http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2014; 37(2): 135–143 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2013.834350
RESEARCH ARTICLE
Evaluation of in vitro and in vivo antioxidant potential of polysaccharides from Aloe vera (Aloe barbadensis Miller) gel Gaurav Kaithwas1, Prashant Singh2, and Daksh Bhatia3 Department of Pharmaceutical Sciences, SB&BT, Babasaheb Bhimrao Ambedkar University, Vidhya Vihar, Lucknow, India, 2Department of Applied Chemistry, Babasaheb Bhimrao Ambedkar University, Vidhya Vihar, Lucknow, India, and 3Department of Pharmacognosy, KIET School of Pharmacy, 13th Km stone, Ghaziabad, Uttar Pradesh, India Abstract
Keywords
In the present study, the antioxidant activity of the polysaccharides from aloe vera (Aloe barbadensis Miller) gel was evaluated, in vitro by five established methods, 1,1-diphenyl-2picrylhydrazyl (DPPH–) radical scavenging, nitric oxide (NO) scavenging, hydrogen peroxide scavenging, superoxide radical (O-2) scavenging and reducing power assay, and in vivo against doxorubicin (DOX)-induced myocardial oxidative stress (OS) in albino wistar rats. The polysaccharides exhibited significant inhibitory activity against DPPH–, superoxide, NO and hydrogen peroxide scavenging assay with significant reducing activity at all concentrations used. DOX-induced (7.5 mg/kg, intravenously) cardiotoxicity manifested biochemically by a significant decrease in blood and tissue glutathione (GSH) along with elevated levels of serum lactate dehydrogenase and creatine phosphokinase. In addition, cardiotoxicity was further confirmed by the significant increase in lipid peroxidation expressed as thiobarbituric acid reactive substances (TBARS), catalase (CAT) and superoxide dismutase (SOD). Administration of aloe vera polysaccharides for 14 days produced a marked protection against cardiotoxicity induced by DOX evidenced by significant reductions in serum lactate dehydrogenase, serum creatine phosphokinase, cardiac TBARS, CAT and SOD along with increased levels of blood and tissue GSH in a dose-dependent manner. The present investigation is the first to establish the antioxidant potency of the polysaccharides from aloe vera against DOX-induced myocardial OS.
Aloe vera gel, doxorubicin, free radicals, oxidative stress, polysaccharides
Introduction Aloe vera (Aloe barbadensis Miller) belongs to the Lileacea family, of which there are approximately 360 species. Aloe vera has been promoted for a large variety of conditions and has come to play a prominent role as a contemporary folk remedy (Volger & Ernest, 1999). The fresh leaves of aloe vera are used to obtain two components, first, bitter yellow latex from a peripheral bundle sheath of aloe, called aloe vera sap, aloe vera juice or aloes. Aloe contains anthraquinone derivatives (aloe emodin) and their glycosides (aloin), which are known for their cathartic effect (Fairbarin, 1980); it also contains amino acids, auxins, gibberellins, minerals, vitamins, an aspirin-like compound, magnesium lactate and various enzymes such as superoxide dismutase (SOD) and catalase (CAT) (Volger & Ernest, 1999). Second, a mucilaginous gel from the parenchymatous tissue in the leaf pulp of aloe vera has been used since early times for the topical treatment of burns and wounds (Choi et al., 2001). As suggested by various researchers, aloe vera gel can be used in skin diseases,
History Received 27 December 2012 Revised 1 May 2013 Accepted 25 May 2013 Published online 13 February 2014
constipation, inflammatory disorders, cancer, ulcer, diabetes and as a free radical scavenger (Hamman, 2008; Noor et al., 2008; Reynolds & Dweck, 1999). The antioxidant and anti-diabetic potential of aloe vera gel was attributed primarily to the presence of polysaccharides (Miranda et al., 2009; Rajasekaran et al., 2005; Yu et al., 2009). Previously, Chun-hui et al. (2007) isolated compositionally two polysaccharides (GAPS-1 and SAPS-1) from aloe vera gel with significant free radical scavenging and antioxidant activities in vitro. Recently, Kaithwas et al. (2011) reported on the significant protection by aloe vera gel against doxorubicin (DOX)-induced oxidative stress (OS). Despite extensive history and immense research, no articles in the literature have discussed the role of polysaccharides from aloe vera gel (AVP) in DOX-induced (anthracycline anticancer drug) OS and cardiotoxicity. The present investigation was therefore undertaken to evaluate the antioxidant potential of AVP and vitamin E (positive reference) (Traber & Packer, 1995) against DOX-induced myocardial OS using albino rats.
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Methods Address for correspondence: Daksh Bhatia, Department of Pharmacognosy, KIET School of Pharmacy, 13th Km stone, Ghaziabad, Uttar Pradesh, India. Fax: +91-8876222878. E-mail: [email protected]
Drugs and chemicals All chemicals used were of analytical grade. 1,1-diphenyl-2picryl-hydrazyl (DPPH) was purchased from Sigma-Aldrich
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Chemical Co. (St. Louis, MO). Sodium nitroprusside, sulphanilamide, napthyl ethylene diamine dihydrochloride, nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), trichloroacetic acid (TCA), thiobarbituric acid (TBA), potassium chloride (KCl), potassium ferricyanide and all solvents were obtained from SD Fine Chemicals (Mumbai, India). Ascorbic acid and vitamin E were procured from Hi Media (Mumbai, India). DOX hydrochloride was received as a gift sample from the Dabur Research Foundation (Ghaziabad, India). Diagnostic kits of lactate dehydrogenase (LDH) and creatine phospokinase (CPK) were purchased from Span Diagnostics Ltd. (Surat, India).
