Optimization of phenolic extraction from Amomum compactum fruits using simplex centroid design

Cardamom (Amomum compactum Sol. Ex Maton) is a medicinal plant that belongs to the family Zingiberaceae. Phenolic compound detected in Cardamom fruit that responsible for several pharmacological activities. Optimization used the simplex centroid design for the yield and phenolic extractions of Cardamom fruit through maceration to optimize the mixing of water, acetone, methanol, and ethanol. The extraction yield is determined by weighing the extracted material compared to the whole sample. Total phenolic content wasmeasured by a spectrophotometric method using Folin-Ciocalteu reagent. The results show that the best for relating extract yield is the linear model, while the quadratic model is the best to connect total phenolic content response. Water (100%) solvent extraction on Cardamom fruits obtained the highest extract yield (10.52%) and the lowest extract yield from 100% ethanol. The extraction of phenolic compoundswith amixture of water (50%)/ethanol (50%) resulted in maximum total phenolic content (168.98 mg GAE/g). However, the 100% ethanol of solvent extraction noted the minimum phenolic content (93.15 mg GAE/g). The results show that we should carefully choose the solventmixture extraction to achieve the extract yield and phenolic extraction goals. This study irst reported an optimization study on phenolic compounds extracted from Cardamom fruit.


Amomum compactum
Sol. Ex Maton, extract yield, phenolic extraction, simplex centroid design, extraction ef iciency A Cardamom (Amomum compactum Sol. Ex Maton) is a medicinal plant that belongs to the family Zingiberaceae. Phenolic compound detected in Cardamom fruit that responsible for several pharmacological activities. Optimization used the simplex centroid design for the yield and phenolic extractions of Cardamom fruit through maceration to optimize the mixing of water, acetone, methanol, and ethanol. The extraction yield is determined by weighing the extracted material compared to the whole sample. Total phenolic content was measured by a spectrophotometric method using Folin-Ciocalteu reagent. The results show that the best for relating extract yield is the linear model, while the quadratic model is the best to connect total phenolic content response. Water (100%) solvent extraction on Cardamom fruits obtained the highest extract yield (10.52%) and the lowest extract yield from 100% ethanol. The extraction of phenolic compounds with a mixture of water (50%)/ethanol (50%) resulted in maximum total phenolic content (168.98 mg GAE/g). However, the 100% ethanol of solvent extraction noted the minimum phenolic content (93.15 mg GAE/g). The results show that we should carefully choose the solvent mixture extraction to achieve the extract yield and phenolic extraction goals. This study irst reported an optimization study on phenolic compounds extracted from Cardamom fruit.

INTRODUCTION
Fruit and vegetable-rich diets are associated with a bene icial function in many human diseases, and the phenolic content of plants is linked to these bene its (Rio et al., 2013). Evidently, because of their bene icial health effects, phenolic compounds have attracted interest in recent years. Phenolic compounds have been extensively studied among the secondary metabolites of different plant species due to their neuroprotection, antioxidant, anti-hyperglycemic, osteoprotection, immunomodulation and anti-in lammatory effects in humans (Chang et al., 2021). Furthermore, various fruits have various phenolic pro iles (Nguyen et al., 2019). Their possible positions in human health vary, therefore.
Amomum compactum Sol. Ex Maton (Cardamom), a common medicinal plant in the family Zingiberaceae, has been widely cultivated for its fruit to produce spice and herb. Cardamom fruits have been used for several applications in medicines, food, and culinary.
Indeed, Cardamom fruits intake is associated with a variety of health ben-e its, including anticancer, antioxidant, antibacterial, antidiabetic, insecticidal and gastro-protective activities (Ashokkumar et al., 2020). Besides being a source of essential oils, cardamom fruits are also among the most common sources of phenolic compounds (Paul and Bhattacharjee, 2018). Phenolics of Cardamom fruits are medicinally crucial because of their antioxidant (Bhatti et al., 2010), antibacterial (Garg et al., 2016), and antimutagenic (Saeed et al., 2014) properties. Different factors, including particle size, temperature, time, solvent polarity, and solvent concentration, usually in luence plant compounds extraction from the plant material (Qomaliyah et al., 2019). Besides that, the solvents of various polarities, different phytochemicals are extracted depending on the chemical composition, as no single solvent can be useful in extracting all the phytochemical compounds present in the plant material. Therefore, the use of a solvent mixture, which can be binary, ternary, or even multi-component combination, is also convenient. In this method, synergistic effects may be observed between solvents, which can yield in the phytochemical's extraction with various compound characteristics. Studies show that the phenolic yield in plant material has an essential impact on solvent polarity combinations (Panzella et al., 2020). Moreover, the value of that is already known; solvent optimization to obtain extracts rich in phenolic compounds from Cardamom fruit is not yet found in the literature.
Optimization of extraction of essential oil compounds (Suttiarporn et al., 2020) from Cardamom fruit has been widely documented, but little attention has been paid to the use of solvent mixtures to optimize phenolic yield. Therefore, this study aimed to determine the effect of different solvent mixtures on the Cardamom fruit maceration process and assess its impact on the yield of extraction and total phenolic content.

