INTRODUCTION:

Interest in the search for new natural antioxidant has grown over the past years because reactive oxygen species (ROS) production and oxidative stress have been shown to be linked to disease such as cancer, cardiovascular disease, osteoporosis and degenerative disease. Such natural antioxidant substances are believed to play a potential role in interfering with the oxidation process by reacting with free radicals, chelating catalytic metals and scavenging oxygen in the biological systems¹.

Free radicals play an important role in some pathogenesis of serious diseases, such as neurodegenerative disorders, atherosclerosis, cataracts, diabetes and inflammation². Oxidation processes caused by reactive oxygen species are a major cause of deterioration of various food products and finally lead to loss of nutritive value or complete spoilage³. Antioxidants can influence the oxidation process through simple or complex mechanisms, including prevention of chain initiation, binding of translational metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, and radical scavenging⁴. Hence there is growing interest in natural poly-phenolic antioxidants, present in medicinal and dietary plants that help assuage oxidative damage⁶. These natural antioxidants not only protect food lipids from oxidation, but also provide health benefits associated with preventing damage due to biological degeneration⁷. The main areas of interest for nutritional and phyto-therapeutic research are nutraceuticals value and antioxidant activity. M. edule is a shrub or a small tree belonging to the family Melastomataceae. It is commonly known as Anjani or Iron wood tree found distributed in coastal region of Deccan Peninsula, eastern part of India and Andaman islands⁷.Traditionally plant is used in the treatment of skin disorder, stomach disorder and snake bite⁸. Many phytoconstituents such as umbelactone,amyrin, sitosterol, tartaric acid, malic acid, oleanolic acid, urseolic acid⁹, tannins¹⁰ were reported. Biological activity such as antimicrobial¹¹, anti-diabetic¹², anti-viral¹³ and wound healing activity¹⁴ were also reported. The antioxidant properties of aqueous and ethanolic extract of the roots of M. edule were determined by radical scavenging activity, super oxide scavenging activity, reducing power and FRAP assay.

MATERIAL AND METHODS:

Plant material:

The plant was collected from Barnawapara forest near Raipur managed by Government of Chhattisgarh State Forest Division. The collected plant was identified and voucher specimen was submitted to the by Department of Botany, Gov. Science College, Durg, Chhattisgarh. The roots were separated from the main plant and dried under shade. Dried roots were subjected to grinding in Willey Grinder to 60 mesh size, and a homogenous brown powder was obtained and stored in an air-conditioned room at 4°C before preparation of the extracts for analysis.

Preparation of extracts:

Preparation of aqueous extract:

Plant material (50 g) was placed in a 500 ml round bottom ﬂask and 250 ml water was added. The ﬂask was connected to a hydro distillation apparatus and the water was boiled for 2 h. Thereafter, the resulting water extract was removed and the extraction process was repeated with fresh extractant a further two times. The water extracts were combined, ﬁltered, reduced in vacuo (45⁰C), freeze dried and then weighed to determine the total extractable compounds (7g). The extract was Stored at 4⁰C for further use.

Preparation of alcoholic extract:

The dried roots were powdered (50g) and extracted with ethanol (95%) in a soxhlet apparatus for 4 h. The extracts were filtered and evaporated to dryness under reduced pressure and controlled temperature (40– 50 ˚C) in a rotary evaporator, then weighed to determine the total extractable compounds (12 g). The Extract was Stored at 4⁰C for further use.

Preliminary Phytochemical screening (Qualitative Analysis):

The preliminary phytochemical studies were performed for testing the different chemical groups present in ethanolic and aqueous extract¹⁵.

Chemicals:

DPPH (1,1 -Diphenyl-2-picrylhydrazyl), ferric chloride, Gallic acid, Ascorbic acid, Quercetin , Folin-Ciocalteu^’s phenol reagent, potassium ferri cyanide, trichloroacetic acid, nicotinamide adenine di-nucleotide (NADH), nitro-blue-tetrazolium (NBT), phenazine-metho-sulphate (PMS) and pyrogallol were purchased from Sigma–Aldrich Chemie (Steinheim, Germany). All other solvents and chemicals were of analytical grade and obtained from Merck, Mumbai (India). The absorbance measurement was done by the ultraviolet-visible spectrophotometer (Shimadzu, 1800).