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Plant material Aloe vera gel was purchased from Nature Forever Living Products International (Scottsdale, AZ). Preparation of aloe vera polysaccharides Aloe vera polysaccharides were prepared according to the method described by Wu et al. (2006), with slight modifications. Briefly, aloe vera gel (300 g) was extracted repeatedly three times with distilled water (2500 mL) at 70–80 C for 2 hours. The filtrate of the obtained extract was condensed in vacuo to syrup (ca. 500 mL), to which cold 95% ethanol (1500 mL) was added. Crude polysaccharide thus obtained was precipitated from the alcoholic liquor during its subsequent standing at 4 C overnight. The precipitate was collected by centrifugation at 12,000 g and repeatedly washed sequentially with possibly less amounts of ethanol, acetone and ether, respectively. Next, 2.40 g of crude polysaccharide was redissolved in distilled water (240 mL) at a concentration of 1% (w/v), followed by filtration. The filtrate was treated with TCA for deprotinization, following the procedure of Yang et al. (1999). After centrifugation at 12,000 g for 10 minutes, the supernatant was then precipitated with 3-fold volumes of 95% ethanol to obtain the crude polysaccharides. In vitro antioxidant activity DPPH radical scavenging activity Reaction mixture (3 mL) containing 0.2 mL of DPPH (100 mM in methanol) and 2.8 mL of methanol solution containing AVP (20–120 mg) was incubated at 37 C for 30 minutes, and absorbance of the test mixture was read at 517 nm using a UV-visible spectrophotometer (UV-V 1700; Shimadzu, Tokyo, Japan). The lower absorbance of the reaction mixture indicated higher free radical scavenging activity (Cotelle et al., 1996). The percentage inhibition of DPPH radical was calculated by comparing the results of the test with those of the controls (not treated with any antioxidant) using the formula shown in equation 1 (Shirwaikar et al., 2004). ðAbsorbance of control Percentage inhibition ¼
Absorbance of testÞ Absorbance of control
100 ð1Þ
Methanol was used as blank. Ascorbic acid (20–120 mg) was taken as a standard. Nitric oxide radical scavenging activity Nitric oxide (NO) radical scavenging was estimated on the basis of Griess Illosvoy reaction using a method followed by Govindarajan et al. (2003). The reaction mixture (3 mL) containing sodium nitroprusside (10 mm, 2 mL), phosphatebuffered saline (PBS: 0.5 mL, pH 7.4) and 0.5 mL of AVP (20–120 mg) or standard ascorbic acid solution (0.5 mL, 20–120 mg) was incubated at 25 C for 150 minutes. After incubation, 0.5 mL of the reaction mixture was mixed with 1 mL of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 minutes for completion of diazotization. Then, 1 mL of naphthyl ethylenediamine dihydrochloride (0.1%, w/v) was added, mixed and allowed to stand for 30 minutes at 25 C. A pink-colored chromophore formed in diffused light. Absorbance of these solutions was measured at 540 nm against the corresponding blank solutions. Percentage inhibition was calculated from the formula in equation 1. Hydrogen peroxide scavenging activity The ability of AVP to quench hydrogen peroxide (H2O2) was determined spectrophotometrically. Different concentrations of AVP (20–120 mg) or ascorbic acid (20–120 mg) were dissolved in 3.4 mL of 0.1 M (pH 7.4) of PBS and mixed with 0.6 mL of 40 mM solution of H2O2. Absorbance of H2O2 at 230 nm was determined 10 minutes later in a spectrophotometer. For each concentration, a separated blank sample was used for background subtraction. Percentage inhibition was calculated from the formula in equation 1 (Ruch et al., 1989). Superoxide radical scavenging activity The 3 mL of reaction mixture consisted of AVP (1 mL, 20– 120 mg), 1 mL of 60 mM of PMS in phosphate buffer (0.1 M, pH 7.4) and 1 mL of 150 mM of NBT in phosphate buffer. Incubation at room temperature for 5 minutes and the resultant color was read spectrophotometrically at 560 nm against a blank. Different concentrations of ascorbic acid (20–120 mg) were used as a positive control. Percentage inhibition was calculated from the formula in equation 1 (Gow-Chin & Hui, 1995). Reducing activity The reducing power of AVP was determined by the method of Jayprakash et al. (2001). Different amounts (20–120 mg) of AVP were mixed with 2.5 mL of phosphate buffer (200 mM, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixtures were incubated for 20 minutes at 50 C. After incubation, 2.5 mL of 10% TCA was added to the mixtures, followed by centrifugation at 650 g for 10 minutes. The upper layer (5 mL) was mixed with 5 mL of distilled water and 1 mL of 0.1% ferric chloride, and absorbance of the resultant solution was measured at 700 nm.
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In vivo studies
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Animals The Wistar strain of albino rats (100–150 g) were obtained from the Central Animal House, Department of Animal Husbandry, Allahabad Agricultural Institute-Deemed University (Naini, Allahabad, India). Animals were housed under standard conditions of temperature (25 1 C) with a 12-hour light-dark cycle and had free access to commercial pellet diet and water ad libitum. Animals were acclimatized for 1 week in the laboratory environment, before experimentation. The study was approved by the institutional animal ethical committee, and experiment was performed in accord with Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines for the Laboratory Animal Ethics, Department of Animal Welfare, Government of India.
Toxicity studies Subacute toxicity study Twenty albino rats (150–200 g) of either sex were divided into two groups. The first group received control vehicle (normal saline) and another group received AVP in a dose of 200 mg/kg (per oral; p.o.) daily. Both groups of rats were kept under similar laboratory conditions and were allowed to take a usual pellet diet and water ad libitum. Animals were observed for their general condition, gross behavior, body weight and so on. All animals were sacrificed on the 15th day; the viscera were removed and examined for gastric erosion in the stomach. Acute toxicity studies Mean lethal dose (LD50) was calculated for AVP after administration through the oral route. Swiss albino mice (25–40 g) of either sex were subjected to fasting overnight, with free access to water. A pilot study was carried out before the main experiment, and based on these findings, doses of 1, 2, 3, 4, 5, 6, 7, 8 and 9 g/kg were selected for the final study. Animals were divided into seven groups of 10 animals each. AVP in the above-mentioned dose was given orally, as a single dose. Mortality was observed over a 24-hour period. The LD50 value was calculated by the arithmetic method of Parmar & Ghosh (1978). In vivo experimental protocol Animals (albino rats, 100–150 g) were randomized and divided into 6 groups of 6 animals each. All test drugs were given every day at the dose levels mentioned for a period of 14 days after the intravenous (i.v.) injection of DOX. To avoid variability in the test volume, a constant dose volume of 1 mL/ kg was used throughout the experiment for all the test drugs used. Groups comprised the following: group 1: normal control (0.9% saline, 1 mL/kg, p.o.); group 2: DOX (7.5 mg/ kg, i.v.); group 3: AVP þ DOX (100 mg/kg, p.o. þ 7.5 mg/kg, i.v.); group 4: AVP þ DOX (200 mg/kg, p.o. þ 7.5 mg/kg, i.v.); group 5: AVP þ DOX (300 mg/kg, p.o. þ 7.5 mg/kg, i.v.) and group 6: vitamin E þ DOX (100 mg/kg, p.o. þ 7.5 mg/kg,
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i.v.). Animals were sacrificed on the 15th day for biochemical evaluation.
Food components and anthropometric measurements Food and water intake was measured manually for a group of animals. Weighed amount of pellet diet was given daily, and the amount left the next day was weighed. Similarly, calibrated water bottles were used to monitor the water intake for a group of animals. Animals were monitored at regular interval for body weights. Biochemical analysis Blood samples were collected from retro-orbital plexus and subjected for blood glutathione (GSH) (Beutlar et al., 1963), serum lactate dehydrogenase (LDH) (Lum & Gambino, 1974) and serum creatine phosphokinase (CPK) (Tietz, 1976) estimations. Rats were sacrificed by decapitation under light ether anesthesia. Hearts were removed and weighed. Isolated heart tissue was subjected to the estimation of protein (Lowry et al., 1951), thiobarbituric acid reactive substances (TBARS) (Ohkawa et al., 1979), GSH (Ellman, 1959; Sedlak & Lindsay, 1968), CAT (Clailborne, 1985) and SOD (Marklund & Marklund, 1974) in tissue.
Statistical analysis All the data are presented as mean standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA), followed by Dunnett’s test for possible significance identification between the various groups. A value of p50.05 was considered statistically significant. Statistical analysis was carried out using GraphPad Prism software (version 3.0; GraphPad Software Inc., San Diego, CA).
Results In vitro antioxidant activity The results of the DPPH and NO scavenging activity of AVP are shown in Figures 1 and 2. AVP demonstrated significant scavenging activity in DPPH and NO assay. In fact, the half-maximal inhibitory concentration (IC50) value for AVP in DPPH assay was less than the minimum dose tested (20 mg/mL). In the NO test, AVP and ascorbic acid demonstrated similar scavenging abilities initially, with AVP demonstrating higher scavenging activity at higher doses. The scavenging ability of AVP in H2O2 assay was better than the standard (ascorbic acid), with maximum of 86.64 and 73.32% inhibition of AVP and ascorbic acid, respectively. AVP exhibited concentration-dependent scavenging activity against hydroxyl radical generated in a Fenton reaction system. The IC50 value of AVP was found to be 2.49 mL/mL, in comparison to standard ascorbic acid (3.68 mL/mL), suggesting a better inhibitory potential of AVP (Figure 3). As evident from Figure 4, AVP exhibited potent superoxide radical scavenging activity, as compared to that of ascorbic acid. The IC50 value indicated that the superoxide free radical scavenging activity of AVP was nearly 64.78 mL/ mL, in comparison to ascorbic acid (86.72 mL/mL).
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Figure 1. Free radical scavenging capacity of AVP and ascorbic acid as determined by the DPPH method. Results are means SD. The experiment was performed in triplicate.
