Antioxidant and Hepatoprotective Activity of Root extract of Baliospermum montanum (Willd) Muell Arg

 

Ahirwar B¹*, Ahirwar D²

¹Institute of Pharmacy, Guru Ghasidas University, Bilaspur (C.G.), India,

²School of Pharmacy, Chouksey Engineering College, Bilaspur (CG) India

 

ABSTRACT:

The hydroalcoholic extract of Baliospermum montanum Muell Arg root was screened for their possible hepatoprotective and antioxidant effect. Carbon tetrachloride (CCl₄) and ethanol were used to produce liver toxicity. The hydroalcoholic extract of plant root was administered and biochemical parameters were estimated. Concurrently, silymarin was used as a standard hepatoprotective agent. The root extract was also studied for in vitro total antioxidant capacity by ABTS assay. The results indicated that biochemical parameter changes produced by CCL₄ and ethanol were restored to normal by the hydroalcoholic extract. The hydroalcoholic extract showed significant (p<0.05) protection of liver cell against CCL₄ and ethanol-induced hepatocellular injury in a dose-dependent manner. The effects of hydroalcoholic extract were comparable with silymarin. Hepatoprotection produced by decreasing the activity of serum enzymes, bilirubin, and lipid peroxidation. The antioxidant potential of the extract may be attributed to the presence of phenolic and flavonoid compounds. The root extract of Baliospermum montanum Muell Arg. exhibited significant antioxidant and hepatoprotective activity.

 

 

 

INTRODUCTION:

The liver regulates many important metabolic functions and its injury or liver dysfunction is a major health problem that remains a global challenge¹. Liver cell injury is caused by various toxic chemicals like certain antibiotics, chemotherapeutic agents, carbon tetrachloride, thioacetamide, excessive alcohol consumption and microbes^2,3. Free radicals stress cause direct injury to tissue and succession of disease ailments. Free radicals are very detrimental in attacking lipids in cell membranes, inducing oxidations that cause membrane damage such as membrane lipid peroxidation and a decrease in membrane fluidity and may also cause DNA mutation leading to cancer⁴. Liver damage is associated with cellular necrosis, increase in tissue lipid peroxidation and depletion in the tissue glutathione levels⁵.

 

 

Ethanol induces a number of deleterious metabolic changes in the liver. Liver injury in chronic alcoholics is due to oxidative stress that leads to fibrosis and impaired liver functions and apoptosis⁶. Traditionally, a number of preparations recommended for the treatment of liver disorder. On ethnopharmacological information and database published which clearly state that plant has the potential to stimulate liver function, protect from damage and help to regenerate hepatic cells⁷. Baliospermum montanum (Willd) Muell is an essential therapeutic plant, which is ordinarily termed as Danti. The plant is a firm monoecious underneath shrub with various shoots from the bottom. In Ayurveda, the root is used to cure jaundice, leucoderma, skin diseases, wounds, and as an anthelmintic⁸. Leaves are found to be useful in asthma, bronchitis⁹ and in treating abdominal tumor¹⁰. Seeds are used as purgative and in gastric complaints¹¹. A decoction of the stem is used to get relief from toothache¹². Extensive uses of the plant, traditionally give the basis to evaluate the antioxidant, hepatoprotective activity and determination of total phenolic and flavonoid content with the growing interest in and demand for nutraceutical products. Recent studies showed that a number of plant products including polyphenolic substances exert potent antioxidant actions that have been associated with lower occurrence of mortality rates in several human diseases. The present study has been undertaken to evaluate the antioxidant and hepatoprotective role of the hydroalcohlic root extract of Baliospermum montanum on CCL₄ and ethanol-induced liver damage in rats.

 

MATERIALS AND METHODS:

Plant material collection:

Dried roots Baliospermum montanum Muell were collected from the Achanakmar-Amarkantak Bioreserve, Chhattisgarh, India. The plant specimen (490/20181000642) was taxonomically identified in the Department of Botany, Guru Ghasidas Vishwavidyalaya (a Central University), Bilaspur, Chhattisgarh, India.

