INTRODUCTION:

The primary organ in the body responsible for the metabolism of both endogenous and foreign substances is the liver. It is crucial for the detoxification and clearance of drugs, and xenobiotics, alcohol, starvation, infections, anaemia, and pharmaceuticals can all harm the liver¹. Hepatotoxicity is the term used to describe damages to the liver caused on by decreased liver function as a result of exposure to a drug or another non-infectious substance². Allopathy medicine has reached such an advanced stage. Despite this, no effective hepatoprotective medicine is available³.

Free radicals can come from both endogenous and exogenous sources. Free radicals are thought to have a role in the origin of a number of degenerative diseases, including coronary artery disease, stroke, rheumatoid arthritis, diabetes, and cancer^4,5. Liver disease is a major challenge faced by the world. Various synthetic drugs for the treatment of liver disease are proven with serious side effects⁶. Due to the antioxidant vitamins and other phytochemicals present in fruits and vegetables, a high intake is linked to a reduced risk of developing certain diseases^7–9. There is a great deal of interest in edible plants that contain antioxidants and health – promoting phytochemicals, in view of their health implication^10,11.

Many compounds and extracts from plants have been evaluated for hepatoprotective and antioxidant effects against chemically-induced liver damage¹². Such an example silymarin, gallic acid, andrographolide, alliin, curcumin, tinosporin, punarnavine, azadirachtin and margolone etc.^6,13–15 of which silymarin compounds, was used as a standard reference and exhibited significant hepatoprotective and antioxidant activity against carbon tetrachloride-induced haptotoxicity in rat models^16,17. As carbon tetrachloride builds up in hepatic parenchymal cells, liver cytochrome P450-dependent monooxygenases convert it to CCl₃¹⁸. One of the principal causes of CCl₄- induced liver injury is lipid peroxidation induced and accelerated by free radical derivatives of CCl₄¹⁹.

In several plants, including carrots, Gymnadeniaconopsea, and Gastrodiaelata Blume, 4-hydroxybenzyl alcohol (HBA), a naturally occurring phenolic chemical, is present. 4-Hydroxybenzyl alcohol (HBA) is the major active constituent of Gastrodiaelata Blume, which has been used in China for centuries as a traditional herbal medicine to treat headache, epilepsy, vertigo and other neurological disorders.

MATERIAL AND METHODS:

Drugs and Chemicals:

Analytical grades chemical was used in this study. Standard drug Silymarin (Micro labs, Bangalore) was obtained as gift sample. CCL₄ was procured from from Thermo Fisher Scientific India Pvt. Ltd. For estimation of biochemical parameter like AST, ALT, ALP, total protein and total bilirubin (Span Diagnostics limited. Surat, India) were purchased from local market, Bilaspur. Other chemicals and reagents were obtained from central drug store of Pharmacy department, GGV, Bilaspur (C.G).

Experimental animals:

Albino Wistar rats weighing (150±20g) used in this study were obtained from departmental animalHouse. The animals were maintained under standard laboratory conditions of constant temperature (27±2°C), relative humidity (50±10)%, and relative humidity 44–56%, light and dark cycles of 10 and 14 hour respectively for 1 week before and during the experiments. Animals were provided with standard laboratory pellet diet (Kapila Pashuahaar, Kanpur India) and the food was withdrawn 18– 24h before the experiment though water was given ad libitum. All studies were performed in accordance with the guide for the care and use of laboratory animals, as adopted and promulgated by the Institutional Animal Care Committee, CPCSEA, India (Reg. no. 994/GO/Re/S/06/CPCSEA)^20,21.

Determination of hepatoprotective activity:

Rats were divided into five groups consisting of six animals in each group and treated for 7 days as follow: group I served as normal control and was daily received normal saline Group II served as CCl₄-hepatotoxicity control and was orally given normal saline for seven days. Group III served as standard group and received reference drug silymarin (100mg/kg per day p.o.) for seven days prior to CCl₄ intoxication²². The animals of groups IV and V received the test drug 4– hydroxybenzyl alcohol at a dose of 25 and 50mg/kg b.w. p.o. respectively. On the last day of the treatment, the animals of groups II–V received a single dose of CCl₄ (1:1 mixture in olive oil) at 1ml/kg b.w. intraperitoneal after 1hr of the treatment. Twenty-four hours after CCl₄injection, all of the animals were anesthetized with ketamine and blood samples collected immediately by cardiac puncture. The livers were removed quickly and dissected to two halves, one for biochemical analysis and the other for histopathological studies

