A comparative experimental evaluation of anti-inflammatory activity of Premna obtusifolia Linn and Premna latifolia Roxb leaves in Charles foster rats.

Agnimantha, a classical Ayurvedic drug is one among the dashamoolas, i.e., group of ten plants whose roots form the useful part. It is a main ingredient of many Ayurvedic preparations. Various source plants from the genus Clerodendrum and Premna are used in different regions of the country. In this study, two species of Premna were selected and evaluated for anti-inflammatory activity against carrageenan induced rat hind paw edema. The selected animals were divided into four groups and test drugs were administered orally in the dose of 9ml/kg. Group I received vehicle (distilled water), Group II received leaf decoction of Premna obtusifolia Linn, Group III received leaf decoction of Premna latifolia Roxb and Group IV received indomethacin as standard anti-inflammatory drug. Test drugs and indomethacinwere administered one hour prior to the injection of 0.05 ml of 1% suspension of carrageenan into the sub plantar region of the left hind paw of rats. Study findings indicate that leaves of both the species of Premna have anti-inflammatory potential, P. latifoliabeing superior to P. obtusifolia.

Introduction

Agnimantha is an important Ayurvedic medicinal plant forming an ingredient of important formulations like dasamoolarishta1, dasamoola kwatha2, dasamoola shatpala ghrita3, maharasnadi kwatha4, varunadi kwatha5, narayana taila6, dasamoolahareetaki7 etc. The root of Agnimantha is used in inflammatory conditions like rheumatoid arthritis, rhinitis8, abscess9 and is also indicated in Shotha (inflammation)10, Vatavyadhi (Neurological disorders)11, Prameha (Diabetes mellitus)12, Medoroga(obesity)13 and Agnimandya (loss of appetite)14. Leaf is indicated in the treatment of Arshas(hemorrhoids) in the form of Avagaha Sweda (sitz bath) to reduce inflammation and relieve pain15. Ayurvedic scholars of modern era consider leaf also as one of the useful part for internal and external use16.

Premna obtusifolia Linn (Syn P. integrifolia Linn; P. serratifolia Linn), Premna latifolia Roxb, Clerodendrum phlomidis Linn, Clerodendrum inerme (L) Gaertn. belonging to the family Verbenaceae, are used as various source plants for Agnimanthain different parts of the country. P. obtusifolia is the source of the drug in Eastern, Central and Southern parts of India, P. latifolia in North and Western regions17. C. phlomidis is considered to be the source plant in Gujarat and other parts of North India18 and C. inerme is also considered to be the source plant for Kshudra Agnimantha19 (another variety of Agnimantha) in certain parts of South India.

P. obtusifolia leaf decoction is reported to be used in the treatment of colic, rheumatism, neuralgia20. P. latifolia is leaf is diuretic and indicated in dropsy21. Studies have shown methanolic extract of the roots of P. obtusifolia to have significant anti inflammatory activity on carrageenan-induced rat hind paw edema, histamine induced wheal formation, and acetic acid-induced mouse vascular permeation22. The root and leaves of P.obtusifolia has been reported to contain an alkaloid premnazole, which is a proven anti inflammatory agent23. Leaves also contain flavonoids and sterols24. The leaves of P.latifolia is composed of flavone glycosides and premnalin25.

Further, the root of P.obtusifolia is listed among the 70 medicinal plant species in high trade (≥ 100 MT/year) being procured from the tropical forest26. For sustainable harvesting of medicinal plants which are in high trade, research on the efficacy of alternate plant part, like leaf instead of root has to be taken up. Hence the present study was under taken to evaluate anti-inflammatory activity of leaves of two species of Premna and to compare them. The presence of significant activity could be a useful evidence to use leaves as substitute to roots wherever it is used for inflammation.

Materials and Method

Test drugs

Fresh leaves of P. obtusifolia (Picture 1), P. latifolia (Picture 2), were collected from Udupi district,Karnataka,India during the month of November 2008 and the botanical identity was confirmed by botanist of the Institute.The fresh leaves were shade dried at room temperature and made into coarse powder. Fresh decoction was prepared by boiling 1part of drug in 8 parts of water till it was reduced to one quarter27.

