Abstract
This study investigates the extraction of phenolic compounds and antioxidant activity from spent coffee ground (SCG) and coffee cherry pulp (CCP) using subcritical water extraction combined with high-pressure carbon dioxide (CO₂). The objective was to optimize extraction conditions to maximize total phenolic content (TPC) and DPPH radical scavenging activity. Using Design Expert V.13 and Central Composite Design (CCD), key parameters including extraction time (30–60 min), temperature (180–220 °C), and solid-to-water ratio (0.024–0.027 g/mL) were systematically analyzed. The optimal conditions for SCG were determined to be 198 °C, 0.027 g/mL solid-to-water ratio, and 60 min, yielding a TPC of 217.26 mg GAE/g DW and a DPPH value of 23.28 µMol TE/g DW. For CCP, the best extraction conditions were 189 °C, 0.024 g/mL solid-to-water ratio, and 54 min, resulting in a TPC of 230.13 mg GAE/g DW and a DPPH value of 32.63 µMol TE/g DW. The results indicate that CCP exhibited higher phenolic content and antioxidant activity than SCG, emphasizing its potential for valorization. Furthermore, Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and Thin-Layer Chromatography (TLC) analyses confirmed the presence of bioactive compounds such as quinic acid, theobromine, and caffeine. These findings demonstrate the effectiveness of subcritical water and CO₂ extraction in enhancing the recovery of bioactive compounds from coffee byproducts. This optimized method provides a sustainable and solvent-free approach to extracting high-value phenolic compounds, with potential applications in functional food, nutraceutical, and pharmaceutical industries.
Keywords: Spent coffee grounds, Coffee cherry pulp, Subcritical water-CO2 extraction, Phenolic optimization
Subject terms: Chemical engineering, Environmental sciences
Introduction
Coffee, one of the most widely consumed beverages globally, is not only prized for its rich flavor and invigorating aroma but also appreciated for its potential health benefits attributed to its bioactive compounds. Among these compounds, phenolic compounds have received considerable attention due to their antioxidant properties and potential health-promoting effects. Phenolic compounds are a diverse group of secondary metabolites found abundantly in coffee beans, contributing to their flavor, aroma, and health-related properties1. Coffee production generates significant quantities of waste, including coffee grounds and coffee pulp, which are rich sources of bioactive compounds such as phenolic compounds known for their antioxidant properties. Efficient extraction of these compounds is essential for their utilization in various industries. Sub-critical water extraction, conducted under conditions where water remains in a liquid state at elevated temperatures and pressures, presents a promising method for extracting these valuable compounds without the need for organic solvents2. The incorporation of high-pressure carbon dioxide (CO2) into subcritical water extraction creates a unique extraction environment that enhances efficiency through multiple mechanisms. When CO2 dissolves in water, it forms carbonic acid, which lowers the pH and improves the extraction of phenolic compounds through enhanced cell wall disruption and increased solubility. This combined system allows precise pressure control (30–40 bar) without mechanical compression, while CO2’s high diffusivity enables better penetration into plant matrices. Additionally, this environmentally friendly approach has shown success in extracting various bioactive compounds while maintaining their stability3.
The optimal conditions for maximizing the recovery of phenolic antioxidants from spent coffee grounds (SCG) and cherry pulp have been a focus of research. Tran et al. identified that aqueous extraction at 100 °C for 60 min with a sample to solvent ratio of 1:100 g/mL resulted in high recovery yields, comparable to 50% aqueous acetone extraction4. This suggests that there might be a research gap in exploring alternative solvents or solvent combinations under synergistic extraction conditions that could further enhance the yield and efficiency of phenolic and antioxidant recovery. The use of hydrolysis, employing water as the sole extraction solvent, represents an eco-friendly approach to extracting antioxidant phenolic compounds from SCG5. This indicates an opportunity for research into integrating and hydrolysis with CO2 extraction methods to optimize the recovery process in an eco-friendly manner, potentially improving the sustainability of the extraction process while maximizing the yield. Tran et al. demonstrated that microwave-assisted extraction could efficiently recover bioactive compounds from coffee pulp waste under optimized conditions, leading to high antioxidant capacities4. This highlights a potential research gap in combining microwave-assisted extraction with response surface methodology to optimize both phenolic content and antioxidant recovery from SCG and cherry pulp, potentially offering a more efficient and scalable extraction process. The research gap, therefore, lies in exploring and optimizing synergistic extraction methods that combine the advantages of aqueous and CO2 extractions, potentially integrated with eco-friendly technologies like hydrolysis or microwave-assisted extraction. The goal would be to enhance both the efficiency and sustainability of extracting phenolic compounds and antioxidants from SCG and cherry pulp, leveraging response surface methodology for process optimization.
Subcritical water extraction (SWE) is a sustainable and efficient technique that utilizes high-temperature and high-pressure water (but below its critical point of 374 °C and 22.1 MPa) to extract bioactive compounds from plant and food by-products. This method has gained significant attention for its ability to enhance the solubility and selectivity of polar and semi-polar compounds while eliminating the need for organic solvents. Studies have demonstrated the effectiveness of SWE in extracting phenolic compounds, flavonoids, and caffeine from coffee by-products such as coffee pulp and SCG. For instance, SWE was successfully applied to Arabica coffee pulp, resulting in a high yield of anthocyanins, which possess strong antioxidant activity6. Similarly, another study found that optimizing SWE conditions, including temperature (160–180 °C), extraction time (35–55 min), and solid-to-liquid ratio (14.1–26.3 g/L), significantly improved the recovery of chlorogenic acids and caffeoylquinic acids from SCG, demonstrating its potential in producing functional ingredients for food and nutraceutical applications7.
