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| RESEARCH ARTICLE |
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| Year : 2013 | Volume
: 45
| Issue : 6 | Page : 575-580 |
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Neuroprotective activity of gossypin from Hibiscus vitifolius against global cerebral ischemia model in rats
VM Chandrashekhar1, S Ganapaty2, A Ramkishan3, M Laxmi Narsu4
1 Department of Pharmacology, Hanagal Shri Kumareshwar College of Pharmacy, Bagalkot, Karnataka, India
2 Pharmacognosy and Phytochemistry Division, College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India
3 CDSCO, Subzonal Office, Ahmedabad, Gujarat, India
4 Center of Biotechnology, Institute of Science and Technology, JNT University, Hyderabad, Andhra Pradesh, India
| Date of Submission | 20-Nov-2012 |
| Date of Decision | 26-Nov-2012 |
| Date of Acceptance | 18-Sep-2013 |
| Date of Web Publication | 14-Nov-2013 |
Correspondence Address:
V M Chandrashekhar
Department of Pharmacology, Hanagal Shri Kumareshwar College of Pharmacy, Bagalkot, Karnataka
India
 Source of Support: This study was supported by a research grant VGST/P-8/CISE/2011-2/1151 from Vision Group on Science and Technology, Department of IT, BT, Science and Technology, Govt. of Karnataka, Bangalore, Karnataka (V.M.C)., Conflict of Interest: None  | Check |
DOI: 10.4103/0253-7613.121367
Objectives: The objective of this study is to evaluate the neuroprotective effect of gossypin (isolated from Hibiscus vitifolius) against global cerebral ischemia/reperfusion (I/R) injury-induced oxidative stress in rats.
Materials and Methods: Sprague Dawlet rats of wither gender were used in the study. Evaluation of cerbroprotective activity of bioflavonoid gossypin (in 5, 10 and 20 mg/kg oral doses) isolated from H. vitifolius was carried out by using the global cerebral I/R model by bilateral carotid artery occlusion for 30 min, followed by 24 h reperfusion. The antioxidant enzymatic and non-enzymatic levels were estimated along with histopathological studies.
Result: Gossypin showed dose-dependent neuroprotective activity by significant decrease in lipid peroxidation (P < 0.001) and increase in the superoxide dismutase, catalase, glutathione and total thiol levels in gossypin treated groups when compared to control group. Cerebral infarction area was markedly reduced in gossypin treated groups when compared to control group.
Conclusion: Gossypin showed potent neuroprotective activity against global cerebral I/R injury-induced oxidative stress in rats.
Keywords: Cerebroprotective, flavonoids, gossypin, ischemia/reperfusion, natural products, oxidative stress
How to cite this article:
Chandrashekhar V M, Ganapaty S, Ramkishan A, Narsu M L. Neuroprotective activity of gossypin from Hibiscus vitifolius against global cerebral ischemia model in rats. Indian J Pharmacol 2013;45:575-80 |
How to cite this URL:
Chandrashekhar V M, Ganapaty S, Ramkishan A, Narsu M L. Neuroprotective activity of gossypin from Hibiscus vitifolius against global cerebral ischemia model in rats. Indian J Pharmacol [serial online] 2013 [cited 2023 Jun 2];45:575-80. Available from: https://www.ijp-online.com/text.asp?2013/45/6/575/121367 |
| » Introduction | |  |
Cerebrovascular diseases include some of the common disorders such as ischemic stroke, hemorrhagic stroke, cerebrovascular anomalies etc. They cause 2 million deaths each year and are a major cause of disability. Stroke has been ranked third most common cause of death world-wide and cerebrovascular diseases are considered to be the second most frequent cause of projected deaths in the year 2020. [1]
Cerebral infarction induced brain damage is closely associated with inflammatory responses especially infiltration of circulating neutrophils to ischemic tissue, [2] which are potential sources of reactive oxygen species (ROS) when activated. [3] In the global ischemia model in rats, blood supply to the brain is reduced by occluding the common carotid arteries with a reduction in blood pressure. [4] The restriction of blood flow causes mitochondrial failure and adenosine triphosphate (ATP) depletion, resulting in excessive glutamate release into the synapse. Excitotoxicity is associated with a massive increase in both cytoplasmic Ca 2+ and ROS production. The electrochemical gradient produced by this chain is used by the ATPase to synthesize ATP. However, mitochondria are not thermodynamically perfect and some of these electrons inevitably leak out of the pathway, leading to production of O 2 - . This leads to brain injury and neuronal death. [5] Neuroprotective agents with minimum risk of adverse effects are required and natural sources are probably ideal to develop safe and effective agents for the management of stroke. In spite of great number of clinical trials, no neuroprotective agent has yet been shown to be effective in the treatment of stroke. [6] Flavonoids from plants are reported to have anti-oxidant activity, but their protection against ischemia/reperfusion (I/R) induced oxidative stress is less explored.
