Investigation of the Phytoconstituents and Antioxidant Activity of Diospyros malabarica Fruit Extracts

Diospyros malabarica (Deshi Gab) belonging to the family “Ebenaceae” grows well in the humid tropical climate of Bangladesh. In order to investigate the phytoconstituents both qualitatively and quantitatively, the seed and flesh extracts of D. malabarica were prepared using two polar solvents (i.e., water and 70% ethanol) and a nonpolar solvent (i.e., hexane). The maximum yield was obtained for aqueous and ethanolic seed extracts indicating that most of the phytoconstituents present in D. malabarica fruit are polar. The qualitative phytochemical analysis of the extracts revealed the presence of diverse amount of phytoconstituents in the extracts. On the other hand, the quantitative phytochemical analysis for phenols, tannins, flavonoids, alkaloids, saponin, proteins, reducing sugar and vitamin C revealed that the maximum amount of phenols, tannins, flavonoids and reducing sugar were present in aqueous seed extract. However, the maximum amount of total protein and vitamin C was found in ethanolic seed extract. D. malabarica seed powder contained more amount of alkaloids

To tackle the menace of various free radicals and reactive oxygen species, potential antioxidants having ability to scavenge free radicals by donating their redox hydrogen are necessary [9]. Although the free radicals can be scavenged by both the natural (plant derived) and synthetic (chemically synthesized) antioxidants, the natural antioxidants are getting preference because they do scavenge free radicals without any side effects [14]. Several studies reported that the synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) show toxicity to human through DNA damaging and other toxic effects [15] [16]. The toxicity of these chemically synthesized antioxidants has necessitated the search of new natural antioxidants. Previously published reports showed that the plant-derived antioxidants have been known to protect human from several chronic diseases such as inflammation, autoimmune diseases, cancer, and tumor formation by inhibiting the propagation of free radical reactions [17]. Hence the present study was conducted to investigate the presence of natural antioxidants in different parts of Diospyros malabarica fruit. D. malabarica belonging to the family "Ebenaceae" grows well in the humid tropical climate of Bangladesh. It is considered to be medicinally important because different parts of it, for instance-bark, showed anti-diabetic [18], anti-diarrheal [19], and anti-inflammatory activity [2]. Previous studies also re- ported that D. malabarica is traditionally used for the treatment of ulcer, dysentery, intermittent fever and irregularities in the menstrual cycle [18] [20]. Furthermore, the unripe fruits are usually used as traditional medicine for the treatment of diarrhea and dysentery as well as to paint the undersides of boats to provide protection in water and thus act as a preservative. The infusion of the unripe fruit is also used to harden ropes and render them more durability in water [2]. Since extensive studies on different parts of D. malabarica fruits have not been carried out yet, different parts of D. malabarica fruits may contain pharmacologically active phytoconstituents with excellent antioxidant propensity.
Herein, we prepared D. malabarica seed and flesh extracts using water, 70% ethanol (i.e., polar solvent), and hexane (i.e., non-polar solvent) to extract both polar and nonpolar phytochemicals. The phytoconstituents of the extracts were determined both qualitatively and quantitatively. The antioxidant activity of the extracts was investigated through DPPH free radical scavenging assay, FRAP assay, and reducing power assay.

Sample Collection and Preparation
The unripe fruits of D. malabarica were collected from the local market of Mymensingh district, Bangladesh (24.7471˚N, 90.4203˚E) and were brought to the laboratory for analysis. After washing properly with water, the flesh and seed parts of the collected D. malabarica fruits were separated carefully and cut into small pieces. Both the flesh and seeds were then dried in a hot air oven at 60˚C to remove solvents (i.e., ethanol, hexane, and water) and condense the phytoconstituents. Thereafter, the dried chips were ground into coarse powder using a blender and the powders were stored in hermetically sealed containers to store the extracts in aseptic conditions as well as to protect them from air, moisture and contaminants, with necessary markings in a cool, dark and dry place.

