Ameliorative potential of a traditionally used plant Fraxinus micrantha against oxidative stress and paracetamol-induced hepatotoxicity
Hasandeep Singh1, Sarabjit Kaur1, Saroj Arora2, Balbir Singh1
1 Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab, India
2 Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, Punjab, India
|Date of Submission||31-May-2019|
|Date of Decision||12-Aug-2019|
|Date of Web Publication||28-Nov-2019|
Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar - 143 005, Punjab
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Liver disorders are one of the serious health issues. The treatment of liver disorders and their associated complications must be done with care due to the adverse effects of present-day medications. Therefore, there is an urgent need to develop effective and non-toxic herbal drugs for hepatoprotection. Objective: The objective of the present study is to investigate the hepatoprotective potential of traditionally used plant Fraxinus micrantha against paracetamol (PCM)-induced hepatotoxicity in rats. Methods: Hepatotoxicity was induced by a standardized single oral dose of paracetamol (PCM) at 3 g/kg body weight. Standard drug (silymarin 50 mg/kg) and test drugs (chloroform and methanol extracts) were administered orally to rats for 7 days. All the animals were sacrificed on the 8th day, and blood was withdrawn by retro-orbital vein puncture, collected in fresh centrifugation tubes, and centrifuged at 10,000 rpm for 10 min to separate serum for various estimations. Livers were isolated immediately for various biochemical and histopathological studies. Results: Treatment with chloroform extract (200, 400, and 800 mg/kg) and methanol extract (200 and 400 mg/kg) ameliorated the elevated levels of hepatic markers, but a significant reduction in these levels was observed by treatment with methanol extract at 800 mg/kg. In addition, treatment with chloroform and methanol extracts resulted in the amelioration of oxidative stress along with the histopathological changes. High-performance liquid chromatographic analysis of methanol extract of F. micrantha revealed the presence of various polyphenols such as rutin, naringenin, kaempferol, physicion, quercetin, and gallic acid. Conclusion: The results of the present study revealed that the observed hepatoprotective effect of methanol extract of F. micrantha may be due to the presence of various polyphenols and also provides pharmacological evidence for the use of F. micrantha bark in folk medicine for the treatment of liver diseases.
Keywords: Fraxinus micrantha, hepatoprotection, histopathology, polyphenols
|How to cite this article:|
Singh H, Kaur S, Arora S, Singh B. Ameliorative potential of a traditionally used plant Fraxinus micrantha against oxidative stress and paracetamol-induced hepatotoxicity. Phcog Mag 2019;15, Suppl S3:433-9
|How to cite this URL:|
Singh H, Kaur S, Arora S, Singh B. Ameliorative potential of a traditionally used plant Fraxinus micrantha against oxidative stress and paracetamol-induced hepatotoxicity. Phcog Mag [serial online] 2019 [cited 2021 Sep 26];15, Suppl S3:433-9. Available from: http://www.phcog.com/text.asp?2019/15/66/433/271652
- Fraxinus micrantha, a traditionally used medicinal plant exhibited potential hepatoprotective and antioxidant activity against PCM induced hepatotoxicity The bioactive methanol extract of F. micrantha showed the presence of various polyphenols (gallic acid, rutin, naringenin, quercetin, kaempferol and physocion) in HPLC analysis suggesting that these compounds may be responsible for the hepatoprotective activity of plant.
Abbreviations used:ALP: Alkaline phosphatase; AOPPs: Advanced oxidative protein products; CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals; GSH: Reduced glutathione; HPLC: High-performance liquid chromatography; IAEC: Institutional Animal Ethics Committee; SGOT: Serum glutamic-oxaloacetic transaminase; SGPT: Serum glutamic-pyruvic transaminase; TBARS: Thiobarbituric acid reactive substances; TrxR: Thioredoxin reductase.
| Introduction|| |
The liver is a prime and most significant organ of the body associated with detoxification and helps in maintaining the physiological and metabolic homeostasis of an organism., The liver plays an imperative role in the regulation of various physiological processes, secretion, and metabolism. Damage to hepatocytes or hepatotoxicity occurs mainly due to the oxidative stress, multidimensional functions, and various xenobiotics which lead to the distortion of liver functions. Several metabolic disorders are associated with significant hepatotoxicity and can even lead to death. Major risk factors pertinent to cirrhosis, hepatitis, and hepatotoxicity include xenobiotics, pollutants, free radicals, food additives, and alcohol. Liver diseases remain one of the most serious health problems, and their management is still a challenge to present-day medications. The treatment of liver diseases and their associated disorders is necessary and therefore must be done with extensive care. Although the currently available therapies offer liver protection and stimulate liver functions or help in the regeneration of hepatic cells, yet their usage is associated with many adverse effects and can be toxic at particular dose. Therefore, there is an urgent need for the development of effective, non-toxic, indigenous, and inexpensive botanical sources for hepatoprotective crude or purified hepatoprotective drugs.
