Curcuma xanthorrhiza Roxb. includes important spices originating from Indonesia which have been distributed to various other countries ranging from Southeast Asia to Europe. This plant is often referred to as “Temulawak” or “Javanese turmeric.” In Indonesia, C. xanthorrhiza is one of the most famous plants because of its use in the treatment of various diseases. Traditionally, it is used to treat fever in children, rheumatism, diarrhea, appetite enhancer, increasing immunity, arthritis, and to overcome constipation. C. xanthorrhiza is often used in combination with other medicinal plants to form a mixture of herbal products to increase stamina and immunity. The potential pharmacological activities of C. xanhtorrhiza include antibacterial, antioxidant, anti-inflammatory, anticancer, antihypertensivity, and immunomodulatory activity. The phytochemical profile obtained from various literature indicate the presence of xanthorrhizol compounds, which are typical compounds of C. xanthorrhiza, amino acids, carbohydrates, curcuminoid compounds such as octahydrocurcumin, curcumin, dihydrocurcumin, and hexahydrocurcumin,[1,5] and vanillin compounds such as isoborneol, camphor, and trans-caryophyllene.
Natural phenolics induce positive health effects, especially through its antioxidant activity. This compound can reduce oxygen concentration and prevent the formation of hydroxyl radicals and rather become non-radical species. This phenolic uniqueness encourages the continued development of optimum methods to extract the phenolic compounds. Meanwhile, the plant properties and characteristics of their components, including solubility and stability, should be considered when developing methods to extract certain compounds. The complex nature of polyphenols makes it a challenging to extract polyphenol chemicals and other useful components. Solvent, concentration, and extraction technique are the main determinants of extraction performance. Therefore, the selection of an appropriate solvent, taking its polarity into account, is of great relevance, both in pure and mixed solvent systems because chemical compounds with different polarities have different antioxidant potentials. There is a substantial correlation between the antioxidant ability of the extract and solvent used.[10,11] The commonly used solvents for the extraction of medicinal plants are water, ethanol, acetone, aether, chloroform, and hexane, each of which has a different level of polarity. Polyphenol antioxidants in plants are more soluble in aqueous solutions of organic solvents such as ethanol, methanol, and acetone. Selecting a mixture of extraction solvents based on the changes in solvent proportions within the system by using an experimental mixture design, such as a simplex centroid, is one approach.
In addition to solvent extraction, extraction techniques also consider time efficiency, cost, and amount of raw materials, especially with the aim of commercial development. Mixture design is a statistical-based method that has become a widely used method in recent years to increase the extraction yield of metabolite compounds. It is more advantageous than previous methods because it can identify the synergistic effect of the interaction between the experimental variables with a smaller number of experiments in a relatively short time period. This mixture design has been previously used to determine the synergistic effect of phenolic and antioxidant extraction with mixed solvents.[13,15] The simplex centroid design (SCD) is the most commonly used method to optimize formulas for the solvent factors of three or more mixed components.[14,16]
To date, no research has been conducted for the optimization of extraction solvents for C. xanthorrhiza phenolic compounds involving a SCD. Thus, the main objective of this work is to maximize the extraction of phenolic compounds with antioxidant activity of C. xanthorrhiza using a SCD with four different polarity solvents. This study provides new findings for the development of compound extraction methods for C. xanthorrhiza.
MATERIALS AND METHODS
The rhizome of C. xanthorrhiza was collected from the garden of the Tropical Biopharmaceutical Cultivation Conservation Unit of Tropical Biopharmaca Research Center, IPB University (6º3’49”S and 106º42’57”E). The samples were cleaned, dried, and air-dried for five days, and then ground into powder using a mill disk. The powder was passed through an 80-mesh sieve and the sample was used for the extraction process.
Simple-centroid design using the design expert program
The model was designed to optimize the extraction of phenolics with their antioxidant activity based on the SCD using Design Expert® 13.0 software (Stat-Ease Inc, Minneapolis, USA). Four solvents used for C. xanhtorrhiza rhizome extraction with various polarities were pure water (A), acetone (B), methanol (C), and ethanol (D). The design consisted of 15 treatments consisting of single solvent, binary, ternary, and quaternary mixtures, as shown in Table 1.
