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The cellular metabolism and exogenous species leads to the formation of free radicals. The biocomponents such as DNA present in the cell reacts with these species and get damaged known as the oxidative damage which leads to mutagenesis,aging and formation of carcinogens. This damage is caused by the abstraction and addition reaction leading to the formation of sugar free radicals having carbon centres and OH- and H- radicals of heterocyclic bases. Antioxidants have the ability to be as free radical scavangers and thus are able to save the human body from the negative impacts of many diseases. The extracts of morina longifolia with methanol showed most inhibition of 101.01% of nitric acid scavanging activity whereas the extract with chloroform showed the least inhibition of 42% at the concentration of 500 ug.

The effective impact of antioxidant potential were shown by the flavonoid and phenolic ectract of the plant. The antioxidant potential of methanolic extract can be due to its ability  to donate electrons and direct free radical scavenging properties. Hence the extract of plant with methanol can be taken as ta source that might be used in medicinal and food industry.

The present investigation is aimed to determine anti-oxidant and hemolytic activity of different extracts of aerial parts of M. longifolia.

Studies on atherosclerosis reveal the probability that the disease may be due to free radical reactions involving diet-derived lipids in the arterial wall and serum to yield peroxides and other substances. These compounds induce endothelial cell injury and produce changes in the arterial walls [4]. Various human diseases such as Alzheimer’s disease, aging, cancer, inflammation, rheumatoid arthritis and atherosclerosis [5, 6] also initiated by free radicals. Antioxidants act as radical scavengers, and are able to protect the human body from these diseases [7, 8]. Antioxidants are synthesized within the body and can also be extracted from the food such as fruits, vegetables, seeds, nuts and oil. Natural antioxidants from many fruits, vegetables and medicinal plants have attracted a great deal of public and scientific attention because of their anti-carcinogenic potential and many other pharmacological effects. Recent epidemiological studies have indicated that diets rich in fruits and vegetables and phytochemicals such as polyphenols, vitamin C, volatile oils and flavonoids are correlated with reduced incidence of cardiovascular and chronic diseases and of certain cancers [9, 10]. Flavonoids can be used for the antimicrobial, antioxidant and cytotoxic activities of some Yemeni medicinal plants [11]. A great number of natural medicinal plants have been tested for their antioxidant activities and results have shown that the crude extracts or isolated pure compounds from them were more effective antioxidants in vitro than BHT or vitamin E[12]. So, medicinal plants can be a potential source of natural antioxidants. Free radicals, however, are not always harmful. They also serve useful purposes in the human body. Several observations indicate that Contribution of various studies have shown that a major role for the maturation of cellular structures has been played by the oxygen radicals in living systems. Furthermore, white blood cells release free radicals to destroy invading pathogenic microbes as part of the body's defense mechanism against disease[13]. Hence it is not possible to completely eliminate the free radicle as it will also be harmful to the plant. Plant extracts which showed hemolytic activity cannot be used as anti-oxidants. This investigation will reveal the anti-oxidant and hemolytic activity of different extracts of aerial parts of M. longifolia.

