MEMBRANE STABILIZING ACTIVITY AND ANTIOXIDANT EFFECT OF SIDA CORDATA (BURM F.) BIOSS AND RHODODENDRON ARBOREUM SM. LEAVES

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Received on 05 August, 2013; received in revised form, 11 September, 2013; accepted, 17 December, 2013; published 01 January, 2014

MEMBRANE STABILIZING ACTIVITY AND ANTIOXIDANT EFFECT OF SIDA CORDATA (BURM F.)BIOSSAND RHODODENDRON ARBOREUM SM. LEAVES

Deepak Kumar*¹, Shefali Arora ², Manoj Kumar ¹, Manoj Kumar Thakur ¹and Abhay Pratap Singh ¹

Department of Pharmaceutical Chemistry, Dolphin (PG) Institute of Biomedical and Natural Sciences ¹, Dehradun, Uttarakhand, India

Departmant of Chemistry, University of Petroleum and Energy Studies ², Dehradun, Uttarakhand, India

ABSTRACT:

Purpose: To investigate membrane stabilizing activity and antioxidant activity off different extracts of leaves of Sida cordata and Rhododendron arboreum.

Methods: Preliminary phytochemical analysis of different extracts of leaves of Sida cordata and Rhododendron arboreum were performed for presence of phytoconstituents. In vitro membrane stabilizing activity was done by using RBC membrane stabilising activity and antioxidant activity was done by using DPPH method.

Results: Preliminary phytochemical analysis showed that methanol extract are very rich in phytoconstituents as they showed the presence of alkaloid, carbohydrate, glycosides, proteins and amino acid, Phenolic compounds. All extract are investigated for their in vitro membrane stabilizing activity and antioxidant effect. Methanol and acetone extract of Sida cordata and Rhododendron arboreum showed maximum effect in comparison with other extracts.

Conclusion: From the above study it could be concluded that the leaves of Sida cordata and Rhododendron arboreum showed protective effect on inflammation and oxidant.

Keywords:

Sida cordata, Rhododendron arboreum, DPPH, Membrane Stabilizing activity, Erythrocyte, Ascorbic acid

INTRODUCTION:Sida cordata (Burm F.) Biossis a small weed found throughout India, usually on the road sides and other waste places. A procumbent, diffuse, much branched hairy herb with a very short main stem and long slender trailing branches that occasionally root at places of contact with the soil; leaves long-petioled, cordate to roundish with stellate hairs; flowers yellow, solitary or in pairs in the axils; fruits schizocarp located within the persistent calyx; seeds brownish, glabrous.

The roots are sweet, sour, astringent, bitter thermogenic and tonic and useful in fever, uropathy and arthritis. The bark of root is used in leucorrhoea and gonorrhoea and hyperdiuresis. The leaves are good for diarrhoea. The flower and ripe fruits refrigerant and are useful in relieving burning sensation ¹. Rhododendron arboreum is one of the most stately and impressive rhododendron species. It is extremely variable in stature, hardness, flower color and leaf characteristics. Its species named Arboreum means tree like. Originally discovered in north central India, the plant is found in the Himalayas from Kashmir to Bhutan & in the hills of Assam & Manipur at altitudes of 1200-400 m. It grows at elevations of 4500 to 10,500 ft & grows up to 40 to 50 ft high sometimes attaining over 100 ft.

This is an evergreen much branched tree up to 14 m in height & 2.4 m in girth. Flowering season is from March- April/ June-September bearing deep red or crimson to pale pink flowers. Rhododendron arboreum is an evergreen shrub or small tree with a showy display ofbright red flowers. Rhododendron is the national flower of Nepal & is known as (Laligurans) & the state tree of Uttarakhand (India). It is called ‘Burans, Bras, Buras or Barah ke-phool’ in local dialect. It is widely popular for the processed juice of its flowers which havegained market popularity as rhodojuice / sharbat. The plant is found in the Himalayas from Kashmir eastwards to Nagaland. Various parts of the plant exhibited medicinal properties & is used for the treatment of various ailments ².

The selection of these plants for evaluation was based on their traditional usages. From literature survey it indicated that few works are carried out on both plants.

Therefore in present study, we investigated the Phytochemical, membrane stabilizing activity and antioxidant activity of Sida cordata(Burm F.)Bios andRhododendron arboreum Sm. Leaves.

EXPERIMENTAL

Collection & identification of leaves of Sida cordata (Bern. F.) Bioss.: Leaves of Sida codata (Burn.f.)Bioss. were collected from locality of Dehradun (India). Plant material was authenticated by S. K. Srivastava (Scientist D/HOD), in Botanical Survey of India, Northern regional centre, Dehradun (BSI). Authenticated specimen no is- Acc. no.113678.

Leaves of Rhododendron arboreum Sm were collected from Dhanaulti road, Mussoorie, Dehradun (India). Plant material was authenticated by S. K. Srivastava (Scientist D/HOD), in Botanical Survey of India, Northern regional centre, Dehradun (BSI). Authenticated specimen no is- Acc. No. 113680.

Extraction and Phytochemical analysis: The collected plant Material was washed with water to remove other undesirable material and dried under shade. The air-dried leaves (100 gm) of Sida cordata and Rhododendron arboreum were crushed.

The crushed leaves extracted with different solvents of increasing polarity viz. petroleum ether, chloroform, acetone, methanol by hot percolation method using Soxhlet Apparatus. The extract was evaporated till dryness to obtain residue. These extracts were concentrated under reduced pressure.

