|Year : 2014 | Volume
| Issue : 3 | Page : 170-176
Protection of zonisamide induced memory impairment by tulsi extract and piracetam on mice
Shraddha J Bennadi, KL Krishna
Department of Pharmacology, Jagadguru Sri Shivarathreeswara College of Pharmacy, Jagadguru Sri Shivarathreeswara University, Mysore, Karnataka, India
|Date of Web Publication||13-Aug-2014|
K L Krishna
Department of Pharmacology, Jagadguru Sri Shivarathreeswara College of Pharmacy, Jagadguru Sri Shivarathreeswara University, Sri Shivarathreeshwara Nagar, Mysore - 570 015, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Memory impairment is the major adverse effects associated with antiepileptic drug therapy. This study was designed to assess the memory impairment activity of zonisamide (ZNS), an antiepileptic drug, in mice. Memory deficit potential of ZNS was compared with phenytoin (PHT), a standard antiepileptic known for its memory impairment activity. The protective effect of Ocimum sanctum extract (OS) and piracetam (PIR) on memory impairment induced by ZNS was also assessed. Materials and Methods: ZNS was administered orally for 29 days and the extent of memory deficit was evaluated by Morris water maze (MWM) test on maximal electro shock-induced epileptic mice. The animals were observed for escape latency time (ELT) and time spent in target quadrant (TSTQ) on MWM test. The brain acetylcholinesterase level was estimated to determine the brain acetylcholine concentration. Result: Chronic administration of ZNS has shown memory deficit in mice and this was significantly restored by co-administration of OS extract and PIR. PIR showed best nootropic activity, whereas OS showed good nootropic as well as synergistic anti-convulsant activity. Conclusion: This study reveals that chronic administration of ZNS produces memory impairment in mice, which can be significantly minimized by co-administration of OS extract and PIR without compromising on ZNS antiepileptic potency. These results provide evidence for potential corrective effect of nootropics in cognitive deficit associated with ZNS.
Keywords: Antiepileptic, cognition impairment, Ocimum sanctum, piracetam,zonisamide
|How to cite this article:|
Bennadi SJ, Krishna K L. Protection of zonisamide induced memory impairment by tulsi extract and piracetam on mice. Int J Health Allied Sci 2014;3:170-6
|How to cite this URL:|
Bennadi SJ, Krishna K L. Protection of zonisamide induced memory impairment by tulsi extract and piracetam on mice. Int J Health Allied Sci [serial online] 2014 [cited 2019 Sep 21];3:170-6. Available from: http://www.ijhas.in/text.asp?2014/3/3/170/138600
| Introduction|| |
Impaired memory is among the most common complaints of patients with epilepsy, and multiple factors contribute to memory impairment. Although the causes of cognitive impairment in patients with epilepsy have not been completely elucidated, three factors are clearly involved; the underlying etiology of epilepsy, the effects of seizures themselves and the central nervous system effects of antiepileptic drugs.  For an optimum antiepileptic therapy, it is desirable to have complete seizure control without interfering cognitive effects. Since the treatment for epilepsy lasts for a long period, it is worth evaluating the effect of nootropics included in the antiepileptic regimen as an add-on therapy to exert its protective effect on cognitive impairment. Nootropics represents a class of psychotropic agent, which has facilitatory effect on intellectual performance, learning capability and memory. The underlying mechanism of nootropic agent can be attributed to its anti-acetylcholinesterase (AChE) property, metabolic enhancement, anti-inflammatory, vasodilatation and antioxidant activity. 
Zonisamide (ZNS) is a synthetic 1,2-benzisoxazole- 3-methanesulfonamide with anticonvulsant properties. The sulfamoyl group on ZNS was expected to suppress seizures in a manner similar to another sulfonamide analog, acetazolamide, through inhibition of carbonic anhydrase. Memory deficit is one of the major adverse effects of ZNS as is that of any antiepileptic drug. 