Test for Flavonoids (Alkaline Reagent Test)
Few drops of 10% NaOH were added to 1 ml of the extracts. The addition of NaOH to the solution initiated the formation of intense yellow color and then the solution became colorless upon addition of few drops of diluted HCl which indicated the presence of flavonoids [21].

Test for Amino Acids and Proteins (Xanthoproteic Test)
2 -6 drops of concentrated HNO 3 were added to 1 ml of the extracts. Then concentrated NaOH solution was added to neutralize the solution. The appearance of yellow or orange color indicated the presence of protein and amino acids in the sample extracts [21].

Test for Cardiac-Active Glycosides (Keller-Killani Test)
2 ml of glacial acetic acid containing one drop of ferric chloride (FeCl 3 ) solution was added to 5 ml of the extracts. Then 1 ml concentrated sulfuric acid was added to the mixture. Brown ring was formed at the interface which indicated the presence of deoxy sugar of cardenolides. A violet ring may appear beneath the brown ring and a greenish ring may also form gradually throughout the acetic acid layer [24].

Test for Terpenoids (Salkowski Test)
5 ml of the extracts were mixed properly with 2 ml of chloroform. Then 3 ml concentrated sulfuric acid was also added to the solution. The appearance of the reddish brown colour at the interface indicated the presence of terpenoids [13].

Test for Xanthoproteins
Few drops of concentrated HNO 3 and NH 3 solution were mixed separately with 1 ml of the extracts. Formation of reddish orange precipitate indicated the presence of xanthoproteins [25].

Test for Quinones
1 ml of alcoholic potassium hydroxide solution was mixed separately with 1 ml of each of the extracts. The formation of color ranging from red to blue gave the proof of the presence of quinones [26].

Determination of Total Phenolic Content (TPC)
The content of total phenolic compounds in D. malabarica seed and flesh extracts was determined by Folin Ciocalteu Reagent (FCR) method as described by Polash, et al. [22] with little modification. Here, gallic acid was used as a standard. The stock solution of aqueous, ethanolic and hexane extracts of D. malabarica seed and flesh were diluted to different concentrations with saline solution (0.9% NaCl). 0.1 ml of the different concentrations of standards and sample extracts were taken into different falcon tubes. 500 µl FCR (10% v/v) and then 400 µl of 7.5% sodium carbonate were added to each tube. The mixtures were incubated for 1.5 hour at room temperature. The absorbance of the mixtures was measured at 765 nm by using UV-vis spectrophotometer (Optizen POP, Korea) against blank containing distilled water. Different concentrations of gallic acid were used to prepare a standard calibration curve that was used to determine the TPC value of the extracts. The TPC of the sample extracts were expressed as microgram of gallic acid equivalent per milliliter (µg GAE/ml).

Estimation of Total Tannin Content (TTC)
Total tannin content (TTC) of D. malabarica seed and flesh extracts was determined according to the protocol described by Tambe and Bhambar [27] with little modification and tannic acid was used as standard. Different concentrations of D. malabarica extracts were prepared with saline solution (0.9% NaCl). In this experiment, 0.1 ml of the different concentrations of standards and sample extracts were taken into different falcon tubes. Then 7.5 ml of distilled water was added to each tubes. 0.5 ml of 100% Folin-Ciocalteu's reagent was added to the above mixtures followed by and the addition of 1 ml 35% Na 2 CO 3 and 0.9 ml of distilled water. The mixtures were incubated for 30 min at room temperature.
Finally, the absorbance of the mixtures was measured at 725 nm by UV-vis spectrophotometer (Optizen POP, Korea) against a blank containing distilled water. The TTC of the extracts was expressed as microgram of tannic acid equivalent per milliliter (µg TAE/ml).

