Pharmacokinetic and pharmacodynamic interaction of hydroalcoholic extract of Ocimum sanctum with valproate

YEBEH-05462; No of Pages 7 Epilepsy & Behavior xxx (2017) xxx–xxx

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Pharmacokinetic and pharmacodynamic interaction of hydroalcoholic extract of Ocimum sanctum with valproate Sudhir Chandra Sarangi ⁎, Dipesh Joshi, Ritesh Kumar, Thomas Kaleekal, Yogendra Kumar Gupta Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India

a r t i c l e

i n f o

Article history: Received 22 June 2017 Revised 10 August 2017 Accepted 10 August 2017 Available online xxxx Keywords: Epilepsy Ocimum sanctum Interaction Valproate Neurobehavioral

a b s t r a c t For effective control of seizures, antiepileptic drugs (AEDs) are administered at higher dose which is associated with several adverse effects. This study envisaged antiepileptic and neuroprotective potential of Tulsi, a commonly used herb for its immunomodulatory property. The optimal dose of Ocimum sanctum hydroalcoholic extract (OSHE) was determined using maximal electroshock seizure (MES)- and pentylenetetrazol (PTZ)-induced seizure models in Wistar rats (200–250 g) after administering OSHE (200–1000 mg/kg) orally for 14 days. For interaction study, OSHE optimal dose in combination with maximum and submaximal therapeutic doses of valproate was administered for 14 days. Serum levels of valproate were estimated using HPLC for pharmacokinetic study. For pharmacodynamic interaction, antiepileptic effect on above seizure models, neurobehavioral effect using Morris water maze, passive avoidance and elevated plus maze tests, and antioxidant capacity were assessed. Ocimum sanctum hydroalcoholic extract 1000 mg/kg was found to be optimal providing 50% protection against both MES- and PTZ-induced seizures. Combination of OSHE with valproate did not alter antiepileptic efficacy of valproate significantly. However, the combination showed better memory retention potential in neurobehavioral tests and protection against oxidative stress compared with valproate-alone-treated groups. Pharmacokinetic parameters did not reveal any significant change in combination group compared with valproate alone. Ocimum, although having per se antiepileptic action, did not affect antiepileptic action of valproate in combination. However, combination treatment has an edge over valproate alone—better neurobehavioral function and reduced oxidative stress—predicting adjuvant potential of Ocimum in epilepsy treatment. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Epilepsy is one of the most prevalent noncommunicable neurologic conditions worldwide (lifetime prevalence was 7.60 per 1000 persons) [1] and also in India (10 million people, 1% of population) [2]. Despite availability of several newer AEDs, 30% of patients with epilepsy still suffer from uncontrolled seizures and many experience sudden deaths [3]. For effective control of seizure, antiepileptic drugs (AEDs) are administered alone or in combination for years together, which is associated with several adverse drug effects. Cognitive dysfunction is commonly seen with all major AEDs at therapeutic doses [4,5]. Valproate (VPA), a first-line and broad spectrum AED, is associated with adverse effects like hepatotoxicity, thrombocytopenia, gastrointestinal irritation, weight gain, transient alopecia, and neurological adverse effects including cognitive problems, ataxia, sedation, tremor, reversible parkinsonism, and dementia [6,7]. As epilepsy needs chronic treatment and VPA is a potent inhibitor of cytochrome P450 (CYP) enzyme system, there is a possibility of clinically significant drug interaction of VPA with other drugs and ⁎ Corresponding author at: Department of Pharmacology, All India Institute of Medical Sciences, New Delhi 110029, India. E-mail address: [email protected] (S.C. Sarangi).

nutritional supplements. These facts definitely put forth the need for drugs having per se or adjuvant role in suppressing epileptogenesis with the capacity for reducing cognitive impairment. In Ayurveda, Ocimum sanctum (Tulsi) (also called as Ocimum tenuiflorum) is known as ‘The Incomparable One’ and ‘The Queen of Herbs’. Daily consumption of Tulsi is said to prevent disease and stresses of daily life and promote general health, wellbeing, and longevity [8]. The anticipatory potentials of Tulsi have been enumerated as chemoprotective, antistress [9], anticonvulsant [10], anxiolytic [11], antiulcer, antidiabetic [12], analgesic, antioxidant [13], anticancer, immunomodulatory, and antiinflammatory agent [14]. There are few previous in vivo studies [10,15–17] which have thrown light upon the anticonvulsant potential of Ocimum extracts using different species (Ocimum basilicum, Ocimum gratissimum, and Ocimum sanctum) in the maximal electroshock seizure (MES) and pentylenetetrazol (PTZ) model. However, there are only few evidences comparing Ocimum with standard antiepileptic drugs; moreover, the combined effect of administering these drugs together has not been characterized in terms of seizure control, neurocognition parameters, and pharmacokinetic interaction. Thus, there is a need for thorough investigation of this widely exploited plant's potential as an antiepileptic and neurocognitive beneficial agent. This study was performed to find

