Chemical composition , anti-inflammatory and analgesic activities of the petrol ether extract of Pinellia cordata

The analgesic and anti-inflammatory effect of the petrol ether extract of Pinellia cordata (PEPC) was investigated using the xylene-induced ear edema and acetic acid-induced writhing test in mice. PEPC was administered intragastrically (i.g.) at 200, 400 and 600 mg/kg. It had significant anti-inflammatory and analgesic activities with dose dependent manner. The chemical components of PEPC were analyzed by gas chromatography-mass spectrometry (GC/MS), and the main components were melissane (20.1%), -sitosterol (13.9%) and linoleic acid (10.1%). These data may afford scientific basis on the traditional use of the plant for painful and inflammatory conditions.


INTRODUTION
There are multiple reports of natural products having antimicrobial, anti-inflammatory and analgesic activities, and the essential oils, sesquiterpenes and flavonoids might be their active components (Akin et al., 2010;Lawal et al., 2010;Lu et al., 2009;Owolabi and Omogbai, 2007).In China, many traditional Chinese medicines have been used to treat pains or inflammation caused by conditions such as arthritis and tendinitis.
Pinellia cordata is a medicinal plant (Araceae family) native to southeast China.The plant is marked with arrowhead leaves and grows along forest stream or steep rock near stream (Figure 1).The stem tuber of P. cordata (Chinese name "Dishuizhu") contains a variety of constituents, including alkaloids, which have a variety of pharmaceutical properties.Though it is a little poisonous, it was traditionally used as a magical herb in east China for treating all kinds of pain, stomachache, snake bite poisoning, injury, arthritis, rheumatism, cancerous tumors and skin diseases.Capsules containing this crude drug were clinically used as analgesic and anti-inflammatory agents in Zhejiang Province (Chiang Su New Medicinal College, 2003;Institute of Botany, 1979).The present study was therefore planned to investigate the chemical *Corresponding author.E-mail: hbkcn@163.com.Fax: +86021-81871301.composition, analgesic and anti-inflammatory effects of the petrol ether extract of P. cordata.

MATERIALS AND METHODS
Adult ICR mice weighing 18-22g were obtained from Animal Center of Second Military Medical University.Mice were housed in cages and maintained in an air-conditioned room with controlled temperature (21 ± 1°C) and humidity (55 ± 15%), and were kept on a 12 h light/dark cycle.They were fed with standard diet and water and acclimated 3 days before they were used.All procedures involving animals were conducted in accordance with the European Community guidelines for the use of experimental animals and approved by the Second Military Medical University Committee on Animal Care and Use.The animals were deprived of food for 15 h before the experiment, with free access to drinking water.Unless indicated, equal number of female and male animals was used in each experiment.In all experiments, animals were randomly divided into groups of ten animals, and each animal was used only once in the experiment.

Protocol
Each group was vehicle treated (intragastrically, i.g.), with PEPC (200, 400 and 600 mg/kg).As a reference, indomethacin (10 mg/kg, i.g.) (Chengdu Pharmaceutical Factory, Chengdu, China) was used as the standard drug.2% Tween 20 solution (TW, Merck) in physiological saline was used as a vehicle.The dosages of PEPC were estimated based on our preliminary experiment and recommended oral dosage for the folk use in human.Acetic acid and xylene (Sinopharm chemical reagent Co. Ltd., China) were used as the inducer drug.
Herbal material and its extract P. cordata was collected in August 2009 from Zhejiang Province, China and was authenticated by Professor Hanchen Zheng, Department of Pharmacognosy, Second Military Medical University.
A voucher specimen (No.H038#) was deposited in the herbarium of the Department of Pharmacognosy, School of Pharmacy, Second Military Medical University.Air-dried powdered material weighing 1 kg was macerated and soaked in three volumes (w/v) of petroleum ether (30 to 60°C) for 24 h at room temperature.The process was repeated three times and filtrates were pooled and concentrated at 45°C under reduced pressure in a rotary evaporator and yielded 22.1 g of petroleum ether extract (PEPC).PEPC was kept in a refrigerator and was freshly dissolved in the vehicle before each experiment.

