Immunomodulatory Activity of Aqueous Extract of Achillea Wilhelmsii C

Indian Journal of Experimental Biology Vol. 47, August 2009, pp. 668-671

Immunomodulatory activity of aqueous extract of Achillea wilhelmsii C. Koch in mice

Fariba Sharififar1, Shirin Pournourmohammadi2 & Moslem Arabnejad1 Department of1Pharmacognosy and 2Toxicology-Pharmacology, Research Center of Pharmaceutics, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran Received 4 December 2008; revised 16 April 2009

Immunomodulatory activity of aqueous extract of Achillea wilhelmsii (25, 50 and 100 mg/kg body weight for 5 days) was evaluated on body weight, relative organ weight, delayed type of hypersensitivity (DTH) response and haemagglutination titre (HT) in female Swiss albino mice. No significant body weight gain differences were recorded in various groups of animals. Significant increase in relative organ weight of spleen at 100 mg/kg was observed. No elevation in the levels of liver function test (LFT) enzymes and kidney relative weight was observed in tested doses of the plant. The extract of A. wilhelmsii elicited a significant increase in the DTH response at the dose of 100 mg/kg. In the HT test, plant extract showed stimulatory effect in all doses, however this changes were significant at 50 mg/kg. No mortality was occurred in tested doses. Overall, A. wilhelmsii showed a stimulatory effect on both humoral and cellular immune functions in mice.

Keywords: Achillea wilhelmsii, DTH, Haemagglutination titre, Immunity response, Toxicity

The immune system is involved in the etiology as C. Koch (Asteraceae), locally known as well as pathophysiologic mechanisms of many “Boomadaran” (Lavender cotton), is widely found in diseases. Modulation of the immune responses to different parts of Iran. Top flowerings of the plant are alleviate the diseases has been of interest for many used as antiflatulence, spasmolytic, antinociceptive years1. Medicinal plants are a rich source of and diuretic14. A. wilhelmsii contains volatile oils, substances which are claimed to induce flavonoids, terpenoids, alkaloids, saponins and paraimmunity, the non-specific immunomodulation of sesquiterpenlactones15. Antihypertensive and essentially granulocytes, macrophages, natural killer hypolipidemic effects of this plant have already been cells and complement functions2 Because of the reported16. However, there is no scientific data on the concerns about the side effects of conventional in vivo immunomodulatory activity of this plant. medicine, the use of natural products as an alternative Therefore the present study has been undertaken to to conventional treatment in healing and treatment of explore the immunomodulatory activity of various various diseases has been on the rise in the last few doses of aqueous extract of A. wilhelmsii top decades3. Medicinal plants serve as therapeutic flowerings in animal models. alternatives, safer choices, or in some cases, as the only effective treatment. A large number of these Materials and Methods plants and their isolated constituents have shown Plant extract ⎯ The plant was collected from beneficial therapeutic effects, including anti-oxidant, Bidkhoon, Kerman during July 2006. The plant was anti-inflammatory, anti-cancer, anti-microbial, and 4-10 identified and authenticated by Dr. Mirtajaldini, immunomodulatory effects . Some of the plants Bahonar University, Kerman, Iran. A voucher with established immunomodulatory activity are specimen of the plant materials (KP1155) was Viscum album, Panax ginseng, Asparagus racemosus, deposited at the Herbarium of Department of Azadirachta indica, Tinospora cordifolia, Polygala 11-13 Pharmacognosy of School of Pharmacy, Kerman senega, and Ocimum santum . Achillea wilhelmsii University of Medical Sciences, Kerman, Iran. ______Aqueous extract of the top flowerings of A. wilhelmsii *Correspondent author was obtained with maceration method and freeze Telephone: 00983413205001 Fax: 00983413205003 dried. The extracts with suitable adsorbent were E-mail: [email protected] stored in the refrigerator until the time of experiments. SHARIFIFAR et. al: IMMUNONODULATORY ACTIVITY OF ACHILLEA WILHELMSII EXTRACT 669

