|Year : 2011 | Volume
| Issue : 2 | Page : 51-54
Anticonvulsant activity of Passiflora incarnata: No role of chrysin
Bhupinder Singh, Awanish Mishra, Rajesh Kumar Goel
Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab, India
|Date of Web Publication||25-Nov-2011|
Rajesh Kumar Goel
Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala - 147002, Punjab
| Abstract|| |
The earlier studies on Passiflora incarnata have pointed out the possible role of chrysin for its anticonvulsant activity. But role of chrysin in anticonvulsant property of P. incarnate seems to be controversial due to its poor bioavailability reported by different studies. Therefore, this study was designed to investigate the role of chrysin in anticonvulsant property of different extracts of P. incarnata used for medicinal purpose, viz. aqueous (PIAE), hydroethanolic (PIHE), and methanolic extracts (PIME). These extracts were prepared from dried leaves of P. incarnata using water, methanol, and 50% ethanol to obtain PIAE, PIME, and PIHE, respectively. The extracts were standardized with reference to chrysin by HPLC-UV method. Different doses (150, 300 and 600 mg/kg; i.p.) of PIAE, PIME, PIHE, and chrysin (1 mg/kg) were administered 30 min before the PTZ injection (75 mg/kg) to evaluate anticonvulsant effect. HPLC estimation has shown the higher amount of chrysin present in PIME followed by PIAE and PIHE. Significant dose-dependent delay in onset of convulsions was observed in PIHE and PIAE treated mice when compared with PTZ convulsive mice, while PIME treatment has not shown delay in onset of convulsions. Chrysin (1 mg/kg, i.p.), as well, did not produce significant increment in onset of convulsions. These results revealed that PIME containing significant amount of chrysin lacks anticonvulsant effect, while PIAE and PIHE, with insignificant chrysin content, have shown significant anticonvulsant effect. Thus, this study suggests that chrysin is not responsible for anticonvulsant effect of P. incarnata.
Keywords: Anticonvulsant, chrysin, HPLC-UV, passifloraceae
|How to cite this article:|
Singh B, Mishra A, Goel RK. Anticonvulsant activity of Passiflora incarnata: No role of chrysin. J Pharm Negative Results 2011;2:51-4
| Introduction|| |
Passiflora is an exotic genus and many of the species are cultivated for their beautiful ornamental flower, indigenous, native to South-East America, with white and purple flowers. Passiflora incarnata is most popularly being used for the medication of insomnia, anxiety, and convulsions, since antiquity. , The major constituents of P. incarnata are flavonoides (apigeni, luteolin, quercetin, kaempferol; C-glycosyl flavonoids: vitexin, isovitexin, orientin, chrysin, etc.), indole alkaloids (harman, harmol, harmine, harmalol, and harmaline), and γ-benzo-pyrone derivative (maltol). The central nervous system (CNS) depressant properties of P. incarnata have been attributed either to the presence of chrysin  or maltol.  In some studies, harmala alkaloids have been suggested as the main bioactive constituents for its CNS-depressant effect, due to its monoamine oxidase inhibitory property. 
Current literature supports the anticonvulsant effect of hydroethanolic extract of P. incarnata may be due to chrysin. , But the role of chrysin in anticonvulsant property of P. incarnata seems to be controversial due to its poor bioavailability as reported by different pharmacokinetic studies. , These studies suggest that chrysin is poorly bioavailable in humans due to its extensive presystemic intestinal as well as hepatic glucuronidation and sulphation and seems not to exert any pharmacological response.  Thus, this study was envisaged to explore the anticonvulsant effect of different extracts of P. incarnata used ethnomedically, viz. aqueous (PIAE), hydroethanolic (PIHE), and methanolic extracts (PIME). All extracts were standardized with reference to chrysin content.
| Materials and Methods|| |
Drugs and chemicals
All standard chemicals used in this study were of analytical grade. Pentylenetetrazole (PTZ) was procured from SIGMA-ALDRICH, Co., St. Louis, MO, USA; diazepam from Jackson Laboratories Ltd., Amritsar, India; chrysin from Acros Organics, Geel, Belgium; DMSO from Qualigens Fine Chemicals, Mumbai, India; carboxy methyl cellulose, ethanol, and methanol from S.D. Fine Chem Ltd., Mumbai, India.
