|Year : 2015 | Volume
| Issue : 3 | Page : 311-315
Hepatoprotection by active fractions from Desmostachya bipinnata stapf (L.) against tamoxifen-induced hepatotoxicity
Kalpana Pravin Rahate1, A Rajasekaran2
1 SKB College of Pharmacy, RTM Nagpur University, Kamptee, Maharashtra, India
2 Research Scholar, Karpagam University, Coimbatore, Tamilnadu, India
|Date of Submission||24-May-2014|
|Date of Decision||06-Jun-2014|
|Date of Acceptance||12-Apr-2015|
|Date of Web Publication||18-May-2015|
Dr. Kalpana Pravin Rahate
SKB College of Pharmacy, RTM Nagpur University, Kamptee, Maharashtra
Source of Support: None, Conflict of Interest: None
Objective: The aim was to evaluate the effect of the polyphenolic fraction of Desmostachya bipinnata Stapf (PFDB) (Poaceae) on tamoxifen (TAM)-induced liver damage in female Sprague-Dawley rats.
Materials and Methods: The roots of Desmostachya bipinnata were extracted in 70% methanol, and the polyphenolic fraction was isolated. Protection of BRL3A cells against ethanol-induced damage was determined by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Hepatotoxicity was induced in rats by oral administration of TAM (45 mg/kg/day) for 21 days. The PFDB was administered to experimental animals at two selected doses (100 and 200 mg/kg/day) during the treatment. The serum levels of various biochemical parameters and the antioxidant enzymes were examined by standard procedures.
Results: A dose-dependent increase in percentage viability was observed when ethanol-exposed BRL3A cells were treated with PFDB. Both the treatment groups upon pretreatment with PFDB exhibited a significant (P ≤ 0.05) protective effect by lowering serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, triglycerides, cholesterol, urea, uric acid, bilirubin and creatinin levels and improving protein level in serum in dose-dependent manner, which was comparable to that of silymarin group. In addition, PFDB prevented elevation of reduced glutathione, glutathione peroxidase, superoxide dismutase and catalase in the TAM-intoxicated rats in concentration-dependent manner and significantly (P < 0.05) reduced the lipid peroxidation in the liver tissue. The biochemical observations were supplemented with histopathological reports, which showed the attenuation of hepatocellular necrosis.
Conclusions: The results of this study strongly indicate that the polyphenolic fraction of the plant roots has a potent hepatoprotective action against TAM-induced hepatic damage in rats.
Keywords: Biochemical, Desmostachya bipinnata stapf, hepatotoxicity, histopathological
|How to cite this article:|
Rahate KP, Rajasekaran A. Hepatoprotection by active fractions from Desmostachya bipinnata stapf (L.) against tamoxifen-induced hepatotoxicity. Indian J Pharmacol 2015;47:311-5
|How to cite this URL:|
Rahate KP, Rajasekaran A. Hepatoprotection by active fractions from Desmostachya bipinnata stapf (L.) against tamoxifen-induced hepatotoxicity. Indian J Pharmacol [serial online] 2015 [cited 2020 Apr 8];47:311-5. Available from: http://www.ijp-online.com/text.asp?2015/47/3/311/157130
| » Introduction|| |
Hepatic system is very vital organ system which clears toxins from the body, processes food nutrients and is involved in the body's metabolic activities. It has become a frequent and primary target for several toxicants.  Excessive exposure to xenobiotics, fat accumulation, alcohol misuse, viral infection, iron or copper accumulation and free radicals generated during metabolism lead to hepatic damage and cause jaundice, cirrhosis and fatty liver, which remain one of the serious health problems.
Drug-induced injury is a potential complication of several medicines. Tamoxifen (TAM) citrate, (1-[4-(2-dimethyl-aminoethoxy) phenyl]-1,2-diphenyl-1-butene), is a nonsteroidal antiestrogen drug that is used in the treatment and prevention of all stages of hormone-dependent breast cancer.  It was revealed that, TAM in high dose is a known liver carcinogen in rats, which are due to oxygen radical overproduction, which occurs during TAM metabolism. Lipid peroxidation (LPX) via subtraction of hydrogen from unsaturated fatty acids forms carbon-cantered lipid radicals.  This molecule adds molecular oxygen to form lipid peroxy radicals, thereby initiating the process of LPX. Furthermore, TAM has been shown to potentiate nitrous oxide production in breast cancer patients through enhancement of nitric oxide synthase II expression. 
