Evaluation of Protective Effect of Alstonia scholaris Leaf Extract against Lead Induced Hepatotoxicity

INTRODUCTION

Lead is one of the earliest heavy metals discovered by human. Due to its unique properties like low melting point, softness, malleability, ductility, and resistance to corrosion, lead has wide application in day to day life such as building materials, ceramics, glass, water pipes, paints, protective coatings, acid storage batteries, gasoline additives, solder, ammunition, jewelry, toys, cosmetics and traditional medicines.¹ Mainly the exposure to lead occurs through the respiratory and gastrointestinal systems. Absorbed lead (inhaled or ingested) is accumulated in soft tissues. It is evident from the autopsy studies of the individuals exposed to lead that the largest depot among the soft tissues is hepatic tissue containing 33% lead. Lead can disturb the normal biochemical process by causing liver damage. Serum biomarkers and antioxidants level are the regular parameters for the evaluation of lead induced hepatotoxicity.²

It was evident from various studies that acute and persistent lead poisoning was responsible for changes in hepatic xenobiotic metabolism, cholesterol metabolism, hepatic cell proliferation, and DNA synthesis. Lead exposure is responsible for adverse effects on hepatic microsomal cytochrome P-450 and related enzymatic activity in rat liver.³

Herbal medicine has now become an integral part of standard healthcare, as they are used both in traditional and scientific research. Herbal medicines are rich in natural constituents, which are responsible for promoting health and reducing illness.⁴

Polyphenolic compounds structurally contain many phenolic groups and are widely available in different plants, fruits and vegetables. Their beneficial effect against many diseased conditions affecting different major organs already has been well established. Beneficial effects of various polyphenols in lead toxicity have already been noticed. Polyphenols also have been reported for detoxification and removal of lead.⁵

In ayurveda plenty of herbs are enlisted to cure different ailments. The plant Alstonia scholaris also known as Devils tree or Dita Bark tree belonging to Apocynaceae family, has been used in various traditional medication such as Ayurvedic, Unani and Sidhha/Tamil for the treatment of diseases and ailments of human being.⁶

The bark of Alstonia scholaris is used as laxative, antipyretic, astringent and cardiotonic. Apart from that, it is used for the treatment of pruritis, leprosy, skin diseases, bronchitis, asthma and ulcer. Traditionally the leaves are used to treat malaria, diarrhea and dysentery. Fruits are used as anthelmintic and to treat epilepsy.⁷ Potent phytoconstituents such as alkaloids, coumarins, flavonoids, polyphenols are the main contributors for these beneficial activities.⁸ So the study has been designed to evaluate protective effect of Alstonia scholaris against lead induced hepato toxicity.

METERIALS AND METHODS

Chemicals:

Lead acetate was purchased from Loba chemicals (India). All other chemicals used in the study were of analytical grade and obtained from Agape Diagnostics LTD, Kochi, and Himedia Laboratories.

Experimental animal:

Wistar albino rats of either sex weighing 150- 200 g were procured from Srinivas College of Pharmacy animal house. They were maintained under standard conditions (temperature 22 ± 2 °C, relative humidity 50 ± % and 12 h light/dark cycle). The animals were housed in sanitized polypropylene cages containing sterile paddy husk as bedding, and had free access to standard pellet diet and water. The protocol was approved by Institutional Animal Ethics Committee. All the animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the “National Academy of Sciences” and published by the “National Institute of Health”. All the procedures were performed in accordance with Institutional Animal ethics committee constituted as per the direction of the committee for the purpose of control and supervision of experiments on animals (CPCSEA), under ministry of animal welfare division, Government of India, New Delhi, India.

Ethical considerations:

All efforts were made to minimize animal suffering and to reduce the number of animals used. The animals received humane care and all the experiments were conducted strictly in accordance with the approved guidelines by the “Institute Animal Ethics Committee” (IAEC) regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) according to Government of India accepted principles for lab animals use and care. The study protocol was approved by Institutional Animal Ethics Committee (IAEC), Srinivas College of Pharmacy, Mangalore (Approval No: SCP/IAEC/ F150/P139/2018).