Despite its advantages, SWE presents certain challenges that must be addressed for large-scale applications. One limitation is its high energy demand, as maintaining elevated temperature and pressure requires substantial energy input and specialized equipment8. Additionally, while SWE is effective for extracting phenolic compounds, it may not be suitable for highly volatile or thermolabile compounds, as prolonged exposure to high temperatures can lead to degradation of sensitive bioactive molecules9. Furthermore, the selectivity of SWE can vary depending on extraction parameters, necessitating careful optimization of solvent conditions to maximize compound stability and yield7. Response Surface Methodology (RSM) has been widely applied in optimizing food industry processes, including extraction techniques, to systematically evaluate the interactions between multiple processing parameters and enhance extraction efficiency10. Nonetheless, SWE remains a promising green extraction technology, aligning with sustainability principles by reducing chemical solvent use while enhancing the recovery of valuable bioactive compounds from coffee industry waste.
RSM is a powerful statistical technique widely used in experimental design, optimization, and analysis of complex systems and processes. Originally developed in the mid-20th century by George E.P. Box and colleagues, RSM has since become a cornerstone methodology in various fields, including engineering, chemistry, agriculture, and food science11–13. RSM enables researchers to efficiently explore and optimize the relationship between multiple variables or factors and one or more response variables of interest. By systematically varying the levels of input variables within a defined experimental domain, RSM allows for the characterization of the response surface, which represents the relationship between input variables and the response(s) of the system. Through mathematical modeling and statistical analysis of experimental data, RSM facilitates the identification of optimal process conditions or design parameters that maximize desired responses or minimize undesirable ones14. RSM enables the design, analysis, and optimization of experiments by exploring the complex interactions between multiple extraction parameters, such as temperature, extraction time, and the ratio of solid to water15. By employing RSM, researchers can identify optimal conditions for maximizing the extraction efficiency of phenolic compounds and antioxidant activity. Following extraction, the crude extracts can be further characterized to identify and quantify individual compounds16,17. In recent years, there has been increasing interest in optimizing extraction processes to enhance the yield and bioactivity of phenolic compounds from SCG.
The objective of this research is to optimize the extraction process for obtaining key compounds from SCG and CCP, focusing on maximizing the content of phenolic acids and enhancing DPPH radical scavenging activity through water extraction and high-pressure carbon dioxide (CO2) extraction techniques. Utilizing Design Expert V.13 software and a Central Composite Design (CCD), the study aims to investigate the impact of various parameters including extraction time (ranging from 30 to 60 min), extraction temperature (between 180 °C and 220 °C), and the solid-to-water ratio (from 0.024 to 0.027 g/mL) on the total phenolic content (TPC) and DPPH radical scavenging activity. By comparing predictive equations with actual experimental outcomes, the research seeks to identify deviations in TPC and DPPH radical scavenging activity from both SCG and coffee pulp, and establish optimal extraction conditions that yield the highest phenolic content and antioxidant activity.
Methodology
Preparation of materials
The coffee grounds and pulp used in this study were sourced from Arabica coffee provided by Doi Chaang Original Coffee Company, located in Chiang Rai, Thailand. The materials were dried using a hot air oven at 105 °C for 1 h. After drying, they were ground finely using an electric grinder and sieved through a 425-micron mesh. The processed materials were then stored in zip-lock bags for further use.
All chemicals used were of analytical grade. Folin-Ciocalteu reagent, gallic acid (≥ 98%), sodium carbonate (≥ 99%), 2,2-diphenyl-1-picrylhydrazyl (DPPH, ≥ 98%), ascorbic acid (≥ 99%), and methanol (HPLC grade, ≥ 99.9%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol (AR grade, ≥ 99.5%) was obtained from RCI Labscan (Bangkok, Thailand). Deionized water was produced using a Millipore water purification system.
Experimental design
The experimental design was carried out using Design Expert V.13 software, employing the Central Composite Design method. Three independent variables were selected: extraction time (min), extraction temperature (°C), and the ratio of solid to water (g/mL). Each variable was tested at five different levels, as shown in Table 1, resulting in a total of 19 experiments for both SCG and CCP.
Table 1.
Variable of experimental design.
| Variables | Symbols | Levels | ||||
|---|---|---|---|---|---|---|
| − α | − 1 | 0 | 1 | + α | ||
| Time (min) | A | 19.77 | 30 | 45 | 60 | 70.22 |
| Temperature (°C) | B | 166.36 | 180 | 200 | 220 | 233.63 |
| Ratio of solid to water (g/mL) | C | 0.0229 | 0.0240 | 0.0255 | 0.0270 | 0.0280 |
Subcritical water extraction using carbon dioxide
The extraction process involved weighing 5 g of the material and placing it into the extraction apparatus (Fig. 1). DI water was added, and carbon dioxide gas was introduced into the extraction tank. The temperature and extraction time were set according to the experimental design, with the internal pressure maintained between 30 and 40 bar. After the extraction, the solids were filtered using No.2 filter paper, and the extracts were concentrated using a rotary evaporator at 40 °C and 72 mbar. The final extracts were stored in cryo-vials at -20 °C.
Fig. 1.
Subcritical water extraction using carbon dioxide setup.
Analysis of cloud extracts
Total phenolic content analysis
The total phenolic content was determined using the Folin-Ciocalteu method. A standard calibration curve was prepared using gallic acid solutions at concentrations ranging from 0 to 100 mg/L in ethanol. The Folin-Ciocalteu reagent and 7% (w/v) sodium carbonate solution were also prepared. For sample analysis, 0.05 g of the extract was dissolved in 20 mL of ethanol and thoroughly mixed. Then, 0.2 mL of the sample solution was transferred into a test tube, followed by the addition of 2.5 mL of distilled water and 0.2 mL of Folin-Ciocalteu reagent. The mixture was allowed to react for 5 min, and subsequently, 2 mL of 7% sodium carbonate solution was added. The reaction mixture was vortexed and incubated in darkness at room temperature for 90 min. After the incubation period, the absorbance of the sample was measured at 760 nm using a UV-Vis spectrophotometer. The total phenolic content was calculated based on the standard calibration curve and expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW)18,19.