Picture 1

Premna obtusifolia Linn

Picture 2

Premna latifolia Roxb

Animals

Charles Foster's albino rats of either sex weighing between 180 ± 20 g were used in the study. The animals were obtained from the animal house attached to SDM Ayurvedic College, Udupi, Karnataka. The experiment was carried out in accordance with the Institutional Animal Ethics Committee (IAEC).

Dose fixation

Dosage was calculated by extrapolating the human dose of decoction (100ml) as per the posology of Kwatha kalpana28 to animal dose (9ml/kg) based on the body surface area ratio by referring to the table of Paget and Barnes29.

Anti-inflammatory activity

The selected animals were randomly divided into four groups of six animals each. Group I received distilled water and served as control. The calculated dose of test drugs A and B were administered to the groups II and III respectively. Fourth group was administered with standard anti-inflammatory drug indomethacin in the dose of 20mg/kg. The anti-inflammatory activity (Carrageenan induced paw edema) was carried out by following method of Winter et al30. The rats were weighed and a mark was made at the tibio-tarsal junction. Then they were dosed orally with the control drug, the test drugs and standard drug one hour before the injection of 0.05 ml of 1% suspension of Carrageenan into the sub plantar region of the left hind paw. The volume of the injected paw was measured by liquid displacement in a Plethysmograph31 immediately after carrageenan injection and again at the interval of 15, 30, 60and 120 min. Each time the paw was dipped till the fixed mark to restore the constant paw volume and the edema volume was measured.

Statistical analysis

The data were expressed as Mean ± SEM. Students “t” test for unpaired data has been used for analyzing the data generated during the study. ‘P’ value less than 0.05 is considered as statistically significant.

Results

A gradual and time dependant increase in paw edema was noticed in control group and it has reached to maximum extent at 2h (Table – 1A & 1B). An apparent and statistically significant inhibition of paw edema was observed in P. latifolia treated group at 15minutes, 60 minutes and 120 minutes and non-significant inhibition at 30minutes. In contrast, pretreatment with P. obtusifolia significantly inhibited paw edema only at 60 and 120minutes. The standard anti-inflammatory drug indomethacin has also significantly inhibited paw edema only at 60 and 120minutes. The result shows that P. latifolia is having better anti-inflammatory activity in terms of onset of action and is better than that of indomethacin.

Table 1A

Effect on Carrageenan induced paw edema

Table 1B

Effect on Carrageenan induced paw edema

Discussion

The present experimental study was carried out to elicit anti-inflammatory activity of leaves of source plants of Agnimantha to revalidate the classical actions delineated. In this study, two species of Premna which are widely used as putative source of classical Agnimantha were compared for anti-inflammatory activity.

Carrageenan induced rat paw edema is commonly used in animal models for acute inflammatory agents and is of biphasic event. The initial phase is attributable to the release of histamine, 5-HT and various kinins in the first hour injection of carrageenan. A more pronounced second phase is related to the release of prostaglandin-like substances in 2h32.

Inhibition of edema observed in inflammatory models in present study, may be attributed to the ability of Premna species to inhibit one or more of the phlogistic mediators, antagonizing their interaction with their respective receptors or it may be due to general mechanism like increasing the membrane stability in the cell. Among the two species, P. latifolia is having the property of suppressing both phases i.e., inhibition of histamine, kinin etc, release as well as suppression of prostaglandin-like substances, while P. obtusifolia is effective only in second phase.

Preliminary phytochemical analysis of drugs shows the presence of phenol and saponins33. Premnazole in leaves showed significant antiinflammatory activity in reducing cotton pellet-induced granuloma formation in rats23. The phyto-constituents like isoxazole alkaloid – Premnazole, flavonoid luteolin and β –sitosterol in P. obtusifolia, premnalin and flavone glycosides in P. latifolia may be responsible for observed activity profile.

However further evaluation on different models representing inflammation is necessary to know exact mechanism as well as to identify phyto-constituent responsible for it and also comparitive evaluation of roots and leaves of these sources on different activities reported for Agnimantha are needed to substantiate the substitution of roots with leaves.

Conclusion

Both the species of Premna are having significant anti-inflammatory activity and among the two species, P.latifolia was found to have better activity. Since ayurvedic classical literature supports the use of leaves of Agnimantha and leaves of both the source plants having shown significant anti inflammatory potential, there is a good chance that roots could be substituted by leaf. Further studies are needed to compare the roots and leaves in different activities attributed to Agnimantha for complete substitution of roots by leaves.