Measurement of serum biochemical markers:

The collected blood was allowed to clot and serum was separated at 2500rpm for 15min and the biochemical parameters like serum enzymes: aspartate aminotransferase (AST, U/L), serum glutamate pyruvate transaminase (ALT, U/L)²³, serum alkaline phosphatase (ALP, KA/dl)²⁴, total bilirubin (mg/dL)²⁵ and total protein (TP) levels were assayed using diagnostic kits.

Measurement of SOD, CAT and GSH in liver homogenate:

Liver samples were homogenized in phosphate buffer (5mM, pH 7.4) to give a 10% (w/v) liver homogenate and then centrifuged at 4000rpm for 15 min at 4°C. The supernatant of the liver homogenate was used for the superoxide dismutase (SOD)²⁶ and catalase (CAT)²⁷ and estimation of glutathione (GSH) levels by a colorimetric method.

Histopathological studies:

One animal from each group was utilized for this purpose. The liver specimens obtained from the control and treated groups of animals were fixed in 10% buffered formalin for 24h. The formalin fixed liver samples were stained with haematoxylin-eosin for photomicroscopic observations of the liver histopathological architecture (. Photographs of each of the slides were taken at 40 magnification.

Statistical analysis:

Results expressed as mean±SEM (n=5); data analysed using One-way ANOVA followed by Newman-Keuls comparison test using GraphPad Prism 9 (GraphPad Software Inc., CA, USA); and results were considered significant when P < 0.05.

RESULT:

Effects of extracts on serum biochemical parameters

The results observed in pre-treatment of 4– hydroxybenzyl alcohol and silymarin with respect to induction of hepatotoxicity using CCl₄were given in Tables 1. The activity of the enzymes AST, ALT, ALP and Total bilirubin (Table 1) were significantly increased in the CCl₄group compared to the control group (p<0.05). The TP levels decreased considerably (p < 0.05) in the toxic group. 4 – hydroxybenzyl alcohol (25 and 50mg/kg) caused significant (p<0.05) reductions in CCl₄-elevated levels of AST, ALT, ALP and total bilirubin in dose dependant manner. 4 – hydroxybenzyl alcohol (25 and 50mg/kg) administration resulted in a significant improvement in TP level in a dose-dependent manner and succeeded to normalize TP level at the highest dose tested.

The pretreatment of 4 – hydroxybenzyl alcohol and Silymarin exhibited inhibition of CCl₄ induced increase in the levels of all the four biochemical parameters, resulting in significant restoration towards their control values (shown in Table 1). Further reduction of serum TP brought towards normal values bypretreatment 4 – hydroxybenzyl alcohol and Silymarin.

Table 1: Effect of 4 – hydroxybenzyl alcoholon serum biochemical parameters in CCl₄-induced hepatotoxicity in rats.

S. No.

AST

IU/l

ALT

IU/l

ALP

KA/dl

Total bilirubin

mg/dl

Total protein U/L

Normal control

66.3±

3.7

29.67±

0.84

81.5±

1.1

1.3±

0.06

8.3±

0.46

Toxic control

290.5±28.4

177.3±

3.9

338.0±15.1

2.7±

0.16

5.1±

0.37

Silymarin

73.6±

4.5***

38.03±

0.29***

107.7±6.2***

1.3±

0.12***

7.5±

0.13***

Test 1

63.6±

4.9*

66.0±

6.8

71.5±

6.0*

1.8±

0.21**

7.4±

0.02*

Test 2

64.8±

9.9***

48.5±

4.5***

68.0±

6.6***

1.5±

0.11***

7.7±

0.06***

All values expressed as mean ± SEM; One-wayAnova followed by Newman-Keuls Multiple Comparison Test. Significantly different at *p<0.05, ** p<0.01, ***p<0.001 as compared to the control group. Normal control = saline, Toxic control = CCl4, Test 1 = 4-hydroxybenzyl alcohol (25 mg/kg), Test 2 = 4-hydroxybenzyl alcohol (50 mg/kg)

Effects of extracts on SOD, CAT and GSH levels:

CCl₄ significantly reduced the activity of SOD (from 76.75 to 54.59 𝜇g/mg) and CAT (from 33.16 to 20.25 𝜇g/mg) in the rats’ livers. Pretreatment of Silymarin and 4-hydroxybenzyl alcohol in rats increased the activities of SOD (Figure 1) and CAT (Figure2) in all treated groups. Again, the 4-hydroxybenzyl alcohol at the higher dose (50 mg/kg) yielded the best antioxidant effect with a very good recovery (Table 2). The activity of GSH significantly decreased (from 7.84 to 2.99 𝜇g/mg) in the rats’ livers following hepatic injury due to CCl₄ exposure. The highest recovery of GSH (at 7.91𝜇g/mg protein) (Figure 3) was again observed in animals receiving test drug and the group of animals that received the highest dose (50 mg/kg) had the best antioxidant effects (Table 2).

Table 2: Effect of 4-hydroxybenzyl alcoholon in vivo antioxidant enzymes level

S. No.

SOD

(𝜇g/mg tissue)

CAT

(𝜇g/mg protein)

GSH

(𝜇g/mg protein)

Normal control

76.75 ±

0.06

33.17 ±

0.17

7.84 ±

0.06

Toxic control

54.59 ±

1.71

20.26 ±

1.45

2.99 ±

0.75

Silymarin

66.24 ±

3.73 **

35.74 ± 4.37***

4.99 ±

0.77*

Test 1

64.66 ±

4.80

45.50 ±

2.80

7.60 ±

1.06**

Test 2

67.68 ±

6.36 ***

45.65 ±

4.39***

7.91 ± 0.97***

All values expressed as mean ± SEM; significantly different at *p<0.05, ** p<0.01, ***p<0.001 as compared to the control group. Normal control = saline, Toxic control = CCl4, Test 1 = 4-hydroxybenzyl alcohol (25 mg/kg), Test 2 = 4-hydroxybenzyl alcohol (50 mg/kg)

Figure 1: SOD level in rats after treatment with 4-hydroxybenzyl alcohol

All values expressed as mean ± SEM;significantly different at *p<0.05, ** p<0.01, ***p<0.001 as compared to the control group. Normal control = saline, Toxic control = CCl4, Test 1 = 4-hydroxybenzyl alcohol (25 mg/kg), Test 2 = 4-hydroxybenzyl alcohol (50 mg/kg)

Figure 2: CAT level in rats after treatment with 4-hydroxybenzyl alcohol

All values expressed as mean ± SEM;significantly different at *p<0.05, ** p<0.01, ***p<0.001 as compared to the control group. Normal control = saline, Toxic control = CCl4, Test 1 = 4-hydroxybenzyl alcohol (25 mg/kg), Test 2 = 4-hydroxybenzyl alcohol (50 mg/kg)

Figure 3 GSH level in rats after treatment with 4-hydroxybenzyl alcohol

All values expressed as mean ± SEM;significantly different at *p<0.05, ** p<0.01, ***p<0.001 as compared to the control group. Normal control = saline, Toxic control = CCl4, Test 1 = 4-hydroxybenzyl alcohol (25 mg/kg), Test 2 = 4-hydroxybenzyl alcohol (50 mg/kg)

Histopathological studies:

The histopathological evaluation of CCl4 toxicity in all the groups was examined and shown in figure. The description is as follows; in Figure 4 section of rat liver treated with normal saline (Normal control) group shows liver parenchyma with intact architecture which is the normal appearance in Figure 5 section of liver in toxicant control group shows partially effaced architecture. Some of the hepatocytes exhibit toxic toxicity-related apoptotic changes, perivenular mononuclear inflammatory infiltration, and scattered inflammatory infiltration within the parenchyma. In figure 6 section of liver in silymarin treated group shows liver parenchyma with intact architecture. Some of the central veins exhibit sinusoidal diffuse congestion. In figure 7 and 8 section of liver in test drug treated (4-hydroxybenzyl alcohol) group’s shows intact architecture, few regenerative hepatocytes, sinusoidal congestion and scattered mononuclear inflammatory cells which is like silymarin treated group.

