Anti-inflammatory activity of two varieties of Pippali (Piper longum Linn.).

The present study has beenundertaken to evaluate the anti-inflammatory activity of two varieties of Pippali in acute and sub-acute experimental models of inflammation in albino rats. Four different market samples of each variety of Pippali were procured from different regions of India. The samples collected from South India which have given more extractive values were selected for screening of anti-inflammatory activity. Randomly selected animals were divided into four groups of six animals each. The test drugs were administered orally at a dose of 200 mg/kg and the activity was compared with standard anti-inflammatory drugs in both models. Among the two different test samples studied, it was found that Chhoti variety of Pippali suppressed inflammation of both acute and sub acute phase, while Badi variety of Pippali only of acute phase. Thus for the therapeutic utility, Chhoti variety of Pippali may be considered over the Badi variety.

Introduction

Pippali (Piper longum Linn.) is one of the prime Rasayana (rejuvenator) drugs in Ayurveda and is widely used to treat various diseases especially for the treatment of respiratory disorders.[1] The root of this plant is known as Pippali Mula in Ayurveda and its fruits (Spike) are mainly used for Rasayana purpose. Several biological activities like immunostimulatory, anti-ulcer, anti-amebic, anti-oxidant, hepatoprotective and anti-inflammatory activities were reported on the fruit of this plant.[2, 3] In Ayurvedic formulary of India, Pippali is being used in 324 formulations and it is one of the ingredients of the Trikatu churna.[4]

However, there are two types of Pippali available in the market in the name of Chhoti Pippali and Badi Pippali. Though both the varieties are used for therapeutic purposes, Chhoti Pippali is more preferred by physicians. It is a well-established fact that the Pippali is one of the most important drugs in the treatment of Tamaka Shwasa (bronchial asthma). The drug which is supposed to be used for the treatment of asthma should have anti-inflammatory activity. Thus, the present pharmacological study has been undertaken to screen the anti-inflammatory activity of two varieties of Pippali in different experimental models to ascertain which variety is having better anti-inflammatory effect.

Materials and Methods

Procurement, identification and authentication of different samples of Pippali fruits

Four different market samples of each variety of Pippali were procured from different regions of India viz., East zone (Kolkotta), West zone (Jamnagar), North zone (Jaipur) and South zone (Thiruvananthapuram). Identification and authentication of these materials was done on the basis of organoleptic characters, exomorphology and pharmacognostic study at Pharmacognosy Laboratory. Further, these samples were subjected to preliminary phytochemical tests. Both Chhoti and Badi varieties of Pippali samples collected from South India, which gave higher extractive values (in terms of water soluble, methanolic and dichloromethane extractives), were selected for pharmacological screening.[5] The test drugs were made into fine powder (120 mesh size) and stored in air tight glass jar till the commencement of pharmacological study.

Animals

Wistar strain albino rats of either sex weighing between 180-200 g were selected for the study from the animal house attached to our institute. They were housed at 25 ± 3°C with constant humidity 50 – 60%, on a 12 h natural day and night cycles. They were fed with Amrut brand rat pellet feed supplied by Pranav Agro Industries and tap water ad libitum. The experiments were carried out in accordance with the directions of the Institutional Animal Ethics Committee (IAEC/02/2007/MD/04).

Dose selection and schedule

The dose of the test drugs was calculated by extrapolating the human dose to animals (550 mg/kg) based on the body surface area ratio by referring to the standard table of Paget and Barnes (1969).[6] The fine powder of both the varieties of Pippali was suspended in distilled water (55 mg/ml) and administered orally at a volume of 1 ml/100 g body weight. The drugs were administered orally with the help of a gastric catheter of suitable size sleeved on to a syringe nozzle. The animals of water control groups received equal volume of distilled water.

Statistical analysis

Students “t” test for unpaired data has been used for analyzing the data generated during the study. The values of drug treated groups were compared with water control group. P value less than 0.05 is considered as statistically significant.

Study protocol

Carrageenan-induced paw edema

The selected animals were weighed and randomly divided into three groups of six each. First group received distilled water and served as the control group. The second and third groups received test drugs Badi variety and Chhoti variety, respectively. Fourth group was administered with standard anti-inflammatory drug phenylbutazone in the dose of 100 mg/kg.[7] The vehicles and test drugs were administered to the respective groups for five consecutive days.

