Chinese Journal of Natural Medicines 2014, 12(9): 06630671
Chinese Journal of Natural Medicines
Antioxidant and hepatoprotective effects of Boswellia ovalifoliolata bark extracts Bandari Uma Mahesh1, Shweta Shrivastava2, Rajeswara Rao Pragada1, V.G.M. Naidu2*, Ramakrishna Sistla2, 3* 1
Department of Pharmacology, A.U. College of Pharmaceutical Sciences, Andhra University, Vishakhapatnam, Andhra Pradesh,
India -530003 2 Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education & Research (NIPER-Hyderabad), Balanagar, Hyderabad, Andhra Pradesh, India-500037; 3 Pharmacology Division, Indian Institute of Chemical Technology, Habsiguda, Hyderabad, Andhra Pradesh, ndia-500007 Available online September 2014
[ABSTRACT] Paracetamol (PCM) hepatotoxicity is related to reactive oxygen species (ROS) formation and excessive oxidative stress; natural antioxidant compounds have been tested as an alternative therapy. This study evaluated the hepatoprotective activity of an alcoholic extract of Boswellia ovalifoliolata (BO) bark against PCM-induced hepatotoxicity. BO extract also demonstrated antioxidant activity in vitro, as well as scavenger activity against 2, 2-diphenyl-1-picrylhydrazyl. Administration of PCM caused a significant increase in the release of transaminases, alkaline phosphatase, and lactate dehydrogenase in serum. Significant enhancement in hepatic lipid peroxidation and marked depletion in reduced glutathione were observed after parac intoxication with severe alterations in liver histology. BO treatment was able to mitigate hepatic damage induced by acute intoxication of PCM and showed a pronounced protective effect against lipid peroxidation, deviated serum enzymatic variables, and maintained glutathione status toward control. The results clearly demonstrate the hepatoprotective effect of BO against the toxicity induced by PCM. [KEY WORDS] Paracetamol; Boswellia ovalifoliolata; Hepatoprotection; Oxidative stress; Liver function tests; Lipid peroxidation
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2014)09-0663-09
Introduction Paracetamol (PCM) is a safe and effective analgesic and antipyretic drug at therapeutic doses. However, an overdose of PCM can result in severe liver injury [1]. The mechanism of PCM-induced liver injury has been studied for several decades. Early results revealed the formation of a reactive metabolite (N-acetyl-p-benzoquinone imine, NAPQI), which is responsible for liver injury through depletion of glutathione (GSH) and binds to cellular proteins [2]. Hepatotoxicity induced by PCM is considered to involve liver cytochrome P450s (CYPs) including CYP2E1, CYP3A4, and CYP1A2 [3]. NAPQI, the toxic metabolite of PCM, inhibits mitochondrial [Received on] 31-May-2013 * [ Corresponding author] D.r Sistla Ramakrishna: Email: sistla@iict. res.in; V.G.M. Naidu: Email: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
oxidative phosphorylation, depletes adenosine triphosphate (ATP), and produces selective mitochondrial oxidant stress [4]. Liver damage is associated with cellular necrosis, an increase in lipid peroxidation, and depletion in glutathione (GSH) level. In addition, serum levels of many biochemical markers like serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), triglycerides, cholesterol, bilirubin, and lactate dehydrogenase (LDH) are elevated [5]. A concept has emerged that mitochondrial dysfunction and damage is a central event responsible for liver injury caused by PCM. Faced with the threat of liver damage caused by PCM, scientists have attempted to find effective chemicals or drugs to treat PCM-induced hepatotoxicity. In the 1970s, N-acetylcystenine (NAC) was introduced to treat patients with PCM-induced liver failure. NAC is still used in the clinic [6], and is the only drug approved for the treatment of PCM poisoning. However, NAC in the treatment of PCM-induced liver damage has considerable limitations, because the therapeutic window of this drug is quite narrow [7]. Thus, it is urgent and critically important to identify
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and explore effective drugs to treat PCM-induced hepatotoxicity. Natural products (NPs), one of the most important resources in drug discovery, have been widely investigated worldwide and have the advantages of abundant resources [8]. Boswellia ovalifoliata N.P. Balakr. & A.N. Henry (BO) is a narrow endemic, endangered, and threatened medicinal plant species. It is deciduous, medium-sized tree belonging to the family Burseraceae. This tree harbors on the Tirumala hills of the Seshachalam hill range of the Eastern Ghats of India [9].The plant is used by tribal people like the Nakkala, Sugali, Chenchu, and indigenous communities to treat a number of aliments. The stem bark is used to reduce rheumatic pains. A stem bark decoction is given orally to reduce the pains. An equal mixture of the gum and stem bark, one tea spoon full, is given daily with sour milk on an empty stomach for a month to cure stomach ulcers [9, 10]. Preliminary investigations conducted in this laboratory have shown very good antioxidant properties of boswellia bark compared to the standard antioxidants. However, no result has so far been reported on the hepatoprotective activity. The present study aimed to examine the hepatoprotective properties of the bark extracts of BO in drug-induced hepatotoxicity.
Material and Methods Materials PCM was a gift sample from Sri Krishna Pharmaceuticals, Hyderabad, India. Liv.52 was purchased from The Himalaya Drug Co., Bangalore, India. Every 2.5 mL of Liv.52 syrup contains an extract of the following: Capparis spinosa (17 mg), Cichorium intybus (17 mg), Solanum nigrum (8 mg), Cassia occidentalis (4 mg), Terminalia arjuna (8 mg), Achillea millefolium (4 mg), and Tamarix gallica (4 mg). SGPT, SGOT, and ALKP Kits were procured from Siemens Ltd, Worli, Mumbai, Acacia gum, DMSO, and sulphanilamide were purchased from Loba Chemie (Mumbai, India). Thiobarbituric acid (TBA), malonaldehyde-bis-dimethyl acetal (MDA), 2, 2-diphenyl- 1-picrylhydrazyl (DPPH), gallic acid, rutin, ethylenediamine tetraacetic acid (EDTA), trichloroacetic acid (TCA), ascorbic acid, and 2, 7-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma (St. Louis, MO, USA), and N-(1-naphthyl)-ethylene-diamine dihydrochloride (NED) was from S.D. Fine Chem Ltd., Mumbai, India. Plant material Dried bark materials of BO were collected in the month of November in the Tirumala Tirupathi hills. The plant material was authenticated by Assistant Professor K. Madhava chetty, Department of Botany, S.V. University, Tirupathi, Andhra Pradesh, India. A voucher specimen No. 816 of the plant is deposited at the herbarium of the S.V. University. Preparation of plant extract The bark materials of BO were shade-dried and ground to coarse powder with high speed mechanical blender. The powdered material of bark was extracted successively with n-hexane, ethyl acetate, ethanol, and water. The hexane, ethyl acetate, and alcohol extracts were filtered, concentrated in a rotavapor (Rota-