Gossypin is a bioflavnoid (gossypin-8-O glucoside; 3, 5, 7, 3,4-pentahydroxy-8-O-glucosylflavone), naturally occurring in plants belonging to the family of Malvaceae. [7] It is reported to have antioxidant, anti-inflammatory, [8],[9] anticonvulsant [10] and anti-cancer activity. [11] In the present study, we investigated the neuroprotective effect of gossypin against global cerebral ischemia model in rats.
| » Materials and Methods | | |
Animals
Sprague-Dawley rats of either gender (200-250 g) and female mice (20-25 g for acute toxicity study) were obtained from the central animal house of H.S.K College Pharmacy and Research Centre, Bagalkot. The animals were housed at room temperature (22-28°C) with 65 ± 10% relative humidity, 12 h light/dark cycle and fed standard diet and water ad libitum. The study was approved and conducted as per the norms of the Institutional Animal Ethics Committee (HSKCP/IAEC, Clear 2009-10/1-8, Dated 28/11/2009). The animals were randomized into six groups of 9 animals each and allowed to acclimatize for 1 week before the experiment. Of the 9 in each group, three rats were separately used for measurement of the infarct area.
Isolation and characterization gossypin from Hibiscus vitifolia
The fresh flowers of Hibiscus vitifolius were collected from the Andhra University campus, Visakhapatnam and authenticated by Prof. M. Venkaiah, Taxonomist, Department of Botany, Andhra University, Visakhapatnam. A voucher specimen (SG-HV-15) was deposited in the College of Pharmaceutical Sciences herbarium, Andhra University.
The isolation of gossypin was carried out using yellow part of the petals of H. vitifolius flowers (1.5 kg), which were separated and extracted thrice by refluxing each time with methanol (4 l each) for 3-4 h and concentrated to a small volume under vacuum. This was refrigerated for 24 h and a large amount of yellowish solid separated out, which was filtered and washed with methanol. The resultant solid was crystallized with aqueous methanol, when a yellow crystalline solid was obtained. Nearly 50% yield, m.p. 228-230°C, was analyzed for C 21 H 20 O 13 (m/e 480 M). Infrared (IR) spectrum exhibited bands at 3175(OH), 1657/cm (C=O) and 1597 (C=C). The 1 H nuclear magnetic resonance (NMR) spectrum exhibited peaks for glucosyl protons as a multiplet between 3.30-4.50 ppm. A singlet at 6.20 ppm indicated a C-H proton at 6 th position. The aromatic protons were shown in 6.80-8.00 ppm region. The C-5 OH proton was observed as a singlet at 12.3 ppm. IR : 1597, 1657, 3175/cm. 1 H-NMR (DMSO-d 6 ) (d ppm): 3.3-3.95 (m, 6H, glucosyl protons), 4.5 (d, J = 7.8 Hz, 1H, C-1 glucosyl proton), 6.2 (s, 1H, C-6 proton), 6.8-7.22 (d, 1H, J = 8.5, C-5' proton), 7.7 (d, J = 8.5, 1H, C-6' proton), 8.0 (d, J = 2.4, 1H, C-2' proton), 8.95 (bs, 2H, C-3, C-7-OH), 12.3 (s, 1H, C-5 OH). MS m/z (%):480 (M+ , 10), 154 (100), 136 (80), 107 (40); Anal. calcd. for C 21 H 20 O 13 ; C, 46.7; H, 4.50; found: C, 46.4; H, 4.20. [12]
Based on the above ultraviolet, IR and NMR data, the compound was characterized as 2-(3,4-Dihydroxyphenyl)-3, 5, 7-trihydroxy-8-{(3, 4, 5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-2-pyranyl)oxy}-4H-4chromenone (gossypin).
Acute toxicity
The acute toxicity study was performed as per the method described by OECD 425 guidelines. The mice were also observed for 14 days for the other signs of toxicity, such as excitation, tremors, twitches, motor co-ordination, righting reflex and respiratory changes. No mortality was observed even at doses of 2000 mg/kg body weight.
