Vol. 29, No. 5, (2020), pp. 5956 - 5962
Optimization of Antioxidant Extraction from the Cacao Bean Shell
Amalia Noviyanty1*, Asriani Hasanuddin2, AbdulRahim1, Gatot Siswo Hutomo1
1
Faculty of Agriculture, Tadulako University, City of Palu, Indonesia
2
Livestock Study Program, Faculty of Animal Husbandry and Fisheries, Tadulako University,City of Palu, Indonesia
Email: [email protected]
Abstract
This research was conducted to determine optimal conditions for antioxidant extractionfrom cacao bean shellsaccording tosolvent type,solvent/shell ratio,and extraction time. Solvents tested included75% ethanol, 85%ethanol, 95% ethanol, ethyl acetate, and a solution of acetone and water at a ratio of 7:3. Solvent/shell ratios tested were 2:1; 3:1; 4:1; 5:1; 6:1; 7:1, and 8:1. Extraction timesof 1.0, 1.5, 2.0, 2.5,and 3.0h were applied. The data obtained were analyzed using a completely randomized design applied to the observation oftotal phenolics and antioxidant activity.The largest quantities of phenolics and highest antioxidant activity, i.e., 99.07 ppm and 49.99 ppm, respectively, were obtained with95% ethanol. Various ratios of the highest-value solvents for total phenolics and antioxidant activity, i.e., 122.7 ppm and 55.92 ppm,were obtained at the solvent ratios of 6:1 (v/b) and 6:1 (v/b), respectively.Various extraction times for the highest values for total phenolics and antioxidant activity, i.e., 120.23 ppm and 51.35 ppm, were obtained at 2 h.95% ethanol (95%),a solvent/shell ratio of6:1 (v/b), and2h ofextractionproduced the most total phenolics and antioxidant activity,i.e.,120.23 ppm and 51.35 ppm, respectively.
Keywords: Cacao bean shell,Extraction of antioxidant,Optimum condition.
1. Introduction
Biologically, an antioxidant is a compound capable of counteracting or reducing the negative effects of oxidants in the bodyby donating an electron to an oxidant.For example, antioxidant compounds in cacao extracts can inhibit cancer cell growth by 70%
by blocking the flow of cells at the second growth phase or G21.
The active compounds can be separated from a plant using an extraction process that is determined by solvent type, solvent ratio, and extraction time.The solvent type in the extraction process affects the acquisition of the active substance2.Thesolvent ratiois applied in accordance with the solubility of the solute. The smaller the solubility of the solute to the solvent, the greater the ratio of solvent to solids, and vice versa.Extraction time has a significant influence on extraction.Too long or too short an extraction time can affect the physical and chemical properties of the extracted material.
More studies have been conducted on antioxidant compounds in the cacao bean than on antioxidant compounds in the bean’sshell. Antioxidant compounds have been found in cacao shell, but little information is available on the optimal conditions for antioxidant extraction from theshells.The objective of this study is to determine the optimal conditions of antioxidant extraction on cacao bean shells.
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2. Materials and Methods
Materials and Tools
Cacao bean shells were provided by Industri Rumah Coklat, on Jalan S.Parman, Palu, Central Sulawesi. Other experimental materials included 95% ethanol, 85% ethanol, 75%
ethanol, 96% ethanol, gallic acid, Folin–Ciocalteu reagent, 20% Na2CO3 solution, and 50µM 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution. The tools used were ordinary scales, jars, analytical scales, filter paper, blenders, water baths, Erlenmeyer flasks, round- bottom measuring flasks equipped with reverse coolers, rotary evaporators, test tubes, shaker machines, N2 gas, ultraviolet-visible (UV–Vis) spectrophotometers, drop pipettes, and writing instruments.
Place of Experiment
The research was conducted at the Laboratory of Agro-Industry, Faculty of Agriculture and Chemistry Laboratory, Faculty of Mathematics and Natural Sciences, Tadulako University, Palu.
Data Collection Technique
Procedures of Extract Sample Preparation
Cacao bean shellpowder (50g) was put into a 500 mLErlenmeyer flask, to which250 mL of solvent was added (for a 5:1 v/b ratio). The mixture was shaken over a shuffle machine (shaker) at 200 rpm for 2 h and reserved for 24 h. The mixture was then filtered to separate the filtrate from the residue. The obtained filtrate was evaporated using a rotary evaporator. Evaporation of the solvent was enhanced with N2. The obtained extract was frozen in a freezer. The obtained concentrated extract was used for the analysis of extracted total phenolics and antioxidants. All extraction processes were replicated and repeated three times.
Experimental Design
In this study, the observed parameters were solvent type, solvent ratio, and extraction time.
1)
Solvents,including75% ethanol, 85% ethanol, 95%ethanol,ethyl acetate, acetone:water (7:3) were tested at a fixed solvent ratioof 5:1 (v/b) and an extraction timeof 2 h. The most effective solvent type was selected based on thetotal extracted phenolics andantioxidant activity.
