Hepatoprotective activity of Chloroform and Ethyl acetate extract of Dipteracanthus patulus against Paracetamol induced Hepatotoxicity in rats through Antioxidant mechanism

 

Yuvaraja K R¹, Santhiagu A^2*, Jasemine S³, Gopalasathees Kumar K⁴

¹Assistant Professor, Department of Pharmaceutical Chemistry, KMCH College of Pharmacy, Coimbatore, Tamil Nadu, India (Affiliated to The Tamil Nadu Dr. M.G.R. Medical University).

²Bioprocess Lab, School of Biotechnology, National Institute of Technology, Calicut, Kerala, India.

³Director, One Beat College of Medical Sciences, Bhira, Uttar Pradesh, India.

⁴Department of Pharmacy, Annamalai University, Chidambaram,Tamil Nadu, India.

ABSTRACT:

Dipteracanthus patulus (DP) is an important medicinal plant and popularly known as black weed. Dipteracanthus patulus belongs to Acanthaceace family and very important indigenous medicinal plant. The earlier study reported that hepatoprotective activity leaves of DP, hence there is no report on hepatoprotective activity of whole plant of DP and the hepatoprotective phytoconsituents like lupeol, β-carotene, β-sitosterol and rutin are already reported in this plant material. Therefore this current research is aimed to evaluate the hepatoprotective effect of whole plant of Dipteracanthus patulus. The paracetamol induced rat model was used to evaluate the hepatoprotective activity and Silymarin was used for standard. The treatment with the ethyl acetate and chloroform extracts of Dipteracanthus patulus to the paracetamol induced liver toxic rats shows the significant reduction of SGOT, SGPT, ALP, total cholesterol, triglycerides, urea and uric acid levels. However the CAT, GSH, GPx and SOD levels were significant increase in ethyl acetate and chloroform extracts of Dipteracanthus patulus treated paracetamol induced liver toxic rats. Results of current research concluded that the hepatoprotective activity of Dipteracanthus patulus through antioxidant mechanism.

 

 

INTRODUCTION:

Dipteracanthus patulus (DP) is an important medicinal plant and popularly known as black weed. Dipteracanthus patulus belongs to Acanthaceace family and very important indigenous medicinal plant, which present in important indigenous medicinal plant, which present in remedy for ear disease and believed to be anti-cancer against the nasopharynx region, slightly hypoglycemic, anti-inflammatory and anti-microbial and also the leaves are eaten as vegetable¹. Dipteracanthus patulus (Jacq.) leaves are used for treating itches, insect bites, paranychia, venereal diseases, sores, tumours and rheumatic complaints².

 

 

The whole plant of DP is shown in Figure 1. In Tamil it is known as Kiranthinayagamor Kayappacchililai and commonly distributed on wasteland in Tamil Nadu. It grows in tropical Africa, Arabia, south west India, Srilanka and Pakistan. Its flowering season is September to march. In folklore medicine, it has been documented that DP is used for curing the eyesore by introducing the extract into eyelid^8,9. In addition, the leaves of various species of Dipteracanthus were used in various infectious diseases Phytochemical analysis of DP showed the presence of lyoniresinol-9’-O-β-D-glucoside, 5,5-dimethoxy-lariciresinol-9-O-β glucopyranoside, β-sitosterol, lupeol, α-ethyl galactose, apiginin-7-O-rutinoside, α-D-glucose, β-D-glucose and β-D-fructose Other phytochemical studies report the presence of ascorbic acid, phenolic compounds, tannin, lycopene, carotenoid and α-tocopherol in DP From our laboratory, we have quantitatively analysed compounds, β- carotene and β-sitosterol^3,4. The earlier study reported that hepatoprotective activity leaves of DP, hence there is no report on hepatoprotective activity of whole plant of DP and the hepatoprotective phytoconsituents like lupeol, β-carotene, β-sitosterol and rutin are already reported in this plant material^5,6. Therefore this current research is aimed to evaluate the hepatoprotective effect of whole plant of DP.

 

 

 

Figure 1: Morphology of Dipteracanthus patulus

 

 

 

MATERIALS AND METHODS:

Materials:

Paracetamol (PCM) was purchased from Sigma Aldrich, USA. Silymarin (SLY) was obtained from Sigma–Aldirch, USA. All other reagents and chemicals were used under analytical grade.