Piracetam (PIR) is considered as an intelligence booster and central nervous system stimulator with no known toxic or addictive capacity. It enhances cognition under conditions of hypoxia and also enhances memory and some kinds of learning in normal humans. At a neuronal level, PIR modulates neurotransmission in a range of transmitter systems (including cholinergic and glutaminergic), has neuroprotective properties and improves neuroplasticity.  It has been shown to have a specific anti-amnesic activity in many experimental paradigms. ,,]
Many herbs such as Withania somnifera and Ocimum sanctum (OS)  are reported to have nootropic as well as anticonvulsant activity. It is interesting to note that OS has recently been reported to possess anticonvulsant activity and can be used as a nootropic agent in the treatment of various cognitive disorders. The active constituents of the herb include volatile oil, chiefly eugenol and β-caryophyllene, flavonoids, and a number of other components present in fixed oil.  OS is reported to possess antioxidant and antistress properties as well. Thus, combination of anti-inflammatory, antioxidant, antistress and neuroprotective role of OS could all be leading to the net memory enhancing effect. It seems that OS improved learning and memory probably because of enhanced cholinergic transmission. ,
Based on the above facts, the present study was conceived to assess the memory impairment property of ZNS using mice as experimental animals. The possible protective effect of nootropic herb, OS extract, and PIR against ZNS-induced memory impairment was also assessed using Morris water maze (MWM) task in MES-induced convulsions.
| Materials and methods|| |
Animals and experimental design
Adult Albino mice (25-30 gm) of either sex were procured from Animal House, JSS Medical College, Mysore-15, Karnataka, India. They were acclimatized for laboratory condition for 7 days and randomly divided into 5 groups of 6 animals each. The project was approved from Institutional Animal Ethical Committee of JSS College of Pharmacy, Mysore (proposal number 089/2011).
Drugs and chemicals
All chemicals used for the study were procured from various suppliers and all were analytical grade. The drugs, phenytoin (PHT), ZNS and PIR, were procured from Abbott Laboratories, India, Sun Pharmaceuticals and UCB India Pvt. Limited, India, respectively.
OS extract (tulsi dry extract) was obtained from Sri Ganesh Herbals, Bangalore India. The hydroalcoholic extract was prepared using dry leaves of OS by the manufacturer and certificate analysis was supplied with the extract. As per the certificate of analysis, the extract complies with the standard. Main active phytoconstituents present in the extract were tannins, volatile oils and fatty materials, and ursolic acid.
Preliminary phytochemical tests
The preliminary phytochemical screening was carried out on hydroalcoholic extracts of OS in order to find out the presence of various phytoconstituents. ,,
Memory impairment activity of ZNS on mice
The pretrained mice were divided in to 5 groups of 6 animals each and treatment was given for 29 days as shown in [Table 1]. For training, the mice were released into the water and allowed 60 seconds to find the platform. If a mouse was not able to find the platform after 60 seconds, it was guided by putting our finger on top of the platform. The mouse was allowed to sit there for 10 seconds. Then, the animals were placed in the heated cage for 60 seconds to warm up and the next trial was started. The mice were returned to their home cage after trials were completed. In general, animals received 3 trials per day with 5 minutes inter-trial interval for 7 days or until the performance was stable. Time to find the hidden platform was considered as escape latency time (ELT). The platform in the water maze was kept at the same position throughout the test to assess the effect on spatial reference memory, which served as an index of learning. Time spent in target quadrant (TSTQ), which is defined as the time spent in the quadrant that previously contained the hidden platform was also measured. Mean time spent in all the three quadrants i.e., Q1, Q2 and Q3 was recorded and the time spent in the target (Q4) quadrant (TSTQ) in search of the missing platform provided served as an index of retrieval.  MES was induced after 2 hours and 1 hour administration of nootropic agent and ZNS, respectively, and duration of convulsions and extent of memory deficit were noted on 8 th , 15 th , 22 nd and 29 th day of study period.
|Table 1: Treatment schedule for assessing the memory impairment activity of ZNS|
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Anticonvulsant activity of ZNS by MES method
Tonic and clonic convulsions were induced in mice by employing MES (45 mA for 0.2 s) using an electroconvulsiometer (INCO, Ambala, India) via crocodile ear clips. Different stages of convulsions: (a) flexion, (b) tonic extensor, (c) clonic convulsions, (d) stupor and (e) recovery or death were noted; time spent by the animal in each phase of the convulsions was recorded. ,
Estimation of Brain AChE Activity
On the 29 th day, the animals were euthanized by cervical dislocation carefully to avoid any injuries to the brain tissue. The whole brain AChE activity was measured using Ellman method. 