Plant Material

The aerial parts of Morina longifolia Wall were collected from Dagan Dhar, District Doda, (at an altitude of 3500-3800 meters), Kashmir Himalaya, in September 2021. The plant species was identified by Dr. Tasir Sharief, Department of Botany, Govt. Degree College Doda J&K. A voucher specimen (DCD/BOT 08/2021) was deposited in the Department of Botany. Extraction The air-dried and powdered aerial parts of Morina longifolia Wall (1.0 kg) was washed with light petroleum ether (60-800). A small part of petroleum free mass was extracted with 90% ethanol. The solvent was evaporated off under reduced pressure. The remaining petroleum free mass was sequentially extracted with chloroform, ethyl acetate and methanol. Each extract were concentrated under reduced pressure over vacuum evaporator. Few grams of each extract were subjected to series of purification as per pharmacological profiles. The purified extracts i.e., crude ethanolic extract (MLE), chloroform extract (MLC), ethyl acetate extract (MLEA) and methanol extract (MLM) were subjected to biological studies and the active extracts were subjected to column chromatography. Determination of Antioxidant Activity The antioxidant activity of MLE, MLC, MLEA and MLM of aerial parts of M. logifolia was determined by using Diphenyl picryl hydrazyl (DPPH) radical scavenging, Nitric oxide (NO) radical scavenging, and reducing power methods. Determination of Reducing Power/Potential (RP) The antioxidant properties of the extracts were measured by modified reducing power method by Oyaizu, [14]. Stock solutions of the extracts (concentration 1mg/ml) were made by using DMSO. Five different concentration of extract (100 µg, 200 µg, 300 µg, 400 µg and 500 µg) were mixed with 1.5 ml of sodium phosphate buffer (0.2 M, pH 6.6) and 1.5 ml 1% of potassium ferricyanide solution. The mixture was then incubated at 50°C for 20 minutes. After incubation 2.5 ml of 10% TCA solution is added and the solution mixture is centrifuged at 1000 rpm for 10 minutes. After centrifugation the upper layer (1.5 ml) was taken and mixed with 1.5 ml of distilled water and 300 µl of 0.1% of ferric chloride and then the absorbance was measured at 700 nm. In an identical manner solution of ascorbic acid (200 mg/mL) was also incubated. Higher absorbance indicates higher reducing power. Reducing power of all the extracts were measured as ascorbate equivalent. DPPH Radical Scavenging Assay The DPPH radical scavenging assay was performed by the modified method of Ohinishi et al., [15]. Stock solutions of the extracts (concentration 1mg/ml) were made by using DMSO. Ascorbic acid was used as a control. DPPH stock concentration was made as 19 mg/ 500 ml of ethanol. Five different concentrations of each extracts (100µg, 200µg, 300µg, 400µg and 500µg) were made and 2ml of each were mixed with 3 ml of DPPH solution. The volume of the solution mixture was made up to 3.5 ml by adding DMSO. Similarly 2ml ethanolic solutions of ascorbic acid (200μg/mL) were mixed with 3ml of DPPH solution which was used as standard. The final solution mixture was then kept for incubation in dark for 30 minutes at room temperature. After the incubation period the absorbance was taken at 517 nm. DPPH blank or control was measured by adding 500 µl DMSO and 3 ml DPPH and then taking the absorbance at 517 nm. Experiment blank was made by using DMSO only. The percentage inhibition was measured by using the following formula.

Nitric Oxide Radical Scavenging Activity

The nitric oxide radical scavenging activity was measured by using Griess’ reagent. 5ml of each extract solutions of different concentrations (100, 200, 300, 400, & 500 µg/L) in standard phosphate buffer solution (pH 7.4) were incubated with 5ml of sodium nitroprusside solution (5mM) in standard phosphate buffer (pH 7.4) at 25°C for 5 hours. In an identical manner 5ml of ascorbic acid solution (200 μg/mL) in standard phosphate buffer solution (pH 7.4) was also incubated with 5ml of sodium nitroprusside solution (5mM) in standard phosphate buffer (pH 7.4). Control experiments without the test compound but with equivalent amount of buffer were also conducted. After incubation, 1ml of the incubation mixture was mixed with 1ml of Griess’ reagent (Sulphanilamide 1%, O-phosphoric acid 2% and naphthyl ethylene diamine dihydrochloride 0.1%) and the absorbance was measured at 546nm [16]. From the absorbance the percent scavenging activity was calculated using the following formula:

Hemolytic Assay

Hemolytic effect of different extracts on human and rat erythrocytes was evaluated by using washed erythrocytes (RBCs). For the preparation of human and rat erythrocytes, the method of Malagoli [17] was followed. Blood samples from Charles foster strain rats were collected (each weighing 130–180 g) in citrated tubes. The cells were then washed three times with 20 mM Tris–HCl containing 144 mM NaCl (pH 7.4) and a 2% erythrocyte suspension was prepared. Human erythrocytes were obtained from the peripheral blood (O positive) of a healthy volunteer. The blood was used within 24 h after bleeding and washed three times in nine volumes of sterile 0.85% NaCl saline solution. After each washing, cells were centrifuged 150g for 5 min and the supernatant was discarded. The final pellet was diluted 1:9 (v/v) in sterile 0.85% NaCl saline solution and then in 1:24 (v/v) sterile Dulbecco’s phosphate buffer saline (D-PBS), pH 7.0 containing 0.5 mM boric acid and 1 mM calcium chloride. The hemolytic activity of the extracts was tested under in vitro conditions in 96-well plates. Each well received 100 ml of 0.85% NaCl solution containing 10 mM CaCl2. The first well served as negative control containing solvent only. In the second well, 100 ml of extracts of various concentrations (5–500 mg ml) were added. The last well served as positive control containing 20 ml of 0.1% Triton X-100 in 0.85% saline. Each well then received 100 ml of a 2% suspension of rat and human erythrocytes in 0.85% saline containing 10 mM CaCl2. After 30-min incubation at room temperature, cells were centrifuged and the supernatant was used to measure the absorbance of the liberated hemoglobin at 540 nm. The average value was calculated from triplicate assays and the IC50 values and 95% confidence interval (CI 95%) were obtained by non-linear regression analyses. All the experiments were done in triplicates and repeated three times. The values were expressed in mean ± SEM.

DPPH Scavenging Activity

The results of DPPH scavenging activity of different extracts are given in table 1. It is evident from the table that methanol extract of M. longifolia showed maximum scavenging activity (56.22% at the concentration 500 µg when compared with other extracts. The DPPH scavenging power of all the extracts increased with increase in concentration. The order of DPPH radical scavenging assay was found to be MLM > MLEA > MLE > MLC. The DPPH scavenging activity of ascorbic acid at 200µg/mL was found to be 84.56% whereas the DPPH scavenging activity of MLM, MLEA, MLE and MLC at the concentration of 500µg/mL was found to be 98.67%, 88.18%, 56.84% and 27.59%, respectively.

Niric Oxide Scavenging Activity

The methanol extract showed maximum inhibition of (101.03%) of nitric oxide radical scavenging activity whereas chloroform extract showed minimum inhibition  43.11% at the concentration of 500µg (table 1) whereas  ascorbic acid showed 91.04% inhibition at the concentration of 200 mg/mL indicate that the scavenging capacity of methanol extract was comparable to that of ascorbic acid. The order of nitric acid scavenging power was found to be MLM > MLEA > MLE > MLC. Furthermore it was observed that nitric oxide scavenging power of different extracts was increased with increase in concentration.

Reducing Power Ability

The results of reducing ability of the various extracts are given in table 1. It was found that the reducing power of these extracts increased with increasing concentration. The values are represented as absorbance. The ethyl acetate extract was found most effective in reduction of Iron (III) and the value was 0.608 O.D at the concentration of 500 mg of the extract. Ascorbic acid showed 0.430 O.D. at the concentration of 200mg. The order of reducing activity was found as MLEA > MLM > MLE > MLC at 500mg concentration. The reducing power of each extract was found to be increased with increasing concentration.

Hemolytic Activity 

To check the potent of the plant matter regarding antioxidant and anticancer activity hemolytic assays were carried out because plants showing hemolytic essays are not potent for such activities. In addition, these data also may reveal some information about the mechanism of cytotoxicity.  In-vitro hemolytic activity on rat and human erythrocytes of various extracts obtained from aerial parts of M. logifolia was performed (Table 2). The total hemolysis was obtained using 20 μl of Triton X-100 (0.1%) and 1 hour incubation. The IC₅₀ and 95 % confidence interval (CI 95%) were obtained by non-linear regression analyses. IC₅₀ values lower than 200μg/ml was considered active. None of the extracts possessed any hemolytic activity against rat or human erythrocytes. 

