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Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats
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Research Paper
Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats Dharmendra Singh a,b, P.V. Arya b, Ashutosh Sharma c, M.P. Dobhal c, R.S. Gupta a,n a
Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur 302 055, India Department of Zoology, Dyal Singh College, University of Delhi, Lodhi Road, New Delhi 110 003, India c Department of Chemistry, University of Rajasthan, Jaipur 302 055, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 19 July 2014 Received in revised form 8 December 2014 Accepted 16 December 2014
Ethnopharmacological relevance: α-Amyrin- a pentacyclic triterpene is widely distributed in nature and Q2 isolated from a variety of plant sources and pharmacologically showed a wide spectrum of activity including anti-inflammatory, anti-ulcer, anti-hyperlipidemic, anti-tumor, and hepatoprotective actions. Aim of the study: To explore a new potent as hepatomodulator from natural sources, α-amyrin- a pentacyclic triterpene isolated from the ethanol extract of the stem bark of Alstonia scholaris Linn., was evaluated against CCl4-induced hepatic oxidative stress through antioxidant status in wistar albino rats. Materials and methods: Experimental rats, hepato-oxidatively stressed by CCl4 (0.2 ml/kg b wt/twice a week, intra-peritoneally), were concurrently received α-amyrin (20 mg/kg body weight/day, orally) for 30 consecutive days. Hepatomodulatory potential was assessed by using the serum- markers like γ-glutamyl transpeptidase (GGT), aspartate and alanine transaminases (AST, ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), acid phosphatase (ACP), sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GDH), and total bilirubin, total protein, glutathione reduced (GSH), ceruloplasmin, β-carotene, vitamin C and vitamin E in serum concomitantly with the hepatic-antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GST), and 5´-nucleotidase, acid ribonuclease, glucose-6-phosphatase, succinic dehydrogenase and cytochrome-P-450 in liver tissue whereas lipid peroxidation (LPO) was estimated in both serum and liver contents. Results: The assessment of all biochemical parameters registered a significant (P o0.001) hepatic oxidative stress in CCl4 treated rats, which was considerably recovered near to almost normal level in rats co-administered with α-amyrin at the dose level of 20 mg/kg body weight/day for 30 consecutive days. The histoarchitectural examination of liver sections from treated groups further corroborated the hepatomodulatory potential of α-amyrin and compared with standard drug-silymarin. Conclusions: These findings indicate that the modulatory potential of α-amyrin against hepatic oxidative stress possibly involve mechanism related to its ability to block the P-450 mediated CCl4 bioactivation through selective inhibitors of ROS (reactive oxygen species) as antioxidants brought about significant inhibition of the formation of LPO suggesting possible involvement of O●2 , HO2, HO●2 , H2O2 and OH. Therefore this study suggests that the use of α-amyrin as a hepatomodulatory potent to feasibility for a promising liver curative drug. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: α-Amyrin Antioxidants Carbon tetrachloride Oxidative stress Marker enzymes
1. Introduction Liver plays an important role in drug elimination and detoxification, but in turn, it can be subjected to damage by xenobiotics, alcohol consumption, malnutrition, infection, anemia and certain drugs (Mamat et al., 2013). Herbs have attracted a great deal of
n
Corresponding author. Tel.: þ 91 141 2711228; fax: þ 91 141 2701137. E-mail address: [email protected] (R.S. Gupta).
interest as physiologically functional foods and as a source for the development of liver curing drugs. Experimental and clinical researches have confirmed the efficacy of few plants like Silybum marianum (Milk thistle), Picrorhiza kurroa (Kutkin), Curcuma longa (turmeric), Camellia sinensis (green tea) and Glycyrrhiza glabra (licorice) etc. (Luper, 1999; Kumar et al., 2012). In spite of significant advances in medicinal plant research and rapid strides in modern medicine, there is a continuous need for more precise, safe and effective treatments of liver diseases. In recent years, there has been a shift towards therapeutic evolution
http://dx.doi.org/10.1016/j.jep.2014.12.025 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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D. Singh et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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of herbal products in liver diseases by carefully synergizing the strengths of the traditional systems of medicine with that of the modern concept of evidence based medicinal evolution. Alstonia scholaris Linn. R. Br. (Apocynaceae) commonly known as “Sapthaparna” has been used for centuries in Ayurvedic medication for the treatment of numerous sicknesses as medicinal plant and largely encountered in the tropical and sub-tropical regions of the world including India. In India, the therapeutic use of Alstonia scholaris has been described in both codified and noncodified drug systems for the treatment of malarial fever, hepatitis, leprosy, menstrual disorder, rheumatism, tuberculosis, gastrointestinal troubles, cancer, snake bites and dog bite sicknesses (Khyade et al., 2014). Preclinical studies of the bark and leaves have shown that it possesses anti-microbial, anti-diarrhoeal, anti-plasmodial, antioxidant, anti-inflammatory, hepatoprotective, anti-stress, immunomodulatory, analgesic, anti-ulcer, wound healing, anti-cancer, chemopreventive, radiation protection, radiation sensitization, and chemosensitization activities. The diverse pharmacological observations are supposed to be due to the presence of alkaloids, flavonoids, phenolic acids and terpenes etc. (Lin et al., 1996; Baliga, 2012). α and β-Amyrin are well studied pentacyclic triterpenes and are abundantly occurred in Alstonia species and other plants like Protium heptaphyllum, which have been offered, for hepatoprotective activity (Oliveira et al., 2005), antihyperglycemic and hypolipidemic activity (Santos et al., 2012) etc. We isolated α-amyrin, as a major constituent from the chloroform extract of Alstonia scholaris stem bark. Further, various pharmacological activities of α-amyrin have been conducted in mammals. However, no attention has been paid to its modulatory potential against hepatic oxidative stress through possible mechanism of action and by the antioxidant status. Hence, the present study aimed to express the modulatory potential of α-amyrin, using CCl4-induced hepatic oxidative damage in rats.
2. Materials and methods 2.1. Plant material Fresh stem bark of Alstonia scholaris was collected from the University Campus, University of Rajasthan, Jaipur and authenticated by the Department of Botany, University of Rajasthan, Jaipur (Herbarium Sheet no. RUBL-19939).
2.2. Extraction and fractionation of plant material The stem bark (3 kg) was shade-dried, grinded to powder and extracted with ethanol for 48 h exhaustively. The filtrate obtained was concentrated under reduced pressure in a rotary evaporator (Labmate (Asia) Pvt. Ltd., Chennai, India) at o40 1C, which yielded a semi-solid brown mass. The brownish extract was washed with petroleum ether to remove the fatty portion. The petroleum ether was removed under reduced pressure. Fat free extract was then extracted with chloroform. The chloroform soluble portion afforded 30 gm of crude extract after the removal of solvent. The obtained crude extract was subjected to column chromatography. For this purpose, a column (1.2 m 5 cm) filled with 800 g Si-gel G (60–120 mesh) was used. The column was eluted with different solvents using mixtures of petroleum ether and chloroform in the order of increasing polarity. The C1–C6 fractions were obtained, respectively. All these fractions were tested for their hepatomodulatory potent against oxidative stress (data not shown).