Animals ⎯ Swiss albino female mice (20 - 25 g) Delayed type of hypersensitivity response ⎯ The were bred and maintained under standard laboratory DTH response was determined using the method of conditions (25° ± 2 °C; 12:12 h (photoperiod). Raisuddin et al.18 with some modifications. On the Commercial pellet diet and water were given ad day of termination of the treatment, animals were libitum. All experiments performed were in compliance immunized with 1×109 SRBC, subcutaneously. On with the Kerman University of Medical Sciences, the next day, the animals were again received 1×108 Animal Experimentation Ethics Committee (AEEC) cells in the left hind footpad by injection. The same guidelines that follow Iran government regulations. volume of normal saline was injected to the right Dosage ⎯ The plant extract was suspended in footpad as trauma control for nonspecific swelling. normal saline and was administered i.p for 5 days at Increase in footpad thickness was measured 24 h after doses of 25, 50 and 100 mg/kg body weight. The dose the challenge using dial caliper. Dexamethasone volume was 0.2 ml. Control animals received the (0.2 mg/kg/day for 5 days) was used as positive same volume of normal saline. group. Body weight, lymphoid organ weight ⎯ Animals Liver function and blood parameters tests ⎯ were divided into four groups (I–IV). Each group Activities of serum glutamate oxalate transaminase comprised of a minimum of six animals. Group I (SGOT), serum glutamate pyruvate transaminase (control) received normal saline; groups II- IV (SGPT) and blood parameters (RBC, WBC and Hb) received plant extract in 25, 50 and 100 mg/kg body were estimated using the kits (Span Diagnostics, Surat, weight dose respectively. The animals were India). For this purpose, four groups of animals humanized 24 h after the last dose. Body weight gain (one control + three treatments) as described above were (percentage) and relative organ weight used and treated for 5 days with respective doses of (organ weight/100 g of body weight) of kidney, liver plant extract. Different blood parameters (RBC, WBC, and spleen were determined for each animal. Hb) were determined in four groups of animals (one Assessment of humoral immune functions ⎯ control+ three treatments) as described above. Animals of all the groups treated in the above- Statistical analysis ⎯ Statistical analysis was described manner were challenged with 0.2 ml of 10% performed using one-way ANOVA, followed by sheep red blood cells (SRBC), i.p on the 10th day of Tukey’s test. The significance in difference was initiation of experiment and the haemagglutinin titre accepted at P <0.05. The values are expressed as was studied as per Bin-Hafeez et al.17. means±S.E. Haemagglutinin titre (HT) assay ⎯ Haemagglutinin titre assay was performed using the procedure of Bin- Results Hafeez et al. 17. On the fifth day after immunization, Effect of the plant extract on body weight and animal was anesthesied and the blood was collected lymphoid organ weight ⎯ None of the studied doses of from cardiac of each mouse for serum preparation. A. wilhelmsii extract showed toxicity or mortality in the Serume was diluted in 50 μl PBS (pH 7.2) with twofold extract-treated animals. No significant difference in the serial dilution in 96-well microtitre plates and mixed body weight gain were recorded in various groups of with 50 μl of 1% SRBC suspension in PBS. Plates animals. The extract did not alter the relative weight of were kept at room temperature for 2 h. The value of kidney and liver in tested dose, however a significant antibody titre was considered the highest serum increase was observed in the relative weight of spleen dilution showing visible aemagglutination. in group IV (P<0.05) (Table 1).

Table 1 ⎯ Effect of different doses of aqueous extract of A. wilhelmsii on the relative organ weight (g) of mice. [Values are mean ± SE from 6 mice in each group].