 

Drugs and Chemicals:

Ethanol and carbon tetrachloride was purchased from Merck India Ltd. and Silymarin was purchased from Sigma Aldrich Co. Standard kit of Serum glutamate pyruvate transaminase (SGPT), Serum glutamate oxaloacetate transaminase (SGOT) and Alkaline phosphatase (ALP) were obtained from Span Diagnostics Ltd. All the other reagents and solvents used for the experiments were of analytical grade (Fischer Scientific, India).

 

Preparation of Extract:

The air-dried roots of Baliospermum montanum Muell were pulverized to get a coarse powder. 100 gm of the powdered material was taken and suspended in 1L 50% ethanol and subjected to maceration at room temperature for seven days. The extract was filtered through a muslin cloth and the filtrate was evaporated under reduced pressure and vacuum dried. The yield of the extract was 2.8% with reference to the dry starting material.

 

In-vivo Hepatotoxicity Study:

Animals:

Wistar rats (110-120g) of either sex were selected for the study. They were housed in the polypropylene cages in an air-conditioned area at 22°C ± 3 and 59 to relative humidity with 12 hour light & dark cycle. All the animals had free access to standard diet and clean water ad libitum. The experiments were conducted according to the Institutional Animal Ethics Committee (IAEC) regulations approved by the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA).

 

Acute Oral Toxicity Studies:

Wister albino rats were divided into five groups of six animals each. The first group served as normal control. The hydroalcoholic extract was administered orally to different groups at the dose level of 250, 500, 1000, and 2000 mg/kg p.o. body weights. All animals were observed for toxic symptoms and mortality for 72 h¹³. No mortality was observed up to a dose level of 2000 mg/kg body weight. Hence the extract was found to be safe up to the dose levels of 2000 mg/kg body weight.

 

Carbon tetrachloride-induced hepatotoxicity:

The rats were grouped randomly into six groups, containing six animals each. Group I, the control group, received the vehicle (normal saline). Group II served as a negative control group and received CCl₄ (2 ml/kg p.o., 1:1 with olive oil) for 5 days. Group III was positive control group treated with standard drug silymarin at 100 mg/kg body weight. Group IV and V were treated with plant extract at 250 and 500 mg/kg body weight along with CCl₄ (2 ml/kg p.o) and group VI treated with 500 mg/kg dose of extract, respectively for five days¹⁴. On the 6^th day of the treatment, blood was collected retro-orbitally under light ether anesthesia and allowed to clot for 30 - 40 min. Serum was separated by centrifugation at 37°C and was used for estimation of various biochemical parameters like Serum glutamate pyruvate transaminase (SGPT), Serum glutamate oxaloacetate transaminase (SGOT), Alkaline phosphatase (ALP) and total bilirubin. After collections of blood, animals were sacrificed under deep ether anesthesia and liver was collected, washed, blotted and weighed immediately³.

 

Ethanol-induced hepatotoxicity:

The rats were grouped randomly into six groups, each containing six animals. Group I, is the control group, received the vehicle (normal saline). Group II served as negative control group, received ethanol (4g/kg p.o., 40% v/v) for 21 days. Group III was positive control group treated with standard drug silymarin at 100 mg/kg body weight. Group IV and V were treated with plant extract at the dose levels of 250 and 500 mg/kg body weight along with ethanol (4g/kg p.o., 40% v/v) and group VI treated with 500 mg/kg dose of extract, respectively. On the 22^ndday of the treatment, blood was collected retro-orbitally under light ether anesthesia and blood was allowed to clot for 30-40 min. Serum was separated by centrifugation at 37°C and was used for estimation of various biochemical parameters like SGOT, SGPT, ALP, and total bilirubin. After collection of blood, animals were sacrificed under deep ether anesthesia and liver was collected, washed, blotted and weighed immediately^15,16.

 

HPTLC Fingerprinting:

The hydroalcoholic extract of roots was subjected to HPTLC analysis to identify the presence of phenolic and flavonoid compounds. Distinct chemoprofile of the extract was developed using HPTLC. The precoated TLC plate of silica gel 60 F254 (Merck) of 0.2 mm thickness was used as stationary phase. 5 µL of a sample of the hydroalcoholic extract was spotted in the form of a band using Linomat V (Camage, Switzerland). TLC pattern of the extract was developed using Toluene: Chloroform: Ethanol (4: 4: 1 v/v/v). In this analysis, plant extract was derivatized with NP reagents at 366 nm. Each determination was carried out in duplicate. In order to calibrate the method, stock solutions of the reference compound were prepared in methanol at different concentration levels¹⁷.

