membrane stabilizing activity - a possible mechanism ...

Rashid et al., IJPRD, 2011; Vol 3(3): May 2011 (1 - 5)

International Standard Serial Number 0974 – 9446

[Research Article]

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MEMBRANE STABILIZING ACTIVITY - A POSSIBLE MECHANISM OF ACTION FOR THE ANTIINFLAMMATORY ACTIVITY OF TWO BANGLADESHI MEDICINAL PLANTS: MESUA NAGASSARIUM (BURM.F.) AND KIGELIA PINNATA (JACK) DC. Mohammad A. Rashid2*,Md. Al Amin Sikder 1, Mohammad. A. Kaisar2, Md. Kowser Miah1, Md. Masud Parvez1, A.K.M. Nawshad Hossian1, 1

Department of Pharmacy, State University of Bangladesh, Dhaka-1205, Bangladesh Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh Email:[email protected]

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ABSTRACT In this study, the methanol extracts and their different organic soluble fractions i.e. pet-ether, carbon tetrachloride, chloroform and aqueous soluble fractions of Mesua nagassarium and Kigelia Pinnata were subjected to study for membrane stabilizing activity. The extractives inhibited heat- as well as hypotonic solution-induced haemolysis of erythrocytes in vitro. The pet-ether soluble fraction (PESF) of M. nagassarium inhibited 49.19 % and 23.73% whereas petether soluble fraction (PESF) of K. pinnata demonstrated 65.7% and 32.03% inhibition of hemolysis of RBC caused by hypotonic solution and heat, respectively. Here acetyl salicylic acid was used as reference standard at 0.10 mg/mL.

Correspondence to Author

Dr. Mohammad Abdur Rashid Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh Email [email protected] Key Words Mesua nagassarium, Kigelia Pinnata, membrane stabilizing activity.

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International Journal of Pharmaceutical Research & Development

INTRODUCTION Mesua nagassarium (Bengali - nagesar, nageswar, Family- Clusiaceae) is a medium-sized or fairly large evergreen flowering tree up to 36 m tall. A mixture of pounded kernels and seed oil isolated from this plant is used for poultice as wounds. The seed-oil is used for treating itch and other skin eruptions, dandruff and rheumatism. The flowers are known to be useful for the treatment of severe colds, bleeding haemorrhoids, dysentery with mucus, excessive thirst, excessive perspiration, cough and digestion, rheumatism and tumors iron induced lipid peroxidation1,2. On the other hand plants Kigelia Pinnata belonging to Bignoniaceae family, is a flowering plant and used against dysentery, venereal diseases and as a topical application on wounds and abscesses. The bark of this plant is used in the treatment of venereal diseases and has a reputation for the treatment of dysentery3. The erythrocyte membrane resembles to lysosomal membrane and as such, the effect of drugs on the stabilization of erythrocyte could be extrapolated to the stabilization of lysosomal membrane4. Therefore, as membrane stabilizes that interfere in the release and or action of mediators like histamine, serotonin, prostaglandins, leukotrienes etc.5. Previously K. Pinnata was reported as anti inflammatory agent in both acute and chronic inflammatory models6 while xanthones isolated from M. nagassarium when given orally or intraperitonially have been shown to exhibit significant anti-inflammatory activity in normal as well as adrenolectomised rats7. Results of these studies indicated the possibility that methanol extracts of the leaves of both plants and their different partitionates interfered in the release and/or action of mediators like histamine, serotonin, prostaglandins, 5 leukotrienes, and etc. MATERIALS METHODS Plant materials The leaves of M. nagassarium and K. Pinnata were collected from Dhaka and Voucher specimen (DACB 35158 and 34998, respectively) for each of the Available online on www.ijprd.com