DPPH assay
For the DPPH assay (2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay), extract concentrations ranging from 0.78 to 100 mg/mL were used. Each sample was mixed with 0.5 mM DPPH solution (550:550 µl) and incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm. Methanol was used as a blank, and 1 mg/mL ascorbic acid was used as a positive control. The percent inhibition was calculated using the formula Eq. (1):
 |
1 |
The antioxidant capacity of the samples was expressed in terms of Trolox equivalent antioxidant capacity (TEAC) and reported as micromoles of Trolox equivalents per gram of dry weight (µMol TE/g DW). This methodology ensures a comprehensive analysis of the phenolic content and antioxidant capacity of the coffee extracts, providing valuable insights for further applications20,21.
LC-MS/MS analysis
The analysis of samples was conducted using an LC-MS/MS system, specifically the X500 QTOF equipped with an electrospray ionization (ESI) source, operated in both positive and negative ionization modes. Mass spectra were recorded within the m/z range of 100–1,000 amu. The optimized ESI conditions included a capillary voltage of + 3,500 V, a dry gas temperature of 350 °C, a dry gas flow rate of 10 L/min, a nebulizer pressure of 30 psig, and a spectral acquisition rate of 4 Hz. Fragmentation was performed using auto MS/MS with collision energies set at 10 V, 20 V, and 40 V, with nitrogen as the collision gas. Chromatographic separation was achieved using a Phenomenex Luna C18(2) column (5 μm, 150 × 4.6 mm) with a mobile phase consisting of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). A linear gradient was applied, increasing solvent B from 10 to 90% over 30 min at a flow rate of 0.5 mL/min. The injection volume was set at 5 µL. Data acquisition and processing were performed using SCIEX OS 2.0.0 software, and compound identification was achieved by comparing retention times, molecular masses, and fragmentation patterns with standard compounds and the METLIN metabolite database22.
Nuclear magnetic resonance (NMR) spectroscopy
NMR spectra were recorded on a JEOL JNM-ECZ500R/S1 (500 MHz) spectrometer using chloroform-d (CDCl₃) as the solvent. ¹H NMR spectra were acquired at 500.16 MHz, with an acquisition time of 1.75 s, a relaxation delay of 5 s, and 256 scans. ¹³C NMR spectra were recorded at 125.77 MHz with proton decoupling, an acquisition time of 0.83 s, a relaxation delay of 2 s, and 10,000 scans. DEPT-135 was performed with a J-coupling constant of 140 Hz to differentiate CH, CH₂, and CH₃ groups. Data processing and peak assignments were performed using JEOL Delta software.
Thin-layer chromatography (TLC)
All chemicals used in this study were of analytical reagent (AR) grade to ensure the accuracy and reliability of the experimental procedures. The solvents used included n-hexane (95%) from KEMAUS, chloroform (99.8%) from Fisher Chemical, dichloromethane from RCI Labscan, ethyl acetate from RCI Labscan, and methanol from RCI Labscan. For Thin-Layer Chromatography (TLC), silica gel 60 F₂₅₄ TLC plates (20 × 20 cm) from Supelco were used. In addition, silica gel 60 (particle size: 0.040–0.063 mm) from Millipore was used for column chromatography. The chromatography column used in this study was made of borosilicate glass with a diameter of 2.5 cm and a length of 50 cm, equipped with a bottom stopcock for controlled solvent flow. The use of high-purity chemicals, standardized chromatography materials, and a well-defined column system ensured consistency and precision in the separation and identification of bioactive compounds throughout the study.
To optimize the separation of bioactive compounds from CCP, various solvent systems were tested using TLC. The solvent systems consisted of different combinations of hexane (C₆H₁₄), chloroform (CHCl₃), dichloromethane (CH₂Cl₂), ethyl acetate (C₄H₈O₂), and methanol (CH₃OH). The TLC procedure was conducted by preparing a mobile phase for each solvent system and developing the TLC plates under controlled conditions.
TLC plates were prepared by cutting them to the required dimensions, and a baseline was drawn 1 cm from the edge to guide sample application. The CCP extract was spotted on the TLC plates using a fine capillary tube, ensuring precise application and a uniform 5 mm spacing between spots. The TLC chamber was saturated with the respective solvent system before placing the plates inside for development.
After the solvent front reached the appropriate height, the plates were removed and dried. The separation of compounds was visualized under UV light at 254 nm, and the migration behavior of the compounds was recorded for further analysis. This experimental setup allowed for a systematic evaluation of different solvent systems to determine their efficiency in separating bioactive compounds from CCP extracts.
Results and discussion
Subcritical water extraction with carbon dioxide
The study involved the extraction of coffee grounds and coffee pulp using the Design Expert software, employing a Central Composite Design method. Three variables were considered: extraction temperature, extraction time, and the solid-to-water ratio. The pressure in the extraction tank was maintained at no less than 30 bar. The results of TPC and antioxidant capacity (measured using the DPPH assay) for coffee grounds and coffee pulp are presented in Tables 2 and 3, respectively.
Table 2.
Results of spent coffee ground extraction.
| RUN | Time (min) | Temperature (°C) | Ratio (g/mL) | Yield (%) | TPC (mg GAE/g DW) | DPPH (µMol TE/g DW) |
|---|---|---|---|---|---|---|
| 1 | 45 | 166.36 | 0.0255 | 27.51 | 83.79 | 14.89 |
| 2 | 19.77 | 200 | 0.0255 | 34.50 | 167.27 | 14.20 |
| 3 | 45 | 200 | 0.0255 | 25.25 | 158.36 | 14.92 |
| 4 | 30 | 180 | 0.027 | 35.97 | 103.72 | 15.02 |
| 5 | 70.22 | 200 | 0.0255 | 25.22 | 177.17 | 9.83 |
| 6 | 45 | 200 | 0.0255 | 30.99 | 207.27 | 8.20 |
| 7 | 45 | 200 | 0.0229 | 24.54 | 169.34 | 6.97 |
| 8 | 30 | 220 | 0.027 | 25.46 | 177.99 | 16.74 |
| 9 | 45 | 200 | 0.0255 | 24.93 | 180.97 | 15.33 |
| 10 | 45 | 200 | 0.028 | 28.39 | 219.40 | 13.26 |
| 11 | 45 | 233.63 | 0.0255 | 21.06 | 156.37 | 10.40 |
| 12 | 60 | 220 | 0.024 | 24.24 | 157.95 | 7.03 |
| 13 | 30 | 220 | 0.024 | 21.93 | 165.11 | 12.17 |
| 14 | 45 | 200 | 0.0255 | 25.11 | 190.07 | 15.26 |
| 15 | 60 | 180 | 0.024 | 30.24 | 133.00 | 19.47 |
| 16 | 60 | 180 | 0.027 | 35.38 | 170.61 | 14.95 |
| 17 | 60 | 220 | 0.027 | 20.01 | 155.74 | 9.46 |
| 18 | 45 | 200 | 0.0255 | 21.90 | 145.19 | 10.62 |
| 19 | 30 | 180 | 0.024 | 39.85 | 133.89 | 31.74 |
Table 3.