Gas chromatography-mass spectrometry (GC/MS) analysis
GC/MS analysis was carried out on a Thermo Focus DSQ GC-MS instrument (Thermo, USA).The analytes were separated on a 30 m × 250 mHP-5 capillary column coated with a 0.25 m film of 5% phenyl methylpolysiloxane.Split ratio was 30:1.The column oven temperature was programmed at 15°C min −1 from an initial temperature of 60 to 300°C, which was held for 5 min.The injection and ion source temperatures were 250°C.Helium (99.999%) was used as carrier gas at a flow rate of 1 ml.min −1 .The ionizing electron energy was 70 eV and the mass range scanned was 40 to 400 amu in full-scan acquisition mode.Compounds were identified using the NIST Mass Spectral Search Program (National Institute of Standards and Technology, USA.)

Assay of xylene -induced inflammation in mice
One hour after giving the medicine for the last time, the mice's right ear was embrocated with xylene according to the method described by literature (Zhou et al., 2008).The edema was measured 1 h after xylene treatment, after which mice were sacrificed and a plug was excised from both the ears.The ear swelling rate (SR) was measured as follows: Where, WR is the weight of right (treated) ear and WL is the weight of the left (untreated) ear.The anti-inflammatory activity was expressed as inhibitory ratio (IR) or percent tumefaction reduction in treated mice compared to the controls.The IR of the treated group was calculated as follows: Where, A is the edema of vehicle group and B is the edema of drug group.Each value was the mean of individual determinations in 10 mice.

Abdominal constriction induced by acetic acid
The writhing test was carried out following the method described by literature (Rabanal et al., 2005).Mice were randomly divided into each group (n = 10), and groups of overnight fasted mice were treated with PEPC, vehicle or indomethacin 1 h before an intraperitoneal injection of 0.7% acetic acid at 0.1 ml/10 g body weight.The number of writhing was counted for each animal, starting 3 min after acetic acid injection over the period of 12 min.

Statistical analysis
Results were expressed as mean ± S.E.M. Statistical analyses were performed with one-way ANOVA followed by Student's t-test.P values less than 0.05 were considered to be statistically significant.
However, constriction induced by acetic acid was considered as a non-selective anti-nociceptive model, and for the chemical diversity of the natural products, herb extracts possesses both peripheral and central analgesic activity (Sánchez-Mateo et al., 2006).The crude drug of P. cordata has been used to treat abdominal pain, waist ache and headache in folk tradition.In order to clarify whether the activities are due to the blockade of the effect or the release of PGs that excite pain nerve endings, more animal models including formalin, hot plate and tail flick test are needed in further study.In the xylene-induced ear edema test, the positive control drug indomethacin at 10 mg/kg exhibited significant inhibitory effect.The inhibition was up to approximately 28.7%.PEPC exerted obvious anti-inflammatory activity in a dose-dependent manner.At the dose of 200, 400 and 600 mg/kg, the edema reductions were 2.6, 14.2 and 30.8%, respectively.At the dose of 600 mg/kg, the potency of PEPC was comparable to that of 10 mg/kg indomethacin (Figure 2).Indomethacin is a known nonselective COX inhibitor.In xylene-induced ear edema acute inflammation model, PEPC might acts in part through the inhibition of the inflammatory mediators of the acute phase of inflammation like indomethacin.
The results demonstrate the anti-inflammatory properties of PEPC and provided evidence of this herbal medicine for the treatment of inflammatory diseases in Chinese.The PEPC is a complex mixture of fatty acids and sterols, which comprises a great part of the fraction.It was reported that stigmasterol and sitosterol could   significantly and dose-dependently reduce formalininduced pain; acetic acid induced abdominal constrictions (Al-Yousuf et al., 2002;Erazo et al., 2006).Oils with higher a-linolenic acid content produced a greater inhibition in carrageenan and arachidonic acid induced paw edema models (Singh et al., 2008).Due to complexity of the chemical compounds of PEPC, the activity of the PEPC might be related to the combination of the major chemical compounds such as linolenic acid and sitosterol.However, to interpret the mechanism and the relationship between chemical composition and pharmacological effects, more animal models, such as rat paw edema model and hot plate test, should be conducted by using single compounds including alkaloids from P. cordata,

Conclusion
Our results indicate that PEPC has analgesic and anti-inflammatory effects, which may provide some pharmacological evidence for its folkloric use and further exploitation.These activities might be related to the combination of the major chemical compounds such as linolenic acid and sitosterol in the PEPC.In further exploring the activity and mechanism of action of PEPC, the active principle could be isolated and further molecular and pharmacological experiments warranted.

Table 1 .
Compounds identified by GC/ MS from the petrol ether extracts of rhizoma of Pinellia cordata N. E. Brown.