Figure 2. NO radical scavenging activity of AVP and ascorbic acid. Results are means SD. The experiment was performed in triplicate.
For measurements of reducing ability, the Fe3þ–Fe2þ transformation was investigated in the presence of AVP. The reducing capacity of a compound may serve as a significant indicator of its potent antioxidant activity. Similar to the antioxidant activity, the reducing power of AVP increased with increasing dosage (Figure 5).
In vivo studies Toxicity studies Results from toxicity studies revealed that AVP was well tolerated up to 5 g/kg, whereas 100% mortality was observed at 9 g/kg when given by the oral route. The LD50 value of AVP was found to be 6.1 g/kg. Administration of AVP for a longer duration of time in subacute toxicity studies did not reveal any untoward effect on behavior, body weight, normal reflexes and visceral appearance in rats. AVP did not
produce any ulcerogenic effect. Results from the toxicity studies suggest that it is also well tolerated.
Experimental studies Food component measurements depicted that food and water intake in the control rats were 135.22 2.11 g/day/group and 208.10 6.17 mL/day/group, respectively (Table 1). All rats injected with DOX (7.5 mg/kg, i.v.) showed a significant (p50.05) decrease in food and water intake along with a decrease in body weight, as compared to control animals. Significant reduction in the ratio of heart weight/body weight in toxic control was observed (p50.05), in comparison to normal control. Animals treated with DOX exhibited a significant increase in serum LDH (621.54 14.52 IU/L) and CPK (72.47 5.13 IU/L) activities, in comparison to control (Table 2).
DOI: 10.3109/01480545.2013.834350
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Figure 3. H2O2 scavenging activity of AVP and ascorbic acid. Results are means SD. The experiment was performed in triplicate.
Figure 4. Superoxide radical scavenging capacity of AVP determined by the PMS/NADH-NBT method. Results are means SD. The experiment was performed in triplicate.
However, concomitant administration of AVP and vitamin E significantly altered the same in a dose-dependent manner. In our study, we found that blood and tissue GSH levels in normal control animals were 3.21 0.12 mg% and 13.01 0.40 mmol/gwt of tissue, respectively (Table 2). DOX decreased the level of tissue and blood GSH and treatment, with AVP significantly helped to normalize the same (Table 2). In the hearts of DOX-treated rats, there was a significant increase in the TBARS generation (30.15 1.21 nmoles of malondialdehyde [MDA]/mg of protein), in comparison to the control (8.43 0.44 nmoles of MDA/mg of protein), indicating reactive oxygen species (ROS) generation and OS (Table 3). DOX treatment raised the SOD level
(76.19 1.88 units of SOD/mg of protein), in comparison to normal control (49.94 2.87 units of SOD/mg of protein; Table 3). CAT activity in DOX-treated animals was increased to 201.11 4.89 nmoles of H2O2/min/mg of protein, in comparison to normal control (i.e. 52.21 2.85 nmoles of H2O2/min/mg of protein; Table 3). Dose-dependent restoration of SOD and CAT level was observed after AVP treatment in our study.
Discussion and conclusion DPPH is a relatively stable free radical and the assay determines the ability of AVP to reduce DPPH radical to
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Figure 5. Reducing power of AVP. Results are means SD of triplicate measurements.
AVP
0.9
Absorbance (700nm)
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
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0 0
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40
60
80
100
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Concentration (µg/ml)
Table 1. Effect of AVP, vitamin E and DOX on food components and anthropometric measurements in different groups. S. no. Group Group Group Group Group Group
Treatment 1 2 3 4 5 6
Food intake (g)
a
0.33 0.01a 0.23 0.01 0.31 0.01b 0.33 0.01a 0.35 0.01a 0.33 0.01a
208.50 6.17 165.00 17.65 185.25 4.40a 218.18 4.40a 224.14 10.9a 201.50 7.42a
135.22 2.11 70.00 10.32 107.92 9.45a 120.08 8.29a 128.12 5.52a 125.64 5.60a
Normal control Toxic control (DOX, 7.5 mg/kg, i.v.) AVP (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (200 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (300 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) Vitamin E (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.)
Heart wt. 100 (g) Body wt.
Water intake (mL)
a
Results are reported as mean SD (n ¼ 6). Statistical significance compared to toxic control on the basis of ANOVA, followed by Dunnett’s test (ap50.001; bp50.01; cp50.05). Table 2. Effect of AVP, vitamin E and DOX on blood and tissue GSH, serum LDH and CPK levels in different groups.
S. no. Group Group Group Group Group Group
1 2 3 4 5 6
Treatment
Blood GSH (mg%)
Tissue GSH (mmol/gwt of tissue)
Serum LDH (IU/L)
Serum CPK (IU/L)
Normal control Toxic control (DOX, 7.5 mg/kg, i.v.) AVP (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (200 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (300 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) Vitamin E (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.)
3.21 0.12a 1.06 0.07 1.41 0.21c 2.77 0.24a 3.33 0.21a 2.98 0.12a
13.01 0.40b 9.11 0.22 9.50 0.21 11.11 0.31b 12.76 0.39a 13.21 0.11a
245.14 3.41a 621.54 14.52 505.21 12.65b 321.14 2.35a 257.16 15.17a 214.21 11.45a
37.46 4.14a 72.47 5.13 57.40 2.62b 41.98 2.74a 37.18 2.49a 39.08 3.48a
Results are reported as mean SD (n ¼ 6). Statistical significance compared to toxic control on the basis of ANOVA, followed by Dunnett’s test (ap50.001; bp50.01; cp50.05). Table 3. Effect of AVP, vitamin E and DOX on myocardial TBARS, CAT and SOD levels in different groups.
S. no. Group Group Group Group Group Group
1 2 3 4 5 6
Treatment
TBARS (nmoles of MDA/mg of protein)
CAT (nmoles of H2O2/min/mg of protein)
SOD (units of SOD/mg of protein)
Normal control Toxic control (DOX, 7.5 mg/kg, i.v.) AVP (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (200 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (300 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) Vitamin E (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.)
8.43 0.44a 30.15 1.21 24.78 0.12c 14.14 0.15a 11.30 0.24a 8.31 0.52a
52.21 2.58a 201.11 4.89 82.94 5.41a 61.94 2.25a 50.63 3.39a 51.50 3.80a
49.94 2.87a 76.19 1.88 69.58 0.75c 53.41 0.08b 43.21 0.47a 49.19 2.14a
Results are reported as mean SD (n ¼ 6). Statistical significance compared to toxic control on the basis of ANOVA, followed by Dunnett’s test (ap50.001; bp50.01; cp50.05).
the corresponding hydrazine by converting the unpaired electrons to paired ones. AVP exhibited significant antioxidant activity in an amount-dependent manner (Figure 1). Antioxidants can act by converting the unpaired electrons to
paired ones (Soares et al., 1997). Scavenging of DPPH radical by AVP demonstrates the correlation of the extent of the scavenging by hydrogen or electron donation of a preformed free radical with antioxidant activity (Morelle et al., 1998).