 

Preparation of plant material:

The whole plant of Dipteracanthus patulus was collected locally Sattankulam at Thuthukudi District in Tamil Nadu state in India. The collected herbal plant was authenticated by Dr. V. Chelladurai, Survey of Medicinal and Aromatic plants Unit Siddha, CCRAS (Central Council for Research in Ayurvedic Sciences), Palayamkottai, Tamil Nadu, India, and voucher specimen was deposited in the Deportment of school of Biotechnology, NITC, Calicut, Kerala for future reference. After removing of the earthy matters, the plant material was washed and shade dried and powdered using a micro pulverizer and the powder passed through sieve no. 40#¹¹.

 

Extraction of Plant material:

Powdered plant material was subjected to continuous hot extraction method by using soxhlet apparatus¹⁰. Increasing orders of polarity solvents (petroleum ether, chloroform, ethyl acetate and methanol) were used. 1 kg of finely powdered plant material was extracted with 5 L of petroleum ether in a soxhlet apparatus for 72 h, after the successive extraction, the obtained marc was further extracted with solvents of increasing order of polarity (Chloroform, Ethyl acetate and Methanol)¹². After extraction the extracts were separately concentrated under vacuum using rotary vacuum evaporator at 40 C until get viscous solid mass. The obtained crude extracts were weighed and stored at 4 C for the further analysis¹³.

 

Experimental animals:

Male albino Wistar rats (150–200g) used in the present study were procured from Sri Venkateshwara Enterprises, Bangalore, India. All the animals were kept under standard laboratory conditions (temperature 25±2 ⁰C and 12 h light/12 h dark cycle) and acclimatized for 1 week before commencement of the experiment. They were allowed to have free access to standard pellet food (National Institute of Nutrition, Hyderabad India) and water ad libitum.⁷ The protocol used in the present study was approved by Institutional Ethical Committee of KMCH College of Pharmacyin accordance with the guidelines for the care and use of laboratory animals set by CPCSEA. Institutional Animal Ethics Committee approval No: KMCRET/PhD/01/2013-14.

 

Acute oral toxicity study:

The OECD-423 guidelines was used for perform acute oral toxicity study for fixing the dose for in vivo study. The rats were fasted overnight with free excess of water and were grouped into four groups consisting of 3 animals each, to which the extract was administered orally at the dose level of 5mg/kg, 50mg/kg, 300mg/kg and 2000mg/kg body weight. They were observed for mortality; toxic symptoms such as behavioral changes, locomotor activity, convulsions; direct observation parameters such as tremor, convulsion, salivation, diarrhoea, sleep, coma, changes in skin and fur, eyes and mucous membrane, respiratory, circulatory, autonomic and CNS, somatomotor activity etc. periodically for 30 min during first 24 h and specific attention given during first 4 h daily for a total period of 14 days.

 

In vivo hepatoprotective activity:

Based on the preliminary study conducted by our lap, CEDP (Chloroform Extract of Dipteracanthus patulus) and EEDP (Ethyl acetate Extract of Dipteracanthus patulus) were selected for in vivo evaluation of hepatoprotective activity. Male wistar albino rats were divided in to seven groups of 6 animals each and received orally the following treatment for seven days. Group I (normal control) received 1% sodium carboxymethyl cellulose (1ml/kg); Group II (PCM control) was given PCM (1g/kg). Group III received both SLY (100mg/kg) and PCM (1g/kg). Group IV and V were administrated with 200 and 400 mg/kg of CED respectively, Group VI and VII were administrated with 200 and 400 mg/kg of EEDPrespectively¹⁴.

 

Table 1: Grouping of animals for hepatoprotective activity

GROUPS	TREATMENT
Group I	Normal control
Group II	Paracetamol 1g/kg
Group III	Paracetamol 1g/kg +Silymarin 50mg/kg
Group IV	Paracetamol 1g/kg + CEDP200mg/kg
Group V	Paracetamol 1g/kg + CEDP400mg/kg
Group VI	Paracetamol 1g/kg + EEDP200mg/kg
Group VII	Paracetamol 1g/kg +EEDP400mg/kg

 

All the treatments were done orally by means of a gastric tube and continued for 7 days. On eighth day of the experiment all the animals were anesthetized and blood samples were collected from retro-orbital plexus. The blood samples were allowed to clot for 1 h at room temperature and the serum was separated by centrifugation at 2500rpm at 4^oC for 15 min. Then the rats were sacrificed and the liver was excised, washed and perfused with ice-cold normal saline. The perfused liver samples were then homogenized in ice bath using homogenizer in chilled 10mM Tris–HCl (pH 7.4) to yield 10% (w/v) liver homogenate for the estimation of biochemical parameters. A small portion of the liver tissue was fixed in formalin for histopathological examination^15-17.