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plant sample has been deposited in Bangladesh National Herbarium for future reference. The leaves were sun dried for several days and then oven dried for 24 hours at considerably low temperature (not more than 400C) for better grinding. The powdered materials of M. nagassarium and K. Pinnata (500 gm each) were macerated in 2.5 L and 2.0 L of methanol, respectively for 7 days and then filtered through a cotton plug followed by Whatman filter paper number 1. The extracts were concentrated with a rotary evaporator at low temperature (40-45 ºC) and reduced pressure. The concentrated methanolic extracts were partitioned by modified Kupchan method8 and the resultant partitionates i.e., pet-ether (PESF), carbon tetrachloride (CTCSF), chloroform (CSF), and aqueous (AQSF) soluble fractions were used for the experimental processes for both plants. Effect on hemolysis Erythrocyte suspension Whole blood was collected from rats under ether anesthesia and heparin was added to prevent clotting. The blood was washed three times with 0.9% saline. The volume of saline was measured and reconstituted as a 40% (v/v) suspension with isotonic buffer solution (pH 7.4) which contained in 1 L of distilled water: NaH2PO4. 2H2O, 0.26 g; Na2HPO4, 1.15 g; NaCl, 9 g (10 mM sodium phosphate buffer). Hypotonic solution-induced hemolysis The membrane stabilizing activity of the extract was assessed by using hypotonic solution induced mice erythrocyte hemolysis designed by Sikder et al., (2010)9. To prepare the erythrocyte suspension whole blood was obtained using syringes (containing anticoagulant EDTA) from mice through cardiac puncture. The blood was centrifuged and blood cells were washed three times with solution (154 mM NaCl) in 10 mM sodium phosphate buffer (pH 7.4) through centrifugation for 10 min at 3000 g. The test sample consisted of stock erythrocyte (RBC) suspension (0.50 mL) mixed with 5 mL of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffered saline (pH 7.4) containing either the extract (2.0 mg/mL) or acetyl salicylic acid (0.1 mg/mL). The control sample consisted of 0.5 mL of 2


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RBCs mixed with hypotonic-buffered saline alone. The mixture was incubated for 10 min at room temperature, centrifuged for 10 min at 3000 g and the absorbance of the supernatant was measured at 540 nm. The percentage inhibition of either hemolysis or membrane stabilization was calculated using the following equation% Inhibition of hemolysis = 100 x (OD1-OD2/OD1) where, OD1 = Optical density of hypotonic-buffered saline solution alone (control) and OD2 = Optical density of test sample in hypotonic solution

produced 49.19 % and 73.19% inhibition of hemolysis of RBC caused by hypotonic solution and acetyl salicylic acid (0.10 mg/mL), respectively. The chloroform (CSF) and carbon tetrachloride (CTCSF) soluble extractives also revealed significant inhibition of hemolysis of RBC (Table 1). On the other hand, at a concentration of 2.0 mg/mL, the pet ether (PESF) and aqueous (AQSF) soluble partitionates of methanol extract of K. pinnata demonstrated 65.7% and 56.48 % inhibition of hemolysis of RBC induced by hypotonic solution, respectively (Table 2).

Heat-induced hemolysis Aliquots (5 ml) of the isotonic buffer containing 2.0 mg/mL of different extractives of both plants were put into two duplicate sets of centrifuge tubes5. The vehicle, in the same amount, was added to another tube as control. Erythrocyte suspension (30 µL) was added to each tube and mixed gently by inversion. One pair of the tubes was incubated at 54oC for 20 min in a water bath. The other pair was maintained at 0-5oC in an ice bath. The reaction mixture was centrifuged for 3 min at 1300 g and the absorbance of the supernatant was measured at 540 nm. The percentage inhibition or acceleration of hemolysis in tests and was calculated according to the equation: % Inhibition of hemolysis = 100 x [1- (OD2-OD1/ OD3OD1)]

Besides the pet-ether soluble partitionate of methanol extract of fruits of M. nagassarium at concentration 2.0 mg/ml, protected 23.73% the lysis of human erythrocyte membrane whereas pet-ether soluble partitionate of methanol extract of leaves of K. pinnata protected 32.03% lysis of human erythrocyte membrane induced by heat, as compared to the standard acetyl salicylic acid (30.23% at conc. 0.10 mg/mL). At a concentration of 2.0 mg/mL, the aqueous soluble fraction (AQSF) of methanol extract of leaves of K. pinnata exerted 28.55 % inhibition of haemolysis of RBC. To confirm the membrane stabilizing activity of both M. nagassarium and K. pinnata observed in the above mentioned models, experiments were performed on the erythrocyte membrane. A possible explanation for the stabilizing activity of the extractives due to an increase in the surface area/volume ratio of the cells which could be brought about by an expansion of membrane or shrinkage of the cell, and an interaction with membrane proteins10. The present investigation suggests that the membrane stabilizing activity of M. nagassarium and K. pinnata may be playing a significant role in its anti-inflammatory activity.

