ANTI-INFLAMMATORY, ANTI-ARTHRITIC AND ANALGESIC ACTIVITY OF THE ALCOHOLIC EXTRACT OF THE PLANT URGINEA INDICA KUNTH.

ANTI-INFLAMMATORY, ANTI-ARTHRITIC AND ANALGESIC ACTIVITY OF THE ALCOHOLIC EXTRACT OF THE PLANT URGINEA INDICA KUNTH.

Md. Mominur Rahman ¹, Jakir Ahmed Chowdhury*², Razibul Habib ¹, Barun Kanti Saha ³, A.D.M Salauddin ⁴ and Mohammad Kaisarul Islam ²

Department of Pharmacy, International Islamic University ¹, Chittagong, Bangladesh

Faculty of Pharmacy, University of Dhaka ², Dhaka-1000, Bangladesh

Institute of Food Science and Technology, Bangladesh Council of Scientific and
Industrial Research (BCSIR) ³, Bangladesh

Department of Pharmacy, University of Science and Technology ⁴, Chittagong, Bangladesh

ABSTRACT

This study was designed to present the observation of the pharmacological properties to the bulb of the plant. The extract of the bulb of Urginea indica Kunth. were collected by using of alcoholic extraction. The anti-inflammatory action of the alcoholic Extract of the bulb of the plant Urginea indica was evaluated in rats (female) against carrageenan induced edema i.e., using plethysmographic method. Besides this method, this extract was also assessed for Cotton pellet test and Hot plate test for anti-inflammatory and analgesic effects respectively. The effects of the extract were compared with the classical anti-inflammatory drug - Ibuprofen. The crude extract and the standard drug were orally administered. A significant anti-inflammatory effect was produced with the   Alcoholic Extract of the plant part. This effect was then compared with the effect from the classical anti-inflammatory drug.

Analgesic activity

INTRODUCTION: Urginea indica Kunth., Fam: Liliaceae (bengali: banpiaaj, janglipiaaj), is a perennial plant with fibrous roots proceeding from the base of a large, tunicated, nearly globular bulb, 4 to 6 inches long, the outer scales of which are thin and papery, red or orange-brown in color. It is also known as Maritime Squill, Scilla maritima (Linn.), Urginea maritime, White-squill, Red-squill ¹. The flowers are in bloom in April and May and are followed by oblong capsules. It is a very variable plant, the bulb differing greatly in size and color, and the leaves of the flower presenting similar varieties, which has led to the formation of several species, about twenty-five species having been described. Two varieties of Squill, termed respectively white and red, are distinguished by druggists.  In the first named, the bulb scales are whitish or yellowish in color, whereas the red species has deep, reddish brown outer scales and yellowish white inner scales,    covered   with   a   pinkish   epidermis,    intermediate   forms   also occurring. No essential difference exists in the medicinal properties of the two kinds.  Merck, in 1879, separated the three bitter glucosidal substances Scillitoxin, Scillipicrin and Scillin.

Other constituents are mucilaginous and saccharine matter, including a peculiar mucilaginous carbohydrate named Sinistrin, an Inulin-like substance. The bulb has antifungal ^{2, 3}, antiangiogenic and proapoptotic activity ⁴, asthma ⁵, emmenagogue,   antihelmintic,   purgative,   alexiteric, useful   in paralysis, bronchitis, dropsy, rheumatism, renal calculi, leprosy, skin diseases, headache, disease of nose, internal pains, scabies ⁶. Three novel flavonoid glycosides, 5,6-dimethyoxy-3', 4''- dioxymethylene- 7- O- (6''- beta- D- glucopyranosyl-beta-D-glucopyranosyl) flavanone, 5, 4'-dihydroxy-3-O-alpha-L-rhamnopyranosyl-6-C-glucopyranosyl-7-O-(6''-para-coumaroyl-beta-D-glucopyranosyl) flavone and 5, 4'-dihydroxy-3-O-(2''''' - beta-glucopyranosyl-alpha-L-rhamnopyranosyl)- 6- C- glucopyranosyl- 7- O- (6''- para-coumaroyl- beta- D- glucopyranosyl) flavone were isolated from the 1-butanol soluble fraction of the bulbs of the plant Urginea indica ⁷.