Discussion

In complex systems, various different mechanisms may contribute to oxidative processes such as generation of different reactive oxygen species from various target structures such as lipids, proteins and carbohydrates. Therefore, it is essential to characterize the extracts by different antioxidant assays [18]. In the present study, the antioxidant assays such as DPPH radical scavenging, nitric

oxide scavenging capacity and measurement of RP together with haemolytic assay were employed to assess the antioxidant activities of different extracts obtained from aerial parts of M. longifolia Wall. Phenolic substances and flavonoids have been shown to be responsible for the antioxidant activity of many of the plants and have been ascribed to various properties like anticancer, antidiabetic, antiaging and prevention of cardiovascular diseases [9, 10].

The ethyl acetate, methanolic and crude alcoholic extracts was found have maximum DPPH and nitric oxide scavenging and reducing power as compared with chloroform extract.  The methanol and ethyl acetate extracts were found to be having the maximum antioxidant activity which may be due to the presence of high amount of flavonoids and phenolic compoundss. This revealed the fact that the extracts obtained from polar solvents was found to be having the maximum antioxidant activity in comparison with the one obtained from non-polar solvents [19]. Ramzi et al., also reported that flavonoids could be responsible for the antimicrobial, antioxidant and cytotoxic activities of some Yemeni medicinal plants [11].

DPPH is a free radical and accept an electron or hydrogen radical to become stable diamagnetic molecule [20]. Among the different extracts, the acetone extract was showing the maximum DPPH scavenging activity.  This was also reported in Lotus seeds where the organic solvent extracts have high levels of DPPH activity [21]. It has been found that antioxidant molecules such ascorbic acid, tocopherol, flavonoids, and tannins reduce and decolourise the DPPH due to their hydrogen donating ability [22].

The principle behind reducing power assay is based on its electron donating activity, which is an important mechanism of phenolic antioxidant action [23]. The ethyl acetate extract was found most effective in reduction of Iron (III) and the value was 0.55 O.D at the concentration of 500 mg of the extract. The order of reducing power was found to be MLEA > MLM > MLE > MLC at 500mg concentration. The reducing potential is very important aspect for the estimation of antioxidant activity. Reducing agents like ascorbic acid can react directly with peroxides and with certain precursors, thereby preventing the formation of peroxide. Therefore the antioxidant activity of M. logifolia extracts might be due to the reduction of superoxide anions, inactivation of free radicals or complexion with metal ions or combination of the three. The anti-oxidant activity might be due to presence of flavonoid or phenolic compounds [24]. Nitric oxide is a free radical produced in the mammalian cells and is involved in regulation of various physiological processes. However excess production of nitric oxide is associated with several diseases [25]. Methanolic extract of M. longifolia aerial parts has demonstrated dose dependent radical scavenging activity against NO free radicals. Hemolytic assays were performed because compounds possessing potent antioxidant and anticancer activity may not be useful in pharmacological preparations if they possess hemolytic effect. None of the extracts were found to possess hemolytic activity (Table 2).

The result of the present study showed that all the extracts exhibited different extent of antioxidant activity but methanol extract showed a higher potency than other extracts of M. longifolia and demonstrated dose dependent antioxidant activity comparable with Ascorbic acid. In all the methods, maximum antioxidant activity was found at the dose of 500 μg/mL.

The result of the present study showed that all the extracts exhibited different extent of antioxidant activity but methanol extract showed a higher potency than other extracts of M. longifolia and demonstrated dose dependent antioxidant activity comparable with Ascorbic acid. In all the methods, maximum antioxidant activity was found at the dose of 500 μg/mL.

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