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Please cite this article as: Sarangi SC, et al, Pharmacokinetic and pharmacodynamic interaction of hydroalcoholic extract of Ocimum sanctum with valproate, Epilepsy Behav (2017), http://dx.doi.org/10.1016/j.yebeh.2017.08.018

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S.C. Sarangi et al. / Epilepsy & Behavior xxx (2017) xxx–xxx

out the per se antiepileptic effect of Ocimum and its pharmacokinetic and pharmacodynamic interaction with valproate. 2. Materials and methods 2.1. Experimental design This experimental study for exploration of antiepileptic potential of Ocimum sanctum hydroalcoholic extract (OSHE) was conducted in rats using standard acute seizure models, i.e., maximal electroshock seizure (MES)- and pentylenetetrazol (PTZ)-induced seizure models. In the first phase, optimal dose of OSHE was screened using four different doses, i.e., 200, 400, 800, and 1000 mg/kg administered orally for 14 days. In the second phase, the optimal dose of OSHE from the first phase was used to determine pharmacokinetic and pharmacodynamic interaction of OSHE with valproate. 2.2. Animals Adult Wistar rats (200–250 g), obtained from the Central Animal Facility of the All India Institute of Medical Sciences, New Delhi were housed in polyacrylic cages (38 × 23 × 10 cm) under standard laboratory conditions with dark and light cycle (approximately 12 h:12 h). They had free access to standard pellet diet and tap water ad libitum. The experiment was started after getting approval from the Institutional Animal Ethics Committee (IAEC), AIIMS, New Delhi (Ethics approval no. 917/IAEC/16) and conducted in accordance with CPCSEA guidelines, Department of Animal Welfare, Government of India. 2.3. Drugs and chemicals Pentylenetetrazol and glutathione were obtained from Sigma Inc., USA. Valproate was obtained from HiMedia Laboratories Pvt. Ltd., Mumbai. Chemicals used in HPLC were of HPLC grade. All other chemicals and solvents were obtained from Merck (India) and were of analytical grade. 2.3.1. Doses of drugs The standardized Ocimum sanctum hydroalcoholic extract (OSHE) was obtained from Natural Remedies Pvt. Ltd., Bangalore, India as a gift sample. Ocimum sanctum hydroalcoholic extract phytochemical analysis by HPLC has shown combine presence of ursolic acid and oleanolic acid 2.8% w/w. Four doses of OSHE, viz. 200, 400, 800, and 1000 mg/kg were used for the study. A freshly prepared suspension was made in distilled water before administering OSHE orally daily for 14 days. Two doses of valproate were used. One dose was equivalent to the maximum recommended human therapeutic dose (MHRD) designated as VPA-M and the other was half of it, i.e., submaximal therapeutic dose (VPA-SM). Maximum recommended human therapeutic dose of valproate is 60 mg/kg [18]. Equivalent dose for rat was calculated as per the following formula: rat dose in mg/kg = human dose in mg/kg × 6.2, where 6.2 is the conversion factor considering body surface area [19,20]. Accordingly, the doses used in rats were 370 mg/kg (VPA-M) and 185 mg/kg (VPA-SM). Valproate solution was freshly prepared in distilled water before oral administration daily for 14 days. A time gap of 30 min was maintained between administration of OSHE and valproate, and the maximum volume administered each time was 0.4 ml/100 g animal. 2.4. Pharmacodynamic studies 2.4.1. Assessment of anticonvulsant action 2.4.1.1. Maximal electroshock seizure (MES)- and pentylenetetrazol (PTZ)induced seizure. Seizure was induced on the 14th day, 60 min after the