Estimation of Total Flavonoid Content (TFC)
Total flavonoid content (TFC) in D. malabarica seed and flesh extracts was determined by aluminium chloride (AlCl 3 ) colorimetric assay [12]. Here, the flavonoid content was expressed as catechin equivalent. 1 ml of the different concentrations of standard and sample extracts were taken into different tubes. Then 4 ml of distilled water was added to each tube followed by the addition of 0.3 ml of 5% sodium nitrite (NaNO 2 ) and incubated for 5 minutes. Then 0.3 ml of 10 % aluminium chloride (AlCl 3 ) was added to the above mixtures and incubated for 6 minutes at room temperature followed by the addition of 2 ml of 1M sodium hydroxide (NaOH). Immediately 2.4 ml of distilled water was added to each tube to make the total volume up to 10 ml. The solutions were mixed well and the absorbance of the mixtures was measured at 510 nm by UV-vis spectrophotometer (Optizen POP, Korea) against a blank containing distilled water. The TFC of the sample extracts was expressed as microgram of catechin equivalent per milliliter (µg CE/ml).

Determination of Total Alkaloids Content
Total alkaloids content in seed and flesh parts of D. malabarica was quantita- hours at room temperature. After incubation, the mixtures were filtered and the volume of the filtrate was reduced to one-quarter of its original volume through evaporation. To this concentrated sample, 32% ammonium hydroxide (aqueous NH 3 ) was added drop-wise in order to precipitate the alkaloids. When the precipitation was completed, the whole solution was allowed to settle and the precipitate was collected by filtration using a pre-weighed filter paper. The percentage of total alkaloid content was calculated as:

( )
Weight of the residue Percentage of total alkaloids % 100 Weight of sample taken = × The alkaloid content of seed and flesh powder of D. malabarica was expressed as mg of alkaloid present per 100 g of sample powder.

Determination of Total Saponin Content
Total saponin content of D. malabarica seed and flesh extracts was estimated by double extraction gravimetric method described by Ezeabara, et al. [29] with few modifications. Briefly, 5 g of the powdered fruit parts were mixed with 50 ml of 20% aqueous ethanol solution and the mixtures were incubated at 55˚C for 90 minutes in a water bath with periodic agitation. After incubation, the mixtures were filtered. The residue was also extracted and filtered in the same manner.
Both the extracts were then mixed together and the combined extract was reduced to 40 ml through evaporation at 90˚C. The concentrated extract was transferred to a separating funnel containing 40 ml dichloromethane and shaken vigorously. When the aqueous and organic layer were partitioned, the aqueous layer was separated. If the aqueous layer is not clear, re-extraction and partitioning should be done. The extracted aqueous portion was transferred to separating funnel where it was washed with 60 ml of 100% ethanol. After that, 50 ml of 5% NaCl solution was added to the mixture. After partitioning, the upper aqueous layer was separated from the funnel and evaporated at 60˚C in a preweighed glass dish. After complete evaporation, the dish was dried and reweighed.
Saponin content was determined from the following formula:

Estimation of Total Protein Content
Total Protein content of D. malabarica seed and flesh extracts were determined

Estimation of Total Reducing Sugar Content
Total reducing sugar (carbohydrates) content of D. malabarica seed and flesh extracts was determined by Nelson-Somogyi method with little modification [32]. In this method, glucose was used as a standard. 1 ml of different dilutions of standards and sample solution were mixed properly with 1 ml of copper reagent. These solutions were boiled at 90˚C for 15 minutes and then cooled. After cooling down, 1 ml of arsenomolybdate color reagent was added to the mixture and the solutions were mixed well. The optical density was measured at 520 nm with UV-vis spectrophotometer (Optizen POP, Korea) against a blank containing reagent and distilled water. The glucose content of sample extracts of various dilutions were expressed as microgram of glucose equivalent per ml of sample extracts (µg Glucose/ml).