2.3. Isolation and identification of active compound The column was eluted with petroleum ether and chloroform in the ratio of 7:3 (v/v). The purified compound-A from the fraction C3 was obtained as colorless crystals (0.85 gm, 0.28% yield), showing better hepatomodulatory potent against oxidative stress than other fractions (data not shown). Its melting point was 184–186 1C and recrystallized by ethanol. It developed yellow color with tetra nitro methane (TNM) indicating unsaturation of compound and showed the presence of triterpenoid group. The structure of purified compound-A was identified by mass spectrometry (Agilent 1100 Series LC/MSD API-ES spectrometer, Agilent Technologies Inc., Santa Clara, CA, USA), infrared analyses (FTIR8400S Spectrophotometer, Shimadzu, Japan) and Nuclear Magnetic Resonance (NMR) (JEOL AL-300 MHz Spectrometer, JEOL USA Inc., Peabody, MA, USA.), using 1H NMR (500 MHz) and 13C NMR (125 MHz) analyses in CDCl3 solution and by comparison of spectral data with those available in the literature. 2.4. Structure elucidation and characterization of active compound The mass spectrum of compound-A showed the molecular ion peak at m/z 426 [M þ ] corresponding to its molecular formula C30H50O. MS (m/z): 427 [Mþ 1] þ , 426 [M] þ , 409, 341, 327, 313, 271, 218, 203, 135, 119. The IR spectrum υmax [KBr] of above compound confirmed the presence of hydroxyl group by showing a broad absorption at 3400. Other characteristic appearance of absorption band at 1380 and 1368 confirmed the presence of gem dimethyl group [QC(CH3)2] deformation and C–O stretching was also established by an absorption at 1050.The 4CQCo stretching was analyzed by the absorption at 1600 and 1550. The 1H NMR spectrum (δ ppm, CDCl3) of the isolated compound showed absorption at 5.15 which was assigned to the olefinic proton at C–12 position. Other sharp absorptions at 1.1 (s, 3H, C–27), 1.05 (s, 3H, C–28), 0.93 (s, 3H, C–23), 0.87 (d, 3H, C–29), 0.84 (d, 3H, C–30), 0.80 (s, 3H, C–25), 0.77 (s, 3H, C–26) and 0.73 (s, 3H, C–24) for eight methyl groups. The hydroxyl group was analyzed by an absorption band at 4.43 on account of C–3 atom. The signals observed from 1.28 to 2.15 were ascertained to remaining 23 protons. 1 H NMR (δ ppm, CDCl3): 4.43 (t, 1H, C–3), 5.15 (d, 1H, C–12), 0.93 (s, 3H, C–23), 0.73 (s, 3H, C–24), 0.80 (s, 3H, C–25), 0.77 (s, 3H, C–26), 1.1 (s, 3H, C–27), 1.05 (s, 3H, C–28), 0.87 (d, 3H, C–29), 0.84 (d, 3H, C–30), and 1.28–2.15 ppm (remaining 23 protons). In the 13C NMR spectrum (δ ppm, CDCl3), the absorptions were observed at 38.61 (C–1), 24.1 (C–2), 79.41 (C–3), 38.01 (C–4), 55.72 (C–5), 19.03 (C–6), 23.86 (C–7), 41.10 (C–8), 48.15 (C–9), 37.43 (C–10), and 18.2 (C–11). The values of other carbons were assigned at 43.4 (C–14), 29.12 (C–15), 37.43(C–16), 33.21(C–17), 59.53 (C–18), 40.25 (C–19), 38.97 (C–20), 31.68 (C–21), and 40.97 (C–22). Two signals at 125.04 and 140.1 were assigned to olefinic carbons i.e. C–12 and C–13. 13 C NMR (δ ppm, CDCl3): 38.61 (C–1), 24.1 (C–2), 79.41 (C–3), 38.01 (C–4), 55.72 (C–5), 19.03 (C–6), 23.86 (C–7), 41.10 (C–8), 48.15 (C–9), 37.43 (C–10), 18.2 (C–11), 125.04 (C–12), 140.1 (C–13), 43.4 (C–14), 29.12 (C–15), 37.43(C–16), 33.21(C–17), 59.53 (C–18), 40.25 (C–19), 38.97 (C–20), 31.68 (C–21), 40.97 (C–22), 27.91 (C–23), 16.78 (C–24), 14.89 (C–25), 16.90 (C–26), 23.81 (C–27), 28.53 (C–28), 23.46 (C–29), and 21.52 (C–30). Methyl groups attached to C–23, C–24, C–25, C–26, C–27, C–28, C–29 and C–30 were appeared at 27.91, 16.78, 14.89, 16.90, 23.81, 28.53, 23.46 and 21.52 respectively. These spectral data of immersions were also showed to be a good agreement with the reported values for α-amyrin (Rahman
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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After acclimatization of 15 days, the animals were divided into the following groups of 06 rats in each group: Group I: Vehicle treated rats were kept on normal diet and served as control for 30 consecutive days. Group II: Rats were intoxicated with carbon tetrachloride (CCl4) at the dose level – 0.2 ml/kg body weight twice a week, intraperitoneally for 30 consecutive days. Group III: Rats were orally received α-amyrin at the dose level – 20 mg/kg body weight/day, and CCl4 as group II, for 30 consecutive days, simultaneously. Group IV: Rats were orally received silymarin at the dose level – 20 mg/kg body weight/day, and CCl4 as group II, for 30 consecutive days, simultaneously.
Fig. 1. Chemical structure of α-amyrin.
2.11. Assessment of liver function et al., 1990; Sirat et al., 2010; Dias et al., 2011). Therefore, on the basis of 1H NMR, 13C NMR and mass spectral studies of compoundA from fraction C3 was characterized as α-amyrin (3β-hydroxy-urs12-en-3-ol) (Fig. 1) having molecular formula C30H50O and experimentally used for hepatomodulatory potential to assess its possibility as a promising therapeutic agent. 2.5. Chemicals All chemicals were analytical grade and chemicals required for all biochemical assays were obtained from Sigma Chemicals Co., USA. 2.6. Standard drug Silymarin was purchased from MP Biomedicals, France and used as reference standard drug for oral administration to rats during experimentation for 30 consecutive days (Singh et al., 2009). 2.7. Animal model Colony bred healthy, adult male albino rats (wistar strain) (Rattus norvegicus) weighing 175–200 g were used in the present study. The rats were housed in polypropylene cages under controlled conditions of temperature (23–26 1C), humidity (60–70%) and light (12 hrs light/dark cycle). They were provided with a nutritionally adequate standard laboratory diet (Lipton, India Ltd.) and tap water ad libitum. 2.8. Ethical aspects The study was approved by the ethical committee (Protocol no: 1678/Go/a/12/CPCSEA/93) of the University Department of Zoology, Jaipur, India. Indian National Science Academy, New Delhi, (INSA, 2000) guidelines were followed for maintenance and use of the experimental animals. 2.9. Chronic toxicity α-Amyrin was administered to the trial groups in graded doses ranging up to 100 mg/kg body weight/day and the rats were observed for any signs of mortality and behavioral infirmities for 30 days afterwards. Its LD50 value was found to be higher than 100 mg/kg body-weight in the experimental animals (data not shown). The minimum dose level of α-amyrin viz. 20 mg/kg body weight/day was used for oral administration to rats during experimentation (Singh et al., 2009).
After 24 h of last dose delivery, the rats of each group were anesthetized with ether and blood was collected by cardiac puncture in heparinized vials. Serum-GGT, AST, ALT, LDH, ALP, ACP, SDH, GDH, total bilirubin and total protein were determined using diagnostic kits. GGT (batch no. 34004), AST (batch no. 61105), ALT (batch no. 60805) kits were purchased from Accurex Biomedical Pvt. Ltd., Mumbai, India. LDH (lot no. 6854), ALP (lot no. 7093), ACP (lot no. 6666), SDH (lot no. 6810), GDH (lot no. 6988), total bilirubin (lot no. 6801), and total protein (lot no. 6808) kits were purchased from Span Diagnostic Ltd., Surat, India, alongwith various serumantioxidant markers such as GSH (Moron et al., 1979), vitamin C (Omaye et al., 1971), vitamin E (Baker et al., 1980), ceruloplasmin (Ravin, 1961), β-carotene (Bradley and Hombeck, 1973) and lipid peroxidation (Ohkawa et al., 1979), respectively. After the collection of blood, liver was immediately excised, washed with cold saline, blotted, weighed and a part of it was minced and homogenized in ice-cold 1.15% w/v KCl in a Potter Elvehjem Teflon glass homogenizer for 1 min to make a 10% w/v liver homogenate. Lipid peroxidation (LPO) (Ohkawa et al., 1979), succinic dehydrogenase (Green et al., 1955), glucose-6-phosphatase (Baginski et al., 1974) and 5´-nucleotidase (Aronson and Touster, 1974) were measured in the liver homogenate. The activities of hepatic lysosomal enzyme-acid ribonuclease (De Duve et al., 1955), antioxidant enzymes like SOD (Marklund and Marklund, 1974), CAT (Aebi, 1984), GPx (Paglia and Velentine, 1967), GR (Carlberg and Mennervick, 1985) and GST (Habig et al., 1974) were also determined. A liver microsomal fraction was prepared (Schneider and Hogeboom, 1950) and the cytochrome P-450 content in this fraction was measured from a reduced carbon monoxide difference spectrum (Omura and Sato, 1964), respectively. 2.12. Histoarchitectural examination of liver Remaining part of the liver was fixed in Bouin's fixative for 24 h after that dehydrated in ethanol series (50–100%), cleared in xylene, and embedded in paraffin using the standard microtechnique (Gray, 1952). Sections of the liver (5 mm) stained with alum haematoxylin and eosin (H–E) for histopathological changes and images were captured by using a light microscope (Nikon-DS-L1-5M). 2.13. Statistical analysis The results obtained in the present study were expressed as the mean 7SEM for each parameter and statistically processed by applying Student ‘t’-test. P-values o0.05 were considered as significant.