Due to lack of reliable liver protection drugs in modern medicine, a large number of medicinal preparations are recommended for the treatment of liver disorders. Hence, it is essential to unfold the scientific basis of the antihepatotoxic nature of the traditionally useful herbal drugs. It is reported that hepatoprotective and antioxidant activity by scavenging free radicals are probably due to phenolic/flavonoid compounds present in the plant.  Desmostachya bipinnata Stapf (family: Poaceae), a perennial grass, is commonly known as "Sacrificial Grass" in English and "Darbha" in Sanskrit. It pacifies pitta, asthma. In Indian traditional medicine, it is used as diuretic and in diarrhoea, dysentery, menorrhagia, jaundice, skin diseases, burning sensation and excessive perspiration.  The plant showed the presence of kaempferol, quercetin, quercetin-3-glucoside, trycin, trycin-7-glucoside,  and 4'- methoxy quercetin-7-O-glucoside.  It is reported to exhibit antiulcerogenic,  antihelicobacter,  antidiarrheal,  analgesic, antipyretic and anti-inflammatory effects.  In our work, we made an attempt to study the hepatoprotective activity of the polyphenolic fraction of this plant.
| » Materials and Methods|| |
All chemicals were obtained from S D Fine Chemicals, Mumbai. 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Silymarin (Standard) were purchased from Sigma Chemical Co., St. Loius, MO, USA.
The plant D. bipinnata Stapf (Poaceae) was collected in the month of October from different places of Tuticorin in Tamil Nadu. The plant was authenticated by The Joint Director, Botanical Survey of India, Coimbatore, India (Authentication No: BSI/SRC/5/23/Tech. 681).
Isolation of Polyphenolic Fraction of Desmostachya Bipinnata
The root powder of D. bipinnata Stapf (Poaceae) (1.4 Kg) was extracted in 70% methanol (2 × 3 L) in soxhlet apparatus. Fractionation of the extract (125 g) was carried out by column chromatography gradient elution using benzene, chloroform, ethyl acetate and methanol (100:0, 80:20, 60:40, 40:60, 20:80, 0:100). Separation of the active constituents was monitored by running thin-layer chromatography of all the fractions using number of mobile phases and the fractions showing similar movements were pooled into three major fractions, E 1 ( 6.9 g), E 2 ( 13.6 g) and E 3 ( 15.2 g). High performance thin-layer chromatography (HPTLC) screening of these fractions revealed the presence of two flavonoids in fractions E 1 and E 3 (apigenin, luteolin and luteolin, quercetin, respectively) and three flavonoids in fraction E 2 (apigenin, luteolin and quercetin). Hence, fraction E 2 was selected to determine hepatoprotective potential.
In vitro Hepatoprotective Effect on BRL3A Cells
The screening of hepatoprotective activity was based on the protection of BRL3A cells against ethanol-induced damage determined by estimating mitochondrial synthesis using the tetrazolium assay.  The BRL3A cells were routinely grown and subcultured as monolayers in Dulbecco's modification of Eagle medium supplemented with 10% fetal bovine serum. The cells were harvested and plated at approximately 10,000 cells/well in 96 well-microtiter plates and left to rest for 24 h at 37°C in a humidified atmosphere of 5% CO 2 . The cells were then exposed to toxicant (medium containing 100 mM ethanol) along with/without a polyphenolic fraction of Desmostachya bipinnata (PFDB). Concentrations of PFDB >650 μg/ml and that of standard drug Silymarin >250 μg/ml were found to be toxic to the cells; hence, concentrations in the range of 125-500 μg/ml were used for PFDB and 250 μg/ml were used for the standard Silymarin. At the end of the period, cytotoxicity was assessed by estimating the viability of the BRL3A cells by the MTT reduction assay. After 24 h incubation, the test solution from each well was removed by aspiration and replaced with 50 μl of MTT. The plates were gently shaken and incubated for 3 h at 37°C in a humidified 5% CO 2 atmosphere. The supernatant was removed, and 100 μl propanol was added and the plates were gently shaken to solubilise the formed formazan. The absorbance was measured using a microplate reader at 540 nm. 