Estimation of Vitamin C
Vitamin C content of D. malabarica seed and flesh extracts was determined by 2,4-dinitrophenyl hydrazine method according to the protocol of Kapur, et al. [33] with little modification where 800 µg/ml ascorbic acid prepared with 5% trichloroacetic acid (TCA) was used as standard. 500 µl of different dilutions of standards and sample extract solutions were mixed properly with 100 µl of 2, 4-Dinitrophenyl hydrazine-thiourea-copper (DTC) solution and incubated at 37˚C for 3 hours in water bath. After incubation, 750 µl of ice-cold 65% sulfuric acid was added and the solutions were mixed thoroughly. The solutions were then kept at room temperature for 30 minutes. Finally, the optical density of the mixture was measured at 530 nm with UV-vis spectrophotometer (Optizen POP, Korea) against a blank containing reagent and 5% TCA. The ascorbic acid content of sample extracts of various dilutions was expressed as microgram of ascorbic acid equivalent (AAE) per ml of sample extracts (µg AAE/ml).

DPPH Free Radical Scavenging Activity Assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of the aqueous, ethanolic, and hexane extracts of D. malabarica were determined according to the method described by Manzocco, et al. [34] with few modifica-Advances in Bioscience and Biotechnology

Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay of D. malabarica seed and fruit extracts was performed according to a method established by Benzie and Strain [35] [36] with little modification. Here the antioxidant activity was measured as the equivalent of the known antioxidant agent (i.e., ascorbic acid). For the preparation of FRAP reagent, the three solutions (i.e., 300 mM acetate buffer prepared with sodium acetate and glacial acetic acid, 10mM 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl and 20 mM FeCl 3 •6H 2 O in distilled water solutions) were mixed freshly at a ratio of 10:1:1 and pre-warmed at 37˚C before using performing the reducing activity assay. Then the different concentrations of standards and sample extracts (0.2 ml) were taken into different tubes. FRAP reagent (1.5 ml, pre-warmed at 37˚C) was added to each tube and incubated for 4 min at 37˚C. Finally, the absorbance of the mixtures was measured at 593 nm using a UV-vis spectrophotometer (Optizen POP, Korea) against a blank containing FRAP reagent and distilled water. The antioxidant activity of the sample extracts was expressed as microgram of ascorbic acid (AA) equivalent per milliliter (µg AAE/ml).

Reducing Power Assay
The antioxidant potential of D. malabarica seed and fruit extracts was also performed by ferric reducing power assay according to Afsar, et al. [37] and Bag, et al. [15] with little modification. For this experiment, gallic acid was used as

Statistical Analysis
Statistical analysis was carried out by GraphPad Prism 5.0 using one-way analysis of variance (ANOVA) test and the significance of the difference between means was determined by Duncan's multiple range test at (P < 0.05) significant level. All the analysis was carried out multiple times and the result was represented in mean ± SEM.

Yield Coefficient
The percentage of yield indicates the amount of extracts obtained through the extraction procedure expressed in gram (g) of extracts obtained from per 100 gram (g) of crude powder and presented in Table 1.
The highest yield was obtained in aqueous seed extracts (40.6 gm per 100 gm of crude powder) and the lowest yield was obtained in hexane flesh extracts (1.8 gm per 100 gm of crude powder). The higher yield in aqueous as well as in ethanolic extracts indicate that most of the phytoconstituents present in D. malabarica fruit are polar. As a result, the phytoconstituents were easily extracted by polar solvent (i.e., water and ethanol). The low yield in hexane extracts revealed that very low amount of nonpolar phytoconstituents are present in both the seed and flesh extracts of D. malabarica (Table 1). On the other hand, greater yield in seed extracts was obtained when compared to that of flesh extract which indicates that D. malabarica seed extracts are excellent source of phytoconstituents.