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DOI: 10.3109/01480545.2013.834350
NO is an important chemical mediator generated by endothelial cells, macrophages, neurons and so on and involved in the regulation of various physiological processes. Excess concentration of NO is associated with pathogenesis of several diseases (Ross, 1993). Oxygen reacts with excess NO to generate nitrite and peroxynitrite anions, which act as free radicals (Sainani et al., 1997). In the present study, AVP competed with oxygen to react with NO and thus inhibited the generation of anions (Figure 2). Superoxide is biologically important because it can be decomposed to form stronger oxidative species, such as singlet oxygen and hydroxyl radicals (Korycka-Dahl & Richardson, 1978). Superoxides are produced from molecular oxygen resulting from oxidative enzymes (Sainani et al., 1997) of the body as well as by nonenzymatic reaction, such as autoxidation by catecholamines (Hemmani & Parihar, 1998). In the present study, it was found that AVP inhibited the superoxide radicals in a dose-dependent manner (Figure 4). In the PMS/nicotinamide adenine dinucleotide (NADH)-NBT system, superoxide anions derived from dissolved oxygen and AVP decreased absorbance at 560 nm, which indicates the consumption of superoxide anions in the reaction mixture by AVP. Hydrogen peroxide is a weak initiator of lipid peroxidation (LPO) (Cohen & Heikkila, 1974). However, it produces active oxygen species as a result of its ability to generate highly reactive hydroxyl radicals through the Fenton reaction (Namiki, 1990). The ability of AVP extract to scavenge H2O2 could also reflect its ability to inhibit the formation of hydroxyl radical in vivo. Various mechanisms, including reducing capacity, prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging, have been claimed to explain antioxidant activities (Diplock, 1997). Reducing the capacity of a compound may serve as a significant indicator of its potential antioxidant activity (Leskovar et al., 2004; Sreekanth et al., 2003). In this study, AVP exhibited an effective reducing capacity at all concentration points (Figure 5). Regarding the in vitro antioxidant potential of AVP, it was considered worthwhile to evaluate its effect against DOX-induced myocardial OS. Despite their potential insidious cardiotoxicity, DOX (anthracycline antibiotic) is often used in the treatment of a wide range of human malignancies. Acute cardiovascular effects develop within minutes or an hour after i.v. injection of DOX and are characterized by hypotension, tachycardia and various arrhythmias, effects which are clinically manageable. Life-threatening chronic effects, such as cardiomyopathy and heart failure, usually develop after several weeks to months of treatments. However, new and low-toxicity anthracycline derivatives or liposome-encapsulated drugs have influenced anthracyclineinduced cardiotoxicity in many ways. Several hypotheses explain the mechanism of anthracycline-induced cardiotoxicity and most of them suggest a crucial role of free radicals and calcium ions (Minotti et al., 1999). In biological systems, DOX is known to produce highly ROS, such as superoxide and hydroxyl radicals (Doroshow et al., 1980), having potential to damage intracellular components. Cardiac muscles are particularly greatly susceptible to the damage
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caused by these ROS because they contain very low levels of ROS-detoxifying enzymes, such as SOD, GSH peroxidase (GPx) and CAT (Takacs et al., 1992); therefore, antioxidants play a critical role in the inactivation of free radicals (Mansat-de Mas et al., 1999). Clinical reports show that patients with advanced cancer develop less limb muscle force, compared to controls (Stone et al., 1999) and have impaired physical performance when undergoing DOX-based chemotherapy (Elbl et al., 2006). Impaired muscle function is commonly attributed to the loss of muscle mass, because cancer cachexia and muscle wasting are known clinical problems (Kotler, 2000). The development of delayed DOX cardiomyopathy in the rat is accompanied by profound anorexia, dramatically reducing the caloric intake, which is a proven fact in many studies. Concomitant administration of AVP significantly increased the DOX-induced reduction in food and water intake along with ratio of heart weight/body weight, indicating that AVP can alter the OS arising as a result of DOX treatment and therefore can help to restore caloric intake in the treatment groups. DOX, by virtue of its quinone structure, acts as an electron acceptor, with the help of various flavoprotien enzymes. Mitochondrial enzymes have been shown to activate DOX to form the semiquinone radical and superoxide anion. Serum LDH and CPK enzyme activities are important to measure both early and late phases of cardiac injury, although these enzymes are nonspecific for measuring cardiac injury, but, together, they could be an indicator of myocardial injury (Kemp et al., 2004). Earlier reports have confirmed that DOX-induced cardiotoxicity is a secondary event subsequent to LPO of cardiac membranes as a result of massive necrosis of cardiac cells in the heart, leading to increase in leakage of LDH and CPK from cardiac myocytes to serum. Concomitant treatment with AVP or vitamin E produced a significant decrease in serum LDH and CPK levels, which could be possibly explained by the fact that AVP exerts anti-LPO activity, causing stabilization of cardiac membranes from the peroxidative damage, thus preventing the leakage of LDH and CPK. GSH acts as an antioxidant coenzyme of GPx, which utilizes it to reduce peroxides or superoxides, yielding oxidized GSH. DOX significantly decreased the level of tissue and blood GSH, which corroborated with earlier studies (Gustafson et al., 1993). Decrease in levels of GSH represents its increased utilization by cardiac cells as a result of OS, and treatment with AVP and vitamin E has significantly helped to restore levels of GSH; this effect could be attributed either as a result of decreased OS or increased biogenesis of GSH. The antioxidant enzymes, SOD and CAT, constitute the major supportive team of defense against free radicals. SOD forms H2O2 and molecular oxygen by scavenging the superoxide radicals, generated as a result of redox cycling of DOX (Gustafson et al., 1993). Previously, most of the studies have reported on the decreased levels of SOD, resulting from OS (Singh et al., 2008) in toxic controls. However, our study showed that the activity of the SOD was significantly increased in DOX-treated animals. Theoretically, it seems that SOD activity decreased with the progression of OS, but here we suggest that as observed increased level of
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SOD could be a consequence of physiological compensatory mechanisms to combat OS. Treatment with AVP significantly normalized SOD levels in a dose-dependent manner, comparable to standard vitamin E, suggesting the possible antioxidant potential of AVP in counteracting DOX-induced OS. Another antioxidant enzyme, besides GPx activity as a defense against H2O2 formations, is CAT enzyme. CAT existing in mammalian cells catalyzes the dismutation of H2O2 (produced from the scavenging effect of SOD) to water and molecular oxygen. Thus, a simultaneous increase in CAT activity is essential to increase SOD activity to counteract the deleterious effects of DOX, which was observed in our study. Concomitant administration of AVP altered CAT levels significantly toward normalization. In vivo measurement of reactive free radicals is difficult because of their high reactivity, short half-life and very low concentration. Thus, the indirect methods usually used for measuring secondary products of OS, such as TBARS, a convenient assay of LPO, are employed. LPO may produce tissue damage and finally cause various cardiovascular complications. Increased free radicals produced may react (Li & Singal, 2000) with polyunsaturated fatty acids in cell membranes, leading to LPO. DOX has been reported to increase MDA, a stable product of LPO in heart tissues. The observation suggests that DOX and its metabolites produces free radical species that attack lipid components, leading to LPO, and coadministration of AVP significantly prevented the increase in the TBARS levels in DOX-treated animals, which was comparable to the standard vitamin E. The pattern of changes were observed in food component intake, anthropometric measurements and biochemical parameters. The existing experimental evidences suggested that DOX-induced OS was primarily the result of the production of free radicals (superoxide and hydroxyl radical) in the heart tissue, having potential to damage various intracellular components. Treatment with AVP proves significant protection from DOX-induced OS, reflecting its antioxidant potential. Our result indicates that AVP possesses significant free radical scavenging activity in vitro and exhibits potent antioxidant potential in DOX-induced OS in vivo. Published data indicate that plant polysaccharides in general have antioxidant activities and can be explored as novel potential antioxidants (Jiang et al., 2005). The potential free radical scavenging mechanism of polysaccharides is poorly understood. This could be attributed to two important factors; first, huge structural diversity of these polysaccharides has given a major hindrance in the structure-activity relationship establishment. Second, most of the hitherto used sulfated polysaccharides or complex mixtures or complete extract containing a large number of other molecules with their own activities. However, the present work describes the purified polysaccharides, thus conclusively proves their antioxidant activity. In conclusion, AVP exhibits a potent antioxidant potential, which supports it to be used as an excellent food supplement. However, further studies are required to confirm the activity of the other compound and to elucidate the molecular and cellular mechanism of AVP.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.