Samples preparations
Cardamom fruits were collected from a local farmer in Bogor, West Java, Indonesia (6 • 43'30.4"S; 106 • 41'40.6"E; altitude of 1267 m) and identi ied in Tropical Biopharmaca Research Center, Bogor Agricultural University, Indonesia (BMK0472052020). Before being milled into powder and prepared for future use, the samples were sun-dried for ive days to a constant weight. Analytical grades are both solvents (water, acetone, methanol, and ethanol) and reagents used and supplied by both Sigma-Aldrich (Sigma Co., USA) and Merck. Equipment, such as the weighing balance, was well-calibrated before use according to the manufacturers' requirements.

Experimental design
The simplex centroid design under mixture method, provided by Design-Expert program (version 11.0), was used to assess the in luence of the solvent composition (a mixture of water (A), acetone (B), methanol (C), and ethanol (D)) on the phenolic extraction from Cardamom fruits. The component levels (water, acetone, methanol, and ethanol) were 0 and 100 percent ( Table 1). The program then produced ifteen experimental runs, which re lect a mixed percentage of solvent composition by different portions.

Extraction procedure and determination of extract yield and total phenolic content
Extracts were made by the maceration method in a 100 ml extraction unit containing the selected solvents (water, acetone, methanol, and ethanol), based on the simplex centroid design as presented in Table 2. Brie ly, 10 g of sample mixtures were extracted with 100 ml of a solvent mixture. Under light cover, the samples were stirred at 140 rpm for 30 min. Then, the sample mixtures were macerated for 24 h in the darkroom. The solution was iltered using Whatman No. 4 ilter paper. The extraction process was repeated, and solutions were collected. Finally, each sample's solutions were concentrated in rotary vacuum evaporator (HAHNVAPOR, Korea) to give the actual extract yield. The extracts were collected and weighed to determine the percentage of extraction yield.
The extracts obtained were examined for total phenolic content. Total phenolic extracts were quantitated with the Folin-Ciocalteu reagent adapted from previously reported method (Khumaida et al., 2019). Gallic acid has been used as an external standard. Brie ly, extract sample (10 ml) was blended with 10 ml of 10% Folin-Ciocalteu, 160 ml of distilled water, and 20 ml of 10% Na 2 CO 3 . Mixtures were then incubated for 30 min at room temperature. Finally, absorption samples or standards were eventually measured using the microplate reader (Epoch BioTek, USA) at 750 nm. Results expressed as gallic acid equivalent (mg GAE/g extract).

Statistical data analysis
The data response from Cardamom fruits extraction experiments were statistically analyzed using Design-Expert software version 11.0 (Stat-Ease Inc., Minneapolis, USA).