Global cerebral I/R induced oxidative stress
Sprague-Dawley rats of either gender (250-300 g) were divided into 6 groups of 9 rats each of which 3 rats were separated for infarction area measurements. The neuroprotective activity of gossypin was assessed at three doses, 5, 10 and 20 mg/kg body weight. [13] All the groups were fed with gossypin or vehicle (1% tween 80) for 10 days prior to the experiment and treated as follows: [14]
Group I: Normal saline (10 ml/kg, p.o.), no ischemia (n = 6).
Group II: Normal saline (10 ml/kg, p.o.), bilateral carotid artery (BCA) occlusion for 30 min (n = 6).
Group III: Normal saline (10 ml/kg, p.o.), BCA occlusion for 30 min, followed by reperfusion for 24 h (n = 6).
Group IV: Gossypin (5 mg/kg, p.o.), BCA occlusion for 30 min, followed by reperfusion for 24 h (n = 6).
Group V: Gossypin (10 mg/kg, p.o.), BCA occlusion for 30 min, followed by reperfusion for 24 h (n = 6).
Group VI: Gossypin (20 mg/kg, p.o.), BCA occlusion for 30 min, followed by reperfusion for 24 h (n = 6).
Induction of ischemia
Animals of group were subjected to BCA occlusion under ketamine anesthesia (45 mg/kg, i.p). Animals were placed on their back, both carotid arteries were exposed and occluded by atraumatic clamps. Temperature was maintained around 37 ± 0.5°C throughout the surgical procedure and artificial ventilation (95% O 2 and 5% CO 2 ) provided with a respirator. [14]
Preparation of post-mitochondrial supernatant
After 24 h of reperfusion, 6 animals of each group were decapitation, the brain removed and washed in cooled 0.9% of saline, kept on ice and subsequently blotted on filter paper, then weighed and homogenized as 10% w/v in cold phosphate buffer (0.05 M, pH 7.4). The homogenates were centrifuged at 10,000 rpm for 10 min at 4°C (MPW-350R, Korea) and post-mitochondrial supernatant (PMS) kept on ice until assayed. The supernatant was used for the estimation of antioxidant enzymes, i.e., superoxide dismutase (SOD), [15] catalase (CAT), [16] reduced glutathione (GSH), [17] total thiols [18] and lipid peroxidation (LPO) markers. [19]
SOD
SOD activity was determined based on its ability to inhibit the auto-oxidation of epinephrine to adrenochrome at alkaline pH. [15] Briefly, 25 μl of the supernatant obtained from the centrifuged brain homogenate was added to a mixture of 0.1 mM epinephrine in carbonate buffer (pH 10.2) in a total volume of 1 ml and the formation of adrenochrome was measured at 295 nm. The SOD activity (U/mg of protein) was calculated by using the standard plot.
CAT
CAT activity was assayed by the method of Calibore. [16] Briefly, the assay mixture consisted of 1.95 ml phosphate buffer (0.05 M, pH 7.0), 1 ml hydrogen peroxide (0.019 M) and 0.05 ml homogenate (10%, w/v) in a total volume of 3 ml. Changes in absorbance were recorded at 240 nm. CAT activity was calculated in terms of nM H 2 O 2 consumed/min/mg protein.
GSH
GSH was estimated in various tissues by the method of Sedlak and Lindsay. [17] Briefly, 5% tissue homogenate was prepared in 20 mM ethylenediaminetetraacetic acid (EDTA), pH 4.7 and 100 μl of the homogenate or pure GSH was added to 0.2 M tris-EDTA buffer (1.0 ml, pH 8.2) and 20 mM EDTA, pH 4.7 (0.9 ml) followed by 20 μl of Ellman's reagent (10 mmol/l DTNB in methanol). After 30 min of incubation at room temperature, absorbance was read at 412 nm. Samples were centrifuged before the absorbance of the supernatants was measured. [17],[18]
Total thiols
This assay is based on the principle of formation of relatively stable yellow color by sulfhydryl groups with DTNB. Briefly, 0.2 ml of brain homogenate was mixed with phosphate buffer (pH 8), 40 μl of 10 mM DTNB and 3.16 ml of methanol. This mixture was incubated for 10 min and the absorbance was measured at 412 nm against appropriate blanks. The total thiol content was calculated by using ε =13.6 × 10 3 /M/cm. [18]
LPO
Thiobarbituric acid reactive substances (TBARS) in the homogenate were estimated by using standard protocol. Briefly, the 0.5 ml of 10% homogenate was incubated with 15% 2, 4, 6-trichloroanisaole, 0.375% 2, 4, 6-tribromoanisole and 5 N HCl at 95°C for 15 min, the mixture was cooled, centrifuged and absorbance of the supernatant measured at 512 nm against appropriate blank. The amount of LPO was determined by using ε =1.56 × 10 5 /M/cm and expressed as TBARS nmoles/mg of protein. [19]
Measurement of cerebral infarct area
Three animals in each group were used for estimation of the infarct area. Evaluation of infarct area was done by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining method. Following I/R after 24 h, animals were decapitated and the brains were removed. After the brains were placed briefly in cold saline, four coronal brain slices (2 mm thick) were made, incubated in phosphate buffered saline (pH 7.4) containing 2% TTC at 37°C for 10 min and kept in neutral-buffered formalin overnight. The images of the TTC stained sections were acquired by scanning by a high resolution scanner (Hewlett Packard Sacnjet 6100 C/T) and cerebral infarction volume and area was measured treatment groups and control group. [20]
Histopathological studies
Brains from control and experimental groups were fixed in 10% of formalin and embedded in paraffin wax and cut into longitudinal section of 5 μm thickness. The sections were stained with hemotoxylin and eosin dye for histopathological observation.