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RESEARCH ARTICLE
Evaluation of in vitro and in vivo antioxidant potential of polysaccharides from Aloe vera (Aloe barbadensis Miller) gel Gaurav Kaithwas1, Prashant Singh2, and Daksh Bhatia3 Department of Pharmaceutical Sciences, SB&BT, Babasaheb Bhimrao Ambedkar University, Vidhya Vihar, Lucknow, India, 2Department of Applied Chemistry, Babasaheb Bhimrao Ambedkar University, Vidhya Vihar, Lucknow, India, and 3Department of Pharmacognosy, KIET School of Pharmacy, 13th Km stone, Ghaziabad, Uttar Pradesh, India Abstract
Keywords
In the present study, the antioxidant activity of the polysaccharides from aloe vera (Aloe barbadensis Miller) gel was evaluated, in vitro by five established methods, 1,1-diphenyl-2picrylhydrazyl (DPPH–) radical scavenging, nitric oxide (NO) scavenging, hydrogen peroxide scavenging, superoxide radical (O-2) scavenging and reducing power assay, and in vivo against doxorubicin (DOX)-induced myocardial oxidative stress (OS) in albino wistar rats. The polysaccharides exhibited significant inhibitory activity against DPPH–, superoxide, NO and hydrogen peroxide scavenging assay with significant reducing activity at all concentrations used. DOX-induced (7.5 mg/kg, intravenously) cardiotoxicity manifested biochemically by a significant decrease in blood and tissue glutathione (GSH) along with elevated levels of serum lactate dehydrogenase and creatine phosphokinase. In addition, cardiotoxicity was further confirmed by the significant increase in lipid peroxidation expressed as thiobarbituric acid reactive substances (TBARS), catalase (CAT) and superoxide dismutase (SOD). Administration of aloe vera polysaccharides for 14 days produced a marked protection against cardiotoxicity induced by DOX evidenced by significant reductions in serum lactate dehydrogenase, serum creatine phosphokinase, cardiac TBARS, CAT and SOD along with increased levels of blood and tissue GSH in a dose-dependent manner. The present investigation is the first to establish the antioxidant potency of the polysaccharides from aloe vera against DOX-induced myocardial OS.
Aloe vera gel, doxorubicin, free radicals, oxidative stress, polysaccharides
Introduction Aloe vera (Aloe barbadensis Miller) belongs to the Lileacea family, of which there are approximately 360 species. Aloe vera has been promoted for a large variety of conditions and has come to play a prominent role as a contemporary folk remedy (Volger & Ernest, 1999). The fresh leaves of aloe vera are used to obtain two components, first, bitter yellow latex from a peripheral bundle sheath of aloe, called aloe vera sap, aloe vera juice or aloes. Aloe contains anthraquinone derivatives (aloe emodin) and their glycosides (aloin), which are known for their cathartic effect (Fairbarin, 1980); it also contains amino acids, auxins, gibberellins, minerals, vitamins, an aspirin-like compound, magnesium lactate and various enzymes such as superoxide dismutase (SOD) and catalase (CAT) (Volger & Ernest, 1999). Second, a mucilaginous gel from the parenchymatous tissue in the leaf pulp of aloe vera has been used since early times for the topical treatment of burns and wounds (Choi et al., 2001). As suggested by various researchers, aloe vera gel can be used in skin diseases,
History Received 27 December 2012 Revised 1 May 2013 Accepted 25 May 2013 Published online 13 February 2014
constipation, inflammatory disorders, cancer, ulcer, diabetes and as a free radical scavenger (Hamman, 2008; Noor et al., 2008; Reynolds & Dweck, 1999). The antioxidant and anti-diabetic potential of aloe vera gel was attributed primarily to the presence of polysaccharides (Miranda et al., 2009; Rajasekaran et al., 2005; Yu et al., 2009). Previously, Chun-hui et al. (2007) isolated compositionally two polysaccharides (GAPS-1 and SAPS-1) from aloe vera gel with significant free radical scavenging and antioxidant activities in vitro. Recently, Kaithwas et al. (2011) reported on the significant protection by aloe vera gel against doxorubicin (DOX)-induced oxidative stress (OS). Despite extensive history and immense research, no articles in the literature have discussed the role of polysaccharides from aloe vera gel (AVP) in DOX-induced (anthracycline anticancer drug) OS and cardiotoxicity. The present investigation was therefore undertaken to evaluate the antioxidant potential of AVP and vitamin E (positive reference) (Traber & Packer, 1995) against DOX-induced myocardial OS using albino rats.
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Methods Address for correspondence: Daksh Bhatia, Department of Pharmacognosy, KIET School of Pharmacy, 13th Km stone, Ghaziabad, Uttar Pradesh, India. Fax: +91-8876222878. E-mail: [email protected]
Drugs and chemicals All chemicals used were of analytical grade. 1,1-diphenyl-2picryl-hydrazyl (DPPH) was purchased from Sigma-Aldrich
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Chemical Co. (St. Louis, MO). Sodium nitroprusside, sulphanilamide, napthyl ethylene diamine dihydrochloride, nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), trichloroacetic acid (TCA), thiobarbituric acid (TBA), potassium chloride (KCl), potassium ferricyanide and all solvents were obtained from SD Fine Chemicals (Mumbai, India). Ascorbic acid and vitamin E were procured from Hi Media (Mumbai, India). DOX hydrochloride was received as a gift sample from the Dabur Research Foundation (Ghaziabad, India). Diagnostic kits of lactate dehydrogenase (LDH) and creatine phospokinase (CPK) were purchased from Span Diagnostics Ltd. (Surat, India).
