Optimization of Antioxidant Extraction from Freeze-dried Pulp , Peel , and Seed of Burmese grape ( Baccaurea ramiflora Lour . ) by Response Surface Methodology


 This study aimed to attain the optimum condition necessary for extracting the maximum yield of antioxidants from the freeze-dried pulp, peel, and seed of Burmese grape using response surface methodology (RSM). Solvent (ethanol) concentration (%), temperature (°C), and time (min) were taken as independent variables by factorial screening for the extraction procedure. After extraction, the antioxidant activity of all samples was determined employing 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, total phenolic compounds (TPC), and ferric reducing antioxidant power (FRAP) assay. The experiment's optimum conditions were 80% solvent concentration, 69.01°C temperature, and 30 min for pulp. The optimum extraction conditions were found at 80°C for 29.39 min incubation time using 52.12% concentrated solvent for seed. For peel, the solvent concentration of 41.62% was found optimum when the temperature of 50°C and 30 min incubation time were used. The actual values of TPC, FRAP, and DPPH for freeze-dried pulp, peel and seed extracts were close to the predicted values, which confirms the models’ validity. The Analysis of Variance (ANOVA) showed that the models were significant for TPC, DPPH, and FRAP values of peel, pulp, and seed at different levels (p < 0.001 to p < 0.05). The composite desirability of pulp, seed, and peel were 0.94, 0.98, and 0.85, respectively, which suggest that the developed model could be effectively used for antioxidants’ extraction from freeze-dried pulp, peel, and seed of Burmese grape.


INTRODUCTION
Bangladesh has some exceptional natural features like 6-8 hours of constant daily sunlight over the year; therefore, this country is boasted with a great variety of fruits. About 70 different types of nutritious fruits are found in this tropical country. Among them, 3.01% of the land is occupied by minor fruits like Baccaurea ramiflora. But they produce more than 8.38% of the total fruit production of Bangladesh. In Bangladesh, the fruit plant is grown mainly in homestead conditions [1]. The tropical foods of Bangladesh are very rich in antioxidants. But some of them are appropriately utilized where some others are underexploited fruits; some natives only consume those. Burmese grape (Baccaurea ramiflora Lour.) is one of them. In Bangladesh, it is commonly known as lotkon. Geographically, Lotkon is grown in the sub-Himalayan track of South-east Asia region [2].

Kingdom Plantae
Division Magnoliophyta

Class Magnoliopsida
Order Malpighiales

Baccaurea ramiflora
The lotkon tree is semi-evergreen, reaching 8-10 meters with thick, broad, and ellipse-shaped leaves. In the summer season, this fruit grows on the tree stems and branches [3]. The yellow to 4 purple color fruit has leathery pericarp and a diameter of 2-3 cm. The pulp inside is pale rose in color and embeds 3-4 seeds inside it [4]. This mild acidic fruit is mainly consumed fresh (Pradhan et al., 2015). There are numerous scientific shreds of evidence that plant and its product, like-fruits, peel, leaves, and seeds contain different bio-active compounds which offer countless health benefits and protection against degenerative diseases [5][6][7][8].
Previous studies revealed that consuming nutritious vegetables and fruits inversely affects the risk of degenerative disorders associated with cardio and cerebrovascular disorder morbidity and various kinds of cancers [910]. This is mainly due to the available antioxidant compounds, e.g., flavonoids, carotenoids, and polyphenols in the fruits and vegetables. These compounds have some positive effects on the human body [11], and play an important role in balancing the ratio of free radicals and scavenging free radicals [12].
The ecological and traditional diversity refers that the plant in Bangladeshi locality might have a crucial source of pharmaceutical ingredients [1]. These fruits are taken mainly for a great source of vitamin A, C, and several other nutrients. It is rich in polyphenols and flavonoid contents [4].
The lotkon fruit has three different parts, e.g., seed, juice/pulp, and peel. Besides pulp, it was reported that the seeds and peel of other fruits are rich in natural antioxidants [7,13]. The enhanced various utilizations of the fruits meet the nutritional challenge. Lotkon fruits have been consumed since ancient times for health and medicinal purposes. However, it is significant that a tiny portion of lotkon fruit is edible, which means a large part of lotkon produce wastage [14,15]. These waste materials are not easily degradable and thus produce environmental pollution.
The use of lotkon seeds and skins may be a possible source of antioxidants and can help control ecological pollution if appropriately used. Several previous efforts were made to estimate the antioxidant activity of the freeze-dried seed, pulp, and skin of Burmese grape [16].
5 However, the process should be optimized before extraction because factors like extraction time, temperature, solvent concentration, solvent type, pressure, solid-to-liquid ratio, and pH can significantly affect the extraction process of fruits [17]. Solvent extraction methods have been considered as the easiest and simple method for antioxidant determination from plants' parts. In general, the solvent polarity, solvent concentration, extraction time, and extraction temperature are the most controlling factors [18][19]. Soong and Barlow (2004) used an identical extraction method for jackfruit, avocado, longan, mango, and tamarind to compare antioxidant activity between the edible portion and seeds [20]. However, according to Eberhardt et al. (2000), it would be difficult to establish a universally optimized extraction protocol due to the complex internal matrix and diversity of natural sources' antioxidant compounds [21]. Hence, the optimum extraction protocol is anticipated to differ with the type of fruits, and between the edible and non-edible portions. Very few previous research works were done to optimize the antioxidant activity of freeze-dried peel, pulp, and seed extract of Burmese grape to the best of our knowledge. Hence, this research work aimed to assess the antioxidant activity of lyophilized peel extract, pulp extract, and seed extract of lotkon samples in this context. The effects of extraction time, extraction temperature, and solvent concentration on the extraction of antioxidants from lotkon pulp, peels, and seeds were also determined.

