B Kumar, K Lakshman, V Narayan Swamy, S Khan, M Tripathi, L Deepa
amrycard powder, antibacterial activity, antioxidant activity, dpph, erythromycin
B Kumar, K Lakshman, V Narayan Swamy, S Khan, M Tripathi, L Deepa. Free Radical Scavenging and Antibacterial Activities of Amrycard Powder (A Ayurvedic Formulation). The Internet Journal of Alternative Medicine. 2008 Volume 7 Number 1.
Free radicals are implicated for more than eighty diseases including diabetes mellitus, arthritis, cancer, ageing, etc. in treatment of theses diseases; antioxidant therapy has gained an utmost importance. Current research is now directed towards finding naturally occurring antioxidant of herbal drugs. Antioxidant activity of methanol extract of Amrycard powder was evaluated by using Phosphomolybdenum assay, DPPH radical scavenging assay, superoxide radical scavenging assay and ABTS assay. The total phenolic, total tannins, and total flavonoids content were determined. Antibacterial activity was also studied against
Reactive oxygen species (ROS), sometimes called active oxygen species, are various forms of activated oxygen, which include free radicals such as superoxide ions (O-), and reactive hydroxyl radicals (OH-), as well as non-free radical species such as hydrogen peroxide (H2O2) . In living organisms various ROSs can form in different ways, including normal aerobic respiration, stimulated polymorphonuclear leukocytes and macrophages, and peroxisomes. These appear to be the main endogenous sources of most of the oxidants produced by cells. Exogenous sources of free radicals include tobacco, smoke, ionizing radiation, certain pollutants, organic solvents, and pesticides , Free to their deterioration . In addition, reactive oxygen species have been implicated immunodeficiency syndrome, heart disease, stroke, arteriosclerosis, diabetes, and cancer . When produced in excess, ROSs can cause tissue injury. However, tissue injury can itself cause ROS generation . Nevertheless, all aerobic organisms, including human beings, have antioxidant defenses that protect against oxidative damages, and numerous damage removal and repair enzymes to remove or repair damaged molecules . However, this natural antioxidant mechanism can be inefficient, and hence dietary intake of antioxidant compounds is important . There are some synthetic antioxidant compounds, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), commonly used in processed foods. However it has been suggested that these compounds have some side effects . In addition, it has been suggested that there is an inverse relationship between dietary intake of antioxidant rich food and the incidence of human disease .
Plant products are being used as source of medicine since long. The medicinal properties of plants have been investigated in recent scientific developments through the world, due to their potent antioxidant activities, no side effects and economic viability . Flavonoids and phenolic compounds widely distributed in plants which have been reported to exert multiple biological effect, including antioxidant, free radical scavenging abilities, anti-inflammatory, anticarcinogenic, etc. they were also suggested to be a potential iron chelator . Among natural antioxidants, phenolic antioxidants are in the forefront since all the phenolic classes (simple phenolics, phenolic acids, anthocyanins, hydroxycinnamic acid derivatives, and flavonoids) have the structural requirements of free radical scavengers and antioxidants.
Amrycard powder traditional used for the treatment of diabetics, it consists of Tej patra (
Materials and Methods
Preparation of Amrycard powder
Tej patra, Bilv patra, Gular patra, Jamun patra, Methi beej, Giloe, and Neem Patra made were reduced to fine powder and passed through the Sieve no.100 and mixed in geometric proportion and packed in well-closed container.
Chemicals and Drugs
Folin Ciocalteu’s reagent (SD Fine Chemicals, India, glacial acetic acid and sodium dodecy sulphate (SDS). 1.1 diphenyl-2-picryl hydrazyl (DPPH), foline denix reagent, ammonium molybdate, 2.2’-azubibis (3-ethylbenzothiazoline-6-sulfonate) ABTS HIMEDIA, India. trichloroacetic acid, thiobarbituric acid E.Merck India, India. Erythromycin (Ranbaxy lab. India)
Bacterial strains were obtained from Microbial type culture collection (MTCC)
Preparation of Extract
Amarycard powder was extracted with methanol by maceration process. The different concentrations (1, 2, 4, 6, 8 and 10 mg/100 l) were prepared with DMSO for antimicrobial activity.
