Assessment of Bioactive Activity of Blackberry Seed Extract

The aim of this study was to determine the activity of the blackberry (Rubus laciniatus extracts. Total phenolic and total antioxidant activity of blackberry seed extracts were assessed with respect to two extracting solvents (absolute ethanol and absolute acetone) and two extracting techniques (hot extraction at 40 ̊C and cold extraction at 26 Proximate analysis showed that blackberry seeds contained 12.12% moisture, 8.00% protein, 0.74% fat, 2.15% ash and 76.99% total carbohydrate. The antioxidant activities of blackberry seeds determined by evaluating total phenolic activity by gallic acid equivalent (GAE), total antioxidant capacity by ascorbic acid equivalent (AAE) content and ferric reducing powder assay. The tested sample showed variable amount of total phenolic content (33.21-56.56 mg GAE/g of dry extract), total antioxidant capacity (75.46-82.42 reducing power (0.23-0.26) depending on different extraction techniques and solvents used. Higher amount of extract, total antioxidant capacity and reducing power were obtained for hot extraction technique. As compared to cold extraction technique higher extract yields (7.48% vs 7.19% for ethanol and 4.35% vs 4.28% for acetone) and other properties were obtained by hot extraction for a given solvent Ethanol extract with hot extraction technique total phenolic content as 56.56 mg GAE/g of dry | Volume – 2 | Issue – 5 | Jul-Aug 2018 6470 | www.ijtsrd.com | Volume Journal of Trend in Scientific and Development (IJTSRD) International Open Access Journal

The aim of this study was to determine the bioactive laciniatus) seed extracts. Total phenolic and total antioxidant activity assessed with respect to two extracting solvents (absolute ethanol and g techniques (hot C and cold extraction at 26˚C). Proximate analysis showed that blackberry seeds contained 12.12% moisture, 8.00% protein, 0.74% fat, 2.15% ash and 76.99% total carbohydrate. The activities of blackberry seeds were total phenolic activity by total antioxidant (AAE) content . The tested sample showed variable amount of total phenolic content E/g of dry extract), total 82.42 AAE/g) and 0.26) depending on different extraction techniques and solvents used. Higher extract, total antioxidant capacity and re obtained for hot extraction As compared to cold extraction technique, (7.48% vs 7.19% for ethanol and and other properties were obtained by hot extraction for a given solvent.
with hot extraction technique gave total phenolic content as 56.56 mg GAE/g of dry extract, total antioxidant capacity as 82.42 AAE/g and reducing power as 0.26 at extract concentration of µg/ml. The overall observations of the present experiment indicated that hot ethanol extract of blackberry seeds have strong antioxidant property and bioactive activity.

INTRODUCTION
Naturally available fruits are gre antioxidant compounds like polyphenols, phenolic acids and flavonoids which protect people from different degenerative diseases like cardiovascular diseases and cancer by scavenging free radicals such as peroxide, hydro peroxide of et al., 2012 and Subramanion Antioxidants are also helpful to prevent or inhibit oxidation processes in human body as well as in food products. Natural antioxidants are available in almost all edible plant products and are stable parts of nutrition. According to Haleem the beneficial properties of phenolic compounds are attributed due to their antioxidant activity. Besides playing the role as a functional component, antioxidants also help the food products in retaining their sensorial quality, e.g. color, texture well as their nutritional quality through preventing the oxidation of essential fatty acids (Coda et al., Natural antioxidant compounds isolated from different sources are now receiving a special attention (Ghosal et al., 2003) and are good alternatives for synthetic antioxidants to the fresh or processed foods (Arabshahi-Delouee and Urooj, 2006). There is a growing demand for natural antioxidants because of toxicological and carcinogenic effects of synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) on animals (Amarowicz et al., 2010). The artificial antioxidant compounds (e.g. BHA and BHT) have a limited allowance for food due to their potential cancerogenicity (Jayaprakasha et al., 2003). Rec consumers often reject food products that are enriched with synthetic antioxidants because some of the synthetic antioxidants may exhibit toxicity and require high manufacturing costs but show lower efficiency than natural antioxidants (Saha et al., 2011).