The cloudy supernatant liquid (0.5 ml) was pipetted out into a 25 ml volumetric flask and dilution was made with a freshly prepared dithiobisnitrobenzoic acid (DTNB) solution (10 mg DTNB in 100 ml of phosphate buffer, pH 8.0). Two 4 ml portions were pipetted out into 2 test tubes from the volumetric flask. Substrate solution (1 ml) (75 mg of acetylcholine iodide per 50 ml of distilled water) was pipetted out into both tubes and incubated for 10 min at 300°C. Phosphate buffer was used for zeroing the colorimeter. The resulting yellow color is due to reduction of DTNB by certain substances in the brain homogenate and due to non-enzymatic hydrolysis of substrate. After having calibrated the instrument, change in absorbance per minute of the sample was recorded at 420 nm. ,
All data are expressed as mean ± SEM and were analyzed by one-way ANOVA followed by Tukey's post-hoc test by using Graph Pad prism version 5.0 software. The differences between the groups were considered significant if P < 0.05.
| Results|| |
Preliminary phytochemical and antioxidant activity of OS extract
Preliminary phytochemical analysis of hydroalcoholic extract of OS revealed the presence of phytochemicals such as alkaloids, flavonoids, triterpenoids, tannins, volatile oils, carbohydrates and saponins.
Memory impairment activity of ZNS
When ZNS was administered orally for 29 days on mice with MES-induced convulsions, the extent of memory deficit was increased as the treatment increases; ELT on 8 th day was found to be 33.47 ± 3.93 seconds, whereas on 29 th day it was increased to 41.80 ± 0.93 seconds. The ELTs on 15 th day and 22 nd day were found to be 34.06 ± 2.15 and 38.34 ± 5.69 seconds, respectively; the ELT was significantly increased when compared to the basal reading (21.25 ± 2.16 s). The same quantum of memory impairment was produced by PHT, whereas the maximum increase in ELT was found to be 41.72 ± 1.06 seconds on 29 th day and the increase was significant when compared to the basal reading of 20.85 ± 0.52 seconds. It was observed that co-administration of OS extract at a dose 200 mg/kg body weight with ZNS in MES-induced convulsions resulted in a significant decrease of ELT; on 8 th day, it was found to be 21.41 ± 1.43 seconds, whereas on 29 th day it was further decreased effectively to 7.81 ± 0.5 seconds. ELTs on 15 th and 22 nd days were found to be 17.95 ± 0.87 and 13.73 ± 0.65 seconds, respectively, when compared to the basal reading of 22.04 ± 3.45 seconds. PIR, which is known for its nootropic activity, also showed a significant decrease in ELT when co-administered with ZNS; on 8 th day, it was found to be 19.48 ± 2.03 seconds, whereas on 29 th day it was significantly decreased to 9.82 ± 1.33 seconds. ELTs on 15 th and 22 nd days were found to be less when compared to the basal reading. The extent of prevention of memory deficit (produced by ZNS) was same by both nootropic tested and both reversed the ZNS-induced memory impairment in mice [Table 2].
|Table 2: Effect of herbal nootropic OS and PIR on ZNS-induced memory deficit in mice (escape latency time in seconds)|
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TSTQ was another parameter used for evaluation of memory deficit property of ZNS. It was observed that chronic administration of ZNS in mice for 29 days with MES-induced convulsions resulted in a significantly decreased TSTQ from 17.19 ± 0.96 seconds on the 8 th day to 11.52 ± 1.68 seconds on the 29 th day, which is significantly decreased when compared to the basal reading (33.16 ± 1.32 s), whereas TSTQs on 15 th and 22 nd days were found to be 14.63 ± 0.94 and 13.58 ± 0.99 seconds, respectively. Memory impairment of ZNS was found to be very much similar to PHT, which showed a decrease in the TSTQ from 23.49 ± 1.03 seconds on 8 th day to 11.47 ± 1.08 seconds on 29 th day, which is significant compared to the basal reading (32.15 ± 1.25 s). The co-administration of OS extract with ZNS resulted in a significant improvement in TSTQ on 29 th day when compared to the ZNS alone-treated group. TSTQs on 15 th and 22 nd days were found to be improving (30.53 ± 0.24 and 32.37 ± 3.06 s, respectively), which is a significant improvement as compared to the basal reading of 33.69 ± 2.37 seconds. The above results show that the OS extract has potent memory improving activity on ZNS-induced memory deficit, which is similar to that produced by well-known nootropic, PIR. Co-administration of PIR in combination with ZNS resulted in a significant increase in TSTQ (35.18 ± 0.90 s) on the 29 th day of treatment from 30.15 ± 1.11 seconds on the 8 th day as compared to the ZNS-treated group. These results demonstrate the memory deficit property of ZNS and corrective potential of OS extract and PIR when tested chronically on mice [Figure 1].