Preparation of extracts
The extraction process was performed by mixing 10 g of dry C. xanthorrhiza powder with 100 mL of solvent (1:10 w/v) with the extraction solvents water, acetone, methanol, and ethanol, as single, binary, ternary, and quaternary mixtures according to the compositions presented in Table 1. Mixtures were stirred for 30 min at 140 rpm, and macerated for 48 h in a dark room. The mixtures were then filtered and concentrated using a rotary evaporator (HAHNVAPOR, Korea). A total of 15 extracts were obtained, and their analytic responses were analyzed for total phenolic content (TPC), antioxidants in the 2,2-diphenyl-picryl hydrazyl radical scavenging (DPPH), and ferric reducing antioxidant power (FRAP) methods.
According to the procedure, TPC was measured using the Follin–Ciocalteu method. In a 96-well microplate, 160 μL of distilled water was added, along with 10 μL of sample extract, 10 μL of 10% Folin–Ciocalteu reagent, and 20 μL of 10% Na2CO3 solution. The mixture was incubated in the dark at room temperature for 30 min. The absorbance was measured at a wavelength of 750 nm using a microplate reader (Epoch BioTek, USA). The standard curve was adjusted using gallic acid, and the resulting TPC was expressed as milligrams of gallic acid equivalent per gram dry weight (mg GAE/g DW).
Antioxidant activity assay
Scavenging activity of DPPH radical
Determination of the antioxidant activity of DPPH (2,2-diphenyl-picryl hydrazyl) was performed according to the method of Calvindi et al. (2020). First, 125 μM DPPH reagent (in methanol) was prepared. For the test, 100 μL of sample extract was added into a 96-well microplate (BIOLOGIX), and 100 μL of 125 M DPPH reagent was added. This was incubated for 30 min in the dark at room temperature. The absorbance was measured at a wavelength of 517 nm using a microplate reader (BMG Labtech, Germany). The standard curve was adjusted to Trolox, and the activity was expressed as micromoles of Trolox equivalent per gram dry weight (μmol TE/g DW).
Ferric reducing antioxidant power assay
Iron-reducing antioxidant strength was determined according to the method described by Calvindi et al. (2020). FRAP reagent consisting of an acetate buffer solution pH 3.6, TPTZ (2,4,6-tri-pyridyl-s-triazine) (10 mM in 40 mmol HCl), and 20 mmol FeCl3 was combined at a ratio of 10:1:1 (v/v/v). In a 96-well microplate (BIOLOGIX), 10 μL of sample extract and 300 μL of FRAP reagent were added. This was incubated for 30 min in the dark at 37°C. The absorbance was measured at a wavelength of 593 nm using a microplate reader (BMG Labtech, Germany). The standard curve was adjusted to Trolox, and the activity was expressed as micromoles of Trolox equivalent per gram dry weight (μmol TE/g DW).
Data analysis was performed using the Design Expert 13.0 program (Stat-Ease Inc, Minneapolis, USA). The selection of optimal condition results was selected based on the highest desirability value closest to 1.00.
RESULTS AND DISCUSSION
Sample extraction optimization
SCD is part of the mixture design which is a method to optimize solvent systems and has the advantage of not requiring a large amount of solvent, requiring a short time period, and having a high success rate by looking at the desirability value. C. xanthorrhiza was extracted using four pure solvents (water, acetone, methanol, and ethanol) as well as a mixture of solvents whose proportions were designed based on the SCD. Table 1 shows the effect of the different solvents on each response variable for TPC, and antioxidant activity as measured by the DPPH and FRAP methods. From the data, we can deduce that the TPC content varied from 10.332 to 36.293 mg GAE/g DW, the DPPH antioxidant activity varied from 4.004 to 28.949 μmol TE/g DW, and the FRAP antioxidant values varied from 7.867 to 192.576 μmol TE/g DW.
