Studies on neuropharmacological and analgesic effects of Periploca aphylla extract in mice

The current study was carried out on a crude extract of stem of Periploca aphylla. It is found in the regions of Jhal magsi, Khuzdar and Pujgur in Balochisatn. P. aphylla has numerous therapeutic uses in traditional medicine. It is used for the relief of pain, ulcer, skin disorders, tumors and for extensive range of other diseases. In neuropharmacological studies mice treated with the crude extract of P. aphylla showed sedative effects in open field, cage crossing, traction and rearing test particularly at the dose of 500 mg/kg as compared with standard drug diazepam. In force induced swiming test increase in immobility time was observed at the dose of 250 and 500 mg/kg. Results were significantly comparable with standard drug diazepam. The administration of the crude extract of P. aphylla produced significant analgesic effects particularly at the dose of 500 mg/kg in the acetic acid induced writhing test and the formalin test. In conclusion, results suggests that the methanolic extract of P. aphylla possesses sedative effects and potent analgesic effects. keywords: Analgesic; Periploca aphylla; Sedative Introduction Plants are vital components of healthy life since they deliver us food and medicines that are safe and effective. Medicinal plants play an important role due to the presence of wide variety of bioactive compounds in them and having various biological and pharmacological activities [1]. About 6000 medicinal plant species exist in Pakistan, and Balochistan which is the largest


Introduction
Plants are vital components of healthy life since they deliver us food and medicines that are safe and effective.Medicinal plants play an important role due to the presence of wide variety of bioactive compounds in them and having various biological and pharmacological activities [1].About 6000 medicinal plant species exist in Pakistan, and Balochistan which is the largest province of Pakistan, has many medicinal plants [2].Periploca aphylla is a medicinal plant of Balochistan and is used by the local people to cure various diseases.It is found in the regions of Jhal Magsi, Pujgur and Khuzdar in Balochistan [3].P. aphylla belongs to the genus Periploca (Asclepiadaceae), which has twelve species.In Pakistan three species have been found.Genus Periploca is medicinally very important [4].P. aphylla is called as "Bata" or "Barara."Locally.Part used is milky juice from the stem and is used for swellings and tumors.It is also employed to treat flu, cough, and swollen joints.P. aphylla is also used for the treatment of constipation, ulcer and skin diseases.Numerous chemical compounds isolated from P.aphylla have shown antibacterial activities and αglucosidase (type VI) inhibitory effect [5, 6].

Studies on phytochemical constituents of P. aphylla show the occurrence of steroids [7],
flavonoids, terpenoids, cardiac glycoside, tannins, betacyanin, coumarin and amino acids.Current research work was carried out to explore the effect of P. aphylla on central nervous system (CNS) and its ability to inhibit the pain as previously no data was available on nueropharmacological and analgesic activities of the plant.

Plant material
Plant material (stem) was collected from Jhal magsi District of Balochistan, Pakistan.Plant was identified by Mrs. Bushra Aziz Khan, Assistant Professor, Department of Pharmacognosy, Faculty of Pharmacy and Health Sciences, University of Balochistan.Voucher specimen No. J-213 was submitted in herbarium of Pharmacognosy department.Plant material was dried under shade and soaked in methanol for 15 days under reduced pressure by using rotary evaporator.After evaporation of solvent, dark green residue was obtained.

Animals
Male and female (both) albino mice, weighing about 20-25 gram were collected from the CASVAB (Centre of advance studies in vaccinology and biotechnology) University of Balochistan, Quetta.Animals were kept under standard environments, temperature was maintained at 25±1 °C, in 12 hours light/ 12 hours dark cycle.The animals had free access to water and feed.Prior to experimentation, the animals were adjusted to Lab environment for 7 days.

Open field activity
The total area of apparatus was 76 X 76 cm 2 , and the height of the walls was 42 cm.Floor of apparatus was separated by means of lines into twenty five equal squares.Mice were placed in center of the apparatus (one at a time).Observation was to count (for 10 minutes) the number of squares crossed by the mice with four paws.

Analgesic activity Acetic acid induced writhing test
In this test abdominal constriction was induced in mice (weighing about 25-28 g).Acetic acid (0.6% in 0.9% saline) 0.1ml/10g was administered I/P after 30 minutes of test drug administration.
Control animals received 2ml of Distilled water and positive control received standard analgesic drug Aspirin (300 mg/kg).Total numbers of abdominal constrictions were counted for 30 minutes [18].Formalin test Twenty microliter of 2.5% formalin, made in distil water, was injected beneath surface of the right hind paw of the mice.Observation (number and time spent on licking and biting) was made for 30 minutes.In first phase (neurogenic pain), mice were observed from 0 to 5 minutes.In second phase mice were observed form 15-30 minutes [18].