Results of Cherry pulp coffee extraction.
| RUN | Time (min) | Temperature (°C) | Ratio (g/mL) | Yield (%) | TPC (mg GAE/g DW) | DPPH (µMol TE/g DW) |
|---|---|---|---|---|---|---|
| 1 | 45 | 166.36 | 0.0255 | 44.83 | 198.52 | 23.90 |
| 2 | 19.77 | 200 | 0.0255 | 32.49 | 197.65 | 21.31 |
| 3 | 45 | 200 | 0.0255 | 19.92 | 126.53 | 15.42 |
| 4 | 30 | 180 | 0.027 | 28.67 | 190.38 | 25.08 |
| 5 | 70.22 | 200 | 0.0255 | 30.91 | 196.44 | 16.99 |
| 6 | 45 | 200 | 0.0255 | 30.86 | 210.95 | 23.07 |
| 7 | 45 | 200 | 0.0229 | 38.44 | 210.37 | 29.21 |
| 8 | 30 | 220 | 0.027 | 29.15 | 190.24 | 22.43 |
| 9 | 45 | 200 | 0.0255 | 30.80 | 186.44 | 19.15 |
| 10 | 45 | 200 | 0.028 | 29.75 | 187.25 | 23.73 |
| 11 | 45 | 233.63 | 0.0255 | 27.69 | 173.34 | 19.58 |
| 12 | 60 | 220 | 0.024 | 27.65 | 164.03 | 18.26 |
| 13 | 30 | 220 | 0.024 | 30.09 | 172.68 | 22.19 |
| 14 | 45 | 200 | 0.0255 | 25.75 | 155.69 | 18.37 |
| 15 | 60 | 180 | 0.024 | 30.41 | 197.51 | 23.16 |
| 16 | 60 | 180 | 0.027 | 32.15 | 171.01 | 28.70 |
| 17 | 60 | 220 | 0.027 | 18.30 | 109.29 | 13.09 |
| 18 | 45 | 200 | 0.0255 | 32.52 | 158.92 | 16.56 |
| 19 | 30 | 180 | 0.024 | 34.16 | 185.20 | 29.72 |
Table 2 shows the experimental results for the extraction of coffee grounds. Among the experiments, run 10 yielded the highest TPC at 219.40 mg GAE/g DW under the conditions of 45 min extraction time, 200 °C extraction temperature, and a solid-to-water ratio of 0.028 g/mL. The highest DPPH value was observed in Run 19, with 31.74 µMol TE/g DW, achieved with 30 min extraction time, 180 °C extraction temperature, and a solid-to-water ratio of 0.024 g/mL. The extraction yield for all experiments ranged from 20.01 to 39.85%. To optimize conditions for high TPC and DPPH values, ANOVA was conducted to identify the relationship between the variables.
Table 3 presents the extraction results for coffee pulp. The highest TPC was 210.95 mg GAE/g DW, observed in Run 6, which used 45 min extraction time, 200 °C extraction temperature, and a solid-to-water ratio of 0.0255 g/mL. The highest DPPH value was 29.72 µMol TE/g DW, found in Run 19 under conditions of 30 min extraction time, 180 °C extraction temperature, and a solid-to-water ratio of 0.024 g/mL. The extraction yields ranged from 18.30 to 44.83%.
Development of predictive models and optimization
Using design Expert V.13, the relationships between the three variables: extraction time (A), extraction temperature (B), and solid-to-water ratio (C) were analyzed. A quadratic model was used, with a significance threshold of p < 0.0523,24. For coffee grounds, the model for TPC yielded an R² of 0.9490, an adjusted R² of 0.8163, and a predicted R² of 0.9084. The significant variables affecting TPC were B, C², A²B, and A²C, where B is the solid-to-water ratio, C² is the square of extraction time, A²B is the interaction between extraction temperature and solid-to-water ratio, and A²C is the interaction between extraction temperature and extraction time. The predictive equation for TPC in coffee grounds is:
 |
2 |
For DPPH, the quadratic model gave an R² of 0.9698, an adjusted R² of 0.8913, and a predicted R² of -5.4727. The significant variables affecting DPPH were C, AC, BC, A², B², and C². The predictive equation for DPPH in coffee grounds is:
 |
3 |
For CCP, the TPC model yielded an R² of 0.9793 and an adjusted R² of 0.9256. The significant variables were A, AC, A², B², and C². The predictive equation for TPC in coffee pulp is:
 |
4 |
For DPPH in coffee pulp, the quadratic model resulted in an R² of 0.9927 and an adjusted R² of 0.9738. The predictive equation for DPPH in coffee pulp is:
 |
5 |
Table 4 presents the p-values of various parameters in the quadratic models for Total Phenolic Content and DPPH radical scavenging activity for both SCG and CCP. For SCG, the TPC model showed significant influences from the solid-to-water ratio (B, p = 0.0035), the quadratic term of time (C², p = 0.0022), and the interaction between temperature and time (A²C, p = 0.0400). These findings indicate that optimizing the solid-to-water ratio and accounting for the interactions between variables are crucial for maximizing phenolic extraction. The DPPH model for SCG identified significant factors including the solid-to-water ratio (C, p = 0.0002) and the interactions AC (p = 0.0256) and BC (p = 0.0024). Additionally, the quadratic terms of temperature (A², p = 0.0475) and time (C², p = 0.0496) were significant, underscoring the importance of these parameters in enhancing antioxidant activity. For CCP, the TPC model highlighted the significance of temperature (A, p = 0.0174), the interaction between temperature and solid-to-water ratio (AC, p = 0.0068), and the quadratic terms of temperature (A², p < 0.0001) and solid-to-water ratio (B², p = 0.0055). These results suggest that precise control of temperature and its interactions is vital for optimal phenolic content extraction. The DPPH model for CCP revealed that the solid-to-water ratio (B, p < 0.0001), the interactions AC (p < 0.0001) and BC (p = 0.0004), and the quadratic terms A² (p = 0.0055) and B² (p < 0.0001) were significant. This indicates the critical role of the solid-to-water ratio and its interactions in achieving the highest antioxidant activity.