In different fruit and beverage industries, during the processing of juice and jam, a large amount of their by-products (mostly of the seeds) is produced. Currently, industrial management have show on developing value added products from t generated from both the food and agricultural processing industries (Balasundram et al., waste products including seeds, peels, stalks, stems, and leaves of plants contain substantial amount of phenolic which can be used as important and cheap sources of natural antioxidants for pharmaceutical, cosmetic, and food application (Bucic 2009).
Plant phenolic compounds are a major source of natural antioxidants and are distributed in fruits, seeds, leaves, vegetables, barks, roots, and flowers of plants (Marinova andYanishlieva, 2003 Lin, 2000). Antioxidant compounds have been identified in the seeds of grape , citrus (Baydar et al., 2006), mango (Maisuthisakal, 2009), canola , and sunflower (Zhao 2011). Singh and Rajini (2004) mentioned that researcher are seeking newer sources of antioxidants especially from plants while Prasad noticed that in recent years, underutilized fruits ar getting special focused in research and development. But due to lack of commercial applications, fruit seeds have not received much attention as antioxidant sources. In Bangladesh, blackberry (Rubus laciniatus important seasonal fruit and the annual production is International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 @ IJTSRD | Available Online @ www.ijtsrd.com | Volume -2 | Issue -5 | Jul-Aug 2018 well as their nutritional quality through preventing the et al., 2012). Natural antioxidant compounds isolated from different sources are now receiving a special attention e good alternatives for synthetic antioxidants to the fresh or processed foods ). There is a growing demand for natural antioxidants because of toxicological and carcinogenic effects of synthetic hydroxyanisole (BHA), ene (BHT) on animals 2010). The artificial antioxidant compounds (e.g. BHA and BHT) have a limited allowance for food due to their potential 2003). Recently consumers often reject food products that are enriched with synthetic antioxidants because some of the synthetic antioxidants may exhibit toxicity and require high manufacturing costs but show lower efficiency 2011).
In different fruit and beverage industries, during the processing of juice and jam, a large amount of their (mostly of the seeds) is produced. Currently, industrial management have shown interest on developing value added products from the waste generated from both the food and agricultural et al., 2006). The waste products including seeds, peels, stalks, stems, and leaves of plants contain substantial amount of which can be used as important and cheap sources of natural antioxidants for pharmaceutical, c, and food application (Bucic-Kojic et al., Rubus laciniatus) is an important seasonal fruit and the annual production is approximately 22,000 metric ton. After consumption of blackberry fruits, the seeds are considered as wastage. But its seeds are great source of antioxidants including ellagic acid, tannins, ella acid, anthocyanins, and cyanidins .
Antioxidants have become a popular research topic because they cannot be generated by the human body and hence have to be taken through the diet. Many fruits and vegetables have been sources of antioxidants. Since a large portion of the human diet is based on fruit and vegetables, it is important to understand the biological and biochemical interactions between these dietary antioxidants and living systems. Seed extra higher antioxidant activity than fruit extracts. There is a growing demand for natural antioxidants because of its safety to human health and so a suitable solvent and extraction technique are needed to preserve the antioxidant properties. Thus, the purpose of this study was to determine the effect of the solvents used in extraction techniques on the antioxidant activity of blackberry seeds while based on above views study has been taken with objectives: (a) to analyze chemical and proximate composition of blackberry seed, (b) to identify the effect of extraction solvent and techniques on the antioxidant activity of blackberry seed and (c) to compare among the effect of extraction methods on the antioxidant activity of blackberry seed. Antioxidants have become a popular research topic because they cannot be generated by the human body and hence have to be taken through the diet. Many fruits and vegetables have been found to be rich sources of antioxidants. Since a large portion of the human diet is based on fruit and vegetables, it is important to understand the biological and biochemical interactions between these dietary antioxidants and living systems. Seed extracts showed higher antioxidant activity than fruit extracts. There is a growing demand for natural antioxidants because of its safety to human health and so a suitable solvent and extraction technique are needed to preserve the , the purpose of this study was to determine the effect of the solvents used in extraction techniques on the antioxidant activity of blackberry seeds while based on above views this the following specific e chemical and proximate composition of blackberry seed, (b) to identify the effect of extraction solvent and techniques on the antioxidant activity of blackberry seed and (c) to compare among the effect of extraction methods on blackberry seed. Preparation of sample First the seeds were separated and sun dried under the sun properly. Then they were grinded with lab grinder. After that they were taken out and fine powder of blackberry seeds were separated by passing the powder through 30 mesh size sieve. The powdered sample was then stored in a poly bag and kept in refrigerator for further analysis.