 

Total phenolic content:

Total phenolic content was estimated in extract using Folin-Ciocalteu reagents. Gallic acid was used as a standard. 100 μL of standard/extract solution was mixed with 125μL Folin-Ciocalteu reagent and 750 μL of sodium carbonate. The solution was adjusted to a final volume of 5 mL with deionized water. The absorbance was measured after incubation for 90 min at 760 nm with a UV-spectrophotometer. All determinations were carried out in triplicate. The concentration of phenolic compounds in extracts was determined from the gallic acid calibration curve. The total content of phenolic compounds in the extracts was expressed as gallic acid equivalents (GAE) mg/g of dried extract¹⁸.

 

Total Flavonoid content:

Aluminum chloride colorimetric method was used to estimate total flavonoid content in plant extract. Quercetin was used to prepare the calibration curve (y= 0.814x – 0.026, r² = 0.995). Sample 0.5 mL (0.1 mg/mL) was mixed with 0.1 mL of AlCl₃ (10%), 01mL of potassium acetate (1 M) and 1.5 mL of methanol (95%). The solution was adjusted with distilled water to a final volume of 5 mL and mixed thoroughly. The mixture was incubated in dark for 60 min at room temperature. The total flavonoid content of the extract was expressed as quercetin equivalents µg/g of dry extract with the absorbance was measured at 415 nm¹⁹.

 

ABTS Assay (Total Antioxidant capacity):

This assay was based on the ability of an antioxidant to interact with ABTS radicals (2, 2- azino-bis thylbenzthiazoline – 6-sulfonic acid). The assay was performed following the manual instruction (Sigma; Aldrich, MO, USA MAk 187) provided with the total antioxidant capacity assay. In brief, Cu⁺² reagent, assay diluents, protein mask, and Trolox standard, 1 μmol, 10 μL of sample, 90 μL of HPLC water were transferred to each well in a 96 well plate. The absorbance was measured at 570 nm and Trolox equivalents in terms of μM/mg of the sample were calculated from the standard curve of Trolox (y= 0.076x-0.75, r² =0.996).

 

Statistical analysis:

The data were expressed as mean±S.E.M. (n=6). Results were analyzed statistically by one –way ANOVA followed by students t test. The difference was considered significant if p<0.05.

 

RESULTS AND DISCUSSION:

Liver cell damage affected by many toxicants such as carbon tetrachloride, thioacetamide, and chronic alcohol intake and microbes is usual. Enhanced lipid per oxidation during metabolism of ethanol may result in the development of hepatitis leading to cirrhosis²⁰. There is a lack of reliable hepatoprotective drugs in modern medicines to prevent and treat drug-induced liver damage. Traditional medicine system is the combination of knowledge, skills, and practices based on the theories, beliefs, and experiences indigenous to different cultures that are used to maintain health along with prevention and diagnosis of ailments²¹. The use of natural antioxidants present in food and other biological materials have attracted considerable interest due to their presumed safety, nutritional and therapeutic value. Antioxidants derived from the fruits, vegetables, spices, and cereals are very effective and reduce interference with the physiology to use free radicals constructively²². The search for natural antioxidants for nutrition, beauty, and medical consumptions has to turn out to be a chief industrial and systematic research trial throughout in past two decades and hence, efforts to gain extensive knowledge regarding the power of antioxidants from plants and to tap their potential²³. In recent years, many studies have been shown that diets containing a high content of phytochemicals can provide protection against various diseases. Hence, an attempt has been made in this research to explore the antioxidant and hepatoprotective effect of Baliospermum montanum root extract.