Plant material and preparation of extracts
P. incarnata leaves were collected from Neha nursery, Gurgaon (Haryana, India). The botanical identity of the plant material was verified, and specimens were deposited at the Herbarium, Department of Botany, Punjabi University, Patiala, Punjab, India (reference voucher no. 51026).
The leaves of P. incarnata were shade dried and cryogrounded to obtain coarse powder. For the preparation of PIHE, 50 g of shade dried and powdered leaves were added to 500 ml of 50% ethanol (v/v) and were macerated at room temperature for 12 h, and second maceration was performed after 12 h. After filtration the extract was dried using rotavapor. Similarly, PIME and PIAE were prepared by using 100% methanol and 100% water (w/w), respectively, by cold maceration process.  The extracts were stored in airtight container at 4C till further studies.
Standardization of chrysin by RP-HPLC-UV method
For standardization, different extracts were directed to liquid-liquid partitioning. Initially the 500 mg extracts were dissolved in water and defatted with petroleum ether and water fraction again partitioned with ethyl acetate. The ethyl acetate fraction was dried and 100 μg/ml solution of these fractions was used for the chrysin estimation.
The HPLC analysis was carried out using Waters HPLC system (Milford, MA, USA) consisting of 515 binary pumps, 2487 dual wavelength UV detector, and Rheodyne manual injector. The chromatographic separation was achieved on reversed-phase analytical column (150 × 4.6 mm i.d., 5 μm; Agilent, USA). The data were acquired and processed in Empower Pro® Operating System (Waters® , Milford, MAUSA). The mobile phase consisted of methanol:water (85:15). Mobile phase was filtered through a 0.45-μm membrane (Millipore, USA) and degassed (Transsonic T 570/H, Elma, Germany). The flow rate was set to 0.5 ml/min and was detected at 269 nm for determination of chrysin. The injection volume was 20 μl, and the peaks were identified by comparison with the retention times of chrysin.
Male Swiss Albino mice weighing 25-35g obtained from Central Research Institute Kausali, Himachal Pradesh, were used for this study. The animals were housed in standard cages and maintained at room temperature with natural day and night cycles. The animals were allowed free access to food (standard laboratory diet) and water during study. All experiments were carried out between 07:00 and 16:00 h. The experimental protocol was duly approved by Institutional Animal Ethics Committee and care of the animals was carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forest, Government of India.
Male Swiss Albino mice were used in this study and divided into 12 groups, each containing six mice. For the induction of convulsions in all mice, PTZ (75 mg/kg; in warm saline) was administered intraperitoneally. Group 1 served as the control group (normal saline; i.p.), group 2 as standard drug pretreatment group (diazepam 4 mg/kg; i.p.), group 3 as chrysin pretreatment (1 mg/kg; i.p.), groups 4-6: PIHE extract pretreatment (150, 300, and 600 mg/kg; i.p.), groups 7-9 as PIAE extract pretreatment (150, 300, and 600 mg/kg; i.p.), and groups 10-12 as PIME extract pretreatment (150, 300, and 600 mg/kg; i.p.).
The onset of convulsions was recorded, and the resultant seizures were scored as follows: stage 0 (no response); stage 1 (hyperactivity, restlessness, and vibrissae twitching); stage 2 (head nodding, head clonus, and myoclonic jerks); stage 3 (unilateralor bilateral limb clonus); stage 4 (forelimb clonic seizures); and stage 5 (generalized clonic seizures with falling). The mortality in the animals was noted and expressed in terms of percentage mortality.