Estimation of Total Phenolic Content
The total phenolic content of the extract was estimated according to the method described by Singleton and Rossi. From the stock solution (1 mg/ml) of the Amrycard powder extract, suitable quantity was taken into a 25 ml volumetric flask and mixed with 10 ml of water and 1.5 ml of Folin Ciocalteu’s reagent. After 5 min, 4 ml of 20% (W/v) sodium carbonate solution was added and volume was made up to 25 ml with double distilled water .The absorbance was recorded at 765 nm, after 30 min. Percentage of total phenolics was calculated from calibration curve of Gallic acid (50-250 µg) plotted by using same procedure and total phenolics were expressed as % Gallic acid .
Estimation Total Tannins
To the 5 ml of Folin Denis reagent (100g of sodium sulphate+20g of phosphomalybdic acid +50 ml of phosphoric acid and 750ml of distilled water was refluxed or boiled for 2 hrs and make up the volume 1000 ml with distilled water) mixed 10 ml of 35% sodium carbonate and add different concentrations of methanolic extract of Amrycard powder. Then make up the volume to 100 ml in volumetric flask with distilled water. Incubate reacting mixture for 30 min at room temperature and absorbance was recorded at 760 nm. Percentage of total tannins was calculated from calibration curve of tannic acid (100-1000 µg) plotted by using same procedure and total tannins were expressed as % tannic acid .
Estimation of Total Flavonoids
In a 10 ml volumetric flask add 4 ml of water 1 ml of methanolic extract of Amrycard powder keep aside for 5 min. then add 3 ml of 5% sodium nitrite and 0.3 ml of 10% aluminum chloride allow the reaction for 6 min. again add 2 ml of 1M sodium hydroxide. Know make up the volume to 10 ml with distilled water and measure the absorbance of pink chromogen at 510 nm. Percentage of total flavonoids was calculated from calibration curve of Quercetin (100-1000 µg) plotted by using same procedure and total flavonoids were expressed as % Quercetin .
The assay is based on the reduction of MoVI to MoV by the extracts and subsequent formation of a green phosphate/MoV complex in acidic pH. Separately extracts and formulations were mixed with 3ml of reagent solution (0.6M sulphuric acid, 28mM sodium phosphate and 4mM ammonium molybdate), incubated at 95°C for 90 min, cooled to room temperature, and absorbance measured at 695nm. The antioxidant activity was expressed as the number of equivalents of ascorbic acid (ASC) using standard plot .
Assay for Antiradical Activity with DPPH
Antiradical activity was measured by a decrease in absorbance at 516 nm in spectrophotometer (Shimadzu model 1601) of methonalic solution of coloured DPPH brought about the sample. A stock solution of DPPH (1.3 ml/ml in methanol) was prepared such that 75 ml of it in 3 ml of methanol gave an initial absorbance of 0.9. This stock solution was used to measure the antiradical activity. For steady state measurements, 100 mm DPPH in methanol was added to extracts and formulation (2-1000mg/ml) in methanol, mixed well and kept in dark for 20 min. decrease in the absorbance in the presence of different concentration of methanolic extract of Amrycard powder was noted. IC50 was calculated from % inhibition. Ascorbic acid was used as positive control .
ABTS Radical Scavenging Assay
For steady state measurements, 100 mM ABTS•- (prepared by the reaction of 2 mM [ABTS2-] was mixed with 0.17 mM potassium persulphate in 20 mM phosphate buffer pH 7.4, kept overnight before use) and mixed with methanolic extract (2-1000 mg/ml) and decrease in absorbance was measured at 734 nm .
