.32 ± 0.18 110.33 ± 0.19 79.98 ± 0.24 Ibuprofen (10 mg/kg) 60.67 ± 0.32*** 68% 30.56 ± 0.32*** 72% 30.10 ± 0.45*** F. montana extract 82.75 ± 0.51*** 56% 40.49 ± 0.43*** 63% 42.25 ± 0.54*** (200 mg/kg) F. montana extract 73.99 ± 0.63*** 61% 35.93 ± 0.58*** 67% 38.06 ± 1.1*** (400 mg/kg) Results are expressed as mean ± SEM. The results were considered statistically signiﬁcant when *P 6 0.05, **P 6 0.01, ***P 6 0.001 compared with normal control.

inhibition in weight of dry cotton pellets, while the extract at showed a signiﬁcant positive correlation with a coefﬁcient of the dose of 200 mg/kg had shown 56% inhibition in weight correlation 0.989. of wet cotton pellets and 63% inhibition in weight of dry cotton pellets when compared to that of control group animals. 3.6. Hydrogen peroxide scavenging activity of F. montana

3.5. Reducing power of F. montana The activity of F. montana on superoxide radical scavenging is shown in Fig. 3. The scavenging ability of F. montana on Fig. 2 shows the reductive capabilities of F. montana compared hydrogen peroxide is compared with that of standard ascorbic to the standard ascorbic acid. The reducing power increased acid. The IC50 value of F. montana on hydrogen peroxide scav- with increasing concentration of F. montana extract. The cor- enging activity was found to be 225.28 ± 1.79 lg/ml, whereas relation between concentration and reducing power of Ascor- the IC50values for ascorbic acid was found to be 98.27 bic acid showed a significant positive correlation with a ± 0.25 lg/ml. The correlation between concentration and coefficient of correlation 0.957 and also the correlation hydrogen peroxide radical scavenging of Ascorbic acid showed between concentration and reducing power of F. montana a significant positive correlation with a coefficient of

1.4 90 80 1.2 r=0.989** r=0.957** 70 1 60 r=0.984** 0.8 50 r=0.989*** F. montana 0.6 F. montana 40 Ascorbic acid Absorbance Ascorbic acid % inhibition 30 0.4 20 0.2 10 0 0 100 200 300 400 500 100 200 300 400 500 Concentration (μg/ml) Concentration (μg/ml)