|Figure 1: Effect of herbal nootropic-OS and PIR on ZNS induced memory deficit in mice (time spent in target quadrant in seconds). *Significant when compared with normal animals (P < 0.05). #Significant when compared with ZNS-treated animals (P < 0.05)|
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Estimation of brain AChE level
It was observed that chronic administration of ZNS with MES-induced convulsions in mice resulted in a significant increase of AChE (140.34 ± 1.91 μmoles) as compared to the normal group (81.47 ± 1.14 μmoles). The level of AChE in PHT-treated animals was also increased (191.32 ± 6.67 μmoles) significantly when compared to normal group animals. This shows both ZNS and PHT increased AChE level, which means there was a decreased level of acetylcholine (responsible for the memory) in ZNS- and PHT-treated animals. When OS extract was co-administered with ZNS, a significantly decreased AChE level (92.21 ± 0.26 μmoles) was seen as compared to the ZNS-treated group. The same was observed when PIR was co-administered with ZNS and activity was found to be significant [Figure 2].
|Figure 2: Effect of herbal nootropic-OS and PIR on ZNS induced memory deficit in mice (Brain AChE level in μmoles) *Significant when compared with normal animals (P < 0.05) #Significant when compared with ZNS-treated animals (P < 0.05)|
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This study clearly shows that ZNS administered for 29 days produced significant memory deficit when tested on MWM by all parameters employed, whereas OS extract as well as PIR have demonstrated potent memory improving activity on ZNS-induced cognitive impairment in mice. By co-administering OS and PIR, the adverse effect produced by ZNS can be minimized to a greater extent without compromising on its antiepileptic potency (discussed below).
Anticonvulsant activity of ZNS on MES method
The anticonvulsant potency of ZNS in presence and absence of OS and PIR was evaluated at different day intervals by MES-induced convulsions on mice. Flexion, extension, clonus, stupor, death or recovery was evaluated in MES-induced convulsion [Table 3]. When ZNS was administered orally, it showed significant protection in extension phase and decreased the duration of flexion, clonus and stupor phases as compared to normal group animals. It was also observed that when OS extract was co-administered along with ZNS, it resulted in 100% protection in extension phase (0.00 ± 0.00) and decreased duration of flexion (1.90 ± 0.32 s), (clonus 7.06 ± 0.35 s) and stupor (16.56 ± 2.68 s), which is significant when compared to the normal group. The results show that when OS extract was co-administered with ZNS, it produced synergistic effect in extension phase and reduced the duration of flexion, clonus and stupor when compared to ZNS only-treated mice. Similarly when PIR was co-administered along with ZNS, it did not produce potentiating effect in extension phase but reduced the duration of flexion, clonus and stupor similar to ZNS-treated group. The result clearly shows that ZNS retains its protection on MES-induced convulsion even when co-administered with nootropic herb OS and PIR; even the activity potentiated with OS extract [Table 3].
|Table 3: Anticonvulsant activity of ZNS in presence of OS and PIR on mice (MES-induced convulsions)|
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| Discussion|| |
Epilepsy is the most common disorder generally occurring in all age groups, and many patients with epilepsy suffer from memory deficit. Antiepileptic drugs are commonly associated with cognitive effects, including impaired attention, vigilance and psychomotor speed, which are frequently observed in children with epilepsy syndromes. ZNS is one of the newer anti-epileptic drugs used for the adjuvant therapy of partial seizures causing memory impairment in nearly 6% of the patients under its treatment. The exact mechanism of action of ZNS on how it causes memory impairment is unknown. The probable mechanism may be that it alters the cholinergic system by reducing the acetylcholine levels in the brain like PHT.  Many herbs have both nootropic as well as anticonvulsant activity, Ocimum sanctum Linn. is one of them, ,,, which can be used to improve memory deficit caused by anticonvulsant drugs as well as potentiate the anticonvulsant activity.
The protective effect of OS extract and PIR on ZNS-induced memory deficit was evaluated by using MWM test. The results and observations of the present study showed that when ZNS was administered for 29 days along with MES-induced convulsions [Table 1], it significantly increased the ELT by 157.5%, 160.28%, 180.42% and 196.70% and decreased TSTQ by 193.24%, 226.65%, 244.18% and 287.89% on 8 th , 15 th , 22 nd and 29 th day of treatment, respectively when compared to normal values (0 th day). The result clearly demonstrates that the ZNS adversely affected cognitive impairment in the MWM task in mice. These findings show that the ZNS treatment in mice induces memory-impairing effects. Our study supports the available literature regarding the memory impairment activity of ZNS.
In the present study, the extent of memory impairment induced by ZNS was compared with that of PHT. The data obtained clearly demonstrates that, memory impairment produced by ZNS was very much similar to PHT, an antiepileptic drug, which has been proven to produce memory impairment in animal model. 