Preparation of ethanolic leaf extract:

About a kilogram of fresh leaves of Alstonia scholaris are collected from the local areas of Mangalore. It was authenticated at the Department of Botany, University College, Mangalore. The leaves were first washed free of sand and debris, then dried in shade and ground to powder. A quantity of the powder (500 g) was weighed and soxhlet extracted with ethanol (95%) for 18 h. The extract was concentrated to a small volume by the rotator evaporator apparatus and then dried at room temperature. The extract obtained was in the form of thick paste and dark green in color.⁹

Experimental design:

The rats were randomly divided into five groups of six each. The different groups were assigned as follows:

Group I : normal control; received saline

Group II: Toxic control; received lead acetate 200 mg/Kg, p.o. for 8 weeks

Group III: Alstona scholaris low dose; received lead acetate 200 mg/kg, p.o.and ethanolic extract of Alstonia scholaris 200 mg/kg,p.o.

Group IV: Alstonia scholaris higher dose; received lead acetate200 mg/kg, p.o. and ethanolic extract of Alstonia scholaris 300 mg/kg, p.o.

All the drug preparations were done in distilled water followed by administering orally once daily for 8 weeks. Hepatotoxicity was induced by administering lead acetate (200 mg/ kg) once daily orally. The animals were given light ether anesthesia on last day after 24 h of treatment, blood was collected through retro orbital puncture; serum was separated and analyzed for various biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphate (ALP), total bilirubin and lipid levels such as total cholesterol (TC), trigycerides (TG), low density lipoprotein (LDL), high density lipoprotein (HDL) levels by semi-autoanalyser (Mispa-plus). Then animals were sacrificed, the livers were removed and homogenized for the estimation of various antioxidants level like super oxide dismutase (SOD), catalase, Lipid peroxidation (LPO) and reduced glutathione (GSH). Remaining livers were embedded in formaline in saline solution (10%) and histopathological studies were carried out.^{10, 11}

Preparation of Liver Tissue Homogenate:

The liver lobules were dissected out gently and rinsed with ice cold physiological saline solution (0.9% NaCl) for removing blood, mucus and other debris from them. The sliced liver was immediately homogenized in ice-cold 0.1 M sodium phosphate buffer (pH 7.4) at 1-4 °C to give a 10% (w/v) homogenate. The homogenates were centrifuged twice at 10000 rpm for 15 minutes at 4 °C. The supernatants were subjected for the estimation of SOD, Catalase, GSH and LPO.¹²

Histological studies:

Liver sections were prepared from the remaining half of the liver samples in each group, stained with Hematoxylin and Eosin (H&E) and changes in histology were observed.¹²

Statistical analysis:

All data were expressed as mean ± SEM. The statistical significance between groups was compared using one-way ANOVA, followed by Tukey-Kramer multiple comparisons tests. p<0.05 was considered significant.

RESULTS

Effect on serum enzymes

Effect on AST, ALT, ALP and Bilirubin (Table no. 1)

Toxic control group revealed extremely significant (P <0.001) increase in serum AST, ALT, ALP and bilirubin levels compared to normal control group.

Treatment groups of AS200, AS300 showed extremely significant (P <0.001) decrease in AST, ALT, ALP and bilirubin levels compared to toxic control.

Effect on TC, TG, HDL, LDL (Table no. 2)

Toxic control group exhibited extremely significant (P ≤ 0.001) increase in TC and TG and LDL levels compared to normal control group. Treatment groups of AS200, AS300 showed extremely significant (P ≤ 0.001) decrease TC and TG and LDL levels compared to toxic control group.

Toxic control group showed extremely significant (P <0.001) decrease in HDL values compared to normal control. Treatment groups of AS200, AS300 showed extremely significant (P <0.001) increase in serum HDL values.

Effect on antioxidants in Liver tissue homogenate (HTH)

Effect on SOD, Catalase and GSH (Table no. 3)

Toxic control group exhibited extremely significant (P <0.001) decrease in SOD, Catalase and GSH activity compared to normal control group.