administration of last treatment, i.e., OSHE or valproate. Maximal electroshock seizure test was carried out as described [21] by delivering suprathreshold electrical stimulus (current intensity: 70 mA, duration: 0.2 s) via ear clip electrodes using electroconvulsiometer (Ugo Basile, Germany). Animals were observed for occurrence, latency, and duration of tonic hind limb extension (THLE), i.e., the hind limbs of animals outstretched 180° to the plane of the body axis. Pentylenetetrazol test was carried out as described [21]. Pentylenetetrazol was prepared freshly in normal saline and administered at a dose of 60 mg/kg, i.p. This dose of PTZ is considered as 100% convulsant dose with minimal mortality in rats [22]. The latency to myoclonic jerks and occurrence and the latency and duration of generalized tonic–clonic seizures (GTCS) with loss of righting reflex were noted. Animals were observed for 60 min after seizure induction. 2.4.2. Neurobehavioral tests During neurobehavioral study, only one animal was tested at a time. These studies were performed in a calm and quiet room devoid of any external interference like high volume noise and bright light. The rats were deprived of food 12 h before the behavioral testing, as this is known to enhance their motivation to perform the test [23]. 2.4.2.1. Morris water maze test. Morris water maze (MWM) test was performed as described earlier [24]. Acquisition trials were carried for 4 days. On the fifth day, a spatial probe trial of 60-s duration was done to detect spatial memory of the animal. Latency to reach the target quadrant and latency to reach the platform during the probe trial was noted. This test was performed from the 10th day to the 14th day, and a probe trial was repeated a day after seizure induction, i.e., on the 15th day. 2.4.2.2. Passive avoidance test. Memory retention deficit was evaluated by a step through passive avoidance (PA) apparatus [23]. On the acquisition trial, initial latency (IL) to enter the dark chamber was recorded. Rats exhibiting an initial latency time of more than 60 s were excluded from further experiments. After 24 h, transfer latency (TL) was measured in the same way as in the acquisition trial, but foot shock was not delivered. This test was performed on the 13th and 14th days, and TL was also measured a day after seizure induction, i.e., on the 15th day. 2.4.2.3. Elevated plus maze test. Acquisition and retention of memory processes were assessed using EPM [23]. On the 1st day, transfer latency (TL) was recorded. Twenty-four hours later, the retention TL was measured in the same manner. If a rat did not enter the enclosed arm within 60 s, the TL was assigned 60 s. This test was performed on the 13th and 14th days, and TL was also measured a day after seizure induction, i.e., on the 15th day. 2.4.3. Oxidative stress The oxidative stress markers malondialdehyde (MDA), reduced glutathione levels (GSH), and superoxide dismutase (SOD) were estimated in the brain cortical tissue homogenate of a rat made in phosphate buffer (pH 7.4). The rats were euthanized by decapitation under ether anesthesia on the 15th day after neurobehavioral assessment, and their brains were quickly removed, cleaned by rinsing with chilled normal saline and stored at −80 °C for analyses within a week. 2.4.3.1. Measurement of lipid peroxidation. Malondialdehyde (MDA) level was estimated as described [25] with little modification. To 1 ml of brain cortical tissue homogenate, the reagents acetic acid 1.5 ml (20%) pH 3.5, 1.5 ml thiobarbituric acid (0.8%), and 0.2 ml sodium dodecyl sulfate (8.1%) were added. The mixture was then heated at 100 °C for 60 min and cooled and 5 ml of n-butanol: pyridine (15:1% v/v) was added. The mixture was vortexed vigorously and centrifuged at 4000 rpm for 10 min. After centrifugation, the organic layer was withdrawn,

Please cite this article as: Sarangi SC, et al, Pharmacokinetic and pharmacodynamic interaction of hydroalcoholic extract of Ocimum sanctum with valproate, Epilepsy Behav (2017), http://dx.doi.org/10.1016/j.yebeh.2017.08.018

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and absorbance was measured at 532 nm using spectrophotometer (Specord 200, Analytic Jena AG, Germany). Tetraethoxy propane was used as external standard. 2.4.3.2. Measurement of reduced glutathione. Glutathione reduced concentration was estimated by the method described [26] with little modification. A homogenate measuring 0.2 ml was centrifuged with equal volume of 5% trichloroacetic acid. To 0.1 ml of the supernatant, 2 ml of phosphate buffer (pH 8.4), 0.5 ml of 5′5 dithiobis (2-nitrobenzoic acid) (DTNB), and 0.4 ml of double distilled water were added. The absorbance was read at 412 nm within 15 min after vortex using spectrophotometer. Glutathione was used as external standard. 2.4.3.3. Measurement of superoxide dismutase (SOD). Superoxide dismutase activity was determined as described [27] with little modification. A homogenate measuring 0.2 ml was centrifuged with equal volume of 5% trichloroacetic acid. To 0.1 ml of the supernatant, 2.85 ml of 50 mM Tris\\HCl buffer (pH 8.5), and 0.05 ml of 4 μM pyrogallol (in 10 mM HCl) were added. Superoxide dismutase activity was determined as percentage inhibition of autoxidation of pyrogallol by observing the increase in absorbance at 420 nm for 3 min at the interval of 30 s using spectrophotometer. 2.5. Pharmacokinetic studies Valproate maximal dose alone and VPA-M + OSHE were administered to two separate groups of animals for 14 days. Blood samples were collected on the 14th day at multiple time points, i.e., 0 (trough sample), 1, 2, 3, 4, 8, 12, and 24 h with respect to administration of drugs to obtain the area under curve (AUC). Serum was separated and kept at − 80 °C until estimation by HPLC (Shimadzu UFLC 20A series, Japan). Valproate level was estimated with slight modification of the process described [28]. Briefly, protein precipitation was done by adding acetonitrile to the serum sample. The sample was vortexed for 1 min and supernatant transferred to autosampler vials from where it (50 μl) was injected into the HPLC system. Separation was achieved on C-18 column (Merck, Germany) and detected with photodiode array detector at wavelength 210 nm. The mobile phase consisted of acetonitrile and orthophosphate buffer in the ratio of 38:62. Chromatography was performed at 50 °C with flow rate of 1.2 ml/min. Different concentrations of standard valproate in serum were used to prepare calibration curve. The concentrations of test samples were obtained