Optimization of Cardamom fruits extract
Extract yield response of solvent optimization of Cardamom fruits extraction are presented in Table 2. Extract yield ranged from 2.77 to 10.52% in actual value and varied from 3.01 to 9.96% in predicted value. The maximum extract yield (10,52%) was provided by 100% water composition (component A) of the experiment run 11. Run 14 indicates an extract yield of 8,68%, consisting of 100% acetone composition (component B), but the solvent's use might not be seen as cost-ef icient compared to a water solvent.  Table 2). The experimental Run 7 was a mixture of water (50%) and ethanol (50%) (components A and D) that gave the highest total phenolic content (168.98 mg GAE/g), even though the mixture of acetone (50%) and methanol (50%) (component B and C) under Run 9 also provided relatively good phenolic content of 139.91 33 mg GAE/g but the use of the two solvents cannot be considered a cost-ef iciency.
Higher polar compounds are generally readily extracted by water, whereas strongly hydroxylated phenolic compounds such as catechins are more soluble in alcohols such as ethanol and methanol (Loarce et al., 2020). As a pure solvent, acetone was inef icient in recovering the compounds of interest. Thus, considering that non-polar solvents, such as acetone, chloroform, and ethyl acetate, have a greater af inity with low-polarity compounds (Wakeel et al., 2019), it is believed that some of these phytochemical compounds have been detected in the Cardamom fruits extracts analyzed. On the other hand, the combination of various solvents has created fascinating synergistic results. The highest yield obtained from polar solvents in this study can suggest that some of the Cardamom components contain several polar metabolites, resulting in high outcomes. Polyphenol, such as phenolics and lavonoids, are reported as essential polar compounds in cardamom fruits (Deepa et al., 2013). Besides, the yield of non-polar solvents in this work is relatively low so that they are no better than polar solvents. A relatively high phenolic yield was provided by experimental runs involving the mixture of non-polar and polar solvents, especially methanol, which may indicate the in luence of polar solvents and the af inity of Cardamom fruit components with non-polar solvents (Amma et al., 2015). For phenolic compounds, extraction well recommended polar solvents such as methanol (Zhang et al., 2020), but they were not well-preferred when the extract intended for human and medicinal purposes. Hence, this study preferred polar solvents such as water and ethanol.

Analysis of variance (ANOVA)
The ANOVA of extract yield and total phenolic content responses of optimization of Cardamom fruit extraction is presented in Table 3. ANOVA of the indings showed signi icant for extract yield and not signi icant for total phenolic content of Cardamom fruits affected by mixture solvent extraction at p < 0.05. Water/ethanol mixture (AD component) is a vital model term and has powerful effects on phenolic yield based on high F-value and low p-value. The determination coef icient (R 2 ) of extract yield and total phenolic content were 0.6849 and 0.7797, respectively. These analyses indicated the reliability of the data distribution in the experiment. The Adeq Precision of extract yield and total phenolic content were 10.3849 and 5.4189, respectively. This result showed Adeq Precision exceeding four. Therefore, this study indicates an adequate signal encouraging model use (Araromi et al., 2017).
The linear model generated describing the impact of interactions between the A (water), B (acetone), C (methanol), and D (ethanol) on the extract yield and total phenolic content is de ined as the coded value in equation 1 and 2, respectively. In extract yield response, the value of the equations' coef icients was 9.96, 6.59, 5.51, and 3.01 obtained for water, acetone, methanol, and ethanol, respectively. This analysis indicates that the solvents have positive effects on the extract yield of Cardamom fruits. Water > acetone > methanol > ethanol is shown in order of impact and this is consistent with extract yield 10.52%, 8.68%, 5.97%, and 2.77% (      (2) where A = water, B = acetone, C = methanol, and D = ethanol The relation between real and expected plots of values ( Figure 1A and Figure 1C respectively for extract

Optimization solution of extract yield and total phenolic content of Cardamom fruits
The experimental set of variables tested the best numerical optimizing solution is typically chosen based on the desirability of 1.00 as desirable as the case may be (Khalafyan et al., 2019). The maximum yield of extract and total phenolic content was obtained from 100% water (10.52%) and a mixture of water-ethanol (168.98 mg GAE/g). The optimum solution was obtained at 71% desirability, which gives 8.045% for extract yield and 146.132 mg GAE/g for total phenolic content with 72% water and 28% ethanol solvents their optimum volume (Table 4). Figure 2A demonstrates the response surface plot for the effect of the interaction of water (A), acetone (B), and methanol (C) on extract yield while retaining ethanol at 25%. On the other hand, the interaction between water (A), acetone (B), and methanol (C) on the total phenolic content, while the ethanol is 25%, is shown in Figure 2B. The curvature existence of all surface plots in Figure 2 shows the reciprocal interactions between the solvents examined for extract yields and total phenolic content of Cardamom fruits extraction.

CONCLUSIONS
The optimization of the mixing of four solvents (water, acetone, methanol, and ethanol) for the extraction of the phenolic from Cardamom fruits using the maceration method is reported for the irst time in this work. The optimization experiments were based on the simplex centroid design, which indicates that 100% water and a mixture of water-ethanol (50%-50%) is better suited for maximum extract yield (10.52%) and total phenolic content (168.98 mg GAE/g), respectively, of Cardamom fruits extraction. The software's proposed optimization approach shows that the best possible result is obtained from mixtures of 72% water and 28% ethanol, with the desirability of 71%. This research has shown the ability to extract phenolic from cardamom fruits with suitable solvents.