Hepatoprotective and Antioxidant Effects of Mucuna pruriens Leaves Against Paracetamol-Induced Hepatotoxicity in Albino Wistar Rats

Abstract

The present study was planned to evaluate the hepatoprotective and antioxidant potential of Mucuna pruriens leaves and to evaluate its therapeutic efficacy in managing hepatotoxicity. Rats weighing 200–250 g were assigned into six groups (n = 6), normal saline only (control), normal saline (NS), Hydroalcoholic extract of Mucuna pruriens leaves (HAMP) (100, 200 and 400 mg/kg) and Silymarin (100 mg/kg) treated orally for five days, paracetamol (PCM) was administered via intra-peritoneal route, biochemical and histology parameters were determined in serum and liver. This research revealed that treatment with HAMP reversed the elevation of liver enzymes such as AST, ALT & ALP, liver SOD, GSH, and CAT were elevated in HAMP and Silymarin groups. The hepatic histological lesions in HAMP were reduced in a dose-dependent manner. This research shows that HAMP attenuates the deleterious effect of paracetamol-induced liver toxicity in rats.

Keywords

Introduction

Herbal medicines which formed the basis of health care throughout the world since the earliest days of mankind are still attracting more and more attention within the context of health care provision and health care reform. Medicinal plants are distributed worldwide, but they are most abundant in tropical countries22. India has 2.4% of world's area with 8% of global biodiversity. One fifth of all the plants found in India are used for medicinal purpose. The world average stands at 12.5% while India has 20% plant species of medicinal value, and which are in use. According to Hamilton 2003, India has about 44% of flora, which is used medicinally. Although it is difficult to estimate the number of medicinal and aromatic plants present worldwide, the fact remains true that India with rich biodiversity ranks first in percent flora, which contains active medicinal ingredient. Over the past decade, interest in drugs derived from higher plants, especially the Phyto therapeutic, has increased substantially and so its market is growing. It is estimated that about 25% of all modern medicines are directly or indirectly derived from higher plants. The liver is a vital organ responsible for numerous physiological functions, including metabolism, detoxification, synthesis of proteins, and bile secretion. Weighing approximately 1.2 to 1.5 kg, it is located in the upper right and part of the upper left quadrants of the abdominal cavity¹beneath the diaphragm. Anatomically, the liver comprises four lobes—right, left, caudate, and quadrate—encapsulated in connective tissue and covered by the peritoneum. The functional unit of the liver is the lobule, a hexagonal structure centred around a central vein and surrounded by portal triads. Blood from the hepatic portal vein and hepatic artery flows through sinusoids lined with hepatocytes and Kupffer cells. Kupffer cells, specialized macrophages, clear aged red blood cells, while hepatocytes perform most of the liver's essential roles, such as nutrient metabolism, detoxification, and bile formation ^{2, 3}

The liver executes a wide range of functions essential for homeostasis. It metabolizes carbohydrates, lipids, and amino acids; synthesizes plasma proteins like albumin and globulins; stores glycogen, triglycerides, vitamins (A, B12), and trace elements (iron, copper); detoxifies xenobiotics and hormones; and excretes bile acids, bilirubin, and cholesterol derivatives. Given its metabolic versatility, the liver is particularly vulnerable to toxic insults and infections. Liver diseases represent a significant global health burden and are among the leading causes of morbidity and mortality worldwide. According to the World Health Organization (WHO), liver cirrhosis alone accounted for approximately 1.3 million deaths globally in 2015, making it one of the top 20 causes of death. Hepatocellular carcinoma (HCC), a primary malignancy of the liver, is now the third leading cause of cancer-related deaths, particularly prevalent in regions such as Asia and sub-Saharan Africa, where hepatitis B and C are endemic. Hepatotoxicity results from direct toxicity of primary compounds or their reactive e metabolites or from immunologically-mediated responses affecting hepatocytes, biliary epithelial cells, and/or liver vasculature. According to the European Medicines Agency (EMEA), liver toxicity has been one of the most frequent reasons for pharmacovigilance safety reports and the withdrawal of approved medicinal agents from the market¹⁹. The occurrence and burden of liver dysfunction/damage is on the increase. More than 900 drugs have been implicated as a cause of liver injury. Drugs account for 5% of hospitalizations, 10% of cases of acute hepatitis, 50% of acute liver failures, and chronic liver disease and cirrhosis account for about 2% of mean in 17countries with nearly 40,000 deaths per year. According to Ostapowicz et al, more than 75% of cases of idiosyncratic drug reactions result in liver transplantation or death. Hence present study was planned to evaluate the hepatoprotective and antioxidant potential of Mucuna pruriens leaves and to evaluate its therapeutic efficacy in managing hepatotoxicity.