Where, OD1 = test sample unheated, OD2 = test sample heated and OD3 = control sample heated Statistical analysis Three replicates of each sample were used for statistical analysis and the values are reported as mean ± SD. RESULTS AND DISCUSSION The extractives of M. nagassarium and K. pinnata, at concentration 2.0 mg/mL, significantly protected the lysis of erythrocyte membrane induced by hypotonic solution and heat as compared to the standard, acetyl salicylic acid (0.10 mg/mL). The petether soluble fraction (PESF) of M. nagassarium Available online on www.ijprd.com

Figure 1: Comparison of activity of different extractives of M. nagassarium on hypotonic solution and heatinduced hemolysis of erythrocyte membrane Figure 2: Comparison of activity of different extractives of K. pinnata on hypotonic solution and heat-induced hemolysis of erythrocyte membrane

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Table 1: Effect of extractives of M. nagassarium on hypotonic solution and heat-induced hemolysis of erythrocyte membrane

Inhibition of hemolysis by different extractives of M. nagassarium

80 70

Table 2: Effect of extractives of K. pinnata on hypotonic solution and heat-induced hemolysis of erythrocyte membrane

Inhibition of hemolysis by different extractives of K. pinnata

80

PESF

70

ASA

60

ASA AQSF

60

PESF 50 40

CTCSF

30

Inhibition of hypotonic solution induced hemolysis

CSF ASA

Inhibition of heat induced hemolysis

MEAP PESF CSF

20 10

CSF

50

CTCSF

MEAP

40

CTCSF PESF

30

ASA CSF

Inhibition of heat induced hemolysis

AQSF 20

AQSF

Inhibition of hypotonic solution induced hemolysis

CTCSF MEAP

MEAP

10

AQSF 0

0

0

2

4

6

0

8

Figure 1: Comparison of activity of different extractives of M. nagassarium on hypotonic solution and heat-induced hemolysis of erythrocyte membrane

2

4

6

8

Figure 2: Comparison of activity of different extractives of K. pinnata on hypotonic solution and heat-induced hemolysis of erythrocyte membrane

Table 1: Effect of extractives of M. nagassarium on hypotonic solution and heat-induced hemolysis of erythrocyte membrane Treatment

Concentration (mg/mL)

Hemolysis inhibition (%) Heat induced

Hypotonic solution induced

MEAP

2.0

16.44±0.66

30.42±0.81

PESF

2.0

23.73±0.35

49.19±1.11

CTCSF

2.0

21.8±0.62

40.16±0.31

CSF

2.0

17.93±0.75

34.63±1.45

AQSF

2.0

8.46±0.55

21.55±0.47

Acetyl salicylic acid

0.1

30.23±1.02

73.19±0.88

The average values of three calculations are presented as mean ± S.D. (standard deviation). Available online on www.ijprd.com

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Table 2: Effect of extractives of K. pinnata on hypotonic solution and heat-induced hemolysis of erythrocyte membrane. Treatment

Concentration (mg/mL)

Hemolysis inhibition (%)

Heat induced Hypotonic solution induced MEAP 2.0 19.69±0.61 45.28±0.65 PESF 2.0 32.03±0.45 65.7±1.21 CTCSF 2.0 22.79±0.69 46.81±0.81 CSF 2.0 24.57±0.84 47.27±1.04 AQSF 2.0 28.55±0.59 56.48±0.84 Acetyl salicylic acid 0.1 30.23±1.02 73.19±0.88 The average values of three calculations are presented as mean ± S.D. (standard deviation). REFERENCES

6. Carey MW, Rao NV, Kumar BR and Mohan GK, Antiinflammatory and analgesic activities of methanolic extract of Kigelia pinnata DC flower, Ethnopharmacological communication, 130(1), 2010, 179 -182. 7. Gopalkrishnan C, Shankarnarayanan D and Nazimuddin Sk, Anti-inflammatory and CNS depressant activities of xanthones from Calophyllum inophylum and Mesua ferrea. Indian Journal of Pharmacology, 12(5), 1980, 181-191. 8. Van Wagenen BC, Larsen R, Cardellina,JH, Ran dazzo D, Lidert ZC and Swithenbank C, Ulosantoin, a potent insecticide from the sponge Ulosa ruetzleri. Journal of Organic Chemistry, 58, 1993, 335-337. 9. Sikder MA, Rahman MA, Islam MR, Kaisar MA, Rahman MS and Rashid MA, In vitro antioxidant, Reducing Power, Free Radical Scavenging and membrane stabilizing activities of Spilanthes calva. Bangladesh Pharmaceutical Journal, 13 (1), 2010, 63-67. 10. Abe H, Katada K, Orita M, Nishikibe M, Effects of calcium antagonists on the erythrocyte membrane. Journal of Pharmacy Pharmacology, 43, 1991, 22-26.

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