INTRODUCTION:

Acute liver failure (ALF) occurs following sudden extensive loss of liver cell mass, resulting in hepatic encephalopathy (HE) and coagulopathy, and can lead to multiple organ failure with a high associated mortality rate. Previous studies have highlighted the major contribution of paracetamol (acetaminophen) as a cause of ALF in the UK, North America and Europe. However, there are significant differences in the epidemiology of paracetamol-induced ALF in North America compared with the UK.

Data from the USA, covering 1990–99, suggested paracetamol overdose was responsible for 56000 emergency department visits, 26000 hospital admissions and 458 deaths each year. Approximately 12 650 (22.6%) emergency department visits, 2240(8.6%) admissions and 100(21.8%) deaths were due to unintentional paracetamol ingestion^1-3. Against this background the US Acute Liver Failure Study group reported that unintentional overdose was the most common pattern of ingestion in patients with ALF, responsible for 48% of all overdoses, and reported similar outcomes between intentional and unintentional overdoses. These data contrast with the pattern of overdose reported in the UK. In the King’s College Hospital series, 92% of paracetamol-induced ALF occurred after ingestion at a single time point with suicidal intent. Contradictory data have also been presented regarding the outcome of ALF induced by accidental (unintentional) overdose of paracetamol, with increased mortality or similar outcomes, being reported ⁴. Lastly, in those series reporting increased mortality in patients following accidental paracetamol poisoning, it is unclear if this excess mortality is associated with this pattern of overdose per se or other clinical features such as organ failure or alcohol consumption prevalent in this population⁵. Paracetamol-related hepatotoxicity is now the most common cause of the potentially devastating clinical syndrome of acute liver failure in many western countries. Most such instances are the consequence of ingestion of large paracetamol overdoses, often taken at a single time-point with suicidal or parasuicidal intent⁵. Cases of severe hepatotoxicity following modest overdoses of up to 10g daily, or even after ingestion of recommended doses (up to 4g daily), have also been reported in patients taking paracetamol for therapeutic purposes, mostly over several days to weeks, in association with concomitant chronic alcohol exposure or use of other cytochrome P450 enzyme-inducing drugs. In patients who develop liver damage following moderate paracetamol overdose up to 10g daily, recent fasting and nutritional impairment have been identified as key precipitants⁶.

A. paniculata contains diterpenes, lactones, and flavonoids. Flavonoids mainly exist in the root, but have also been isolated from the leaves. "e aerial parts contain alkanes, ketones, and aldehydes. Although it was initially thought that the bitter substance in the leaves was the lactone andrographolide, later investigations showed that the leaves contained two bitter principles – andrographolide and a compound named kalmeghin. Four lactones – chuanxinlian A (deoxyandrographolide), B (andrographolide), C (neoandrographolide) and D (14-deoxy-11, 12-didehydroandrographolide) – were isolated from the aerial parts⁷.

Green unripe fruits contain glycoalkaloids and their eating is a toxic to human being as well as livestock that include solamargine, solasonine, solanine, α and β-solamagrine, solasodinsolanidine (0.09-0.65%). The former two also found in leaves. Solanine is found in all parts of the plants, with the level increasing as the plant matures, though it is apparently modified by soil type and climate⁸.

The medicinal plant Phyllanthus niruri Linn. (Euphorbiaceae), its wide variety of phytochemicals and their pharmacological properties. The active phytochemicals, flavonoids, alkaloids, terpenoids, lignans, polyphenols, tannins, coumarins and saponins, have been identified from various parts of Phyllanthus niruri. Extracts of this herb have been proven to have therapeutic effects in many clinical studies⁹.