Group Relative organ weight (g) Spleen Liver kidney A. wilhelmsii extract (25 mg/kg) 0.5 ± 0.09 4.9 ± 0.3 1.1± 0.2 A. wilhelmsii extract (50 mg/kg) 0.51± 0.08 5.2± 0.5 1.1 ± 0.1 A. wilhelmsii extract (100 mg/kg) 0.65± 0.16* 5.2 ± 0.7 1.1 ± 0.1 Control 0.37 ± 0.07 4.6 ± 0.6 1.1 ± 0.2 * P<0.05 when compared with control 670 INDIAN J EXP BIOL, AUGUST 2009

Effect of plant extract on humoral immunity test groups, however a significant decrease in RBC parameters ⎯ In haemmaglutination test, doses of 25, and Hb were observed (P<0.05) (Table 2). 50 and 100 mg/kg showed titre values of 1:4.0, 1:5.1 and 1:4.0, respectively, while the titre value of control Discussion was 1:2.0. The increase in the titre values was Boomadarn has primarily been described as an significant (P<0.05) in 50 mg/kg. anti-spasmodic and anti flatulence herb in traditional Effect of plant extract on cell-mediated immunity medicine. This plant has been suggested for treatment 16 parameters (DTH) ⎯ Plant extract at dose of of hypertension . In the present study, Boomadaran 100 mg/kg elicited a significant increase in DTH showed an overall stimulatory effect on the immune response in comparison to control animals (P<0.05) functions in mice. Stimulatory effects were observed (Fig.1). In this experiment, dexamethasone has on both humoral and cellular immunity. In HT test, decreased DTH response significantly in comparison the plant showed an increase response in all doses, but to control (P<0.05). this increase was significant only in dose 50mg/kg. Effect of plant extract on liver enzymes and blood This activity could be due to the presence of parameters ⎯ There was no significant elevation in flavonoids or saponins which augment the humoral the levels of SGOT and SGPT as a result of treatment response, by stimulating the macrophages and with A. wilhelmsii at any of the doses used in this B-lymphocytes subsets involved in antibody 19 study (P<0.05) (Table 2). No significant differences synthesis . It appears that 50 mg/kg is the optimum in blood parameters and WBC were recorded between dose in mice in humoral immunity. An increase in dose might have induced down-regulation of immune functions. Estimation of the LFT enzymes did not reflect any toxicity which was concomitant with any significant increase in relative weight of liver. All doses of the plant decreased the SGPT, however this difference was significant in 100 mg/kg dose which may be due to the hepatoprotective effect of the plant. Results of the present study also revealed no significant difference in the blood parameters. In DTH test, the DTH response, which directly correlates with cell-mediated immunity (CMI), was found to be the highest at the maximum dose (100 mg/kg) tested in the extract. The mechanism behind this elevated DTH during the CMI responses could be due to sensitized T-lymphocytes. When challenged by the antigen, they are converted to lymphoblast and secrete a variety of molecules Fig. 1⎯ The effects of various doses of aqueous extract of A. including proinflammatory lymphokines, attracting wilhelmsii on delayed type hypersensitivity (DTH) response in more scavenger cells to the site of reaction20. An mice immunized with sheep red blood cells in comparison to increase in DTH response indicates a stimulatory dexamethasone and control [values are mean ± SE of footpad thickness from 6 mice in each group. * P<0.05 when compared effect of the plant which has occurred on the with the control animals] lymphocytes and accessory cell types required for the

Table 2 ⎯ The effect of aqueous extract A. wilhelmsii on liver enzymes, WBC and blood parameters. [Values are mean ± SE from 6 mice in each group].

Group Treatment SGPT(IU/L) SGOT(IU/L) WBC(103/mm3) Hb(g/dL) RBC(106/mm3) I A. wilhelmsii extract (25 mg/kg) 50.2 ± 17.4 141.7 ± 39 4.8 ± 1.0 11.8 ± 1.4* 7.8 ± 0.6* II A. wilhelmsii extract (50 mg/kg) 44.6 ± 25.6 115.0 ± 29.2 3.3 ± 1.1 13.8 ± 0.7 9.0 ± 0.7 III A. wilhelmsii extract (100 mg/kg) 42.9 ± 25* 107.8 ± 25.5 4.6 ± 2.4 13.7 ± 1.3 9.0 ± 0.7 IV Control 72.8 ± 17 106.6 ± 20 4.9 ± 0.6 15.0 ± 1.6 9.7± 1.3 *P<0.05 when compared with control animals (significantly different) SHARIFIFAR et. al: IMMUNONODULATORY ACTIVITY OF ACHILLEA WILHELMSII EXTRACT 671

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