Fraxinus micrantha is one of the ashes of olive family, Oleaceae, found in Asia mainly in India and Nepal. It is usually found in Himachal Pradesh and Uttar Pradesh region in India.F. micrantha have been studied since time immemorial for its therapeutic and economical value globally. The inner bark of the plant has been used traditionally by the local inhabitants of Dharchula, Himalayas, as an infusion for the treatment of liver enlargement, jaundice, and other liver diseases. Previous studies have reported that ethanol extract of Fraxinus rhynchophylla ameliorated the CCl4-induced liver fibrosis in rats through its antioxidant potential. Furthermore, the leaves and stem bark extract of Fraxinus angustifolia also attenuated the paracetamol- and streptozotocin-induced liver injuries and diabetes. These results suggest that hepatoprotective and antidiabetic activities pertain to the presence of various antioxidant compounds in the plant. Younis et al. have also reported that methanol extract of leaves of Fraxinus xanthoxyloides showed protection against CCl4-induced liver damage due to its antioxidant constituents.In vitro antioxidant activities of extracts of F. floribunda and F. angustifolia have also been reported., The methanol extract of F. micrantha aerial parts also showed marked anti-inflammatory activity in carrageenan-induced inflammation model in mice. It was further shown that oxidative stress and various inflammatory processes might be the causes of various liver disorders., Handa et al. 1986 have shown the effect of some coumarin and flavonoid components of Fraxinus spp. in liver protection. These components cause an increase in the bile secretion and showed low toxicity and marked choleretic effects in various animal models. Based on these earlier findings for the use of Fraxinus species in liver disorders, it was envisaged to explore F. micrantha bark for its hepatoprotective ability in PCM-induced hepatotoxicity.
| Methods|| |
Plant material and extraction
The dried bark of F. micrantha was procured from Sri Venkateswara University, Tripura, and the identity of the plant was confirmed by Dr. K. Madhava Chetty, Assistant Professor, Department of Botany, Sri Venkateswara University, Andhra Pradesh. A voucher specimen number 711 has been deposited in the same department's herbarium. The plant material was coarsely powdered and subjected to successive Soxhlet extraction using different solvents in increasing order of their polarity, viz. petroleum ether, chloroform, methanol, and finally, the marc was digested with water to obtain the aqueous extract. Each extract was concentrated using rotary evaporator (IKA Works Inc., North America) and stored at low temperature, which was further used for pharmacological studies.
Drugs and chemicals
Paracetamol, 1, 1, 3, 3-tetramethoxypropane (Sigma-Aldrich, Bangalore, India), reduced glutathione (GSH) (Loba Chemie, Mumbai, India), thiobarbituric acid (Loba Chemie, Mumbai, India), tris buffer (Merck Specialities, Mumbai, India), glacial acetic acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate (Thermo Fisher Scientific, Mumbai, India) were used in the present study.
Male Wistar rats weighing 200–250 g procured from National Institute of Pharmaceutical Education and Research with no prior drug treatment were employed in the present study. Animals were housed in the animal house of Guru Nanak Dev University with free access to water and laboratory pellet chow diet. The rats were exposed to normal cycle of light and dark. The experimental protocol number 226/CPCSEA-2015-35 was duly approved by the Institutional Animal Ethics Committee of Guru Nanak Dev University, and animals were taken care of as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals.
Preliminary phytochemical screening of extracts
The extracts prepared from the powdered bark of F. micrantha were subjected to preliminary phytochemical screening using standard methods for different classes of phytoconstituents.
Acute toxicity studies
The acute toxicity studies were carried out according to OECD guidelines. The extracts were found to be safe up to 2000 mg/kg. No behavioral, biochemical, and histopathological changes were observed with no mortality. The animals were observed for 14 days, and there was no change in the weight and feed on animals.
Plasmid DNA nicking assay
DNA protective activity was measured using plasmid nicking assay given by Lee et al. 2002. The methanol extract prepared from F. micrantha bark was evaluated for its antioxidant effect by its ability to protect plasmid DNA from the devastating effect of hydroxyl radicals generated by Fenton's reagent.