The extracts of leaves of Sida cordata and Rhododendron arboretum undergo various qualitative phytochemical tests. They showed their presence and absence in the different solvent systems. The different extracts of leaves of Sida cordata and Rhododendron arboreum were tested for various phytoconstituents viz., alkaloid, carbohydrate, sterols and Terpenoids, Phenolic compound and tannins, Protein and amino acid, saponin etc.

Membrane stabilizing activity and antioxidant activity:

Membrane stabilizing activity of different extracts: The membrane stabilizing ativity of the extracts was determined according to the method of Shinde et al and Sikdar et al ^{3, 4}.

Effect on haemolysis: Erythrocyte suspension: Whole blood was collected from goat under ether anesthesia. NIH was used to prevent clotting. NIH solution was made by adding 5.5 g Trisodium citrate, 2 g citric acid, 6.125 g dextrose in 250 ml distilled water. 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 100ml of distilled water: NaH₂PO₄.2H₂O, 0.26 g; Na₂HPO₄, 1.15 g; NaCl, 9 g (10 mM sodium phosphate buffer). The isotonic buffer solution was composed of 154 mM NaCl in 10 mM sodium phosphate buffer (pH 7.4).

Hypotonic solution-induced haemolysis: Stock erythrocyte suspension (30 µl.) was mixed with 5 ml of the hypotonic solution containing the different extracts of Sida cordata at concentrations of 1000 µg/ml, 1500 µg/ml, 2000 µg/ml and 2000 µg/ml, 2500 µg/ml, 3000 µg/ml for Rhododendron arboreum , while the control sample was mixed with drug free solution. The mixtures were incubated for 10 min at room temperature, and centrifuged at 3000 rpm for 10 min.

All the experiments were performed in triplicates and the absorbance (O.D.) of the supernatant was measured at 560 nm.

Acetyl salicylic acid (Aspirin) was used as a reference standard at a concentration 1000 µg/ml, 1500 µg/ml and 2000 µg/ml.

Calculation: The percentage inhibition or acceleration of haemolysis in tests and standardwas calculated according to the equation:

% acceleration or inhibition of hemolysis = 100x [ 14 OD1-OD2OD1"> ]

Where, OD ₁= Optical density of hypotonic saline solution + blood (control) and

OD₂= Optical density of test sample in hypotonic saline solution + blood

Antioxidant activity of leaves extract:

Antioxidant activity ^{5, 6}:

DPPH Method:

Mechanism of DPPH method: The molecule of 1,1-diphenyl-2-picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl; DPPH:1) is characterised as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, so that the molecules do not dimerise, as would be the case with most other free radicals. The delocalisation also gives rise to the deep violet colour, characterised by an absorption band in ethanol solution centred at about 517 nm. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form (2) with the loss of this violet colour (although there would be expected to be a residual pale yellow colour from the picryl group still present).
Methodology of DPPH method:

Preparation of DPPH: DPPH is a highly oxidisable compound. It oxidized in light, so DPPH is prepared in dark. Weigh accurately 20 mg DPPH and dissolved in 100 ml of solvent. Generally Methanol and for some cases Ethanol is used as a solvent for DPPH.

Preparation of test and standard ascorbic acid solution: Ascorbic acid is a strong antioxidizing agent. It is taken as standard. Standard solution of ascorbic acid and test sample of different extracts of leaves of Sida cordata and Rhododendron arboreum is prepared viz. 50 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, 500 μg/ml. 3 ml of different concentration of test sample of different extracts of Sida cordata and Rhododendron arboreum and standard solution of ascorbic acid were mixed with 1 ml of DPPH solution in dark. The prepared solution of test sample and standard were incubated for 30 minutes then measure absorbance with the help of U.V. Spectrophotometer at 517 nm.

Calculation: We calculate the % activity of individual concentration of individual extract from the following formula:

%Activity=

Abs. of Control – Abs. of Individual Conc. X 100

Abs. of Control

Abs. = Absorbance.

RESULT AND DISCUSSION: From phytochemical analysis, there is presence of steroids in petroleum ether extract; Saponin is present in petroleum ether and chloroform extract. Alkaloid, carbohydrate and Phenolic compounds showed in acetone extract. Methanol extract was the richest extract for phytoconstituents. It contains all tested phytoconstituents viz. Alkaloids, Carbohydrate, glycosides, proteins and amino acid, Phenolic compounds.

The membrane stabilizing action of different extracts of leaves of Sida cordata and Rhododendron arboreum observed in this model, experiments were performed on the erythrocyte membrane. Sida cordata and Rhododendron arboreum was found to inhibit the haemolysis of erythrocytes induced by hypotonic solution showed in Tables 1-4.

TABLE 1: ABSORBANCE OF DIFFERENT EXTRACT OF SIDA CORDATA LEAVES ON HEMOLYSIS

Concentration (μg/ml)	Absorbance of Extracts at 560 nm				Standard drug
Concentration (μg/ml)	Pet. ether	Chloroform	Acetone	Methanol	Acetyl Salicylic acid (Aspirin)
1000	1.003±0.002	0.809±0.003	0.597±0.002	0.462±0.003	0.442±0.002
1500	0.925±0.003	0.755±0.003	0.567±0.004	0.430±0.005	0.361±0.002
2000	0.914±0.002	0.725±0.004	0.524±0.006	0.412±0.007	0.342±0.001
Control	1.051±0.001	0.936±0.005	1.030±0.008	0.847±0.006	0.919±0.002