Qualitative Phytochemical Screening
The results of qualitative phytochemical screening of D. malabarica seed and flesh extracts are presented in Table 2.
The seed and flesh extracts of D. malabarica prepared using distilled water, and 70% ethanol showed the presence of high amount of tannin, reducing sugar, protein, cardiac-active glycosides, and quinones. In addition, these extracts also  showed the appearance of moderate amounts of phenol, terpenoids, saponins, flavonoids, alkaloids, and glycosides (Table 2). On the other hand, hexane seed, and hexane flesh extracts showed negative results in almost all the qualitative tests which indicates the absence of nonpolar phytoconstituents in the D. malabarica seed and flesh extracts.

Determination of Total Phenol, Tannin and Flavonoid Content
Total phenol content (TPC), total tannin content (TTC) and total flavonoid  (Figures 1-3). For all extracts, the concentration of TPC, TTC and TFC increased as the amount of the plant extracts was increased. The amount of TPC, TTC, and TFC in all the extracts always follow the same order: Aqueous seed > ethanolic seed > ethanolic flesh > aqueous flesh >> hexane extracts. This indicates that most of the phenolics, tannins and flavonoids were extracted through polar solvents (i.e., water and ethanol). The phenol, tannin, and flavonoid contents in aqueous seed extract were significantly higher than that of aqueous flesh extract. Similarly, the phenol, tannin, and flavonoid contents in ethanolic seed extract were significantly higher than that of ethanolic flesh extract. Although the differences between aqueous seed and ethanolic seed extracts were very minuscule in terms of their phenol, tannin, and flavonoid contents (Figures 1-3). The highest TPC was found in aqueous seed extract (23 µg GAE/ml) and the lowest TPC was found for hexane flesh extract (1.15 µg GAE/ml) at a concentration of 100 µg/ml ( Figure 1).
Again, the highest TTC was found in aqueous seed extract (369 µg TAE/ml) and the lowest TTC was found in hexane seed extract (10.72 µg TAE/ml) at a concentration of 500 µg/ml ( Figure 2). Furthermore, the highest TFC was found in aqueous seed extract (110 µg CE/ml) and the lowest TFC was found in hexane seed extract (2.81 µg CE/ml) at a concentration of 1000 µg/ml (Figure 3).

Determination of Total Alkaloid and Total Saponin Content
Total alkaloid and total saponin content of D. malabarica seed and flesh extracts were estimated by gravimetric analysis method ( Table 3). The total amount of     Advances in Bioscience and Biotechnology that is, alkaloid content of seed was significantly higher than that of flesh.
Therefore, D. malabarica seed is an excellent source of alkaloid. However, the amount of saponin was 0.42 gm and 0.74 gm in 100 gm of each of the seed and flesh powder of D. malabarica, respectively, that is, saponin content of flesh was not significantly higher than that of seed. Hence, D. malabarica fruit is a moderate source of saponin.

Estimation of Total Protein, Reducing Sugar and Vitamin C Content
Total protein, total reducing sugar, and total vitamin C content of D. malabarica seed and flesh extracts were determined in a dose dependent manner ( Figures   4-6). Ethanolic seed extract showed the highest amount of total protein and total vitamin C content, whereas aqueous seed extracts showed the highest amount of total reducing sugar content. In all concentrations, the higher amount of proteins, reducing sugars, and vitamin C were extracted with polar (i.e., water and ethanol) solvents which indicate that most of the proteins, reducing sugars and vitamin C present in D. malabarica seed and flesh were polar in nature. The protein, reducing sugar, and vitamin C content of the aqueous seed extract was significantly higher than that of the aqueous flesh extract. On the other hand, the protein, reducing sugar, and vitamin C content of ethanolic seed extract was significantly higher than that of the ethanolic flesh extract. The differences of protein, reducing sugar and vitamin C content between aqueous seed and ethanolic seed extracts were insignificant (Figures 4-6). The highest protein content was obtained in ethanol seed extract (920 μg BSAE/ml) and the lowest protein content was obtained in aqueous flesh extract (156 μg BSAE/ml) at a concentration of 600 μg/ml (Figure 4). The highest reducing sugar content was obtained in aqueous seed extract (694 μg GE/ml) and the lowest reducing sugar content was  The graph shows vitamin C content of four different concentrations (i.e., 125 µg/ml, 250 µg/ml, 500 µg/ml and 1000 µg/ml) of both the seed and flesh extracts expressed in µg AAE/ml. Here, AF = aqueous flesh extract, AS = aqueous seed extract, EF = ethanolic flesh extract, ES = ethanolic seed extract, HF = hexane flesh extract and HS = hexane seed extract. Data were analyzed by GraphPad Prism 5.0 using ANOVA and significance level was determined (*p < 0.05, ***p < 0.001).
obtained in aqueous flesh extract (194 μg GE/ml) at a concentration of 250 μg/ml ( Figure 5). The highest vitamin C content was obtained in ethanolic seed extract (23 μg AAE/ml) and the lowest vitamin C content was obtained in aqueous flesh extract (5 μg AAE/ml) at a concentration of 250 μg/ml ( Figure 6).
Hexane extracts showed very little or almost absence of protein, reducing sugar as well as ascorbic acid.

