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Research Paper
Journal of Pharmacy And Pharmacology
Anticonvulsant activity of Aloe vera leaf extract in acute and chronic models of epilepsy in mice Naveen Rathora, Tarun Arorab, Sachin Manochab, Amol N. Patilb, Pramod K. Medirattac and Krishna K. Sharmac a Department of Pharmacology, University College of Medical Sciences, Delhi, bDepartment of Pharmacology, Vardhman Mahavir Medical College, New Delhi, cDepartment of Pharmacology, Sharda Institute of Medical Sciences and Research, Greater Noida, Uttar Pradesh, India
Keywords Aloe vera; ICES; kindling; MES; mice; pentylenetetrazole Correspondence Tarun Arora, Department of Pharmacology, Vardhman Mahavir Medical College, Safdarjung, New Delhi 110029, India. E-mail: [email protected] Received May 5, 2013 Accepted October 15, 2013 doi: 10.1111/jphp.12181
Abstract Objectives The effect of Aloe vera in epilepsy has not yet been explored. This study was done to explore the effect of aqueous extract of Aloe vera leaf powder on three acute and one chronic model of epilepsy. Methods In acute study, aqueous extract of Aloe vera leaf (extract) powder was administered in doses 100, 200 and 400 mg/kg p.o. Dose of 400 mg/kg of Aloe vera leaf extract was chosen for chronic administration. Oxidative stress parameters viz. malondialdehyde (MDA) and reduced glutathione (GSH) were also estimated in brain of kindled animals. Key findings In acute study, Aloe vera leaf (extract) powder in a dose-dependent manner significantly decreased duration of tonic hind limb extension in maximal electroshock seizure model, increased seizure threshold current in increasing current electroshock seizure model, and increased latency to onset and decreased duration of clonic convulsion in pentylenetetrazole (PTZ) model as compared with control group. In chronic study, Aloe vera leaf (extract) powder prevented progression of kindling in PTZ-kindled mice. Aloe vera leaf (extract) powder 400 mg/kg p.o. also reduced brain levels of MDA and increased GSH levels as compared to the PTZ-kindled non-treated group. Conclusions The results of study showed that Aloe vera leaf (extract) powder possessed significant anticonvulsant and anti-oxidant activity.
Introduction The current challenge in anti-epileptic therapy is need for drugs that can prevent or retard the underlying neuronal damage and at same time have less side effects such as sedation, ataxia and cognitive impairment.[1] Aloe vera (taxonomically called as A. barbadensis) has been used in herbal medicine in many cultures since ages. As a herbal remedy, A. vera whole-leaf extract is advertised for detoxification; it is claimed to cure constipation, help flush out toxins and wastes from the body, promote digestion, and reduce the risk of illnesses.[2] Experimentally, it has been demonstrated to have antidiabetic, antiinflammatory, anti-oxidant, immunomodulatory, antineoplastic and neuroprotective properties.[3] A. vera contains various potentially active constituents like aloin, aloeemodin, vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids and amino acids.[4] Many studies of the immunological effects of extracts from plants of
Aloe have focused on the clear mesophyll gel of the A. vera leaf and on its major storage carbohydrate, acetylated mannan and acemannan.[5] Acemannan is known to have diverse biological activity, including immunomodulatory and antitumor attributes. The anticancer biological mechanism of acemannan may be exerted through pluripotent effector cells, such as macrophages, as Aloe extracts are known to induce macrophage activating activity.[6] In addition, A. vera has been demonstrated to have antidiabetic and anti-inflammatory properties.[7,8] The anti-oxidant effect of A. vera has been proved to be beneficial in diabetes9 and cancer.[10] One potential area where effect of A. vera has not been explored is neurological disorders such as epilepsy. There is some evidence in literature on the effect of A. vera on neuronal activity. Bouthet and coworkers have demonstrated that A. vera increases proliferation in cultured ‘neuron-like’ rat adrenal pheochromo-
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cytoma cells treated in suspension.[11] Another study provides evidence of direct concentration-dependent depressive effect of A. vera on the neurotransmission process with respect to presynaptic and postsynaptic mechanisms.[12] Moreover, a recent study has reported that A. vera improves anti-oxidant activity within the hippocampus and cerebral cortex leading to improvement of motor and memory behavioural tasks in diabetic mice.[13] In view of the earlier evidence on effect of A. vera on hippocampus and presynaptic and postsynaptic mechanisms, the fact that generation of seizures involve changes in synaptic transmission of excitatory and inhibitory neurotransmitters across the synapses in cerebral cortex, hippocampus and thalamus,[14] along with the learning and memory loss because of increased oxidative stress seen with both epilepsy and some anti-epileptic drugs (AEDs) in patients with chronic epilepsy, supplements the available evidence on anti-oxidant effect of A. vera. In addition, some AEDs like lamotrigine have been shown to have beneficial anti-epileptic and anti-oxidant effect in epilepsy.[15] Therefore, this study was done to find out the effect of A. vera in various epileptogenesis models and the confirmation of its beneficial anti-oxidant effect.
Materials and Methods Animals Healthy Swiss albino inbred mice of either sex weighing 25–30 g were used. The inbred strain was used, as the data obtained in inbred animals are more consistent and reproducible. The animals were procured from the Central Animal House of University College of Medical Sciences. Animals were housed in groups of six mice per cage (43 × 28.6 × 15.5 cm) with free access to pellet diet and water ad libitum. Care of the animals was taken as per ‘Committee for the Purpose of Control and Supervision of Experiments on Animals’, India, and the study was approved by Institutional Ethics Committee-Animal Research, UCMS (IEC-AR/11/2008, dated 26 September 2008). The recommendations from the Declaration of Helsinki and the internationally accepted principles in the care and use of experimental animals had been adhered to during the study. Adequate measures were taken to avoid any pain or discomfort to the animals during handling or experimentation. The animals were divided into treatment and control group by simple randomisation, and all experiments were performed between 9:30 am and 6:30 pm in the neuropharmacology laboratory of the department.
Preparation of Aloe vera leaf extract A. vera leaf extract was obtained from M/s Indo World Trading Corporation, New Delhi, India (Batch No.: IWTC/ 478
711/9432). As per the literature provided by the manufacturer, the gel obtained from A. vera leaf was mixed with double distilled water in the ratio 1 : 1, mechanically shaken at room temperature, concentrated in the evaporator followed by lyophilisation to obtain a brown powder. The characterisation of a sample of the brown powder extract by the spectrophotometer (IP66 Method) revealed 3.14% aloin. The A. vera leaf gel contains about 98% water. As per gas chromatography-mass spectrometry (GC-MS), various compounds identified in A. vera leaf gel extract were phenolic acids/polyphenols such as phenol (14.32 ppm), vanillic acid (58.6 ppm), homovanillic acid (18.55 ppm), protocatechuic (163.21 ppm), 3,4-dihydroxyphenylacetic (7.54 ppm), 5-methoxyprotocatechuic (2.5 ppm), syringic (25.54 ppm), sinapic (32.68 ppm), p-coumaric (450.87 ppm), isoferulic (52.90 ppm), ferulic (88.67 ppm), Aloe-emodin (87.79 ppm), 4-phenyllactic (11.02 ppm), 4-ethylphenol (10.12 ppm), hydrocinnamic (37.50 ppm), p-salicylic (186 ppm), benzoic (870.1 ppm), hydro-p-coumaric (15.31 ppm), alcohols such as 2-butanol (13.65 ppm), glycerol (340.9 ppm), phenylethanol (86.56 ppm), aldehydes such as benzaldehyde (56.34 ppm), m-tolualdehyde (18.21 ppm), organic acids such as lactic (148 ppm), glycolic (93.1 ppm), furoic (57.43 ppm), succinic (383 ppm), 2-methylsuccinic (62.1 ppm), malic (46.7 ppm), tartaric (18.3 ppm), isonicotinic (40.21 ppm), alkanes such as 1,3dihydroxybutane (10.22 ppm), pyrimidines such as uracil (697.23 ppm), thymine (429.76 ppm), indoles such as indole3-acetic acid (2.80 ppm), alkaloids such as hypoxanthine (27.65 ppm), ketones such as acetophenone (8.02 ppm), sterols such as cholestanol (24.32 ppm), β-sitosterol (1604.5 ppm), dicarboxylic acids such as azelaic acid (0.02 ppm) and undecanedioic acid (0.04 ppm).[16] For the purpose of study, A. vera leaf extract powder was dissolved in normal saline to prepare suspensions of 100, 200 and 400 mg/kg. The drug solutions were prepared freshly each time and administered intragastrically. The dosing schedule used was once per day. All drugs were administered in a volume of 10 ml/kg/dose, and the animals were tested 1 h after oral drug administration. The animals were divided into following treatment groups in acute study.