Antioxidant assays:

Determination of total phenolics:

The total phenolics content was deliberate through the method described by Kima¹⁶ et al. The sample mixture contains an aliquot (1 ml) of appropriately diluted extracts or standard solutions of gallic acid (20, 40, 60, 80 and 100 mg/l) which was added to a 25 ml volumetric ﬂask containing 9 ml of ddH₂O, for reagent blank we have used ddH₂O. Now add 1ml of Folin and Ciocalteu’s phenol reagent to the above mixture and shaken. After 5 min, 10 ml of 7% Na₂CO₃ solution was added with mixing. The solution was then immediately diluted to volume (25 ml) with ddH₂O and mixed thoroughly and incubates for 90 min at 23⁰C. Now absorbance was taken for extracts and blank at 750 nm. For both fractions of extract of M. edule the total phenolic contents were expressed as mg gallic acid equivalents (GAE)/100 g fresh sample. All samples were analyzed in three replications.

Total flavonoid content:

Total ﬂavonoid content was determined by method described by Jin and Yin¹⁷. According to this method aliquots was prepared by dissolving of 0.1 g of extracts 1 ml deionized water. Takeout 0.5 ml of sample solution and was mixed with 1.5 ml of 95% alcohol, 0.1 ml of 10% aluminum chloride hexahydrate (AlCl₃.6H₂O), 0.1 ml of 1 M potassium acetate (CH₃COOK), and 2.8 ml of deionized water. The mixture was kept for incubation at room temperature for 40 min. After the reaction time absorbance was measured at 415 nm against a deionized water blank on spectrophotometer (Shimadzu-1800). Quercetin was chosen as a standard (0–50 mg/l). Total ﬂavonoids of fruits were expressed as mg quercetin equivalents (QE) /100 g fresh matter from herbs. Absorbance was determined in triplicate for both extracts respectively.

Total Carotenoids content assay:

The total carotenoid content was determined according to the method of Kocacaliskan and Kadioglu¹⁸ with slight modification. The carotenoids are the compounds with proven antioxidant activity. 10mg of the decoction sample of the roots was dissolved with 10ml distilled water. The absorbance was measured at 450nm the UV-Vis light spectrophotometer. The total carotenoid content was calculated using following equation:

Total carotenoid = 4.07 A₄₅₀– [(0.0435 × Chlorophyll a) + (0.367 × Chlorophyll b)]

Measurements of the DPPH Radical Scavenging Activity:

Myagmar¹⁹ et al. describe the DPPH radical scavenging activity which was measured with spectrophotometer (Shimadzu-1800). DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become stable diamagnetic molecule. In scavenging activity the reaction mixture was contained 1.0 ml of 0.1 mM DPPH-ethanol solution, 0.95 ml of 0.05 M Tris-HCl buffer (pH 7.4) and 50 µl of the different concentration of fraction of extracts as well as for standards (0-100µg/ml). After 30 sec the absorbance was measured in spectrophotometer at 517 nm and the free radical scavenging activity was calculated in the form of IC₅₀ value according to the standard equation using ascorbic acid as control. Lower value of absorbance indicates the higher free radical scavenging activity of the reaction mixture. The reduction capability of DPPH radicals was determined by the decrease in its which is induced by antioxidants. The IC₅₀ value is the concentration where 50% inhibition occurs. In the blank control, the sample was substituted with deionized water. In the positive control, the sample was substituted with Ascorbic acid.