vac, Heidolf, Germany) at reduced pressure below 40 C. In vitro antioxidant assays DPPH radical scavenging The extracts from BO were tested for their ability to scavenge free radicals by DPPH radical scavenging assay [11]. Varying concentrations of the extracts were made up to 1 mL with respective solvents. A sample of the extract (20 µL) at each concentration was added to 0.1 mmol·L–1 DPPH in ethanol (200 µL) and incubated in the dark at room temp for 30 min. Absorbance was read at 517 nm using a multimode detection reader (Molecular Devices, Sunnyvale, CA, USA ). Ascorbic acid, curcumin, and Trolox were employed as standards. Analyses were run in triplicate and percentage free radical scavenging activity (% FRSA) was calculated using the formula % FRSA = (A control –A extracts)/A control 100 ABTS radical cation decolorization assay ABTS radical cation scavenging activity was determined according to the method of Kraus et al [12]. ABTS.+ was produced by reacting 2 mmol·L–1 ABTS in water with 2.45 mmol·L–1 potassium persulfate, and the mixture kept in the dark at room temp for 12 h. The concentration of ABTS.+ solution was adjusted to give an absorbance of 0.750 ± 0.05 at 734 nm in phosphate buffer pH 7.4. The diluted ABTS.+ solution (200 µL) was added to extracts (20 µL) at solution different concentrations (3–1 000 µg·mL–1). The ability of the extracts to neutralize ABTS.+ was measured at 734 nm of minutes incubation. Reducing capacity Reducing power is based on the ability of plant extract to reduce Fe(III) to Fe(II). Plant extracts were mixed with 1% potassium ferricyanide at 50 C for 20 min, and the clear supernatant was treated with FeCl3 (0.1%) and the absorbance measured at 700 nm [13]. Total phenolic content The total phenolic content was determined by the method of Scalbert et al [14]. The total phenol content was expressed in milligram of gallic acid equivalents/g extract. Determination of total flavonoids The total flavonoid content was determined by the method of Brighente et al [15], and was expressed as rutin equivalent/g extract. Hepatoprotective activity Test animals Male Wistar albino rats weighing between 180–200 g were obtained from Genotox, Hyderabad, India. The animals were grouped and maintained under temperature-controlled environment with a 12:12 light/dark cycle with standard conditions of temperature [(25 ± 2) C] and relative humidity (30%–70%) during the experimental period. The animals were fed with standard pellet diet and fresh water ad libitum. All the animals were acclimatized to laboratory conditions for a week before commencement of the experiments. All procedures described were reviewed and approved by the Institutional Animal Eth-
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ics Committee (IICT/PHARM/SRK/22/02/2012/ 17 and 15, dated 22/02/2012). The animal experiments were conducted in accordance with the CPCSEA guidelines. Acute oral toxicity An acute oral toxicity study was performed as per OECD guidelines for the testing of chemicals, Test No. 423 (OECD, 2001; Acute oral toxicity – acute toxic class method). Wistar rats (n = 6) of either sex, selected by a random sampling technique, were used for the acute toxicity study. Three animals were kept overnight with access to water, but not food, after which the alcoholic extract of BO bark was administered orally at a dose level of 2 000 mg/kg body weight by gastric intubation and the animals were observed for 14 days. If mortality was observed in two out of three animals, then the dose administered was identified as a toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was observed again, the procedure was repeated for lower doses (300, 50, and 5 mg/kg body weight). Test compound preparation Alcoholic bark extracts of BO were weighed and made into a suspension with gum acacia (2%) in de-ionized water. PCM: PCM powder (3 g·kg–1) was weighed and made into a suspension with gum acacia (2%) in de-ionized water. Experimental design Hepatic injury was induced in rats by oral administration of a single dose of PCM (3 g·kg–1) [16]. Liv.52, a known hepatoprotective agent was used as reference standard [17]. Animals were divided into four groups (I–IV) of six animals in each. Animals were grouped as follows: Group-I: Served as control group, given normal food and water daily for 7 days. Group-II: PCM control group, given water daily for 7 day followed by single dose of PCM (3 g·kg–1, p.o.) on day 7. Group-III: Reference standard group, pre-treatment with Liv.52 (1 mL·kg–1 syrup, p.o.) for 7 days followed by single dose of PCM (3 g·kg–1, p.o.) on day 7. Group-IV: Pre-treatment with alcoholic bark extract of BO (400 mg·kg–1, p.o.) for 7 days, followed by single dose of PCM (3 g·kg–1, p.o.) on day 7. PCM was administered to the Group II-IV animals after overnight fasting. The biochemical parameters were estimated 24 h after PCM insult. Serum sample preparation At the end of the experimental period, blood samples were collected using heparinized capillary tubes from the retro orbital plexus of the rats of all groups. Blood samples were allowed to clot and then centrifuged (Biofuge Stratos, Germany) at 4 000 r·min–1 for 15 min at 4 °C to separate the sera, which were stored at –80 °C until further analysis. Serum was used for estimation of SGOT, SGPT, LDH, and ALP using corresponding kits from Siemens, India with an Auto Blood analyzer (Siemens, Dimension Xpand plus; USA). Tissue preparation and homogenization Animals were sacrificed by cervical dislocation with light ether anesthesia and liver tissues were excised. Tissues were washed thoroughly with ice-cold normal saline and weighed.
A portion of the tissue were stored in formalin and used for histopathological analysis. Remaining tissues were stored at – 80 ºC and used for further estimations. Tissues were cut in to small pieces and homogenized (Heidoph, Silent Crusher S, Germany) in ice-cold phosphate buffer saline (PBS) (0.05 mol·L–1, pH 7) to obtain 1 : 9 (W/V) (10%) whole homogenate. Homogenate was mixed with equal volume of 10% trichloroacetic acid (TCA) and centrifuged at 5 000 r·min–1 for 10 min and supernatant was used for the determination of MDA and GSH. The remaining homogenate was centrifuged at 17 000 g for 60 min at 4 C, and supernatants were used for the measurement of antioxidant parameters like GSH, CAT, SOD, and ROS. Assessment of oxidative stress parameters Estimation of hepatic lipid peroxidation (MDA) The concentration of MDA in liver homogenate was used as an index of lipid peroxidation, and was determined based on the reaction with thiobarbituric acid [18]. MDA levels were quantified at an extinction coefficient of 1.56 × 105 M–1·cm–1 and expressed as nanomoles of MDA per g of tissue. Estimation of reduced glutathione (GSH) Tissue GSH concentration was measured by the method of Ellman [19] and was expressed as µmol/g tissue. Estimation of catalase (CAT) Catalase activity in liver tissue was determined by measuring the rate of decomposition of hydrogen peroxide at 240 nm [20] and the activity was expressed as U/mg protein. Estimation of superoxide dismutase (SOD) Total SOD activity (cytosolic and mitochondrial) was determined using an SOD assay kit (Sigma-Aldrich Co., St Louis, MO, USA) according to the following manufacturer specifications. Estimation of liver total reactive oxygen species (ROS) Reactive oxygen species (ROS) levels in the liver tissue were determined fluorometrically by using 2, 7-dichlorofluorescein diacetate (DCF-DA) according to the published method [21]. Briefly, to a volume of 100 µmol·L–1 DCF-DA dissolved in DMSO (10 µL), 90 µL of liver homogenate supernatant was added and incubated for 30 min at room temp in the dark. After incubation, the volume was made up to 3 mL using PBS (0.1 mol·L–1, pH 7.4) and the fluorescence was measured at an excitation wavelength of 488 nm and emission wavelength of 525 nm using microplate reader (Spectra Max Plus, M4, Molecular Devices, Sunnyvale, CA, USA). The result was expressed as a percentage change fluorescence, where the normoxia group was taken as 100%. Histological analysis All liver samples were processed and embedded in paraffin. Sections were cut at 5 µm thicknesses on a rotary microtome, mounted and stained with hematoxylin and eosin. These sections were evaluated for histological changes under light microscopy (Nikon E800 research microscope, Tokyo, Japan). Statistical analysis All results were expressed as mean ± SEM. The intergroup variation between various groups was measured by one way analysis of variance (ANOVA) using the Graph Pad Prism, version 5.0 and the comparisons between groups were
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conducted by “Bonferroni's Multiple Comparison Test”. Results were considered statistically significant when P < 0.05.