Extraction procedure
For cold extraction, two clean and dried conical flasks were taken. 25 gm of dried blackberry was taken in each conical flask. Then 200ml of ethanol was added to one conical flask and 200 ml of acetone was added to another one. After that both of the conical flasks were put in a continuous shaker machine at a constant temperature of 26 hours.
For hot extraction, the above procedure was followed but using a temperature controlled shaker machine at 40˚C for 48 hrs. Then all the obtained acetone and ethanol extract were filtered through a filter paper and the filtrate was then dried in a vacuum oven at reduced pressure and at a temperature of 40 dried extract of blackberry seed sample was weighed in an electric balance and stored in vial for further analysis.

Methodolgy for determination of Total Phenolic Content (TPC)
The TPC of the extracts was determined by following the modified Folin-Ciocaltu method (Imran and Khan, 2014). In short, 1.0 ml of each extract (1 mg/ml) was mixed with 5 ml Folin-Ciocaltu reagent (1 distilled water) and 4 ml of 7.5% sodium carbonate. The mixture was vortexed for 15 sec and allowed to stand for 30 min at 40˚C for color development. Then the absorbance was measured at 765 nm with a International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 First the seeds were separated and sun dried under the sun properly. Then they were grinded with lab grinder. After that they were taken out and fine powder of blackberry seeds were separated by passing the powder through 30 mesh size sieve. The powdered ample was then stored in a poly bag and kept in The blackberry seeds were analyzed for their chemical component like moisture, ash, fat, protein and total carbohydrate. AOAC (2005) was used for determination of moisture content while AOAC AOAC method 7.045 (2005) for crude fat content and protein was Kjeldahl method described by Carbohydrate content of the samples rmined as total carbohydrate by subtracting the measured protein, fat, ash and moisture from 100 For cold extraction, two clean and dried conical flasks were taken. 25 gm of dried blackberry seed powder was taken in each conical flask. Then 200ml of ethanol was added to one conical flask and 200 ml of acetone was added to another one. After that both of the conical flasks were put in a continuous shaker machine at a constant temperature of 26˚C for 48 For hot extraction, the above procedure was followed but using a temperature controlled shaker machine at C for 48 hrs. Then all the obtained acetone and ethanol extract were filtered through a filter paper and acuum oven at reduced pressure and at a temperature of 40˚C. The dried extract of blackberry seed sample was weighed in an electric balance and stored in vial for further for determination of Total Phenolic The TPC of the extracts was determined by following Ciocaltu method (Imran and Khan, 2014). In short, 1.0 ml of each extract (1 mg/ml) was Ciocaltu reagent (1:10 v/v in distilled water) and 4 ml of 7.5% sodium carbonate. The mixture was vortexed for 15 sec and allowed to C for color development. Then the absorbance was measured at 765 nm with a spectrophotometer. Gallic acid was used as a stan for the calibration curve. While preparing the standard curve the concentration of gallic acid were 0, 0.25, 0.50, 0.75, 1.00 mg per ml solutions of gallic acid in methanol: water (50:50, v/v). The standard curve was prepared by using the method as me except the sample was absent there. According to the obtained data, the concentration vs. absorbance curve of gallic acid was drawn which is shown in Figure 1.