 

Hepatitis induced by ethanol and CCl₄ shows many metabolic and morphological aberrations in the liver of experimental animals similar, to those observed in human viral hepatitis^24,25. All these toxicants produce liver injury by a multiple step mechanism. In particular, the peroxidation of endogenous lipids has been shown to be a major factor in the cytotoxicity of hepatocytes, while oxidative damage is generally attributed to the formation of highly reactive hydroxyl radical (OH), stimulator of lipid peroxidation and source for destruction and cell membrane damage^26,27,28. In the present study, rats treated with ethanol and CCl₄ toxicants developed severe hepatocellular injuries which were observed by significant (p<0.05) elevation of SGOT, SGPT, ALP, AST, ALT, and total bilirubin activities when compared with the normal control group. Animals treated with a hydroalcoholic extract of Baliospermum montanum root prevented the elevation of serum enzymes suggesting that their hepatoprotective activity might be due to its effect against cellular leakage and loss of functional integrity of the cell membrane^29,30 in a dose-dependent manner. The activity of extract at the dose of 500 mg/kg was comparable to the silymarin. Results showed that pre-treatment of rats with Baliospermum montanum extract effectively protected the animals against CCl₄ and ethanol-induced hepatic destruction, as evidenced by decreased serum SGOT, SGPT, AST, ALT, and ALP activities in dose-dependently (Table 1, figure 1, 2). The tendency of these enzymes to return to normality in the extract administered group is a clear manifestation of anti-hepatotoxic effects of the extract. The reduction in the levels of SGPT and SGOT toward the normal value indicated that the extract protected the structural integrity of hepatocytes cell membrane by stimulating the regeneration process of damaged cells.^31,32 Reduction in ALP levels implies that the extract restored the stability of the biliary function during injury with CCl₄ and ethanol³³.

 

Table 1 Effect of hydroalcoholic extract on biochemical parameters in ethanol induced hepatotoxicity in rats

Treatment	SGPT U/L	SGOT U/L	ALP IU/L	Total Bilirubin mg/dl
Group-I: Normal Saline (1mL/kg, p.o)	60.15±2.79	159.01±2.79	188.71±2.64	0.56±0.01
Group-II: ethanol (4gm/kg), 40% v/v	95.43±5.2*9	254.31±2.63*	243.18±11.45*	5.28±0.03*
Group-III: Silymarin (100 mg/kg p.o.) + Ethanol 4gm/kg (40% v/v) p.o.	64.8123.40*	169.81±2.78*	192.53±4.90*	0.63±0.05*
Group VI : ALE6 (250 mg/kg p.o.) + Ethanol 4gm/kg (40% v/v) p.o.	89.80±4.38	191.28±4.17	213.31±4.29	1.08±0.06
Group V : ALE6 (500 mg/kg p.o.) + Ethanol 4gm/kg (40% v/v) p.o.	67.23±5.36*	177.19±2.01*	195.29±2.17*	0.83±0.02*
Group VI: ALE6 (500 mg/kg)	69.19±2.48*	163.16±4.45*	193.35±3.11*	0.75±0.04*

ALE = Hydroalcoholic extract, * p<0.05

 

Figure 1 Effect of hydroalcoholic extract on biochemical parameters in CCl₄ induced hepatotoxicity in rats

 

ALE = Hydroalcoholic extract, * = p<0.05

 

Figure 2 Effect of hydroalcoholic extract on biochemical parameters in CCl₄ induced hepatotoxicity in rats

The treatment with CCl₄ and ethanol significantly increased the liver weight and volume compared to the normal group. Pretreatment with silymarin and hydroalcoholic extract of Baliospermum montanum significantly prevent the increase in liver weight and volume induced by CCl₄ and ethanol (Table 2).

 

Plant materials rich in phenolics are increasingly being used in the food industry because they retard oxidative degradation of lipids and improve the quality and nutrition value of food due to their hydroxyl groups confer scavenging ability³⁴. The observed hepatoprotective activity of the root extract may be due to the presence of phenolic compounds (Table 3) found in the previous preliminary phytochemical screening as these compounds are believed to have the ability for scavenging and stabilizing lipid oxidation^{35, 36} and some studies suggested a correlation between phenolic content, antioxidant and hepatoprotective activities³⁷

 

Table 2 Effect of hydroalcoholic extract on total lever weight and volume

Treatment	Mean liver weight (µ/100 g)	Mean liver volume (ml/100 g)
Group-I : Normal Saline (1mL/kg, p.o)	4.11 ± 0.11	5.94 ± 0.14
Group-II: Ethanol 4 gm/kg, p.o.)	8.07 ± 0.32	8.79 ± 1.15
Group-III: Silymarin (100 mg/kg p.o.) + ethanol, 4 gm/kg, p.o.)	4. 89 ± 0.42*	6.01 ± 0.77*
Group VI: ALE6 (250 mg/kg + ethanol, 4 gm/kg, p.o.)	6.59 ± 0.10	7.11 ± 0.14
Group V: ALE6 (500 mg/kg + ethanol, 4 gm/kg, p.o.)	5.15 ±0.11*	6.63 ± 0.09*
Group VI : ALE6 (500 mg/kg)	5.87 ± 0.91*	6.71 ± 0.03*

