EVALUATION OF HEPATOPROTECTIVE POTENTIAL OF ALSTONIA SCHOLARIS STEM BARK AGAINST CCL4 INDUCED HEPATOTOXICITY – AN IN-VIVO SCREENING

EVALUATION OF HEPATOPROTECTIVE POTENTIAL OF ALSTONIA SCHOLARIS STEM BARK AGAINST CCL4 INDUCED HEPATOTOXICITY – AN IN-VIVO SCREENING

Jaspreet Kaur Sodhi *, Birendra Shrivastava and Hardarshn Singh Lamba

R. Institute of Pharmacy, Ghaziabad, Uttar Pradesh, India.

ABSTRACT: The hepatoprotective potential of ethanolic extract of stem bark of Alstonia scholaris belonging to the family Apocynaceae and commonly known as Devil’s tree or Saptaparni was assessed against carbon tetrachloride-induced hepatotoxicity in Wistar rats. It is an evergreen tropical tree native to the Indian sub-continent and Southeast Asia and contains various phytoconstituents like alkaloids, triterpenoids, flavonoids, steroids, and phenolic acids which have shown promising therapeutic potential. The hepatoprotective effect was assessed by evaluating the biochemical parameters like serum alanine transaminase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) and total bilirubin and histopathological studies of the liver. Rats treated with ethanolic extracts of Alstonia scholaris in the dose of 150 mg/kg and 300 mg/kg, evinced a considerable reduction in CCl₄ induced augmented serum enzyme levels (ALT, AST, ALP, and total bilirubin) and also a considerable increase in total protein levels that was lowered by hepatotoxic compound used, comparable with Silymarin (standard drug). Alstonia scholaris was also able to significantly prevent the rise in MDA level as evident by the TBARS test for in-vivo lipid peroxidation. Histopathology of the extract-treated groups showed a lessening of the pathogenesis and revealed a marked reduction in hepatic injuries that was equivalent to the Silymarin-treated group.

Keywords: Alstonia scholaris, Ethanolic extract, Carbon tetrachloride, Hepatoprotective activity

INTRODUCTION: Liver is the largest internal organ and also the largest gland of the body that is found in the upper right-hand part of the abdominal sac, below the diaphragm and on top of the stomach, right kidney and the intestines. Weighing about three pounds, it has a cone-like structure that is soft and pinkish-brown in colour ¹.

It consists of two main lobes- the left and the right lobes, consisting of 8-sections containing thousands of lobules. These lobules join with tiny ducts that further attach with bigger ducts forming a common hepatic duct that transports the bile juices made by the liver cells to the gallbladder and the duodenum through a common bile duct. Almost 13% of the body’s blood flows through the liver at any period of time ².

“Hepatocytes- the regenerative cells” form the main tissue cells of the liver that constitute around 70-80% of the cytoplasmic mass. They are polygonal cells having a single round nucleus and a distinguished nucleolus ³.

The liver performs dual roles as a metabolic and biochemical transformation factory. The portal vein receives blood containing substances, and via the hepatic artery, oxygen-rich blood is received ⁴. Thus a liver is classified as a “bidirectional biofilter” ⁵ which performs a vast variety of functions.

As the liver functions’ in eliminating substances via the portal circulation, thus it becomes prone to relentless assault by external compounds, leading to hepatic disorders ⁶. Liver disorders stay one of the significant dangers to general wellbeing and thus pose serious health issues worldwide ⁷.

Under experimental conditions, hepatic injury can be caused either by partial hepatectomy or by various hepatotoxins like carbon-tetrachloride (CCl₄), thioacetamide, D-galactosamine, chloroform, paracetamol, arsenic, ethyl alcohol and pyridine ⁸.