Induction of hepatotoxicity
Hepatotoxicity was induced by a standardized single oral dose of PCM at 3 g/kg body weight. PCM was freshly prepared and administered to overnight-fasted rats.
Nine groups, each comprising six male Wistar albino rats (200–250 g), were used in the present study. Standard drug (silymarin) and test drugs (chloroform and methanol extracts) were administered orally to rats for 7 days. Tween 80 (0.5% aqueous) was used as a vehicle to prepare the suspension of various test doses.
Group I (normal): Hepatotoxicity was not induced to any animal of this group. Group II (PCM intoxicated): Hepatotoxicity was induced by a single oral dose of paracetamol (PCM) at 3 g/kg. Group III (positive control): Rats with hepatotoxicity were treated with silymarin (50 mg/kg) for 7 days orally. Group IV–VI (chloroform extract – 200, 400, and 800 mg/kg): After induction of hepatotoxicity, chloroform extract of F. micrantha (200, 400, and 800 mg/kg p. o.) was administered for 7 consecutive days. Group VII–IX (methanol extract – 200, 400, and 800 mg/kg): The methanol extract of F. micrantha (200, 400, and 800 mg/kg p. o.) was administered to PCM-intoxicated rats for 7 consecutive days.
Blood was withdrawn under anesthesia (phenobarbital sodium, 40 mg/kg i.p.) by retro-orbital vein puncture, collected in fresh centrifugation tubes, and centrifuged at 10,000 rpm for 10 min to separate serum for various estimations. All the animals under study were sacrificed by cervical dislocation on the 8th day after overnight fasting. Livers were isolated immediately for various biochemical and histopathological studies.
Estimation of liver marker enzymes
Serum parameters such as serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT), alkaline phosphatase, albumin, total cholesterol, and triglycerides were estimated using commercially available kits (Erba Lachema). The concentrations were expressed in milligrams per deciliter.
Estimation of oxidative stress in liver tissues
The quantitative measurement of thiobarbituric acid reactive substances (TBARS) was performed as per previously described method. The results were expressed as nanomoles per milligram of protein. GSH level in tissue was determined using a previously standardized method, and the results were expressed as micrograms of GSH per milligram of protein. The liver advanced oxidative protein products (AOPPs) were determined as standardized by Witko-Sarsat et al. The results of the assay were expressed as micromole per milligram of protein.
Estimation of liver thioredoxin reductase activity
Liver thioredoxin reductase (TrxR) activity was performed according to the method of Holmgren and Bjornstedt. One unit of activity was defined as 1 μM 2-nitro-5-thiobenzoate (TNB) formed per minute per milligram of protein.
The liver tissues were preserved in 10% formalin in a stoppered container. The prepared slides were then examined with the help of a binocular microscope, and pictures were taken with the help of camera fixed on microscope.
High-performance liquid chromatography of methanol extract of Fraxinus micrantha
The methanol extract was subjected to high-performance liquid chromatography (HPLC) at a concentration of 10 mg/ml using HPLC grade acetonitrile and 0.1% formic acid. HPLC apparatus was fitted with RP-18e Chromolith column (5 μm Merck 100–4.6), an efficient degasser, and a photodiode array detector (Shimadzu). Analysis of different compounds was carried out using an isocratic system. Injection volume was 5 μl, flow rate was 0.8 ml/min, and column temperature was 35 degrees.
All the results were expressed as mean ± standard error mean. Data were statistically analyzed by one-way ANOVA followed by Tukey's test using GraphPad Prism Version 7 software. GraphPad Software, 2365 Northside Dr. Suite 560, San Diego, CA. P < 0.05 was considered to be statistically significant.
| Results|| |
Preliminary phytochemical screening of extracts
A preliminary phytochemical screening study showed the presence of lipids and steroids in hexane extract and alkaloids in chloroform extract. Methanol extract gave positive tests for alkaloids, glycosides, and flavonoids. Aqueous extract contained carbohydrates and proteins.