Results were expressed as mean ± SEM. The significance of anticonvulsant effect was determined by one-way analysis of variance test followed by Dunnett's test except percentage mortality data that were subjected to chi-square test, and the results were regarded as significant at P < 0.05.
| Results|| |
In RP-HPLC-UV method chrysin showed retention at 4.3 min and different extracts were compared with this for the estimation of chrysin. Different extracts of P. incarnata have shown varied amount of chrysin. Maximum content of chrysin was found to be present in PIME followed by PIAE and PIHE [Figure 1] and [Table 1].
|Figure 1: RP-HPLC-UV chromatogram of chrysin in different extracts of Passiflora incarnata|
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PTZ (75 mg/kg; i.p.) treatment significantly induced convulsion in the mice and the average onset of convulsion (85 s), while PIHE and PIAE treatment significantly delayed the onset of convulsions induced by PTZ, dose dependently. In contrast, PIME and chrysin did not show the delay in onset of convulsions. The latency in onset of convulsions with treatment of PIHE (600 mg/kg) and PIAE (600 mg/kg) was found to be equipotent to that of diazepam (4 mg/kg) [Table 2].
|Table 2: Effect of different extracts of Passiflora incarnata on seizure severity score and %mortality in PTZ-induced convulsions|
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The PTZ treatment showed maximum seizure severity score and followed by 100% mortality, while there was a dose-dependent significant reduction in seizure severity score, and mortality was observed in PIHE- and PIAE-treated mice. PIHE (300 and 600 mg/kg) and PIAE (600 mg/kg) had shown no mortality, whereas reduced mortality has been observed at lesser doses of PIHE and PIAE. PIME-treated mice did not produce any significant protection [Table 2].
| Discussion|| |
Different extracts of P. incarnata have been reported to possess CNS-depressant properties like decoction, methanolic extract, and hydroethanolic extract. ,, The CNS-depressant properties can be through different phytoconstituents such as harmala alkaloids, chrysin, and maltol. In contrast, some pharmacological investigations reported that neither the harmala alkaloids nor the flavonoids are likely the active components.  Till date, there is no lucid scientific consensus regarding the active phytoconstituent of P. incarnata for its CNS-depressant activity.
The finding of this study revealed PIHE to have the most significant anticonvulsant effect followed by PIAE, whereas PIME was found to be ineffective as anticonvulsant. These findings are in line with earlier studies in which passipay (hydroethanolic extract) had been reported to possess anticonvulsant effect. ,
But interestingly, in our study the HPLC analysis of extracts revealed the maximum amount of chrysin in PIME, while much lesser amount was available in PIAE and PIHE. Thus, suggesting that chrysin may not be the active phytoconstituent responsible for anticonvulsant effect of P. incarnata. Even in this study intraperitoneal injection of chrysin (1 mg/kg) failed to exert anticonvulsant effect, while some literature evidence suggests the intracerebroventricular route of chrysin for its anticonvulsant activity.  This does not reflect that chrysin do not possess anticonvulsant property but it appears to be a matter of route of administration. It may be due to its extensive presystemic hepatic metabolism following intraperitoneal administration of extracts in our study, which reduces its bioavailability. ,,
The anticonvulsant effect of hydroethanolic and aqueous extract of P. incarnata may be justified based on the recent reports of presence of γ-amino butyric acid (GABA; an inhibitory amino acid), which is recently been identified as dominant amino acid in these types of extracts of P. incarnata.  In cold extraction, maximum amount of GABA was extracted using 44% ethanol,  suggesting that the anticonvulsant effect of PIHE and PIAE may be due to GABA, thus supporting our findings. Therefore, based on above discussion it can be concluded that chrysin may not be the active phytoconstituent responsible for anticonvulsant effect of P. incarnata. But improved formulations of the methanolic extract with better bioavailability of chrysin can enhance the clinical usefulness of P. incarnata.
| Acknowledgement|| |
The authors acknowledge the supports provided by the Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, India, to execute the study and All India Council for Technical Education for granting research fellowship to Mr. Bhupinder Singh.
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[Table 1], [Table 2]