Paracetamol (PCM)

PCM is a nonsteroidal anti-inflammatory drug (NSAID) used for analgesic, antipyretic and anti-inflammatory activity⁸. It is most commonly used OTC (over the counter) drug. Although it is safe, effective and well tolerated drug when used in therapeutic doses, it’s over dose is a common means of self-poisoning worldwide. Currently PCM is the most common cause of ALF (acute liver failure) in both United States and United Kingdom, with a trend to increasing incidence in the United States. In overdose, paracetamol produces a centrilobular hepatic necrosis that can be fatal. The initial phases of paracetamol toxicity were described way back in 1970’s at Dr. Gillette’s laboratory). Following an oral dose, the drug is well absorbed from the gastrointestinal tract, reaching a peak plasma concentration within 30-60 minutes⁸. The drug is metabolized in the liver, 80% of an administered dose (therapeutic dose), undergoes glucuronide conjugation and sulphate conjugation; the remaining drug is metabolically activated by cytochrome P450 to form a reactive metabolite, N-Acetyl P Benzo-quinoneimine (NAPQI) which in turn conjugates with glutathione to form mercapturic acid and is eliminated in urine⁹. More recently, the cytochromes 2EI, 1A2, 3A4 and 2A6 have been reported to oxidize paracetamol to the reactive metabolite. After a toxic dose of paracetamol, total hepatic GSH is depleted by as much as 90% and as a result, the metabolite covalently binds to cysteine groups on protein forming acetaminophen protein adducts. Although covalent binding on proteins has been shown to be an excellent relation of its toxicity, a number other events have been shown to occur and are likely important in the initiation and repair of toxicity.

The leaves of Mucuna pruriens are used as remedy for various diseases such as diabetes, arthritis, dysentery, and cardiovascular diseases. Phytochemicals are bioactive compounds found in plants that work with nutrients and dietary fiber to protect against diseases. They are nonnutritive compounds (secondary metabolites) that contribute to flavor color. Many phytochemicals have antioxidant activity and reduce the risk of many diseases, for example, alkyl sulfide (found in onions and garlic), carotenoids (from carrots), and flavonoids (present in fruits and vegetables). Reactive oxygen-free radicals (ROS) have been implicated in many diseases and in aging process. These free radicals, which cause tissue damage via oxidative stress, are generated by aerobic respiration, inflammation, and lipid peroxidation. Antioxidant systems minimize or prevent deleterious effects of the ROS²⁵. Fresh leaves of Mucuna pruriens were collected from the UP East region and authenticated by a botanist. Verification was performed at the Botanical Survey of India, Pune. All reagents used were of analytical grade, and instruments were validated as per SOP³⁰. Leaves were shade-dried, coarsely powdered, and stored in airtight containers.

Extraction of Plant Material

Hydroalcoholic extraction (ethanol: water, 70:30) was employed. 300 g of dried powder was extracted using Soxhlet apparatus at 64°C for 72 h. The extract was concentrated under reduced pressure at 40°C using a rotary evaporator and stored at 4°C.

Figure 1: Soxhlet extraction of Mucuna pruriens leaves

Preliminary Phytochemical Screening

Standard phytochemical tests were performed to detect alkaloids, carbohydrates, glycosides, saponins, proteins, flavonoids, tannins, steroids, fixed oils, and amino acids. Specific tests included Mayer’s, Wagner’s, Dragendorff’s, Hager’s, and Legal’s tests for alkaloids; Molisch, Fehling’s, Benedict’s, and Iodine tests for carbohydrates; Borntrager’s and modified Borntrager’s for glycosides; Liebermann-Burchard and Salkowski for steroids; Foam test for saponins; Ninhydrin test for proteins; Shinoda test for flavonoids; and saponification for fixed oils.