2) Seven solvent ratios (2:1; 3:1; 4:1; 5:1; 6:1; 7:1, and 8:1[v/b]) were tested, with the most effective solvent determined by the first step. The most effective solvent ratio was selected based on the total extracted phenolics and antioxidant activity.
3) The most effective solvent type as determined in the first step,andthe most
effective solvent ratio as determined in the second step,were tested for1.5, 2.0,
2.5, 3.0, and 3.5 h. The most effective extraction time was selected based on
the total extracted phenolics and antioxidant activity.
Vol. 29, No. 5, (2020), pp. 5956 - 5962 Statistical Analysis
Experiment results were reported as means ± standard deviation for replicatesof solvent extraction and the triplicate of assays. All results were analyzed usinga completely randomized designfor one-way analysis of variance with a BNJ test, which is used to determine the significant differences between the means at the 5% level.
3. Results
The results of the variance analysis showed that solvent type had a significant effect on the totalphenolics extractedfrom cacao bean shells.The BNJ 5% test (Fig. 1) showed that a total phenolics content of 99.07 ppm, which wasproduced using95% ethanol (v/b),was the highest total phenolics value and was no different from treatment with acetone:water (7:3).The solvent type presented a significant effect on the IC50 value of extracts of cacao bean shells. The BNJ 5% test (Fig. 2) showedthat the highest IC50 value (49.89 ppm) was associated with the use of 95% ethanol (v/b) and was comparable to treatment using acetone:water (7:3).
Fig 1. Total Phenolics for Different Solvent Type/Cacao Bean Shells
Fig 2. IC50 Value for Antioxidant Activity for Different Solvent Type/Cacao Bean Shells (v/b)
Results for total phenolics obtained (Fig. 3) show that an increase in the solvent shell ratiowill also increase thetotal phenolics. The lowest phenolics total (96.17 ppm) was found with solvent/shell ratio of 2:1 (v/b),whereas the highest phenolics total (122.70 ppm) was obtained with a solvent/shell ratio of 6:1 (v/b).The solubility of phenolic compounds in the cacao bean shell increased with the use of a solvent.The results of the variance analysis show that the solvent ratio had a significant effect on the total phenolics of a cacao bean shell. The BNJ 5% test results (Fig. 3) show that the highest phenolics total was obtained with a solvent/shell ratio of 6:1 (v/b), which differedlittle from a 7:1 ratio.
Fig 3. Total Phenolics for Different Solvent Ratio/Cacao Bean Shells
Fig 4. IC50 Value of Antioxidant
Activity for Different Solvent
Ratio/Cacao Bean Shells (v/b)
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Total phenolics obtained (Fig. 5) indicates that an increase in extraction time will increase the phenolics total. The lowest phenolics total(95.16 ppm) was found with 1h of extraction,whereas the highest total (120.23 ppm) was obtained with 2h of extraction.Figure 6 shows that the highest IC50 value (51.35 ppm) was obtained with2h of extraction,whereas the lowest IC50 value (63.12 ppm)was obtained at 1h.
Fig 5. Total Phenolics for Different Extraction Times.
Fig 6. IC50 Antioxidant Activity at Different Extraction Times
4. Discussion
The results of total phenolics in Figure 1 show that 95% ethanol has a level of polarity that is almost equal to the extract of the cacao bean shell. It was more effective in dissolving the phenolic compounds contained in the cacao bean shell compared with the solvent ethyl acetate, whose polarity levels are below 85% ethanol, 95% ethanol, and acetone:water (7:3). A compound will dissolve in solvents that have the same polarity 3. Polar solvents are able to more effectively dissolve phenol so the levels in the extract become high. Flavonoids are polyphenol compounds (hydroxyl groups) that are polar and soluble in polar solvents such as ethanol, methanol, water, acetone, butanol, dimethylformamide, and dimethyl sulfoxide 4.
Phenolic compounds are not only soluble in acetone solvents but also in ethyl acetate solvents. Phenolicsare included in compounds that are polar, making the phenolic easily soluble in a polar solvent 5. The ethyl acetate solvent is semi-polar, having little ability to attract polar compounds. Phenolics are acidic because of the nature of the H+ ion that escapes easily. Whether the extract produced with acetone:water (7:3) have a high antioxidant activitywas unknown. The results of the antioxidant activity show that acetone:water (7:3) solventdemonstrated low antioxidant activity compared with 95%
ethanol solvents (Fig. 2). As a result, 95% ethanol provided the optimal solvent.
Figure 2 shows that the antioxidant activities of cacao bean shell extracts at IC50 values ranged from 49.89 ppm to 274.16 ppm. Using 95% ethanol produces the highest antioxidant activity compared with other solvents.The amount of antioxidant activity is characterized by the IC50 value, which is the concentration of sample solution needed to inhibit 50% of DPPH free radicals. Smaller IC50 values indicate higher antioxidant activity.
The results show that95% ethanol produces an IC50 value below 50 ppm, whereas an ethyl acetate solvent produces an IC50 value above 200 ppm for antioxidant activity.