Table 4.
The p-values of various parameters in the quadratic models for TPC and DPPH.
| Variable | SCG | CCP | ||
|---|---|---|---|---|
| TPC | DPPH | TPC | DPPH | |
| Model | 0.0202 | 0.0059 | 0.0024 | 0.0002 |
| A-Temperature | 0.0660 | 0.1090 | 0.0174 | 0.3412 |
| B-Ratio | 0.0035 | 0.6846 | 0.1247 | 0.0059 |
| C-Time | 0.0556 | 0.0002 | 0.8715 | 0.0575 |
| AB | 0.5884 | 0.5292 | 0.0711 | 0.0304 |
| AC | 0.4687 | 0.0256 | 0.0068 | 0.0016 |
| BC | 0.1738 | 0.0024 | 0.5286 | 0.0004 |
| A2 | 0.1059 | 0.0475 | < 0.0001 | 0.0055 |
| B2 | 0.0809 | 0.0421 | 0.0055 | < 0.0001 |
| C2 | 0.0022 | 0.0496 | 0.0127 | 0.0002 |
| ABC | 0.6707 | 0.7283 | 0.3782 | 0.0041 |
| A2B | 0.0036 | 0.9583 | 0.0398 | 0.0137 |
| A2C | 0.0400 | 0.0005 | 0.6144 | 0.8120 |
| AB2 | 0.0836 | 0.6141 | 0.0076 | 0.0002 |
| Lack of fit | 0.9509 | 0.0003 | 0.0050 | 0.3600 |
Optimization using Design Expert identified the optimal conditions for extraction. For coffee grounds, the optimal conditions were 198 °C, 0.027 g/mL solid-to-water ratio, and 60 min extraction time. For coffee pulp, the optimal conditions were 189 °C, 0.024 g/mL solid-to-water ratio, and 54 min extraction time.
The response surface plots for TPC and DPPH radical scavenging activity from spent coffee ground illustrate the significant effects of extraction time, temperature, and solid-to-water ratio. For TPC (Fig. 2), the quadratic model yielded an R² of 0.9490, indicating a strong fit. Significant variables included the solid-to-water ratio (B), the square of extraction time (C²), the interaction between temperature and solid-to-water ratio (A²B), and the interaction between temperature and extraction time (A²C). The optimal conditions were 198 °C, a solid-to-water ratio of 0.027 g/mL, and 60 min, resulting in a TPC of 217.26 mg GAE/g DW. This shows that higher temperature and solid-to-water ratio, combined with sufficient extraction time, maximize phenolic content extraction19,20.
Fig. 2.
Response surface of TPC from spent coffee ground for each variable.
Similarly, for DPPH radical scavenging activity (Fig. 3), the quadratic model had an R² of 0.9698, suggesting a strong correlation. Significant variables were the solid-to-water ratio (C), interactions between time and temperature (AC), and temperature and solid-to-water ratio (BC). Under optimal conditions, the DPPH value was 23.29 µMol TE/g DW, demonstrating that a well-balanced solid-to-water ratio and maintaining high temperature and adequate time are crucial for maximizing antioxidant activity22,25,26. These results underscore the importance of optimizing these variables to enhance the efficiency of phenolic compound extraction and antioxidant activity from SCG.
Fig. 3.
Response surface of DPPH from spent coffee ground for each variable.
The response surface plots for TPC and DPPH radical scavenging activity from CCP indicate significant influences from extraction time, temperature, and solid-to-water ratio. For TPC (Fig. 4), the quadratic model achieved an R² of 0.9793, demonstrating a very high correlation. Key variables included extraction time (A), interactions between time and solid-to-water ratio (AC), and the squared terms of all three variables (A², B², C²). The optimal conditions identified were 189 °C, a solid-to-water ratio of 0.024 g/mL, and 54 min, resulting in a TPC of 230.13 mg GAE/g DW. This suggests that lower temperatures and shorter extraction times are more effective for extracting phenolics from CCP compared to SCG20,27,28.
Fig. 4.
Response surface of TPC from CCP for each variable.
Similarly, the response surface plot for DPPH radical scavenging activity (Fig. 5) revealed an R² of 0.9927, indicating an excellent fit. Significant factors included extraction time (A), the interaction between time and temperature (AC), and the squared terms of time and solid-to-water ratio (A², C²). The optimal conditions produced a DPPH value of 32.64 µMol TE/g DW. This demonstrates that maintaining a lower temperature and solid-to-water ratio, alongside adequate extraction time, maximizes the antioxidant potential of CCP. These findings underscore the importance of optimizing extraction parameters specific to the material being processed to achieve the highest yield of bioactive compounds.
Fig. 5.
Response surface of DPPH from CCP for each variable.