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from the standard curve using the area of peak in the chromatogram of test samples. 2.6. Statistical analysis All data are represented as mean ± SD. Analysis of variance (ANOVA) followed by Bonferroni's Post Hoc test was applied to compare different groups. Independent sample T-test was applied as statistical test to compare between two individual groups. A P-value b 0.05 was considered as statistically significant. Phoenix® WinNonlin® 7.0 (Certara USA, Inc.) was used to calculate PK parameters. 3. Results 3.1. Effect of different treatments on seizure induction All rats in the control group experienced seizure (Table 1). Though OSHE 800 and 1000 mg/kg provided 50% protection against both MES-induced tonic hind limb extension (THLE) and PTZ-induced generalized tonic–clonic seizure (GTCS), the comparative better outcome with respect to latency and duration of seizure, resulted in selection of optimum dose of Ocimum as 1000 mg/kg, which was then used in combination with valproate. Though Ocimum extract has shown per se antiepileptic potential (50% protection) at 800 and 1000 mg/kg doses, its combination with valproate did not alter antiepileptic efficacy of valproate significantly. 3.2. Effect of different treatments on neurobehavioral test performance in MES model In all the neurobehavioral tests, i.e., EPM test, passive avoidance test and Morris water maze (MWM) test, the comparison of control group with drug-treated groups did not show any significant difference. However, in passive avoidance test, OSHE 1000 mg/kg-treated group (OSHE 1000), and the combination groups (OSHE + valproate) showed significant increase in transfer latency (TL) before as well as after seizure induction compared with valproate-alone-treated groups indicating better fear memory retention potential of OSHE. In MWM test, OSHE 1000 group and the combination groups (OSHE + valproate) have taken significantly less time to reach platform compared with both VPA-M (P b 0.05) and VPA-SM (P b 0.01) groups, revealing better learning memory retention (Table 2).

Table 1 Effect of OSHE and valproate on seizure induction. Groups

MES model

PTZ model

% Protection

Latency to THLE (s)

Duration of THLE (s)

% Protection

Latency to GTCS (s)

Duration of GTCS (s)

Control OSHE 200 OSHE 400

0 (0/6) 33 (2/6) 17 (1/6)

5.2 ± 1.17 5.8 ± 1.30 6.8 ± 2.16

6.5 ± 1.05 4.0 ± 2.28 3.8 ± 2.14

0 (0/6) 0 (0/6) 17 (1/6)

63.17 ± 12.38 63.0 ± 9.75 69.5 ± 15.69

OSHE 800

50 (3/6)

5.3 ± 1.15

4.0 ± 4.43

50 (3/6)

268.0 ± 175.00

OSHE 1000

50 (3/6)

8.3 ± 1.53

2.8 ± 3.19

50 (3/6)

334.0 ± 403.71

VPA-M

100 (6/6)

–

–

83 (5/6)

177.0 ± 0

VPA-SM

67 (4/6)

7.0 ± 1.41

67 (4/6)

112.0 ± 19.80

Comb-M

83 (5/6)

7.0 ± 0.00

67 (4/6)

74.5 ± .71

Comb-SM

67 (4/6)

6.0 ± 1.41

1.3 ± 2.16 P0(0.023) 0.7 ± 1.63 P0(0.005) 1.5 ± 2.34 P0(0.033)

50 (3/6)

79.3 ± 15.53

23.0 ± 4.19 15.0 ± 3.00 11.0 ± 4.08 P0(0.028) 8.3 ± 6.95 P0(0.002) 5.6 ± 7.79 P0(0.000) 1.6 ± 3.58 P0(0.000) 3.7 ± 5.82 P0(0.000) 1.7 ± 2.66 P0(0.000) 6.5 ± 5.97 P0(0.000)

Values are expressed as Mean ± SD. THLE: tonic hind limb extension. GTCS: generalized tonic–clonic seizure, OSHE 1000: Ocimum 1000 mg/kg, VPA-M: valproate maximal dose (370 mg/kg), VPA-SM: valproate submaximal dose (185 mg/kg), Comb-M: VPA-M + Ocimum 1000 mg/kg, Comb-SM: VPA-SM + Ocimum 1000 mg/kg. P0 is P-value vs. control group on basis of ANOVA followed by post hoc Bonferroni test.