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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D. Singh et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
11.76 71.25 38.53 71.87a 18.96 71.19a 20.21 71.55a
42.34 7 1.84 137.29 7 2.26a 50.417 1.65a 55.30 7 0.96a
7.89 7 0.03 16.42 7 1.21a 10.06 7 0.87b 11.98 7 0.43b
0.65 7 0.09 1.83 7 0.17a 0.727 0.10a 0.94 7 0.12b
6.56 7 0.31 3.08 7 0.28a 6.98 7 0.33a 6.93 7 0.21a
The results of biochemical parameters revealed that the administration of carbon tetrachloride (CCl4) to rats caused significant (P o0.001) oxidative stress as evidenced by marker enzymes and antioxidant defense system through liver and serum contents (Tables 1 and 2, and Figs. 2–9). The treatment with CCl4 elevated hepatic enzymatic activities of 5´-nucleotidase, acid ribonuclease and attenuated succinic dehydrogenase, glucose-6-phosphatase, with a concurrent decline in hepatic cytochrome P-450 level (Table 2). The significant (P o0.001) protection against CCl4 induced alterations in these parameters which was achieved with the oral treatment of α-amyrin and silymarin (Table 2). The significantly equal protection was showed in α-amyrin and silymarin treated groups against CCl4 induced hepatic aberrations. The activities of GSH, ceruloplasmin, β-carotene, vitamin C and vitamin E in serum were significantly (P o0.001) decreased in CCl4 treated rats (Figs. 2 and 3). Simultaneous treatment with α-amyrin and silymarin afforded a significant (P o0.001) protection against CCl4-induced alterations in the serum antioxidant levels (Figs. 2 and 3). The degree of protection was not significantly different in the both α-amyrin and silymarin treated groups. Carbon tetrachloride caused a significant (P o0.001) decline in the hepatic antioxidant enzymes such as SOD, CAT, GPx, GR and GST in comparison to normal controls (Figs. 4–7). In contrast, oral treatment to rats with α-amyrin and silymarin, at the dose level of 20 mg/kg body weight/day, showed a significant (P o0.001) elevating effect on CCl4-induced depletion in the levels of hepatic antioxidant enzymes (Figs. 4–7). The elimination of hepatic oxidative stress by α-amyrin and silymarin was statistically similar in nature (Figs. 4–7). The rats treated with CCl4, developed a significant (P o0.001) elevation in the level of lipid peroxidation as in the both serum and liver contents (Fig. 8). In contrast, treatment with α-amyrin and silymarin showed a significant (P o0.001) lowering effect on CCl4-induced elevation of lipid peroxidation in both serum and liver contents (Fig. 8). The lowing effect of lipid peroxidation by αamyrin and silymarin treatment was found to be similar statistically after 30 consecutive days of experimentation. The histopathological study of CCl4-induced rats liver sections when compared to normal hepatic architecture (Fig. 9A) showed intense centrilobular necrosis and vacuolization in the hepatocytes (Fig. 9B). The oral treatment with α-amyrin and silymarin, along with CCl4, showed signs of protection against CCl4 toxicity to a considerable extent as evident from the formation of normal hepatic cords and absence of necrosis and vacuoles in the liver histopathological examinations of the experimental rats (Fig. 9(C) and (D)). Levels of Significance:Data are mean 7 SEM (n¼ 6). a¼ Po 0.001 Gp. II compared with control (Gp. I). a¼ Po 0.001; b ¼ Po 0.01 Gp. III compared with Gp. II. a¼ Po 0.001; b ¼ Po 0.01 Gp. IV compared with Gp. II.
81.20 72.93 165.93 73.20a 86.79 72.41a 87.48 72.01a 107.42 7 2.53 300.87 7 5.92a 120.94 7 2.03a 127.487 2.07a 124.337 1.47 335.55 7 3.50a 138.38 7 1.69a 131.177 1.52a 8.337 0.30 38.90 7 2.17a 13.317 0.89a 14.90 7 1.76a Control (Group I) CCl4 (Group II) CCl4 þα-amyrin (Group III) CCl4 þSilymarin (Group IV)
20.79 71.29 63.69 72.26a 25.79 71.18a 30.47 71.74a
Total Bilirubin (mg/100 ml) GDH (IU/L) SDH (IU/L) ACP (KAU) LDH (IU/L) ALT (IU/L) AST (IU/L) GGT (IU/L)
Alp (KAU)
3. Results
Treatment design
Table 1 Showing hepatomodulatory potential of α-amyrin and silymarin in wistar albino rats through their serum marker parameters.
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Total protein (gm/dL)
4
4. Discussion Toxic species are the main cause for oxidative damage of hepatocytes which are usually metabolites. In case of CCl4 intake by experimental animals, the toxic species are altered the liver metabolism through oxidative damage and also caused fatty degeneration, fibrosis, hepatocellular death, and carcinogenicity (Weber et al., 2003; Manubolu et al., 2014). It is now generally accepted that the hepatotoxicity by CCl4 is the result of reductive dehalogenation, which is catalyzed by P-450 and forms the highly reactive trichloromethyl free radical (CCl●3). This then readily interact with molecular oxygen to form the trichloromethyl peroxy radical (CCl3OO●). Both radicals are capable of binding to proteins or lipids or of abstracting a hydrogen atom from an unsaturated lipid, which initiate lipid peroxidation and
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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D. Singh et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 2 Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their hepatic biochemical parameters. Treatment design
5´-Nucleotidase (μ mole inorganic phosphate released/ min/mg protein)
Acid ribonuclease (Units/mg protein)
Glucose-6-phosphatase (μ mole inorganic phosphate released/min/ mg protein)
Succinic dehydrogenase (μ mole Cytochrome P450 (n mole/mg indophenol reduced/min/mg protein) protein)
Control (Group I) CCl4 (Group II) CCl4 þ α-amyrin (Group III) CCl4 þ Silymarin (Group IV)
2.89 70.21 5.14 70.32a 3.16 70.24a
14.37 71.33 33.72 73.87a 16.42 71.52b
8.247 0.62 4.747 0.43a 7.78 7 054a
0.76 70.08 0.3370.03a 0.72 70.06a
4.687 0.32 2.017 0.12a 4.08 7 0.32a
3.22 70.28a
17.85 72.13b
7.157 0.48b
0.65 70.04a
3.87 7 0.37a
Levels of Significance: Data are mean 7SEM (n ¼6). a¼ Po 0.001 Gp. II compared with control (Gp. I). a¼ Po 0.001; b¼ Po 0.01 Gp. III compared with Gp. II. a¼ Po 0.001; b¼ Po 0.01 Gp. IV compared with Gp. II.
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(n mole/mg protein)
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10 8 6
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Fig. 2. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their serum non-enzymatic antioxidants. Data points with letter notation (a) are statistically significant at a¼ Po 0.001.
Fig. 4. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their liver enzymatic antioxidant. Data points with letter notation (a) are statistically significant at a ¼P o 0.001.
a
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Fig. 3. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their serum non-enzymatic antioxidants. Data points with letter notation (a) are statistically significant at a¼ Po 0.001.
liver damage and by play a significant role in the pathogenesis of diseases (Williams and Burk, 1990; Weber et al., 2003). Therefore, the suppression of P-450 can result in a reduction in the level of the reactive metabolites, and correspondingly, less tissue injury. The metabolic activation of CCl4 is believed to be mediated through P-450 2E1 (Zangar et al., 2000; Murthy et al., 2014). The inhibitory effect of CCl4 on cytochrome P-450 level was also compensated by α-amyrin and silymarin through maintenance of its normal level. The role of α-amyrin in the protection of
Fig. 5. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their liver enzymatic antioxidant. Data points with letter notation (a) are statistically significant at a ¼P o 0.001.