Results Antioxidant assays DPPH and ABTS.+ scavenging spectrophotometric methods are used for determining the antioxidant activity of the selected compounds. When an antioxidant is added to the DPPH and or ABTS.+ radicals, there is a degree of decolorisation owing to the presence of antioxidants which reverses the formation of DPPH radical and ABTS.+. Fig. 1a illustrates the % DPPH free radical scavenging activity of the extracts from the bark of BO and compared with standard antioxidants like ascorbic acid, trolox, and curcumin. The results from the DPPH test showed that alcoholic extracts from the bark extracts have dose-dependent ability to quench free radicals. Based on the EC50 values (Fig. 1b),
the DPPH radical scavenging effect of bark extracts of BO and standard antioxidants decreased in the order of BB Alcoh> ascorbic acid> trolox> curcumin. As seen in the Fig. 2a, alcoholic extracts of BO showed significant ABTS.+ scavenging activity in a concentration-dependent manner. Based on the EC50 values (Fig. 2b), ABTS.+ scavenging activity of the bark extracts of BO and standard antioxidants decreased in the order of BB Alcoh > BB EtOAc ≥ curcumin > ascorbic acid > trolox. Electron donating capacity for bioactive compounds is an indicator of its reducing activity. The reducing power for bioactive compounds can be measured by monitoring the conversion of the yellow Fe3+/ ferricyanide complex to the perl’s Prussian blue ferrous form. As seen in Fig. 3, alcoholic and ethyl acetate extracts from the bark of BO had effective reducing power when compared to that of standards. Total phenolic and flavonoid
Fig. 1 In vitro antioxidant activity determined by the DPPH method. (a) DRC of alcoholic (BB Alcoh) and ethyl acetate (BB EtOAc) bark extracts of BO compared to ascorbic acid, curcumin, and Trolox; (b) EC50 values of alcoholic and ethyl acetate extracts of BO compared to ascorbic acid, curcumin, and Trolox, determined by the DPPH method. Data represents mean ± SEM (n = 3). Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". ***P < 0.001, **P < 0.01, *P < 0.05
Fig. 2 In vitro antioxidant activity determined by the ABTS method (mean ± SEM, n = 3). (a) DRC of alcoholic (BB Alcoh) and ethyl acetate (BB EtOAc) bark extracts of BO compared to ascorbic acid, curcumin, and Trolox; (b) EC50 values of alcoholic and ethyl acetate extracts of BO compared to ascorbic acid, curcumin and Trolox. Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". ***P < 0.001, **P < 0.01, *P < 0.05
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Fig. 3 In vitro antioxidant activity of hexane (BB Hex), alcoholic (BB Alcoh), and ethyl acetate (BB EtOAc) bark extracts of BO compared to ascorbic acid, curcumin, and trolox, determined by reducing power method (mean ± SEM, n = 3)
content of plant extracts are considered to be correlated with their antioxidant activity. Therefore total phenol and flavonoid contents in the bark extracts were determined and the results were expressed in terms of gallic acid and rutin equivalents, respectively. The total phenolic content for ethyl acetate and
alcoholic extracts from the bark of BO were found to be (154.7 ± 2.6) and (305.5 ± 0.55) mg/g gallic acid equivalents, respectively, whereas the total flavonoid content for the ethyl acetate and alcoholic extracts from the bark of BO were found to be (6.4 ± 0.11) and (23.2 ± 0.47) mg/g rutin equivalents, respectively. Hepatoprotective activity Acute oral toxicity The alcoholic extract of the BO bark did not cause any mortality up to 2 000 mg·kg–1, and hence 1/5th of the maximum dose administered (i.e., 400 mg·kg–1, p.o.) was selected for the present study. Effect of alcoholic bark extracts on liver function Animals after PCM administration exhibited toxic response with significant alterations in liver marker enzymes. As compared to the control values, PCM significantly enhanced SGPT, SGOT, ALP, and LDH levels in the blood circulation (***P < 0.001). As evidenced by Bonferroni's Multiple Comparison Test after administration, the alcoholic extracts of the bark of BO showed a protective effect on PCM-induced liver injury by remarkably preventing the elevation of serum levels of SGPT, SGOT, and LDH at a dose of 400 mg·kg–1 when compared to the PCM-treated group, as shown in Fig. 4.
Fig. 4 Effect of alcoholic bark extracts of BO on serum biochemical parameters. (a) SGPT; (b) SGOT; (c) ALP; and (d) LDH. Bars are mean ± SEM (n = 6) for each group. Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". *= compared with vehicle control and # =PCM treated groups. ***P < 0.001, **P < 0.01, *P < 0.05; ###P < 0.001, ##P < 0.01，#P < 0.05. NC: Vehicle control, PCM: Paracetamol control, Paracetamol (3 g·kg–1, p.o.) on day 7; PCM + Liv.52: Reference standard group, pre-treatment with Liv.52 (1 mL·kg–1 syrup, p.o.) for 7 days followed by single dose of PCM (3 g·kg–1, p.o.) on day 7; PCM + BB: Pre-treatment with alcoholic bark extract of BO (400 mg·kg–1, p.o.) for 7 days followed by single dose of PCM (3 g·kg–1, p.o.) on day 7. Rats were sacrificed 24 h after PCM intoxication
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Effect of alcoholic bark extracts on GSH, catalase, and SOD in the liver tissue PCM treatment resulted in significant (***P < 0.001) reduction for the activity of GSH, catalase, and SOD. Treatment with BO caused a significant (###P < 0.001) increase in PCM-diminished activity of GSH, catalase and
SOD enzymes. The protective effect of alcoholic extracts of bark of BO for GSH and SOD at a dose of 400 mg·kg–1 was comparable and not significantly (*P > 0.05) different from the effect of Liv.52 (standard), whereas for catalase treatment with BO caused a significant (+++P < 0.001) increase compared to Liv.52 (standard).
Fig. 5 Effect of alcoholic bark extracts of BO on GSH, catalase, and SOD in the liver tissue. Bars are mean ± SEM (n = 6) for each group. Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". * = compared with vehicle control and # =PCM treated groups. ***P < 0.001, **P < 0.01, *P < 0.05; ###P < 0.001, ##P < 0.01, #P < 0.05. Group and treatment details are the same as described in Fig. 4
Effect of alcoholic bark extracts on lipid peroxidation & ROS The inhibitory effects of the alcoholic bark extract on PCM-induced peroxidative damage and ROS are shown in Fig. 6. Hepatic LPO and ROS were enhanced after PCM administration significantly (P < 0.001). Treatment with the alcoholic bark extract significantly inhibited the LPO and ROS (P < 0.001) and reduced hepatic peroxidative stress, which is an indication of its antioxidative effect. Histopathological study Light microscopic study of normal liver showed normal histology of hepatic cells with a well-preserved nucleus and cytoplasm, and hepatocytes were arrayed in well-formed cords around the central vein. Administration of PCM induced severe morphological and histopathological deformations in the liver. Lymphocytic infiltration around the central
vein, sinusoidal congestion, and ballooning of hepatocytes were also observed. Treatment with the alcoholic bark extract (400 mg·kg–1) recovered the histological architecture in comparison to the groups which were exposed to PCM. Treatment with the bark extract showed more or less normal lobular pattern with a better cord arrangement of well-formed hepatocytes with prominent nucleus, and maintained the central vein.