MATERIALS AND METHOD: The whole plant of Urgenia indica was weighed 39 kg. The leave and roots were cut out from the bulb of plant and then the weight was 24 kg. The bulb of the plant was sliced transversely and was subjected to air dry for four days. The air dried sliced pieces were then transferred into oven for drying. After final drying the weight was found 9 kg. Oven dried crispy pieces were finally crushed to form powder. After crushing the weight was found 4.5 kg. The plant was collected from Joydevpur in August 2009 and was taxonomically identified by a Scientific Officer, Bangladesh National Herbarium (BNH) and one voucher specimen has been deposited there.

The experiments were carried out on albino rates (Swiss Strain) were obtained from Animal House of BCSIR, Chittagong. 150-200g rats were used. The rats were housed in plastic cages. They were maintained at room temperature under conditions of natural light and dark schedule. The rats were fed with Vat chow - prepared according to the formula developed by the Bangladesh Council for Scientific and Industrial Research (BCSIR), Chittagong. They were allowed to drink normal water; the drug and extract were administered with the help of feeding needle.

In our study, extraction of dried and powdered plant (4.5 kg) of U. indica was done by cold extraction process by using methanol as a solvent ⁸. The air dried and pulverized plant material (141.86 gm) was cold extracted with methanol. And after that the fractions were evaporated by roto-dryer at low temperature (40- 50°C) to dryness. Crude alcoholic extract was subjected to anti-inflammatory activity, anti-arthritic activity and analgesic activity study. Ibuprofen was bought from retail pharmacy whose strength was checked within the laboratory and used as standard. Solutions of the drug were prepared according to various dosages regimens of distilled water. Doses of Ibuprofen were selected as reported in different literature and pilot experiments were carried out in the study. All drugs were administered orally.

Among the many methods used for screening and evaluation of anti-inflammatory activity, the most commonly employed techniques is based upon the ability of such agents to inhibit the acute inflammatory edema produced in the hind paw of the rat following injection of a phlogistic agent, like Carrageenan; a mucopolysaccharide derived from Iris sea moss, Chondrus carrageenin (Sigma, Japan) was prepared as 0.1% solution in water for injection.

Method for the study on Inflammatory Activity: Acute inflammation was induced as described by Winter et al ⁹. A volume of 0.05 ml 1% carrageenan was injected into the planter aponeurosis of the right hind paw of the rats. The rats were orally administered with solution of the extract or Ibuprofen and 1 hour prior to carrageenan injection to observed preventive effects. The volumes of the treated paw of the experimental rats were measured with Plethysmometer Cat. No.-7140. Measurements were recorded immediately after carrageenan injection and then on hourly basis during the study period. The mean increase in volume of the injected paw of each group at 0, 1^st, 2^nd, 3^rd and 4^th hour after carrageenan injection was calculated to observe the activity of standard and the extract. For standard drug testing, increase in paw-volume 4 hours after carrageenan administration was considered as a measurement of effect. The percentage of edema reduction with test drug and extracts were calculated and recorded for comparison against the control group.

Method for the study of Anti-Arthritic Activity: The method was based on that of Meir et al. ¹⁰ and was carried out male rats weighing 150-160 gm with some minor modification. Surgical grade cotton was rolled in pellet (10±0.5 mg) and sterilized at 120°C for two hours in an electric oven. These were soaked in 1% solution of crystalline penicillin just before use. The cotton pellets were implanted at the plantar surface under skin of abdomen. Treated animals received the test drugs one hour before the implantation and thereafter daily for 7 days.

Control animals received the vehicle only. On eight day rats were sacrificed and pellets with granulomatous tissues over them were recovered. These were cut into two part and dried to a constant weight at 60°C for 24 hours and carefully reweighed. Mean increase in weight of the pellets for control and animals were calculated.