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Plant material Aloe vera gel was purchased from Nature Forever Living Products International (Scottsdale, AZ). Preparation of aloe vera polysaccharides Aloe vera polysaccharides were prepared according to the method described by Wu et al. (2006), with slight modifications. Briefly, aloe vera gel (300 g) was extracted repeatedly three times with distilled water (2500 mL) at 70–80 C for 2 hours. The filtrate of the obtained extract was condensed in vacuo to syrup (ca. 500 mL), to which cold 95% ethanol (1500 mL) was added. Crude polysaccharide thus obtained was precipitated from the alcoholic liquor during its subsequent standing at 4 C overnight. The precipitate was collected by centrifugation at 12,000 g and repeatedly washed sequentially with possibly less amounts of ethanol, acetone and ether, respectively. Next, 2.40 g of crude polysaccharide was redissolved in distilled water (240 mL) at a concentration of 1% (w/v), followed by filtration. The filtrate was treated with TCA for deprotinization, following the procedure of Yang et al. (1999). After centrifugation at 12,000 g for 10 minutes, the supernatant was then precipitated with 3-fold volumes of 95% ethanol to obtain the crude polysaccharides. In vitro antioxidant activity DPPH radical scavenging activity Reaction mixture (3 mL) containing 0.2 mL of DPPH (100 mM in methanol) and 2.8 mL of methanol solution containing AVP (20–120 mg) was incubated at 37 C for 30 minutes, and absorbance of the test mixture was read at 517 nm using a UV-visible spectrophotometer (UV-V 1700; Shimadzu, Tokyo, Japan). The lower absorbance of the reaction mixture indicated higher free radical scavenging activity (Cotelle et al., 1996). The percentage inhibition of DPPH radical was calculated by comparing the results of the test with those of the controls (not treated with any antioxidant) using the formula shown in equation 1 (Shirwaikar et al., 2004). ðAbsorbance of control Percentage inhibition ¼
Absorbance of testÞ Absorbance of control
100 ð1Þ
Methanol was used as blank. Ascorbic acid (20–120 mg) was taken as a standard. Nitric oxide radical scavenging activity Nitric oxide (NO) radical scavenging was estimated on the basis of Griess Illosvoy reaction using a method followed by Govindarajan et al. (2003). The reaction mixture (3 mL) containing sodium nitroprusside (10 mm, 2 mL), phosphatebuffered saline (PBS: 0.5 mL, pH 7.4) and 0.5 mL of AVP (20–120 mg) or standard ascorbic acid solution (0.5 mL, 20–120 mg) was incubated at 25 C for 150 minutes. After incubation, 0.5 mL of the reaction mixture was mixed with 1 mL of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 minutes for completion of diazotization. Then, 1 mL of naphthyl ethylenediamine dihydrochloride (0.1%, w/v) was added, mixed and allowed to stand for 30 minutes at 25 C. A pink-colored chromophore formed in diffused light. Absorbance of these solutions was measured at 540 nm against the corresponding blank solutions. Percentage inhibition was calculated from the formula in equation 1. Hydrogen peroxide scavenging activity The ability of AVP to quench hydrogen peroxide (H2O2) was determined spectrophotometrically. Different concentrations of AVP (20–120 mg) or ascorbic acid (20–120 mg) were dissolved in 3.4 mL of 0.1 M (pH 7.4) of PBS and mixed with 0.6 mL of 40 mM solution of H2O2. Absorbance of H2O2 at 230 nm was determined 10 minutes later in a spectrophotometer. For each concentration, a separated blank sample was used for background subtraction. Percentage inhibition was calculated from the formula in equation 1 (Ruch et al., 1989). Superoxide radical scavenging activity The 3 mL of reaction mixture consisted of AVP (1 mL, 20– 120 mg), 1 mL of 60 mM of PMS in phosphate buffer (0.1 M, pH 7.4) and 1 mL of 150 mM of NBT in phosphate buffer. Incubation at room temperature for 5 minutes and the resultant color was read spectrophotometrically at 560 nm against a blank. Different concentrations of ascorbic acid (20–120 mg) were used as a positive control. Percentage inhibition was calculated from the formula in equation 1 (Gow-Chin & Hui, 1995). Reducing activity The reducing power of AVP was determined by the method of Jayprakash et al. (2001). Different amounts (20–120 mg) of AVP were mixed with 2.5 mL of phosphate buffer (200 mM, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixtures were incubated for 20 minutes at 50 C. After incubation, 2.5 mL of 10% TCA was added to the mixtures, followed by centrifugation at 650 g for 10 minutes. The upper layer (5 mL) was mixed with 5 mL of distilled water and 1 mL of 0.1% ferric chloride, and absorbance of the resultant solution was measured at 700 nm.
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In vivo studies
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Animals The Wistar strain of albino rats (100–150 g) were obtained from the Central Animal House, Department of Animal Husbandry, Allahabad Agricultural Institute-Deemed University (Naini, Allahabad, India). Animals were housed under standard conditions of temperature (25 1 C) with a 12-hour light-dark cycle and had free access to commercial pellet diet and water ad libitum. Animals were acclimatized for 1 week in the laboratory environment, before experimentation. The study was approved by the institutional animal ethical committee, and experiment was performed in accord with Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines for the Laboratory Animal Ethics, Department of Animal Welfare, Government of India.
Toxicity studies Subacute toxicity study Twenty albino rats (150–200 g) of either sex were divided into two groups. The first group received control vehicle (normal saline) and another group received AVP in a dose of 200 mg/kg (per oral; p.o.) daily. Both groups of rats were kept under similar laboratory conditions and were allowed to take a usual pellet diet and water ad libitum. Animals were observed for their general condition, gross behavior, body weight and so on. All animals were sacrificed on the 15th day; the viscera were removed and examined for gastric erosion in the stomach. Acute toxicity studies Mean lethal dose (LD50) was calculated for AVP after administration through the oral route. Swiss albino mice (25–40 g) of either sex were subjected to fasting overnight, with free access to water. A pilot study was carried out before the main experiment, and based on these findings, doses of 1, 2, 3, 4, 5, 6, 7, 8 and 9 g/kg were selected for the final study. Animals were divided into seven groups of 10 animals each. AVP in the above-mentioned dose was given orally, as a single dose. Mortality was observed over a 24-hour period. The LD50 value was calculated by the arithmetic method of Parmar & Ghosh (1978). In vivo experimental protocol Animals (albino rats, 100–150 g) were randomized and divided into 6 groups of 6 animals each. All test drugs were given every day at the dose levels mentioned for a period of 14 days after the intravenous (i.v.) injection of DOX. To avoid variability in the test volume, a constant dose volume of 1 mL/ kg was used throughout the experiment for all the test drugs used. Groups comprised the following: group 1: normal control (0.9% saline, 1 mL/kg, p.o.); group 2: DOX (7.5 mg/ kg, i.v.); group 3: AVP þ DOX (100 mg/kg, p.o. þ 7.5 mg/kg, i.v.); group 4: AVP þ DOX (200 mg/kg, p.o. þ 7.5 mg/kg, i.v.); group 5: AVP þ DOX (300 mg/kg, p.o. þ 7.5 mg/kg, i.v.) and group 6: vitamin E þ DOX (100 mg/kg, p.o. þ 7.5 mg/kg,
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i.v.). Animals were sacrificed on the 15th day for biochemical evaluation.
Food components and anthropometric measurements Food and water intake was measured manually for a group of animals. Weighed amount of pellet diet was given daily, and the amount left the next day was weighed. Similarly, calibrated water bottles were used to monitor the water intake for a group of animals. Animals were monitored at regular interval for body weights. Biochemical analysis Blood samples were collected from retro-orbital plexus and subjected for blood glutathione (GSH) (Beutlar et al., 1963), serum lactate dehydrogenase (LDH) (Lum & Gambino, 1974) and serum creatine phosphokinase (CPK) (Tietz, 1976) estimations. Rats were sacrificed by decapitation under light ether anesthesia. Hearts were removed and weighed. Isolated heart tissue was subjected to the estimation of protein (Lowry et al., 1951), thiobarbituric acid reactive substances (TBARS) (Ohkawa et al., 1979), GSH (Ellman, 1959; Sedlak & Lindsay, 1968), CAT (Clailborne, 1985) and SOD (Marklund & Marklund, 1974) in tissue.
Statistical analysis All the data are presented as mean standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA), followed by Dunnett’s test for possible significance identification between the various groups. A value of p50.05 was considered statistically significant. Statistical analysis was carried out using GraphPad Prism software (version 3.0; GraphPad Software Inc., San Diego, CA).
Results In vitro antioxidant activity The results of the DPPH and NO scavenging activity of AVP are shown in Figures 1 and 2. AVP demonstrated significant scavenging activity in DPPH and NO assay. In fact, the half-maximal inhibitory concentration (IC50) value for AVP in DPPH assay was less than the minimum dose tested (20 mg/mL). In the NO test, AVP and ascorbic acid demonstrated similar scavenging abilities initially, with AVP demonstrating higher scavenging activity at higher doses. The scavenging ability of AVP in H2O2 assay was better than the standard (ascorbic acid), with maximum of 86.64 and 73.32% inhibition of AVP and ascorbic acid, respectively. AVP exhibited concentration-dependent scavenging activity against hydroxyl radical generated in a Fenton reaction system. The IC50 value of AVP was found to be 2.49 mL/mL, in comparison to standard ascorbic acid (3.68 mL/mL), suggesting a better inhibitory potential of AVP (Figure 3). As evident from Figure 4, AVP exhibited potent superoxide radical scavenging activity, as compared to that of ascorbic acid. The IC50 value indicated that the superoxide free radical scavenging activity of AVP was nearly 64.78 mL/ mL, in comparison to ascorbic acid (86.72 mL/mL).
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Figure 1. Free radical scavenging capacity of AVP and ascorbic acid as determined by the DPPH method. Results are means SD. The experiment was performed in triplicate.