Preparation of Animals

Albino Wistar rats (200–250 g) were procured from the animal house of Saraswati Higher Education and Technical College of Pharmacy, Varanasi. Animals were maintained under standard conditions (25 ± 1°C, 12-hour light/dark cycle, 44–56% humidity) and given standard feed and water ad libitum. Animals were fasted for one hour before experiments.

Acute Toxicity Study

Mice fasted for 12 h were administered M. pruriens extract intraperitoneally at doses of 200, 300, 500, and 1000 mg/kg. Animals were monitored for signs of toxicity and behavioral changes for 2 h and then for 14 days for delayed effects. LD?? was determined using log dose-probit analysis.

Pharmacological Evaluation of Extract

The assessment of extract from a pharmacological perspective is crucial for determining their safety, effectiveness, and therapeutic capabilities, thereby establishing a scientific basis for their use in clinical settings. A thorough evaluation of the pharmacodynamic and pharmacokinetic qualities aids in understanding the mechanisms of action and potential adverse effects linked to the active components found in extract.

Table 1: Experimental Design

Groups	Treatments
Group-A	Normal saline (NS) only (10 mL/kg, p.o.)
Group-B	Paracetamol (50 mg/kg, i.p.)
Group-C	Paracetamol (50 mg/kg, i.p.) plus 100 mg/kg,p.o. HAMP extracts
Group-D	Paracetamol (50 mg/kg, i.p.) plus 200 mg/kg, p.o. HAMP extracts
Group-E	Paracetamol (50 mg/kg, i.p.) plus 400 mg/kg, p.o. HAMP extracts
Group-F	Paracetamol (50 mg/kg, i.p.) plus 100 mg/kg, p.oSilymarin (Standard drug)

Rats from various groups were sacrificed on day 6 of the treatment regimen after being sedated with light ether. This was done twenty-four (24) hours following the last treatment. The blood was drawn from the retro-orbital plexus and placed in a vial without anticoagulants. The separated serum sample was centrifuged at 5,000 rpm for 10 minutes, which was then used for biochemical parameters analysis using standard diagnostic kits. Theliver was immediately taken out and washed with ice-cold saline. The blood and liver samples were assessed for the Histopathological study:

Histopathological study

After the rats were sacrificed, the liver was removed. The liver was used for histological investigations after being blotted without blood or tissue fluids. The liver tissue was fixed in 5% formalin for 48 hours, dehydrated by passing through various ethyl alcohol-water combinations, and immersed first in paraffin, then in xylene. Then, using a microtome, slices measuring 15 mm thick were cut and put on a glass slide. After being stained for 3–5 minutes with 10% haematoxylin³¹, the liver slices were rinsed under running water to make the staining more pronounced. The sections were then counterstained for 2 minutes with 10% eosin. The slices were examined using a photomicroscope at a magnification × 400, and the desired spots were photographed

Assessment of antioxidant activity of Mucuna pruriens extract:

DPPH free radical-scavenging activity:

The sample extract was taken 0.1 mL, added 1.9 mL of ethanol PA, and 1 mL of 0.4 M DPPH. Mix using a vortex for 30 seconds and cover the tube with aluminium foil then stand for 30 minutes at room temperature. The absorbance was measure at a wavelength of 517 nm. The blank used was an ethanol sample without the addition of DPPH. Ascorbate acid in different concentrations was applied as a standard and final results were shown as milligrams of ascorbate acid equivalents antioxidant capacity (AAEAC) per 1 g of dry weight60, 61, 62 (DW).

RESULTS AND DISCUSSION

Collection and Identification

The leaves of Mucuna pruriens was collected from East region of Uttar Pradesh. The initial identification was based on its organoleptic and morphological characteristics, and the verification was conducted by Botanical Survey of India, Pune.