Sample collection and preparation of sample extracts
Lotkon fruits were purchased from the Agora super shop, Subidbazar, Sylhet, Bangladesh.
Different parts of lotkon fruits were separated by maintaining the hygienic condition. All the parts of the Burmese grape were then freeze-dried. For this, the samples were firstly frozen in a blast freezer at −18 °C followed by lyophilization in a freeze dryer ((Model-LYOQUEST-55) at 6 20 °C under vacuum condition. The dried parts were then powdered by laboratory type hammer mill and passed through a sieve of 1 mm size. The freeze-dried powders from peel, pulp, and seed of Burmese grape were extracted using 40-80% concentrated ethanol. Figure 01 illustrates the schematic overview of the research design.

Extraction procedure
Firstly, 0.5 g of sample (dry powder) was taken for each extraction.

Total phenolic contents (TPC)
The total polar phenolic compound was determined by the modified Folin-ciocalteu reagent (FCR) method described by Zzaman et al. (2021) with some moderations [22]. In a 10 mL flask, 0.5 mL of the ethanolic extract was placed. After that, 0.5 mL Folin-Ciocalteu reagent was added to the extracts. Following this, 1 mL of NaCO3 solution was added, and the volume was made up to 10 mL with double-distilled water. The supernatant's absorbance was taken after keeping the mixture for 1 hr, using a UV Spectrophotometer (PG Instruments Ltd, Model -T60 U) at a wavelength of 725 nm against a reagent blank. The gallic acid (GA) calibration curve 7 (y=2692.4x; R² = 0.98) was prepared by fitting the absorbance versus its corresponding pure gallic acid solutions, where the water blank was taken as zero (0). The results were calculated in mg GAE/100 grams of sample.

Ferric reducing antioxidant power (FRAP)
The ferric reducing antioxidant power (FRAP) was measured using the updated method of Where, yr represents the measured responses variables, while xi and xj are the levels of independent variables. ao is a constant (predicted response at the center), ai, aii, and aij are the linear, quadratic, and two-factor interactive coefficient of the model, respectively. All the statistical significance tests were based on the total error criteria with a confidence level of 95%. 9

RESULTS AND DISCUSSIONS
The linear models result from the response surface of TPC, FRAP and DPPH radical scavenging property for freeze-dried lotkon pulp, seed, and peel against different runs have been illustrated in Table 02.