Please cite this article as: Sarangi SC, et al, Pharmacokinetic and pharmacodynamic interaction of hydroalcoholic extract of Ocimum sanctum with valproate, Epilepsy Behav (2017), http://dx.doi.org/10.1016/j.yebeh.2017.08.018

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S.C. Sarangi et al. / Epilepsy & Behavior xxx (2017) xxx–xxx

Table 2 Effect of OSHE and valproate on neurobehavioral test performance in MES model. Groups

EPM test

PA test

MWM test

TL before (s)

TL after (s)

TL before (s)

TL after (s)

Lat TQ before (s)

Lat TQ after (s)

Lat PF before (s)

Lat PF after (s)

Control OSHE 200 OSHE 400 OSHE 800

11.4 ± 3.69 13.6 ± 3.26 19.8 ± 9.91 18.4 ± 9.65

9.3 ± 2.59 12.2 ± 4.37 15.5 ± 7.20 13.5 ± 10.35

163.9 ± 149.55 80.5 ± 122.91 92.1 ± 122.99 82.5 ± 121.82

150.4 ± 135.92 69.3 ± 106.88 78.2 ± 124.69 66.3 ± 89.84

6.9 ± 1.78 8.4 ± 7.20 12.1 ± 5.14 8.4 ± 4.96

15.7 ± 11.53 15.7 ± 4.24 38.9 ± 18.03 15.0 ± 13.82

33.6 ± 25.32 26.4 ± 21.45 43.9 ± 20.90 18.9 ± 11.11

OSHE 1000

19.3 ± 2.24

21.8 ± 14.39

249.3 ± 55.43 P#(0.006)

7.9 ± 3.37

VPA-M VPA-SM Comb-M

16.1 ± 1.94 14.3 ± 2.58 16.4 ± 3.11

15.4 ± 6.83 15.5 ± 9.17 13.1 ± 2.03 P#(0.001)

39.3 ± 46.45 158.7 ± 131.01 197.3 ± 140.71 P*(0.001)

5.8 ± 3.26 4.4 ± 1.51 4.3 ± 1.39

3.6 ± 1.63 5.4 ± 3.42 4.5 ± 2.05

7.3 ± 1.19 P*(0.048) P#(0.004) 28.8 ± 19.36 34.6 ± 23.52 7.3 ± 4.22 P#(0.02)

Comb-SM

16.4 ± 5.66

12.2 ± 2.35 P#(0.000)

300.0 ± 0.00 P*(0.023) P#(0.000) 105.6 ± 114.79 131.4 ± 154.06 300.0 ± 0.00 P*(0.023) P#(0.000) 267.5 ± 72.58 P#(0.009)

5.6 ± 1.39 11.2 ± 3.05 16.9 ± 5.71 3.2 ± 1.34 P*(0.045) 5.3 ± 1.91

184.3 ± 116.79

3.3 ± 0.66 P#(0.016)

4.5 ± 2.03

10.8 ± 4.77 P*(0.015) P#(0.001) 22.0 ± 24.08 18.7 ± 16.07 9.0 ± 4.25 P*(0.025) P#(0.001) 9.1 ± 5.53 P#(0.000)

7.7 ± 6.69 P#(0.021)

Values are expressed as Mean ± SD. EPM: elevated plus maze; PA: passive avoidance; MWM: Morris water maze; ‘before’ means before seizure induction (14th day); ‘after’ means after seizure induction (15th day); TL: transfer latency. Lat TQ: latency to reach target quadrant, Lat PF: latency to reach platform. OSHE 1000: Ocimum 1000 mg/kg, VPA-M: valproate maximal dose (370 mg/kg), VPA-SM: valproate sub-maximal dose (185 mg/kg), Comb-M: VPA-M + Ocimum 1000 mg/kg, Comb-SM: VPA-SM + Ocimum 1000 mg/kg. P* is P-value vs. VPA-M, P# is P-value vs. VPA-SM on basis of T-test.

3.3. Effect of different treatments on neurobehavioral test performance in PTZ model As in MES-induced seizure model, the comparison of control group with drug-treated groups did not show any significant difference in EPM test, PA test, and MWM test. However, in EPM test OSHE 800, OSHE 1000, and Comb-M groups showed significant (P b 0.01) decrease in latency to enter in enclosed arm as compared with VPA-M group indicating better memory retention potential of OSHE in these groups. Ocimum sanctum hydroalcoholic extract 1000 mg/kg-treated group showed significant increase in transfer latency (TL) in PA test and took significantly less time to reach PF in MWM test both before as well as after seizure induction compared with valproate-alone-treated groups indicating better memory retention potential of OSHE as compared with valproate-alone-treated groups (Table 3). 3.4. Effect of different treatments on oxidative stress parameters in MES model In MES model, the levels of MDA in OSHE 400 and OSHE 800 groups were significantly (P b 0.01) less as compared with control and

valproate-alone groups. Ocimum sanctum hydroalcoholic extract 1000 group also had significantly (P b 0.01) decreased MDA levels as compared with control group. Combination of OSHE with valproate resulted in significantly higher GSH levels as compared with Control, VPA-M, and VPA-SM groups indicating protective effect of Ocimum against oxidative stress. In SOD activity test, though the differences were not statistically significant, slightly higher protection against oxidative stress was seen in combination of OSHE with valproate groups than other groups (Table 4).

3.5. Effect of different treatments on oxidative stress parameters in PTZ model In PTZ model, there was no significant difference with respect to MDA levels among the groups. Glutathione reduced levels were significantly (P b 0.01) higher in all drug-treated groups compared with control group; however, there was no significant difference among drug-treated groups. In SOD activity test, OSHE 400 and Comb-M groups showed significantly higher protection against oxidative stress as compared with VPA-SM group (Table 4).