Acute study Group 1 was control group and administered physiological saline Group 2 was given A. vera 100 mg/kg p.o. Group 3 was given A. vera 200 mg/kg p.o. Group 4 was given A. vera 400 mg/kg p.o. Phenytoin (25 mg/kg i.p.) and diazepam (5 mg/kg i.p.) were kept as standard groups in maximal electroshock seizure (MES) and pentylenetetrazole (PTZ) model, respectively.
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Epileptogenesis models in acute study Maximal electroshock seizure test The MES test was performed as described by Swinyard et al.[17] Electroconvulsion was produced using electroconvulsiometer. An alternating current (50 mA, 0.2-s duration) was delivered via ear-clip electrodes, and duration of tonic hind limb extension (THLE) was measured for comparison in between the groups. Increasing current electroshock seizure test The ICES test, as proposed by Kitano et al.[18] was used to evaluate the effect of A. vera on convulsions. Starting with a current of 2 mA, electroshock was delivered to each mouse via ear-clip electrodes as a single train of pulses (for 0.2 s) with linearly increasing intensity of 2 mA/2 s. The current at which THLE occurred was recorded as the seizure threshold current. If no THLE was observed up to a current of 30 mA, electroshock was terminated, and this cut-off current was used in the analysis. Pentylenetetrazole-induced convulsions PTZ is a powerful central nervous system stimulant believed to be acting by GABAergic inhibition.[19] The administration of the compound causes stimulation of the cortex leading to tonic-clonic convulsions. Injection of PTZ in a dose of 60 mg/kg was given subcutaneously in acute study, and the latency to and duration of clonic convulsions were recorded for a period of 30 min.
Epileptogenesis model in chronic study Pentylenetetrazole-induced kindling Repeated subconvulsive dose of PTZ once every 48 h for 4–6 weeks is known to induce a permanent change in the epileptogenic sensitivity of brain such that initial subconvulsive stimuli become capable of evoking fully developed epilepsy.[20] This model is clearly implicated in development of chronic epileptogenesis as in humans and has strong predictive value. One of the proposed mechanisms of human epilepsy is the initial sensitisation of neuronal cells because of some extraneous or genetic insult that in turn progresses to involve more neuronal cells and ultimately leads to full-blown tonic-colonic seizures (equivalent to kindling in animals).[14] Chemical kindling is the development of seizures following repeated administration of a convulsant substance at a dose that is insufficient to produce convulsions following a single administration. Kindling produced by PTZ in mice has characteristics very similar to those described for electrical kindling, that is, the response to PTZ is proportional to the
Anticonvulsant effect of Aloe vera in mice
stimulus intensity. The PTZ kindling model is generally used as a laboratory model of human partial complex epilepsy.[21] In addition, kindling is a model of epilepsy that has the advantages of both an epileptogenic and a spontaneous seizure model.[21] Because spontaneity and recurrence of seizures are the basic features of human epilepsy, chronic models like kindling are advantageous over acute models.[21] The inhibition of such tonic-clonic seizures (induced by subconvulsive dose of PTZ) by diazepam, carbamazepine and various other AEDs is a standard model for testing the drugs effective in chronic epilepsy.[15,21] The development of epileptogenesis in PTZ was given in a dose of 35 mg/kg intraperitoneally on alternate days at the same time (thrice a week), and the animals were observed for appearance of seizure activity. The evaluation of seizure activity was done as follows: Score 0 – No change Score 1 – Hyperactivity, restlessness, vibrissae twitching Score 2 – Head nodding, head clonus, myoclonic jerks Score 3 – Unilateral or bilateral limb clonus Score 4 – Forelimb clonic seizures Score 5 – Generalised clonic seizures with loss of righting reflex and whole body extension. When the animal had seizure score of 4 at three consecutive injections of PTZ at 72-h interval, animal was taken as kindled, and treatment was discontinued. To study the effect of A. vera on development of kindling, animals were divided in two groups as follows: Group 1: received PTZ (35 mg/kg i.p.) alone on alternate days (three times a week) Group 2: treated with A. vera (400 mg/kg p.o. daily) along with PTZ (three times a week). In another set of experiment, to find out latency to and duration of clonic convulsions in PTZ-kindled animals, they were subdivided in another two groups (n = 10). Group 1: were treated with PTZ (35 mg/kg i.p.) Group 2: were treated with A. vera (400 mg/kg p.o.) along with PTZ (35 mg/kg i.p.) Assessment of oxidative stress Assessment of oxidative stress was done in kindled mice by measuring the levels of malondialdehyde (MDA) and reduced glutathione (GSH) in brain. MDA: this (indicator of lipid peroxidation) was estimated as described by Ohkawa et al.[22] GSH: this was estimated by the method described by Ellman.[23]
Statistical analysis The data were expressed as mean ± standard error of mean, and results were analysed by one-way analysis of variance followed by Tukey’s t test in acute study and measurement
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of oxidative stress. Wilcoxon signed-rank test and Freidman test followed by Mann–Whitney test for intergroup comparison, and unpaired t test were used for analysing data obtained from chronic study at significance level 5%.
treated group, seizure threshold current was increased significantly to 26.0 ± 0.54 mA (P < 0.001) as compared with the control group (Table 1). Effect of Aloe vera on pentylenetetrazole-induced latency and duration of clonic convulsion
Results Acute study Effect of Aloe vera on maximal electroshock seizure test A. vera in the dose of 100 mg/kg did not significantly modify the duration of THLE as compared with the control group. However, administration of A. vera in doses of 200 and 400 mg/kg significantly (P < 0.001) decreased the duration of THLE to 10.45 ± 0.73 s and 7.02 ± 0.32 s, respectively, as compared with the control group. None of the mice in the phenytoin group showed THLE (Table 1). Effect of Aloe vera on increasing current electroshock seizure test
Chronic study
The seizure threshold current in the control group was 13.50 ± 0.73 mA that was not significantly increased by A. vera 100 mg/kg (15.25 ± 0.53 mA). However, A. vera in doses of 200 and 400 mg/kg significantly increased the seizure threshold current, respectively. In the phenytoin
Table 1
A. vera in a dose of 100 mg/kg did not significantly increase the latency to onset of clonic convulsions. While A. vera in doses of 200 and 400 mg/kg significantly increased the latency to onset of clonic convulsion (P < 0.001 and P < 0.001, respectively) as compared with the control group. Diazepam exhibited complete protection against clonic convulsions. The duration of clonic convulsions in A. vera 100 mg/kg treated group was not significantly decreased as compared with the control group. In A. vera 200 mg/kg group, the duration was significantly (P < 0.05) reduced to 15.13 ± 0.30 s, while in Aloe vera 400 mg/kg group, it was reduced to 5.50 ± 1.69 s (P < 0.001) as compared with the control group (Table 2).
Effect of Aloe vera on development of pentylenetetrazole-induced kindling measured by kindling score A. vera treatment of 400 mg/kg/day p.o. along with PTZ 35 mg/kg intraperitoneally on alternate days had significant
Effect of Aloe vera on maximal electroshock seizure test and increasing current electroshock seizure test in mice
Group
Treatment
Duration of tonic hind limb extension (s)
Seizure threshold current (mA)
Control (Group1) Aloe vera (Group 2) Aloe vera (Group 3) Aloe vera (Group 4) Phenytoin (Group 5)
Normal saline (10 ml/kg) 100 mg/kg p.o. 200 mg/kg p.o. 400 mg/kg p.o. 25 mg/kg i.p.