 

Estimation of Serum biochemical parameters:

The estimation of Serum Glutamic Pyruvate Oxaloacetate (SGOT), Serum Glutamic Pyruvate Transaminase (SGPT), Serum Alkaline Phosphatase (ALP), serum urea, uric acid, creatinine, serum total cholesterol, total triglyceride, high-density lipoproteins (HDL)- cholesterol level and low-density lipoproteins (LDL) cholesterol level were done by using standard Enzymatic (Span Diagnostics, India) [18-21].

 

Estimation of in vivo antioxidant markers:

The tissue were weighed and 10% tissue homogenate was prepared with 0.025m Tris-HCl buffer, P^H 7.5. After centrifugation at 10,000×g for 10 min, the clear supernatant was used to measure thiobarbituric acid reactive substances (TBARS). For the estimation of non-enzymatic and enzymatic antioxidants, tissue was minced and homogenized (10% w/v) in 0.1 M phosphate buffer (P^H 7.0) and centrifuged for 10 min, the resulting supernatant was used for enzyme assays²². After treatment with plant extracts and standard, the rats were sacrificed, liver were isolated and washed with normal saline and stored for 12 h for in vivo antioxidant studies. The separated liver was homogenized with motor driven Teflon coated homogenizer with 0.1 M Tris-HCl buffer (pH 7.4) to get 10% homogenate. Catalase (CAT)²³, reduced glutathione (GSH)²⁴, Superoxide dismutase (SOD), Glutathione peroxidase (GPx) and lipid peroxidation (LPO)²⁵ levels were estimated using standard methods.

 

Histopathological studies:

At the end of the study the livers were dissected out, excised and rinsed in an ice cold saline solution. A small portion of liver was fixed in 10% neutral formalin, dehydrated in alcohol and then implanted in paraffin. Microtome sections of 4–5μm thickness were made by using a rotary microtome. The sections were stained with haematoxylin–eosin (H&E) dye to observe histopathological changes²⁶.

 

Statistical analysis:

All the results were expressed as Mean±SEM. The statistical analysis was carried out by one-way ANOVA followed by Dunnett’s multiple comparison tests using Graph pad Prism-5 software. P < 0.05 was considered as significant.

 

RESULTS AND DISCUSSION:

Acute oral toxicity study:

The results of acute oral toxicity study were shown in Table 1. From the acute toxicity studies of ethyl acetate and chloroform extracts of Dipteracanthus patulus was observed that there was no mortality up to the dose of 2000 mg/kg. Hence the dose fixed for study as (1/10^th) 200 mg/kg as low dose and (1/5^th) 400 mg/kg as high dose for CEDP and EEDP.

 

Serum biochemical parameters:

The high dose administration of paracetamol caused the necrosis of liver thereby increases the activities of SGPT, SGOT and ALP in blood²⁷. The SGPT, SGOT and ALP levels of various treatment groups were shown in Table 2.

 

Table 2: Acute toxicity study of ethyl acetate and chloroform extracts of Dipteracanthus patulus

 

Table 3: Effect of CEDP and EEDP on serum biochemical parameters of paracetamol induced hepatotoxicity rats

Indexes	Control	PCM 1gm	PCM+SILY 50mg	PCM+CEDP 200mg	PCM+ CEDP 400mg	PCM+EEDP 200mg	PCM+EEDP 400mg
SGPT(U/L)	66.16±7.98	398±3.07###	261±3.47***	255±2.25**	349±1.98***	350±3.08***	356.6±3.34***
SGOT(U/L)	99±2.09	153±5.08###	125±3.96***	137±3.32**	128±2.03**	146.5±1.96***	135.1±2.65***
ALP(U/L)	170.51±11.6	318.61±3.95#	250±1.68***	282±3.04**	219±1.69**	283±2.47***	228.8±3.09**
HDL (mg/dl)	53 ±3.90	28 ±1.28##	47±2.97***	35.15±4.05***	37±1.28***	35±3.03***	36±2.64***
TG (mg/dl)	103 ±2.08	379 ±3.81##	203±3.95***	196.1±1.48***	185 ±3.33***	243.7±1.89***	201.7±2.05***
TC (mg/dl)	80 ±2.03	347±1.22##	113±1.98***	151±5.07***	142±2.99***	180±3.66***	156.6±2.33***
TB (mg/dl)	0.8 ±0.01	2.8 ±0.01##	1.3±0.02***	1.48±0.03***	1.37±0.01***	1.58±0.01***	1.42±0.02**