O-methylpongamol, lanceolatin B, (+) purpurin, maackiain5 Flowers Delphinidin chloride, cyaniding, karanjin, kanjon, Leaves Lupeol, Rutin, Rotenoid, triterpenoid and beta-sitosterol, Pods and seeds Purpurin, purpuritenin A, B, tephroglabrin, tepurinidiol, purpureamethide, O-methylpongamol, sitosterol, Rotenoid, Diketone-pongamol, isolonchocarpin, furanoflavones karanjin and kanjone, flavanone purpurin. A flavonoid, lanceolarin B, Root Purpurenone, purpurin, dehydroisoderricin, maackiain, new epoxflavanone; pongamol, flemichapparins B and C, rutin, methylkaranjic acid, sitosterol, spinasterol, lanceolatin A, lanceolatin B5, Stem 7-O- [ Beta-D-glucopyranosyl-(1-4)-O-BETA-D-galactopyranoside, Aerial parts Tephrosin, pongaglabol, semiglabrin¹⁰.

Most of the known chemical constituents in H. antidysentrica have been found in the stem, bark, leaves and a few in the seeds as well. The major constituents are steroidal alkaloids, flavonoids, triterpenoids, phenolic acids, tannin, resin, coumarins, saponins and ergostenol. The 68 alkaloids which have been discovered from various parts of H. antidysenterica¹¹.

The plant mainly contains alkaloids, glycosides, steroids, sesquiterpenoid, aliphatic compound, essential oils, mixture of fatty acids and polysaccharides. The alkaloids include berberine, bitter gilonin, non-glycoside gilonin gilosterol. The major phytoconstituent in Tinospora cordifolia include tinosporine, tinosporide, tinosporaside, cordifolide, cordifol, heptacosanol, clerodane furano diterpene, diterpenoid furanolactone tinosporidine, columbin and b-sitosterol. Berberine, Palmatine, Tembertarine, Magniflorine, Choline, and Tinosporin are reported from its stem¹².

MATERIALS AND METHOD:

Animal:

A total number of 24 healthy albino wistar rats of 20 weeks and weighing 200±40grams were housed into cages for 15 days. They were housed in a controlled room temperature environment (25±2⁰C) and light with alternate 12-hour light/dark cycles. The animals had free access to rat’s pellets (Keval sales corporation) and water ad libitum. After 15 days of acclimatization, rats were randomly divided into four experimental groups of six rats each groups.

Ethanol induced hepatotoxicity:

Rats were divided into four groups each containing 6 animals. Group first is normal group received the normal saline 10ml/kg daily for 21^th days. Group second negative control this group received ethanol 12 ml/kg bw per day is standard dose of this group is Silymarin (100 mg/kg) daily for 21^th days and in 21^th day administered ethanol. Group third is standard dose of this group is Silymarin (100mg/kg) daily for 21^th days and in 21^th day administered ethanol. Silymarin and ethanol is administered orally. Fourth group is treated with ethanol + livina (Herbal capsule). Animals were sacrificed by anaesthesia after 16 hours of ethanol administration, the liver perfused with saline was dissected out and processed immediately for biochemical parameters¹³.

Table 1: Ethanol induced hepatotoxicity

Groups	Treatment groups
Group I	Normal saline 1ml/kg bw
Group II	Inducer Ethanol 12mL/kg bw (70%)
Group III	Ethanol 12ml/kg bw + silymarin (100mg/kg bw)
Group IV	Ethanol 12mL/kg bw (70%) + Livina (200mg/kg bw)

Biochemical studies:

The biochemical parameters were determined after 24 hour fasting after administration of last dose of treatment. Blood was obtained from all animal by puncturing retro-orbital plexus. The blood sample is allowed to for 45 minute at room temperature. Serum was separated by centrifugation at 7200rpm at 4⁰C for 15 minute and utilized for the estimation of various bio-chemical parameters namely SGPT, SGOT, ALP, and serum bilirubin.

ALP (Alkaline phosphatase):

At pH 10.3 Alkaline phosphatase (ALP) catalyses the hydrolysis of colourless p-Nitrophenyl phosphate (pNPP) to yellow coloured p-Nitrophenol and phosphate. Change in absorbance due to yellow colour formation is measured kinetically at 405nm and is proportional to ALP activity in the sample¹².