Figure 4: Control group showing normal hepatocytes

Figure 5: CCl₄treated hepatotoxic group shows that hepatic cells damage and congestion of the liver

Figure 6: Hepatocytes in group treated with Standard (Silymarin)

Figure 7 4-Hydroxybenzyl alcohol of 25 mg/kg shows that few regenerative hepatocytes, sinusoidal congestion and scattered mononuclear inflammatory cells

Figure 8 4-Hydroxybenzyl alcohol of 50 mg/kg shows more reduced regenerative hepatocytes, sinusoidal congestion and scattered mononuclear inflammatory cells

DISCUSSION:

There are many factors which are responsible for the liver damage or injuries such as chemicals and drugs.

Carbon tetrachloride is a chemical hepatotoxin used for inducing hepatotoxicity in animal model. CCl4 is metabolized by cytochrome P450 system and converted to trichloromethyl and trichloromethylperoxy radicals which initiates peroxidation of polyunsaturated fatty acid constituents of various membranes with secondary damage, severe enzymatic disturbances, and increases MDA production^28,29.

Due to metabolic activation, the hepatotoxic drug CCl4 causes selective toxicity to the liver cells, maintaining their semi-normal metabolic activity. It also causes the functional and morphological changes in the cell membrane which may leads to cell death¹⁸. In cytoplasm the AST ALT concentration is higher in the hepatic cell and AST in particular exists in mitochondria³⁰. Due to the damage caused to hepatic cells, the leakage of plasma causing an increased levels of hepatospecific enzymes in serum. Elevated serum enzyme levels such as AST and ALT are a sign of cellular leakage and functional integrity of cell membrane in liver³¹ The hepatprotective index of a drug can be assessed by its capability to decrease the injurious effects or to preserve the normal hepatic physiological mechanisms, which have been induced by a hepatotoxin. The serum AST, ALT and ALP levels measurements serve as a means for the indirect assessment of condition of liver.

4-Hydroxybenzyl alcohol (HBA) is phenolic compound occurred naturally, found in many plants, including carrots, Gymnadeniaconopseaand Gastrodiaelata Blume. 4-Hydroxybenzyl alcohol (HBA) is the major active constituent of Gastrodiaelata Blume, which has been used in China for centuries as a traditional herbal medicine to treat headache, epilepsy, vertigo and other neurological disorders. The phenolic compound exhibits a pleiotropic pharmacological profile, exerting various beneficial effects on the central nervous system. These include antioxidant, hepatoprotective, anxiolytic, sedative and anti-apoptotic actions.

The pre-treatment of the animals with 4-Hydroxybenzyl alcohol with respect to intoxication with CCl₄controlled the AST, ALT and ALP levels when compared with the toxic group. Bilirubin higher concentration in serum is a sign for increased erythrocyte degeneration rate³². The higher levels of bile in serum are a result of the liver's impaired ability to excrete bile as a result of the hepatotoxin's damage to the liver³³. The oral administration of 4-Hydroxybenzyl alcohol at a dose of 25 and 50mg/kg b.w. effectively reduced the serum TB levels. Due to defective protein biosynthesis in liver the TP levels will be depressed in hepatotoxic conditions³⁴. The endoplasmic reticulum's polyribosomes are disrupted and disassociated during CCl4 intoxication, which lowers the amount of protein that is synthesised. The pre-treatment of 4-Hydroxybenzyl alcohol well restored the proteins synthesis by protecting the polyribosomes.

CONCLUSION:

In the present study, the 4-Hydroxybenzyl alcohol significantly reduced the elevated levels of above-mentioned serum marker enzymes. Hence, at this point it is concluded that the 4-Hydroxybenzyl alcohol possesss hepatoprotective activity. Histopathological findings confirm this study by demonstrating the plant's considerable action. Additionally, it was noted that hepatocytes were regenerating, which indicates hepatoprotective activity. Finally based on improvement in serum marker enzyme levels, physical parameters, functional parameters, and histopathological studies, it is concluded that the 4-Hydroxybenzyl alcohol possesses hepatoprotective activity and thus supports the traditional application of the same in the context of contemporary science.

CONFLICT OF INTEREST STATEMENT:

The authors declare that there are no conflicts of interest.

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Received on 04.05.2023 Modified on 06.08.2023

Research J. Pharm. and Tech 2023; 16(12):6022-6027.

DOI: 10.52711/0974-360X.2023.00977