Effects of Food Processing on Resveratrol and Total Phenolic Content in Melinjo ( Gnetum gnemon L . ) Seeds

Objective: Trans-resveratrol is a phenolic compound in the stilbene group present in Melinjo (Gnetum gnemon L.) seeds. In Indonesia, these seeds are consumed by roasting, frying (i.e., emping), and boiling. This study aimed to evaluate the effects of food processing on trans-resveratrol and total phenolic compound levels in melinjo seeds. Methods: Melinjo seed was roasted at 145oC for 10 and 20 min, boiled at 100oC for 5, 15, and 30 min, and fried at 160-170oC for 2 and 4 min. Samples were refluxed with 96% ethanol. The determination of trans-resveratrol levels was performed using high-performance liquid chromatography (HPLC), and total phenolic content was determined using Folin-Ciocalteu method. Results: Based on the results, it was found that trans-resveratrol and total phenolic content levels in Melinjo powder decreased from 36% to 10% (trans-resveratrol) and 15% to 4% (total phenolic) after 20 min of roasting, and in fried empings, it decreased 60%, 68%, and 92% (trans-resveratrol) and 41%, 45%, 97% (total phenolic) after 5, 15, and 30 min of boiling, respectively. Conclusion: There were significant changes in trans-resveratrol levels and total phenolic content in Melinjo seeds before and after various cooking processes (i.e., roasting, boiling, and frying).

in Indonesian soup (i.e., Sayur Asem) or boiled with vegetables and spices.As a foodstuff, Melinjo seeds have also been reported to have no toxic effects when consumed over long periods of time. 13od processing that involves heat, including roasting, baking, boiling, and frying, affect chemical compounds in some plants and foodstuffs.Previous studies have shown that the roasting process alters structural and chemical compounds by decreasing liquid content, modifying lipids, and causing discoloration and flavor changes. 14Additionally, one study reported that trans-resveratrol level in roasted peanuts was less than in raw peanuts, 15 while another study reported that trans-resveratrol levels in raw blueberries were higher than in roasted blueberries. 16However, research of food processing on the concentration of trans-resveratrol and total phenolic content in Melinjo seeds has not been reported.The current study was conducted to determine the food processing effect by roasting, boiling, and frying and its duration on Melinjo seeds on trans-resveratrol and phenolic content.

Plant Material
Melinjo (Gnetum gnemon L.) seeds were achieved from Pandeglang traditional market, Banten Province, Indonesia and authenticated at the Herbarium Bogoriense, Bogor, and West Java, Indonesia.The voucher specimen was deposited at Herbarium of Pharmacognosy-Phytochemistry, Faculty of Pharmacy, Universitas Indonesia, Depok, and West Java, Indonesia.

Chemical Materials and General Equipment
The chemicals were used in this study, such as ethanol, aqua DM, ethyl acetate, n-hexane were purchased from PT. Smart Lab Indonesia, Indonesia.Acetonitrile, acetic acid, Folin-Ciocalteau reagent, sodium carbonate, methanol pro analysis were obtained from PT. Merck, Germany via PT Elo Karsa Utama, Indonesia.The equipment were used including oven vacuum (Jisico, Korea), rotary vacuum evaporator (Buchi, Switzerland), high-performance liquid chromatography (HPLC) (Shimadzu SPD-20A, Japan), and microplate reader (Versa Max, USA).

Food Processing
Fried Emping Raw emping was fried in cooking oil for 2 and 4 min at 160-170⁰C and the oil removed by refluxing with n-hexane.Both raw the fried emping were ground into powder.

Roasted Melinjo seed powder
Melinjo seeds were peeled to remove the outer shell and to expose the yellowish white inner seed before being ground into powder with a blender.This powder was inserted into a drying cabinet equipped with lights for seven days; after which time, the powder was removed for use in the experiment.Melinjo seed powder with and without eggs was roasted in an oven at 145 o C for 10 and 20 min and cooled to room temperature.

Boiled Melinjo Seeds
Melinjo seeds with and without skin were boiled in water at 100°C at a ratio of 1:5 (seeds: water) for 5, 15, and 30 min before removal and milling to powder.

Extract Preparation
All samples were refluxed 17 using 96% ethanol (Merck, Germany) for 1 h.The same procedure was repeated three times.The solvent was removed using a rotary vacuum evaporator at 50°C, and the samples were dried using an oven vacuum at 50°C.

Determination of Resveratrol Content Using High-Performance Liquid Chromatography (HPLC)
Trans-resveratrol levels were determined using HPLC according to the Suoto method. 18with modification.Reverse phase HPLC was performed using a C18 column (5 µm particle size, 4.6 x 150 mm, Zorbax).The mobile phase was conducted in acetonitrile: water (25:75) solution (Merck, Germany), with the pH adjusted to 3 using acetic acid.Ultraviolet detection was performed at 306 nm, the flow rate was determined to be 1 ml/min, and the injection volume was determined to be 20 µl.