Analysis of Total Phenolic Content
The Folin-ciocalteu reagent (FCR) method was used to estimate the total phenolic content (TPC) in The analysis of variance (ANOVA) was performed for second-order quadratic equation regression models with the response of total phenolic content for freeze-dried pulp, seed, and peel of Burmese grape. The results from Table 3 show that the models are highly significant (p<0.01). The co-efficient of determination (R 2 ) value of TPC describes that these models could effectively describe 97.37%, 97.31%, and 95.25% data for pulp, seed, and peel, respectively.
The three-dimensional response surface, along with the contour plot for total phenolic content is presented in Figure 2.  Table 2). The response surface graph of Figure 2a represented that the extraction of TPC in pulp was increased diagonally by maintaining high temperature and time. It was also found that increased extraction time favors polyphenolic compounds extraction. The combination of more than 75 °C temperature and 20 min was found TPC to be greater than 14 mg GAE/100g DM (Figure 2b).
Beyond considering time and solvent concentration for seed, the extraction of TPC tends to decrease with an increase in temperature of up to 65 °C; above it, TPC tends to increase with temperature.
The 3D response surface graph indicates that the TPC decreases with time but increases with the solvent concentration. In contrast, the maximum extraction of TPC from peel was found from 50-60% solvent concentration and 20-25 minutes. However, the temperature had a proportional relation with TPC, and solvent concentration had a quadratic relationship (Figure 2c).
Most other authors also stated that increasing extraction temperature increases extraction, diffusion coefficient, and solute solubility. However, TPC can be degraded above a particular temperature for different samples [17,25]. A previous study reveals that some phenolics bonded by protein might cause TPC loss in the pulp at higher temperature [26]. Dorta et al. (2012) also demonstrated a consistent result in mango peel [27]. Moreover, the heat can enhance the recovery of phenolic compounds. Shi et al. (2003) described that enhanced extraction temperature loosens the plant tissue and interrupts the association between phenolic compounds and proteins, or polysaccharides, therefore, increase the solubility, which also improves the diffusion rate [28]. It was also observed that time has the most considerable effect on TPC yield.
Inevitably, solute solubility and phenolic extraction increase with time. It might be because of the time required to increase the medium's solubility and subsequently dissolve out from the dried powder.

Analysis of Ferric Reducing Antioxidant Power (FRAP)
Ferric reducing antioxidant power (FRAP) was found higher in seed in contrast with pulp and peel.     On the other hand, the model obtained from the response surface assay of FRAP for pulp and seed showed that the extraction temperature and time were significant. Therefore, an increase in these variables can promote the growth of FRAP. The response surface graph illustrated that with the rise in temperature and time, the FRAP value was increased for pulp as well as seed ( Figure 3a). The contour plot indicates that the maximum FRAP in pulp was achieved when the temperature and time were maintained at 80 °C and 30 min, respectively.
The combination of 80 °C and 30 min extraction time was required to get a FRAP concentration of more than 3500 mg AAE/100g DM (Figure 3b). In peel, the highest FRAP value was achieved at about 60% solvent concentrations, and FRAP value were reduced at temperature more than 60 °C (Figure 3c). Maybe, it is due to the denaturation of the phenolic compounds that shows FRAP activities. Figure 3c [31].

DPPH radical scavenging activity
The scavenging activity of DPPH radical (%) was found highest in the 14 th run (59.06 %) for pulp, 3 rd run for seed (62.63%), and 1st run for skin (50.78%) as shown in Table 02 The results of analysis of variance (ANOVA) ( , 10, and 11 shows that the models for seed and peel were insignificant (p>0.05); that is, the seed and peel models were correct. However, for pulp, it was not appropriate. Between 60-70 °C temperature and more than 26 minutes, the DPPH scavenging effect was found to be more than 66% for pulp (Figure 4a). At higher temperatures and extended extraction time, maybe the cells' breakdown is more as reflected by the higher antioxidant activity. It might be the reason behind the increase in DPPH activity with an increase in time and temperature ( Figure 4). Figure 4b demonstrates that in the seed, the DPPH activity reached the highest peak at 65 °C using 60% solvent concentration. Scavenging activity decreased when the temperature was increased further. The combination was found to be 60-70 °C temperature and 28-30 min time from the contour plot ( Figure 4b). As like as the FRAP response, a negative slope was found for the peel. This demonstrated that, at 60% solvent concentration and 50-52 °C temperature, the DPPH scavenging effect was found to be more than 45% (Figure 4c) reported similar results for antioxidant extraction from moringa leaves and jackfruit pulp and seed [7,12].

Optimization of the extraction process
An optimization study was done based on the findings mentioned above to determine the best operating condition to extract from the pulp, seed, and peel. The target was set to maximize FCR, FRAP, and DPPH; where the feasibility of the study was also taken into account. Table 6 provides information about the target and fit values of TPC (mg GAE/100g DM), FRAP (mg AAE/100g DM), and DPPH radical scavenging activity (%) of freeze-dried pulp, seed, and peel of Burmese grape.

CONCLUSIONS
Response surface methodology was applied to estimate the optimum operating conditions to extract bioactive compounds, namely by determining FCR, FRAP, and DPPH scavenging effect in Burmese grapes (Baccaurea ramiflora Lour.). Quadratic models were used to predict the response of the parameters. The response surface model, contour plot, and the optimization study were performed to find the optimum conditions for the pulp, seed, and peel portion of the berry. were satisfactory. This model may be efficiently used for the optimization of antioxidants' extraction process from Burmese grapes.