14.54 ± 0.37 13.14 ± 0.55 10.45 ± 0.73a 7.02 ± 0.32a 00 ± 00a,b
13.50 ± 0.73 15.25 ± 0.53 16.25 ± 0.45c 18.0 ± 0.54d 26.0 ± 0.54d,e
Values are expressed as mean ± standard error of the mean, n = 10 mice in each group. aP < 0.001 as compared with the control group. bP < 0.001 as compared with the Aloe vera 400 mg/kg group. cP < 0.05 as compared with the control group. dP < 0.001 as compared with the control group. e P < 0.001 as compared with the Aloe vera 400 mg/kg group.
Table 2
Effect of Aloe vera on pentylenetetrazole (60 mg/kg s.c.) induced latency and duration to onset of clonic convulsions in mice
Group
Treatment
Latency to onset of clonic convulsion (s)
Duration of clonic convulsions (s)
Control (normal saline) (Group 1) Aloe vera (Group 2) Aloe vera (Group 3) Aloe vera (Group 4) Diazepam (Group 5)
10 ml/kg p.o. 100 mg/kg p.o. 200 mg/kg p.o. 400 mg/kg p.o. 5 mg/kg i.p.
642.38 ± 27.09 683.75 ± 18.61 874.88 ± 17.26a 1083.75 ± 31.37b 0.0 ± 0.0b
19.50 ± 0.73 18.25 ± 0.59 15.13 ± 0.30c 5.50 ± 1.69d 0.0 ± 0.0d,e
Values are expressed as mean ± standard error of the mean, n = 10 mice in each group. aP < 0.05 as compared with the control group. bP < 0.001 as compared with the control group. cP < 0.05 as compared with the control group. dP < 0.001 as compared with the control group. eP < 0.001 as compared with the Aloe vera 400 mg/kg group.
480
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Anticonvulsant effect of Aloe vera in mice
6
--- PTZ alone --- Aloe vera + PTZ
Mean seizure score
5
4
3 *
* 2 *
*
*
*
*
*
1
0 1
3
5
8
10
12
15 17 Days
19
22
24
26
29
31
Figure 1 Effect of chronic administration of Aloe vera (400 mg/kg p.o.) on pentylenetetrazole-induced kindling score. *P = 0.000 at week 2, week 3 and week 4 as compared with pentylenetetrazole group as per Mann–Whitney U test.
Table 3
Effect of Aloe vera on development of pentylenetetrazole (PTZ)-induced kindling measured by kindling score
Group PTZ PTZ + Aloe vera
Treatment 35 mg/kg i.p. 400 mg/kg p.o. + 35 mg/kg i.p.
Week 1
Week 2
Week 3
Z = −1.831
Z = −2.812 , Z = −3.162b, Z = −4.061c a
Week 4
Z = −2.821 Z = −3.162b, Z = −4.059c a
Z = −2.807a, χ2 = 30d Z = −3.162b, Z = −4.065c, χ2 = 30d
P= 0.005 at week 2, week 3 and week 4 as per Wilcoxon signed-rank test. bP=0.002 at week 2, week 3 and week 4 as per Wilcoxon signed-rank test. cP=0.000 at week 2, week 3 and week 4 as compared with PTZ group as per Mann–Whitney U test. dP=0.000 at week 4 as compared with week 1 as per Freidman test.
a
protective effect on the development of kindling. There was a significant increase in the kindling score in the PTZkindled nontreated group at week 4 compared with week 1 (χ2 = 30, P = 0.000), while the kindling score also showed a similar trend in A. Vera-treated PTZ-kindled group (χ2 = 30, P = 0.000) as per Freidman test for intragroup comparison (Figure 1, Table 3). Comparative analysis of the kindling score within PTZ-kindled non-treated group at week 1, week 2, week 3 and week 4 also showed a significant increase in the kindling score with increasing time (P = 0.005 at week 2, week 3 and week 4) as per Wilcoxon signed-rank test with similar trends appearing in the A. vera-treated PTZ-kindled group (Figure 1, Table 3). The kindling score in A. vera-treated PTZ-kindled group at end of second, third and fourth week was significantly different (P = 0.000, z = −4.061 at 2 weeks; P = 0.000, z = −4.059 at 3 weeks; and P = 0.000, z = −4.065) than the kindling score in PTZ-kindled nontreated group as per Mann–Whitney U test for intergroup comparison (Figure 1, Table 3).
Effect of Aloe vera on the latency to onset and duration of clonic convulsions in pentylenetetrazole-kindled mice Aloe vera (400 mg/kg p.o.) significantly (P < 0.05) increased the latency to onset of clonic convulsions in PTZ-kindled mice as compared with the kindled mice that received PTZ alone. A. vera significantly (P < 0.05) decreased the duration of clonic convulsions in PTZ-kindled mice as compared with the kindled mice that received PTZ alone (Table 4). Effect of Aloe vera on brain levels of malondialdehyde (nmol/g wet brain tissue) in pentylenetetrazole-kindled mice There was significant (P < 0.001) increase in the brain MDA levels of PTZ-kindled group as compared with the control group. The MDA levels in the A. vera-treated kindled group were significantly (P < 0.001) lower as compared with the PTZ group (Table 5).
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Table 4
Naveen Rathor et al.
Effect of Aloe vera on the latency and duration to onset of clonic convulsion in the pentylenetetrazole (PTZ)-kindled mice
Group
Treatment
Latency to onset of clonic convulsion (s)
Duration of clonic convulsion (s)
PTZ Aloe vera + PTZ
35 mg/kg i.p. 400 mg/kg p.o. + 35 mg/kg i.p.
355.30 ± 24.73 453.90 ± 25.18a
19.10 ± 1.04 13.80 ± 1.63b
Values are mean ± standard error of the mean, n = 10 mice in each group. aP < 0.05 as compared with the PTZ group. bP < 0.05 as compared with the PTZ group.
Table 5
Effect of Aloe vera on brain levels of MDA and glutathione in PTZ-kindled mice
Group
Treatment
MDA (nmol/g wet brain tissue)
GSH (μg/g wet brain tissue)
Control PTZ Aloe vera + PTZ
Normal saline (10 ml/kg) 35 mg/kg i.p. 400 mg/kg/day p.o. + 35 mg/kg i.p.
231.70 ± 12.77 430.60 ± 11.80a 301.90 ± 14.86b,c
218.30 ± 6.92 109.70 ± 7.25d 177.60 ± 7.20e,f
GSH, glutathione; MDA, malondialdehyde; PTZ, pentylenetetrazole.Values are expressed as mean ± standard error of the mean, n = 10 mice in each group. aP < 0.001as compared with the control group. bP < 0.05 as compared with the control group. cP < 0.001as compared with the PTZ group. d P < 0.001 as compared with control group. eP < 0.05 as compared with the control group. fP < 0.001as compared with the PTZ group.
Table 6
Effect of Aloe vera on cyclooxygenase-inhibitory activity in formalin-induced inflammatory response
Group
Treatment
Duration of licks/bites in early phase (s)
Duration of licks/bites in late phase (s)
Control Aloe vera Aloe vera
Normal saline, 200 mg/kg p.o. 200 mg/kg p.o. 400 mg/kg p.o.
52.43 ± 4.12 51.25 ± 4.24 51.14 ± 4.30
52.43 ± 4.12 35.18 ± 5.15a 18.19 ± 4.17b
Values are expressed as mean ± standard error of the mean, n = 10 mice in each group. aP < 0.05 as compared with the control group. bP < 0.001 as compared with the control group.