Working reagent preparation:

Prepare “Working reagent” by reconstituting one vial of Reagent 2 (pNPP substrate) with 5.5mL Reagent 1 (AMP buffer) were added¹¹.

To 20μL of animal serum sample added 1000μL of Reagent A and mixed well. The mixture is incubated at the assay temperature of 37⁰C for 1minute. The absorbance was read after 30 seconds. Repeat reading after every 30 seconds i.e. upto 120 seconds at 405nm wavelength. The mean absorbance change per minute was calculated.

AST (Aspartate Transaminase) or SGOT (Glutamic-oxalacetic transaminase):

Aspartate Transaminase (AST) catalyses the transamination of L-Aspartate and α-ketoglutarate to form L-Glutamate and Oxaloacetate. In subsequent reaction, Malate Dehydrogenase (MDH) reduces Oxaloacetate to Malate with simultaneous oxidation of Nicotinamide Adenine Dinucleotide (reduced) (NADH) to Nicotinamide Adenine Dinucleotide (NAD). The rate of oxidation of NADH is measured kinetically by monitoring the decrease in absorbance at 340nm and is directly proportional to AST activity in the sample. Lactate Dehydrogenase (LD) is added to enzyme system to prevent endogenous pyruvate interference, which is normally present in the serum¹⁴.

Working reagent preparation:

To 100μL of animal serum sample added 1000μL of Reagent 1 and mixed well. The mixture is incubated at the assay temperature of 37⁰C for 1minute. Then added 1mL of Reagent 2 and mixed well. The absorbance read after 60 seconds. Repeat reading after every 30 second i.e. up to 120 seconds at 340nm wavelength. The mean absorbance change per minute was calculated.

Calculation:

AST activity (IU/L) = Δ A/minute × Kinetic factor

Where Δ A/minute = Change in absorbance per minute, Kinetic factor (K) = 1768.

ALT (Alanine Transaminase) or Glutamic-pyruvic transaminase (SGPT)

Alanine Transaminase (ALT) catalyses the transamination of L-Alanine and α-Ketoglutarate to form pyruvate and L-Glutamate. In subsequent reaction, Lactate Dehydrogenase (LD) reduces pyruvate to Lactate with simultaneous oxidation of Nicotinamide Adenine Dinucleotide [reduced] (NADH) to Nicotinamide Adenine Dinucleotide (NAD). The rate of oxidation of NADH is measured kinetically by monitoring the decrease in absorbance at 340nm. LD rapidly and completely reduces endogenous sample pyruvate during the initial incubation period, so that it does not interfere with the assay.

Working reagent preparation:

To 100μL of animal serum sample added 1000μL of working ALT Reagent 1 and mixed well. The mixture is incubated at 37⁰C for 1minute. The absorbance was read after 60 seconds. Repeat reading after every 30 seconds i.e. up to 120 seconds at 340nm wavelength. The mean absorbance change per minute was calculated.

Calculation:

ALT activity (IU/L) = Δ A/minute × Kinetic factor

Where Δ A/minute = Change in absorbance per minute, Kinetic factor (K) = 1768.

Total Bilirubin:

Total bilirubin in serum or plasma is determined using jendrassik and Grof method by coupling with diazotized sulfanilic acid after addition of caffeine, sodium benzoate and sodium acetate. A blue azobilirubin is formed in alkaline Fehling solution II, which is measured photometrically¹⁵.

Reagent preparation:

Mix Reagent 1 and Reagent 2 in the ratio of 4 + 1 (e. g. 400µl of sulfanilic acid solution and 100µl of sodium nitrite solution) to make a diazo solution. The mixing ratio should be observed exactly. Always use freshly prepared diazo solution. The absorbance was read at 546 nm wavelength¹⁵.