Considering the selection of a suitable model, the response variables were analyzed based on the analysis of variance (ANOVA). ANOVA was used as an evaluation model that matched several criteria measured with a 95% confidence interval [Table 2]. The response to TPC and antioxidant activity of DPPH showed a special cubic model (P-value >0.05), while the antioxidant FRAP showed a linear model (P-value <0.05). The performance of each model was determined through the coefficient R2 and according to the literature the coefficient of determination (R2) shows conformity if it exceeds 70%, it can also be interpreted with the value of R2 getting closer to 1.00, meaning that the model is more suitable to the actual data or is significant.[20,21] From the results obtained in this study, the coefficient values of R2 were TPC (0.9808), DPPH (0.9583), and FRAP (0.7872). These values provide a good representation of 98.08%, 95.83%, and 78.72% of the response variability of TPC, DPPH, and FRAP, respectively, indicating that the selected or recommended model was suitable. Apart from the R2 coefficient, the adjusted R2 can be used to determine the suitability of the experimental results with the theoretical results. In addition, the coefficient of variation was considered to be <10%, this value is a measure of deviation from the average value indicating the precision of the test. The adeq precision generated from the ANOVA was 6.6590 (TPC), 4.4649 (DPPH), and 12.3225 (FRAP); the adeq precision is the ratio to error, where if the ratio is >4 it indicates an adequate signal and the model can be used to navigate the design space.
Efficiency of the solvents on the TPC
The effects of the solvent system on the TPC were observed and the ternary mixture of water, acetone, and methanol showed the optimum results with up to 36.293 mg GAE/g DW extracted [Figure 1]. The TPC measurement was based on the Follin–Ciocalteu reaction using a standard gallic acid curve. Gallic acid is a phenolic compound derived from hydroxybenzoic acid, which is relatively stable, thus it is effective and suitable for use as a standard curve.
The equation shown below can be used to predict the response of each treatment factor. By default, the highest mixture component is coded as +1 and the lowest as zero to negative. The coded equations are useful to identify the relative impact of the factors by comparing the factor coefficients. In equation 1, it is observed that the ternary mixture of water, acetone, and methanol can increase the extraction up to +573.36, followed by a binary mixture of methanol and ethanol (+80.25), but there is an antagonistic effect that occurs at several factors (equation 1).
TPC = 13.00A + 20.77B + 20.28C + 10.29D + 2.11AB – 20.04AC + 49.20AD – 19.71BC + 18.14BD + 80.25CD + 573.36ABC – 105.28ABD – 73.11ACD – 267.81BCD (1)
Where, A: water, B: acetone, C: methanol, and D: ethanol.
The extraction solvent is an important factor to obtain the TPC extract, this is because it is influenced by the solubility of these compounds in the extraction solvent and polarity of the solvent itself. The results of this study showed that the ternary mixture of water, acetone, and methanol was better than any pure solvent alone [Figure 1]. The solubility of the phenolic compounds present in the plant material has a better solubility with a higher polarity. According to Harborne (1998), the diversity of chemical structures of polar to nonpolar phenolic compounds such as phenolic acids and flavonoid glycosides is extracted using methanol. In the literature, curcumin is a natural polyphenolic compound derived from C. xanthorrhiza, which tends to be nonpolar and is suitable for extraction using acetone and ethanol. Other phenolic compounds contained in the rhizome of C. xanthorrhiza, such as vanillin and dehydro-6-gingerdion are polar.
Efficiency of the solvents on the antioxidant activity
Antioxidant activity is a complex process that often involves many processes and is governed by numerous variables that cannot be fully explained by one approach. To elucidate the various mechanisms of antioxidant action, it is imperative to perform various types of antioxidant capacity measurements.[26,27] DPPH and FRAP testing are the most commonly used methods to measure antioxidant activity. These methods have different reaction mechanisms; therefore, the results obtained depend on the method used. In this study, these methods were used to optimize the extraction of C. xanthorrhiza by evaluating its antioxidant properties.
To optimize the radical activity of DPPH, it was observed in the results of the SCD analysis [Figure 2] that the optimum point was obtained with a ternary mixture of water, acetone, and methanol with an activity value of 28.949 mmol TE/g DW. These results provide information that the antioxidants compounds such as phenolic groups are mostly extracted with a solvent combination of water, acetone, and methanol. In addition, it was observed from equation 1 that the solvent can increase the activity synergistically (+566.06) compared to other treatments. Equation 2, which is shown below, can be used to predict the response of each treatment factor. By default, the highest mix component is coded as +1 and the lowest as zero to negative. The coded equations are useful to identify the relative impact of factors by comparing the factor coefficients.