Effect of Aloe vera on brain levels of reduced glutathione in pentylenetetrazole-kindled mice There was significant (P < 0.001) decrease in the brain GSH levels of the PTZ-kindled group as compared with the control group. The GSH levels in the A. vera-treated kindled group were significantly (P < 0.001) increased as compared with the PTZ group (Table 5). Effect of Aloe vera on cyclooxygenase-inhibitory activity in formalin-induced inflammatory response The effect of A. vera leaf extract powder was also studied on cyclooxygenase (COX)-inhibitory activity in formalininduced inflammatory response. Results showed that doses of 200 and 400 mg/kg significantly decreased the duration of lick/bite responses in late phase from 48 s in nontreated group to 18 s in 400 mg/kg A. vera group and 35 s in 200 mg/kg A. vera group. (Table 6)
Discussion In this study, efforts were made to investigate the antiepileptic potential of A. vera using both acute and chronic 482
animal models of epilepsy. The results of this study showed that A. vera significantly decreased the duration of THLE in MES (grand mal) model and significantly increased the seizure threshold current at which THLE occurred in ICES (grand mal) model of convulsion in a dose-dependent manner. These results were similar to previous studies showing anti-epileptic activity of phenytoin (standard drug) in the earlier models.[18,24] Anticonvulsant activity was also assessed by PTZ-induced acute convulsive model. A. vera-treated group showed an increase in the latency to onset and decrease in duration of convulsions as compared with the control group. These results were similar to the effect of phenytoin, diazepam and other AEDs in PTZinduced acute convulsions.[25] A. vera has been shown to inhibit NMDA-induced retinal ganglionic cell apoptosis.[26] Considering the role of NMDA in epilepsy and its modulation by lamotrigine, the beneficial effect of A. vera in epileptic models could be explained by its property of modulating similar NMDA receptors in brain cells. However, this needs to be confirmed in follow-up studies. In the model of chemical kindling (chronic group), effect of A. vera was studied on development and expression of convulsions in PTZ-kindled mice. The process of epileptogenesis and longterm use of certain anti-epileptic drugs has been shown to
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cause increase in reactive oxygen species (ROS) that leads to oxidative stress and neuronal damage in patients with epilepsy.[27] So, parameters of oxidative stress like MDA and GSH were also included to find out the effect of A. vera in modulating oxidative activity in epileptic animals. In addition considering the published evidence of A. vera leaf extract on COX inhibition, an inflammatory model of chronic pain (formalin induced) was also evaluated for COX-inhibitory activity of A. vera leaf extract in various doses. Dose of 400 mg/kg of A. vera was chosen in kindling for chronic administration as this dose demonstrated maximum absolute increase in seizure threshold in acute studies. A. vera treatment of 400 mg/kg/day daily along with alternate day PTZ had significant protective effect on the development of kindling in mice, and there was a statistically significant decrease in the kindling scores at week 2, week 3 and week 4 along with a significant increase in the latency to onset and decrease in duration of clonic convulsions as compared with PTZ-kindled nontreated group. These results were similar to the effect of phenytoin, carbamazepine and other AEDs in PTZ-induced kindling model.[17] A. vera has also been shown to inhibit COX enzyme. The results of our study also showed that doses of 200 and 400 mg/kg of A. vera leaf extract powder significantly decreased the duration of lick/bite responses in late phase from 48 s in nontreated group to 18 s in 400 mg/kg A. vera-treated group and 35 s in 200 mg/kg A. vera-treated group. A radical scavenging glycoprotein from A. vera gel that inhibits COX-2 and thromboxane A2 synthase has been isolated.[28,29] Various studies have shown that COX inhibitors have got protective effect in electroshock-induced and PTZ-induced convulsion in animal models, so anticonvulsant property of A. vera could also be explained by its COX enzyme inhibiting potential.[30–32] However, this needs to be confirmed in future studies using suitable controls. The assay of MDA is often considered as an index of freeradical generation. The results of this study showed that A. vera-treated PTZ-kindled group showed significant decrease in the MDA levels as compared with the PTZkindled nontreated group. GSH is the most prevalent and important intracellular anti-oxidant. This compound is able to scavenge both singlet oxygen and hydroxyl radicals. The results of this study showed that A. vera-treated group exhibited a significant increase in GSH levels in mice brain as compared with PTZ-kindled nontreated group, suggesting an anti-oxidant effect of A. vera. The process of epileptogenesis and long-term use of certain AEDs has been shown in previous studies to cause increase in ROS leading to oxidative stress and neuronal damage in patients with epilepsy.[33] There is evidence for increased lipid peroxidation (index for MDA) in PTZ-induced kindling in rats,[34] and certain
Anticonvulsant effect of Aloe vera in mice
AEDs like carbamazepine themselves have been shown to cause increase in oxidative stress by reducing the levels of GSH.[27,34] On the other hand, lamotrigine has shown antioxidative potential in some previous studies.[35] Our results were similar to anti-oxidant effect of lamotrigine in similar epileptic animal models.[17] In accordance with existing literature that COX enzyme leads to formation of hydroxyl free-radical and peroxynitrite free-radical owing to peroxidative activity[36,37] and considering the results of some previous studies showing anti-oxidant effect of A. vera[7] and inhibition of cyclooxgenase enzyme by it[28] could also explain the anti-oxidative mechanism of A. vera. Besides this, several low-MW compounds present in aloe gel have been shown to be capable of inhibiting the release of reactive oxygen free-radicals from activated human neutrophils.[38] Hence, anti-oxidant property of A. vera could prevent the neuronal damage in epileptic patients that itself is partly due to increased oxidative stress. Previous reports of HPLC analysis on A. vera leaf extract have identified two diastereomers of Aloin, that is, Aloin A and Aloin B along with Aloe-emodin. Aloe-emodin itself is derived from oxidation of Aloin. The levels of Aloin B at 1.335 ppm were found to be higher than Aloin A at 0.868 ppm that were higher than levels of Aloin-emodin at 0.200 ppm.[39] Further structural elucidation studies of these chromones identified them as 8-C-glycosyl-7-O methyl-(S) aloesol, isoaloeresin D and aloeresin E by reverse phase HPLC and headspace GC-MS.[40] Because the active constituent of A. vera leaf extract in our study was Aloin (3.4%) and considering the previous evidence on beneficial effects of aloin in inflammation, diabetes and infections,[7,8,38] both the anti-epileptic and anti-oxidant activity could be attributed to aloin; however, this needs to be followed up vigorously in future studies.
Conclusion Considering the significant anticonvulsant (in acute models), anti-epileptic (chronic model) and anti-oxidative effect of A. vera leaf powder extract, the results of our study provided the evidence that it could be used as an alternative to AEDs in patients of both grand mal and absence epilepsy, and might prevent the neuronal damage in such patients. In addition, because the leaf powder extract was derived from the lyophilisation of gel (central part), other parts of A. vera leaf (peripheral layer) need to be studied for their antiepileptic effects in future studies.
Declarations Acknowledgements The Authors would like to thank M/s Indo World Trading Corporation, New Delhi for providing A. vera leaf extract
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powder and the technical and animal house staff of Department of Pharmacology for providing the animals and material support.