Figure 2. NO radical scavenging activity of AVP and ascorbic acid. Results are means SD. The experiment was performed in triplicate.
For measurements of reducing ability, the Fe3þ–Fe2þ transformation was investigated in the presence of AVP. The reducing capacity of a compound may serve as a significant indicator of its potent antioxidant activity. Similar to the antioxidant activity, the reducing power of AVP increased with increasing dosage (Figure 5).
In vivo studies Toxicity studies Results from toxicity studies revealed that AVP was well tolerated up to 5 g/kg, whereas 100% mortality was observed at 9 g/kg when given by the oral route. The LD50 value of AVP was found to be 6.1 g/kg. Administration of AVP for a longer duration of time in subacute toxicity studies did not reveal any untoward effect on behavior, body weight, normal reflexes and visceral appearance in rats. AVP did not
produce any ulcerogenic effect. Results from the toxicity studies suggest that it is also well tolerated.
Experimental studies Food component measurements depicted that food and water intake in the control rats were 135.22 2.11 g/day/group and 208.10 6.17 mL/day/group, respectively (Table 1). All rats injected with DOX (7.5 mg/kg, i.v.) showed a significant (p50.05) decrease in food and water intake along with a decrease in body weight, as compared to control animals. Significant reduction in the ratio of heart weight/body weight in toxic control was observed (p50.05), in comparison to normal control. Animals treated with DOX exhibited a significant increase in serum LDH (621.54 14.52 IU/L) and CPK (72.47 5.13 IU/L) activities, in comparison to control (Table 2).
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Figure 3. H2O2 scavenging activity of AVP and ascorbic acid. Results are means SD. The experiment was performed in triplicate.
Figure 4. Superoxide radical scavenging capacity of AVP determined by the PMS/NADH-NBT method. Results are means SD. The experiment was performed in triplicate.
However, concomitant administration of AVP and vitamin E significantly altered the same in a dose-dependent manner. In our study, we found that blood and tissue GSH levels in normal control animals were 3.21 0.12 mg% and 13.01 0.40 mmol/gwt of tissue, respectively (Table 2). DOX decreased the level of tissue and blood GSH and treatment, with AVP significantly helped to normalize the same (Table 2). In the hearts of DOX-treated rats, there was a significant increase in the TBARS generation (30.15 1.21 nmoles of malondialdehyde [MDA]/mg of protein), in comparison to the control (8.43 0.44 nmoles of MDA/mg of protein), indicating reactive oxygen species (ROS) generation and OS (Table 3). DOX treatment raised the SOD level
(76.19 1.88 units of SOD/mg of protein), in comparison to normal control (49.94 2.87 units of SOD/mg of protein; Table 3). CAT activity in DOX-treated animals was increased to 201.11 4.89 nmoles of H2O2/min/mg of protein, in comparison to normal control (i.e. 52.21 2.85 nmoles of H2O2/min/mg of protein; Table 3). Dose-dependent restoration of SOD and CAT level was observed after AVP treatment in our study.
Discussion and conclusion DPPH is a relatively stable free radical and the assay determines the ability of AVP to reduce DPPH radical to
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Figure 5. Reducing power of AVP. Results are means SD of triplicate measurements.
AVP
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Table 1. Effect of AVP, vitamin E and DOX on food components and anthropometric measurements in different groups. S. no. Group Group Group Group Group Group
Treatment 1 2 3 4 5 6
Food intake (g)
a
0.33 0.01a 0.23 0.01 0.31 0.01b 0.33 0.01a 0.35 0.01a 0.33 0.01a
208.50 6.17 165.00 17.65 185.25 4.40a 218.18 4.40a 224.14 10.9a 201.50 7.42a
135.22 2.11 70.00 10.32 107.92 9.45a 120.08 8.29a 128.12 5.52a 125.64 5.60a
Normal control Toxic control (DOX, 7.5 mg/kg, i.v.) AVP (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (200 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (300 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) Vitamin E (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.)
Heart wt. 100 (g) Body wt.
Water intake (mL)
a
Results are reported as mean SD (n ¼ 6). Statistical significance compared to toxic control on the basis of ANOVA, followed by Dunnett’s test (ap50.001; bp50.01; cp50.05). Table 2. Effect of AVP, vitamin E and DOX on blood and tissue GSH, serum LDH and CPK levels in different groups.
S. no. Group Group Group Group Group Group
1 2 3 4 5 6
Treatment
Blood GSH (mg%)
Tissue GSH (mmol/gwt of tissue)
Serum LDH (IU/L)
Serum CPK (IU/L)
Normal control Toxic control (DOX, 7.5 mg/kg, i.v.) AVP (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (200 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (300 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) Vitamin E (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.)
3.21 0.12a 1.06 0.07 1.41 0.21c 2.77 0.24a 3.33 0.21a 2.98 0.12a
13.01 0.40b 9.11 0.22 9.50 0.21 11.11 0.31b 12.76 0.39a 13.21 0.11a
245.14 3.41a 621.54 14.52 505.21 12.65b 321.14 2.35a 257.16 15.17a 214.21 11.45a
37.46 4.14a 72.47 5.13 57.40 2.62b 41.98 2.74a 37.18 2.49a 39.08 3.48a
Results are reported as mean SD (n ¼ 6). Statistical significance compared to toxic control on the basis of ANOVA, followed by Dunnett’s test (ap50.001; bp50.01; cp50.05). Table 3. Effect of AVP, vitamin E and DOX on myocardial TBARS, CAT and SOD levels in different groups.
S. no. Group Group Group Group Group Group
1 2 3 4 5 6
Treatment
TBARS (nmoles of MDA/mg of protein)
CAT (nmoles of H2O2/min/mg of protein)
SOD (units of SOD/mg of protein)
Normal control Toxic control (DOX, 7.5 mg/kg, i.v.) AVP (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (200 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) AVP (300 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.) Vitamin E (100 mg/kg, p.o.) þ DOX (7.5 mg/kg, i.v.)
8.43 0.44a 30.15 1.21 24.78 0.12c 14.14 0.15a 11.30 0.24a 8.31 0.52a
52.21 2.58a 201.11 4.89 82.94 5.41a 61.94 2.25a 50.63 3.39a 51.50 3.80a
49.94 2.87a 76.19 1.88 69.58 0.75c 53.41 0.08b 43.21 0.47a 49.19 2.14a
Results are reported as mean SD (n ¼ 6). Statistical significance compared to toxic control on the basis of ANOVA, followed by Dunnett’s test (ap50.001; bp50.01; cp50.05).
the corresponding hydrazine by converting the unpaired electrons to paired ones. AVP exhibited significant antioxidant activity in an amount-dependent manner (Figure 1). Antioxidants can act by converting the unpaired electrons to
paired ones (Soares et al., 1997). Scavenging of DPPH radical by AVP demonstrates the correlation of the extent of the scavenging by hydrogen or electron donation of a preformed free radical with antioxidant activity (Morelle et al., 1998).