Statistical analysis
All the values are expressed as mean ± standard error of mean and were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (post hoc) and the value P < 0.05 is considered to be statistically significant.
| » Results | | |
Antioxidant enzyme estimation
The control group (Group III; normal saline with I/R) showed a significant decrease (P < 0.01) in SOD levels when compared to normal group (Group I). The gossypin treated groups in I/R induced oxidative stress showed significant (P < 0.001) increase in the SOD levels in a dose dependent manner in brain homogenate when compared to control group. The results of dose effects relationships are presented in [Figure 1]a. | Figure 1: Effect of gossypin on antioxidant levels in ischemia/ reperfusion (I/R) induced oxidative stress in rats. Data is expressed as mean ± standard error of mean, n = 6, aP < 0.05, bP < 0.01 as compared to normal group (group I). *P < 0.05, ***P < 0.001 as compared to I/R control group (group III)
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Group III showed a significant reduction (P < 0.01) in CAT levels as compared to normal group (Group I). The gossypin treated groups in I/R induced oxidative stress in rats showed a significant (P < 0.001) increase in the CAT, levels in brain homogenate as compared to control group. The results of dose effects relationships are presented in [Figure 1]b.
Non-enzymatic antioxidant estimation
Group III showed a significant reduction (P < 0.01) in GSH levels as compared to the normal group while both group II and III demonstrated a significant decrease (P < 0.05) in levels of total thiols as compared to group I. The gossypin treated groups in I/R induced oxidative stress in rats showed a significant (P < 0.001) increase in the GSH and total thiols levels in brain homogenate as compared to control group. The results of dose effects relationships are presented in [Figure 1]c and d.
LPO estimation
Significant increase (P < 0.01) in the level of TBARS was seen in group II and III as compared to group I. The gossypin treated animals showed significantly (P < 0.001) reduced TBARS levels in a dose dependent manner in brain homogenate as comparison to control group. The results of dose effects relationships are presented graphically in [Figure 2]. | Figure 2: Effect of gossypin on lipid peroxidation markers in ischemia/ reperfusion (I/R) induced oxidative stress in rats. All data are expressed as mean ± standard error of mean, n = 6, aP < 0.01 as compared to normal group (group I). ***P < 0.001 as compared to I/R control group (group III)
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Measurement of cerebral infarction area
Analysis of the cerebral infarction area in hisopathological slides revealed that gossypin treated groups showed neuroprotection as compared to control group especially in caudal and rostral side of hippocampus, as shown in [Figure 3] and [Figure 4]. A large infarction area is observed mainly in the caudal and rostral side of hippocampus in the group III (arrow), which is markedly reduced in the groups with gossypin (d-f) [Figure 3]. In [Figure 4], marked infiltration of neutrophils, increase in intracellular space and decrease in density of cells was observed with the architecture completely altered in group III. In contrast, there is a significant reversal of neuronal damage observed in gossypin treated groups. | Figure 3: Neuroprotective activity of gossypin in global cerebral I/R induced oxidative stress in rat. (a) normal; (b) normal saline+ ischemia for 30 min; (c) normal saline + ischemia for 30 min followed by 24 h reperfusion (I/R) and (d) gossypin (5 mg/kg) + I/R; (e) gossypin (10 mg/kg) + I/R; (f): gossypin (20 mg/kg). A large infraction area observed in the caudal and rostral side of hippocampus in the brain of I/R treated rats shown with an arrow. The infraction was markedly reduced in the rat brains treated with gossypin (d-f). I/R= Ischemia/reperfusion. Arrows indicated the presence of infarction
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 | Figure 4: Neuroprotective activity of gossypin in global cerebral I/R induced oxidative stress in rat. (a) normal saline 10 ml/kg, (b) control (normal saline 10 ml/kg + ischemia 30 min followed by 24 h reperfusion, (c) gossypin (5mg/kg) + I/R, (d) gossypin (10 mg/kg) + I/R, (e) gossypin (20 mg/kg). In b, marked infiltration of neutrophils, increased intracellular space, altered architecture and hemorrhage and neuronal cell death was observed. In contrast, marked reversal of neuronal damage was observed in gossypin treated groups (c-e). Arrow indicates the caudal and hippocampus regions of brain. I/R= Ischemia/reperfusion