Discussion In the present study, the alcoholic bark extract of BO was observed to exhibit a hepatoprotective effect as demonstrated by a significant decrease in SGOT, SGPT, ALP, and LDH concentrations, and by preventing liver histopathological changes in rats with PCM-induced hepatotoxicity. Moreover, the alcoholic bark extract of BO enhanced the activities of
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Fig. 6 Effect of the alcoholic bark extract of BO on MDA and ROS in the liver tissues. Bars are mean ± SEM (n = 6) for each group. Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". *= compared with vehicle control and # =PCM treated groups. ***P < 0.001, **P < 0.01, *P < 0.05; ###P < 0.001, ##P < 0.01, #P < 0.05. Group and treatment details are the same as described in Fig. 4
Fig. 7 Photomicrographs of liver sections stained with haematoxylin and eosin (H&E). (a) liver of control rats showing normal histology; (b) Liver of rats after oral administration of paracetamol showing damage in the hepatocytes, characterized by the presence of hydropic degeneration, perinuclear vacuolization, and dilatation in blood sinusoids; (c) Liver of rats pre-treated with Liv.52 (1 mL/kg syrup, p.o.); (d) Liver of rats pre-treated with alcoholic extract of BO (400 mg·kg–1, p.o.) demonstrating normal histological appearance of the liver with normal polyhedral hepatocytes. Group and treatment details are the same as described in Fig. 4. (CV: centrilobular vein, *: perinuclear vacuolization, #: hydropic degeneration)
GSH, antioxidant enzymes (SOD, CAT), and diminished the amount of lipid peroxide against the PCM-induced hepatotoxicity in these animals, suggesting that the reduction of
oxidative stress in this scenario likely plays a role in the mechanism of its hepatoprotective effects. PCM is an antipyretic and analgesic drug, which is widely used to cure fever, headache, and other pains, and is readily available without prescription. When taken at toxic doses, it becomes a potent hepatotoxin, generating fulminated hepatic and renal tubular necrosis which is lethal in humans and experimental animals [5, 22]. The laboratory features of hepatotoxicity induced by PCM resemble other kinds of acute inflammatory liver disease with prominent increases of SGOT, SGPT, ALP, and LDH levels [23-24]. In the present study, the serum levels of the hepatic enzymes SGOT, SGPT, ALP, and LDH were increased, and reflected the hepatocellular damage in the PCM-induced hepatotoxicity animal model. The alcoholic bark extract of BO at a concentration of 400 mg/kg, however, could lower the SGPT, SGOT, ALP, and LDH levels in these PCM-intoxicated animals. In addition, the examination of liver function correlated the histopathological changes from the photomicroscopy observation. The centrilobular hepatic necrosis, Kupffer cells, ballooning degeneration, and infiltrating lymphocytes were displayed as a result of PCM intoxication. Treatment with an alcoholic bark extract of BO prevented these histopathological changes. Thus, these results suggested that the inhibition of liver function markers elevation and liver damage may participate in the protective effect of the alcoholic bark extract of BO against PCM-induced hepatotoxicity [25-26]. The metabolic activation and biochemical mechanisms of hepatotoxicity induced by PCM have been reviewed, and it has been shown that an overdose of PCM can cause centrilobular hepatic necrosis, liver function failure, and death in humans, as well as in experimental animals [27]. However, when PCM is taken at toxic doses, the compound is converted to a toxic form, NAPQI. NAPQI is an electrophilic intermediate which is oxidized by cytochrome P450 and converted to a highly reactive and toxic metabolite, as in cases of
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PCM overdose [28]. NAPQI can rapidly react with glutathione (GSH) and lead to a 90% total hepatic GSH depletion in cells and mitochondria, which can result in hepatocellular death and mitochondrial dysfunction. In addition, NAPQI can increase the formation of ROS and reactive nitrogen species (RNS), such as superoxide anion, hydroxyl radical, and hydrogen peroxide, and nitrous oxide, and peroxynitrite, respectively [29]. Excess levels of ROS and RNS can attack biological molecules such as DNA, protein, and phospholipids, which leads to lipid peroxidation, nitration of tyrosine, and depletion of the antioxidant enzymes, that further results in oxidative stress [30]. In the assessment of liver damage by PCM the determination of enzyme levels such as SGPT, SGOT, ALP, and LDH is most commonly used. Hepatocellular necrosis or membrane damage releases the enzymes into circulation and hence they can be measured in the serum. A high level of SGOT indicates liver damage, such as viral hepatitis, cardiac infarction, and muscle injury. SGOT catalyzes the conversion of alanine to pyruvate and glutamate, and is released in a similar manner [31]. Therefore, SGPT is more specific to the liver, and is thus a better parameter for detecting liver injury. Elevated levels of serum enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver [32-33]. On the other hand, a serum level of ALP is related to the function of hepatic cells. An increase in serum level of ALP is due to increased synthesis in the presence of increasing biliary pressure [34]. In the present study, the data suggested that high dosage of PCM in the liver could lead to decreased levels of GSH, antioxidant enzymes (SOD and CAT), and present a significant level of hepatotoxicity in the course of the treatment. However, the alcoholic bark extract of BO could raise the levels of GSH, SOD, and CAT against the PCM-induced oxidative stress mediated by ROS and RNS. Furthermore, the level of MDA was increased in the group receiving PCM administration, but pretreatment with the alcoholic bark extract of BO reduced the amount of MDA. This result indicated that decreasing the formation of lipid peroxidation is also one of the events in preventing the oxidative toxicity by PCM. The reversal may be because of the prevention of the leakage of intracellular enzymes by its membrane- stabilizing activity. This is in agreement with the commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and the regeneration of hepatocytes.
radical scavengers and reduce or inhibit the oxidative stress induced by PCM administration. Further studies are, however, needed to isolate the specific components responsible for the antioxidant action of this extract and to establish their mechanism of action.
Acknowledgements The authors are thankful to Dr. A. Kamal, Project Director, NIPER-Hyderabad for financial support, and to Dr. K. Madhava Chetty, Sri Venkateswara University, Tirupathi for authentication of the plant material.