Acompound is a powerful antioxidant if the IC50 value is 0–50 ppm, a strong antioxidant has an IC50 of 51–100 ppm, whereas a moderate antioxidant has anIC50 of 101–150
Vol. 29, No. 5, (2020), pp. 5956 - 5962 ppm, and a weak antioxidant has an IC50 of 151–200 ppm6. Extracts that have an IC50 value of less than 200 ppm are classified as having strong antioxidant activity7.
Whether the extract produced usinga solvent/shell ratio of 7:1 (v/b) had high antioxidant activity as well was difficult to determine. Results of the study on antioxidant activity (Fig. 4) show that solvent/shell ratio of 6:1 (v/b) had the best antioxidant activity compared with 7:1 (v/b). Therefore, a solvent/shell ratio of 6:1 (v/b) was optimal. The higher the solvent ratio leads to a higher average total produced phenolics 8 and the more solvents that are added, the greater the solvent’s ability to dissolve the material. As a result, more components of the material can be extracted by the solvent.
The results of the variance analysis showed that solvent type has a significant effect on the IC50 value of the extract of cacao bean shells. The results of BNJ 5% test (Fig. 4) showedthat an IC50 value of 55.92 ppm was found with the use of a solvent/shell ratio of 6:1 (v/b) which comes with the highest IC50 value and is different from all of the treatments.The greater the ratio between the sample with the solvent, the lower the resulting antioxidant activity (IC50 is greater). This may be due to a large sample ratio. If the concentration of the solution is near the equilibrium point, the phenolic compound becomes difficult to extract. When the ratio of the sample and the solvent is small, the time required to reach the equilibrium point is longer, and the phenolic concentration in the liquid becomes so dilute that lower levels of antioxidants are produced9.
Figure 5 shows that an increase in extraction time will increase the total phenolics obtained. The lowest total phenolics (95.16 ppm) was found with the use of 1h of extraction time,whereas the highest phenolics total (120.23 ppm) wasobtained after 2h.Figure 5 also shows that an increase in up to 2h of extraction time increases the total compound content of cacao bean shell phenolics. The solubility of phenolic compounds in cacao bean shells increaseswith extraction time.The longer the extraction time, the longer the contact between the material with the solvent, making the solvent capable of attracting more compound in materials.
However, at 2.5h of extraction, there was a decrease in total phenolics. It can be assumed that at 2 h, extraction reached its optimal point, so further increases in time beyond 2 h did not affect the extraction process of the targeted phenolics compound. Fick’s second law states that the diffusion process will reach an equilibrium stage after a certain time. At that time, even though the extraction time is increased, it will not increase the amount of the extracted material 10.The results of the variance analysis show that extraction time has a significant effect on the total phenolics of cacao bean shells. The results of BNJ 5% test (Fig. 5) show that the highest phenolics total was obtained using a 2h extraction period, which was not different from a 2.5h extraction.
A question of interest iswhether the extract produced with 3h of extraction will have high antioxidant activity. The results of the study on antioxidant activity (Fig. 6) show that a 2h extraction time produced greater amount of antioxidant activity compared with 2.5h of extraction. The use of 2h of extraction to extract the cacao bean shell produced optimalresults.Figure 6 shows that the highest IC50 value (52.37 ppm) was obtained at 2h of extraction,whereas the lowest IC50 (63.08 ppm) was obtained at 1h of extraction. A 2h extraction time was associated with the highest antioxidant activity 11.At 2h of extraction and longer, the IC50 value decreased because the longer the extraction time, the longer the contact between the solvent and the material 7. Therefore, mass deposition by diffusion will occur until the solution concentrations inside and outside of the material extraction reach equilibrium.
Vol. 29, No. 5, (2020), pp. 5956 - 5962 The results of the variance analysis showed that solvent type had a significant effect on the IC50 value of cacao bean shell extract. The results of BNJ 5% tests (Fig. 6) show that an IC50 value of 51.35 ppm was produced with a 2h extraction time, and it is not different from 5h and 3htreatment extraction times.All samples used in tests of extraction time had IC50 values of between 52.37 and 63.08 ppm. In accordance with the IC50 value parameter,
5. Conclusion
The extraction parameters of solvent type, solvent ratio, and extraction time had significant effects on thetotal phenolics contents and antioxidant activity of cacao bean shell extract. The highesttotal phenolics value (120.23 ppm) and greatestantioxidant activity (51.35 ppm)were associated with95% ethanol at a solvent/shell ratio of 6:1 (v/b) for 2h.
Acknowledgments
The authors would like to thank the Directorate of Research and Community Service of the Directorate General for Strengthening Research and Development at the Ministry of Research, Technology and Higher Education.
Ethical Clearance: Taken from Faculty of Agricultural, Tadulako University committee.
Source of Funding: Doctoral Dissertation Research Grant from DP2M DIKTI with the contract No: 097/ SP2H/ LT/DRPM/2018, dated March 26, 2018.
Conflict of Interest: None
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