Percentage yield

The leaves of Mucuna pruriens was demonstrated the percentage yield as 53.60% respectively.

Phytochemical screening

Phytochemical screening of hydroalcoholic extract of Mucuna pruriens leaves was done as previously described by Khandelwal K. R. The presence of selected phytochemical constituents such as saponins, tannins, flavonoids, cardiac glycosides, terpenoids, and alkaloids, was investigated.

Table 2: Phytochemical composition of hydroalcoholic leaves extracts of Mucuna pruriens

Test performed	Hydroalcoholic extract
Alkaloids
Mayer’s reagent	-
Wagner’s reagent	-
Saponins
Foam test	+
Borntrager’s test	+
Tannins
Lead acetate solution	+
Ferric chloride solution	+
Flavonoids
Shinoda test	+
Cardiac glycosides
Legal test	+
Terpenoids
Tin and thionyl chloride test	+

Acute toxicity study:

The M. pruriens extract administered by the intraperitoneal route, no deaths were recorded at the lowest dose of 200 mg/kg while mortality was 100% at the highest dose of 1000 mg/kg. The LD50 was estimated to be 373.04 mg/kg. The animals that received the extract showed writhing and convulsion, especially at the higher doses.

Pharmacological Evaluation of Extract

Effect of HAMP extract on Paracetamol induced hepatotoxicity on serum ALT, AST and ALP

The effect of Paracetamol, HAMP and SLM on ALT, AST and ALP in serum is presented in Table 3. The group admin­istered with NS+PCM showed a significant (p<0.05) ele­vation in serum ALT, AST and ALP relative to the control. Treatment with graded doses of HAMP (100, 200 and 400 mg/kg) showed a decrease in serum ALT, AST and ALP, with a significant reduction (p<0.05) at HAMP 400 mg/ kg when compared with NS + Paracetamol group. However, the group administered with SLM 100mg/kg + PCM showed a noticeable reduction (p<0.05) in serum ALT, AST and ALP in comparison with NS + PCM treated group (Table 3).

Table 3: Effect of Mucuna pruriens on Paracetamol induced alterations in serum AST, ALT and ALP content.

Groups	AST (U/l)	ALT (U/l)	ALP (U/l)
Group-A (Control group)	7.00± 1.51	6.03±0.86	10.33±1.65
Group-B	19.00±0.58	12.65±0.86	25.00±0.57
Group-C	17.37±1.20	09.23±0.88	22.65±0.33
Group-D	13.00±0.58	09.00±0.58	17.00±0.58
Group-E	10.00±0.58	8.50±0.76	13.33±1.45
Group-F	7.00±0.58	7.33±0.67	11.33±0.66

All the values have been expressed as Mean ±S.E.M, test employed one-way ANOVA by Dennett’s test (n=6); significantly different from the control at *(P<0.05), ** (P<0.01), *** ( P<0.001) and ns (non-significant) when compared to the control group.

Figure 2: Effect of HAMP extract on serum AST level

Figure 3: Effect of HAMP extract on serum ALT level

Figure 4: Effect of HAMP extract on serum ALP level

Histopathological study:

The histological results are established in Fig.8. The micro­scopic examination established no observable lesion of he­patic tissue from the normal saline-treated group (Fig. 8A). On the other hand, NS+PCM-treated group showed atrophy of cords, random hepatocellular coagulative necrosis and inflammation relative to the normal saline-treated group, which showed hepatotoxicity (Fig. 8B). HAMP 100 mg/ kg + PCM treatment demonstrated moderate peri-acinar hepatocellular coagulation necrosis (Fig. 8C). The HAMP 200 mg/kg + PCM-treated group showed an improvement in liver histological changes when compared with the NS+ PCM-administered group but still exhibited moderate centrilobular cord atrophy (Fig. 8D). Similarly, the group treated with HAMP 400 mg/ kg + PCM improved the liver histological changes com­pared with NS+ PCM by demonstrating mild cord atrophy (Fig. 58). However, the liver of the group treated with SLM 100 mg/kg + PCM showed no observable lesion (Fig. 8F).