Method for the study of Analgesic Activity: The method was originally described by Woolfe and Mac Donald (1944) has been modified by several investigators ^11-13 where the study was conducted on mice by the "Hot-Plate" (Scorel - DS37, UGO Basile, Italy). Hot plate was maintained at a constant temperature of 55.0±0.5°C. Each mouse was placed on the hot surface and the time of response of this thermal stimuli, indicated by the licking of hind and/or for paws or by licking of the legs or by trying to jump out, was recorded and the results were compared with that of control group. All data from each treated and control group was analyzed by using t-test.

RESULT AND DISCUSSION:

Anti-inflammatory Activity Study: Twenty rats were selected for total study and all the rats were classified into four groups as – Gr-A (Normal control: Rats with no drug and served as normal control), Gr-B (Anti-inflammatory control: 3.0 ml of water is given orally. After one hour this group was served with carrageenan solution (1%) at the rate of 0.5 mg/kg body weight, injected into the planter aponeurosis), Gr-C (Sample treatment: 3.0 ml of alcoholic extract of Urginea indica (at dose of 1.5 gm/kg, oral) was given to each rat. After one hour this group was served with carrageenan solution (1%) at the rate of 0.5 mg/kg body weight, injected into the planter aponeurosis) and Gr-D (Drug treatment: 3.0 ml Ibuprofen at dose of l.5 gm/ kg was given to each rat orally. After one hour each rat of this group was served with carrageenan solution (1%) at the rate of 0.5mg/kg body weight, injected into the planter aponeurosis and served as positive control). The results are depicted in table 1, fig. 1 & 2.

TABLE 1: ANTI-INFLAMMATORY EFFECT OF URGINEA INDICA

n = 05; AEUI means Alcoholic Extract of U. Indica and Ibuprofen is standard drug. Results are expressed as Mean ± SEM. Unpaired t-test was performed as the test of significance. **p<0.002, ***p<0.001.NB: As value of t is greater than p0.00l. Hence, difference is highly significance at level (p<0.001) 85 significance at level (p<0.005).

FIG. 1: DETERMINATION OF ANTI-INFLAMMATORY ACTIVITY BY CARRAGEENAN INDUCED ANTI-INFLAMINATION TEST

FIG. 2: ANTI-INFLAMATORY ACTIVITY TEST ON THE BASIS OF % INHIBITION

Anti-arthritis activity test by Cotton Pellet Method: Eighteen rats were selected for the study and grouped into equal three classes as- Gr-A (Anti-inflammatory control: 3.0 ml of water is given orally. After one hour this group was served with carrageenan solution (1%) at the rate of 0.5 mg/kg body weight, injected into the planter aponeurosis), Gr-B (Sample treatment: 3.0 ml of alcoholic extract of Urginea indica (at dose of 1.5 gm/kg, oral) was given to each rat.

After one hour this group was served with carrageenan solution (1%) at the rate of 0.5 mg/kg body weight, injected into the planter aponeurosis) and Gr-D (Drug treatment: 3.0 ml Ibuprofen at dose of l.5 gm/ kg was given to each rat orally. After one hour each rat of this group was served with carrageenan solution (1%) at the rate of 0.5mg/kg body weight, injected into the planter aponeurosis and served as positive control). The results are depicted in table 2, fig. 3 & 4.

TABLE 2: INHIBITION OF GRANULOMATOUS TISSUE GROWTH ON COTTON PELLETS IN RATS

n = 6; AEUI means Alcoholic Extract of U. Indica and Ibuprofen is standard drug. Results are expressed as mean ± SEM. Unpaired t-test was performed as the test of significance. ***p<0.001

FIG. 3: COMPARISON OF ANTI-ARTHRITIS ACTIVITY

FIG. 4: ANTI-ARTHRITIS ACTIVITY ON THE BASIS OF % INHIBITION

Analgesic activity test by Hot Plate Method: Thirty rats were selected for the study and grouped into equal three classes as- Gr-A (Anti-inflammatory control: 3.0 ml of water is given orally. After one hour this group was served with carrageenan solution (1%) at the rate of 0.5 mg/kg body weight, injected into the planter aponeurosis), Gr-B (Sample treatment: 3.0 ml of alcoholic extract of Urginea indica (at dose of 1.5 gm/kg, oral) was given to each rat.

