Assessment of anti-nociceptive efficacy of costus speciosus rhizome in swiss albino mice.

Present study attempts to evaluate the anti-nociceptive activity of the aqueous and ethanol extracts of Costus speciosus rhizome (CPA and CPE) in Swiss albino mice. The maceration extracts were evaluated for anti-nociceptive activity by acetic acid-induced writhing and tail flick method in mice. The anti-nociceptive screening revealed significant peripheral anti-nociceptive actions of both extracts against acetic acid induced writhing in mice. Aqueous extract (CPA) significantly inhibited writhes at the dose of 75 and 150 mg/kg body weight, while ethanol extract (CPE) produced significant protection at the dose of 150 mg/kg body weight. However, in tail flick method only the ethanol extract (CPE) showed significant central analgesic action, while aqueous extract was totally ineffective. The present investigation demonstrates that the rhizome extracts of C. speciosus exhibited significant anti-nociceptive effects in Swiss albino mice.

Introduction

Costus speciosus (J. Konig) Smith (Zingiberaceae), called Kembuka in Sanskrit, wild ginger in English is an erect plant, up to 2.7 meters high; root stock tuberous; stem sub-woody at the base occurring in the moist and wet evergreen areas of the Indo-Malayan region and Sri Lanka. Within India it occurs from Central and Eastern Himalayas to Southern India. Rhizomes have anti-fertility, anabolic properties. Traditionally, it is indicated in the treatment of cough, fever, skin diseases, snake bite, anemia and inflammation. The rhizomes also possess anti-cholinesterase, anti-inflammatory, stimulant, depurative and anthelmintic effects[1]. The juice of fresh tips of young branches is instilled in case of otitis[2]. It is used mixing with sugarcane juice along with other herbs to cure jaundice[3]. It is also used in arthritis and applied as a paste[4]. Its rhizome contains a complex mixture of saponins viz. dioscin, gracillin and β-sitosterol-β-D-glucoside. Tigogenin and diosgenin from rhizomes and stems have been isolated. Also, α-amyrin stearate, β-amyrin and lupeol have been isolated from its rhizomes. Isolation of palmitates has been reported from leaves. The seed fat contains palmitic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, gadoleic acid, and behenic acid. Two new quinines dihydrophytilplastoquinone and its methyl derivatives along with α-tocopherolquinone have been isolated from seeds. It is also reported to isolate diosgenone, prosapogenin B of dioscin, cycloartanol, 25-en-cycloartenol and octacosanoic acid along with diosgenin from the rhizome[56]. Previous workers reported several pharmacological studies on this plant[7-10]. The present study was undertaken to evaluate the anti-nociceptive activity of C. speciosus rhizome in Swiss albino mice.

Materials and Methods

Plant material

The rhizomes of mature plants were collected from places in and around Dibrugarh, Assam, India during the month of October-November 2007 and identified as Costus speciosus (J. Konig) Smith (Zingiberaceae), vide authentication letter no. BSI/EC/Tech./2007/581 by Dr. T. M. Hynniewta, taxonomist at Botanical Survey of India, Eastern Circle, Shillong, India. A voucher specimen herbarium of the said plant was preserved for future reference. The rhizomes were first washed thoroughly under running water to remove traces of soil particles adhered to it. Then it was subjected to shed drying at temperature 21-24 °C for about three to four weeks to remove the resident moisture. After this the rhizomes were cut to small pieces and ground mechanically into a coarse powder.

Drugs and Chemicals

The following drugs and chemicals were obtained from the sources specified: Glacial acetic acid, from Ranbaxy Fine Chemicals Ltd., New Delhi; Ibuprofen from Recon, Bangalore, India; Morphine sulphate, from Sigma Chemical Co., USA. All other chemicals were of analytical grade obtained commercially. Double-distilled water from all-glass still was employed throughout the study.

Preparation of extracts

The powdered plant materials were extracted with distilled water and 95% v/v ethanol separately by maceration at room temperature (24-26°C) for 7 days. Then the extracts were filtered and concentrated using rotary vacuum evaporator at 45°C. The semisolid mass thus obtained were stored in desiccator until further use. The extracts were denoted as CPE (ethanol extract) and CPA (aqueous extract).

Animals

Adult male albino mice of Swiss strain weighing 20 ± 2 g were procured from registered breeders (Rita Ghosh & Co., Kolkata) and maintained under standard laboratory conditions (temperature 25 ± 2°C with dark and light circle 14/10 h). They were allowed free access to standard dry pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The mice were acclimatized to laboratory condition for 10 days before commencement of the experiment.

Acetic acid-induced writhing test [11]

The animals were divided into six groups (n = 8). The first group of animals (which served as control) received 0.9% normal saline 10 ml/kg body weight p. o. The second group of animals (which served as standard) received ibuprofen at a dose of 50 mg/kg body weight p. o. The remaining four groups received the test extracts (CPE, CPA) at the doses of 75 mg and 150 mg/kg body weight p. o. respectively.

30 Minutes after administration of normal saline, ibuprofen and test extracts acetic acid was administered (1 % v/v, 0.2 ml, irrespective of body weight, i. p.) to all groups. The number of writhing was noted during a period of 10 minutes.