Superoxide Radical Scavenging Activity
The assay was based on the capacity of the methanol extract of Amrycard powder to inhibit blue formazan formation by scavenging the superoxide radicals generated in riboflavin-light-NBT system . The reaction mixture contained 50 mM phosphate buffer pH 7.6, 20 µg riboflavin, 12 mM EDTA, and NBT 0.1 mg/3 ml was added in the sequence. Reaction mixture was initiated by illuminating the reaction mixture with different concentrations (2-1000 µg) of methanol extract of Amrycard powder for 90 sec. immediately after illumination, the absorbance was measured at 590 nm, IC50 was calculated. Methanol was used for blank reading. Ascorbic acid was used as positive control.
The antibacterial activity was evaluated by employing 24 hrs cultures of
Preliminary phytochemical screening of the Amrycard powder showed the presence of phenolics, flavonoids, tannins, and steroids. Subsequent quantification showed shoed the presence of amount of total phenolics, tannins and total flavonoids were showed in Table 1.
The Phosphomolybdenum assay is based on the reduction of MoVI to MoV by antioxidant compounds and a formation of green phosphate/MoV complex with a maximal absorption at 695 nm and it was efficient to extend its application to plants polyphenols . Total antioxidant capacity of Chitrakadi vati was found to be 3.25 µg.
The methanolic extract was able to reduce the stable radical DPPH to the coloured diphenylpricrylhydrazine. Both extracts and formulation exhibited a concentration-dependent DPPH radical scavenging activity but the formulation showed more potent activity when compared to extracts (Figure 1).Methanolic extract of Amrycard powder was found to have comparable activity to standard ASC (IC50–3.6 g/ml) in scavenging 100M ABTS (Figure 2). For kinetic studies, the concentration of ABTS was kept at 100 M. In the absence of the formulation, the ABTS signal did not show any decay and remained stable. However, in the presence of the methanolic extract of formulation the absorption due to the ABTS decayed completely in 20 seconds. This absorption time plot was fitted to a single exponential function to get observed decay rate constant, which was found to increase with increasing concentration of extract. The superoxide radical activity of the ayurvedic formulation increased with the increase in concentration (Figure 3). The IC50 value for formulation was found to be 482µg.
Antibacterial activity of different concentration of methanol extract of Amrycard powder was measured in terms of Zone of Inhibition. It revealed that significant antibacterial activity was showed against bacterial strains like
Polyphenols and flavonoids used for the prevention and cure of various diseases, which is mainly associated with free radicals . Free radical scavenging of phenolic compounds is an important property underlying their various biological and pharmacological activities. Flavonoids and phenolic compounds possess antioxidant activity. Phenolic compounds are known to be powerful antioxidant agents. Since Amrycard powder contains good amount of total phenolics (1.35µg) it was thought of interest to screen it for its possible antioxidant activity. The antioxidant activity may result from the neutralization of free radical initiating oxidation processes, or from the termination of radical chain reactions. For this reason, the above mentioned methods of antioxidant activity estimation were used. Proton-radical scavenging action is an important attribute of antioxidants, which is measured by DPPH radical scavenging assay. The method is based on the reduction of alcoholic DPPH solution in the presence of hydrogen-donating antioxidant due to the formation of the non-radical form DPPH-H by the reaction resulting in a color change from purple to yellow. As seen from the results extract Amrycard powder exhibited a more potent activity. Superoxide radicals are generated during the normal physiological process mainly in mitochondria. Although superoxide anion is by itself as weak oxidant, it gives rise to the powerful and dangerous hydroxyl radicals as well as singlet oxygen both of which contribute to the oxidative stress  the capacity of different concentration of extract to scavenge superoxide radical reveals that the extract of formulation possess superoxide dismutase like activity. Although the activity was found to be lower than scavenging activity of ascorbic acid in entire dosage ranges.
The antibacterial activity of Amrycard powder showed significant activity against
Authors are thankful to K.V. Naveen Kiran, Chairman, Sri K.V.College of Pharmacy, Chickballapur, Karnataka (India) for providing their help in the successful completion of the work.
1000µg of formulation ≡µg of gallic acid, tannic acid and quercetin