INTRODUCTION:
Free radicals play an important role in biological process such as host defence, over-production of hydroxyl radical (.OH), hydrogen peroxide (H2O2), superoxide anions (O2--) and nitric oxide (NO) which contributes in cancer, AIDS, diabetes, hypertension etc. [1,2]. However, Antioxidant reduced oxidation of other molecules as lipids, proteins by inhibition of oxidative chain reaction [3]. When lipids oxidized with free radical reaction then it lead for degradation of foods and that can minimize by antioxidant [4]. Plant’s phytochemicals viewed a good alternative natural antioxidant and found to be useful for free radical pathology and food industry.
Oxidation of food is influence for destruction of food, loss of nutritional value and rate of oxidation can be reduced by antioxidants. Now, phytochemicals are going to improve quality of food with slow oxidative degradation of lipids [5,6].
Aloe is widely distributed plant and traditionally used as cosmetics and pharmaceutical purpose in all over. It’s used for treatment of wounds, burns and constipation from thousands of years [7]. It has pharmacological properties such as hypoglycaemic effect on diabetic mellitus, antibacterial, antifungal, anticancerous, anti-inflammatory, immune-stimulant, and antioxidant [8]. Aloe juice has bitter taste and contains aloin, anthreaquinone, isobarbaloin, emodin, ester of cinnamic acid that play an important role in treatment of cancer, ulcer, and diabetes. Presence of phytochemicals such as alkaloid, carbohydrate, tannin, steroid, tri-terpenoids, glucose, and galactose was confirmed in extracts of Aloe by HPTLC method [9]. There are many ingredients isolated and studied for their biological activities. Pharmacological ingredients as anthreaquinone, aloe-emodin, and barbaloin prominent free radical scavenging activity. Phenolic compound such as alkaloids and flavonoids are natural occurring phytochemical and expressed antimicrobial, anti-mutagenic, anti-inflammatory and hepatoprotective [10,11].
The present investigation is employed for extraction of leaves and isolation of phenolic compound as flavonoids from Aloe vera and study their phytochemical screening, antimicrobial, antioxidant and Nitrous oxide scavenging activity.
MATERIAL AND METHOD:
Crude Drug Extraction:
Aloe vera’s leaves were collected near to Chandigarh, India and identified by Gaurav Srivastav (Botanist) and dried at room temperature in absence of light after washed. Obtained powder (100gm) after milling was subjected to extraction with methanol & water in ratio of 60:40 at Soxhlet for 48hrs. The obtained extract was concentrated in oven at 600C and store at 40C for further used.
Phytochemical Screening:
A minimum amount of extracts were used for phytochemical screening for compound as total phenol, alkaloids, flavonoids, tannins, Saponins, steroids, terpenoids, carbohydrate and glycosides. According to selected method with little modification, 2.0gm of plant extract was dissolved in 10ml double distilled water and filtered with filter and further used for phytochemical screening [12].
|
Component Exit Test |
Protocol for Test |
Result for Confirmation |
|
Alkaloids |
1.0ml extract + 3.0 drops of Dragendoff’s reagent |
Orange-red precipitate |
|
Flavonoids |
1.0ml extract + few drops of dil. NaOH |
Intense yellow colour |
|
Total phenol |
1.0ml extract + 2.0ml water + few drops of 10% FeCl3 |
Blue-green colour |
|
Tannins |
100mg solvent free extract + 1ml 5% FeCl3 |
Bluish- black precipitate |
|
Saponins |
1.0ml extract + 20ml water + agitation for 15min. |
1cm layer of foam |
|
Steroids |
1.0ml extract + 10ml CHCl3 + 10ml conc. H2SO4 |
Upper layer – red and lower layer – yellow-green |
|
Terpenoids |
5.0ml extract + 2.0ml CHCl3 + 3.0ml conc. H2SO4 |
Reddish brown precipitate |
|
Carbohydrate |
2.0ml extract + 2 drops of Molish reagent (95% α-Napthol in ethanol) + 2.0ml conc. H2SO4 |
Purple colour at junction |
|
Glycosides |
5.0ml extract + 2.0ml CHCl3 + 2.0ml CH3COOH |
Violet, blue to green colour |
Determination of Total Phenolic Component:
Total amount of phenolic contents was determined using method by Folin Ciocalteu's phenol reagent with slight modification. 1.0ml of sample was mixed with 2.0ml of Folin Ciocalteu's phenol reagent (10%). After 3 min, 1.0 ml of saturated Na2CO3 (~35 %) was added to mixture and made up to 10 ml by adding distilled water. The reaction was kept in the dark for 90 min, observed under UV-Vis spectrophotometer at 760nm absorbance. Tannic acid was used as a standard with varied concentration from 50 ppm to 200 ppm. A calibration curve was constructed with different concentrations of Tannic acid (0.01- 0.1mM) as standard. The results were expressed as mg of Tannic acid equivalents/g of extract and same procedure was done with extract and calculated as mean value ± SD (n = 3) [13].
Determination of Flavonoid Content:
Flavonoids content of isolated crude extract was determined by method with slight modification. Take 0.5ml of the sample (Extract) containing 1.25ml of distilled water. Then added 0.075ml of 5 % sodium nitrite solution and allowed to stand for 5 min. Added 0.15ml of 10 % aluminium chloride, after 6 min 0.5ml of 1.0M sodium hydroxide were added and the mixture were diluted with another 0.275ml of distilled water. The absorbance of the mixture at 510nm was measured immediately. The flavonoid content was expressed as mg of Quercetin equivalents/g sample and same procedure were done with Extract of all plants and calculated as mean value ± SD (n=3) [13].