Plasmid DNA nicking assay
The methanol extract of F. micrantha showed no protection at the lowest concentration. However, a dose-dependent protection was observed at higher concentrations which showed a significant hydroxyl radical scavenging activity in plasmid DNA nicking assay [Figure 1].
|Figure 1: Inhibition of DNA scission by methanol extract of Fraxinus micrantha at different concentrations showing percentage amount of supercoiled DNA conserved against OH radicals created by Fentons's reagent (FR). Lane 1: Normal control; Lane 2: Positive control (rutin); Lane 3: Negative control (Fenton's reagent); Lane 4-8: Fenton's reagent + concentrations of extract (50, 100, 250, 500, and 1000 μg/ml)|
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Effect of various interventions on hepatocellular enzyme levels in PCM-induced hepatotoxicity
PCM-induced hepatotoxicity resulted in a significant increase in serum SGPT, SGOT, ALP, Albumin (ALB), triglycerides, and cholesterol levels as compared to the control group. Administration of chloroform and methanol extracts (200, 400, and 800 mg/kg) attenuated the PCM-induced hepatocellular enzyme alterations [Figure 2] and [Figure 3]. However, methanol extract (800 mg/kg) exhibited better protection than chloroform extract in PCM-induced hepatotoxicity.
|Figure 2: The effects of chloroform and methanol extract on serum glutamic-pyruvic transaminase, serum glutamic-oxaloacetic transaminase, and alkaline phosphate levels. Values are expressed as mean ± standard error mean (n = 6).aP < 0.05 versus normal control;bP < 0.05 versus PCM (hepatotoxic)|
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|Figure 3: The effects of chloroform and methanol extract on albumin, triglycerides, and cholesterol levels. Values are expressed as mean ± standard error mean (n = 6).aP < 0.05 versus normal control;bP < 0.05 versus PCM (hepatotoxic)|
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Effect of various interventions on oxidative stress markers in PCM-induced hepatotoxicity
Liver damage by PCM resulted in a significant increase in oxidative stress (decrease in the levels of GSH and increase in the levels of TBARS and AOPPs) when compared to the control group. Oral administration of chloroform and methanol extracts (200, 400, and 800 mg/kg) showed protection against PCM-induced hepatotoxicity, but a significant reduction in oxidative stress was observed in methanol extract at 800 mg/kg [Table 1].
|Table 1: Effect of various interventions of F. micrantha on tissue biomarker changes|
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Effect of various interventions on liver thioredoxin reductase in PCM-induced hepatotoxicity
The levels of TrxR were significantly decreased in PCM-intoxicated group, which were reversed by the treatment with chloroform and methanol extracts (200, 400, and 800 mg/kg) for 7 days. Moreover, methanol extract at 800 mg/kg showed better protection [Table 1].
Histopathological studies were carried out for liver tissues and witnessed all the altered biochemical changes. Normal heptic architecture was observed in normal group [Figure 4]A. As was expected, the histopathology examination of the liver revealed that PCM destroyed the hepatic architecture with necrosis and multiple portal-central bridging [Figure 4]B. Photomicrographs of the liver of silymarin (50 mg/kg)-treated animals showed near-normal architecture with mild inflammation [Figure 4]C. Group IV to VI animals treated with chloroform extract (200, 400, and 800 mg/kg) showed focal and portal inflammation with spotty necrosis [Figure 4]D, [Figure 4]E, [Figure 4]F]. Photomicrographs show confluent necrosis and inflammation in methanol extract at doses of 200 and 400 mg/kg groups [Figure 4]G and [Figure 4]H)]. However, mild inflammation and normal hepatic architecture were observed in 800 mg/kg methanol extract-treated group [Figure 4]I.
|Figure 4: (A) Photomicrographs of liver showing normal hepatic architecture. (B) Necrosis and multiple portal-central bridging by PCM hepatotoxicity. (C) Near-normal architecture with mild inflammation in silymarin-treated group. (D-F) Focal and portal inflammation in animals treated with chloroform extract (200, 400, and 800 mg/kg). (G and H) Photomicrographs show confluent necrosis and inflammation in methanol extract at doses of 200 and 400 mg/kg groups,. (I) Mild inflammation and normal hepatic architecture in 800 mg/kg methanol extract-treated group (H and E, ×100 and Reticulin, ×100)|
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High-performance liquid chromatography of bioactive methanol extract of Fraxinus micrantha
HPLC results of the bioactive methanol extract revealed the presence of rutin, naringenin, kaempferol, Physicion, quercetin, and gallic acid [Table 2].