After one hour this group was served with carrageenan solution (1%) at the rate of 0.5mg/kg body weight, injected into the planter aponeurosis) and Gr-D (Drug treatment: 3.0 ml Ibuprofen at dose of l.5 gm/kg was given to each rat orally. After one hour each rat of this group was served with carrageenan solution (1%) at the rate of 0.5 mg/kg body weight, injected into the planter aponeurosis and served as positive control). The results are depicted in table 3, fig. 5.

  TABLE 3: ANALGESIC ACTIVITY STUDY ON MICE BY HOT PLATE TEST

n=10; AEUI means Alcoholic Extract of U. Indica and Ibuprofen is standard drug. Results are expressed as mean 11.65± SEM. Unpaired t-test was performed as the test of significance. **p<0.005, ***p<0.001. N.B: As value of is greater than t_0.001. Hence, difference is highly significance at level (p<0.001).

FIG. 5: COMPARISON OF ANALGESIC EFFECT BY HOT PLATE METHOD

CONCLUSION: In this experiment it has been shown that the alcoholic extract of the bulb of Urginea indica has high anti-inflammatory activity, good anti-arthritic activity and moderately good analgesic effects. Alcohol is hydrophilic in nature so the extract easily mixed with water. The observation made by the experiment as - inhibition of carrageenan induces edema in rats and comparative result with anti-inflammatory effect shows that the root of Urginea indica, the extract has fast onset of action and is more effective in early period after administration, the bulb of Urginea indica (alcoholic extract) produce significant anti-arthritic activity, in this experiment it has been shown that the analgesic effect is not better than other two effects.

 REFERENCE

1. Deb DB, Dasgupta S: Studies on Indian squill Urginea-indica. Quarterly Journal of Crude Drug Research 1976; 14: 49-60.
2. Sandhya R, Shenoy MN, Shiva K, Swaminathan S, and Gupta MN: Major Antifungal Activity from the bulbs of Indian Squill Urginea indica is a chitinase. Biotechnology Progress 2006; 22(3): 631–637. DOI: 10.1021/bp050305n
3. Deepak AV, Thippeswamy G, Shivakameshwari MN, Salimath BP: Isolation and characterization of a 29-kDa glycoprotein with antifungal activity from bulbs of Urginea indica. Biochem Biophys Res Commun 2003; 311(3):735-42.
4. Deepak AV, Salimath BP: Antiangiogenic and proapoptotic activity of a novel glycoprotein from indica is mediated by NF-kappaB and Caspase activated DNase in ascites tumor model. Biochimie 2006; 88(3-4): 297-307.
5. Marx J, Pretorius E, BesterMJ: Effects of Urginea sanguinea, a traditional asthma remedy, on embryo neuronal development. J Ethnopharmacol 2006; 104(3): 315-21
6. Bangladesh National Formulary of Unani Medicine. Edited by The Bangladesh Unani and Aurvedic Board, Dhaka, Bangladesh.
7. Sultana N, Akter K, Nahar N, Khan MS, Mosihuzzaman M, Sohrab MH, Krohn K: Novel flavonoid glycosides from the bulbs of Urginea indica Nat Prod Res 2010; 24(11): 1018-26.
8. Trease GE, Evans WC: Trease and Evans' Pharmacognosy, 13th ed. London; Philadelphia: Baillire Tindall 1989.
9. Winter CA, Risley EA, Nuss W: Carrageenan-induced edema in hind paws of the rat as an assay for anti-inflammatory drugs. Proc Soc Exp Biol Med 1962; 111: 544-547.
10. Meier R, Schuler W, Desaulles P: Zur Frage des Mechanismus der Hemmung des Bindegewebswachstums durch Cortisone. Experientia 1950; 6: 469-471.
11. Woolfe G, Donald MA: J Pharm Exp Ther 1944; 80: 300-7.
12. Turner RA: Analgesics. In: Turner RA and Hebborn P. (Eds.) Screening Methods in Pharmacology. Academic Press, New York 1960; 100-102
13. Paget GE, Barnes JM, Laurance DR, Bachrach AC: Evaluation Drug Activities and Pharmacokinetics. New York Academic Press Vol. 1, 1983.
    

    ---