CCl4-mediated loss in cytochrome P-450 content may be considered as an indication of improved protein synthesis in the hepatic cells (Mandal et al., 1993; Murthy et al., 2014). The lipid peroxidative degradation of biomembranes is one of the principle causes of hepatotoxicity induced by CCl4. Because lipid peroxidation is viewed as a complicated biochemical reaction involving free radicals, oxygen, metal ions and a host of other factors in the biological system. Since lipids constitute nearly 60% of the compounds in biomembranes, only major perturbation is bound to affect structure and function of the cell. In recent years, lipids and their derivatives have been recognized as important molecules in signal transduction (Manubolu et al., 2014). The efficacy of any hepatoprotective drug is dependent on its capacity of either reducing the harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4 and
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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a
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Fig. 6. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their liver enzymatic antioxidants. Data points with letter notation (a) are statistically significant at a¼ Po 0.001.
8 7 6 5 4 3 2 1 0
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GST Fig. 7. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their liver enzymatic antioxidant. Data points with letter notation (a) are statistically significant at a¼ Po 0.001.
(n mole MDA/mg protein)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
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Fig. 8. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their serum and tissue lipid peroxidation. Data points with different letter notations (a, b) are significantly different at a¼ Po 0.001; b¼ Po 0.01.
other hepatotoxicants (Mani Senthilkumar et al., 2005). In our present investigation, the measurement of lipid peroxidation in the liver tissue and serum is a convenient method to monitor oxidative cell damage. Inhibition of elevated LPO has been observed in α-amyrin and silymarin treated groups due to its antioxidant and free radical scavenging activities through reestablishment of biomembranes of hepatic parenchymal cells (Singh et al., 2014a). In agreement with results obtained in previous investigations (Bishayee et al., 1995; Murthy et al., 2014; Shaaban et al., 2014), our present study elicited a significant increase in the activities of GGT, AST, ALT, LDH, ALP, ACP, SDH, and GDH with the exposure of CCl4 which indicating considerable hepatocellular injury. Oral
treatment with α-amyrin and silymarin attenuated these increased enzyme activities produced by CCl4 and a subsequent recovery towards normalization of these enzymes strongly suggests the possibility of α-amyrin being able to state the hepatocytes so as to cause accelerated regeneration of parenchymal cells and lysosomes, thus protecting against lysosomal integrity and cell membrane fragility decreasing the leakage of marker enzymes into the circulation (Bishayee et al., 1995; Murthy et al., 2014). Stabilization of serum-total bilirubin and total protein levels by the administration of α-amyrin is further a clear indication of the improvement of functional status of the hepatic cells (Shah et al., 2013; Singh et al., 2014a). The protective role of α-amyrin and silymarin has also been reflected in the normal levels of hepatic glucose-6-phosphatase, 5´-nucleotidase and succinic dehydrogenase activities that were severely altered in the CCl4 intoxicated rats liver. A significant lower level of acid ribonuclease enzyme, as a result of α-amyrin and silymarin treatment against CCl4 induced marked elevation, indicates a protection against the rupture of lysosomes and leakage of this enzyme in situ as a consequence of damage to the lysosomes of CCl4 treated liver cells (Kumar et al., 2007). The antioxidant defense enzymes have been suggestive to playing an important role in the maintaining of physiological levels of oxygen and hydrogen peroxide and eliminating peroxides generated from inadvertent exposure to xenobiotics and drugs. Any natural compound with antioxidant properties may help in maintaining health when continuously taken as components of dietary food, spices or drugs. The increase in the levels of antioxidant profiles i.e. SOD, CAT, GR and GPx by α-amyrin and silymarin may be attributed to have biological significance in eliminating reactive free radicals that may affect the normal functioning of cells. GST is a soluble protein located in the cytosol, and plays an important role in the detoxification and excretion of xenobiotics and drugs (Singh et al., 2000; Huo et al., 2011). Moreover, the GST functionally binds GSH and the endogenous or exogenous substances. Since it increases the solubility of hydrophobic substances, it also plays an important role in the storage and excretion of xenobiotics and drugs. During our experiment, both the α-amyrin and silymarin increased the GST activity and metabolized toxic compounds to nontoxic compounds and protect the liver of experimental rats (Huo et al., 2011; Singh et al., 2014b). Our study further revealed that chronic exposure to CCl4 significantly decreased the activities of non-enzymic antioxidants namely GSH, ceruloplasmin, β-carotene, vitamin C and vitamin E in serum might be responsible for hepatocellular injury. The supplementation of α-amyrin and silymarin was enhanced non-enzymic antioxidants significantly in CCl4 treated rats. GSH, vitamin C and vitamin E exist in their inter convertible forms and participate in the detoxification of the toxic reactive oxygen species. Regeneration to their reduced forms is brought about by reduced glutathione because the detoxification pathway involves GSH conjugation of the trichloromethyl radical, a P-450 2E1mediated CCl4 metabolite (Singh et al., 2014b). Previous studies on the mechanism of CCl4-induced hepatotoxicity have shown that GSH plays a key role in detoxifying the reactive toxic metabolites of CCl4 and that liver necrosis begins when the GSH stores are markedly depleted (Recknagel et al., 1991). GSH is largely mediated through the activity of GST and forms adducts with the toxic metabolites of CCl4 (Singh et al., 2014b). Moreover, GSH contribute to the detoxification of CCl4 and it has been suggested that one of the principle cause of CCl4-induced liver injury is lipid peroxidation caused by its free radical derivatives (Recknagel et al., 1991; Singh et al., 2014b). Vitamin C is considered to be the most important antioxidant in extracellular fluids (Stocker and Frei, 1991; Gupta and Singh, 2006) and also acts to protect membranes against peroxidation by enhancing the activity
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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Fig. 9. Photomicrograph of rat liver sections with haematoxylin-eosin at X100. (A) Showing as control with well brought central vein, hepatic cell with preserved cytoplasm and prominent nucleus; (B) Showing after CCl4 intoxication as marked steatosis (S) of the hepatocytes with ballooning degeneration and distended portal vein, mild periportal fibrosis and necrosis (N); (C) Showing after α-amyrin and CCl4 treatment as considerable reduction in necrosis and fatty changes with pyknotic nuclei and cytoplasmic clearing; (D) Showing after silymarin (standard) and CCl4 treatment as moderately regeneration in hepatocellular architecture. Scale bar, 100 μm.
of α-tocopherol, the chief lipid soluble and chain breaking antioxidant. β-carotene is a potent free radical quencher, singlet oxygen scavenger and lipid peroxidation (Blot et al., 1995; Gupta and Singh, 2006). Histopathological interpretations suggested that the reactive oxygen metabolites and lipid peroxidation may play a role in the various pathological lesions i.e. marked steatosis, pseudolobulation, ballooning degeneration, periportal fibrosis and necrosis with the loss of normal liver architecture. Because of CCl4 toxicity, a toxic reactive metabolite-trichloromethyl free radical was produced which binds covalently to the macromolecules of the lipid membranes of the adipose tissue and causes peroxidative degradation. As a result, fats from the adipose tissue are translocated and accumulated in the hepatocytes (Okuno et al., 1998; Cordeiro and Kaliwal, 2013).The degenerative changes showed minimal or absence with α-amyrin and silymarin treatments. α-Amyrin because of its ability to reduce fat accumulation, scavenge free radicals, interaction with oxidative cascade, quenching oxygen ions, inhibiting oxidative enzymes and lipid peroxidation, and restores the hepatic antioxidant status of the experimental rats.
5. Conclusion It can be concluded that the modulatory potential of α-amyrin against hepatic oxidative stress possibly involves mechanism related to its ability to block the P-450 mediated CCl4 bioactivation through selective inhibitors of ROS (reactive oxygen species) as antioxidants brought about significant inhibition of the formation of LPO suggesting possible involvement of O●2 , HO2, HO●2 , H2O2 and OH. Therefore this study suggests the use of α-amyrin as a hepatomodulatory potent to feasibility for a promising liver curative drug. The appropriate mechanism of action and comparative evaluation of α-amyrin and β-amyrin is in progress and will be reported elsewhere.
Acknowledgments The authors are thankful to the respective authorities for providing the necessary facilities and support.