References 1. Terada K, Inoue Y. Clinical application of newer anti-epileptic drugs. Rinsho Shinkeigaku 2012; 52: 1088–1090. 2. Ayushveda. Aloe Drink – Benefits of Aloe vera Juice. Panchkula, Haryana, India: Ayushveda Infomatics, 2010. Available at. http://www.us.ayushveda .com/aloe-dring-benefits-of-aloe -vera-juice/ (accessed 2 March 2010). 3. Shelton M. Aloe vera, its chemical and therapeutic properties. Int J Dermatol 1991; 30: 679–683. 4. Harlev E et al. Anticancer potential of Aloes: antioxidant, antiproliferative, and immunostimulatory attributes. Planta Med 2012; 78: 843–852. 5. Tizard IR, Ramamoorthy L. Aloes and the immune system. In: Reynolds T, 2nd ed. Medicinal Aromatic Plants – Industrial Profiles. Boca Raton, FL: CRC Press, 2004: 311–332. 6. Zhang L, Tizard IR. Activation of a mouse macrophage cell line by acemannan: the major carbohydrate fraction from Aloe vera gel. Immunopharmacology 1996; 35: 119– 128. 7. Moniruzzaman M et al. In vitro antioxidant effects of Aloe barbadensis miller extracts and the potential role of these extracts as antidiabetic and antilipidemic agents on streptozotocin-induced type 2 diabetic model rats. Molecules 2012; 17: 12851– 12867. 8. Rathor N et al. Acute effect of Aloe vera gel extract on experimental models of pain. Inflammation 2012; 35: 1900–1903. 9. Rajasekaran S et al. Antioxidant effect of Aloe vera gel extract in streptozotocin-induced diabetes in rats. Pharmacol Rep 2005; 57: 90–96. 10. Boudreau MD et al. Clear evidence of carcinogenic activity by a whole-leaf extract of Aloe barbadensis miller (Aloe 484
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phenytoin) in electroshock induced convulsion. Methods Find Exp Clin Pharmacol 2003; 25: 87–90. 33. Sudha K et al. Oxidative stress and anti-oxidants in epilepsy. Clin Chim Acta 2001; 303: 19–24. 34. Rauca C et al. Formation of free hydroxyl radicals after pentylenetetrazole-induced seizures and kindling. Brain Res 1999; 847: 347–351. 35. Pavone A, Cardile V. An in vitro study of new antiepileptic drugs and
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astrocytes. Epilepsia 2003; 4(Suppl 10): 34–39. 36. Haliwell B. Reactive oxygen species and the central nervous system. J Neurochem 1992; 59: 1609–1623. 37. Dhir A et al. Effect of naproxen, a non-selective cyclo-oxygenase inhibitor, on penylenetetrazol-induced kindling in mice. Clin Exp Pharmacol Physiol 2005; 32: 574–584. 38. Hart LA et al. Effects of low molecular constituents from Aloe vera gel on oxidative metabolism and cytotoxic and
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bactericidal activities of human neutrophils. Int J Immunopharmacol 1990; 12: 427–434. 39. Sehgal I et al. Toxicologic assessment of a commercial decolorized whole leaf Aloe vera juice, lily of the desert filtered whole leaf juice with Aloesorb. J Toxicol 2013; 2013: 1–12. 40. Saccù D et al. Aloe exudate: characterization by reversed phase HPLC and headspace GC-MS. J Agric Food Chem 2001; 49: 4526– 4530.
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Research Paper
Journal of Pharmacy And Pharmacology
Anticonvulsant activity of Aloe vera leaf extract in acute and chronic models of epilepsy in mice Naveen Rathora, Tarun Arorab, Sachin Manochab, Amol N. Patilb, Pramod K. Medirattac and Krishna K. Sharmac a Department of Pharmacology, University College of Medical Sciences, Delhi, bDepartment of Pharmacology, Vardhman Mahavir Medical College, New Delhi, cDepartment of Pharmacology, Sharda Institute of Medical Sciences and Research, Greater Noida, Uttar Pradesh, India
Keywords Aloe vera; ICES; kindling; MES; mice; pentylenetetrazole Correspondence Tarun Arora, Department of Pharmacology, Vardhman Mahavir Medical College, Safdarjung, New Delhi 110029, India. E-mail: [email protected] Received May 5, 2013 Accepted October 15, 2013 doi: 10.1111/jphp.12181
Abstract Objectives The effect of Aloe vera in epilepsy has not yet been explored. This study was done to explore the effect of aqueous extract of Aloe vera leaf powder on three acute and one chronic model of epilepsy. Methods In acute study, aqueous extract of Aloe vera leaf (extract) powder was administered in doses 100, 200 and 400 mg/kg p.o. Dose of 400 mg/kg of Aloe vera leaf extract was chosen for chronic administration. Oxidative stress parameters viz. malondialdehyde (MDA) and reduced glutathione (GSH) were also estimated in brain of kindled animals. Key findings In acute study, Aloe vera leaf (extract) powder in a dose-dependent manner significantly decreased duration of tonic hind limb extension in maximal electroshock seizure model, increased seizure threshold current in increasing current electroshock seizure model, and increased latency to onset and decreased duration of clonic convulsion in pentylenetetrazole (PTZ) model as compared with control group. In chronic study, Aloe vera leaf (extract) powder prevented progression of kindling in PTZ-kindled mice. Aloe vera leaf (extract) powder 400 mg/kg p.o. also reduced brain levels of MDA and increased GSH levels as compared to the PTZ-kindled non-treated group. Conclusions The results of study showed that Aloe vera leaf (extract) powder possessed significant anticonvulsant and anti-oxidant activity.
Introduction The current challenge in anti-epileptic therapy is need for drugs that can prevent or retard the underlying neuronal damage and at same time have less side effects such as sedation, ataxia and cognitive impairment.[1] Aloe vera (taxonomically called as A. barbadensis) has been used in herbal medicine in many cultures since ages. As a herbal remedy, A. vera whole-leaf extract is advertised for detoxification; it is claimed to cure constipation, help flush out toxins and wastes from the body, promote digestion, and reduce the risk of illnesses.[2] Experimentally, it has been demonstrated to have antidiabetic, antiinflammatory, anti-oxidant, immunomodulatory, antineoplastic and neuroprotective properties.[3] A. vera contains various potentially active constituents like aloin, aloeemodin, vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids and amino acids.[4] Many studies of the immunological effects of extracts from plants of
Aloe have focused on the clear mesophyll gel of the A. vera leaf and on its major storage carbohydrate, acetylated mannan and acemannan.[5] Acemannan is known to have diverse biological activity, including immunomodulatory and antitumor attributes. The anticancer biological mechanism of acemannan may be exerted through pluripotent effector cells, such as macrophages, as Aloe extracts are known to induce macrophage activating activity.[6] In addition, A. vera has been demonstrated to have antidiabetic and anti-inflammatory properties.[7,8] The anti-oxidant effect of A. vera has been proved to be beneficial in diabetes9 and cancer.[10] One potential area where effect of A. vera has not been explored is neurological disorders such as epilepsy. There is some evidence in literature on the effect of A. vera on neuronal activity. Bouthet and coworkers have demonstrated that A. vera increases proliferation in cultured ‘neuron-like’ rat adrenal pheochromo-
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cytoma cells treated in suspension.[11] Another study provides evidence of direct concentration-dependent depressive effect of A. vera on the neurotransmission process with respect to presynaptic and postsynaptic mechanisms.[12] Moreover, a recent study has reported that A. vera improves anti-oxidant activity within the hippocampus and cerebral cortex leading to improvement of motor and memory behavioural tasks in diabetic mice.[13] In view of the earlier evidence on effect of A. vera on hippocampus and presynaptic and postsynaptic mechanisms, the fact that generation of seizures involve changes in synaptic transmission of excitatory and inhibitory neurotransmitters across the synapses in cerebral cortex, hippocampus and thalamus,[14] along with the learning and memory loss because of increased oxidative stress seen with both epilepsy and some anti-epileptic drugs (AEDs) in patients with chronic epilepsy, supplements the available evidence on anti-oxidant effect of A. vera. In addition, some AEDs like lamotrigine have been shown to have beneficial anti-epileptic and anti-oxidant effect in epilepsy.[15] Therefore, this study was done to find out the effect of A. vera in various epileptogenesis models and the confirmation of its beneficial anti-oxidant effect.
Materials and Methods Animals Healthy Swiss albino inbred mice of either sex weighing 25–30 g were used. The inbred strain was used, as the data obtained in inbred animals are more consistent and reproducible. The animals were procured from the Central Animal House of University College of Medical Sciences. Animals were housed in groups of six mice per cage (43 × 28.6 × 15.5 cm) with free access to pellet diet and water ad libitum. Care of the animals was taken as per ‘Committee for the Purpose of Control and Supervision of Experiments on Animals’, India, and the study was approved by Institutional Ethics Committee-Animal Research, UCMS (IEC-AR/11/2008, dated 26 September 2008). The recommendations from the Declaration of Helsinki and the internationally accepted principles in the care and use of experimental animals had been adhered to during the study. Adequate measures were taken to avoid any pain or discomfort to the animals during handling or experimentation. The animals were divided into treatment and control group by simple randomisation, and all experiments were performed between 9:30 am and 6:30 pm in the neuropharmacology laboratory of the department.