Figures 6 and 7 shows the optimal extraction conditions of coffee ground and coffee pulp respectively, while Table 4 compares the predicted and experimental results. For coffee grounds, the predicted TPC was 184.56 mg GAE/g DW, while the experimental value was 217.26 mg GAE/g DW, resulting in a 15.05% error. For coffee pulp, the predicted TPC was 199.85 mg GAE/g DW, and the experimental value was 230.13 mg GAE/g DW, with a 13.14% error. The predicted yields for both coffee grounds and coffee pulp were fairly accurate, with errors of 3.05% and 13.22%, respectively. The predicted DPPH values for coffee grounds were more accurate than those for coffee pulp, consistent with the desirability values close to 1.000, indicating the models’ reliability in predicting optimal extraction conditions.
Fig. 6.
Optimized condition of spent coffee ground extraction.
Fig. 7.
Optimized condition of CCP extraction.
This study successfully optimized the extraction conditions for phenolic compounds and antioxidant activity from spent coffee ground and CCP using sub-critical water and high-pressure CO2 extraction techniques. Table 5 showed, the optimal conditions of SCG were 198 °C, a solid-to-water ratio of 0.027 g/mL, and 60 min, resulting in a TPC of 217.26 mg GAE/g DW and a DPPH value of 23.28 µMol TE/g DW. For coffee cherry pulp, the optimal conditions were 189 °C, a solid-to-water ratio of 0.024 g/mL, and 54 min, yielding a TPC of 230.13 mg GAE/g DW and a DPPH value of 32.63 µMol TE/g DW. These findings indicate that coffee cherry pulp has a higher potential for phenolic content and antioxidant activity compared to spent coffee ground.
Table 5.
Comparison between predicted and experimental of TPC and DPPH.
| Extracted | TPC | DPPH | ||||
|---|---|---|---|---|---|---|
| Predicted | Experimental | %error | Predicted | Experimental | %error | |
| SCG | 184.56 | 217.26 ± 10 | 15.05 | 25.87 | 23.28 ± 2.59 | 11.12 |
| CCP | 199.85 | 230.13 ± 15 | 13.14 | 24.47 | 32.63 ± 3.82 | 25.01 |
The higher TPC and DPPH values in coffee cherry pulp can be attributed to its richer phenolic content. Phenolic compounds, known for their antioxidant properties, are more abundant in coffee cherry pulp, leading to higher extraction yields. Sub-critical water combined with high-pressure CO2 effectively enhanced the solubility and extraction of these bioactive compounds. The optimized conditions facilitated maximum recovery of phenolics, crucial for antioxidant activity.
The optimal conditions for spent coffee ground resulted in a TPC of 217.26 mg GAE/g DW and a DPPH value of 23.28 µMol TE/g DW, indicating effective extraction and significant antioxidant activity. The standard deviations of ± 10 mg GAE/g DW for TPC and ± 2.59 µMol TE/g DW for DPPH demonstrate good precision. For coffee cherry pulp, the TPC was 230.13 mg GAE/g DW and the DPPH value was 32.63 µMol TE/g DW, reflecting its higher phenolic content and antioxidant potential. The standard deviations of ± 15 mg GAE/g DW for TPC and ± 3.82 µMol TE/g DW for DPPH indicate reliable results.
The findings of this study align with previous research4,5, which reported high phenolic content and significant antioxidant activity in coffee by-products. The successful application of RSM enabled the precise optimization of extraction parameters, ensuring that maximum bioactive compound recovery and antioxidant activity were achieved. The minor variations observed between predicted and experimental values (15.05% error for spent ground coffee and 13.14% for coffee cherry pulp) fall within an acceptable margin, reinforcing the reliability and predictive power of the applied mathematical models. These discrepancies could be attributed to minor variations in sample composition, equipment calibration, or environmental factors during extraction. Nonetheless, the overall agreement between predicted and actual yields validates the robustness of the optimization approach and highlights the potential for scaling up sub-critical water extraction as an efficient method for valorizing coffee by-products.
The subcritical water and CO2 extraction method demonstrated superior efficiency in extracting phenolic compounds and enhancing antioxidant activity from coffee by-products in Table 5. For spent coffee ground, the optimal conditions were 198 °C, a solid-to-water ratio of 0.027 g/mL, and 60 min, resulting in a TPC of 217.26 ± 10 mg GAE/g DW and a DPPH value of 23.29 ± 2.59 µMol TE/g DW. For coffee cherry pulp, the optimal conditions were 189 °C, a solid-to-water ratio of 0.024 g/mL, and 54 min, yielding a TPC of 230.13 ± 15 mg GAE/g DW and a DPPH value of 32.64 ± 3.82 µMol TE/g DW. These findings indicate that coffee cherry pulp has a higher potential for phenolic content and antioxidant activity compared to spent coffee ground.
The results were compared with other studies to highlight the effectiveness of different extraction methods in Table 6. Murthy & Naidu utilized solvent extraction with 50% aqueous acetone, yielding lower TPC and DPPH values than subcritical water and CO2 extraction16. Duangjai et al. used aqueous extraction and achieved comparable antioxidant activity but lower phenolic content22. Bresciani et al. used ethanol extraction with lower yields of both phenolic content and antioxidant activity17. Tran et al. found that aqueous extraction at optimal conditions yielded high phenolic antioxidants, comparable to 50% aqueous acetone extraction4. Ballesteros et al. focused on encapsulating antioxidants using freeze-drying and spray-drying, achieving high TPC similar to the subcritical water and CO2 method5. Honggao et al. used subcritical water with nitrogen, resulting in significantly lower TPC but higher DPPH activity compared to CO230.
Table 6.