Figure 5: Effects of Paracetamol on the liver of control rats and rats subjected to graded doses of HAMP and Silymarin.

(A) Control animals (Normal Saline) show no visible lesions ; (B) Normal Saline+ PCM treated animals show atrophy of cords (blue arrow), random hepatocellular co­agulative necrosis (black arrow) and inflammation (red arrow); (C) HAMP 100 mg/kg + PCM) show moderate peri-acinar hepatocellular coagulation necrosis (black arrow); (D) HAMP 200 mg/kg + PCM) show moder­ate centrilobular cord atrophy (blue arrow).; (E) HAMP 400 mg/kg + PCM) show mild cord atrophy (blue arrow); (F) SLM 100 mg/kg + PCM) show no observable lesion.

DPPH free radical-scavenging activity evaluation

The present study explored the DPPH antioxidant abilities of velvet bean extracts. The assay was inexpensive, simple, and reproducible, thus utilized to assess the antioxidant capacity in vitro. The DPPH - RSA method use 1,1-diphenyl-2-picrylhydrazyl (DPPH) reagent, and the standard used is ascorbic acid, and the DPPH radical mechanism donates a hydrogen atom so that it is reduced by antioxidants. The changes of color from purple to yellow due to the alleviation of DPPH to non-radical molecules. The results of the analysis of variance indicated that the treatment of temperature and time of extraction and their interactions were significant (p<0.05) on DPPH extract of velvet beans antioxidant capacity. The antioxidant capacity of DPPH (mg AAEAC/g) of velvet bean extract can be shown in Table 6. The highest antioxidant capacity of the extract at a temperature of 60°Cfor 360 minutes and the lowest at a temperature of 30°C for 120 minutes, which were 38,30±0,03 mg AAEAC/g and 6,25±0,34 mg AAEAC/g.

Table 4: Antioxidant capacity (DPPH) of velvet bean extract in different extraction temperatures and extraction times

Note: the same letter behind the average value in the same column shows that the effect of different treatments is insignificant (p > 0.05).

The medicinal benefits of plant leaves may be due to the phytochemicals that make up those leaves. The presence of selected phytochemical constituents such as saponins, tannins, flavonoids, cardiac glycosides, terpenoids, and alkaloids, was found in hydroalcoholic extract of M. pruriens leaves. The secondary metabolites (phytochemicals) and other chemical components of medicinal plants account for their medicinal value. The presence of phenolic compounds in Mucuna pruriens leaves is responsible for its' antioxidative characteristics and their value as herbal remedies. The liver is an organ involved in many metabolic processes and susceptible to xenobiotic injury because of its critical role in xenobiotic metabolism. Hepatotoxic medications like acetaminophen can damage the liver. Paracetamol is a well-known NSAID frequently used to treat fever and pain, and there has been much concern regarding the hepatotoxicity effect of Paracetamol. The deposition of Paracetamol, as well as its bioactivation, produces intermediate reactive species, which cause the production of oxidative stress, inflammation, tissue necrosis and deterioration that are likely responsible for liver damage.

Our study investigated the attenuating effects of the HAMP against paracetamol-induced liver injury and its modulating effects on free radicals and biochemical and histological parameters alterations in Wistar rats. The re­sult of our study revealed that the administration of paracetamol can cause hepatic tissue damage and inflamma­tion. Our findings from the hepato-protective potentials of the hydroalcoholic extract of Mucuna pruriens against PCM-induced liver toxicity in rats align with earlier re­search that supported PCM’s hepatotoxic effects (Peter et al. 2017). Also, treatment with graded doses of hydroalcoholic extract of Mucuna pruriens (100, 200 and 400 mg/ kg) and Silymarin 100 mg/kg plus paracetamol reversed the physiological abnormalities caused by paracetamol. This suggests that the HAMP maintained the liver cell mem­branes’ structural integrity, which prevented the release of ALT, AST, and ALP into the bloodstream.  One of the markers of liver impairments is serum bili­rubin, a test for hepatic excretory function. Bilirubin is a by-product produced by the catabolism of heme, which is usually conjugated by the liver to produce bilirubin’s diglucuronide and move from the body through the bile. As a result, increased serum bilirubin is observed when there is liver injury. Serum bilirubin is also increased when there is too much breakdown of erythrocytes in case of hemolytic anemia, in which di­clofenac may be the cause. The heme produced from the breakdown of hemoglobin resulting from red blood cells is catabolized by the reticuloendothelial system (RES) to generate bilirubin.