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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Research Paper
Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats Dharmendra Singh a,b, P.V. Arya b, Ashutosh Sharma c, M.P. Dobhal c, R.S. Gupta a,n a
Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur 302 055, India Department of Zoology, Dyal Singh College, University of Delhi, Lodhi Road, New Delhi 110 003, India c Department of Chemistry, University of Rajasthan, Jaipur 302 055, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 19 July 2014 Received in revised form 8 December 2014 Accepted 16 December 2014
Ethnopharmacological relevance: α-Amyrin- a pentacyclic triterpene is widely distributed in nature and Q2 isolated from a variety of plant sources and pharmacologically showed a wide spectrum of activity including anti-inflammatory, anti-ulcer, anti-hyperlipidemic, anti-tumor, and hepatoprotective actions. Aim of the study: To explore a new potent as hepatomodulator from natural sources, α-amyrin- a pentacyclic triterpene isolated from the ethanol extract of the stem bark of Alstonia scholaris Linn., was evaluated against CCl4-induced hepatic oxidative stress through antioxidant status in wistar albino rats. Materials and methods: Experimental rats, hepato-oxidatively stressed by CCl4 (0.2 ml/kg b wt/twice a week, intra-peritoneally), were concurrently received α-amyrin (20 mg/kg body weight/day, orally) for 30 consecutive days. Hepatomodulatory potential was assessed by using the serum- markers like γ-glutamyl transpeptidase (GGT), aspartate and alanine transaminases (AST, ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), acid phosphatase (ACP), sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GDH), and total bilirubin, total protein, glutathione reduced (GSH), ceruloplasmin, β-carotene, vitamin C and vitamin E in serum concomitantly with the hepatic-antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GST), and 5´-nucleotidase, acid ribonuclease, glucose-6-phosphatase, succinic dehydrogenase and cytochrome-P-450 in liver tissue whereas lipid peroxidation (LPO) was estimated in both serum and liver contents. Results: The assessment of all biochemical parameters registered a significant (P o0.001) hepatic oxidative stress in CCl4 treated rats, which was considerably recovered near to almost normal level in rats co-administered with α-amyrin at the dose level of 20 mg/kg body weight/day for 30 consecutive days. The histoarchitectural examination of liver sections from treated groups further corroborated the hepatomodulatory potential of α-amyrin and compared with standard drug-silymarin. Conclusions: These findings indicate that the modulatory potential of α-amyrin against hepatic oxidative stress possibly involve mechanism related to its ability to block the P-450 mediated CCl4 bioactivation through selective inhibitors of ROS (reactive oxygen species) as antioxidants brought about significant inhibition of the formation of LPO suggesting possible involvement of O●2 , HO2, HO●2 , H2O2 and OH. Therefore this study suggests that the use of α-amyrin as a hepatomodulatory potent to feasibility for a promising liver curative drug. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: α-Amyrin Antioxidants Carbon tetrachloride Oxidative stress Marker enzymes
1. Introduction Liver plays an important role in drug elimination and detoxification, but in turn, it can be subjected to damage by xenobiotics, alcohol consumption, malnutrition, infection, anemia and certain drugs (Mamat et al., 2013). Herbs have attracted a great deal of
n
Corresponding author. Tel.: þ 91 141 2711228; fax: þ 91 141 2701137. E-mail address: [email protected] (R.S. Gupta).
interest as physiologically functional foods and as a source for the development of liver curing drugs. Experimental and clinical researches have confirmed the efficacy of few plants like Silybum marianum (Milk thistle), Picrorhiza kurroa (Kutkin), Curcuma longa (turmeric), Camellia sinensis (green tea) and Glycyrrhiza glabra (licorice) etc. (Luper, 1999; Kumar et al., 2012). In spite of significant advances in medicinal plant research and rapid strides in modern medicine, there is a continuous need for more precise, safe and effective treatments of liver diseases. In recent years, there has been a shift towards therapeutic evolution
http://dx.doi.org/10.1016/j.jep.2014.12.025 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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of herbal products in liver diseases by carefully synergizing the strengths of the traditional systems of medicine with that of the modern concept of evidence based medicinal evolution. Alstonia scholaris Linn. R. Br. (Apocynaceae) commonly known as “Sapthaparna” has been used for centuries in Ayurvedic medication for the treatment of numerous sicknesses as medicinal plant and largely encountered in the tropical and sub-tropical regions of the world including India. In India, the therapeutic use of Alstonia scholaris has been described in both codified and noncodified drug systems for the treatment of malarial fever, hepatitis, leprosy, menstrual disorder, rheumatism, tuberculosis, gastrointestinal troubles, cancer, snake bites and dog bite sicknesses (Khyade et al., 2014). Preclinical studies of the bark and leaves have shown that it possesses anti-microbial, anti-diarrhoeal, anti-plasmodial, antioxidant, anti-inflammatory, hepatoprotective, anti-stress, immunomodulatory, analgesic, anti-ulcer, wound healing, anti-cancer, chemopreventive, radiation protection, radiation sensitization, and chemosensitization activities. The diverse pharmacological observations are supposed to be due to the presence of alkaloids, flavonoids, phenolic acids and terpenes etc. (Lin et al., 1996; Baliga, 2012). α and β-Amyrin are well studied pentacyclic triterpenes and are abundantly occurred in Alstonia species and other plants like Protium heptaphyllum, which have been offered, for hepatoprotective activity (Oliveira et al., 2005), antihyperglycemic and hypolipidemic activity (Santos et al., 2012) etc. We isolated α-amyrin, as a major constituent from the chloroform extract of Alstonia scholaris stem bark. Further, various pharmacological activities of α-amyrin have been conducted in mammals. However, no attention has been paid to its modulatory potential against hepatic oxidative stress through possible mechanism of action and by the antioxidant status. Hence, the present study aimed to express the modulatory potential of α-amyrin, using CCl4-induced hepatic oxidative damage in rats.
2. Materials and methods 2.1. Plant material Fresh stem bark of Alstonia scholaris was collected from the University Campus, University of Rajasthan, Jaipur and authenticated by the Department of Botany, University of Rajasthan, Jaipur (Herbarium Sheet no. RUBL-19939).
2.2. Extraction and fractionation of plant material The stem bark (3 kg) was shade-dried, grinded to powder and extracted with ethanol for 48 h exhaustively. The filtrate obtained was concentrated under reduced pressure in a rotary evaporator (Labmate (Asia) Pvt. Ltd., Chennai, India) at o40 1C, which yielded a semi-solid brown mass. The brownish extract was washed with petroleum ether to remove the fatty portion. The petroleum ether was removed under reduced pressure. Fat free extract was then extracted with chloroform. The chloroform soluble portion afforded 30 gm of crude extract after the removal of solvent. The obtained crude extract was subjected to column chromatography. For this purpose, a column (1.2 m 5 cm) filled with 800 g Si-gel G (60–120 mesh) was used. The column was eluted with different solvents using mixtures of petroleum ether and chloroform in the order of increasing polarity. The C1–C6 fractions were obtained, respectively. All these fractions were tested for their hepatomodulatory potent against oxidative stress (data not shown).