Comparison of extraction methods for TPC and DPPH from coffee byproducts.
| Study | Method | Material | TPC (mg GAE/g DW) | DPPH (µMol TE/g DW) | Key findings |
|---|---|---|---|---|---|
| This study | Subcritical water + CO2 | Spent coffee ground | 217.26 ± 10 | 23.29 ± 2.59 | High phenolic content and antioxidant activity; optimized conditions: 198 °C, 0.027 g/mL, 60 min |
| Coffee cherry pulp | 230.13 ± 15 | 32.64 ± 3.82 | Higher yield in coffee cherry pulp; optimized conditions: 189 °C, 0.024 g/mL, 54 min | ||
| Murthy & Naidu29 | Solvent extraction (acetone) | Coffee pulp | 112.3 | 18.7 | Utilized 50% aqueous acetone; emphasized recovery of functional compounds |
| Duangjai et al.22 | Aqueous extraction | Coffee pulp | 95.2 | 24.6 | Simple water extraction; focused on antimicrobial and antioxidant activities |
| Bresciani et al.17 | Solvent extraction (ethanol) | Coffee silverskin | 156.7 | 21.5 | Ethanol extraction; investigated different coffee byproducts |
| Tran et al.4 | Aqueous extraction | Coffee pulp | 6.68 ± 0.68 | 1.56 ± 0.04 | Optimal conditions: 100 °C, 60 min, 1:100 g/mL, comparable to 50% aqueous acetone extraction |
| Ballesteros et al.5 | Freeze-drying, Spray-drying | Spent coffee ground | 180–220 | – | Encapsulation of antioxidants; focused on process optimization for food application |
| Honggao et al.30 | Subcritical water + N2 | Spent coffee ground | 86.23 | 42.13 | Optimal condition: 179 °C, 36 min, 14.1 g/L and 5 MPa |
The IC₅₀ values in Table 7 indicate the antioxidant activity of coffee byproduct extracts, with lower values signifying higher potency. Subcritical water extraction with CO₂ in this study yielded 160.15 ± 7.03 µg/mL for SCG and 121.31 ± 4.83 µg/mL for CCP, demonstrating moderate to strong antioxidant activity. Compared to aqueous extraction by Duangjai et al. (IC₅₀ = 82 ± 7.8 µg/mL), sub-critical water extraction resulted in slightly weaker antioxidant capacity, though still effective in phenolic compound preservation22.
Table 7.
Comparison of IC₅₀ values of coffee byproducts extracted.
| Study | Method | Material | IC50 (µg/mL) |
|---|---|---|---|
| This study | Subcritical water + CO2 | Spent coffee ground | 160.15 ± 7.03 |
| Coffee cherry pulp | 121.31 ± 4.83 | ||
| Duangjai et al.22 | Aqueous extraction | Coffee pulp | 82 ± 7.8 |
| Kusumocahyo et al.31 | Solvent extraction, Spray-drying |
Coffee pulp (before drying) Coffee pulp (after drying) |
331.93 383.54 |
| Nitchima et al.32 | Hot water extraction | Coffee pulp | 940 |
In contrast, solvent extraction with spray-drying resulted in significantly higher IC₅₀ values (331.93–383.54 µg/mL)31, suggesting compound degradation during drying. Hot water extraction showed the weakest antioxidant activity (IC₅₀ = 940 µg/mL)32, likely due to thermal degradation of phenolics. These findings highlight subcritical water extraction as a promising method, offering a balance between efficiency and compound stability, making it a viable approach for valorizing coffee by-products in food and nutraceutical applications.
Using carbon dioxide instead of nitrogen in subcritical water extraction significantly enhances the extraction efficiency of phenolic compounds. CO2 increases the acidity of the solvent slightly, improving solubility and facilitating the breakdown of cell walls, which releases more phenolic compounds. This results in a more efficient extraction process compared to using N2, which is inert and does not alter the solvent’s pH or pressure. The increased pressure and slight acidity introduced by CO2 make it a more effective choice for extracting phenolic compounds in subcritical water extraction processes33.
Effectiveness and limitations of TLC separation
The results indicate that TLC was effective in fractionating some phenolic compounds (e.g., quinic acid) but had limitations in retaining volatile or highly soluble compounds like caffeine and 5-HMF. In this study, coffee cherry pulp extract was selected for TLC separation due to its higher TPC compared to spent coffee ground extract. Table 8 shows that only CH₂Cl₂:CH₃OH (9.5:0.5) and C₄H₈O₂:CH₃OH (1:1) were effective in separating crude extracts from CCP. This suggests that alternative solvent systems or preparative chromatography techniques might be needed for better compound recovery. Additionally, the differences in compound distribution between SCG and CCP highlight that CCP is a superior source of bioactive compounds, particularly alkaloids and phenolics.
Table 8.
CCP extraction by thin-layer chromatography.
| Experimental | Solvent system | Ratio | Extracted performance |
|---|---|---|---|
| 1 | C6H14:CH3OH | 9:1 | No |
| 2 | CHCl3:CH3OH | 8:2 | No |
| 3 | CH2Cl2:CH3OH | 9.5:0.5 | Yes |
| 4 | C4H8O2:CH2Cl2 | 9:1 | No |
| 5 | C4H8O2:C6H14 | 1:1 | No |
| 6 | CHCl3:C4H8O2 | 7:3 | No |
| 7 | C4H8O2:CH3OH | 1:1 | Yes |
Chemical composition of coffee waste extracts
The chemical composition of coffee waste extracts, including SCG, CCP, and the fraction separated using Thin-Layer Chromatography by CH₂Cl₂:CH₃OH as ratio 9.5:0.5 (FCCP) shows in Fig. 8, was analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS/MS). The identified compounds, their retention times, ionization modes, and relative abundance (area under the curve) are summarized in Table 9.
Fig. 8.
The fraction separated using CH₂Cl₂:CH₃OH (9.5:0.5).
Table 9.