In this study, the paracetamol significantly increased the serum levels of ALT, AST, ALP, and creatinine compared to control. AST and ALT are both sensitive markers of hepatocellular injury and these proteins leak through the liver cell membrane into circulation, causing increased serum levels, sequel to damage to hepatocytes. ALT, a cytosolic enzyme, is primarily present in the liver, while AST, a mitochondrial enzyme, is released from the heart, liver, skeletal muscles, and kidney. ALP is produced in many tissues (especially the bone, liver, intestine, and placenta), and elevated serum levels of this enzyme, due to defective hepatic excretion or increased production by hepatic parenchymal or duct cells, in the absence of bone disease and pregnancy, indicate hepatobiliary disease.  Our study showed high ALT, ALP & AST levels in the NS + PCM treated group compared to the control group, a con­venient indicator of liver diseases. Our results showed a dose-dependent decrease in ALT, ALP & AST in groups administered with graded doses of HAMP extract (100, 200 and 400 mg/kg) and Silymarin plus paracetamol, which indicates that the liver functions were restored to a level comparable to the control group, the results are consistent with earlier findings. The marked changes observed in serum AST, ALT & ALP levels due to paracetamol toxicity were reversed by HAMP extract and Silymarin-administered groups. These findings suggest that the extract of M. pruriens possesses hepatoprotective activity.

In the present study, the histology and the biochemi­cal findings were comparable. Inflammation and hepato­cellular coagulative necrosis were observed in the group administered with normal saline and paracetamol, which is consistent with earlier findings. The group treated with graded doses of HAMP plus paracetamol demonstrated a protective effect by minimizing hepatocellular necrosis and degeneration and reducing the hepatic histological lesions brought on by paracetamol administration. The same protective effect was seen in the liver of the animals treated with Silymarin. DPPH (1,1 diphenyl-2-picrylhydrazil) is a stable free radical widely used to assess the radical-scavenging capacity of natural substances. Antioxidants convert DPPH to DPPH-H by a reaction, which lower absorbance. The discoloration level reveals the antioxidant compounds' scavenging capacity in terms of their capacity to donate hydrogen. Antioxidants found in the leaves of Mucuna pruriens can scavenge the DPPH radical by donating a proton to create the reduced DPPH. In this study, the DPPH radical scavenging activity of the M. pruriens leaves fractions was concentration dependent, contrary to the previous report on the DPPH radical scavenging activity of methanol extract of Mucuna pruriens leaves. The antioxidant activity of the fractions was expressed as IC50, which indicates the fractions' inhibitory concentration (μg/mL) that inhibited the formation of DPPH radicals by 50%. Ascorbic acid was used as a positive control. The antioxidant capacity was 38.15 mg AAEAC/g on DPPH-RSA.

The current work confirms previous research; it was asserted that flavonoids and related polyphenols play a substantial role in the antioxidant activity of medicinal plants. This is caused by the presence of hydroxyl groups and conjugated ring structures. Furthermore, many phenolic compounds can act as antioxidants by neutralizing or stabilizing free radicals implicated in oxidative processes through hydrogenation or by complexing with oxidizing species.

CONCLUSION

This research exhibits evidence indicating the hepatic protection conferred by the hydroalcoholic extract of Mucuna pruriens, mostly by inhibiting the formation of reactive oxygen species and augmenting liver enzymes involved in normal functioning. Moreover, the extract also prevented aberrant biochemical markers and histological changes evoked by PCM-induced liver toxicity. More exploratory research would be needed to give further clinical validation to the hepatoprotective effects of HAMP extracts against paracetamol-induced liver toxicity.

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