The mean writhing scores in each group were calculated and expressed the percentage of protection using the following formula:-

(Control mean - Treated mean/ Control mean) ×100 %.

Tail flick test [12]

Before treatment the basal reaction time for each mouse to radiant heat (Analgesiometer, Techno) was determined by placing the tip of the tail on the radiant heat source (nicrome wire). The strength of the current passing through the naked nicrome wire was kept constant at 6 Amps. The tail withdrawal time in seconds from the heat (flicking response) was considered as nociceptive end point. Any mouse failing to withdraw its tail within 3-5 seconds was excluded from the study. The prescreened animals (reaction time: 3-4 sec) were divided into six groups (n = 8). The first group (which served as control) received 0.9% normal saline 10 ml/kg body weight p.o. The second group (which served as standard) received morphine sulphate at a dose of 5 mg/kg body weight s.c. The remaining four groups received the test extracts (CPE, CPA) at the doses of 75 mg and 150 mg/kg body weight p.o. respectively. The reactions of all groups of mice were determined after 30, 60 and 120 minutes by tail flick method similarly as mentioned above and the latency times (in seconds) were recorded. The mean latency times for each group were calculated.

Statistical analysis

The experimental values were expressed as mean ± standard error of mean (SEM) and statistical significance was analyzed by one-way ANOVA followed by Dunnett's post hoc test of significance. p < 0.05 was considered as statistically significant.

Results

Acetic acid-induced writhing test

The results of acetic acid induced writhing are summarized in Table 1. The ethanol extract showed significant (p < 0.05) inhibition of writhes, at a dose of 150 mg/kg body weight. The aqueous extract showed significant and dose dependent protection at the dose of 75 and 150 mg/kg body weight (p < 0.05 and p < 0.01 respectively) as compared with control group.

Table 1

Influence of C. speciosus on acetic acid-induced writhing in mice

Tail flick test

The results of tail flick test are shown in Table 2. Only the ethanol extract at the dose of 150 mg/kg exhibited significant (p < 0.01, after 60 mins and p < 0.05, after 120 mins) increase in reaction time. Peak analgesic effect was observed after 60 min.

Table 2

Influence of C. speciosus on tail flick test in mice

Discussion

The anti-nociceptive efficacy of C. speciosus extracts was evaluated by both acetic acid induced writhing method and tail flick method in mice to assess peripheral (non-narcotic) and central (narcotic) type of activity respectively[13].

Acetic acid induced writhing is chemically induced nociception by intraperitonial injection of dilute acetic acid solution to mice. The chemical agents can produce nociceptive reactions in mice. Intraperitonial injection of phenyl para quinone, bradykinin or dilute acetic acid (1-3 % v/v) produces pain reaction that is characterized as writhing response. Constriction of abdomen, turning of trunk (twist) and extension of hind limbs (at least one) are considered as writhing reaction to chemically induced pain[1314].

Acetic acid induced writhing test is known as a visceral pain model nociception. Several mediators like kinins, acetylcholine, substance P, calcitonin-gene-related peptide and prostaglandins (PG) take part in visceral pain model nocicepotion and transmission of the nociception from the viscera18. In this test both central and peripheral analgesics are detected. Analgesics of both narcotic (central) e. g. morphine, pentazocin, pethidine and non--narcotic (peripheral) type, e.g. aspirin, ibuprofen, indomethacin can inhibit the writhing response in mice[1314].

The demonstration of significant peripheral anti-nociceptive actions by aqueous extract of stem bark (p < 0.05, at 75 mg/kg, p < 0.01 at 150 mg/kg) can provide a scientific basis for its traditional uses. The ethanol extract (CPE) also showed significant (p < 0.05, at 150 mg/kg) peripheral anti-nociceptive actions and hence, supporting its traditional uses.

The tail flick test is thermally induced nociception model where radiant heat is used as a source of pain. Here, radiant heat (through a hot nicrome wire) is applied to the tail of mice and the withdrawal of tail from the radiant heat source (hot nicrome wire) is considered as flicking response to thermally induced pain. The flicking reaction which is the end point of this test may be mediated as a spinal reflex. Analgesics of only central (narcotic) type, e.g. morphine, pethidine, pentazocine etc can increase the tail flick latency period indicating anti-nociception[1315].

The results of tail flick test clearly indicated that the ethanol extract of stem bark had significant central (narcotic) anti-nociceptive action that was absent in aqueous extract; but the extent of central analgesic effect was much lower than that of morphine sulfate. This means that the ethanol extract of C. speciosus rhizome exerted anti-nociceptive activity involving both peripheral and central mechanisms, whereas the aqueous extract inhibited only the peripheral pain mechanisms in mice.

Conclusion

From the present preliminary study it can be concluded that both the extracts of C. speciosus rhizome possessed significant anti-nociceptive efficacy in Swiss albino mice, the aqueous extract being only peripherally active and the ethanol extract being active both centrally and peripherally. Purification of the extracts and further studies can reveal the exact mechanisms behind the anti-nociceptive effects of C. speciosus rhizome.