DPPH Free Radical Scavenging Assay
DPPH is a stable free radical and that can accept an electron or hydrogen radical quenches DPPH free radicals and converts them into a colourless product [9].
Herein, all the D. malabarica fruit extracts were used for DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging assay and ascorbic acid was used as positive control. The highest scavenging activity was performed by aqueous seed extract, followed by ethanolic seed extract and showed significant free radical scavenging activity even at a very low concentration (5 µg/ml) (Figure 7(A)).
On the other hand, both the aqueous and ethanolic flesh extracts showed significant free radical scavenging activity at 50 µg/ml (Figure 7(A)).
To determine the IC 50 value (i.e., the concentration of the plant extract re-   (Figure 7(B)). Therefore, it was shown that the IC 50 values of aqueous seed, aqueous flesh, ethanolic seed and ethanolic flesh extracts were not significant with respect to ascorbic acid. However, the IC 50 values of both hexane extracts were statistically significant when compared to ascorbic acid. The lowest IC 50 value was obtained for aqueous seed extract (i.e., 44.50 µg/ml) and is the most efficient free radical scavenger. On the other hand, the highest IC 50 value was obtained for hexane flesh extract (i.e., 2359.66 µg/ml) and is the least efficient free radical scavenger.

Ferric Reducing Antioxidant Power (FRAP) Assay
In FRAP assay, the antioxidant activity of D. malabarica fruit extracts was expressed in µg AAE/ml. Here, the reduction of a ferric tripyridyltriazine (Fe 3+ -TPTZ) complex into its ferrous form (Fe 2+ ) in the presence of an antioxidant produces an intense blue color at low pH that can be monitored by measuring the absorbance at 593 nm [38]. Therefore, the change in absorbance is directly related to the reducing power of the electron donating antioxidants present in the reaction mixture [35]. The ferric reducing antioxidant potential (FRAP) for aqueous seed extract was significantly higher than that of aqueous flesh extract.
Similarly, the FRAP for ethanolic seed extract was significantly higher than that of ethanolic flesh extract. However, the difference between aqueous seed and ethanolic seed extracts in terms of ferric reducing antioxidant potential was statistically insignificant (Figure 8). In the current study, the aqueous seed (7.31 µg AAE/ml) and ethanolic seed (5.18 µg AAE/ml) extracts showed significant antioxidant activity when compared to other extracts at a concentration of 25 μg/ml  Figure 8). In addition, the concentration of antioxidant activity increased as the amount of the plant extract (6.25 to 25 μg/ml) was increased.

