INTRODUCTION:
Free radicals or Reactive Oxygen Species (ROS) are formed as physiological by products due to metabolic processes in the body. These radicals are capable of oxidizing various bio-molecules as well as they initiate degenerative diseases1. Superoxide anion, hydroxyl radical and hydrogen peroxide as free radicals play a crucial role in the development of various lifestyle diseases. Oxidative stress is among the causative factors in the development of chronic and degenerative diseases like ageing, diabetes mellitus, cancer, atherosclerosis and other degenerative diseases. These ROS or free radicals
are generated in the human body through exogeneous sources and aerobic respiration. In vivo free radicals play an important role in phagocytosis, energy production and cell growth regulation, but these radicals may also damage the cells. These free radicals/ oxidative stress occur due to imbalance between the antioxidants and oxidants2. Oxidants are species with very short half-life, but high reactivity and damaging activity towards molecules like DNA, proteins and lipids. ROS circulating in the body reacts with the electron of molecules in the body and affect the various enzyme systems and damage them 3. Antioxidants are the substance that terminate free radicals and in turn reduces or prevents the risk of disorders4. Most of the organisms are protected to some extent by free radical damage with the help of antioxidants or enzymes like flavonoids, tannins, polyphenols, phenolic acids, ascorbic acid, super-oxide dismutase, catalase, tocopherol and glutathione. Prior and Cao5 reported that dietary antioxidants as well as antioxidant supplements can protect against the free radical damage. But natural antioxidants are much preferred to protect against oxidative damage caused by free radicals in the human body6. Our body is even safeguarded by natural antioxidant defence, but there is always a need for natural antioxidants7. The phenolic compounds from medicinal plants possess strong antioxidant activity and may help aid in the protection against cell and oxidative damage due to free-radicals8. The well-known radical scavengers are reducing agents, hydrogen donors, and singlet oxygen quenchers and metal chelators9. The most effective natural antioxidants are ascorbic acid, carotenoids and phenolic compounds 10. These antioxidants can inhibit lipid peroxidation by active oxygen species, scavenge free radicals and chelating heavy metal ions11. A plant-based diet is effective against diseases related to chronic oxidative stress. Antioxidants from dietary plants contribute to beneficial health effects3. Medicinal plants play an important role in the ancient and modern health care system. The Indian system of medicine, Ayurveda uses specific components of therapeutic value from plant based drugs or formulations to treat various diseases. Plant based drugs are easily available, cheaper and non-toxic when compared to modern medicine. Medicinal plant constituents with antioxidant activity exerts protection against oxidative damage in the body12.
Naturally, compounds that impart bright colour to fruits act as antioxidants in the body by scavenging the free radicals. Epidemiological studies showed a positive correlation between the intake of fruits and disease prevention as well as ageing. Fruits are thus called as ‘functional foods’, which are capable of promoting good health and preventing diseases. Phenolic flavonoids, lycopene, carotenoids and glucosinolates are antioxidants which are the most thoroughly studied13. Fruits are also called as ‘fountains of youth'14.
Syzygium cumini (L.) Skeels, belongs to the family Myrtaceae, is one of the best-known species distributed in Asia. S. cumini is popularly known as Jamun or Indian blackberry widely used as healers in the traditional medicines like Ayurveda, Unani, and Siddha. Different parts of the plant S. cumini are reported to have several medicinal properties like antidiabetic15, anti-inflammatory16 and free radical scavenging potential17. Jamun is rich source of compounds like anthocyanins, glucoside, ellagic acid, isoquercetin, kaemferol and myrecetin. Jamun are rich in sugar, mineral salts, vitamins C, PP which fortifies the benefits of vitamin C, anthocyanins and flavonoids. Different parts of the Jamun were also reported for its antioxidant, anti-inflammatory, nitric oxide scavenging and free radical scavenging. So an attempt was made in this study to test the antioxidant activity of lyophilized Syzygium cumini (L) Skeels fruit or Lyophilized Jamun Pulp for antioxidant scavenging activity is assessed by enzymatic and non enzymatic assays.