ALE = Hydroalcoholic extrct, * = p<0.05

Table 3 Total phenolic, total flavonoid content and total antioxidant capacity of hydroalcoholic extract

Sample	Total phenolic content [GAE (µg/gm)]	Total flavonoid content [quercetin equivalent (µg/gm)]	Total antioxidant capacity [nmol/µg of extract Trolox equivalent
Hydroalcoholic extract	2845	128.9	16.54

 

Heavy alcohol produces free radicals and accumulation of reactive oxygen species, which cause lipid peroxidation and depletes antioxidant enzymes, creating an imbalance between oxygen radicals and antioxidants³⁸. The antioxidant plant component, phenolic compounds are widely investigated in several medicinal plants. The amount of total phenolic compounds and total flavonoids in the hydroalcoholic extract of the root are shown in Table 3 and expressed 2845µg/g of GAE, and flavonoid 128.9 µg/g of quercetin equivalent. In HPTLC study, hydroalcoholic extract showed the presence of phenolic and flavonoids in HPTLC finger print analysis when derivatized with NP reagent. The phenolic and flavonoids were detected using external standards, quercetin, and gallic acid (Figure 3 A, B, and 4).

 

A                                                                                                   B

Figure 3 (A) Gallic acid standard curve (B) Quercetin standard curve

 

Figure 4 HPTLC analysis of phenolic compound using NP reagent at 366 nm

ABTS is an excellent tool to determine antioxidant activity. The decolorization of ABTS radical reflects the capacity of an antioxidant species to donate electrons or hydrogen atoms to inactivate the radical species³⁹. In the present study, the sample showed a decrease in the absorbance with moderate scavenging activity. The percentage of inhibition of ABTS+ radical is plotted as a function of concentration. The extract exhibited 16.54 nmol/µg of the extract-Trolox equivalent of antioxidant activity (Table 3). Oxidative stress in mammal results from the imbalance between the generation of free radicals and the rate of their suppression by an antioxidant.³⁵ Hepatotoxicity may result in an effect of excessive free radical formation due to exogenous chemicals or metabolic reactions³⁶. In living organisms, reactive oxygen species (ROS) and reactive nitrogen species (RNS) cause deleterious cytotoxic effects to mammalian cells. These free radicals include various forms of activated oxygen and nitrogen such as superoxide anion (O₂), hydroxyl (OH), nitric oxide radicals (NO) and non free radical species such as hydrogen peroxide (H₂O₂), nitrous acid (HNO2) which leads to the generation of free radicals. These free radicals are continuously formed inside the human body as a result of exposure to exogenous chemicals in the environment or due to various endogenous metabolic reactions involving bioenergetic electron transfer and redox enzymes¹³. Antioxidants have been reported to scavenge free radicals by interfering with the oxidation process and chelating metal ions. Thus, oxidative stress is prevented by the action of antioxidants. In the present study, hydroalcoholic extract showed a significant reduction in free radicals suggesting potent free radical scavenging activity and antioxidant property.

 

CONCLUSION:

Results of the present study indicated that the hydroalcoholic extract of root of Baliospermum montanum exhibited a potential hepatoprotective activity against carbon tetrachloride and ethanol-induced hepatotoxicity and validate the traditional use of this plant in hepatocellular injury. Further, the isolation of active principles will be advantageous to produce novel bioactive constituents from this extract, which may possess more significance in the treatment of liver diseases, and to elucidate its exact mechanism of action. Attempts are being made to isolate and characterize the active principle to which the hepatoprotective activity can be attributed.

 

ACKNOWLEDGMENT:

Authors are thankful to Science Engineering and Research Board, New Delhi for financial support (SB/EMEQ-515/2014, SERB, New Delhi) is acknowledged. Also, thank full to home University for providing necessary infrastructure support for carrying out the research work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 14.10.2018          Modified on 19.11.2018

Accepted on 15.03.2019        © RJPT All right reserved