Antioxidant activity tests
The antioxidant activity of different extracts of blackberry seed was determined by following two methods: total antioxidant capacity and reducing power assay. spectrophotometer. Gallic acid was used as a standard for the calibration curve. While preparing the standard curve the concentration of gallic acid were 0, 0.25, 0.50, 0.75, 1.00 mg per ml solutions of gallic acid in methanol: water (50:50, v/v). The standard curve was prepared by using the method as mentioned above except the sample was absent there. According to the obtained data, the concentration vs. absorbance curve of gallic acid was drawn which is shown in Figure 1. The antioxidant activity of different extracts of blackberry seed was determined by following two methods: total antioxidant capacity and reducing determination ts (acetone, ethanol) were weighed and mixed in 50 ml of acetone and ethanol respectively and mixed completely by using sonicator and vortex machine stirring. Thus the concentration of each solution would be 1000 µg/ml (stock solution).
tions were carried out to obtain the concentrations of 20, 40, 60, 80,100 and 250µg/ml from the stock solution of acetone and ethanol extracts for determining antioxidant activities of blackberry seed. Total antioxidant activity of the ed by the phosphomolybdenum assay method which is based on the reduction of Molybdenum, Mo (VI) to Phosphate-Molybdenum, Mo (V) by the extract and subsequent formation of a green phosphate-Mo (V) complex in acidic condition (Matthias et al., 2015). The extract (2.0 mg/ml, 0.3 ml) was allowed to mix up with 3.0 ml of reagent solution (0.6 M H2SO4, 28 mM , 4 mM ammonium molybdate) and the reaction mixture was incubated at 95˚C for 90 minutes. After cooling at room temperature, the absorbance of the solution was measured at 695 nm International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 @ IJTSRD | Available Online @ www.ijtsrd.com using a UV-Visible spectrophotometer against an appropriate blank. The antioxidant activity was expressed as the number of gram equivalents of ascorbic acid. Concentration vs. absorbance (695 nm) curve of ascorbic acid for determination of total antioxidant capacity is shown in Figure 2. The standard curve was prepared by using the same method as mentioned above except the sample. Reducing power assay This assay was determined according to the method reported by Dehpour et al. (2009). Briefly, 10 ml of acetone and ethanol extracts solution of different concentrations was mixed with 2.5 ml of potassium ferricyanide [K 3 Fe(CN) 6 ] (1%; w/v) and 2.5 ml of phosphate buffer (0.2 M; pH 6.6) . The mixture was incubated at 50°C for 20 min. After that the reaction was terminated by the addition of 2.5 ml of trichloroacetic acid (10%; w/v). Then the mixture was centrifuged at 3000 rpm for 10 min supernatant solution (2.5 ml) was mixed with distilled water (2.5 ml) and ferric chloride (0.5 ml; 0.1%, w/v) solution. Then the absorbance was measured at 700 nm against a blank reading. Increased absorbance value of the reaction mixture indicates increased of reducing power of the extracts. Three replicates were made for each of the tested sample.

RESULTS AND DISCUSSION Composition of blackberry seed
The blackberry seed was analysed for the moisture, ash, protein, fat and total carbohydrate obtained average values of triplicate determination are shown in Table 1. Visible spectrophotometer against an appropriate blank. The antioxidant activity was expressed as the number of gram equivalents of ic acid. Concentration vs. absorbance (695 nm) curve of ascorbic acid for determination of total antioxidant capacity is shown in Figure 2. The standard curve was prepared by using the same method as mentioned above except the sample.