DPPH Scavenged (%) = [(1-A _{test 517}/A_{control 517})] X100

Reducing power assay:

The reducing power of both extract of M. edule was determined according to the method reported by Shen Q²⁰. 1.0 ml of sample (20 to 100µg/ml) in PBS buffer (pH = 7.4, 0.02 M) was mixed with 1.0 ml potassium ferricyanide (1.0%, w/v). After this, the mixture was incubated at 50 ⁰C for 20 min. Then 1.0 ml of tri chloro acetic acid (10.0%, w/v) was added to the mixture to terminate the reaction. After that, the solution was mixed with 0.4 ml ferric chloride (0.1%, w/v) for 10 min. Ascorbic acid at various concentrations (20 to 100µg/ml) was used as standard. The absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

Superoxide-radical scavenging assay:

The superoxide scavenging ability of the extract was assessed by the method of Singh²¹ et al. According to this method different concentration of extract (100-1000 µl/ml) was prepared. Different concentrations of extracts were taken and the total volume made up to 400 µl using phosphate buffer (0.1M pH 7.4). The reaction mixture containing 400 µl of different serial dilutions, 1 ml of PMS (60 µM), 1 ml of NADH (677µM) and 100 µl of NBT (144 µM) in phosphate buffer (0.1M pH 7.4), was incubated at room temperature for 5 min. The color was read using spectrophotometer at 560 nm against a blank. All the measurements were taken in triplicate and the mean values were calculated. Percentage SRSA was calculated using the following equation:

% SRSA = [1– Abs _sample560/ Abs _control560] X 100.

Ferric reducing antioxidant power (FRAP) assay:

Ferric reducing antioxidant power assay of the extract of M.edulewas done according to the method of Benzi and Stain (Benziand Stain, 1996) with some modifications. The stock solutions included 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM hydrochloric acid, and 20 mM ferric chloride hexahydrate solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL ferric chloride hexahydrate solution, which was then warmed to 37 °C before use. The solutions of extract and that of troloxwere prepared in methanol (250 μg mL). 10 μL of each of crude extracts was taken in separate test tubes and 2990 μL of FRAP solution was added to each to make a total volume of 3 mL. The plant crude extracts were allowed to react with the FRAP solution in the dark for 30 min. The absorbance of the coloured product (ferrous tripyridyltriazine complex) was measured at 593 nm. Additional dilution was needed if the FRAP value measured was over the linear range of the standard curve. The results are expressed in μmol TE/mL

RESULTS AND DISCUSSION:

Preliminary phytochemical screening

Preliminary phytochemical screening of different fractions of M. edule where showed presence of steroid, triterpenes, flavonoid and polyphenolics (Table 1).

Anti-oxidant assay:

Determination of total phenolic content:

The total amount of phenolics in M. edule extracts determined that there were 94±1.4 and 20.64±0.26 mg gallic acid equivalents of phenolic compounds in 100g of the ethanolic and aqueous extract of M. edule respectively (Figure 1). It may be attributed, in a significant part to antioxidant activities of the M. edule.

Table 1 Phyto-constituents present in M. edule.

Extract / fractions	% yield	Phyto-constituents
Extract / fractions	% yield	Alkaloid	Steroid	Triterpenes	Glycoside	Phenolic	Flavonoid	Tannin	Sugar	Saponins
Ethanolic	24	-	+	+	-	+	+	+	-	-
Aqueous	14	-	+	+	-	+	+	+	-	-

+ Present, - absent.

Table 2 FRAP value, IC₅₀ and EC₅₀for standards and extracts of M. edule.

Method	Parameter	Sample	Result
DPPH	IC ₅₀	Ascorbic acid	61±0.58µg/ml
DPPH	IC ₅₀	Ethanolic extract	77.5±0.78µg/ml
Superoxide radical scavenging activity	IC ₅₀	Gallic acid	679±2.4µg/ml
Superoxide radical scavenging activity	IC ₅₀	Ethanolic extract	894±5.4µg/ml
Reducing power assay	EC₅₀	Ascorbic acid	55±0.97 µg/ml
Reducing power assay	EC₅₀	Ethanolic extract	74±1.4µg/ml
Ferric reducing antioxidant power (FRAP) assay	FRAP value	Ethanolic extract	320±0.89μmol TE mL^-1
Ferric reducing antioxidant power (FRAP) assay	FRAP value	Aqueous extract	41±0.83μmol TE mL^-1

Figure 1 Total phenolic content

Figure 2 Total flavonoid content

Figure 3 DPPH radical scavenging activity

Figure 4 Reducing power assay

Figure 5 Superoxide radical scavenging activity

Determination of total flavonoid content

The total amount of flavonoid content in M. edule extracts found to be 21.56±3.2 and 5.95±0.18 mg quercetin equivalents (QE) of flavonoid content in 100g of the ethanolic and aqueous extract of M. edule respectively (Figure 2).