|Table 2: Polyphenolic compounds detected by high-performance liquid chromatography in methanol extract of Fraxinus micrantha|
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| Discussion|| |
Acetaminophen or paracetamol (PCM) is a derivative of para-aminophenol which is commonly used as an analgesic and antipyretic drug. PCM is a well-known nonsteroidal anti-inflammatory drug which is toxic at larger dose and causes acute liver damage and hepatic necrosis. The mechanism by which PCM causes hepatotoxicity has been attributed to the formation of highly toxic metabolite N-acetyl-p-benzoquinone imine which further causes oxidative stress and depletion of glutathione.,
In the present study, a single oral dose of PCM (3 g/kg) resulted in significant hepatic damage as demonstrated by pertinent biochemical and histopathological changes. PCM treatment significantly increased the levels of SGPT, SGOT, ALP, ALB, triglycerides, and total cholesterol pertinent to liver damage as they leak into the blood due to hepatotoxicity.
Treatment with chloroform extract (200, 400, and 800 mg/kg) and methanol extract (200 and 400 mg/kg) moderately ameliorated the elevated levels of SGPT, SGOT, ALP, ALB, triglycerides, and total cholesterol, but a significant reduction in these levels was observed by treatment with the highest dose of methanol extract (800 mg/kg). This reversal of hepatic biomarkers by F. micrantha treatment may be due to the membrane stabilization, hepatocyte regeneration, and thereby preventing their escape which is in line with previous studies.
It is further supposed that the main culprit owing to the hepatic damage is lipid peroxidation due to the administration of PCM. The increase in the level of TBARS and decreased GSH levels in liver pertains to enhanced lipid peroxidation due to the failure of antioxidative defense mechanism. GSH is the abundant intracellular antioxidant which is involved in the protection against oxidative stress damage and also in diverse detoxification mechanisms. This further leads to the degradation of cellular macromolecules and causes tissue damage. Furthermore, PCM treatment demonstrated the increased levels of AOPPs (products of oxidative modification of proteins) due to oxidative stress modification of proteins, thereby affecting various cellular metabolisms., Moreover, a decline in TrxR levels observed here with PCM-induced toxicity are in line with previous findings of Erkekoglu et al. who documented that inhibition of TrxR in di(2-ethylhexyl) phthalate-induced hepatotoxicity leads to reduction in the activity of enzymes that are dependent on thioredoxin, thereby decrease the free radical scavenging ability, and lead to oxidative stress, apoptosis, and necrosis.
Treatment with chloroform (200, 400, and 800 mg/kg) and methanol extract (200 and 400 mg/kg) resulted in the amelioration of oxidative stress. However, a marked protection against oxidative stress was observed in animals treated with methanol extract 800 mg/kg. These observed improvements in the above oxidative stress markers indicate the protective role of F. micrantha against hepatic damage.
The induction of toxicity by PCM and hepatoprotective effect of F. micrantha extracts as evident from serum and tissue biochemical analysis is further supported by the histopathological observations. PCM intoxication significantly leads to the destruction of hepatic architecture along with necrosis, inflammation, and portal-to-central bridging. Treatment with F. micrantha continuously for 7 days not only reduced the inflammation and necrosis in liver tissues as compared to PCM-intoxicated group but also significantly decreased the morphological changes in liver tissues leading to the normal hepatic architecture.
Phytochemical screening of methanol extract of F. micrantha showed the presence of various polyphenols. The HPLC analysis of methanol extract of F. micrantha revealed the presence of various polyphenols such as rutin, naringenin, kaempferol, physocion, quercetin, and gallic acid [Table 2]. The methanol extract of F. micrantha also showed a significant antioxidant activity in plasmid DNA nicking assay.
Many research groups have demonstrated that free radical scavenging activity is an important mechanism for hepatoprotective activity. Various polyphenols from plants such as Cichorium glandulosum, apple pomace, Folium microcos, and Cleome viscosa are reported to exert protective effects in liver toxicity due to their antioxidative potential.,,, Gowri et al. 2008 have also reported that polyphenols from Commiphora berryi are responsible for hepatoprotection and decrease the oxidative stress. Our results also suggest that the observed hepatoprotective effect of methanol extract of F. micrantha pertains to the presence of various polyphenols. The present study provides a pharmacological evidence for the appropriate use of F. micrantha bark in folk medicine for the treatment of liver diseases.
| Conclusion|| |
The results of the present investigation suggest that the various polyphenols present in the methanol extract of F. micrantha bark might be responsible for the hepatoprotective activity due to their potent antioxidant effect. Further studies are required to ascertain the underlying mechanism of F. micrantha in hepatoprotection.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]