2.3. Isolation and identification of active compound The column was eluted with petroleum ether and chloroform in the ratio of 7:3 (v/v). The purified compound-A from the fraction C3 was obtained as colorless crystals (0.85 gm, 0.28% yield), showing better hepatomodulatory potent against oxidative stress than other fractions (data not shown). Its melting point was 184–186 1C and recrystallized by ethanol. It developed yellow color with tetra nitro methane (TNM) indicating unsaturation of compound and showed the presence of triterpenoid group. The structure of purified compound-A was identified by mass spectrometry (Agilent 1100 Series LC/MSD API-ES spectrometer, Agilent Technologies Inc., Santa Clara, CA, USA), infrared analyses (FTIR8400S Spectrophotometer, Shimadzu, Japan) and Nuclear Magnetic Resonance (NMR) (JEOL AL-300 MHz Spectrometer, JEOL USA Inc., Peabody, MA, USA.), using 1H NMR (500 MHz) and 13C NMR (125 MHz) analyses in CDCl3 solution and by comparison of spectral data with those available in the literature. 2.4. Structure elucidation and characterization of active compound The mass spectrum of compound-A showed the molecular ion peak at m/z 426 [M þ ] corresponding to its molecular formula C30H50O. MS (m/z): 427 [Mþ 1] þ , 426 [M] þ , 409, 341, 327, 313, 271, 218, 203, 135, 119. The IR spectrum υmax [KBr] of above compound confirmed the presence of hydroxyl group by showing a broad absorption at 3400. Other characteristic appearance of absorption band at 1380 and 1368 confirmed the presence of gem dimethyl group [QC(CH3)2] deformation and C–O stretching was also established by an absorption at 1050.The 4CQCo stretching was analyzed by the absorption at 1600 and 1550. The 1H NMR spectrum (δ ppm, CDCl3) of the isolated compound showed absorption at 5.15 which was assigned to the olefinic proton at C–12 position. Other sharp absorptions at 1.1 (s, 3H, C–27), 1.05 (s, 3H, C–28), 0.93 (s, 3H, C–23), 0.87 (d, 3H, C–29), 0.84 (d, 3H, C–30), 0.80 (s, 3H, C–25), 0.77 (s, 3H, C–26) and 0.73 (s, 3H, C–24) for eight methyl groups. The hydroxyl group was analyzed by an absorption band at 4.43 on account of C–3 atom. The signals observed from 1.28 to 2.15 were ascertained to remaining 23 protons. 1 H NMR (δ ppm, CDCl3): 4.43 (t, 1H, C–3), 5.15 (d, 1H, C–12), 0.93 (s, 3H, C–23), 0.73 (s, 3H, C–24), 0.80 (s, 3H, C–25), 0.77 (s, 3H, C–26), 1.1 (s, 3H, C–27), 1.05 (s, 3H, C–28), 0.87 (d, 3H, C–29), 0.84 (d, 3H, C–30), and 1.28–2.15 ppm (remaining 23 protons). In the 13C NMR spectrum (δ ppm, CDCl3), the absorptions were observed at 38.61 (C–1), 24.1 (C–2), 79.41 (C–3), 38.01 (C–4), 55.72 (C–5), 19.03 (C–6), 23.86 (C–7), 41.10 (C–8), 48.15 (C–9), 37.43 (C–10), and 18.2 (C–11). The values of other carbons were assigned at 43.4 (C–14), 29.12 (C–15), 37.43(C–16), 33.21(C–17), 59.53 (C–18), 40.25 (C–19), 38.97 (C–20), 31.68 (C–21), and 40.97 (C–22). Two signals at 125.04 and 140.1 were assigned to olefinic carbons i.e. C–12 and C–13. 13 C NMR (δ ppm, CDCl3): 38.61 (C–1), 24.1 (C–2), 79.41 (C–3), 38.01 (C–4), 55.72 (C–5), 19.03 (C–6), 23.86 (C–7), 41.10 (C–8), 48.15 (C–9), 37.43 (C–10), 18.2 (C–11), 125.04 (C–12), 140.1 (C–13), 43.4 (C–14), 29.12 (C–15), 37.43(C–16), 33.21(C–17), 59.53 (C–18), 40.25 (C–19), 38.97 (C–20), 31.68 (C–21), 40.97 (C–22), 27.91 (C–23), 16.78 (C–24), 14.89 (C–25), 16.90 (C–26), 23.81 (C–27), 28.53 (C–28), 23.46 (C–29), and 21.52 (C–30). Methyl groups attached to C–23, C–24, C–25, C–26, C–27, C–28, C–29 and C–30 were appeared at 27.91, 16.78, 14.89, 16.90, 23.81, 28.53, 23.46 and 21.52 respectively. These spectral data of immersions were also showed to be a good agreement with the reported values for α-amyrin (Rahman
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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After acclimatization of 15 days, the animals were divided into the following groups of 06 rats in each group: Group I: Vehicle treated rats were kept on normal diet and served as control for 30 consecutive days. Group II: Rats were intoxicated with carbon tetrachloride (CCl4) at the dose level – 0.2 ml/kg body weight twice a week, intraperitoneally for 30 consecutive days. Group III: Rats were orally received α-amyrin at the dose level – 20 mg/kg body weight/day, and CCl4 as group II, for 30 consecutive days, simultaneously. Group IV: Rats were orally received silymarin at the dose level – 20 mg/kg body weight/day, and CCl4 as group II, for 30 consecutive days, simultaneously.
Fig. 1. Chemical structure of α-amyrin.
2.11. Assessment of liver function et al., 1990; Sirat et al., 2010; Dias et al., 2011). Therefore, on the basis of 1H NMR, 13C NMR and mass spectral studies of compoundA from fraction C3 was characterized as α-amyrin (3β-hydroxy-urs12-en-3-ol) (Fig. 1) having molecular formula C30H50O and experimentally used for hepatomodulatory potential to assess its possibility as a promising therapeutic agent. 2.5. Chemicals All chemicals were analytical grade and chemicals required for all biochemical assays were obtained from Sigma Chemicals Co., USA. 2.6. Standard drug Silymarin was purchased from MP Biomedicals, France and used as reference standard drug for oral administration to rats during experimentation for 30 consecutive days (Singh et al., 2009). 2.7. Animal model Colony bred healthy, adult male albino rats (wistar strain) (Rattus norvegicus) weighing 175–200 g were used in the present study. The rats were housed in polypropylene cages under controlled conditions of temperature (23–26 1C), humidity (60–70%) and light (12 hrs light/dark cycle). They were provided with a nutritionally adequate standard laboratory diet (Lipton, India Ltd.) and tap water ad libitum. 2.8. Ethical aspects The study was approved by the ethical committee (Protocol no: 1678/Go/a/12/CPCSEA/93) of the University Department of Zoology, Jaipur, India. Indian National Science Academy, New Delhi, (INSA, 2000) guidelines were followed for maintenance and use of the experimental animals. 2.9. Chronic toxicity α-Amyrin was administered to the trial groups in graded doses ranging up to 100 mg/kg body weight/day and the rats were observed for any signs of mortality and behavioral infirmities for 30 days afterwards. Its LD50 value was found to be higher than 100 mg/kg body-weight in the experimental animals (data not shown). The minimum dose level of α-amyrin viz. 20 mg/kg body weight/day was used for oral administration to rats during experimentation (Singh et al., 2009).
After 24 h of last dose delivery, the rats of each group were anesthetized with ether and blood was collected by cardiac puncture in heparinized vials. Serum-GGT, AST, ALT, LDH, ALP, ACP, SDH, GDH, total bilirubin and total protein were determined using diagnostic kits. GGT (batch no. 34004), AST (batch no. 61105), ALT (batch no. 60805) kits were purchased from Accurex Biomedical Pvt. Ltd., Mumbai, India. LDH (lot no. 6854), ALP (lot no. 7093), ACP (lot no. 6666), SDH (lot no. 6810), GDH (lot no. 6988), total bilirubin (lot no. 6801), and total protein (lot no. 6808) kits were purchased from Span Diagnostic Ltd., Surat, India, alongwith various serumantioxidant markers such as GSH (Moron et al., 1979), vitamin C (Omaye et al., 1971), vitamin E (Baker et al., 1980), ceruloplasmin (Ravin, 1961), β-carotene (Bradley and Hombeck, 1973) and lipid peroxidation (Ohkawa et al., 1979), respectively. After the collection of blood, liver was immediately excised, washed with cold saline, blotted, weighed and a part of it was minced and homogenized in ice-cold 1.15% w/v KCl in a Potter Elvehjem Teflon glass homogenizer for 1 min to make a 10% w/v liver homogenate. Lipid peroxidation (LPO) (Ohkawa et al., 1979), succinic dehydrogenase (Green et al., 1955), glucose-6-phosphatase (Baginski et al., 1974) and 5´-nucleotidase (Aronson and Touster, 1974) were measured in the liver homogenate. The activities of hepatic lysosomal enzyme-acid ribonuclease (De Duve et al., 1955), antioxidant enzymes like SOD (Marklund and Marklund, 1974), CAT (Aebi, 1984), GPx (Paglia and Velentine, 1967), GR (Carlberg and Mennervick, 1985) and GST (Habig et al., 1974) were also determined. A liver microsomal fraction was prepared (Schneider and Hogeboom, 1950) and the cytochrome P-450 content in this fraction was measured from a reduced carbon monoxide difference spectrum (Omura and Sato, 1964), respectively. 2.12. Histoarchitectural examination of liver Remaining part of the liver was fixed in Bouin's fixative for 24 h after that dehydrated in ethanol series (50–100%), cleared in xylene, and embedded in paraffin using the standard microtechnique (Gray, 1952). Sections of the liver (5 mm) stained with alum haematoxylin and eosin (H–E) for histopathological changes and images were captured by using a light microscope (Nikon-DS-L1-5M). 2.13. Statistical analysis The results obtained in the present study were expressed as the mean 7SEM for each parameter and statistically processed by applying Student ‘t’-test. P-values o0.05 were considered as significant.