Figure 2 Effect of F. montana on reducing power. Results are Figure 3 Hydrogen peroxide radical scavenging activity of F. expressed as mean ± SEM. The results were considered statisti- montana. Results are expressed as mean ± SEM. The results were cally significant when compared with concentration (**P 6 0.01, considered statistically significant when compared with concen- ***P 6 0.001). tration (**P 6 0.01, ***P 6 0.001). Hepatoprotective and antioxidant activities of Flacourtia montana J. Grah leaf extract 215 correlation 0.989 and also the correlation between concentra- sections of F. montana treatment exhibited prominent protection and reducing power of F. montana showed a significant tion against paracetamol intoxication, which was evidenced positive correlation with a coefficient of correlation 0.984. by less vacuole formation and less disarrangement and degeneration of hepatocytes. 3.7. Total phenolic content of F. montana In addition to hepatoprotective activity, F. montana was investigated in acute and sub acute inflammatory models for The total phenolic content of methanol extract of F. montana anti-inflammatory activity. Carrageenan is a strong chemical was 74.33 ± 4.93 mg gallic acid equivalent/gram extract calcu- used for the release of inflammatory and proinflammatory lated from the gallic acid calibration curve (fig. 4). The total mediators (prostaglandins, leukotrienes, histamine, bradyki- a 34 phenolic contents were calculated using the following linear nin, TNF- , etc). The carrageenan induced paw edema has equation based on the calibration curve of gallic acid; been accepted as a preliminary screening model for investigat- 35 Y = 0.0015X + 0.396, R2 = 0.9808 where Y is the absorbance ing systematic anti-inflammatory agent. The edema forma- and X is the amount of gallic acid in lg. tion by sub plantar injection of carrageenan is a biphasic response; initial phase (1 h) is being due to the release of sero- 4. Discussion tonin and histamine whereas the later phase (over 1 h) is attrib- uted to the release of prostaglandins, the cyclooxygenase products and the continuity between two phases is provided The present study was designed to establish the scientific evi- by kinins.36,37 Methanolic extract of F. montana significantly dences for the usage of F. montana plant in hepatic and inflam- inhibited later phase of edema so it seems to be possible that matory conditions. Hepatoprotective effect was studied in F. montana blocks prostaglandins and cyclooxygenase release paracetamol induced hepatotoxicity and anti-inflammatory in the later phase of acute inflammation. The present results effect in two models: carrageenan induced paw edema and cot- indicate that F. montana (200 and 400 mg/kg) had an efficient ton pellet granuloma formation. therapeutic activity in acute inflammatory conditions. In the As far as liver diseases are concerned, traditional system of cotton pellet granuloma model, inflammation and granuloma medicine is highly hope giving. Paracetamol induced hepato- developed during a period of 7 days. The dry weight of the pel- toxicity is one of the well known and commonly used animal lets correlates with the amount of granulomatous tissue. Pro- models for studying the hepatoprotective property. Adminis- tein synthesis is necessary for the formation of granuloma. tration of paracetamol at a dose of (1–3 g/kg/day, p.o) results Inflammation involves infiltration of macrophages, neu- in hepatic damage and showed a significant elevation of serum trophils and proliferation of fibroblasts, which are the basic level of hepatic enzymes, AST, ALT and ALP. The toxic meta- sources for granuloma formation.38,39 Consequently decrease bolic N-acetyl-p-benzoquinoneimine is an oxidative product of in granuloma weight indicates the suppression of the prolifer- paracetamol formed by the action of cytochrome P-450 and it 30 ative phase, which was effectively inhibited by the methanolic reacts with paracetamol. N-acetyl-p-benzoquinoneimine extract of F. montana. F. montana (200 and 400 mg/kg) (NAPQI), which is one of the metabolites of paracetamol appears to be equally effective to that of Ibuprofen in inhibit- which undergoes metabolism in the liver via the action of cyto- 31–33 ing the dry weight of cotton pellets. Anti-inflammatory activity chrome P450 monooxygenase, is highly responsible for the of flavonoids, triterpenoids and phytosterols has been reported paracetamol toxic effect to the liver. Histopathology of the so it might be possible that these particular phytoconstituents liver shows necrosis of the centrilobular hepatocytes character- from methanolic extract of F. montana could have contributed ized by nuclear pyknosis, eosinophilic and large excessive hep- 34 to this activity. atic lesions. Liver injuries are often associated with a rise in Free radicals are harmful as they can participate in super- serum AST, ALT and ALP levels. In the present study, a sig- fluous side reactions due to their chain reaction properties nificant elevation in serum AST, ALT and ALP levels was which result in cell damage, blemishing of food, degradation observed in paracetamol treated groups indicating hepatocel- of various materials like rubber, gasoline etc. Life threatening lular damage. Pre-treatment with Silymarin (100 mg/kg, p.o.) diseases for example cancer, stroke, and diabetes may occur as and F. montana extract (200 and 400 mg/kg) caused a signifi- a result of the consequences of free radicals. Antioxidants are cant reduction in elevated enzyme levels showing the hepato- those molecules which can halt these chain reactions by remov- protective effect. Histopathological examination of liver 42 ing free radical intermediates. H2O2 is rapidly decomposed into oxygen and water and this may produce hydroxyl radicals (OH-) that can initiate lipid peroxidation and cause DNA 0.9 40 y = 0.0015x + 0.396 damage in the body. The H2O2 scavenging activity of F. mon- 0.8 R² = 0.9808 tana was evaluated and compared with ascorbic acid. The 0.7 maximum percentage scavenging of F. montana extract was 0.6 Absorbance(765nm) observed at 500 lg/ml concentration. 0.5 Phenolic compounds are responsible for antioxidant prop- 0.4 Linear erties due to their ability to donate electrons resulting in the 0.3 (Absorbance(765nm)) conversion of highly reactive free radicals to nonreactive stable 0.2 Absorbance (765nm) 0.1 molecules. Like flavonoids, tannins carry a high degree of free 0 radical scavenging property which may also be the prime rea- 41 0 100 200 300 son behind antioxidant activity. That is why total phenolic Concentration (mg/ml) content determination was important for making a correlation between antioxidant property and amount of phenolics.42 Figure 4 Standard curve of gallic acid standard at 765 nm. From the present study we obtained a notable amount of 216 C. 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