Concentration vs. Absorbance (at 695nm) curve of ascorbic acid for determination of total This assay was determined according to the method . (2009). Briefly, 10 ml of extracts solution of different concentrations was mixed with 2.5 ml of potassium w/v) and 2.5 ml of phosphate buffer (0.2 M; pH 6.6) . The mixture was incubated at 50°C for 20 min. After that the reaction was terminated by the addition of 2.5 ml of trichloroacetic acid (10%; w/v). Then the mixture was centrifuged at 3000 rpm for 10 min. Finally the supernatant solution (2.5 ml) was mixed with distilled water (2.5 ml) and ferric chloride (0.5 ml; 0.1%, w/v) solution. Then the absorbance was measured at 700 nm against a blank reading. Increased absorbance icates increased of reducing power of the extracts. Three replicates were for the moisture, ash, protein, fat and total carbohydrate and the etermination are Composition of blackberry seed  parameters of blackberry seed Two different solvents (absolute acetone and absolute ethanol) and two different techniques (hot extraction and cold extraction) were used for determining the amounts of antioxidant extracts from blackberry seed the results are shown in Table 2.  (2006) reported that this variation might be due to the different availability of extractable components, resulting from various chemical compositions of solvent used.
Results also revealed that between the two techniques (hot and cold extraction), efficiency of the hot extraction is slightly higher than cold extraction (1.02 times for acetone and 1.04 times for ethanol) which indicates that hot extraction system is somewhat more efficient for the recovery of antioxidant components than cold extraction system. The result is more or less similar with the findings of Shon et al investigated that alcoholic solvent and hot extraction method are more efficient to extract antioxidant compounds from Phellinus baumii. Chata (2006) reported that hot extraction performed under 40˚C helps to mix the solvent with solution properly which was responsible to extract the phenolic component by solvent. Yen et al. (2004) concluded that hot extraction at 40˚C does not affect the loss of phenolic content as the temperature is mild temperature.

Amount of total phenolic content (TPC) in blackberry seed
To determine the total phenolic contents (mg gallic acid equivalent per gram of dry extract) in blackberry seed for acetone and ethanol extract a regression equation (1) developed by using standard curve ( Figure 1) and the result is shown in Figure 3. y = 6.975x-0.0152 ……………. (1), where y indicates absorbance at 765 nm and x indicates the concentration of gallic acid in μg/ml. From figure 3, it is seen that HEE gave the highest phenolic content as 56.56 mg Gallic Acid Equivalent (GAE)/gm of dry extract, and is followed by CEE with 38.00 mg GAE/gm of dry extract. HAE gave the total phenolic content with 38.83 mg GAE/gm of dry extract and the lowest phenolic content 33.21 mg GAE/gm of dry extract was given by CAE. From the results it can be shown that, the difference between hot and cold extraction using ethanol is comparatively higher than the difference between hot and cold extraction using acetone. So, the solvent effect is quite high. For cold extraction, TPC from CEE is 1.14 times higher than CAE and TPC from HEE is 1.46 times higher than HAE. These observations can be compared with the findings of Deepika who reported total phenolic content of jackfruit seed International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 @ IJTSRD | Available Online @ www.ijtsrd.com | Volume -2 | Issue -5 | Jul-Aug 2018 plants with ethanol than other solvents used like acetone.  reported that this different availability of extractable components, resulting from various chemical compositions of solvent used.
Results also revealed that between the two techniques (hot and cold extraction), efficiency of the hot extraction (1.02 times for acetone and 1.04 times for ethanol) which indicates that hot extraction system is somewhat more efficient for the recovery of antioxidant components than cold extraction system. The result is more or less et al. (1), where y ance at 765 nm and x indicates the it is seen that HEE gave the highest 56.56 mg Gallic Acid Equivalent (GAE)/gm of dry extract, and is followed by CEE xtract. HAE gave the total phenolic content with 38.83 mg GAE/gm of dry extract and the lowest phenolic content 33.21 mg GAE/gm of dry extract was given by CAE. From the results it can be shown that, the difference between anol is comparatively higher than the difference between hot and cold extraction using acetone. So, the solvent effect is quite high. For cold extraction, TPC from CEE is 1.14 times higher than CAE and TPC from HEE is 1.46 rvations can be compared with the findings of Deepika et al. (2011), who reported total phenolic content of jackfruit seed as 1.45± 0.007 µg GAE/ mg for acetone extract and 2.12±0.009 µg GAE/ mg for ethanol extract. This variation may be due to the fact th often extracted in better amounts in more polar solvents such methanol, ethanol than other solvents such as acetone (Siddhuraju and Becker, 2003;Anwar et al., 2006;Sultana et al., 2007).