Total carotenoid content assay

The total carotenoid contents of the ethanolic extract and aqueous extract of roots M.edule are 43.12±0.75µg and 23.12±0.55µg respectively.

1, 1-diphenyl-2-picryl hydrazyl (DPPH) assay

This assay illustrates a decrease in the concentration of DPPH radical due to the scavenging ability of the soluble constituents in the ethanolic extract of roots of M. edule and the standard ascorbic acid, as a reference compound, presented the highest activity at all concentrations. The IC ₅₀ values were found to be 77.5± 0.78 µg/ml and 61± 0.58 µg/ml for ethanolic extract of roots of M. edule and ascorbic acid respectively (Figure 3). Aqueous extract of not showing any scavenging activity.

Reducing power assay:

All the concentration of ethanolic extract of roots of M. edule showed significant activities when compared to standard ascorbic acid. The EC₅₀ values were found to be 74.34±1.4 µg/ml and 55.12±0.97 µg/ml of ethanolic extract of roots of M. edule and ascorbic acid respectively (Figure 4). Aqueous extract of M. edule not showing significant reducing capacity.

Superoxide radical scavenging activity:

From our studies we found that the ethanolic extract of roots of M. edule showed greater percentage of inhibition with increase in concentrations. The IC ₅₀was found to be 894± 5.4µg/ml and 679± 2.4µg/ml for ethanolic extract of roots of M. edule and gallic acid respectively (Figure 5). Significant superoxide radical scavenging activity not found for aqueous extract.

Ferric reducing antioxidant power (FRAP) assay:

The ferric reducing antioxidant power (FRAP) assay measures the reducing ability of antioxidants against the oxidative effects of reactive oxygen species. In redox reaction electron donating antioxidants can be described as reductants and inactivation of oxidants by reductants. This assay is based on the ability of antioxidants to reduce Fe³⁺ to Fe²⁺ in the presence of tripyridyltriazine (TPTZ), whereby an intense blue Fe²⁺–TPTZ complex with an absorbance maximum at 593 nm is formed. Increasing absorbance indicates an increase in reductive ability. The ethanolic fraction showed the highest FRAPS value (320±0.89μmol TE mL^-1) and aqueous extract showed the lower FRAPS value (41±0.83μmol TE mL^-1). The FRAP value, IC ₅₀ and EC ₅₀are mentioned in (Table 2).

CONCLUSION:

Various chemical constituents are present in the M. umbellate are as tannin, flavonoid, triterpenes and phenolics, which are responsible for antioxidant activity. Phenolic compounds and flavonoids are well known as antioxidant and scavenging agents against free radicals associated with oxidative damage. Total phenolic and flavonoid content was present in higher concentration in ethanolic than aqueous extract of M. edule. The high antioxidant activity exhibited by M. edule extract provided justification for the therapeutic use of this plant in folkloric medicine due to the phytochemical constituents. The present data suggest that this extract could be a potential source of natural antioxidant that could be of great importance for the treatment of radical related diseases and age associated diseases.

ACKNOWLEDGEMENT:

The authors would like to thanks Govt. Science College Durg for identification and management of Apollo College of Pharmacy, Anjora, Durg for providing necessary facilities and support to carry out this work.

CONFLICT OF INTERESTS:

The authors declare that there is no conflict of interests regarding the publication of this paper.

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Received on 07.09.2016 Modified on 08.11.2016

Research J. Pharm. and Tech 2016; 9(10):1547-1551.

DOI: 10.5958/0974-360X.2016.00303.6