Total bilirubin concentration = A × 10.3 mg/d

Serum Glutamic Pyruvic Transaminase Test (SGPT)

Table 2: Serum Glutamic Pyruvic Transaminase Test results

S. No	Treatment Group	Results (Mean±SD)
1.	Normal saline 1ml/kg bw	46.852±29.238
2.	Inducer Ethanol 12ml/kg bw (70%)	168.02±33.134
3.	Ethanol 12ml/kg bw + silymarin (100 mg/kg bw)	60.996±3.307
4.	Ethanol 12ml/kg bw (70%) + Livina (200 mg/kg bw)	55.102±38.87

Fig. 1: Bar graph represents the Serum Glutamic Pyruvic Transaminase test results Serum Glutamic-Oxaloacetic Transaminase Test (SGOT)

Table 2: Serum Glutamic-Oxaloacetic Transaminase Test results

S. No	Treatment Group	Results (Mean±SD)
1.	Normal saline 1ml/kg bw	48.914±3.819
2.	Inducer Ethanol 12ml/kg bw (70%)	99.892±3.307
3.	Ethanol 12ml/kg bw + silymarin (100 mg/kg bw)	55.102±4.095
4.	Ethanol 12ml/kg bw (70%) + Livina (200 mg/kg bw)	53.04±4.183

Fig. 2: Bar graph represents the Serum Glutamic-Oxaloacetic Transaminase test results Alkaline Phosphatase (ALP) Test

Table 4: Alkaline Phosphatase (ALP) Test results

S. No	Treatment Group	Results (Mean±SD)
1.	Normal saline 1ml/kg bw	14.916±5.073
2.	Inducer Ethanol 12ml/kg bw (70%)	47.46±5.073
3.	Ethanol 12ml/kg bw + silymarin (100 mg/kg bw)	17.176±5.8585
4.	Ethanol 12ml/kg bw (70%) + Livina (200 mg/kg bw)	19.888±5.858

Fig. 3: Bar graph represents the Alkaline Phosphatase (ALP)test results Total Bilirubin Test

Table 5: Total Bilirubin Test results

S. No	Treatment Group	Results (Mean±SD)
1.	Normal saline 1ml/kg bw	1.0098±0.283
2.	Inducer Ethanol 12ml/kg bw (70%)	1.133±1.2082
3.	Ethanol 12ml/kg bw + silymarin (100 mg/kg bw)	0.3705±0.392
4.	Ethanol 12ml/kg bw (70%) + Livina (200mg/kg bw)	0.824±0.654

Fig. 4: Bar graph represents the total bilirubin test results

Histopathology:

Group I

Normal saline 1ml/kg bw

Fig. 5: Photomicrographs of liver of rats showing Kuppfer cells (KC), hepatocytes, Central Vein (CV), Sinusoids

Group II

Inducer Ethanol 12ml/kg bw (70%)

Fig. 6: Photomicrographs of liver of rats showing inflammation, necrosis and damaged vascular endothelium

Group III

Ethanol 12ml/kg bw + silymarin (100 mg/kg bw)

Fig. 7: Photomicrographs of liver of rats showing hepatocytes, branch of hepatic artery, kupper cells, blood vessels, bile ductule, sinusoids

Group IV

Ethanol 12ml/kg bw (70%) + Livina (200 mg/kg bw)

Fig. 7: Photomicrographs of liver of rats showing kuppfer cells, bile ductile, Central vein, hepatocytes, endothelium

DISCUSSION:

The study reveals the hepatoprotective activity of Livina capsule in ethanol induced rat model. Treatment of rat with ethanol (12ml/kg bw) increased lipid peroxidation as shown by elevated MDA levels in serum and liver tissues. This situation suggests the induction of oxidative stress in cells. The application of multi herbal medicine (Livina®) significantly reduced the serum MDA level. The treatment also reduced the hepatic and renal MDA levels which indicate that this herbal medicine maintains the normal fluidity of the cell membrane which plays a vital role in cell functioning⁸.

The ability of a hepatoprotective drug to reduce the injurious effects or to preserve the normal hepatic physiological mechanisms, which have been disturbed by a hepatotoxin, is the index of its protective effects. Although serum enzyme levels and ascorbic acid in urine are not a direct measure of hepatic injury, they show the status of the liver. The lowering of enzymes level is definite indication of hepatoprotective action of the drug ¹⁰.