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Conclusions The results demonstrated for the first time the hepatoprotective activity of BO bark extract in rats in vivo. The extract was able to protect the liver from the oxidative stress generated by PCM due to its antioxidant activity, which is probably due to its scavenger activity against several ROS/reactive nitrogen species attributed to the phenolic compounds. These phenolic compounds in the extract act as antioxidants and free
[13]
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Cite this article as: Bandari Uma Mahesh, Shweta Shrivastava, Rajeswara Rao Pragada, V.G.M. Naidu, Ramakrishna Sistla. Antioxidant and hepatoprotective effects of Boswellia ovalifoliolata bark extracts [J]. Chinese Journal of Natural Medicines, 2014, 12(9): 663-671
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Chinese Journal of Natural Medicines
Antioxidant and hepatoprotective effects of Boswellia ovalifoliolata bark extracts Bandari Uma Mahesh1, Shweta Shrivastava2, Rajeswara Rao Pragada1, V.G.M. Naidu2*, Ramakrishna Sistla2, 3* 1
Department of Pharmacology, A.U. College of Pharmaceutical Sciences, Andhra University, Vishakhapatnam, Andhra Pradesh,
India -530003 2 Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education & Research (NIPER-Hyderabad), Balanagar, Hyderabad, Andhra Pradesh, India-500037; 3 Pharmacology Division, Indian Institute of Chemical Technology, Habsiguda, Hyderabad, Andhra Pradesh, ndia-500007 Available online September 2014
[ABSTRACT] Paracetamol (PCM) hepatotoxicity is related to reactive oxygen species (ROS) formation and excessive oxidative stress; natural antioxidant compounds have been tested as an alternative therapy. This study evaluated the hepatoprotective activity of an alcoholic extract of Boswellia ovalifoliolata (BO) bark against PCM-induced hepatotoxicity. BO extract also demonstrated antioxidant activity in vitro, as well as scavenger activity against 2, 2-diphenyl-1-picrylhydrazyl. Administration of PCM caused a significant increase in the release of transaminases, alkaline phosphatase, and lactate dehydrogenase in serum. Significant enhancement in hepatic lipid peroxidation and marked depletion in reduced glutathione were observed after parac intoxication with severe alterations in liver histology. BO treatment was able to mitigate hepatic damage induced by acute intoxication of PCM and showed a pronounced protective effect against lipid peroxidation, deviated serum enzymatic variables, and maintained glutathione status toward control. The results clearly demonstrate the hepatoprotective effect of BO against the toxicity induced by PCM. [KEY WORDS] Paracetamol; Boswellia ovalifoliolata; Hepatoprotection; Oxidative stress; Liver function tests; Lipid peroxidation
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2014)09-0663-09
Introduction Paracetamol (PCM) is a safe and effective analgesic and antipyretic drug at therapeutic doses. However, an overdose of PCM can result in severe liver injury [1]. The mechanism of PCM-induced liver injury has been studied for several decades. Early results revealed the formation of a reactive metabolite (N-acetyl-p-benzoquinone imine, NAPQI), which is responsible for liver injury through depletion of glutathione (GSH) and binds to cellular proteins [2]. Hepatotoxicity induced by PCM is considered to involve liver cytochrome P450s (CYPs) including CYP2E1, CYP3A4, and CYP1A2 [3]. NAPQI, the toxic metabolite of PCM, inhibits mitochondrial [Received on] 31-May-2013 * [ Corresponding author] D.r Sistla Ramakrishna: Email: sistla@iict. res.in; V.G.M. Naidu: Email: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
oxidative phosphorylation, depletes adenosine triphosphate (ATP), and produces selective mitochondrial oxidant stress [4]. Liver damage is associated with cellular necrosis, an increase in lipid peroxidation, and depletion in glutathione (GSH) level. In addition, serum levels of many biochemical markers like serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), triglycerides, cholesterol, bilirubin, and lactate dehydrogenase (LDH) are elevated [5]. A concept has emerged that mitochondrial dysfunction and damage is a central event responsible for liver injury caused by PCM. Faced with the threat of liver damage caused by PCM, scientists have attempted to find effective chemicals or drugs to treat PCM-induced hepatotoxicity. In the 1970s, N-acetylcystenine (NAC) was introduced to treat patients with PCM-induced liver failure. NAC is still used in the clinic [6], and is the only drug approved for the treatment of PCM poisoning. However, NAC in the treatment of PCM-induced liver damage has considerable limitations, because the therapeutic window of this drug is quite narrow [7]. Thus, it is urgent and critically important to identify
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and explore effective drugs to treat PCM-induced hepatotoxicity. Natural products (NPs), one of the most important resources in drug discovery, have been widely investigated worldwide and have the advantages of abundant resources [8]. Boswellia ovalifoliata N.P. Balakr. & A.N. Henry (BO) is a narrow endemic, endangered, and threatened medicinal plant species. It is deciduous, medium-sized tree belonging to the family Burseraceae. This tree harbors on the Tirumala hills of the Seshachalam hill range of the Eastern Ghats of India [9].The plant is used by tribal people like the Nakkala, Sugali, Chenchu, and indigenous communities to treat a number of aliments. The stem bark is used to reduce rheumatic pains. A stem bark decoction is given orally to reduce the pains. An equal mixture of the gum and stem bark, one tea spoon full, is given daily with sour milk on an empty stomach for a month to cure stomach ulcers [9, 10]. Preliminary investigations conducted in this laboratory have shown very good antioxidant properties of boswellia bark compared to the standard antioxidants. However, no result has so far been reported on the hepatoprotective activity. The present study aimed to examine the hepatoprotective properties of the bark extracts of BO in drug-induced hepatotoxicity.