Total antioxidant capacity of blackberry seed
The standard calibration curve of ascorbic acid for the determination of total antioxidant capacity in different solvent extracts of blackberry seed Figure 2 and from those data the following regression equation was developedy = 0.003x -0.020…………... (2), where y is absorbance at 695 nm and x is concentration in µg/ml. By using equation (2) total a different solvent extracts was determined as ascorbic acid equivalent per gram of dry extract (AAE/g) and shown in Figure 4. The highest TAC was obtained from HEE (82.42 AAE/g) while CAE gave lowest value (75.46 AAE/g). Again between extracts, HEE gave slightly high ascorbic acid equivalent (AAE) which was 1.07 times more than CEE and between the acetone extract, HAE gave 1.06 times more AAE than CAE. So in this case we may conclude that, HEE is the most efficient among th Page: 1601 as 1.45± 0.007 µg GAE/ mg for acetone extract and 2.12±0.009 µg GAE/ mg for ethanol extract. This variation may be due to the fact that, phenolics are often extracted in better amounts in more polar solvents such methanol, ethanol than other solvents such as acetone (Siddhuraju and Becker, 2003;Anwar ., 2007).

Total antioxidant capacity of different extracts of
The standard calibration curve of ascorbic acid for the determination of total antioxidant capacity in different solvent extracts of blackberry seed was developed in Figure 2 and from those data the following regression 0.020…………... (2), where y is absorbance at 695 nm and x is concentration in µg/ml. By using equation (2) total antioxidant capacity of different solvent extracts was determined as ascorbic acid equivalent per gram of dry extract (AAE/g) and shown in Figure 4. The highest TAC was obtained from HEE (82.42 AAE/g) while CAE gave lowest value (75.46 AAE/g). Again between the ethanol extracts, HEE gave slightly high ascorbic acid equivalent (AAE) which was 1.07 times more than CEE and between the acetone extract, HAE gave 1.06 times more AAE than CAE. So in this case we may conclude that, HEE is the most efficient among the International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 @ IJTSRD | Available Online @ www.ijtsrd.com solvent extracts tested. These results are in concordance with the findings of Soong and Barlow (2004), who measured the antioxidant capacity in different fruit portions. According to his findings, the seeds were showed a much higher antioxidant activity than the edible portions, e.g. jackfruit seed, tamarind seed, avocado seed contained antioxidant capacity as 7.4, 698, 236 AAE (µmol/g ) while their edible portion contained antioxidant capacity as 3, 17, 4.9 AAE(µmol/g).