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DOI: 10.3109/01480545.2013.834350
NO is an important chemical mediator generated by endothelial cells, macrophages, neurons and so on and involved in the regulation of various physiological processes. Excess concentration of NO is associated with pathogenesis of several diseases (Ross, 1993). Oxygen reacts with excess NO to generate nitrite and peroxynitrite anions, which act as free radicals (Sainani et al., 1997). In the present study, AVP competed with oxygen to react with NO and thus inhibited the generation of anions (Figure 2). Superoxide is biologically important because it can be decomposed to form stronger oxidative species, such as singlet oxygen and hydroxyl radicals (Korycka-Dahl & Richardson, 1978). Superoxides are produced from molecular oxygen resulting from oxidative enzymes (Sainani et al., 1997) of the body as well as by nonenzymatic reaction, such as autoxidation by catecholamines (Hemmani & Parihar, 1998). In the present study, it was found that AVP inhibited the superoxide radicals in a dose-dependent manner (Figure 4). In the PMS/nicotinamide adenine dinucleotide (NADH)-NBT system, superoxide anions derived from dissolved oxygen and AVP decreased absorbance at 560 nm, which indicates the consumption of superoxide anions in the reaction mixture by AVP. Hydrogen peroxide is a weak initiator of lipid peroxidation (LPO) (Cohen & Heikkila, 1974). However, it produces active oxygen species as a result of its ability to generate highly reactive hydroxyl radicals through the Fenton reaction (Namiki, 1990). The ability of AVP extract to scavenge H2O2 could also reflect its ability to inhibit the formation of hydroxyl radical in vivo. Various mechanisms, including reducing capacity, prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging, have been claimed to explain antioxidant activities (Diplock, 1997). Reducing the capacity of a compound may serve as a significant indicator of its potential antioxidant activity (Leskovar et al., 2004; Sreekanth et al., 2003). In this study, AVP exhibited an effective reducing capacity at all concentration points (Figure 5). Regarding the in vitro antioxidant potential of AVP, it was considered worthwhile to evaluate its effect against DOX-induced myocardial OS. Despite their potential insidious cardiotoxicity, DOX (anthracycline antibiotic) is often used in the treatment of a wide range of human malignancies. Acute cardiovascular effects develop within minutes or an hour after i.v. injection of DOX and are characterized by hypotension, tachycardia and various arrhythmias, effects which are clinically manageable. Life-threatening chronic effects, such as cardiomyopathy and heart failure, usually develop after several weeks to months of treatments. However, new and low-toxicity anthracycline derivatives or liposome-encapsulated drugs have influenced anthracyclineinduced cardiotoxicity in many ways. Several hypotheses explain the mechanism of anthracycline-induced cardiotoxicity and most of them suggest a crucial role of free radicals and calcium ions (Minotti et al., 1999). In biological systems, DOX is known to produce highly ROS, such as superoxide and hydroxyl radicals (Doroshow et al., 1980), having potential to damage intracellular components. Cardiac muscles are particularly greatly susceptible to the damage
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caused by these ROS because they contain very low levels of ROS-detoxifying enzymes, such as SOD, GSH peroxidase (GPx) and CAT (Takacs et al., 1992); therefore, antioxidants play a critical role in the inactivation of free radicals (Mansat-de Mas et al., 1999). Clinical reports show that patients with advanced cancer develop less limb muscle force, compared to controls (Stone et al., 1999) and have impaired physical performance when undergoing DOX-based chemotherapy (Elbl et al., 2006). Impaired muscle function is commonly attributed to the loss of muscle mass, because cancer cachexia and muscle wasting are known clinical problems (Kotler, 2000). The development of delayed DOX cardiomyopathy in the rat is accompanied by profound anorexia, dramatically reducing the caloric intake, which is a proven fact in many studies. Concomitant administration of AVP significantly increased the DOX-induced reduction in food and water intake along with ratio of heart weight/body weight, indicating that AVP can alter the OS arising as a result of DOX treatment and therefore can help to restore caloric intake in the treatment groups. DOX, by virtue of its quinone structure, acts as an electron acceptor, with the help of various flavoprotien enzymes. Mitochondrial enzymes have been shown to activate DOX to form the semiquinone radical and superoxide anion. Serum LDH and CPK enzyme activities are important to measure both early and late phases of cardiac injury, although these enzymes are nonspecific for measuring cardiac injury, but, together, they could be an indicator of myocardial injury (Kemp et al., 2004). Earlier reports have confirmed that DOX-induced cardiotoxicity is a secondary event subsequent to LPO of cardiac membranes as a result of massive necrosis of cardiac cells in the heart, leading to increase in leakage of LDH and CPK from cardiac myocytes to serum. Concomitant treatment with AVP or vitamin E produced a significant decrease in serum LDH and CPK levels, which could be possibly explained by the fact that AVP exerts anti-LPO activity, causing stabilization of cardiac membranes from the peroxidative damage, thus preventing the leakage of LDH and CPK. GSH acts as an antioxidant coenzyme of GPx, which utilizes it to reduce peroxides or superoxides, yielding oxidized GSH. DOX significantly decreased the level of tissue and blood GSH, which corroborated with earlier studies (Gustafson et al., 1993). Decrease in levels of GSH represents its increased utilization by cardiac cells as a result of OS, and treatment with AVP and vitamin E has significantly helped to restore levels of GSH; this effect could be attributed either as a result of decreased OS or increased biogenesis of GSH. The antioxidant enzymes, SOD and CAT, constitute the major supportive team of defense against free radicals. SOD forms H2O2 and molecular oxygen by scavenging the superoxide radicals, generated as a result of redox cycling of DOX (Gustafson et al., 1993). Previously, most of the studies have reported on the decreased levels of SOD, resulting from OS (Singh et al., 2008) in toxic controls. However, our study showed that the activity of the SOD was significantly increased in DOX-treated animals. Theoretically, it seems that SOD activity decreased with the progression of OS, but here we suggest that as observed increased level of
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SOD could be a consequence of physiological compensatory mechanisms to combat OS. Treatment with AVP significantly normalized SOD levels in a dose-dependent manner, comparable to standard vitamin E, suggesting the possible antioxidant potential of AVP in counteracting DOX-induced OS. Another antioxidant enzyme, besides GPx activity as a defense against H2O2 formations, is CAT enzyme. CAT existing in mammalian cells catalyzes the dismutation of H2O2 (produced from the scavenging effect of SOD) to water and molecular oxygen. Thus, a simultaneous increase in CAT activity is essential to increase SOD activity to counteract the deleterious effects of DOX, which was observed in our study. Concomitant administration of AVP altered CAT levels significantly toward normalization. In vivo measurement of reactive free radicals is difficult because of their high reactivity, short half-life and very low concentration. Thus, the indirect methods usually used for measuring secondary products of OS, such as TBARS, a convenient assay of LPO, are employed. LPO may produce tissue damage and finally cause various cardiovascular complications. Increased free radicals produced may react (Li & Singal, 2000) with polyunsaturated fatty acids in cell membranes, leading to LPO. DOX has been reported to increase MDA, a stable product of LPO in heart tissues. The observation suggests that DOX and its metabolites produces free radical species that attack lipid components, leading to LPO, and coadministration of AVP significantly prevented the increase in the TBARS levels in DOX-treated animals, which was comparable to the standard vitamin E. The pattern of changes were observed in food component intake, anthropometric measurements and biochemical parameters. The existing experimental evidences suggested that DOX-induced OS was primarily the result of the production of free radicals (superoxide and hydroxyl radical) in the heart tissue, having potential to damage various intracellular components. Treatment with AVP proves significant protection from DOX-induced OS, reflecting its antioxidant potential. Our result indicates that AVP possesses significant free radical scavenging activity in vitro and exhibits potent antioxidant potential in DOX-induced OS in vivo. Published data indicate that plant polysaccharides in general have antioxidant activities and can be explored as novel potential antioxidants (Jiang et al., 2005). The potential free radical scavenging mechanism of polysaccharides is poorly understood. This could be attributed to two important factors; first, huge structural diversity of these polysaccharides has given a major hindrance in the structure-activity relationship establishment. Second, most of the hitherto used sulfated polysaccharides or complex mixtures or complete extract containing a large number of other molecules with their own activities. However, the present work describes the purified polysaccharides, thus conclusively proves their antioxidant activity. In conclusion, AVP exhibits a potent antioxidant potential, which supports it to be used as an excellent food supplement. However, further studies are required to confirm the activity of the other compound and to elucidate the molecular and cellular mechanism of AVP.
Drug Chem Toxicol, 2014; 37(2): 135–143
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.
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