DPPH = 5.39A + 11.72B + 11.51C + 3.94D + 5.36AB – 12.85AC + 27. 71AD – 21.79BC + 22.46BD + 7 4.59CD + 566.06ABC – 6.46ABD – 227.48ACD – 228.95BCD (2)
Where, A: water, B: acetone, C: methanol, and D: ethanol.
Meanwhile, based on Figure 3, the antioxidant results of the FRAP method show that the optimum point was obtained in the single solvent treatment of water with an activity value of 192.576 mmol TE/g DW. These results are consistent when viewed from the coded equation 3, where water can increase the antioxidant activity synergistically (+183.62), followed by methanol (+44.13), acetone (+16.40), and ethanol (+0.1643). This suggests that water solvent extraction of C. xanthorrhiza rhizomes can extract chemicals that are rich in hydrogen donors, allowing them to convert oxidant molecules into more stable compounds.
FRAP = 183.62A + 16.40B + 4 4.31C + 0.1642D (3)
Where, A: water, B: acetone, C: methanol, and D: ethanol.
Antioxidants are important to prevent disorders caused by oxidant damage. One class of polar chemical compounds known as phenolic compounds contains one or more hydrogen donors and function as antioxidants. By absorbing and neutralizing free radicals, the antioxidant activity of phenolic compounds is important. Simple phenols that have a single aromatic rings with one hydroxyl group, polyphenols with two or more subunits such as flavonoids, and tannins, which have three or more phenolic subunits are types of phenolic chemicals. Curcumin is a natural polyphenolic compound derived from C. xanthorrhiza, which has a major function in antioxidant activity.[25,31] According to the study conducted by Taher and Sarmidi (2015), the DPPH antioxidant activity of C. xanthorrhiza extracted with acetone was the highest at 38.75%. This result differed from ours likely because the oleoresin extract was selected for extraction optimization and the infusion extraction technique was used.
Optimized solvents systems
The optimal performance for all response variables was determined by predicting the solvent system using models, contour plots, and desirability analysis. Fifteen solvent systems were selected, including single solvent, and binary, ternary, and quaternary mixtures. Each system was used to evaluate the TPC, and antioxidant activity of DPPH and FRAP, as presented in Table 3. The mathematically predicted responses and optimum conditions were determined based on the highest desirability value closest to 1.00, which indicated the best level of accuracy. This value is a function of the level of confidence in determining the optimal point based on the specified response variables. TPC with antioxidant activity could be most effectively extracted from C. xanthorrhiza with a ternary combination solvent consisting of water, acetone, and methanol in the following proportions: 0.409, 0.307, and 0.284, respectively, with a desirability value of 0.723. There were 34.112 mg GAE/g DW in the TPC, 26.533 mmol TE/g DW in the DPPH, and 92.353 μmol TE/g DW in the FRAP of this extracted mixture.
The results of this study indicate that the use of a SCD as a method to improve the extraction conditions of C. xanthorrhiza rhizomes was suitable. The ANOVA indicated the model for FRAP was significant, and the model for TPC and DPPH was not significant, but the model selection criteria were statistically supported. The developed model was quite accurate, as shown by the R2 values for TPC, DPPH, and FRAP, which were 0.9808, 0.9583, and 0.7872, respectively. The optimum conditions for the extraction of C. xanthorrhiza rhizomes were a ternary combination of solvents including water, acetone, and methanol in the proportions 0.409, 0.307, and 0.284, respectively, and a desirability level of 0.723. This resulted in 34.112 mg GAE/g DW of TPC, 26.533 μmol TE/g DW of DPPH, and 92.353 μmol TE/g DW of FRAP. These results can be used for the development of new drugs, beverages, and functional foods by providing beneficial information for the extraction of the phenolic components of C. xanthorrhiza.
Financial support and sponsorship
This research was funded by Jambi University And IPB University Research Collaborations, grant number 2158/UN21.11/PT.01.05/SPK/2022 and 4014/IT3.L1 /PT.01.03/M/T/2022.
Conflicts of interest
There are no conflicts of interest.
The author gratefully acknowledged the Jambi University and IPB University Research Collaborations for grant (2158/UN21.11/PT.01.05/SPK/2022 and 4014/IT3.L1 /PT.01.03/M/T/2022).
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