Preparation of Aloe vera leaf extract A. vera leaf extract was obtained from M/s Indo World Trading Corporation, New Delhi, India (Batch No.: IWTC/ 478
711/9432). As per the literature provided by the manufacturer, the gel obtained from A. vera leaf was mixed with double distilled water in the ratio 1 : 1, mechanically shaken at room temperature, concentrated in the evaporator followed by lyophilisation to obtain a brown powder. The characterisation of a sample of the brown powder extract by the spectrophotometer (IP66 Method) revealed 3.14% aloin. The A. vera leaf gel contains about 98% water. As per gas chromatography-mass spectrometry (GC-MS), various compounds identified in A. vera leaf gel extract were phenolic acids/polyphenols such as phenol (14.32 ppm), vanillic acid (58.6 ppm), homovanillic acid (18.55 ppm), protocatechuic (163.21 ppm), 3,4-dihydroxyphenylacetic (7.54 ppm), 5-methoxyprotocatechuic (2.5 ppm), syringic (25.54 ppm), sinapic (32.68 ppm), p-coumaric (450.87 ppm), isoferulic (52.90 ppm), ferulic (88.67 ppm), Aloe-emodin (87.79 ppm), 4-phenyllactic (11.02 ppm), 4-ethylphenol (10.12 ppm), hydrocinnamic (37.50 ppm), p-salicylic (186 ppm), benzoic (870.1 ppm), hydro-p-coumaric (15.31 ppm), alcohols such as 2-butanol (13.65 ppm), glycerol (340.9 ppm), phenylethanol (86.56 ppm), aldehydes such as benzaldehyde (56.34 ppm), m-tolualdehyde (18.21 ppm), organic acids such as lactic (148 ppm), glycolic (93.1 ppm), furoic (57.43 ppm), succinic (383 ppm), 2-methylsuccinic (62.1 ppm), malic (46.7 ppm), tartaric (18.3 ppm), isonicotinic (40.21 ppm), alkanes such as 1,3dihydroxybutane (10.22 ppm), pyrimidines such as uracil (697.23 ppm), thymine (429.76 ppm), indoles such as indole3-acetic acid (2.80 ppm), alkaloids such as hypoxanthine (27.65 ppm), ketones such as acetophenone (8.02 ppm), sterols such as cholestanol (24.32 ppm), β-sitosterol (1604.5 ppm), dicarboxylic acids such as azelaic acid (0.02 ppm) and undecanedioic acid (0.04 ppm).[16] For the purpose of study, A. vera leaf extract powder was dissolved in normal saline to prepare suspensions of 100, 200 and 400 mg/kg. The drug solutions were prepared freshly each time and administered intragastrically. The dosing schedule used was once per day. All drugs were administered in a volume of 10 ml/kg/dose, and the animals were tested 1 h after oral drug administration. The animals were divided into following treatment groups in acute study.
Acute study Group 1 was control group and administered physiological saline Group 2 was given A. vera 100 mg/kg p.o. Group 3 was given A. vera 200 mg/kg p.o. Group 4 was given A. vera 400 mg/kg p.o. Phenytoin (25 mg/kg i.p.) and diazepam (5 mg/kg i.p.) were kept as standard groups in maximal electroshock seizure (MES) and pentylenetetrazole (PTZ) model, respectively.
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Epileptogenesis models in acute study Maximal electroshock seizure test The MES test was performed as described by Swinyard et al.[17] Electroconvulsion was produced using electroconvulsiometer. An alternating current (50 mA, 0.2-s duration) was delivered via ear-clip electrodes, and duration of tonic hind limb extension (THLE) was measured for comparison in between the groups. Increasing current electroshock seizure test The ICES test, as proposed by Kitano et al.[18] was used to evaluate the effect of A. vera on convulsions. Starting with a current of 2 mA, electroshock was delivered to each mouse via ear-clip electrodes as a single train of pulses (for 0.2 s) with linearly increasing intensity of 2 mA/2 s. The current at which THLE occurred was recorded as the seizure threshold current. If no THLE was observed up to a current of 30 mA, electroshock was terminated, and this cut-off current was used in the analysis. Pentylenetetrazole-induced convulsions PTZ is a powerful central nervous system stimulant believed to be acting by GABAergic inhibition.[19] The administration of the compound causes stimulation of the cortex leading to tonic-clonic convulsions. Injection of PTZ in a dose of 60 mg/kg was given subcutaneously in acute study, and the latency to and duration of clonic convulsions were recorded for a period of 30 min.
Epileptogenesis model in chronic study Pentylenetetrazole-induced kindling Repeated subconvulsive dose of PTZ once every 48 h for 4–6 weeks is known to induce a permanent change in the epileptogenic sensitivity of brain such that initial subconvulsive stimuli become capable of evoking fully developed epilepsy.[20] This model is clearly implicated in development of chronic epileptogenesis as in humans and has strong predictive value. One of the proposed mechanisms of human epilepsy is the initial sensitisation of neuronal cells because of some extraneous or genetic insult that in turn progresses to involve more neuronal cells and ultimately leads to full-blown tonic-colonic seizures (equivalent to kindling in animals).[14] Chemical kindling is the development of seizures following repeated administration of a convulsant substance at a dose that is insufficient to produce convulsions following a single administration. Kindling produced by PTZ in mice has characteristics very similar to those described for electrical kindling, that is, the response to PTZ is proportional to the
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stimulus intensity. The PTZ kindling model is generally used as a laboratory model of human partial complex epilepsy.[21] In addition, kindling is a model of epilepsy that has the advantages of both an epileptogenic and a spontaneous seizure model.[21] Because spontaneity and recurrence of seizures are the basic features of human epilepsy, chronic models like kindling are advantageous over acute models.[21] The inhibition of such tonic-clonic seizures (induced by subconvulsive dose of PTZ) by diazepam, carbamazepine and various other AEDs is a standard model for testing the drugs effective in chronic epilepsy.[15,21] The development of epileptogenesis in PTZ was given in a dose of 35 mg/kg intraperitoneally on alternate days at the same time (thrice a week), and the animals were observed for appearance of seizure activity. The evaluation of seizure activity was done as follows: Score 0 – No change Score 1 – Hyperactivity, restlessness, vibrissae twitching Score 2 – Head nodding, head clonus, myoclonic jerks Score 3 – Unilateral or bilateral limb clonus Score 4 – Forelimb clonic seizures Score 5 – Generalised clonic seizures with loss of righting reflex and whole body extension. When the animal had seizure score of 4 at three consecutive injections of PTZ at 72-h interval, animal was taken as kindled, and treatment was discontinued. To study the effect of A. vera on development of kindling, animals were divided in two groups as follows: Group 1: received PTZ (35 mg/kg i.p.) alone on alternate days (three times a week) Group 2: treated with A. vera (400 mg/kg p.o. daily) along with PTZ (three times a week). In another set of experiment, to find out latency to and duration of clonic convulsions in PTZ-kindled animals, they were subdivided in another two groups (n = 10). Group 1: were treated with PTZ (35 mg/kg i.p.) Group 2: were treated with A. vera (400 mg/kg p.o.) along with PTZ (35 mg/kg i.p.) Assessment of oxidative stress Assessment of oxidative stress was done in kindled mice by measuring the levels of malondialdehyde (MDA) and reduced glutathione (GSH) in brain. MDA: this (indicator of lipid peroxidation) was estimated as described by Ohkawa et al.[22] GSH: this was estimated by the method described by Ellman.[23]
Statistical analysis The data were expressed as mean ± standard error of mean, and results were analysed by one-way analysis of variance followed by Tukey’s t test in acute study and measurement
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of oxidative stress. Wilcoxon signed-rank test and Freidman test followed by Mann–Whitney test for intergroup comparison, and unpaired t test were used for analysing data obtained from chronic study at significance level 5%.
treated group, seizure threshold current was increased significantly to 26.0 ± 0.54 mA (P < 0.001) as compared with the control group (Table 1). Effect of Aloe vera on pentylenetetrazole-induced latency and duration of clonic convulsion
Results Acute study Effect of Aloe vera on maximal electroshock seizure test A. vera in the dose of 100 mg/kg did not significantly modify the duration of THLE as compared with the control group. However, administration of A. vera in doses of 200 and 400 mg/kg significantly (P < 0.001) decreased the duration of THLE to 10.45 ± 0.73 s and 7.02 ± 0.32 s, respectively, as compared with the control group. None of the mice in the phenytoin group showed THLE (Table 1). Effect of Aloe vera on increasing current electroshock seizure test
Chronic study
The seizure threshold current in the control group was 13.50 ± 0.73 mA that was not significantly increased by A. vera 100 mg/kg (15.25 ± 0.53 mA). However, A. vera in doses of 200 and 400 mg/kg significantly increased the seizure threshold current, respectively. In the phenytoin
Table 1
A. vera in a dose of 100 mg/kg did not significantly increase the latency to onset of clonic convulsions. While A. vera in doses of 200 and 400 mg/kg significantly increased the latency to onset of clonic convulsion (P < 0.001 and P < 0.001, respectively) as compared with the control group. Diazepam exhibited complete protection against clonic convulsions. The duration of clonic convulsions in A. vera 100 mg/kg treated group was not significantly decreased as compared with the control group. In A. vera 200 mg/kg group, the duration was significantly (P < 0.05) reduced to 15.13 ± 0.30 s, while in Aloe vera 400 mg/kg group, it was reduced to 5.50 ± 1.69 s (P < 0.001) as compared with the control group (Table 2).