Chemical composition of coffee waste extracts by LC-MS/MS.
| No. | Retention time | Chemical | m/z | mode | Area | ||
|---|---|---|---|---|---|---|---|
| SCG | CCP | FCCP | |||||
| 1 | 1.23 | Quinic acid | 191.0562 | − | 1.52E + 07 | 1.02E + 07 | n.d. |
| 2 | 1.57 | Maleic acid | 115.0037 | − | n.d. | 9.27E + 05 | n.d. |
| 3 | 2.18 | Theobromine | 181.0719 | + | n.d. | 1.57E + 06 | n.d. |
| 4 | 2.19 | 5-Hydroxymethylfurfural | 127.0389 | + | 1.25E + 07 | n.d. | 1.12E + 05 |
| 5 | 2.31 | Cantharidin | 197.0808 | + | n.d. | 2.572e + 05 | 1.60E + 05 |
| 6 | 2.75 | Theophylline | 179.0579 | ± | n.d. | 1.34E + 06 | 4.00E + 04 |
| 7 | 3.53 | Protocatechuic aldehyde | 137.0246 | ± | n.d. | 8.04E + 05 | 1.12E + 04 |
| 8 | 4.54 | Caffeine | 195.0873 | + | n.d. | n.d. | 1.36E + 08 |
| 9 | 6.58 | 3-N-butyl-4,5-dihydrophthalide | 193.0858 | + | 4.79E + 06 | 1.25E + 06 | n.d. |
The distribution of bioactive compounds varied across different extracts. Quinic acid, a precursor of chlorogenic acid known for its antioxidant properties, was detected in CCP and FCCP but was absent in SCG34. This suggests that coffee cherry pulp retains a higher content of bioactive phenolics compared to SCG. Furthermore, the presence of quinic acid in FCCP indicates that the TLC separation system (CH₂Cl₂:CH₃OH, 9.5:0.5) was effective in isolating this compound. Similarly, 5-Hydroxymethylfurfural (5-HMF), a product of the Maillard reaction, was abundant in SCG and CCP but significantly reduced in FCCP35. This could indicate that TLC fractionation resulted in either degradation or ineffective retention of this compound under the selected solvent system.
Caffeine was detected exclusively in CCP with a notably high intensity (1.36E + 08), confirming that coffee cherry pulp is a rich source of this stimulant36. However, FCCP did not retain caffeine, implying that the TLC solvent system used was not optimal for preserving or recovering this compound. Theobromine and Theophylline, which are structurally similar to caffeine and possess cardiovascular and bronchodilatory effects, were also present in CCP but absent in SCG and FCCP37. This suggests that coffee cherry pulp may serve as a valuable source of these bioactive alkaloids, potentially contributing to its pharmacological applications.
Cantharidin, a bioactive compound with potential antimicrobial and anticancer properties, was detected in CCP and FCCP, suggesting successful retention through TLC separation38. In contrast, 3-N-butyl-4,5-dihydrophthalide, a phthalide derivative with potential biological activity, was primarily found in SCG and FCCP, but in lower concentrations compared to caffeine and methylxanthines39. These findings highlight that CCP is a superior source of bioactive compounds, particularly alkaloids and phenolics, which aligns with previous studies suggesting that coffee pulp, often considered waste, holds potential for nutraceutical and pharmaceutical applications40.
Structural confirmation using NMR analysis
NMR analysis further validated the LC-MS/MS findings. The proton and carbon spectra confirmed the presence of caffeine as a major constituent in FCCP. The spectral peaks closely matched standard caffeine spectra, reinforcing the reliability of the LC-MS/MS identification. The DEPT spectra provided additional confirmation by differentiating between CH, CH₂, and CH₃ groups, which is critical for structural elucidation. Figures 9 and 10 illustrate the carbon NMR spectrum and the standard caffeine spectrum, respectively. These results are consistent with previous studies that have used NMR for identifying methylxanthine compounds in plant extracts41–44.
Fig. 9.
Analytical of NMR—carbon.
Fig. 10.
Standard curve of caffeine by 13 C-NMR analysis.
Conclusion
The extraction of Arabica coffee grounds and pulp using subcritical water combined with carbon dioxide revealed that the phenolic content in coffee pulp was higher than in coffee grounds, as well as showing a higher DPPH value. The yields of the crude extracts were similar, with values of 38.11% for coffee grounds and 39.23% for coffee pulp. The optimal extraction conditions for coffee grounds were found to be an extraction temperature of 198 °C, a solid-to-water ratio of 0.027 g/ml, and an extraction time of 60 min, resulting in a TPC of 217.26 mg GAE/g DW and a DPPH value of 23.28 µMol TE/g DW. For coffee pulp, the optimal conditions were an extraction temperature of 189 °C, a solid-to-water ratio of 0.024 g/ml, and an extraction time of 54 min, yielding a TPC of 230.13 mg GAE/g DW and a DPPH value of 32.63 µMol TE/g DW.
The LC-MS/MS analysis revealed that CCP contains a higher diversity of bioactive compounds compared to SCG, including phenolic acids, methylxanthines, and secondary metabolites. However, the TLC separation system used in this study exhibited limitations in retaining caffeine and 5-HMF, necessitating further refinement for effective compound isolation. The use of DEPT-NMR further strengthened the structural identification of key bioactive compounds. These findings underscore the potential of coffee cherry pulp as a valuable bioresource for functional food and pharmaceutical applications.
Future research should focus on scaling up the subcritical water and CO2 extraction process to evaluate industrial feasibility and energy efficiency. Comparative studies with other agricultural byproducts can help determine the method’s broader applicability. Further characterization of extracted bioactive compounds will provide insights into their health benefits. Exploring the use of these extracts in food and pharmaceutical products, assessing their environmental impact through life cycle analysis, and investigating synergistic effects with other natural antioxidants can lead to novel, more effective formulations.
Acknowledgements
This research was supported by the National Science, Research and Innovation Fund, Thailand Science Research and Innovation (TSRI), through Rajamangala University of Technology Thanyaburi (FRB66E0710H.1) (Grant No.: FRB660012/0168).
Author contributions
V.K. , N.P., K.K., and R.S. conceptualized the study, developed the methodology, and performed the experiments. V.K. also contributed to formal analysis. K.K. assisted with chemical analysis and data validation. T.R. contributed to the experimental design and statistical analysis. N.P. supervised the project, conducted formal analysis, acquired funding, and was responsible for project administration. N.P. and S.S. contributed to experimental planning, conducted formal analysis, provided technical expert. V.K. and R.S. wrote the original draft. N.P., T.R. and S.S. reviewed and edited the manuscript. All authors reviewed the final manuscript and approved its submission.
Data availability
The datasets used during the current study are available from the corresponding author on reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used during the current study are available from the corresponding author on reasonable request.