A comparative anti-inflammatory activity of raw and processed Kupeelu (Strychnos nux-vomica Linn.) seeds on albino rats

Abstract

Seeds of Kupeelu (Strychnos nux-vomica Linn.), a known poisonous drug, is used extensively in various Ayurvedic formulations with great therapeutic significance. Ayurveda recommends the administration of Kupeelu only after passing through specific purificatory procedures in different media like cow's urine (Go mutra), cow's milk (Go dugdha), cow's ghee (Go ghrita), Kanji (thin gruel) etc. Strychnos nux vomica seeds are extensively advocated for nervous debility, paralysis, and weakness of limbs, sexual weakness, dyspepsia, and dysentery and in rheumatism where it can be assumed that besides other properties, Kupeelu may have some sort of anti-inflammatory activity too. In the present study, the powder of raw and processed Kupeelu seeds (processed / purified with Kanji i.e sour gruel) as test drugs were assessed for anti-inflammatory activity by employing Carrageenan and Formaldehyde induced hind paw oedema in Wistar strain albino rats at a dose of 22.5 mg/kg body weight orally. This study reveals that both raw and purified Kupeelu showed presence of highly significant anti-inflammatory activity against formaldehyde induced hind paw oedema, but did not have similar activity against Carrageenan induced hind paw oedema.

Introduction

Kupeelu (Strychnos nux-vomica Linn., Family: Loganiaceae), a well-known plant in Ayurveda, is being used extensively today in different classical formulations with great therapeutic significance¹. Though described under the semi poisonous group², Kupeelu has been used successfully in many diseases after proper purification/processing known as Shodhana in Ayurveda³. Its seeds are mainly used as aphrodisiac, appetizer, anti periodic, digestive, purgative, and stimulant. They are also used in anemia, asthma, bronchitis, and intermittent and malarial fever and in weakness of extremities⁴. The seeds are also used as an ingredient of compound formulations for the treatment of rheumatism in Ayurvedic⁵ and Unani⁶ system of medicine. Sixteen alkaloids have been isolated and identified from the crude nux vomica seed and out of them 80% are strychnine and brucine, as well as their derivatives such as isostrychnine and brucine N-oxide⁷. Indian nuxvomica seeds contain 2.6 to 3% of total alkaloids approximately, of which 1.25 to 2.5% is strychnine and brucine is 1.5 to 1.7%. The seeds also contain chlorogenic acid, a glycoside (loganin), and about 3% of fixed oil⁸.

Either S. nux-vomica or its alkaloids have been reported for its analgesic and anti-inflammatory⁹, anti-oxidant¹⁰, anti-tumor¹¹, anti-snake venom¹², anti-diarrhoeal¹³ and hepatoprotective¹⁴ activities in different modern literatures. However, till date no studies have been reported on the anti-inflammatory activity of the Shodhita (detoxified/processed) seeds powder. Hence, this study has been undertaken with an aim to evaluate the comparative anti-inflammatory activity of raw and processed seeds of Kupeelu through kanji (sour gruel).

Material and Methods

Collection and preparation of drug

Fully matured and botanically authenticated Kupeelu (S. nux-vomica Linn.) fruits were collected from Bankura district, West Bengal, India during the month of December and the seeds were taken out from the fruit pulp, thoroughly washed in tap water and dried under shade. The seeds were subjected to purificatory measures by following the procedure mentioned in Rasatarangini¹⁴. After the purificatory procedure the alkaloid contents of the seeds have been estimated and reported¹⁵. The raw seed powder (KR) and purified seed powder (KKJ) were kept in an airtight glass container and used for the experimental.

Experimental animals

Wister strain albino rats of either sex weighing 200 ± 10g were procured from the animal house attached to our institute. They were housed in polypropylene cages and fed with Amrut brand rat pellet feed supplied by Pranav Agro Industries and tap water given ad libitum. The animals were acclimatized for at least one week in lab conditions before the commencement of experiment in standard laboratory conditions 12 ± 01 hour day and night rhythm, maintained at 25 ± 3°C and 40 to 60 % humidity. Before the test, the animals were made to fast for at least 12 hours. Institutional animal ethics committee had approved the experimental protocol (IAEC/06/09-11/PhD 04) and the care of animals was taken as per the CPCSEA guidelines.