In vivo Hepatoprotective Activity on Tamoxifen-Induced Hepatotoxicity
Female Sprague-Dawley rats (150-200 g) were used for the study. The experimental protocol was approved by the institutional animal ethical committee at KMCH College of Pharmacy, Coimbatore. The study was performed in accordance with CPCSEA guidelines. To establish the safety of the drug, acute toxicity study was carried out as per OECD-423 guidelines. It was observed that all animals tolerated mass test doses and showed no ill effects at LD50 >2000 mg/kg. Dose of PFDB was fixed at 100 and 200 mg/kg, whereas for standard silymarin at 45 mg/kg. The animals were divided into five groups of six animals in each group. Group I served as control received normal saline and was not treated with the toxicant. Group II served as TAM control. Group III received silymarin (100 mg/kg body weight). Group IV and V received PFDB (100 and 200 mg/kg body weight). The animals received these treatments by oral route for 21 days. Except Group I, all other groups received TAM (45 mg/kg body weight) p.o. form day 10-21. On 22 nd day blood was obtained from the animals by puncturing retro-orbital plexus and allowed to clot at room temperature. Serum was separated by centrifugation and utilized for estimation of biochemical parameters including serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), cholesterol, triglycerides (TG), urea, CR, uric acid and bilirubin.  The livers were excised and homogenate was processed for the estimation of LPX. A part of homogenate after precipitating proteins with Trichloroacetic acid was used for estimation of glutathione. The rest of the homogenate was centrifuged and the supernatant was used for estimation of superoxide dismutase (SOD) and catalase (CAT) activities.
The Liver was removed, fixed overnight in 10% buffered formalin and embedded in paraffin. 4-6 μm thick sections were made, dewaxed in xylene, rehydrated in graded alcohol series and washed with distilled water for 2 min. Subsequently, the sections were stained with hematoxylin for 5 min at room temperature. After 15 min, the sections were counterstained with eosin for 2 min, dehydrated in graded alcohol series, washed with xylene and observed under the microscope at ×10 and ×40 magnifications.
The statistical analysis was performed using one-way analysis of variance. The values are expressed as mean ± standard deviation. Comparison of mean values was performed by Duncan's Multiple Range Test (DMRT) using SPSS software (San Diego, USA). P < 0.05 was considered as significant.
| » Results|| |
In vitro Hepatoprotective Effect on BRL3A Cells
The effects of PFDB on BRL3A cells intoxicated with ethanol are reported in [Table 1]. The cells exposed with ethanol showed a percentage viability of 10.302%, while the cells which were pretreated with PFDB showed a dose-dependent increase in percentage viability and the results are highly significant (P < 0.001, when compared to ethanol intoxicated cells). Maximum protection to the cells was offered upon pretreatment with 500 μg/ml PFDB and was found to be 67.0035%.
In vivo Hepatoprotective Activity
In liver function test parameters, a remarkable elevation was observed in serum SGOT, SGPT, ALP, TG, cholesterol, urea, uric acid, bilirubin and creatinin levels in TAM-intoxicated rats. This may be due to extensive hepatic damage caused during TAM treatment. Pretreatment of rats with PFDB effectively protected them against TAM-induced hepatotoxicity, which is evidenced by decreased levels of these parameters in serum [Table 2]. The protective effect was found to be dose-dependent and comparable to standard silymarin treated rats.
|Table 2: Effect of PFDB on biochemical parameters in TAM - intoxicated female Sprague Dawley rats |
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The decrease in antioxidant defence systems of TAM-intoxicated rats render them more susceptible to hepatotoxicity. As shown in [Table 3], the levels of antioxidant enzyme activities in liver homogenates (reduced glutathione [GSH], glutathione peroxidase [GPX], SOD and CAT) were significantly improved upon pretreatment of TAM-intoxicated rats with PFDB which inhibited TAM-induced hepatic injury and thereby the level of oxidative stress due to decreased LPX and enhanced antioxidant enzyme activities.
|Table 3: Effect of PFDB on antioxidant enzyme levels in TAM - intoxicated female Sprague Dawley rats |
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The histopathological studies revealed an extensive hepatic damage in case of TAM group. Silymarin and both the test groups showed a protective effect by decreasing the extent of centrilobular necrosis, feathery degeneration and inflammation when compared to TAM group [Figure 1].
|Figure 1: Effects of polyphenolic fraction of Desmostachya bipinnata on Tamoxifen- induced liver damage in rats-histopathology (stained with hematoxylin and eosin): (a) Normal group, normal cellular architecture with distinct hepatic cells (10×). (b) TAM-intoxicated group, feathery degeneration with necrosis and vacoulation (40×). (c) Silymarin (100 mg/kg) + Tamoxifen-treated group, less necrosis and vacoulation (40×). (d) PFDB (100 mg/kg) + Tamoxifen-treated group (40×). (e) PFDB (200 mg/kg) + Tamoxifen-treated group, increased number of hepatocytes with normal nucleus (40×)|
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| » Discussion|| |
In vitro cell viability and hepatoprotective activity of PFDB was assessed on BRL3A cell lines upon alcohol-induced hepatotoxicity by MTT assay method. The ability of cells to reduce MTT provides an indication of the mitochondrial integrity and activity, which in turn may be interpreted as a measure of viability and/or cell number. The nontoxic concentrations of PFDB tested against ethanol-induced toxicity in BRL3A offered dose-dependent protection to cells.