Table 3 Effect of OSHE and valproate on neurobehavioral test performance in PTZ model. Groups

EPM test

PA test

MWM test

TL before (s)

TL after (s)

TL before (s)

TL after (s)

Lat TQ before (s)

Lat TQ after (s)

Lat PF before (s)

Lat PF after (s)

Control OSHE 200 OSHE 400 OSHE 800

15.7 ± 5.32 18.6 ± 7.10 36.6 ± 27.07 25.7 ± 9.17

9.0 ± 1.87 11.9 ± 2.88 26.9 ± 17.73 13.2 ± 6.16 P*(0.005)

141.8 ± 130.64 183.0 ± 136.29 207.6 ± 106.78 257.9 ± 94.14

100.4 ± 107.27 226.0 ± 102.62 168.3 ± 89.83 250.7 ± 51.20 P*(0.023)

5.6 ± 5.31 12.3 ± 12.01 8.4 ± 8.25 4.3 ± 1.55

5.9 ± 2.18 4.7 ± 2.24 4.6 ± 1.77 3.2 ± 2.89

18.9 ± 14.44 21.2 ± 21.58 15.7 ± 11.29 15.1 ± 12.98

OSHE 1000

22.4 ± 4.64

11.9 ± 4.96 P*(0.003)

243.6 ± 49.37 P*(0.015)

4.5 ± 1.08

6.4 ± 2.83

VPA-M VPA-SM Comb-M

24.2 ± 17.20 20.2 ± 8.46 24.6 ± 9.36

218.5 ± 116.28 106.1 ± 79.12 161.3 ± 107.18

3.7 ± 1.02 3.3 ± 0.73 4.3 ± 1.58

6.2 ± 3.16 3.6 ± 1.25 5.9 ± 3.66

5.9 ± 1.09 P*(0.016) P#(0.000) 21.8 ± 19.30 29.1 ± 15.76 22.5 ± 21.38

Comb-SM

19.9 ± 7.78

28.4 ± 22.62 14.4 ± 10.35 10.2 ± 1.50 P*(0.001) 11.8 ± 8.28

292.6 ± 18.04 P*(0.009) P#(0.009) 218.3 ± 99.52 197.5 ± 107.79 252.4 ± 106.39

24.3 ± 22.95 51.4 ± 17.10 21.2 ± 21.95 4.7 ± 2.82 P#(0.002) P*(0.023) 9.0 ± 3.73 P*(0.027) P#(0.004) 26.1 ± 21.06 36.7 ± 17.37 17.8 ± 12.00

157.3 ± 134.59

129.0 ± 112.46

4.4 ± 1.32

5.8 ± 1.24

11.8 ± 6.31 P#(0.006)

12.9 ± 9.15

Values are expressed as Mean ± SD. Elevated plus maze; PA: passive avoidance; MWM: Morris water maze; ‘before’ means before seizure induction (14th day); ‘after’ means after seizure induction (15th day); TL: transfer latency. Lat TQ: latency to reach target quadrant, Lat PF: latency to reach platform. OSHE 1000: Ocimum 1000 mg/kg, VPA-M: valproate maximal dose (370 mg/kg), VPA-SM: valproate sub-maximal dose (185 mg/kg), Comb-M: VPA-M + Ocimum 1000 mg/kg, Comb-SM: VPA-SM + Ocimum 1000 mg/kg. P* is P-value vs. VPA-M, P# is P-value vs. VPA-SM on basis of T-test.

Please cite this article as: Sarangi SC, et al, Pharmacokinetic and pharmacodynamic interaction of hydroalcoholic extract of Ocimum sanctum with valproate, Epilepsy Behav (2017), http://dx.doi.org/10.1016/j.yebeh.2017.08.018

S.C. Sarangi et al. / Epilepsy & Behavior xxx (2017) xxx–xxx

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Table 4 Effect of OSHE and valproate on oxidative stress parameters in MES and PTZ model. Groups

MES model

PTZ model

MDA (nmol/g tissue)

GSH (μg/g tissue)

SOD (% inhibition)

MDA (nmol/g tissue)

GSH (μg/g tissue)

SOD (% inhibition)

Control OSHE 200

1227.4 ± 47.55 1178.2 ± 63.72

65.8 ± 22.77 71.6 ± 10.74

22.9 ± 4.85 25.0 ± 8.64

1332.3 ± 116.02 1269.6 ± 161.14

28.6 ± 2.05 29.8 ± 2.83

OSHE 400

121.0 ± 12.51 P0(0.000) P1(0.000) P2(0.000) 57.9 ± 4.65

23.2 ± 5.20

1399.7 ± 146.04

24.5 ± 3.58

1134.7 ± 56.32

210.8 ± 22.90 P0(0.000)