Effect of Aloe vera on development of pentylenetetrazole-induced kindling measured by kindling score A. vera treatment of 400 mg/kg/day p.o. along with PTZ 35 mg/kg intraperitoneally on alternate days had significant
Effect of Aloe vera on maximal electroshock seizure test and increasing current electroshock seizure test in mice
Group
Treatment
Duration of tonic hind limb extension (s)
Seizure threshold current (mA)
Control (Group1) Aloe vera (Group 2) Aloe vera (Group 3) Aloe vera (Group 4) Phenytoin (Group 5)
Normal saline (10 ml/kg) 100 mg/kg p.o. 200 mg/kg p.o. 400 mg/kg p.o. 25 mg/kg i.p.
14.54 ± 0.37 13.14 ± 0.55 10.45 ± 0.73a 7.02 ± 0.32a 00 ± 00a,b
13.50 ± 0.73 15.25 ± 0.53 16.25 ± 0.45c 18.0 ± 0.54d 26.0 ± 0.54d,e
Values are expressed as mean ± standard error of the mean, n = 10 mice in each group. aP < 0.001 as compared with the control group. bP < 0.001 as compared with the Aloe vera 400 mg/kg group. cP < 0.05 as compared with the control group. dP < 0.001 as compared with the control group. e P < 0.001 as compared with the Aloe vera 400 mg/kg group.
Table 2
Effect of Aloe vera on pentylenetetrazole (60 mg/kg s.c.) induced latency and duration to onset of clonic convulsions in mice
Group
Treatment
Latency to onset of clonic convulsion (s)
Duration of clonic convulsions (s)
Control (normal saline) (Group 1) Aloe vera (Group 2) Aloe vera (Group 3) Aloe vera (Group 4) Diazepam (Group 5)
10 ml/kg p.o. 100 mg/kg p.o. 200 mg/kg p.o. 400 mg/kg p.o. 5 mg/kg i.p.
642.38 ± 27.09 683.75 ± 18.61 874.88 ± 17.26a 1083.75 ± 31.37b 0.0 ± 0.0b
19.50 ± 0.73 18.25 ± 0.59 15.13 ± 0.30c 5.50 ± 1.69d 0.0 ± 0.0d,e
Values are expressed as mean ± standard error of the mean, n = 10 mice in each group. aP < 0.05 as compared with the control group. bP < 0.001 as compared with the control group. cP < 0.05 as compared with the control group. dP < 0.001 as compared with the control group. eP < 0.001 as compared with the Aloe vera 400 mg/kg group.
480
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Anticonvulsant effect of Aloe vera in mice
6
--- PTZ alone --- Aloe vera + PTZ
Mean seizure score
5
4
3 *
* 2 *
*
*
*
*
*
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0 1
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8
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12
15 17 Days
19
22
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26
29
31
Figure 1 Effect of chronic administration of Aloe vera (400 mg/kg p.o.) on pentylenetetrazole-induced kindling score. *P = 0.000 at week 2, week 3 and week 4 as compared with pentylenetetrazole group as per Mann–Whitney U test.
Table 3
Effect of Aloe vera on development of pentylenetetrazole (PTZ)-induced kindling measured by kindling score
Group PTZ PTZ + Aloe vera
Treatment 35 mg/kg i.p. 400 mg/kg p.o. + 35 mg/kg i.p.
Week 1
Week 2
Week 3
Z = −1.831
Z = −2.812 , Z = −3.162b, Z = −4.061c a
Week 4
Z = −2.821 Z = −3.162b, Z = −4.059c a
Z = −2.807a, χ2 = 30d Z = −3.162b, Z = −4.065c, χ2 = 30d
P= 0.005 at week 2, week 3 and week 4 as per Wilcoxon signed-rank test. bP=0.002 at week 2, week 3 and week 4 as per Wilcoxon signed-rank test. cP=0.000 at week 2, week 3 and week 4 as compared with PTZ group as per Mann–Whitney U test. dP=0.000 at week 4 as compared with week 1 as per Freidman test.
a
protective effect on the development of kindling. There was a significant increase in the kindling score in the PTZkindled nontreated group at week 4 compared with week 1 (χ2 = 30, P = 0.000), while the kindling score also showed a similar trend in A. Vera-treated PTZ-kindled group (χ2 = 30, P = 0.000) as per Freidman test for intragroup comparison (Figure 1, Table 3). Comparative analysis of the kindling score within PTZ-kindled non-treated group at week 1, week 2, week 3 and week 4 also showed a significant increase in the kindling score with increasing time (P = 0.005 at week 2, week 3 and week 4) as per Wilcoxon signed-rank test with similar trends appearing in the A. vera-treated PTZ-kindled group (Figure 1, Table 3). The kindling score in A. vera-treated PTZ-kindled group at end of second, third and fourth week was significantly different (P = 0.000, z = −4.061 at 2 weeks; P = 0.000, z = −4.059 at 3 weeks; and P = 0.000, z = −4.065) than the kindling score in PTZ-kindled nontreated group as per Mann–Whitney U test for intergroup comparison (Figure 1, Table 3).
Effect of Aloe vera on the latency to onset and duration of clonic convulsions in pentylenetetrazole-kindled mice Aloe vera (400 mg/kg p.o.) significantly (P < 0.05) increased the latency to onset of clonic convulsions in PTZ-kindled mice as compared with the kindled mice that received PTZ alone. A. vera significantly (P < 0.05) decreased the duration of clonic convulsions in PTZ-kindled mice as compared with the kindled mice that received PTZ alone (Table 4). Effect of Aloe vera on brain levels of malondialdehyde (nmol/g wet brain tissue) in pentylenetetrazole-kindled mice There was significant (P < 0.001) increase in the brain MDA levels of PTZ-kindled group as compared with the control group. The MDA levels in the A. vera-treated kindled group were significantly (P < 0.001) lower as compared with the PTZ group (Table 5).
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 477–485
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Table 4
Naveen Rathor et al.
Effect of Aloe vera on the latency and duration to onset of clonic convulsion in the pentylenetetrazole (PTZ)-kindled mice
Group
Treatment
Latency to onset of clonic convulsion (s)
Duration of clonic convulsion (s)
PTZ Aloe vera + PTZ
35 mg/kg i.p. 400 mg/kg p.o. + 35 mg/kg i.p.
355.30 ± 24.73 453.90 ± 25.18a
19.10 ± 1.04 13.80 ± 1.63b
Values are mean ± standard error of the mean, n = 10 mice in each group. aP < 0.05 as compared with the PTZ group. bP < 0.05 as compared with the PTZ group.
Table 5
Effect of Aloe vera on brain levels of MDA and glutathione in PTZ-kindled mice
Group
Treatment
MDA (nmol/g wet brain tissue)
GSH (μg/g wet brain tissue)
Control PTZ Aloe vera + PTZ
Normal saline (10 ml/kg) 35 mg/kg i.p. 400 mg/kg/day p.o. + 35 mg/kg i.p.