Statistical analysis
All the calculations were recorded as mean with + SEM.The Dunnett's test was used to determine the significance of difference between the means and values of P<0.01 were considered as highly significant and P<0.05 significant [2].

Phytochemical tests
Results were positive for the presence of tannins, terpenoids, saponins and flavonoids in phytochemical tests (Table 1).

Traction test
In traction test it was observed that, when the oral dose of 500mg/kg was given, the time to travel on iron rod was decreased.Average 11.2+ 0.37 seconds were taken by control group to travel the iron rod.When the dose of 250 and 500mg/kg crude extract of P. aphylla was given, the average time taken to travel the iron rod by mice was 13.4 + 0.68 and 14.8+1.39seconds respectively.While it was 19.4+ 0.92 seconds when the mice were treated with standard drug Diazepam (Table 5).

Analgesic activity Acetic acid induced writhing test
In this test aspirin (300mg/kg) oral dose was used as reference drug.Significant (p<0.05)dose related inhibition of writhes were observed after administration of crude extract of P. aphylla (Table 7).

Formalin test
In this test P. aphylla crude extract showed significant (p<0.05)analgesic effect (as decrease in number of licking and biting and time spent on licking and biting were observed).Results were more significant at 500mg/ kg oral dose as compared with aspirin (Table 8).In animal models, for assessment of antidepressant activity, forced swimming test is applied.In such models anti-depressant activity is calculated by shortening of immobility time.On the other hand, if immobility duration is long, it reflects the depressant activity of the plant extract [21, 25].Crude extract of the plant showed a marked central nervous system depressant activity which was dose dependent, and it may linked to the presence of flavonoids [25].When acetic acid was administered intraperitoneally, it caused pain by abdominal muscle contraction which progressed into hind limb extension and body part elongation.All these effects are mediated by receptor located in peritoneum, [27] it is clear that the sensation of pain was produced by the intra peritoneal injection of acetic acid which causes the release of endogenous substances supposed to initiate the pain sensation.Prostaglandin E2, Prostaglandin F2α, Prostaglandin I2, certain other prostaglandins and peritoneal mast cells have accumulated due to this process which have shown by many studies [27] in peritoneal fluids treated with acetic acid.Capillary permeability is another mechanism by which acetic acid further increases the pain sensation [20].
Crude extracts of P. aphylla has the ability to reduce the writhing in mice induced by acetic acid.This is indicative of the fact that the plant possesses the anti-nociceptive activity [28].There are two distinct phases in Formalin test which reflect different stages in the process of pain.The first phase shows its direct effect on nociceptors which are mainly responsible for non-inflammatory pain.Contrary to this, the second phase exerts its effect on inflammatory pain [29].A significant (P<0.05)analgesic effect was produced in both phases.This indicates that the anti-nociceptive effect of the extract was produced by both inflammatory and neurogenic mechanisms [30].

Conclusion
Current study reveals that crude extracts of P. aphylla possess CNS depressant and analgesic activity.It may be used as alternative medicine for depression and as analgesic agent, however detail studies are required for isolation of active constituents responsible for its pharmacological effect.

Table 1 . Preliminary phytochemical test of methanolic extract of P. aphylla Chemical Constituents Stem extract of P. aphylla
Values are the mean number of open field activities in 10 minutes.All values are mean ± SEM; n=5; * = Significant results (P<0.05),** = highly significant results (P<0.01) Open filed activityFor control group, squares crossed by the mice was 191.6+3.18taken as mean, and 142 + 2.12 and 134.6+2.5 for 250mg/kg and 500mg/kg of mice treated with crude extract of P. aphylla respectively and 94.4+ 1.57 for diazepam.Crude extract of P. aphylla significantly (p<0.05)decreased the open field activity (Table2).

Table 3 . Assessment of cage crossing activity
Values are the mean number of cage crossing activities in 10 minutes.All values are mean ± SEM; n=5; * = Significant results (P<0.05),**= highly significant results (P<0.01)Values are the mean number of rearing activities in 10 minutes.All values are mean ± SEM; n=5; * = Significant results (P<0.05),** = highly significant results (P<0.01)

Table 5 . Assessment of traction test
Values represent the mean  SEM.Statistically significant from control and standard drug.* Significant at p< 0.05, ** highly significant at p< 0.005.