31.1 ± 2.64

67.5 ± 15.42

29.9 ± 4.15

1086.0 ± 91.61

62.0 ± 6.91

29.2 ± 5.04

1212.7 ± 151.80

VPA-SM

1152.4 ± 50.31

48.3 ± 6.14

21.8 ± 2.93

1221.1 ± 65.28

Comb-M

1292.9 ± 24.30

32.1 ± 2.82

1104.3 ± 106.17

Comb-SM

1251.2 ± 71.68

129.8 ± 33.63 P0(0.000) P1(0.000) P2(0.000) 143.0 ± 16.17 P0(0.000) P1(0.000) P2(0.000)

167.2 ± 18.42 P0(0.001) 166.7 ± 3.33 P0(0.000) 153.0 ± 15.46 P0(0.008) 197.1 ± 12.92 P0(0.000)

30.3 ± 1.89

VPA-M

853.2 ± 16.72 P0(0.000) P1(0.000) P2(0.000) 963.9 ± 74.75 P0(0.000) P1(0.004) P2(0.003) 1052.3 ± 72.22 P0(0.001) 1138.1 ± 72.76

72.9 ± 20.04 181.6 ± 52.98 P0(0.000) 152.1 ± 36.58 P0(0.001)

31.6 ± 1.33

1196.6 ± 45.06

147.8 ± 24.10 P0(0.003)

24.3 ± 1.76

OSHE 800

OSHE 1000

32.1 ± 5.26 P2(0.048)

28.9 ± 2.99 23.6 ± 4.03 32.3 ± 5.95 P2(0.037)

Values are expressed as Mean ± SD. MDA: Malondialdehyde, GSH: glutathione reduced, SOD: superoxide dismutase. OSHE 1000: Ocimum 1000 mg/kg, VPA-M: valproate maximal dose (370 mg/kg), VPA-SM: valproate sub-maximal dose (185 mg/kg), Comb-M: VPA-M + Ocimum 1000 mg/kg, Comb-SM: VPA-SM + Ocimum 1000 mg/kg. P0 is P-value vs. control, P1 is P-value vs. VPA-M, and P2 is P-value vs. VPA-SM as per ANOVA.

The oxidative stress parameters analysis revealed that Ocimum alone or in combination with valproate leads to partial protection against oxidative stress caused by valproate and/or seizure. 3.6. Effect of Ocimum on pharmacokinetics of valproate The pharmacokinetic study depicting serum valproate levels at different time intervals by HPLC analysis showed the highest level of valproate (Cmax) at 1 h after treatment in both valproate group and combination group (data not shown). The results show that the serum levels of valproate at each time point is slightly less in valproate + Ocimum group than valproate-alone group, which point towards a possible pharmacokinetic interaction. However, the difference is not statistically significant. Different pharmacokinetic parameters were calculated from the serum levels of valproate at different time points and are tabulated in Table 5. There is no significant difference in area under curve, Cmax, and Tmax. Half-life, though not statistically significant, was little extended in the combination of OSHE + valproate group as compared with valproate alone. Thus, the result of pharmacokinetic data reveals no or minimal pharmacokinetic interaction between OSHE and valproate.

Table 5 Effect of Ocimum on pharmacokinetic parameters of valproate. PK parameter

VPA-M

Comb-M

Cmax (μg/ml) Tmax (h) AUC0–24 h AUC0–∞ T1/2

135.1 ± 23.06 1 894.2 ± 291.67 1334.4 ± 484.49 16.1 ± 2.33

101.3 ± 19.05 1 638.6 ± 151.73 1034.0 ± 131.92 21.3 ± 10.22

Values are expressed as Mean ± SD. PK: pharmacokinetic, VPA-M: valproate maximal dose (370 mg/kg), Comb-M: VPA-M + Ocimum 1000 mg/kg, Cmax: maximum concentration, Tmax: time at which maximum concentration is reached, AUC: area under curve, T1/2: half-life.

4. Discussion In spite of currently available armaments of AEDs, 20–30% of PWE continue to have seizures which affect subjects' quality of life, which put forth the need for novel therapies with better efficacy and lesser adverse effect profile [3,29]. Valproate is a broad spectrum and first line antiepileptic drug and is associated with several adverse effects on chronic administration including cognitive dysfunction [4,20,30,31]. In Ayurvedic practice, Ocimum sanctum (Tulsi) (also called as Ocimum tenuiflorum) has been implicated for its pleiotropic potentials including immunomodulatory and antiinflammatory property [14], antistress [9], anxiolytic [11], and antioxidant action [13]. Few in vivo studies [10,15, 17,32] have shown reduced incidence of seizure, increased latency to seizure occurrence, and decreased duration of seizure due to Ocimum. In this study, we have evaluated antiepileptic and neuroprotective potential of Ocimum per se and its role as an adjuvant with valproate. In MES- and PTZ-induced seizure, both OSHE 800 mg/kg and OSHE 1000 mg/kg groups protected half (50%) of the animals. This is similar to the result obtained earlier [15] in MES model and much higher than the results in PTZ model (just 6–12% protection). In this study, OSHE 200 and 400 groups offered 0 to 17% protection in PTZ model. However, Okoli et al. [32] have shown 50% protection at these doses, which can be attributed to the use of different species of Ocimum (Ocimum gratissimum vs. Ocimum sanctum) and type of extract (methanolic and petroleum ether extract vs. hydroalcoholic extract). Valproate-alone treatment exerted protection against seizure in MES model (100% and 67%, respectively with maximal and submaximal dose) and PTZ model (83% and 67%, respectively with maximal and submaximal dose). The combination of OSHE 1000 with both VPA-M and VPA-SM did not offer any additional protection against seizure compared with valproate alone. This signifies that there is little pharmacodynamic interaction of Ocimum with valproate with respect to antiepileptic effect. That cognitive impairment is commonly observed in PWE may be due to seizure itself or as adverse effect of AED therapy [4,20,30]. Regarding the effect of valproate on cognitive impairment, there are