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| » Discussion | | |
Oxygen maintains brain function and is crucial for life. However, O 2 supplied at concentrations greater than those in normal air is highly toxic. High pressure O2 can lead to production of ROS, [21] which are very important mediators of cell injury and cell death. These radicals are either directly or indirectly involved in various clinical disorders such as neurodegenerative disorders, cancer and atherosclerosis. [22]
In the present study, antioxidant enzyme parameters such as SOD, CAT, GSH, total thiol levels and LPO were estimated in brain homogenate. Gossypin treated groups showed significantly higher extent of protection (P < 0.001) against I/R induced oxidative stress as compared with the control group. Enhanced LPO expressed in terms of TBARS and significantly reduced activity of antioxidant enzymes such as SOD and CAT observed in group III confirms the brain damage due to oxidative stress. Superoxide anion radicals contribute to post-ischemic/reperfusion tissue damage demonstrated in several organs and is associated with pathology of diseases and conditions such as neurodegenerative diseases, I/R injury and inflammation. [23] The SOD and CAT levels were increased significantly and even comparable to normal group after treatment with gossypin in I/R induced rats. Similarly, significantly decreased levels of GSH and total thiols were observed in I/R treated animals. GSH is considered to be a central component in the antioxidant defenses of cells, acts both to directly detoxify ROS and as a substrate for various peroxidases. Dysfunction of the GSH system has been implicated in a number of neurodegenerative diseases [24] and is a potential contributor to oxidative damage following temporary ischemia. In present studies, there is significant (P < 0.001) increased levels of GSH and total thiols in the groups treated with gossypin. The level of LPO is a measure of membrane damage as well as alteration in structure and function of cellular membrane. [25] The LPO level was significantly decreased in the gossypin treated groups, which suggest a free radical scavenging activity.
The cerebral infarction area and histopathological studies revealed the protection against the I/R induced oxidative stress in gossypin treated groups. It has been suggested in other studies that gossypin shows their protection through inhibition of xanthine oxidase inhibition pathway. [26],[27] During ischemia, increased ATP consumption leads to accumulation and purine catabolites hypoxanthine and xanthine, which upon subsequent reperfusion and influx of oxygen are metabolized by xanthine oxidase to produce enormous amount of superoxide radicals and hydrogen peroxide radicals. This leads to brain injury and neuronal death. Oxidative stress, as indicated by elevated xanthine and uric acid levels, is considered to be related to glutamate-mediated excitotoxicity. [28] In biological organisms, ROS are generated by a number of enzymatic systems and inhibition of these enzymes or down regulation of their intracellular expression and activity has also been proposed to be an important mechanism of the gossypin "antioxidant" effects. [29] Gossypin is shown to have anti-inflammatory activity which may also attribute to neuroprotection in ischemia by inhibiting inflammation mediators. In the present study, we observed that direct free radical scavenging activity was responsible for the antioxidant and neuroprotective action of the gossypin. Further, molecular level studies are required to exploit the full therapeutic potential of natural product gossypin.
| » Acknowledgments | | |
This study was supported by a research grant VGST/P-8/CISE/2011-2/1151 from Vision Group on Science and Technology, Department of IT, BT, Science and Technology, Govt. of Karnataka, Bangalore, Karnataka (V.M.C)
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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Direct Surface Analysis Coupled to High-Resolution Mass Spectrometry Reveals Heterogeneous Composition of the Cuticle of Hibiscus trionum Petals |
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| Chiara Giorio, Edwige Moyroud, Beverley J. Glover, Paul C. Skelton, Markus Kalberer | | Analytical Chemistry. 2015; 87(19): 9900 | | [Pubmed] | [DOI] | |
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