Among many natural products, plants serve as a major chemical substance source by acting either directly as drugs or as a key component in synthetic drug formulations. For a successful examination, the right choice of plant species plays an important role. Though arbitrary choice gives a few hints, directed assortment dependent on chemotaxonomic connections and ethnomedical data inferred from traditional medication are bound to yield pharmacologically active constituents ^{9, 10}. The use of herbal drugs in treating liver diseases has long folklore, but proof of its efficacy is inadequate. Additionally, there are worries about the nature of studies testing herbal medicines. Notwithstanding these constraints, various herbal drugs show promising impacts, tentatively in cell culture, animal studies, or clinical preliminaries. Hepatoprotective plants contain chemical components like phenols, coumarins, monoterpenes, glycosides, alkaloids, and xanthenes ¹¹.

One such plant is Alstonia scholaris (family Apocynaceae), popularly known as “Devil’s tree” or “Saptaparni” and is attaining the attention of researcher’s for its pharmacological activities. Various phytoconstituents have been reported in different parts of the plant, such as bark ¹², leaves ¹³, roots ¹⁴, flowers and fruits ¹⁶ such as alkaloids, iridoids, coumarins, flavonoids, leucoanthocyanins, reducing sugars, simple phenolics, steroids, saponins and tannins ¹⁷. It has been reported to possess antimalarial ¹⁸, antimicrobial ¹⁹, free radical scavenging and antioxidant ²⁰, anti-diabetic ²¹, analgesic and anti-inflammatory ²², anticancer and cytotoxicity ^{23, 24}, radioprotective  ²⁵, CNS activity ²⁶, immunostimulating ²⁷, antifertility ²⁸, antidiarrheal ²⁹, bronchodilatory ³⁰, anti-tussive and anti-asthmatic ³¹ activities. Keeping in view the biologically active nature of A. scholaris, the current study aims to assess the hepatoprotective potential of stem bark of A. scholaris against carbon tetrachloride-induced hepatic injury.

MATERIALS AND METHODS:

Chemicals: All the chemicals and solvents utilized in the study were of analytical grade and procured from the local market of Ghaziabad. The assay kits utilized for the estimation of biochemical parameters like ALP, AST, ALT, total bilirubin, total protein, etc., were acquired from ERBA (Himachal Pradesh, India).

Plant Collection and Authentication: The mature stem bark from the woody trunk portion of Alstonia scholaris was procured from the local area of Ghaziabad, Uttar Pradesh, India. It was authenticated by an emeritus scientist, Dr. Sunita Garg of Raw Material Herbarium and Museum (RHMD), Delhi (taxonomic reference number: NISCAIR/RHMD-3653-54).

Preparation of the Extract: Shade dried stem bark was grounded into fine powder with the help of an electric grinder and was subjected to continuous extraction with hot ethanol using Soxhlet apparatus ^{32, 33}. The extract so obtained was filtered, and the filtrate was made solvent-free using a rotary vacuum evaporator and was subjected to preliminary phytochemical analysis ³⁴.

In-vitro Antioxidant Activity: The free radical scavenging activity of ethanolic extract of Alstonia scholaris (EEAS) stem bark was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) against standard ascorbic acid, utilizing the standard method ³⁵. Concisely, the reaction mixture contained various concentrations of the ethanolic extract and DPPH in ethanol. The mixture was shaken, left for 25-30 min at room temperature in the dark, and the reduction of DPPH radical was determined by reading the decrease in absorbance at 517nm using a UV-visible spectrophotometer against the corresponding blank (ethanol). Ascorbic acid was used as a reference compound. All determinations were performed in triplicate ^{36, 37}. The results revealed that EEAS showed the highest antioxidant activity of 80.3% as compared to the standard ascorbic acid. Thus EEAS has proton donating ability and could serve as a free radical inhibitor or scavenger and act as a natural antioxidant.