Please cite this article as: Sarangi SC, et al, Pharmacokinetic and pharmacodynamic interaction of hydroalcoholic extract of Ocimum sanctum with valproate, Epilepsy Behav (2017), http://dx.doi.org/10.1016/j.yebeh.2017.08.018

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S.C. Sarangi et al. / Epilepsy & Behavior xxx (2017) xxx–xxx

dubious opinions. Previous studies have reported reversible cognitive impairment and parkinsonism due to chronic treatment of valproate [31,33]. In this study, valproate did not cause significant cognitive impairment as compared with control group, which can be due to lesser treatment duration (only 14 days). However, combined Ocimum and valproate treatment lead to better memory retention than valproate alone. Our results match with the findings of previous studies which have shown protection of Ocimum against maximal electroshockinduced cognitive dysfunction [34] and attenuation of experimentally induced memory deficit as revealed through passive avoidance task [35,36]. Thus results of learning and memory paradigms indicate beneficial effects of Ocimum and its potential as an adjuvant with valproate. Ocimum has shown antioxidant effect in terms of significantly decreased MDA levels and increased GSH levels as compared with control and valproate-alone-treated groups in MES-induced seizure model and significantly raised GSH levels as compared with control in PTZ-induced seizure model. Earlier studies have also demonstrated antioxidant role of Ocimum by reduction in MDA level in hypoperfusion-induced oxidative stress [37] and decreased lipid peroxidation and increased reduced glutathione content in blood in diabetic rabbits [38]. No changes in SOD levels by Ocimum sanctum have been previously reported [39] which is consistent with this study. Coadministration of Ocimum reduced serum valproate concentration at each time point as compared with valproate treatment alone, though the difference is not significant. Similarly, pharmacokinetic parameters did not differ significantly between valproate-alone group and combined Ocimum and valproate group, though AUC and Cmax (135.1 ± 23.06 vs.101.3 ± 19.05 μg/ml, respectively) are lower, and half-life is little extended in the later one. This partial but nonsignificant kinetic interaction might be responsible for the paradoxical response with respect to seizure control, i.e., both Ocimum and valproate individually have antiepileptic potential, but in combination there is no additional benefit. There are previous studies regarding pharmacokinetic interaction of valproate with herbals other than Ocimum. A study [21] showed coadministration of curcumin (300 mg/kg, p.o.) along with subtherapeutic dose of valproate (150 mg/kg, i.p.) did not cause any significant change in the serum levels of valproate which is in line with this study. Similarly, another study [40] showed Cmax and Tmax to be 29.40 μg/ml and 0.5 h respectively after single oral dose of 150 mg/kg of valproate to rats, and the reason for quite lesser Cmax in this study can be attributed to lower dose of valproate. To the best of our knowledge, this study was the first to report pharmacodynamic and kinetic interaction between Ocimum extract and valproate in epilepsy models. Though Ocimum extract showed per se antiepileptic activity, it did not enhance antiepileptic effect of valproate when used in combination. One of the possible reasons for this might be pharmacokinetic interaction which is evident in this study, though not significant. Ocimum-alone-treated groups have shown better memory retention potential and reduced oxidative stress. The combination therapy of Ocimum and valproate resulted in enhanced learning and memory capacity and partially reduced oxidative stress as compared with valproate-alone-treated groups. These study results suggest that though Ocimum may not enhance the antiepileptic efficacy of valproate, it can be a potential adjuvant to valproate in attenuating its adverse drug effects. Acknowledgment The authors acknowledge Natural Remedies Pvt. Ltd., Bangalore, India for providing the Ocimum hydroalcoholic extract as a gift sample. We also thank Mr. NS Ganeshan for assisting in the estimation of valproate in serum by HPLC. Funding The study was funded by the Department of Pharmacology, AIIMS (Project no. A-428/2016/RS), New Delhi.

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Please cite this article as: Sarangi SC, et al, Pharmacokinetic and pharmacodynamic interaction of hydroalcoholic extract of Ocimum sanctum with valproate, Epilepsy Behav (2017), http://dx.doi.org/10.1016/j.yebeh.2017.08.018