Column Chromatography and Thin Layer Chromatography:
Combined solvent (Toluene: Ethyl acetate: formic acid in ratio of 7:5:1) was used as mobile phase in silica gel column chromatography (200g, 60-200 mesh). 10gm extracts was loaded in glass column with silica gel and 150ml of solvent system (Toluene: Ethyl acetate: formic acid) was used for running sample. For confirmation of separation, thin layer chromatography was performed by taking sample with mobile phase. The analysis of flavonoids was performed on silica gel coated TLC plates using 10µl of sample. The mobile phase for TLC was Toluene: Ethyl acetate: formic acid (7:5:1) and fractions were further screened for flavonoids [14,15].
Spectrophotometer and HPLC:
For determination of flavonoids content, spectrophotometric wavelength was considered on UV/Visible spectrophotometer (Shimadzu) with spectra (200-500nm) using Querecetin dehydrate as standard. 1ml of fraction, 1ml of pure water, 5ml acetate buffer (pH-3.8±0.5), and 3.0ml AlCl3(0.1M) solution added and after shaking kept for 30minuts. Absorbance was measured in 200-400nm of sample [16]. After consideration of wavelength, reversed-phase high performance chromatography was performed for determination of flavonoids in extracts. The 20µl of column chromatography purified liquid extracts was injected into HPLC (reversed phase, C18, 250mm x 416mm, Shimadzu) at 350nm for 16min at 1.0ml/min with HPLC grade Methanol : Water (60:40) mobile phase [17,18].
Antibacterial Activity:
Four pathogenic bacteria namely Escherichia coli (MTCC 25922), Bacillus subtilis (MTCC 3256), Pseudomonas aeuroginosa (MTCC 1688) and Staphylococcus auerus (MTCC 6810) were collected from MTCC, Chandigarh. For preparation of inoculums, they were sub-cultured in test tube containing 10ml nutrient broth and standardized with saline water. Agar well diffusion was used with slight manipulation. 0.5ml (9.0 x 104 CFU/ml) of 48 hrs old cultures of test organisms was inoculated into different sterile petri-plates and 15-20ml sterile media was poured into each petri-plate (90 x 90mm). The petri-plates were gently shaken for proper mixing and allowed to solidify. Thereafter, four wells were punched of 5mm diameter with a sterilized cork borer. For each well 50μl (25 - 100µg/ml) of flavonoids were added. The plates were incubated at 370C for 24 hrs and then zone of inhibition in mm was measured. The experiment was carried out in triplicates and calculated as mean value ± SD (n = 3) [12].
Antioxidant Activity:
The antioxidant activity of the flavonoids and the standard was assessed on the basis of the radical scavenging effect of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH-0.002% in methanol) method. The diluted working solution of the flavonoids (10 mg/ml) was prepared using the respective solvents. 10mg/ml of Ascorbic acid used as reference for compare results of plant extracts. In 3ml of total reaction solution, 2ml of flavonoids/standard solution (200-1000µg/ml) and 1.0ml of DPPH were mixed and allowed to react at 370C for 30 min. afterward, absorbance value were measured at 520nm and converted into percent antioxidant activity. The percentage antioxidant activity was calculated by following formula and calculated as mean value ± SD (n = 3) [19].
Percent (%) inhibition of DPPH activity = (A – B)/A *100
Where
A = Absorbance of the blank and B = Absorbance of the sample
Nitric Oxide Radical Scavenging Activity:
Scavengers of nitric oxide complete with oxygen, leading to reduced production of nitrite ions. Aloe flavonoid was dissolved in distilled water for this quantification. 2ml Sodium Nitroprusside (5mM) in standard phosphate buffer saline (0.025mM, pH-7.4) was incubated with 0.5ml of different concentration (200-1000μg/ml) of flavonoids were incubated at 29oC for 3 hours. Control experiment without the test compounds but with equivalent amount of buffer was conducted in an identical manner. After 3 hours incubated, samples were diluted with 1 ml of Griess reagents (prepared by mixing equal volume of 1% sulphanilamide in 2% phosphoric acid and 0.1% Naphthylethylene diamine dihydrochloride in water) and incubated for 30min. The absorbance of the colour developed during diazotization of Nitrite with sulphanilamide and its subsequent coupling with Napthylethylene diamine hydro-chloride was observed at 550nm on spectrophotometer. Same procedure was done with ascorbic acid, which was standard in comparison to extract. Calculated the % inhibition by formula and plot graph in compared to standard and calculated as mean value ± SD (n = 3) [20,21].
Where,
Acontrol = Absorbance of control reaction
Atest= Absorbance in the presence of the samples of extract.
Statistical Analysis:
The date recorded during investigation, were analysed with applying of Student t-test at excel sheet and signified the data at p<0.05% for organisms and solvents.
RESULT AND DISCUSSION:
Herbal medicines contain many constituents and can be separated by chromatographic techniques. There are many references [14-18] present to characterize the different components and their activity. Present studies reported that Phenolic compounds are well known antioxidant and scavenging agents and flavonoids is one of them. Flavonoids are secondary metabolites of plants and expressed as antimicrobial, antioxidant, scavenging activity, carcinogenesis and other diseases.
Phytochemical Screening:
Qualitative phytochemical screening carried on Aloe vera extracts showed the presence of phenolic compounds, flavonoids, alkaloids, amino acids, carbohydrates, reducing sugar, glycosides, tannins, and saponins. The results revealed the presence of medicinal as well as physiological active compounds [22].