Tamoxifen citrate is an anti-estrogenic drug widely used for the treatment of breast cancer. It was reported that TAM in toxic doses lead to oxidative liver damage as it has been elucidated to be a hepatocarcinogen in rats. ,, It may be more toxic to the liver because it has a much higher affinity for hepatic tissue than for any other tissues.  It was found to produce five DNA adducts in rat liver that appeared to be responsible for carcinogenesis. 
The extent of hepatic damage by TAM-intoxication was assessed by the level of released cytoplasmic transaminases (SGOT, SGPT) and phosphatase in circulation. The rise in the serum levels of aspartate aminotransferase, alanine transaminase and ALP has been attributed to the damaged structural integrity of the liver, because these are cytoplasmic in location and are released into circulation after cellular damage.  The results of this study demonstrate that pretreatment of rats with PFDB effectively protected them against TAM-induced hepatotoxicity. This has been evidenced by decreased serum aminotransferase activity. A remarkable elevation in serum TG, cholesterol, urea, uric acid, bilirubin, creatinin and lipid peroxidase levels was observed in TAM-intoxicated rats, whereas the level of total proteins was reduced due to the TAM-induced hepatotoxicity. The reduction is attributed to the initial damage produced and localised in the endoplasmic reticulum, which results in the loss of P450 leading to its functional failure with a decrease in protein synthesis and accumulation of TG leading to fatty liver. Inhibition of bile acids synthesis from cholesterol leading to increase in cholesterol levels was also resulted due to TAM-intoxication. Suppression of cholesterol levels by PFDB suggests the bile acids synthesis inhibition was reversed. The decrease in antioxidant defence systems of TAM-intoxicated rats render them more susceptible to hepatotoxicity. The levels of antioxidant enzyme activities in liver homogenates (GSH, GPX, SOD and CAT) were significantly improved upon pretreatment of TAM-intoxicated rats with PFDB which inhibited TAM-induced hepatic injury and thereby the level of oxidative stress as it can decrease LPX and enhance antioxidant enzyme activities [Table 3], whereas the level of TBARS were significantly decreased comparable to TAM-intoxicated group. The increase in GPX and SOD activities after PFDB administration to TAM-intoxicated rats, was attributed to induction or mutually protective interactions especially SOD, which is present at low level only but highly inducible under oxidative stress.  The destruction and degradation of phospholipids hydroperoxides are carried out by GPX and this suggested being a pathway of cytoprotection against the deleterious effects of phospholipids hydroperoxides. The decreased GPX level of TAM-intoxicated rats leads to an increase of toxic level to the cells.  The decreased activities of GPX and SOD in liver homogenate of TAM-intoxicated rats may be due to oxidative stress induced inactivation and/or exhaustion.  Furthermore, it was reported that, the decreased GPX activity leads to H 2 O 2 accumulation in the liver which in turn inactivates SOD. , The detoxification of different drugs and xenobiotics in the liver produce GSH.  GSH plays a common role in cellular resistance to oxidative damage as a free radical scavenger as protein-bound glutathione and by the generation of ascorbate or tocopherol in the liver.  The decreased hepatic GSH in TAM-intoxicated rats could be as a result of hexose monophosphate shunt impairment due to TAM-intoxication and thereby NADPH availability is reduced and the ability to recycle GSSG to GSH is decreased.  By blocking oxidative damage through LPX and protein oxidation, PFDB prevented the loss of membrane permeability and dysfunction of cellular proteins and decreased the endogenous level of hydroxyl radical and GSH. The liver sections of the rats treated with normal saline showed normal hepatic architecture, whereas that of TAM-treated group showed total loss of hepatic architecture with intense peripheral central vein necrosis, fatty changes, feathery degeneration, inflammation, and apoptosis. In case of rats treated with silymarin and PFDB (100 mg/kg and 200 mg/kg) a normal hepatic architecture was seen with only moderate accumulation of fatty lobules and mild degree of cell necrosis [Figure 1]a-e. This indicates that PFDB have offered protection against TAM intoxication comparable to that of silymarin.
In summary, this study suggests that the oral administration of D. bipinnata significantly ameliorates TAM hepatotoxicity in rats. The polyphenolic fraction may be protecting the liver by free radical scavenging activity and thus preventing peroxidation of lipids of the endoplasmic reticulum. Moreover, this may be due to the presence of flavonoids in the fraction (whose presence was confirmed by HPTLC fingerprinting). Purification and characterization of the flavonoids present in the polyphenolic fraction are under progress. The study also rules out the presence of any antagonistic effect of the combination indicating a synergistic effect only.
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[Table 1], [Table 2], [Table 3]