Material and Methods Materials PCM was a gift sample from Sri Krishna Pharmaceuticals, Hyderabad, India. Liv.52 was purchased from The Himalaya Drug Co., Bangalore, India. Every 2.5 mL of Liv.52 syrup contains an extract of the following: Capparis spinosa (17 mg), Cichorium intybus (17 mg), Solanum nigrum (8 mg), Cassia occidentalis (4 mg), Terminalia arjuna (8 mg), Achillea millefolium (4 mg), and Tamarix gallica (4 mg). SGPT, SGOT, and ALKP Kits were procured from Siemens Ltd, Worli, Mumbai, Acacia gum, DMSO, and sulphanilamide were purchased from Loba Chemie (Mumbai, India). Thiobarbituric acid (TBA), malonaldehyde-bis-dimethyl acetal (MDA), 2, 2-diphenyl- 1-picrylhydrazyl (DPPH), gallic acid, rutin, ethylenediamine tetraacetic acid (EDTA), trichloroacetic acid (TCA), ascorbic acid, and 2, 7-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma (St. Louis, MO, USA), and N-(1-naphthyl)-ethylene-diamine dihydrochloride (NED) was from S.D. Fine Chem Ltd., Mumbai, India. Plant material Dried bark materials of BO were collected in the month of November in the Tirumala Tirupathi hills. The plant material was authenticated by Assistant Professor K. Madhava chetty, Department of Botany, S.V. University, Tirupathi, Andhra Pradesh, India. A voucher specimen No. 816 of the plant is deposited at the herbarium of the S.V. University. Preparation of plant extract The bark materials of BO were shade-dried and ground to coarse powder with high speed mechanical blender. The powdered material of bark was extracted successively with n-hexane, ethyl acetate, ethanol, and water. The hexane, ethyl acetate, and alcohol extracts were filtered, concentrated in a rotavapor (Rota-
vac, Heidolf, Germany) at reduced pressure below 40 C. In vitro antioxidant assays DPPH radical scavenging The extracts from BO were tested for their ability to scavenge free radicals by DPPH radical scavenging assay [11]. Varying concentrations of the extracts were made up to 1 mL with respective solvents. A sample of the extract (20 µL) at each concentration was added to 0.1 mmol·L–1 DPPH in ethanol (200 µL) and incubated in the dark at room temp for 30 min. Absorbance was read at 517 nm using a multimode detection reader (Molecular Devices, Sunnyvale, CA, USA ). Ascorbic acid, curcumin, and Trolox were employed as standards. Analyses were run in triplicate and percentage free radical scavenging activity (% FRSA) was calculated using the formula % FRSA = (A control –A extracts)/A control 100 ABTS radical cation decolorization assay ABTS radical cation scavenging activity was determined according to the method of Kraus et al [12]. ABTS.+ was produced by reacting 2 mmol·L–1 ABTS in water with 2.45 mmol·L–1 potassium persulfate, and the mixture kept in the dark at room temp for 12 h. The concentration of ABTS.+ solution was adjusted to give an absorbance of 0.750 ± 0.05 at 734 nm in phosphate buffer pH 7.4. The diluted ABTS.+ solution (200 µL) was added to extracts (20 µL) at solution different concentrations (3–1 000 µg·mL–1). The ability of the extracts to neutralize ABTS.+ was measured at 734 nm of minutes incubation. Reducing capacity Reducing power is based on the ability of plant extract to reduce Fe(III) to Fe(II). Plant extracts were mixed with 1% potassium ferricyanide at 50 C for 20 min, and the clear supernatant was treated with FeCl3 (0.1%) and the absorbance measured at 700 nm [13]. Total phenolic content The total phenolic content was determined by the method of Scalbert et al [14]. The total phenol content was expressed in milligram of gallic acid equivalents/g extract. Determination of total flavonoids The total flavonoid content was determined by the method of Brighente et al [15], and was expressed as rutin equivalent/g extract. Hepatoprotective activity Test animals Male Wistar albino rats weighing between 180–200 g were obtained from Genotox, Hyderabad, India. The animals were grouped and maintained under temperature-controlled environment with a 12:12 light/dark cycle with standard conditions of temperature [(25 ± 2) C] and relative humidity (30%–70%) during the experimental period. The animals were fed with standard pellet diet and fresh water ad libitum. All the animals were acclimatized to laboratory conditions for a week before commencement of the experiments. All procedures described were reviewed and approved by the Institutional Animal Eth-
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ics Committee (IICT/PHARM/SRK/22/02/2012/ 17 and 15, dated 22/02/2012). The animal experiments were conducted in accordance with the CPCSEA guidelines. Acute oral toxicity An acute oral toxicity study was performed as per OECD guidelines for the testing of chemicals, Test No. 423 (OECD, 2001; Acute oral toxicity – acute toxic class method). Wistar rats (n = 6) of either sex, selected by a random sampling technique, were used for the acute toxicity study. Three animals were kept overnight with access to water, but not food, after which the alcoholic extract of BO bark was administered orally at a dose level of 2 000 mg/kg body weight by gastric intubation and the animals were observed for 14 days. If mortality was observed in two out of three animals, then the dose administered was identified as a toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was observed again, the procedure was repeated for lower doses (300, 50, and 5 mg/kg body weight). Test compound preparation Alcoholic bark extracts of BO were weighed and made into a suspension with gum acacia (2%) in de-ionized water. PCM: PCM powder (3 g·kg–1) was weighed and made into a suspension with gum acacia (2%) in de-ionized water. Experimental design Hepatic injury was induced in rats by oral administration of a single dose of PCM (3 g·kg–1) [16]. Liv.52, a known hepatoprotective agent was used as reference standard [17]. Animals were divided into four groups (I–IV) of six animals in each. Animals were grouped as follows: Group-I: Served as control group, given normal food and water daily for 7 days. Group-II: PCM control group, given water daily for 7 day followed by single dose of PCM (3 g·kg–1, p.o.) on day 7. Group-III: Reference standard group, pre-treatment with Liv.52 (1 mL·kg–1 syrup, p.o.) for 7 days followed by single dose of PCM (3 g·kg–1, p.o.) on day 7. Group-IV: Pre-treatment with alcoholic bark extract of BO (400 mg·kg–1, p.o.) for 7 days, followed by single dose of PCM (3 g·kg–1, p.o.) on day 7. PCM was administered to the Group II-IV animals after overnight fasting. The biochemical parameters were estimated 24 h after PCM insult. Serum sample preparation At the end of the experimental period, blood samples were collected using heparinized capillary tubes from the retro orbital plexus of the rats of all groups. Blood samples were allowed to clot and then centrifuged (Biofuge Stratos, Germany) at 4 000 r·min–1 for 15 min at 4 °C to separate the sera, which were stored at –80 °C until further analysis. Serum was used for estimation of SGOT, SGPT, LDH, and ALP using corresponding kits from Siemens, India with an Auto Blood analyzer (Siemens, Dimension Xpand plus; USA). Tissue preparation and homogenization Animals were sacrificed by cervical dislocation with light ether anesthesia and liver tissues were excised. Tissues were washed thoroughly with ice-cold normal saline and weighed.
A portion of the tissue were stored in formalin and used for histopathological analysis. Remaining tissues were stored at – 80 ºC and used for further estimations. Tissues were cut in to small pieces and homogenized (Heidoph, Silent Crusher S, Germany) in ice-cold phosphate buffer saline (PBS) (0.05 mol·L–1, pH 7) to obtain 1 : 9 (W/V) (10%) whole homogenate. Homogenate was mixed with equal volume of 10% trichloroacetic acid (TCA) and centrifuged at 5 000 r·min–1 for 10 min and supernatant was used for the determination of MDA and GSH. The remaining homogenate was centrifuged at 17 000 g for 60 min at 4 C, and supernatants were used for the measurement of antioxidant parameters like GSH, CAT, SOD, and ROS. Assessment of oxidative stress parameters Estimation of hepatic lipid peroxidation (MDA) The concentration of MDA in liver homogenate was used as an index of lipid peroxidation, and was determined based on the reaction with thiobarbituric acid [18]. MDA levels were quantified at an extinction coefficient of 1.56 × 105 M–1·cm–1 and expressed as nanomoles of MDA per g of tissue. Estimation of reduced glutathione (GSH) Tissue GSH concentration was measured by the method of Ellman [19] and was expressed as µmol/g tissue. Estimation of catalase (CAT) Catalase activity in liver tissue was determined by measuring the rate of decomposition of hydrogen peroxide at 240 nm [20] and the activity was expressed as U/mg protein. Estimation of superoxide dismutase (SOD) Total SOD activity (cytosolic and mitochondrial) was determined using an SOD assay kit (Sigma-Aldrich Co., St Louis, MO, USA) according to the following manufacturer specifications. Estimation of liver total reactive oxygen species (ROS) Reactive oxygen species (ROS) levels in the liver tissue were determined fluorometrically by using 2, 7-dichlorofluorescein diacetate (DCF-DA) according to the published method [21]. Briefly, to a volume of 100 µmol·L–1 DCF-DA dissolved in DMSO (10 µL), 90 µL of liver homogenate supernatant was added and incubated for 30 min at room temp in the dark. After incubation, the volume was made up to 3 mL using PBS (0.1 mol·L–1, pH 7.4) and the fluorescence was measured at an excitation wavelength of 488 nm and emission wavelength of 525 nm using microplate reader (Spectra Max Plus, M4, Molecular Devices, Sunnyvale, CA, USA). The result was expressed as a percentage change fluorescence, where the normoxia group was taken as 100%. Histological analysis All liver samples were processed and embedded in paraffin. Sections were cut at 5 µm thicknesses on a rotary microtome, mounted and stained with hematoxylin and eosin. These sections were evaluated for histological changes under light microscopy (Nikon E800 research microscope, Tokyo, Japan). Statistical analysis All results were expressed as mean ± SEM. The intergroup variation between various groups was measured by one way analysis of variance (ANOVA) using the Graph Pad Prism, version 5.0 and the comparisons between groups were
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conducted by “Bonferroni's Multiple Comparison Test”. Results were considered statistically significant when P < 0.05.