MATERIAL AND METHODS:
Chemicals and reagents:
Processing of sample
The Jamun fruit samples were collected from Chennai and characterized by the Department of Botany, Women’s Christian College, Chennai. The fruits were thoroughly washed and dried to remove moisture. The pulp along with the skin was separated from the seed and then the pulp was pulverized to make the mixture homogeneous. The pulp was taken in a lyophilizer tray (Penguin, 2014) and pre treated by freezing at – 80 °C in the deep freezer. Later the sample was lyophilized at -40 °C for 48 H and then the powder was stored under refrigeration for further analysis. The extract was prepared by dissolving in DMSO and used for each assay. The antioxidants activity was also assessed by enzymatic and non enzymatic methods as follows:
Non enzymatic methods
DPPH Scavenging activity18
DPPH is an easy, simple, reliable and inexpensive method to measure the antioxidant potential of foods and does not require a special reaction or device. It involves the use of the free radical, 2, 2-Diphenyl-1- picrylhydrazyl (DPPH) to act as free radical scavengers or hydrogen donors. DPPH is a stable and synthetic radical that does not disintegrate in solvent. Free radical scavenging activity of lyophilized pulp extracts of Sygium cumini (L) Skeels fruit pulp was measured at 517nm. Different extracts in solvent with concentration ranging from 100 - 500 µg/ml was prepared and the mixture was shaken vigorously and allowed to stand at room temp for 30 minutes, then, absorbance was measured. Ascorbic acid was used as the standard and experiment were carried out in triplicates. The IC 50 value of the sample was also calculated using inhibition curve. The DPPH scavenging effect was calculated using the following equation:
The percentage inhibition was calculated by using the formula:
% Inhibition (or) % Antioxidant activity = {(absorbance at blank) – (absorbance at test) / (absorbance at blank)} X 100
IC50 of extract was calculated by plotting % inhibition vs concentration
ABTS Radical scavening activity19
ABTS assay measures the relative ability of antioxidant to scavenge the ABTS generated in aqueous phase, as compared with a standard. ABTS was generated by reacting a strong oxidizing agent (eg. Potassium permanganate or potassium per sulphate) with ABTS salt. Reduction of blue green ABTS radical coloured solution by hydrogen donating antioxidant was measured by the suppression of its characteristic long wave absorption spectrum20. The stock solution comprises of 7 mM ABTS solution and 2.45 mM potassium persulfate solution. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 4-16 H at room temperature in the dark. The resulting solution was then diluted with ethanol by mixing 1 ml of freshly prepared ABTS solution. The absorbance was taken after 15 minutes at 734nm using the spectrophotometer. The ABTS scavenging capacity of the extract was compared with that of BHT and percentage inhibition was calculated as
O.D. of control - O.D. of Test
ABTS radical = ---------------------------------------- Χ 100
scavenging O.D. of control
activity (%)
Enzymatic method
Nitric Oxide Scavenging activity21
The nitric oxide scavenging activity was assessed by standard method with modifications using sodium nitroprusside22. A volume of 2 mL of sodium nitroprusside prepared in 0.5 mM phosphate buffer saline (pH 7.4) was mixed with 0.5 mL of extract at various concentrations (0.2-1.0 mg/mL) and incubated at 25°C for 150 minutes. To this 0.5 mL of aliquot solution was added with 0.5 mL of Griess reagent. The mixture was incubated at room temperature for 30 min. Later 100 µL of sample having different concentration 20-100 μg/ml was added. Ascorbic acid was used as standard. The absorbance was read in spectrophotometer at 550nm. The % inhibition was calculated using the formula:
O.D. of control - O.D. of Test
% Inhibition = ---------------------------------------- Χ 100
O.D. of control
Superoxide anion scavenging activity
Radical scavenging activity was measured by the reduction of NBT23. The non-enzymatic superoxide radicals, which reduces (NBT) to form purple formazan. The reaction mixture has phosphate buffer, NBT, PMS with various concentrations of LJP extract. After incubation for 5 min at ambient temperature, the absorbance was measured against an appropriate blank to determine the quantity by absorbance. All tests were performed in triplicates with quercetin as the positive control24. The annihilation activity of free radicals was calculated in % inhibition as per the following relation:
(Absorbance of control – Absorbance of sample)
Inhibition % = -------------------------------------------X 100
Absorbance of control
RESULTS AND DISCUSSION:
Berries are the excellent source of various bioactive compounds, polyphenolics, anthocyanins, proanthocyanins, ellagitannins, flavanols and phenolic acids. Jamun is a seasonal summer fruit, with rich therapeutic value and used to treat various ailments as prescribed in ancient medicine. In this study, lyophilized fruit pulp extract of Syzygium cumini (L) / jamun was prepared in DMSO and tested for its antioxidant potential by radical scavenging activity using different assays like DPPH, ABTS, Nitric oxide scavenging activity and superoxide anion scavenging activity. The results are discussed below:
Fig. 1. DPPH Scavenging activity of LJP
Fig. 2. ABTS Radical Scavenging activity of LJP
The free radical scavenging activity of the Lyophilized Jamun Pulp (LJP) was measured as decolorizing activity following the trapping of the unpaired electron of DPPH as shown in figure 1. The concentration dependent response was observed with LJP at the concentration tested from 20-100µg/mL. The LJP showed better inhibition of 62 % at 100µg/mL. Ascorbic acid included as positive control, inhibited DPPH absorption by 98.3% at 100µg/ml. The concentration of LJP extracts that scavenged 50% of DPPH was observed at 39.8µg/ml, therefore the IC50 value for LJP was 39.8µg/ml. Similar study was done by Aqil et al32, 2012 on unhydrolyzed and hydrolyzed jamun pulp showed better inhibition at 25% and 46% respectively at 50µg/mL and 50% DPPH activity was observed for hydrolyzed pulp at 79 µg/mL and 90 µg/mL. This shows that the LJP possess better antioxidant activity in terms of DPPH radical scavenging activity. Figure 2 shows the scavenging of ABTS radicals by LJP extract with the standard BHT. LJP scavenged the ABTS radical by greater than 94% at 100µg/ml, whereas the standard showed around 100% activity. The lowest concentration of LJP showed 32% inhibition at 20µg/mL. The concentration of extract that scavenged 50% of ABTS radicals during the 30-minute inhibition incubation, obtained by interpolation of plotted data was 63.43 µg/ml. Similar study done by Aqil et al25, 2012 on unhydrolysed and hydrolyzed jamun pulp showed at 52 µg/ml and 80 µg/mL respectively. This shows that LJP extract shows as better ABTS radical scavenging activity.