Figure 4: Total antioxidant capacity of different solvent extracts of blackberry seed
Reducing power assay of different extracts of blackberry seed The data of reducing power assay (as absorbance at 700nm) for different extracts of blackberry seed with hot and cold technique were plotted in a linear graph ( Figure 5 and 6) as absorbance at 700 nm on Y and concentration of extract (µg/ml) on X Figure 5 shows the comparison of reducing power assay of CAE (cold acetone extract at 26 (hot acetone extract at 40˚C) of blackberry seed. It is seen that the reducing power of both CAE and HAE of blackberry seed was found to increase with the increasing extract concentration. absorbance of 0.25 for HAE and 0.23 for CAE were observed at extract concentration of 250µg/ml. Thus HAE gave 1.09 times more reducing power than CAE at 250 µg/ml extract concentration. The results can be compared with the findings of Yeasmen and Islam (2015), who conducted research with tamarind seed and found that the reducing power of bot CAE of T. indica seed was increased with the increased extract concentration. The findings also admitted that the maximum absorbance for HAE and CAE were 0.66 and 0.64 respectively at extract concentration of 500µg/ml. International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 @ IJTSRD | Available Online @ www.ijtsrd.com | Volume -2 | Issue -5 | Jul-Aug 2018 solvent extracts tested. These results are in concordance with the findings of Soong and Barlow (2004), who measured the antioxidant capacity in different fruit portions. According to his findings, the seeds were showed a much higher antioxidant activity than the edible portions, e.g. jackfruit seed, tamarind seed, avocado seed contained antioxidant capacity as 7.4, 698, 236 AAE (µmol/g ) while their edible portion contained antioxidant capacity as 3, 17, 4.9 acity of different solvent extracts of blackberry seed

Reducing power assay of different extracts of
The data of reducing power assay (as absorbance at 700nm) for different extracts of blackberry seed with plotted in a linear graph ( Figure 5 and 6) as absorbance at 700 nm on Y-axis and concentration of extract (µg/ml) on X-axis. Figure 5 shows the comparison of reducing power assay of CAE (cold acetone extract at 26˚C) and HAE ) of blackberry seed. It is seen that the reducing power of both CAE and HAE of blackberry seed was found to increase with the creasing extract concentration. Maximum absorbance of 0.25 for HAE and 0.23 for CAE were 50µg/ml. Thus HAE gave 1.09 times more reducing power than CAE at 250 µg/ml extract concentration. The results can be Yeasmen and Islam (2015), who conducted research with tamarind seed and found that the reducing power of both HAE and seed was increased with the increased extract concentration. The findings also admitted that the maximum absorbance for HAE and CAE were 0.66 and 0.64 respectively at extract Figure 6 shows the comparis assay of CEE (cold ethanol extract at 26 (hot ethanol extract at 40˚C) of blackberry seed. It is seen that the reducing power of both CEE and HEE of blackberry seed increased with the increasi concentration. Maximum absorbance of 0.26 for HEE and 0.23 for CEE were observed at extract concentration of 250µg/ml. It is found that, HEE gave 1.13 times more reducing power than CEE at 250µg/ml extract concentration.   Yeasmen and Islam (2015), who conducted research with tamarind seed and found the maximum absorbance for HEE and CEE were 0 respectively at extract concentration of 500 Comparing the result between the above section it is clearly seen that, reducing power assay for both hot and cold ethanol extracts of blackberry seed is found considerably higher compared acetone extracts (e.g. 0.26 for HEE vs. 0.25 for HAE). In the previous graph it is shown that reducing power of hot ethanol and acetone extract is higher than the reducing power of cold ethanol and acetone extract. Thus hot ethanol and acetone extracts were compared with ascorbic acid (standard) in the next section as far reducing power is concerned. Page: 1602 Figure 6 shows the comparison of reducing power assay of CEE (cold ethanol extract at 26˚C) and HEE C) of blackberry seed. It is seen that the reducing power of both CEE and HEE of blackberry seed increased with the increasing extract m absorbance of 0.26 for HEE and 0.23 for CEE were observed at extract concentration of 250µg/ml. It is found that, HEE gave 1.13 times more reducing power than CEE at 250µg/ml extract concentration.   (2015), who conducted research with tamarind seed and found the maximum absorbance for HEE and CEE were 0.7702 and 0.7328 respectively at extract concentration of 500µg/ml. Comparing the result between the above section it is clearly seen that, reducing power assay for both hot and cold ethanol extracts of blackberry seed is found considerably higher compared to the corresponding acetone extracts (e.g. 0.26 for HEE vs. 0.25 for HAE). In the previous graph it is shown that reducing power of hot ethanol and acetone extract is higher than the reducing power of cold ethanol and acetone extract. d acetone extracts were compared with ascorbic acid (standard) in the next section as far International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 @ IJTSRD | Available Online @ www.ijtsrd.com Comparison of reducing power assay of hot ethanol and acetone extracts with ascorbic acid Figure 7 shows the comparison of hot ethanol and acetone extracts with ascorbic acid (standard) for reducing power assay represented as absorbance at 700 nm. It is seen that for both of the hot extracts, the higher the concentration of extracts the higher reducing power (absorbance at 700 nm) as mentioned earlier and that the reducing power (absorbance at 700 nm) is quite higher for ascorbic acid for each concentration level tested. acetone extracts with standard. The maximum absorbance for ascorbic acid is 1.4 compared to 0.26 and 0.25 for hot ethanol and acetone extract respectively at concentration level of 250µg/ml. Thus it is found that the reducing power for ascorbic acid is undoubtedly higher t extracts -ethanol and acetone. The highest reducing power is given by HEE among the tested extracts (CEE, HAE and CAE) except standard. These results can be compared with the findings of (2013), who conducted research with tamarind seed and showed that at concentration of 500µg/ml ethanol extracts of tamarind seed gave the reducing power as 0.71 which continued to increase with the increasing concentration such that at 600, 700 and 800 µ concentration, the absorbance values were 1.7, 1.

CONCLUSION
The results of the present investigation showed that the ethanol and acetone extracts of blackberry seed, extracted by hot (40˚C) extraction technique exhibited better antioxidant activities and phenolic contents in comparison to cold ethanol and aceton blackberry seed (7.48% vs.7.19% for ethanol and International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 @ IJTSRD | Available Online @ www.ijtsrd.com | Volume -2 | Issue -5 | Jul-Aug 2018 reducing power assay of hot ethanol ascorbic acid (standard) Figure 7 shows the comparison of hot ethanol and acetone extracts with ascorbic acid (standard) for reducing power assay represented as absorbance at 700 nm. It is seen that for both of the hot extracts, the higher the concentration of extracts the higher the reducing power (absorbance at 700 nm) as mentioned earlier and that the reducing power (absorbance at 700 nm) is quite higher for ascorbic acid for each Figure 7: Reducing power assay of hot ethanol and ith standard. The maximum absorbance for ascorbic acid is 1.48 for hot ethanol and acetone extract respectively at concentration level of 250µg/ml. Thus it is found that the reducing power for ascorbic acid is undoubtedly higher than the tested ethanol and acetone. The highest reducing power is given by HEE among the tested extracts (CEE, HAE and CAE) except standard. These results can be compared with the findings of , who conducted research with tamarind seed and showed that at concentration of 500µg/ml ethanol extracts of tamarind seed gave the reducing power as 0.71 which continued to increase with the increasing concentration such that at 600, 700 and 800 µg/ml concentration, the absorbance values were 1.7, 1.9 . The results of the present investigation showed that the ethanol and acetone extracts of blackberry seed, C) extraction technique exhibited better antioxidant activities and phenolic contents in comparison to cold ethanol and acetone extracts of blackberry seed (7.48% vs.7.19% for ethanol and 4.35% vs. 4.28% for acetone). Hot extraction also exhibited more reducing power assay than cold extraction. The obtained data would certainly help to determine the potency of blackberry seed as potential source of natural antioxidant to be used for functional food applications. So, future research program and planning could individual components responsible for activity and ensure their applications for human and animals.

ACKNOWLEDGEMENT:
The authors acknowledge with thanks for the financial assistance received to carry out the study from the National Science and Technology (NST) fellowship under Ministry of Science and Technology Bangladesh. 4.35% vs. 4.28% for acetone). Hot extraction also more reducing power assay than cold extraction. The obtained data would certainly help to determine the potency of blackberry seed as a potential source of natural antioxidant to be used for functional food applications. So, future research could be taken to separate individual components responsible for antioxidant ensure their applications for welfare of