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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11.76 71.25 38.53 71.87a 18.96 71.19a 20.21 71.55a
42.34 7 1.84 137.29 7 2.26a 50.417 1.65a 55.30 7 0.96a
7.89 7 0.03 16.42 7 1.21a 10.06 7 0.87b 11.98 7 0.43b
0.65 7 0.09 1.83 7 0.17a 0.727 0.10a 0.94 7 0.12b
6.56 7 0.31 3.08 7 0.28a 6.98 7 0.33a 6.93 7 0.21a
The results of biochemical parameters revealed that the administration of carbon tetrachloride (CCl4) to rats caused significant (P o0.001) oxidative stress as evidenced by marker enzymes and antioxidant defense system through liver and serum contents (Tables 1 and 2, and Figs. 2–9). The treatment with CCl4 elevated hepatic enzymatic activities of 5´-nucleotidase, acid ribonuclease and attenuated succinic dehydrogenase, glucose-6-phosphatase, with a concurrent decline in hepatic cytochrome P-450 level (Table 2). The significant (P o0.001) protection against CCl4 induced alterations in these parameters which was achieved with the oral treatment of α-amyrin and silymarin (Table 2). The significantly equal protection was showed in α-amyrin and silymarin treated groups against CCl4 induced hepatic aberrations. The activities of GSH, ceruloplasmin, β-carotene, vitamin C and vitamin E in serum were significantly (P o0.001) decreased in CCl4 treated rats (Figs. 2 and 3). Simultaneous treatment with α-amyrin and silymarin afforded a significant (P o0.001) protection against CCl4-induced alterations in the serum antioxidant levels (Figs. 2 and 3). The degree of protection was not significantly different in the both α-amyrin and silymarin treated groups. Carbon tetrachloride caused a significant (P o0.001) decline in the hepatic antioxidant enzymes such as SOD, CAT, GPx, GR and GST in comparison to normal controls (Figs. 4–7). In contrast, oral treatment to rats with α-amyrin and silymarin, at the dose level of 20 mg/kg body weight/day, showed a significant (P o0.001) elevating effect on CCl4-induced depletion in the levels of hepatic antioxidant enzymes (Figs. 4–7). The elimination of hepatic oxidative stress by α-amyrin and silymarin was statistically similar in nature (Figs. 4–7). The rats treated with CCl4, developed a significant (P o0.001) elevation in the level of lipid peroxidation as in the both serum and liver contents (Fig. 8). In contrast, treatment with α-amyrin and silymarin showed a significant (P o0.001) lowering effect on CCl4-induced elevation of lipid peroxidation in both serum and liver contents (Fig. 8). The lowing effect of lipid peroxidation by αamyrin and silymarin treatment was found to be similar statistically after 30 consecutive days of experimentation. The histopathological study of CCl4-induced rats liver sections when compared to normal hepatic architecture (Fig. 9A) showed intense centrilobular necrosis and vacuolization in the hepatocytes (Fig. 9B). The oral treatment with α-amyrin and silymarin, along with CCl4, showed signs of protection against CCl4 toxicity to a considerable extent as evident from the formation of normal hepatic cords and absence of necrosis and vacuoles in the liver histopathological examinations of the experimental rats (Fig. 9(C) and (D)). Levels of Significance:Data are mean 7 SEM (n¼ 6). a¼ Po 0.001 Gp. II compared with control (Gp. I). a¼ Po 0.001; b ¼ Po 0.01 Gp. III compared with Gp. II. a¼ Po 0.001; b ¼ Po 0.01 Gp. IV compared with Gp. II.
81.20 72.93 165.93 73.20a 86.79 72.41a 87.48 72.01a 107.42 7 2.53 300.87 7 5.92a 120.94 7 2.03a 127.487 2.07a 124.337 1.47 335.55 7 3.50a 138.38 7 1.69a 131.177 1.52a 8.337 0.30 38.90 7 2.17a 13.317 0.89a 14.90 7 1.76a Control (Group I) CCl4 (Group II) CCl4 þα-amyrin (Group III) CCl4 þSilymarin (Group IV)
20.79 71.29 63.69 72.26a 25.79 71.18a 30.47 71.74a
Total Bilirubin (mg/100 ml) GDH (IU/L) SDH (IU/L) ACP (KAU) LDH (IU/L) ALT (IU/L) AST (IU/L) GGT (IU/L)
Alp (KAU)
3. Results
Treatment design
Table 1 Showing hepatomodulatory potential of α-amyrin and silymarin in wistar albino rats through their serum marker parameters.
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Total protein (gm/dL)
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4. Discussion Toxic species are the main cause for oxidative damage of hepatocytes which are usually metabolites. In case of CCl4 intake by experimental animals, the toxic species are altered the liver metabolism through oxidative damage and also caused fatty degeneration, fibrosis, hepatocellular death, and carcinogenicity (Weber et al., 2003; Manubolu et al., 2014). It is now generally accepted that the hepatotoxicity by CCl4 is the result of reductive dehalogenation, which is catalyzed by P-450 and forms the highly reactive trichloromethyl free radical (CCl●3). This then readily interact with molecular oxygen to form the trichloromethyl peroxy radical (CCl3OO●). Both radicals are capable of binding to proteins or lipids or of abstracting a hydrogen atom from an unsaturated lipid, which initiate lipid peroxidation and
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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D. Singh et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 2 Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their hepatic biochemical parameters. Treatment design
5´-Nucleotidase (μ mole inorganic phosphate released/ min/mg protein)
Acid ribonuclease (Units/mg protein)
Glucose-6-phosphatase (μ mole inorganic phosphate released/min/ mg protein)
Succinic dehydrogenase (μ mole Cytochrome P450 (n mole/mg indophenol reduced/min/mg protein) protein)
Control (Group I) CCl4 (Group II) CCl4 þ α-amyrin (Group III) CCl4 þ Silymarin (Group IV)
2.89 70.21 5.14 70.32a 3.16 70.24a
14.37 71.33 33.72 73.87a 16.42 71.52b
8.247 0.62 4.747 0.43a 7.78 7 054a
0.76 70.08 0.3370.03a 0.72 70.06a
4.687 0.32 2.017 0.12a 4.08 7 0.32a
3.22 70.28a
17.85 72.13b
7.157 0.48b
0.65 70.04a
3.87 7 0.37a
Levels of Significance: Data are mean 7SEM (n ¼6). a¼ Po 0.001 Gp. II compared with control (Gp. I). a¼ Po 0.001; b¼ Po 0.01 Gp. III compared with Gp. II. a¼ Po 0.001; b¼ Po 0.01 Gp. IV compared with Gp. II.
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Fig. 2. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their serum non-enzymatic antioxidants. Data points with letter notation (a) are statistically significant at a¼ Po 0.001.
Fig. 4. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their liver enzymatic antioxidant. Data points with letter notation (a) are statistically significant at a ¼P o 0.001.
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Fig. 3. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their serum non-enzymatic antioxidants. Data points with letter notation (a) are statistically significant at a¼ Po 0.001.
liver damage and by play a significant role in the pathogenesis of diseases (Williams and Burk, 1990; Weber et al., 2003). Therefore, the suppression of P-450 can result in a reduction in the level of the reactive metabolites, and correspondingly, less tissue injury. The metabolic activation of CCl4 is believed to be mediated through P-450 2E1 (Zangar et al., 2000; Murthy et al., 2014). The inhibitory effect of CCl4 on cytochrome P-450 level was also compensated by α-amyrin and silymarin through maintenance of its normal level. The role of α-amyrin in the protection of
Fig. 5. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their liver enzymatic antioxidant. Data points with letter notation (a) are statistically significant at a ¼P o 0.001.