Treatment groups of AS200, AS300 showed extremely significant (P <0.001) increase in SOD, Catalase and GSH values compared to toxic control group.

Effect on LPO (Table no. 3)

Toxic control group exhibited extremely significant (P <0.001) increase in LPO level compared to normal control group.

Treatment groups of AS200, AS300 showed extremely significant (P <0.001) decrease in LPO level compared to toxic control group.

DISCUSSION

Lead toxicity is probably the most common form of heavy metal intoxication. Lead is known to cause liver damage and increase liver enzyme levels by disturbing the normal biochemical process. Lead-induced hepatic injury is largely due to caspase 3 activation or due to generation of reactive oxygen species (ROS) such as superoxide radicals, hydrogen peroxide, hydroxyl radicals and lipid peroxidation which cause oxidative stress resulting in DNA damage.¹³ The objective of the present study was to evaluate protective effect of Alstonia scholaris leaf extract against lead induced hepatotoxicity.

Natural polyphenolic compounds can detoxify lead from major organs and can revert the detrimental effects of lead. The probable reason by which it may show the protection is scavenging ROS produced by lead and other heavy metals. Moreover, it is already reported from the earler studies that they are also responsible for detoxification by removal of accumulated heavy metals from major organs. Apart from that, Polyphenols attenuated ROS-mediated inflammatory cytokines secretion through ERK/ JNK/p38 pathways which is responsible for protection against lead induced inflammatory reactions.⁵

Lead toxicity is reported to cause hepatocyte or biliary epithelial necrosis, compromise of hepatocyte membrane integrity, and cholestasis. Damage of the membrane of hepatocytes by lead results in release of hepatic enzymes into circulation causing increase in blood level of the enzymes.¹⁴ In this present study also serum level of ALT, AST and ALP significantly increased in lead only treated group when compared with the control rats and other treated groups. This is an indication of impaired liver function.

The cell membrane is the main target of the oxidative damage formed by xenobiotics, including heavy metals. Lead produces oxidative injury by increasing peroxidation of membrane lipids.¹⁴ Alstonia scholaris leaf extract treatment dose dependently exhibited protection against lead-induced liver damage which can be evident by reduced serum concentration of AST, ALT and ALP.

Bilirubin is the chief bile pigment that is formed after the heme breakdown of hemoglobin in red blood cells. It gets transported to the liver and is produced by the liver into the bile. Conjugation of bilirubin is a prerequisite for its excretion into the bile. Structural damage to hepatic cells leads to functional compromisation of the organ. This leads to increased level of serum bilirubin indicating hepatic damage.¹⁵ Bilirubin level is very high in lead only treated group, which reduced in the rats treated with Alstonia scholaris leaf extract in two doses of 200 and 300 mg/kg body weight along with lead.

Lead-induced hyperplasia involves alterations in hepatic cholesterol metabolism that results in simultaneous increase in both liver and serum total cholesterol. Contrary to the general trend of suppression of CYP-450s as discussed above, lanosterol 14a-demethylase (CYP51), an essential enzyme for cholesterol biosynthesis, was found induced in lead mediated liver hyperplasia.¹⁶ Total cholesterol level is very high in lead treated group, the level got decrease in case of rats treated with Alstonia scholaris leaf extract.

Treatment with ethanolic extract of Alstonia scholaris leaf extract reduced the elevated triglyceride levels, suggesting that the extracts prevented lead-induced hyperlipidemia probably due to their hepatoprotective activity.

It was found that administration of lead to rats elevates plasma LDL (low density lipoprotein) and reduces plasma HDL (high density lipoprotein).10 The ethanolic extract of Alstonia scholaris leaf extract shows decrease in LDL level and increase in HDL in serum of rats treated with Alstonia scholaris leaf extract.