231.70 ± 12.77 430.60 ± 11.80a 301.90 ± 14.86b,c
218.30 ± 6.92 109.70 ± 7.25d 177.60 ± 7.20e,f
GSH, glutathione; MDA, malondialdehyde; PTZ, pentylenetetrazole.Values are expressed as mean ± standard error of the mean, n = 10 mice in each group. aP < 0.001as compared with the control group. bP < 0.05 as compared with the control group. cP < 0.001as compared with the PTZ group. d P < 0.001 as compared with control group. eP < 0.05 as compared with the control group. fP < 0.001as compared with the PTZ group.
Table 6
Effect of Aloe vera on cyclooxygenase-inhibitory activity in formalin-induced inflammatory response
Group
Treatment
Duration of licks/bites in early phase (s)
Duration of licks/bites in late phase (s)
Control Aloe vera Aloe vera
Normal saline, 200 mg/kg p.o. 200 mg/kg p.o. 400 mg/kg p.o.
52.43 ± 4.12 51.25 ± 4.24 51.14 ± 4.30
52.43 ± 4.12 35.18 ± 5.15a 18.19 ± 4.17b
Values are expressed as mean ± standard error of the mean, n = 10 mice in each group. aP < 0.05 as compared with the control group. bP < 0.001 as compared with the control group.
Effect of Aloe vera on brain levels of reduced glutathione in pentylenetetrazole-kindled mice There was significant (P < 0.001) decrease in the brain GSH levels of the PTZ-kindled group as compared with the control group. The GSH levels in the A. vera-treated kindled group were significantly (P < 0.001) increased as compared with the PTZ group (Table 5). Effect of Aloe vera on cyclooxygenase-inhibitory activity in formalin-induced inflammatory response The effect of A. vera leaf extract powder was also studied on cyclooxygenase (COX)-inhibitory activity in formalininduced inflammatory response. Results showed that doses of 200 and 400 mg/kg significantly decreased the duration of lick/bite responses in late phase from 48 s in nontreated group to 18 s in 400 mg/kg A. vera group and 35 s in 200 mg/kg A. vera group. (Table 6)
Discussion In this study, efforts were made to investigate the antiepileptic potential of A. vera using both acute and chronic 482
animal models of epilepsy. The results of this study showed that A. vera significantly decreased the duration of THLE in MES (grand mal) model and significantly increased the seizure threshold current at which THLE occurred in ICES (grand mal) model of convulsion in a dose-dependent manner. These results were similar to previous studies showing anti-epileptic activity of phenytoin (standard drug) in the earlier models.[18,24] Anticonvulsant activity was also assessed by PTZ-induced acute convulsive model. A. vera-treated group showed an increase in the latency to onset and decrease in duration of convulsions as compared with the control group. These results were similar to the effect of phenytoin, diazepam and other AEDs in PTZinduced acute convulsions.[25] A. vera has been shown to inhibit NMDA-induced retinal ganglionic cell apoptosis.[26] Considering the role of NMDA in epilepsy and its modulation by lamotrigine, the beneficial effect of A. vera in epileptic models could be explained by its property of modulating similar NMDA receptors in brain cells. However, this needs to be confirmed in follow-up studies. In the model of chemical kindling (chronic group), effect of A. vera was studied on development and expression of convulsions in PTZ-kindled mice. The process of epileptogenesis and longterm use of certain anti-epileptic drugs has been shown to
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cause increase in reactive oxygen species (ROS) that leads to oxidative stress and neuronal damage in patients with epilepsy.[27] So, parameters of oxidative stress like MDA and GSH were also included to find out the effect of A. vera in modulating oxidative activity in epileptic animals. In addition considering the published evidence of A. vera leaf extract on COX inhibition, an inflammatory model of chronic pain (formalin induced) was also evaluated for COX-inhibitory activity of A. vera leaf extract in various doses. Dose of 400 mg/kg of A. vera was chosen in kindling for chronic administration as this dose demonstrated maximum absolute increase in seizure threshold in acute studies. A. vera treatment of 400 mg/kg/day daily along with alternate day PTZ had significant protective effect on the development of kindling in mice, and there was a statistically significant decrease in the kindling scores at week 2, week 3 and week 4 along with a significant increase in the latency to onset and decrease in duration of clonic convulsions as compared with PTZ-kindled nontreated group. These results were similar to the effect of phenytoin, carbamazepine and other AEDs in PTZ-induced kindling model.[17] A. vera has also been shown to inhibit COX enzyme. The results of our study also showed that doses of 200 and 400 mg/kg of A. vera leaf extract powder significantly decreased the duration of lick/bite responses in late phase from 48 s in nontreated group to 18 s in 400 mg/kg A. vera-treated group and 35 s in 200 mg/kg A. vera-treated group. A radical scavenging glycoprotein from A. vera gel that inhibits COX-2 and thromboxane A2 synthase has been isolated.[28,29] Various studies have shown that COX inhibitors have got protective effect in electroshock-induced and PTZ-induced convulsion in animal models, so anticonvulsant property of A. vera could also be explained by its COX enzyme inhibiting potential.[30–32] However, this needs to be confirmed in future studies using suitable controls. The assay of MDA is often considered as an index of freeradical generation. The results of this study showed that A. vera-treated PTZ-kindled group showed significant decrease in the MDA levels as compared with the PTZkindled nontreated group. GSH is the most prevalent and important intracellular anti-oxidant. This compound is able to scavenge both singlet oxygen and hydroxyl radicals. The results of this study showed that A. vera-treated group exhibited a significant increase in GSH levels in mice brain as compared with PTZ-kindled nontreated group, suggesting an anti-oxidant effect of A. vera. The process of epileptogenesis and long-term use of certain AEDs has been shown in previous studies to cause increase in ROS leading to oxidative stress and neuronal damage in patients with epilepsy.[33] There is evidence for increased lipid peroxidation (index for MDA) in PTZ-induced kindling in rats,[34] and certain
Anticonvulsant effect of Aloe vera in mice
AEDs like carbamazepine themselves have been shown to cause increase in oxidative stress by reducing the levels of GSH.[27,34] On the other hand, lamotrigine has shown antioxidative potential in some previous studies.[35] Our results were similar to anti-oxidant effect of lamotrigine in similar epileptic animal models.[17] In accordance with existing literature that COX enzyme leads to formation of hydroxyl free-radical and peroxynitrite free-radical owing to peroxidative activity[36,37] and considering the results of some previous studies showing anti-oxidant effect of A. vera[7] and inhibition of cyclooxgenase enzyme by it[28] could also explain the anti-oxidative mechanism of A. vera. Besides this, several low-MW compounds present in aloe gel have been shown to be capable of inhibiting the release of reactive oxygen free-radicals from activated human neutrophils.[38] Hence, anti-oxidant property of A. vera could prevent the neuronal damage in epileptic patients that itself is partly due to increased oxidative stress. Previous reports of HPLC analysis on A. vera leaf extract have identified two diastereomers of Aloin, that is, Aloin A and Aloin B along with Aloe-emodin. Aloe-emodin itself is derived from oxidation of Aloin. The levels of Aloin B at 1.335 ppm were found to be higher than Aloin A at 0.868 ppm that were higher than levels of Aloin-emodin at 0.200 ppm.[39] Further structural elucidation studies of these chromones identified them as 8-C-glycosyl-7-O methyl-(S) aloesol, isoaloeresin D and aloeresin E by reverse phase HPLC and headspace GC-MS.[40] Because the active constituent of A. vera leaf extract in our study was Aloin (3.4%) and considering the previous evidence on beneficial effects of aloin in inflammation, diabetes and infections,[7,8,38] both the anti-epileptic and anti-oxidant activity could be attributed to aloin; however, this needs to be followed up vigorously in future studies.
Conclusion Considering the significant anticonvulsant (in acute models), anti-epileptic (chronic model) and anti-oxidative effect of A. vera leaf powder extract, the results of our study provided the evidence that it could be used as an alternative to AEDs in patients of both grand mal and absence epilepsy, and might prevent the neuronal damage in such patients. In addition, because the leaf powder extract was derived from the lyophilisation of gel (central part), other parts of A. vera leaf (peripheral layer) need to be studied for their antiepileptic effects in future studies.
Declarations Acknowledgements The Authors would like to thank M/s Indo World Trading Corporation, New Delhi for providing A. vera leaf extract
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powder and the technical and animal house staff of Department of Pharmacology for providing the animals and material support.
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