Determination of Total Phenolic Content (TPC)
TPC was determined using the Folin-Ciocalteau (F-C) method.Each sample weighed 20 grams and was dissolved in a methanol aqua demineralization mixture diluted to 400 ppm; 20 μL of the solution was pipetted and inserted into a 96-well microplate with each sample done in triplicate.Then, 100 μL of an F-C reagent was added, and the mixture was shaken for 1 min and incubated for 4 min.Then, 75 μL of Na 2 CO 3 solution was shaken for 1 min and incubated for 120 min.[21]

RESULTS AND DISCUSSION
In present study, the influence of food processes (including roasting, boiling, and frying) on the difference of trans-resveratrol and polyphenolic content of Melinjo seeds has been done.After the food processes, then each sample was macerated using 96% ethanol.The obtained extract was analyzed trans-resveratrol levels and total polyphenolics content.

Determination of Resveratrol Content
As can be seen in Figure 1, resveratrol level was calculated using a calibration curve and the regression equation y = 167959x + 2341.5 with R value = 0.9991.Retention time was used to identify trans-resveratrol in a sample.The peak of the trans-resveratrol standard is shown at an 8-min retention time.Table 1 shows an increasing level of trans-resveratrol in Melinjo seed powder without egg (36%) compared to powder with egg (12%) after roasting for 10 min.This result was similar to previous research conducted by Rudolf (2003), 22 which showed that trans-resveratrol levels in nuts increased after the roasting and has been patented since 2010. 23rans-resveratrol content decreased by 10% in the sample without eggs and by 24% in the sample with eggs after baking for 20 min, possibly due to stilbene compounds, particularly resveratrol, being damaged. 15Protein in eggs is known to easily denature at high temperatures, with the denaturation product forming aggregates, including ovalbumin. 24These protein aggregates are water insoluble; thus, high temperatures decrease protein solubility. 25he trans-resveratrol level of fried emping is shown in Table 2 and was found to decrease after the frying process, which is similar to previous results for Vaccinium myrtillus L. and Vaccinium corymbosum L. after baking. 16Another study reported that trans-resveratrol level in raw peanuts was higher than in roasted peanut, 26 including grapes, and may be one of the compounds responsible for the health benefits of red wine.Analytical methods for measuring resveratrol in wine and peanuts were adapted to isolate, identify, and quantify resveratrol in several cultivars of peanuts.Aqueous ethanol (80% v/v Trans-resveratrol content in boiled melinjo (Table 1) decreased over time because of the instability of resveratrol.This instability was investigated by Zupancic et al., 27 this paper presents newly determined trans-RSV solubility and stability at various pH and temperatures, and the importance of such data for the studies of novel trans-RSV-loaded nanofibers.In acidic pH trans-RSV was stable, whereas its degradation started to increase exponentially above pH 6.8.Consequently, it is worthwhile to note that special consideration has to be dedicated to long dissolution testing or biological assays on cell lines in order to obtain relevant data.Measurements were done by validated  UV/VIS spectroscopy, HPLC, and newly developed UPLC methods.Specificity was confirmed for HPLC and UPLC method, whereas UV/ VIS spectroscopy resulted in false higher trans-RSV concentrations in conditions under which it was not stable (alkaline pH, light, increased temperature who found that trans-resveratrol stability was dependent on temperature; when trans-resveratrol was heated to over 30 o C, its isomerization form changed from trans-resveratrol to cis-resveratrol. Additionally, as the temperature increased trans-resveratrol degraded into a degradation product.To test the results, the data were analyzed using statistical package for the social sciences (SPSS) using a one-way analysis of variance (ANOVA) method, which indicated that changes in trans-resveratrol level were significant (p < 0.05).

Determination of Total Phenolic Content (TPC)
As can be seen in Table 2, demonstrated TPC in roasted Melinjo seed powder, including changes when eggs were included (p < 0.05).These results supported the results obtained using HPLC.Xu and Chang (2008)  found that temperature treatments, such as heating or roasting, of plants likely evaporated water at an intracellular level, triggering chemical reactions resulting in cell structure changes and increasing the availability of phenolic compounds in plant matrices. 28Similarly, Yang et al.
reported that the TPC of beans roasted for 5 min increased compared to raw beans. 29and Hečimović et al. also reported the comparative study of polyphenols and caffeine in different coffee varieties affected by the degree of roasting. 30The change in TPC in fried emping was also significant (p < 0.05), based on a one-way ANOVA and Tukey's post hoc analysis.
A decrease was expected because the polyphenol group is heat sensitive, which includes resveratrol.Previous studies reported that the TPC in Melinjo seeds decreased during the boiling process, 1 which was supported in the presents study; the TPC in boiled Melinjo seeds decreased with boiling time (Table 2; p< 0.05).This decrease occurred because the heating process in boiling made polyphenolic molecules unstable and ruptured molecule bonds.

CONCLUSION
Based on the results above, Effects of food processing on concentration of trans-resveratrol and total phenolic content in Melinjo (Gnetum gnemon L.) seeds has been performed.There were significant changes in trans-resveratrol levels and total phenolic content in Melinjo seeds before and after various cooking processes (i.e., roasting, boiling, and frying).The difference of trans-resveratrol and phenolic content in each food processing of Melinjo seeds is preliminary data for further study and its effect on pharmacological effects.