Initially left hind paw volumes up to the tibio-tarsal articulation were recorded prior to Carrageenan injection by using plethysmograph.[8] The plethysmograph employed consisted of a 10 ml glass vessel (25 × 65mm) fixed to a 2 ml glass syringe through pressure tubing. About 4 ml of mercury was filled in the syringe and the mercury level was adjusted to zero mark on the micropipette. The space between the zero mark and the fixed mark on the glass vessel was filled with water and few drops of teepol. The initial level of fluid was adjusted and set at zero. The paw was immersed in water exactly up to the tibio-tarsal articulation. The increased level of water in the glass vessel was adjusted to the prefixed mark by releasing the pressure of the connected syringe. The level where water and mercury interface in the micropipette was recorded as paw volume.

On fifth day 1 h after drug administration, edema was produced by injecting 0.1 ml of freshly prepared 1% carrageenan in sterile saline solution to the sub-plantar aponeurosis of the left hind limb. The rats were administered tap water in the dose of 2 ml per 100 g body weight to ensure uniform hydration and hence to minimize variations in edema formation. Paw volume was recorded 3 h after carrageenan injection. Results were expressed as an increase in paw volume in comparison to the initial paw volumes and also in comparison with the control group.

Formaldehyde-induced paw edema

In this model, test conditions and groupings were similar to carrageenan-induced paw edema except the standard anti-inflammatory drug used i.e, diclofenac sodium (5 mg/kg) was used as the standard anti-inflammatory drug. Pedal inflammation was induced by injecting 0.1 ml of 3% formaldehyde solution below the plantar aponeurosis of the right hind paw of the rats. The paw volume was recorded immediately prior to compound administration (0 h) and then at 24 and 48 h after formaldehyde injection. Results were expressed as an increase in paw volume in comparison to the initial paw volumes and also in comparison with the control group.[9]

Results

Data pertaining to effect of Badi variety and Chhoti variety on carrageenan-nduced hind paw edema in rats are given in Table 1. Both the test drugs significantly inhibited the paw edema in comparison to water control group. Among the two varieties, Chhoti variety shows better suppression of paw edema in comparison to Badi variety.

Table 1

Effect on carrageenan-induced paw edema in albino rats

Table 2 shows data related to the effect of Badi variety and Chhoti variety on formaldehyde-induced hind paw edema in rats. Apparent and statistically non-significant edema suppression was observed in Badi variety at both 24 and 48 h of formalin injection. In Chhoti variety administered rats, statistically highly significant suppression of paw edema was observed at both time intervals in comparison to water control group. As expected, in diclofenac sodium treated group, the edema suppression was observed at both 24 and 48 h of formalin injection. The observed suppression of edema in Chhoti variety treated group at both 24 and 48 h is comparable to standard anti-inflammatory drug, especially at 48 h it shows better suppression of paw edema than that of standard drug.

Table 2

Effect on formalin-induced paw edema in albino rats

Discussion

Carrageenan-induced edema is commonly used in animal models for acute inflammatory agent(s) and is believed to be a biphasic event. The initial phase is attributable to the release of various biochemicals, viz. histamine, 5-HT, various kinins in the first hour injection of carrageenan. A more pronounced second phase is related to the release of prostaglandin-like substances in 2 to 3 h.[10, 11]

Both the varieties of Pippali produced a considerable suppression of edema formation against carrageenan-induced paw edema in rats. The observed effect may be due to inhibition of one or more of the phlogistic mediators, antagonizing their interaction with their respective receptors, inhibition of phlogistic mediators or it may be due to general mechanism like increasing the membrane stability in the cell.

Inhibition of formaldehyde-induced pedal edema in rats is one of the most suitable tests to evaluate the anti-proliferative activity of inflammation in which inflammation occurs through proliferation and migration of fibroblast which are mainly concerned with the formation of connective tissue.[12] In this model, the Badi variety suppressed edema at both 24 h and 48 h time intervals in a non significant manner and Chhoti variety produced highly significant suppression of pedal edema which is almost equal to the effect of standard drug at 24 h and more effective than standard drug at 48 h of formaldehyde injection. The mechanism involved here may be attributed to suppression of this phase of inflammation by active constituents present in Chhoti variety of test drug. The suppression of edema by standard reference drug, diclofenac sodium; which is a non-selective COX inhibitor, was also found to be significant. The most remarkable point of this study was that the Chhoti variety at 200 mg/kg produced more inhibition of edema than the standard anti-inflammatory drug, diclofenac sodium.

Pippalı (Piper longum Linn.) fruit contains a number of constituents, including volatile oil, alkaloids, isobutylamides, lignans and esters. Piperine, which is the prime constituent of fruit, is reported to be having significant anti-inflammatory activity.[13, 14] In this study also, Piperine may be responsible for observed anti-inflammatory activity.

Conclusion

This study shows that Chhoti variety of Pippali suppressed inflammation of both acute and sub acute phase while Badi variety of Pippali only of acute phase. Thus for the therapeutic utility, Chhoti variety of Pippali may be considered over the Badi variety.