When there is inappropriate balance between Reactive oxygen species (ROS) metabolites and antioxidant defense, it causes “oxidative stress”. Excessive reactive hydroxyl free radicals Production from superoxide radicals and H₂ O₂ causes increased lipid peroxidation. This can be a contributing factor for remarkable variation in LPO and notable changes in the antioxidant enzymes activity.¹⁶

Estimation of antioxidant enzyme levels are considered as hallmarks of oxidative stress. Lead induced toxicity can cause reduction in function of Superoxide dismutase (SOD), Catalase (CAT). The reduction of SOD and CAT can make the examined hepatic tissue of rat susceptible to oxidative stress, as these enzymes increase the breakdown of reactive oxygen species. These antioxidants levels can come up with a clear manifestation on the level of cellular damage occurring in hepatic tissue.¹¹

Upon lead exposure reduction in SOD and CAT activities may cause increased oxidative changes of the cell membrane as well as intracellular structures. The mechanism for lead induced changes in SOD and CAT can be due to changes in functional groups or through binding to their metal enzyme cofactors.¹⁴

The concentration of reduced Glutathione (GSH) in this study proposes glutathione utilization by glutathione peroxidase (GPx). The GPx causes oxidation of GSH to Glutathione disulfide (GSSG), this oxidation reaction occurs in the presence of (H₂ O₂ ). Coupling of lead to GSH is responsible for GSH- lead complex formation which is eventually removed through the bile, and it has been demonstrated in vivo. Reduction of GSH indirectly may occur due to condensation of two molecules of damniolevulinic acid (δ-ALA) to prophobilinogen by lead. δ- ALA is known to be a powerful lipid peroxidation (LPO) inducer and responsible for formation of reactive oxygen species both in vivo and in vitro. Accumulation of δ- ALA may cause reduction of GSH from lead accumulated cells.₁₇ Treatment with ASLE along with lead decreased the lipid peroxidation in hepatic tissue as compared with lead only treated animals and elevated levels of the antioxidant enzymes (SOD, CAT and GST) and non-enzymatic potential (GSH), support the protective role of Alstonia scholaris extract in lead intoxication.

Hepatotoxicity reduces the serum total protein level due to the damage to the tissues. Alstonia scholaris leaf extract demonstrated increase in serum Total Protein level which indicating its hepatoprotective activity. In this investigation, lead exposure changed hepatic cell histopathology characterized by localized necrosis, vacuolation of hepatocyte, swelling, pyknotic nuclei, leucocytic infiltration, dilation of central vein and sinusoids. (Figure 1).

Lead is recognized to induce hepatic damage. The pathological alterations may cause change in liver function, interfering with the plasma proteins secretion. This causes reduced blood osmotic pressure, along with decreased tissue fluids drainage, which is observed in the tissue by the odema and congestion.¹⁷ Results revealed cellular infiltration also in the liver tissue. Hepatic tissue cellular infiltration is evident by presence of leucocytes and lymphocytes, the important response after any tissue injury. Both the doses of Alstonia scholaris leaf extract exhibited protective effects in hepatic tissue against lead induced toxicity. Alstonia scholaris leaf extract along with lead retained hepatic architecture and was able to diminish the injury and histological alternations in liver.

The observed beneficial effects associated with Alstonia scholaris leaf extract may be due to the presence of potent polyphenolic compounds such as flavonoids and terpenoids. Antioxidant potential associated with Alstonia scholaris leaf extract might be played pivotal role to demonstrate potent hepato-protective activity against lead induced toxicity.

CONCLUSION

From the present study it can be concluded that Alstonia scholaris leaf extract exhibited dosedependent protective effect against lead induced hepatotoxicity. The hepatoprotective effects of leaf extract may be due to presence of natural polyphenols which shows free radical scavenging, anti-inflammatory and chelating effect properties. Biochemical parameters, antioxidants levels and histopathological studies have exhibited that Alstonia scholaris leaf have hepatoprotective activity in a dose dependent manner. Findings of the study can be beneficial for the people chronically exposed to high lead level.

ACKNOWLEDGEMENTS

The authors are thankful to Srinivas College of Pharmacy, Mangalore, Karnataka, for providing necessary facilities for the present research work. The authors are also thankful to Dr Siddaraju M N, M.Sc. PhD Assistant professor, Department of Botany, University College, Mangalore.

DECLARATION OF INTEREST None.