In-vivo Activity: Wistar Rats (either sex), weighing between 300-350 g, were procured from the animal house of Meerut Institute of Engineering And Technology, Meerut, Uttar Pradesh, India. The animals were quarantined and housed in an Animal House Facility for acclimatization for seven days prior to experimentation. Animals were housed in polypropylene cages with dust-free rice husk as a bedding material and maintained under standard laboratory conditions with controlled temperature (23 ± 2°C), humidity (40 ± 10%), and a natural (12 hour each) light-dark cycle. They were fed with a standard rodent pellet diet and water ad libitum. The care of laboratory animals was done as per the guidelines of CPCSEA, Ministry of Forests & Environment, Government of India. The research protocol of this study was approved by the IAEC of Meerut Institute of Engineering And Technology (registration no. IAEC/MIET/2021/27).

Experimental Design:

Acute Toxicological Studies: Wistar rats (n=3) were selected by random sampling technique, and OECD-423 guidelines ³⁸ were followed to study the acute oral toxicity. The ethanolic extract was administered orally, starting with 5 mg/kg body weight up to 2 g/kg b.w. Initially, for the first hour after administering the dose, individual animals were monitored at least once and then regularly throughout the first 24 h, with specific care being given during the first 4 h, every day after that for a total of 14 days.

Hepatoprotective Activity against Carbon Tetrachloride Induced Hepatotoxicity: Hepatotoxicity was induced in rats by intraperitoneal administration of carbon tetrachloride (CCl₄), and Silymarin was used as a reference standard. To study the activity, animals were randomly divided into five groups (n=6 animals in each group). The first group served as normal control and received normal saline only (10 mL/kg of b.w.). The second group served as a disease control group and received CCl₄ dissolved in olive oil (3 mL/kg of b.w.) i.p., twice a week for 4 weeks. The third group was pre-administered with CCl₄ as in group 2 followed by Silymarin (100 mg/kg p.o.) daily for 4 weeks. Groups 4 and 5 received an ethanolic extract of stem bark of Alstonia scholaris in the dose of 150 mg/kg and 300 mg/kg, respectively, daily and 30 min before the administration of carbon tetrachloride, on the days of CCl₄ administration (as in group 2), for 4 weeks. After twenty-four hours of the last dose, the animals were euthanized and liver and blood samples were collected for the biochemical estimations and histopathological evaluations.

Assessment of Liver Function: The biochemical investigation for assessing liver functions was done where estimation of serum enzymes like ALT, AST ³⁹ and ALP ⁴⁰, total bilirubin, total proteins ^{41, 42}, serum albumin ⁴³ and TBARS ^{44, 45} was carried out to study the effect of ethanolic extracts on liver toxicity induced by CCl₄.

Histopathological Analysis: The liver tissues preserved in 10% formalin were dehydrated in graded concentrations of ethanol, immersed in xylene, and then embedded in paraffin. The sections of 4 µm thickness were cut and stained with haematoxylin and eosin ⁴⁶. The slides were observed for gross histopathological changes and neutrophil accumulation.

Statistical Analysis: All obtained values were expressed as mean ± standard error mean (SEM). The significance of the difference was statistically analyzed using a one-way analysis of variance (ANOVA) and P˂0.05 was considered statistically significant. The statistical analysis of the data was performed using GraphPad Prism 5 software (GraphPad Inc., La Jolla CA).

RESULTS AND DISCUSSION: Numerous herbs are said to offer alleviation of hepatic diseases in the Indian medical system. The stated therapeutic reputation of such herbs must be scientifically confirmed. Thus in the current research work, the ethanolic extract of Alstonia scholaris stem bark was investigated for its hepatoprotective study. The percentage yield obtained from the ethanolic extract was found to be 13.9 %, and the preliminary phytochemical evaluation revealed the presence of plant secondary metabolites such as alkaloids, flavonoids, saponins, and carbohydrates, terpenoids and tannins in the ethanolic extract of Alstonia scholaris stem bark Table 1.

TABLE 1: PRELIMINARY PHYTOCHEMICAL SCREENING OF ETHANOLIC EXTRACT OF ALSTONIA SCHOLARIS STEMBARK

+ = Presence of active constituents - = Absence of active constituents.