Fig. 3. Nitric oxide Scavenging activity of LJP
Nitric oxide (NO) is a potent inhibitor of physiological processes. The diffusible free radicals play important role in biological system including anti tumor activities 26. Suppression of released NO may be partially due to direct NO scavenging, as the extracts of Syzygium cumuni decreased the amount of nitrite generated from the decomposition in-vitro27. The scavenging activity of NO depends on the concentration of extract. Figure 3 illustrates a significant decrease in the NO radical due to the scavenging ability of extracts and ascorbic acid was used as the control. The LJP extract showed maximum activity of about 33.3% inhibition at 1000 µg/ml whereas ascorbic acid showed 58% inhibition at the same concentration. The results clearly infers that the LJP extract of Syzygium cumini fruit pulp having NO scavenging activity very lesser compared to standard ascorbic acid.
Fig. 4. Superoxide anion Scavenging activity of LJP
The superoxide anion scavenging activity is depicted in Fig 4. The inhibition activity of LJP extract was compared to standard quercetin. The percentage inhibition of LJP extract and standard quercetin followed dose dependent manner. When compared with the standard quercetin the LJP showed less superoxide scavenging activity. About 38.8% inhibition was observed in LJP extract at 1000 µg/mL concentration. The concentration of LJP extract that reduces superoxide anion during the incubation period is very less. The Superoxide anion radical scavenging activity was found to have maximum activity 47.72 % at a concentration of 10 mg/mL in the methanolic seed extract of Syzygium cumini28. The scavenging activity of the extract was dose dependant as the activity increases with the extract concentration. Jamun appears to be the only fruit that contains the five anthocyanidins present in blueberry along with significant amounts of ellagic acid/ ellagitannins. The anti-carcinogenicity potential of these five anthocyanidins provide additive, if not synergistic effects. Antioxidants have attracted much interest with respect to protective effects against radical scavenging activity involved in many degenerative diseases, including cancer. Multiple antioxidants present in whole fruits is considered to elicit higher activity than any individual compounds. Many studies have shown that both pulp and seed of jamun had high antioxidant capacities. Oxidative damage to the DNA is the initiation of most human cancer28.
CONCLUSION:
The present investigation was made to evaluate the antioxidant potential of lyophilized S. cumini (L) Skeels fruit pulp by enzymatic and non-enzymatic radical scavenging activity. The sample lyophilized jamun pulp possess (LJP) DPPH radical scavenging activity, ABTS radical scavenging activity much higher compared to Nitric oxide scavenging activity and superoxide radical activity. More investigations are required to establish therapeutic significance of antioxidant potential of LJP extract and its products as nutraceuticals. There is increased awareness among the people about the beneficial effects of fruits as nutraceuticals in our daily diet. Seasonal and regional fruits can serve an important therapeutic role or as an ingredient in the balanced diet. For the developing country like India, seasonal and low cost fruits like Jamun play a pivotal role in micronutrient deficiency as well as disease prevention.
ACKNOWLEDGEMENT:
Author thanks Dr. L. Stanley Abraham, Scientist E, and Dr. V. Ganesh Kumar, Scientist E, Centre for Ocean research, Sathyabama University for their valuable suggestions in carrying out this research.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 25.03.2017 Modified on 26.04.2017
Accepted on 30.04.2017 © RJPT All right reserved
Research J. Pharm. and Tech. 2017; 10(4): 986-990.
DOI: 10.5958/0974-360X.2017.00179.2