The present investigation also revealed that the given dose of ethanol (12ml/kg bw) (70%) produced significant elevation in SGPT, SGOT, ALP, bilirubin (direct and total) indicating all impaired liver function and these parameters have been reported to sensitive indicator of liver injury¹¹. The present study reveals that the effect of pre-treatment of livina capsule had been effective in offering protection, which is comparable to Silymarin.

A very important observation with livina capsule dose of 200mg/kg is highly effective in decreasing the elevated level of serum total bilirubin (0.824mg/dl). Biochemical test of Alkaline Phosphatase (ALP) Test showed results decreasing in the level 19.888IU/L. Serum Glutamic-Oxaloacetic Transaminase Test decreasing in the level as compared to induced 53.04 IU/Serum Glutamic Pyruvic Transaminase decreasing in the level as compared to induced and nearest to the standard 55.102 IU/L.

The histopathological studies are the evidence of efficacy of drug as protectant. Simultaneous treatment of Livina capsule with ethanol exhibits less damage to the hepatic cells as compared to the rats treated with ethanol alone. Intralobular veins though are damaged but to a lesser extent. Endothelium is disrupted at places. Hepatic cells adjoining to intralobular vein show atrophy. The sections of the liver treated with Livina capsule and ethanol reveals better hepatoprotective activity. Almost negligible damage to a few hepatocytes present in the close vicinity of intralobular vein is observed. Endothelium lining is almost smooth except one or two places. Hepatocytes show normal appearance only some cells show higher numbers of vacuoles in the cytoplasm. The results of histopathological study also support the results of biochemical parameters.

Ascorbic acid is formed as a metabolite of glucose and galactose in rat liver microsomes via the glucoronic acid pathway and is excreted in urine. The enzyme UDP glucose dehydrogenase and UDP glucuronide transferase are responsible for its formation in the liver microsomes. Its formation and excretion is altered by several drugs and substances that affect the drug metabolizing enzyme systems. Reduction in ascorbic acid excretion in CCl4 treated rats may reflect the inhibition of such enzymes. Alteration in urinary ascorbic acid excretion appears to be reflecting ascorbic acid level in liver.

CONCLUSION:

The plant kingdom represents a rich storehouse of organic compounds, many of which have been used for medicinal purposes and could serve as lead for the development of novel agents having good efficacy in various pathological disorders. Treatment with multi herbal medicine normalized the serum and tissue enzyme activities by suppression of extensive generation of toxic during hepatic injury. So, Livina® capsule composed of various medicinal herbs may be a potent drug that sounds for the prevention of cellular oxidative stress.

The shade dried powder of different herbal plants were used by which livina capsule was prepared consist of bioactive components. These components like carbohydrates, glycosides, flavonoids, alkaloids, triterpenoids and phenolic components are present in the Livina . Possess significant effect on hepatoprotective activity. Experimental results have revealed that Livina capsule have various degrees of hepatoprotective activity depending upon the dose level and the bioactive components present in it.

Thus, from the present study concluded that the Livina capsule levels higher hepatoprotective activity. With respect to this study, the findings showed that the treatment of Livina capsule could maintain or decrease the level of total bilirubin, SGOT, SGPT and ALP. This has given us knowledge of the possible role of enzyme in protecting the liver lesion and reduces in-vivo hepatotoxicity in liver. Present study supports the use of Livina capsule by local healers as traditional medicine in treatment of liver disorder. This effect can be attributed to presence of various bioactive components present on extract and also be due to protective potential of extract confirm the mechanism of cation behind the ethanol induced potential of Livina capsule.

ACKNOWLEDGEMENT:

Authors are very thankful to Dr. Azaz Khan Director, Pinnacale Biomedical Research Institute Bhopal and entire team of PBRI who support to complete this research work successfully.