Dose selection

The dose fixation for the experimental animals was done on the basis of body surface area ratio by referring to the standard table¹⁶. The adult human dose (250 mg per day) was converted to animal dose. On this basis, the rat dose was found to be 22.5 mg/kg. The test drugs were suspended in distilled water with suitable concentration depending upon body weight of animals. The drugs were administered to overnight fasted animals orally with the help of gastric catheter sleeved to syringe nozzle.

Anti-inflammatory activity

1. Carrageenan induced hind paw oedema¹⁷ :The selected animals were weighed and randomly divided into three groups of six each. First group received distilled water and served as control group. The second and third groups received test drugs in the dose of 22.5mg/kg. The vehicle and test drugs were administered to the respective groups for five consecutive days. On fifth day, one hour after drug administration, initially left hind paw volumes up to the tibio-tarsal articulation were recorded prior to Carrageenan injection by using plethysmograph¹⁸ and then oedema was produced by injecting 0.1 ml freshly prepared 1% carrageenan in sterile saline solution to the sub-plantar aponeurosis of the left hind limb. The rats were administered distilled water in the dose of 2 ml per 100 g body weight to ensure uniform hydration and hence to minimize variations in oedema formation. Paw volume was recorded at the interval of 3h and 6h after carrageenan injection. Results were expressed as an increase in paw volume in comparison to the initial paw volumes and in comparison with control group.

2. Formaldehyde induced paw edema in rats¹⁹: The test conditions and groupings were similar to carrageenan induced paw oedema as mentioned above. The drug was administered once daily for five consecutive days. On 5th day, initial left hind paw volumes were recorded with the help of Plethysmometer. One hour after the drug administration, 0.1 ml of 3% formaldehyde solution was injected to sub-plantar aponeurosis of the left hind limb. Paw volumes were measured at 24 h and 48 h after formaldehyde injection as described earlier. Results were expressed as percentage increase in paw volume at various time intervals in comparison to the initial values.

Statistical analysis

Data are expressed as mean ± SEM. Statistical evaluation was carried out by unpaired Student ‘t’ test. ‘p’ value less than 0.05 is considered as statistically significant, the value of p<0.01 or p<0.001 is considered as highly significant.

Results and Discussion

In carrageenan induced paw oedema test the first phase (up to 3 hours after injection of carrageenan) results from the concomitant release of mediators: histamine, serotonin and kinins on the vascular permeability and the second phase (up to 6 hours after injection of carrageenan) is correlated with leukotrienes formation and release²⁰. From the results, as depicted in table - 1, both raw and possessed Kupeelu seeds at the given dose level failed to exhibit any significant response at the first phase. However, both KR and KKJ non-significantly inhibited the second phase of carrageenan induced inflammatory response indicate mild action of test drug on leukotrienes formation and release.

Table 1.

Effect on carrageenan induced paw oedema

The formalin-induced inflammation in the rats foot may be conveniently divided into two parts, the first involving 5-hydroxytryptamine as mediator and the second mediator which is unrelated to 5-hydroxytryptamine²¹. In contrast to results obtained in carrageenan induced paw oedema, both KR and KKJ significantly suppressed paw oedema at both time intervals, which were found to be statistically highly significant. The result shows that decrease in paw volume is nearly equal in both the samples after 24 h whereas the decrease is more in raw drug than the Kanji purified sample after 48h of Formaldehyde administration (Table-2). Thus, the result indicates that Kupeelu seed powder is having significant anti-inflammatory activity on the proliferative phases of inflammation.

Table 2.

Effect on formalin induced paw oedema

Conclusion

Kupeelu (KR) seed powder is having significant anti-inflammatory activity and subjecting through purificatory procedures with Kanji (sour gruel) did not affect its efficacy.