CCl4-mediated loss in cytochrome P-450 content may be considered as an indication of improved protein synthesis in the hepatic cells (Mandal et al., 1993; Murthy et al., 2014). The lipid peroxidative degradation of biomembranes is one of the principle causes of hepatotoxicity induced by CCl4. Because lipid peroxidation is viewed as a complicated biochemical reaction involving free radicals, oxygen, metal ions and a host of other factors in the biological system. Since lipids constitute nearly 60% of the compounds in biomembranes, only major perturbation is bound to affect structure and function of the cell. In recent years, lipids and their derivatives have been recognized as important molecules in signal transduction (Manubolu et al., 2014). The efficacy of any hepatoprotective drug is dependent on its capacity of either reducing the harmful effect or restoring the normal hepatic physiology that has been disturbed by CCl4 and
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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Fig. 6. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their liver enzymatic antioxidants. Data points with letter notation (a) are statistically significant at a¼ Po 0.001.
8 7 6 5 4 3 2 1 0
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GST Fig. 7. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their liver enzymatic antioxidant. Data points with letter notation (a) are statistically significant at a¼ Po 0.001.
(n mole MDA/mg protein)
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Fig. 8. Showing modulatory potential of α-amyrin and silymarin in wistar albino rats through their serum and tissue lipid peroxidation. Data points with different letter notations (a, b) are significantly different at a¼ Po 0.001; b¼ Po 0.01.
other hepatotoxicants (Mani Senthilkumar et al., 2005). In our present investigation, the measurement of lipid peroxidation in the liver tissue and serum is a convenient method to monitor oxidative cell damage. Inhibition of elevated LPO has been observed in α-amyrin and silymarin treated groups due to its antioxidant and free radical scavenging activities through reestablishment of biomembranes of hepatic parenchymal cells (Singh et al., 2014a). In agreement with results obtained in previous investigations (Bishayee et al., 1995; Murthy et al., 2014; Shaaban et al., 2014), our present study elicited a significant increase in the activities of GGT, AST, ALT, LDH, ALP, ACP, SDH, and GDH with the exposure of CCl4 which indicating considerable hepatocellular injury. Oral
treatment with α-amyrin and silymarin attenuated these increased enzyme activities produced by CCl4 and a subsequent recovery towards normalization of these enzymes strongly suggests the possibility of α-amyrin being able to state the hepatocytes so as to cause accelerated regeneration of parenchymal cells and lysosomes, thus protecting against lysosomal integrity and cell membrane fragility decreasing the leakage of marker enzymes into the circulation (Bishayee et al., 1995; Murthy et al., 2014). Stabilization of serum-total bilirubin and total protein levels by the administration of α-amyrin is further a clear indication of the improvement of functional status of the hepatic cells (Shah et al., 2013; Singh et al., 2014a). The protective role of α-amyrin and silymarin has also been reflected in the normal levels of hepatic glucose-6-phosphatase, 5´-nucleotidase and succinic dehydrogenase activities that were severely altered in the CCl4 intoxicated rats liver. A significant lower level of acid ribonuclease enzyme, as a result of α-amyrin and silymarin treatment against CCl4 induced marked elevation, indicates a protection against the rupture of lysosomes and leakage of this enzyme in situ as a consequence of damage to the lysosomes of CCl4 treated liver cells (Kumar et al., 2007). The antioxidant defense enzymes have been suggestive to playing an important role in the maintaining of physiological levels of oxygen and hydrogen peroxide and eliminating peroxides generated from inadvertent exposure to xenobiotics and drugs. Any natural compound with antioxidant properties may help in maintaining health when continuously taken as components of dietary food, spices or drugs. The increase in the levels of antioxidant profiles i.e. SOD, CAT, GR and GPx by α-amyrin and silymarin may be attributed to have biological significance in eliminating reactive free radicals that may affect the normal functioning of cells. GST is a soluble protein located in the cytosol, and plays an important role in the detoxification and excretion of xenobiotics and drugs (Singh et al., 2000; Huo et al., 2011). Moreover, the GST functionally binds GSH and the endogenous or exogenous substances. Since it increases the solubility of hydrophobic substances, it also plays an important role in the storage and excretion of xenobiotics and drugs. During our experiment, both the α-amyrin and silymarin increased the GST activity and metabolized toxic compounds to nontoxic compounds and protect the liver of experimental rats (Huo et al., 2011; Singh et al., 2014b). Our study further revealed that chronic exposure to CCl4 significantly decreased the activities of non-enzymic antioxidants namely GSH, ceruloplasmin, β-carotene, vitamin C and vitamin E in serum might be responsible for hepatocellular injury. The supplementation of α-amyrin and silymarin was enhanced non-enzymic antioxidants significantly in CCl4 treated rats. GSH, vitamin C and vitamin E exist in their inter convertible forms and participate in the detoxification of the toxic reactive oxygen species. Regeneration to their reduced forms is brought about by reduced glutathione because the detoxification pathway involves GSH conjugation of the trichloromethyl radical, a P-450 2E1mediated CCl4 metabolite (Singh et al., 2014b). Previous studies on the mechanism of CCl4-induced hepatotoxicity have shown that GSH plays a key role in detoxifying the reactive toxic metabolites of CCl4 and that liver necrosis begins when the GSH stores are markedly depleted (Recknagel et al., 1991). GSH is largely mediated through the activity of GST and forms adducts with the toxic metabolites of CCl4 (Singh et al., 2014b). Moreover, GSH contribute to the detoxification of CCl4 and it has been suggested that one of the principle cause of CCl4-induced liver injury is lipid peroxidation caused by its free radical derivatives (Recknagel et al., 1991; Singh et al., 2014b). Vitamin C is considered to be the most important antioxidant in extracellular fluids (Stocker and Frei, 1991; Gupta and Singh, 2006) and also acts to protect membranes against peroxidation by enhancing the activity
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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Fig. 9. Photomicrograph of rat liver sections with haematoxylin-eosin at X100. (A) Showing as control with well brought central vein, hepatic cell with preserved cytoplasm and prominent nucleus; (B) Showing after CCl4 intoxication as marked steatosis (S) of the hepatocytes with ballooning degeneration and distended portal vein, mild periportal fibrosis and necrosis (N); (C) Showing after α-amyrin and CCl4 treatment as considerable reduction in necrosis and fatty changes with pyknotic nuclei and cytoplasmic clearing; (D) Showing after silymarin (standard) and CCl4 treatment as moderately regeneration in hepatocellular architecture. Scale bar, 100 μm.
of α-tocopherol, the chief lipid soluble and chain breaking antioxidant. β-carotene is a potent free radical quencher, singlet oxygen scavenger and lipid peroxidation (Blot et al., 1995; Gupta and Singh, 2006). Histopathological interpretations suggested that the reactive oxygen metabolites and lipid peroxidation may play a role in the various pathological lesions i.e. marked steatosis, pseudolobulation, ballooning degeneration, periportal fibrosis and necrosis with the loss of normal liver architecture. Because of CCl4 toxicity, a toxic reactive metabolite-trichloromethyl free radical was produced which binds covalently to the macromolecules of the lipid membranes of the adipose tissue and causes peroxidative degradation. As a result, fats from the adipose tissue are translocated and accumulated in the hepatocytes (Okuno et al., 1998; Cordeiro and Kaliwal, 2013).The degenerative changes showed minimal or absence with α-amyrin and silymarin treatments. α-Amyrin because of its ability to reduce fat accumulation, scavenge free radicals, interaction with oxidative cascade, quenching oxygen ions, inhibiting oxidative enzymes and lipid peroxidation, and restores the hepatic antioxidant status of the experimental rats.
5. Conclusion It can be concluded that the modulatory potential of α-amyrin against hepatic oxidative stress possibly involves mechanism related to its ability to block the P-450 mediated CCl4 bioactivation through selective inhibitors of ROS (reactive oxygen species) as antioxidants brought about significant inhibition of the formation of LPO suggesting possible involvement of O●2 , HO2, HO●2 , H2O2 and OH. Therefore this study suggests the use of α-amyrin as a hepatomodulatory potent to feasibility for a promising liver curative drug. The appropriate mechanism of action and comparative evaluation of α-amyrin and β-amyrin is in progress and will be reported elsewhere.
Acknowledgments The authors are thankful to the respective authorities for providing the necessary facilities and support.
Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i
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Please cite this article as: Singh, D., et al., Modulatory potential of α-amyrin against hepatic oxidative stress through antioxidant status in wistar albino rats. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.12.025i