As the ethanolic extract showed the presence of maximum number of phytoconstituents, thus it was evaluated for hepatoprotective potential. The ethanolic extract of stem bark of Alstonia scholaris was found to be nontoxic up to a dose of 2 g/kg with no signs of mortality in test animals. A best known example of a zone-3 hepatotoxin is carbon tetrachloride which causes necrosis and liver failure. Its metabolism produces a hazardous trichloromethyl radical, which causes injury.

In the present work, it was perceived that the rats treated with carbon tetrachloride developed significant hepatic damage as seen by augmented serum levels of hepato-specific enzymes like ALT (SGPT), AST (SGOT), ALP and total bilirubin and lowered serum total protein levels when compared to normal control. Pre-treatment with Silymarin and EEAS showed good protection against carbon tetrachloride-induced liver toxicity. Tests indicate a significant reduction in augmented serum enzyme levels and a considerable increase in total protein levels with extract-treated animals, evident from Table 2.

In-vivo lipid peroxidation study (TBARS) revealed that the CCl₄ treated group showed a significant increase in malondialdehyde (MDA) level when compared with the normal control group. EEAS was able to significantly prevent this rise in MDA level, as evident from Table 2.

TABLE 2: EFFECT OF ETHANOLIC EXTRACTS OF STEM BARK OF ALSTONIA SCHOLARIS (EEAS) ON ALT, AST, ALP, TOTAL BILIRUBIN, TOTAL PROTEINS, SERUM ALBUMIN, AND LPO LEVELS IN CARBON TETRACHLORIDE-INDUCED HEPATOTOXICITY IN RATS

Values are mean ± SEM (n=6) one-way ANOVA. Where, * represents significant at p<0.05, ** represents highly significant at p< 0.01, *** represents very significant at p<0.001. All values are compared with the toxicant.

Histopathological Studies of the Liver in Carbon Tetrachloride Induced Hepatotoxicity: The histopathological evaluation of carbon tetrachloride toxicity in all the groups was examined and shown in Fig. 1. The rat liver section treated with the normal control group shows liver parenchyma with intact architecture, which is the usual morphology, whereas the architecture of the liver in the disease control group is partially effaced; certain hepatocytes show apoptotic changes, perivenular mononuclear inflammatory infiltration, and scattered inflammatory infiltration within the parenchyma as signs of toxicity. In contrast, the liver section in the Silymarin treated group shows liver parenchyma with intact architecture though some of the central veins show congestion with diffuse sinusoids. Also, the liver sections taken from EEAS treated groups (150 and 300 mg/kg) show lessening of the pathogenesis and revealed a marked reduction in hepatic injuries that is equivalent to Silymarin treated group. Pursuant to these findings, flavonoids and saponins found in EEAS may be responsible for the hepatoprotective action. It is clear from the findings of this study that compared to Silymarin, the ethanolic extract of Alstonia scholaris stem bark also offers a pharmacologically fruitful treatment for a variety of liver ailments, indicating an improvement in the liver's functional status, which was also supported by histopathological findings.

FIG. 1: HISTOPATHOLOGY OF THE RAT LIVER IN CARBON TETRACHLORIDE-INDUCED HEPATOTOXICITY

CONCLUSION: The studies above reveal that treatment with ethanolic extract of A. scholaris stem bark was efficacious in offering protection against hepatotoxicity caused by carbon tetrachloride. The hepatoprotective potential of 300 mg/kg was substantially greater compared to 150 mg/kg. Histological observations corroborated the extract's therapeutic properties.

ACKNOWLEDGEMENTS: The authors would like to thank Dr. Vipin Kumar Garg, Professor and Head of Pharmaceutical Technology at Meerut Institute of Engineering and Technology (MIET), for providing facilities to carry out the pharmacological activity.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

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    How to cite this article:

    Sodhi JK, Shrivastava B and Lamba HS: Evaluation of hepatoprotective potential of Alstonia scholaris stem bark against CCl4 induced hepatotoxicity – an in-vivo screening. Int J Pharm Sci & Res 2022; 13(11): 4459-65. doi: 10.13040/IJPSR.0975-8232.13(11).4459-65.

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