Results Antioxidant assays DPPH and ABTS.+ scavenging spectrophotometric methods are used for determining the antioxidant activity of the selected compounds. When an antioxidant is added to the DPPH and or ABTS.+ radicals, there is a degree of decolorisation owing to the presence of antioxidants which reverses the formation of DPPH radical and ABTS.+. Fig. 1a illustrates the % DPPH free radical scavenging activity of the extracts from the bark of BO and compared with standard antioxidants like ascorbic acid, trolox, and curcumin. The results from the DPPH test showed that alcoholic extracts from the bark extracts have dose-dependent ability to quench free radicals. Based on the EC50 values (Fig. 1b),
the DPPH radical scavenging effect of bark extracts of BO and standard antioxidants decreased in the order of BB Alcoh> ascorbic acid> trolox> curcumin. As seen in the Fig. 2a, alcoholic extracts of BO showed significant ABTS.+ scavenging activity in a concentration-dependent manner. Based on the EC50 values (Fig. 2b), ABTS.+ scavenging activity of the bark extracts of BO and standard antioxidants decreased in the order of BB Alcoh > BB EtOAc ≥ curcumin > ascorbic acid > trolox. Electron donating capacity for bioactive compounds is an indicator of its reducing activity. The reducing power for bioactive compounds can be measured by monitoring the conversion of the yellow Fe3+/ ferricyanide complex to the perl’s Prussian blue ferrous form. As seen in Fig. 3, alcoholic and ethyl acetate extracts from the bark of BO had effective reducing power when compared to that of standards. Total phenolic and flavonoid
Fig. 1 In vitro antioxidant activity determined by the DPPH method. (a) DRC of alcoholic (BB Alcoh) and ethyl acetate (BB EtOAc) bark extracts of BO compared to ascorbic acid, curcumin, and Trolox; (b) EC50 values of alcoholic and ethyl acetate extracts of BO compared to ascorbic acid, curcumin, and Trolox, determined by the DPPH method. Data represents mean ± SEM (n = 3). Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". ***P < 0.001, **P < 0.01, *P < 0.05
Fig. 2 In vitro antioxidant activity determined by the ABTS method (mean ± SEM, n = 3). (a) DRC of alcoholic (BB Alcoh) and ethyl acetate (BB EtOAc) bark extracts of BO compared to ascorbic acid, curcumin, and Trolox; (b) EC50 values of alcoholic and ethyl acetate extracts of BO compared to ascorbic acid, curcumin and Trolox. Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". ***P < 0.001, **P < 0.01, *P < 0.05
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Fig. 3 In vitro antioxidant activity of hexane (BB Hex), alcoholic (BB Alcoh), and ethyl acetate (BB EtOAc) bark extracts of BO compared to ascorbic acid, curcumin, and trolox, determined by reducing power method (mean ± SEM, n = 3)
content of plant extracts are considered to be correlated with their antioxidant activity. Therefore total phenol and flavonoid contents in the bark extracts were determined and the results were expressed in terms of gallic acid and rutin equivalents, respectively. The total phenolic content for ethyl acetate and
alcoholic extracts from the bark of BO were found to be (154.7 ± 2.6) and (305.5 ± 0.55) mg/g gallic acid equivalents, respectively, whereas the total flavonoid content for the ethyl acetate and alcoholic extracts from the bark of BO were found to be (6.4 ± 0.11) and (23.2 ± 0.47) mg/g rutin equivalents, respectively. Hepatoprotective activity Acute oral toxicity The alcoholic extract of the BO bark did not cause any mortality up to 2 000 mg·kg–1, and hence 1/5th of the maximum dose administered (i.e., 400 mg·kg–1, p.o.) was selected for the present study. Effect of alcoholic bark extracts on liver function Animals after PCM administration exhibited toxic response with significant alterations in liver marker enzymes. As compared to the control values, PCM significantly enhanced SGPT, SGOT, ALP, and LDH levels in the blood circulation (***P < 0.001). As evidenced by Bonferroni's Multiple Comparison Test after administration, the alcoholic extracts of the bark of BO showed a protective effect on PCM-induced liver injury by remarkably preventing the elevation of serum levels of SGPT, SGOT, and LDH at a dose of 400 mg·kg–1 when compared to the PCM-treated group, as shown in Fig. 4.
Fig. 4 Effect of alcoholic bark extracts of BO on serum biochemical parameters. (a) SGPT; (b) SGOT; (c) ALP; and (d) LDH. Bars are mean ± SEM (n = 6) for each group. Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". *= compared with vehicle control and # =PCM treated groups. ***P < 0.001, **P < 0.01, *P < 0.05; ###P < 0.001, ##P < 0.01，#P < 0.05. NC: Vehicle control, PCM: Paracetamol control, Paracetamol (3 g·kg–1, p.o.) on day 7; PCM + Liv.52: Reference standard group, pre-treatment with Liv.52 (1 mL·kg–1 syrup, p.o.) for 7 days followed by single dose of PCM (3 g·kg–1, p.o.) on day 7; PCM + BB: Pre-treatment with alcoholic bark extract of BO (400 mg·kg–1, p.o.) for 7 days followed by single dose of PCM (3 g·kg–1, p.o.) on day 7. Rats were sacrificed 24 h after PCM intoxication
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Effect of alcoholic bark extracts on GSH, catalase, and SOD in the liver tissue PCM treatment resulted in significant (***P < 0.001) reduction for the activity of GSH, catalase, and SOD. Treatment with BO caused a significant (###P < 0.001) increase in PCM-diminished activity of GSH, catalase and
SOD enzymes. The protective effect of alcoholic extracts of bark of BO for GSH and SOD at a dose of 400 mg·kg–1 was comparable and not significantly (*P > 0.05) different from the effect of Liv.52 (standard), whereas for catalase treatment with BO caused a significant (+++P < 0.001) increase compared to Liv.52 (standard).