Reducing Power Assay
The reducing power of the extracts may serve as an indicator of their potential antioxidant activity [9]. In this assay, the presence of antioxidant activity in the sample extracts may cause the reduction of Fe 3+ /Ferric cyanide complex to ferrous form which can be monitored spectrophotometrically at 700 nm. The antioxidant potential of the different seed and flesh extracts were estimated from the differences of their absorption at various concentrations. Furthermore, the reducing power of the sample extracts increased with the increase of their concentrations (i.e., 6.25 to 25 μg/ml) ( Figure 9) and gallic acid was used as positive control. The maximum reducing power activity was obtained in the aqueous seed and ethanolic seed extracts followed by ethanolic flesh and aqueous flesh extracts.

Discussion
The present study was carried out to investigate the phytochemical constituents and antioxidant potential of D. malabarica seed and flesh extracts prepared using two polar (i.e., distilled water, and 70% ethanol) solvents and a nonpolar  be effective for the prevention of diverse oxidative stress associated diseases such as cancer, cardiovascular diseases, chronic inflammation and so on [13] [39] [40]. Tannins are also known to have antibacterial [41] [42], antiviral [43], and antitumor activities [43] [44]. Furthermore, plant derived alkaloids are known to show various biological activities including anti-inflammatory [45], antimalarial [46], and antibacterial [47] activity. Saponins are well known for their wide range of pharmacological and medicinal activities such as anti-ulcer, anticancer, antimicrobial and so on [29]. Vitamin C is known to be used as food additive, antioxidant, and reducing agent [33] [48]. Furthermore, previous research on D. malabarica bark reported the potential bioactivity (e.g., antioxidant activity) of the extract [18] [19].
Our data also support the antioxidant activity of D. malabarica fruit extracts. The aqueous and ethanolic extracts of D. malabarica seed and flesh showed free radical scavenging as well as reducing power activity, that is, antioxidant activity.
The extracts showed antioxidant activity according to the following order: aqueous seed extract > ethanolic seed extract > ethanolic flesh extract > aqueous flesh extract >> hexane extracts. According to Aparadh et al. [49] and Hossain et al. [50], antioxidant activity of plant extracts depend on the presence of respective secondary metabolites, such as flavonoids, tannins and phenolics. Another study reported that vitamin C is the indicator of antioxidant activity [30]. The level of phenolics, tannins, flavonoids, and vitamin C in D. malabarica fruit extracts are in the order of aqueous seed > ethanolic seed > ethanolic flesh > aqueous flesh >> hexane extracts (Figures 1-3, and Figure 6). The antioxidant activity of the extracts follow the same trend as the secondary metabolites ( Figures 7-9). Taken together, since these phytoconstituents are mainly responsible for the antioxidant activity, D. malabarica fruit extracts can be an important source of natural antioxidants.

Conclusion
Diospyros malabarica flesh and seed extracts have been prepared using two polar solvents (i.e., water, and ethanol) and a nonpolar solvent (i.e., hexane). The phytoconstituents including total phenol content, total tannin content, total flavonoid content, total protein content, total reducing sugar content, total ascorbic acid content, total saponin content, and total alkaloid content of all the extracts were determined through both qualitative and quantitative analysis. Some other phytoconstituents such as glycosides, terpenoids, resin and so on were determined only through qualitative analysis. D. malabarica flesh and seed extracts obtained from the polar solvents contained a greater amount of phytoconstituents when compared to their nonpolar solvent counterparts. More specifically, aqueous seed extract contained the highest amount of phytoconstituents except total content of protein and vitamin C. Furthermore, aqueous seed extract showed the highest DPPH free radical scavenging activity, ferric reducing antioxidant power (FRAP) activity and reducing power activity assay followed by ethanolic Advances in Bioscience and Biotechnology seed extract. Therefore, D. malabarica seed is rich in different phytoconstituents with numerous therapeutic propensity and can be used as a potential source of natural antioxidants with other therapeutic applications including hepatoprotective efficacy. D. malabarica fruit can further be screened against various disease causing pathogens and can be a potential source of chemical and biologically important drug candidates.