REFERENCES:

1. Muthukumaran P. Hazeena Begum V. Effect of Poorna Chandrodayam Chendooram (PCM - Metallic Drug) on Lipid Profile, Liver Function and Kidney Function Parameters of Rats. Asian Journal of Pharmaceutical Analysis. 2020; 10(1): 27-31

2. Suraj JP. Shivani DP. Pratibha BP. Pranali SP. Ganesh BV. Indryani DR. Evaluation of Standardization Parameters of Ayurvedic Marketed Polyherbal Formulation. Asian Journal of Pharmaceutical Analysis. 2020; 8(4): 220-226

3. Sathish R. Sravan Kumar P. Natarajan K. Sridhar N. Hepatoprotective and Anti Pyretic Activities of Methanolic Extract of Butea Monosperma Lam Stem Bark in Wister Rats Asian J. Pharm. Res. 2011; 1: 130-133.

4. Malathi SR. Ahamed J. Cholarajan A. Tylophora asthmatica L. Prevents Lipid Peroxidation in Acetaminophen Induced Hepato Toxicity in Rats, Asian J. Res. Pharm. Sci. 2011; 1: 71-73.

5. Kanakam V. Goli V. Bhaskar J. Srisailam K. More S. Antioxidant and Hepatoprotective Effects of the Methanol Extract of the Flowers of Tamarindus indica. Asian J. Pharm. Tech. 2011; 1(3): 73-78.

6. Sahu SK. Das D. Tripathy NK. Hepatoprotective activity of aerial part of Glinus oppositifolius L. against Paracetamol-induced Hepatic Injury in Rats. Asian J. Pharm. Tech. 2012; 2: (4): 154-156

7. Anurag N. Pathak AK. Ankur C. Priya S. Yogesh S. Evaluation of a Polyherbal Preparation for Wound Healing Activity. Research J. Pharmacology and Pharmacodynamics, 2010; 2(5): 340-342.

8. Vilas A. Arsul RO. Ganjiwale PG. Yeole. Development and Validation of Phyllanthin by HPTLC in Hepatoprotective Polyherbal Tablet Dosage Form. Asian J. Research Chem. 2011; 4(5): 815-817.

9. Rajesh MG. Latha MS. Hepatoprotective Activity of Glycyrrhiza glabra Linn. on Experimental Liver Damage in Albino Rats. Research J. Pharmacognosy and Phytochemistry, 2010; 2(4): 313-316.

10. Pradnya NJ. Prachali RC, Suhas, S. Pharmacological Evaluation of Livodac Tablet on Ethanol Induced Hepatotoxicity Models in Wistar Rats. Research Journal of Pharmacology and Pharmacodynamics. 2020; 12(2): 91-97

11. Asoh S. Mori T. Nagai S. Yamagata K. Nishimaki K. Miyato Y. Ohta S. Zonal necrosis prevented by transduction of the artificial anti-death FNK protein. Cell Death & Differentiation, 2005; 12(4), 384-394.

12. Badar VA. Thawani VR. Wakode PT. Shrivastava MP. Gharpure KJ. Hingorani LL. Khiyani RM. Efficacy of Tinospora cordifolia in allergic rhinitis. Journal of Ethnopharmacology, 2005; 96(3), 445-449.

13. Bagheri SM. Zare-Mohazabieh F. Momeni-Asl H. Yadegari M. Mirjalili A. Anvari M. Anti liver and hepatoprotective effects of aqueous extract of Plantago ovata seed on -ulcerated rats. Biomedical Journal, 2018; 41(1), 41-45.

14. Bennett WM. Drug nephrotoxicity: an overview. Renal Failure, 1997; 19(2), 221-224.

15. Bhandari U. Shamsher AA. Pillai KK. Khan MSY. Antihepatotoxic activity of ginger ethanol extract in rats. Pharmaceutical Biology, 2003; 41(1), 68-71.

Received on 04.03.2022 Modified on 20.04.2022

Asian J. Res. Pharm. Sci. 2022; 12(3):171-176.

DOI: 10.52711/2231-5659.2022.00029