Fig. 5 Effect of alcoholic bark extracts of BO on GSH, catalase, and SOD in the liver tissue. Bars are mean ± SEM (n = 6) for each group. Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". * = compared with vehicle control and # =PCM treated groups. ***P < 0.001, **P < 0.01, *P < 0.05; ###P < 0.001, ##P < 0.01, #P < 0.05. Group and treatment details are the same as described in Fig. 4
Effect of alcoholic bark extracts on lipid peroxidation & ROS The inhibitory effects of the alcoholic bark extract on PCM-induced peroxidative damage and ROS are shown in Fig. 6. Hepatic LPO and ROS were enhanced after PCM administration significantly (P < 0.001). Treatment with the alcoholic bark extract significantly inhibited the LPO and ROS (P < 0.001) and reduced hepatic peroxidative stress, which is an indication of its antioxidative effect. Histopathological study Light microscopic study of normal liver showed normal histology of hepatic cells with a well-preserved nucleus and cytoplasm, and hepatocytes were arrayed in well-formed cords around the central vein. Administration of PCM induced severe morphological and histopathological deformations in the liver. Lymphocytic infiltration around the central
vein, sinusoidal congestion, and ballooning of hepatocytes were also observed. Treatment with the alcoholic bark extract (400 mg·kg–1) recovered the histological architecture in comparison to the groups which were exposed to PCM. Treatment with the bark extract showed more or less normal lobular pattern with a better cord arrangement of well-formed hepatocytes with prominent nucleus, and maintained the central vein.
Discussion In the present study, the alcoholic bark extract of BO was observed to exhibit a hepatoprotective effect as demonstrated by a significant decrease in SGOT, SGPT, ALP, and LDH concentrations, and by preventing liver histopathological changes in rats with PCM-induced hepatotoxicity. Moreover, the alcoholic bark extract of BO enhanced the activities of
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Fig. 6 Effect of the alcoholic bark extract of BO on MDA and ROS in the liver tissues. Bars are mean ± SEM (n = 6) for each group. Data were analyzed by one-way analysis of variance (ANOVA), followed by "Bonferroni's Multiple Comparison Test". *= compared with vehicle control and # =PCM treated groups. ***P < 0.001, **P < 0.01, *P < 0.05; ###P < 0.001, ##P < 0.01, #P < 0.05. Group and treatment details are the same as described in Fig. 4
Fig. 7 Photomicrographs of liver sections stained with haematoxylin and eosin (H&E). (a) liver of control rats showing normal histology; (b) Liver of rats after oral administration of paracetamol showing damage in the hepatocytes, characterized by the presence of hydropic degeneration, perinuclear vacuolization, and dilatation in blood sinusoids; (c) Liver of rats pre-treated with Liv.52 (1 mL/kg syrup, p.o.); (d) Liver of rats pre-treated with alcoholic extract of BO (400 mg·kg–1, p.o.) demonstrating normal histological appearance of the liver with normal polyhedral hepatocytes. Group and treatment details are the same as described in Fig. 4. (CV: centrilobular vein, *: perinuclear vacuolization, #: hydropic degeneration)
GSH, antioxidant enzymes (SOD, CAT), and diminished the amount of lipid peroxide against the PCM-induced hepatotoxicity in these animals, suggesting that the reduction of
oxidative stress in this scenario likely plays a role in the mechanism of its hepatoprotective effects. PCM is an antipyretic and analgesic drug, which is widely used to cure fever, headache, and other pains, and is readily available without prescription. When taken at toxic doses, it becomes a potent hepatotoxin, generating fulminated hepatic and renal tubular necrosis which is lethal in humans and experimental animals [5, 22]. The laboratory features of hepatotoxicity induced by PCM resemble other kinds of acute inflammatory liver disease with prominent increases of SGOT, SGPT, ALP, and LDH levels [23-24]. In the present study, the serum levels of the hepatic enzymes SGOT, SGPT, ALP, and LDH were increased, and reflected the hepatocellular damage in the PCM-induced hepatotoxicity animal model. The alcoholic bark extract of BO at a concentration of 400 mg/kg, however, could lower the SGPT, SGOT, ALP, and LDH levels in these PCM-intoxicated animals. In addition, the examination of liver function correlated the histopathological changes from the photomicroscopy observation. The centrilobular hepatic necrosis, Kupffer cells, ballooning degeneration, and infiltrating lymphocytes were displayed as a result of PCM intoxication. Treatment with an alcoholic bark extract of BO prevented these histopathological changes. Thus, these results suggested that the inhibition of liver function markers elevation and liver damage may participate in the protective effect of the alcoholic bark extract of BO against PCM-induced hepatotoxicity [25-26]. The metabolic activation and biochemical mechanisms of hepatotoxicity induced by PCM have been reviewed, and it has been shown that an overdose of PCM can cause centrilobular hepatic necrosis, liver function failure, and death in humans, as well as in experimental animals [27]. However, when PCM is taken at toxic doses, the compound is converted to a toxic form, NAPQI. NAPQI is an electrophilic intermediate which is oxidized by cytochrome P450 and converted to a highly reactive and toxic metabolite, as in cases of
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PCM overdose [28]. NAPQI can rapidly react with glutathione (GSH) and lead to a 90% total hepatic GSH depletion in cells and mitochondria, which can result in hepatocellular death and mitochondrial dysfunction. In addition, NAPQI can increase the formation of ROS and reactive nitrogen species (RNS), such as superoxide anion, hydroxyl radical, and hydrogen peroxide, and nitrous oxide, and peroxynitrite, respectively [29]. Excess levels of ROS and RNS can attack biological molecules such as DNA, protein, and phospholipids, which leads to lipid peroxidation, nitration of tyrosine, and depletion of the antioxidant enzymes, that further results in oxidative stress [30]. In the assessment of liver damage by PCM the determination of enzyme levels such as SGPT, SGOT, ALP, and LDH is most commonly used. Hepatocellular necrosis or membrane damage releases the enzymes into circulation and hence they can be measured in the serum. A high level of SGOT indicates liver damage, such as viral hepatitis, cardiac infarction, and muscle injury. SGOT catalyzes the conversion of alanine to pyruvate and glutamate, and is released in a similar manner [31]. Therefore, SGPT is more specific to the liver, and is thus a better parameter for detecting liver injury. Elevated levels of serum enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver [32-33]. On the other hand, a serum level of ALP is related to the function of hepatic cells. An increase in serum level of ALP is due to increased synthesis in the presence of increasing biliary pressure [34]. In the present study, the data suggested that high dosage of PCM in the liver could lead to decreased levels of GSH, antioxidant enzymes (SOD and CAT), and present a significant level of hepatotoxicity in the course of the treatment. However, the alcoholic bark extract of BO could raise the levels of GSH, SOD, and CAT against the PCM-induced oxidative stress mediated by ROS and RNS. Furthermore, the level of MDA was increased in the group receiving PCM administration, but pretreatment with the alcoholic bark extract of BO reduced the amount of MDA. This result indicated that decreasing the formation of lipid peroxidation is also one of the events in preventing the oxidative toxicity by PCM. The reversal may be because of the prevention of the leakage of intracellular enzymes by its membrane- stabilizing activity. This is in agreement with the commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and the regeneration of hepatocytes.
radical scavengers and reduce or inhibit the oxidative stress induced by PCM administration. Further studies are, however, needed to isolate the specific components responsible for the antioxidant action of this extract and to establish their mechanism of action.
Acknowledgements The authors are thankful to Dr. A. Kamal, Project Director, NIPER-Hyderabad for financial support, and to Dr. K. Madhava Chetty, Sri Venkateswara University, Tirupathi for authentication of the plant material.
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Cite this article as: Bandari Uma Mahesh, Shweta Shrivastava, Rajeswara Rao Pragada, V.G.M. Naidu, Ramakrishna Sistla. Antioxidant and hepatoprotective effects of Boswellia ovalifoliolata bark extracts [J]. Chinese Journal of Natural Medicines, 2014, 12(9): 663-671
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