When OS was co-administered with ZNS, it significantly reversed the ELT by 102%, 122.78%, 160.52% and 306.7% and reversed the TSTQ by 87.56%, 90.62%, 96.08% and 106.35% on 8 th , 15 th , 22 nd and 29 th day of treatment, respectively when compared to the basal values (0 th day). The same was observed when PIR was co-administered with ZNS; co-administration revealed significant reversal of the ELT by 108.31%, 117.5%, 153.67% and 293.6% and reversal of TSTQ by 91.50%, 97.57%, 102.03% and 106.76% on 8 th , 15 th , 22 nd and 29 th day of treatment, respectively when compared to the basal values (0 th day).
The extent of memory impairment produced by ZNS is compared with PHT-treated group, already proven for its memory deficit activity, which produced increased ELT by 124.9%, 169.35%, 196.4% and 200.09% and decreased TSTQ by 136.84%, 160.34%, 215.91% and 280.26% on 8 th , 15 th , 22 nd and 29 th day, respectively compared to normal readings (0 th day), produced in similar quantum as produced by ZNS-treated group [Figure 1]. PIR and OS are said to have nootropic effects on acquisition, consolidation and retention of memory in various experimental models. ,,
The central cholinergic system plays an important role in learning and memory,  so the effect of the tested drugs on brain AChE can also be ascertained in brain homogenate. ,, This study revealed that the AChE level in ZNS-treated animal was high (means level of acetylcholine is less). It was observed that administration of ZNS along with MES-induced convulsions resulted in a significantly increased AChE level (72.26%) as compared to the normal group and it was very much similar to PHT-treated animal group (134.83%) [Figure 2]. Co-administration of OS extract with ZNS has significantly decreased AChE value and similarly with PIR co-administered group animals. The precise mechanism by which PIR exerts its nootropic effect is not known. Multiple mechanisms have been suggested, such as an enhancement of oxidative glycolysis, an effect on the Ca 2+ channels and an effect on the cholinergic system. , The nootropic effect of OS extract may be attributed to the antioxidant property. 
Some recent studies report the anticonvulsant effects of OS.  These effects were observed at a dose of 200 mg/kg required to produce nootropic activity. In the present study, we found the potentiating effects in OS against MES-induced convulsions as it has inhibited the extensor duration by 100%, reduced clonus duration by 77.83% and stupor duration by 70.05% when compared to ZNS alone-treated mice. PIR was less effective and its antiepileptic activity was very much similar to ZNS alone-treated animals [Table 3]. ZNS retained its antiepileptic activity when co-administered with PIR while activity was potentiated with OS extract. This study clearly demonstrates the protective effect and synergistic effect of OS on ZNS for grand mal type of epilepsy on which ZNS alone shows less significant antiepileptic activity.
The exact mechanism of action by which OS elicits its nootropic effects is not known but studies state that plant's antioxidant properties contribute favorably to the memory enhancement effect, and beneficial effect on learning and memory may be because of the facilitation of cholinergic transmission in brain. However, further studies are necessitated to identify the exact mechanism of action. The preliminary phytochemical studies show that the OS extract contains flavonoids and polyphenolic compounds, which are responsible for its potent anti-oxidant activity (data not included). Correction of ZNS-induced memory deficit by OS extract may be due to its strong anti-oxidant activity and flavonoids and polyphenolic compounds present in the extract may be responsible.
| Conclusion|| |
In the present study, we conclude that ZNS produces significant memory deficit when administered orally for 29 days on mice, and memory deficit was demonstrated by ELT, TSTQ as well as brain AChE level. The extent of memory deficit was found to be similar to that of PHT. The anticonvulsant activity of ZNS when co-administered with OS extract/PIR was evaluated by MES-induced convulsion to assess any dynamic drug-drug interaction. OS extract and PIR significantly reversed the ZNS-induced memory deficit by MWM test, and ZNS retains its antiepileptic activity on co-administration. The flavonoids and polyphenolic compounds present in the OS extract possess strong anti-oxidant activity, which may be responsible for the above said activity. However, further research is required to investigate the usefulness of these nootropics in various animal models and clinical studies to explore the full potential of OS extract and PIR in correcting ZNS-induced cognitive deficits. Our study gives a platform for the further extensive research on employing nootropics for correction of antiepileptic drug-induced cognitive impairment.
| Acknowledgement|| |
The authors sincerely thank Dr. H.G. Shivakumar, Principal, JSS College of Pharmacy, Mysore, for his support and encouragement. Our gratitude goes to JSS University, Mysore, for providing all the necessary facilities.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]