 

The Serum SGOT, SGPT and ALP levels were increased significantly (p<0.001) in PCM induced rats compared to normal controlled rats which confirms the toxicity of liver. The standard SLY, CEDP and EEDP treated groups significantly (p<0.05) reduced these level compared to PCM induced control group. The liver is the major organ for metabolism of lipids the toxicity of liver may causes the elevation of lipid levels in serum²⁸. The PCM induced rats showed significant increase levels of triglyceride and total cholesterol in blood. The standard SLY, CEDP and EEDP treated groups significantly (p<0.05) reduced these level compared to PCM induced control group. Serum bilirubin level is the key parameter to find the normal liver function²⁹. The PCM induced group shows the significant increase of serum bilirubin level, however treatment with SLY, CEDP and EEDP groups were decreased towards normal when compared with PCM control animals.

 

In vivo antioxidant markers:

Antioxidant enzymes form the first line of defense against ROS (Reactive Oxygen Species) in the cells include GSH, SOD, GPx and CAT which play a key role in scavenging the toxic intermediate of incomplete oxidation³⁰. A reduction in the activities of these antioxidant enzymes can result in lead to initiation and propagation of LPO. The CAT, SOD, GSH and GPx values of extracts and standard treated groups were shown in Table 3. The PCM induced group shows decreased level of CAT, GSH, SOD and GPx which indicates that the oxidative stress induced by PCM. CEDP and EEDP treatment with PCM toxic rats, significantly increased antioxidant CAT, GSH, SOD and GPx levels, it confirms the CEDP and EEDP can reduces oxidative stress during liver toxicity.

 

The results of the histopathological study were shown in Figure 2. Group I Normal control, Liver shows normal lobular architecture, Hepatocytes show no significant pathology, Group II PCM induced control Hepatocytes show cytoplasmic vacuolation. Periportal inflammation, Central vein shows congestion destruction, Group III SLY 50 mg/kg treated PCM control liver shows normal portal tracts, the hepatocytes in the hepatic parenchyma are normal, Group IV CEDP 200mg/kg treated PCM control shows there are epitheloid cell granulomas in the hepatic parenchyma. The portal tracts are normal; Group V CEDP 400 mg/kg treated PCM control liver shows mildhydropic changes. The portal tracts are normal. There is no inflammation or necrosis seen, Group VI EEDP 200 mg/kg treated PCM control- The sinusoids are mildly dilated. The portal tracts are normal. There is no a hydropic change or necrosis and Group VII EEDP 400 mg/kg treated PCM control shows the sinusoids is dilated. The hepatocytes are unremarkable.

 

Table 4: Effect of CEDP and EEDP on in vivo antioxidant markers in liver homogenate of paracetamol induced hepatotoxicity rats.

Treatment Group	Dose /kg	CAT (H2O2 consumed/ min/mg of protein)	SOD (unit/min/mg protein)	GSH (unit/min/mg of protein)	GPx (GSH consumed/ min/mg protein)	LPO (MDA per mg of protein)
Control	1 cmc	276.12±7.16	90.41±3.65	41.67±1.29	115.07±1.16	4.28±0.81
Toxic control (PCM)	1g	91.71±15.15###	37.32±2.11###	19.86±2.13###	41.67±2.19###	85.19±0.91###
Standard (SLY) + PCM	50 mg+1g	219.61±6.76***	69.13±3.15***	36.18±1.76**	91.73±3.12***	10.16±1.21***
EEDP+ PCM	200mg+1g	130.45±0.14**	40.25±0.14**	23.42±0.23***	52.42±0.54**	26.25±0.41**
EEDP+ PCM	400mg+1 g	224.56±1.23**	60.25±0.13**	27.42±0.14***	86.21±2.25**	17.25±1.25**
CEDP+ PCM	200mg+1g	148.24±0.42**	43.25±2.23***	30.25±0.12***	70.45±0.64***	18.04±0.14***
CEDP+ PCM	400mg+1 g	251.02±0.48***	64.25±0.45**	32.14±4.16***	92.41±0.24***	12.04±1.45*

 

Histopathological study:

Figure 2: Histopathology

 

CONCLUSION:

Results of the current study revealed that the plant extracts of Dipteracanthus patulus has significant hepatoprotective activity through antioxidant mechanism against paracetamol induced liver toxic rats.

 

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Received on 14.06.2019         Modified on 05.07.2019

Accepted on 10.08.2019         © RJPT All right reserved