Effect of HPE parameter on extraction efficiency
Figure 1 shows the CGA and caffeine extraction efficiencies at 400 MPa and 25 °C for 2.5 min using three solvents (methanol, ethanol, and water). The extraction efficiency of water reached 16.6%, which was significantly higher than that of methanol and ethanol (9.2% and 7.5%, respectively). The caffeine and CGA concentrations in the water extract were also higher than those in other solvents. Similar results were obtained in the study of Budryn et al. (2009), where a higher CGA content was found in the water extract than in the ethanol extract, although the caffeine content in the ethanol extract was higher than that of the water extract. The extractability of a solvent mainly depends on the following: the solubility of the compound in the solvent system, the mass-transfer kinetics of the product, and the intensity of the solute/matrix interaction. The dielectric constant and polarity of water are obviously higher than those of alcohol. As there are no limitations on the use of water in future applications within the food and pharmaceutical industries, water extraction is considered a preferred extraction method. In addition, the method does not produce toxic waste, unlike organic solvent extraction methods (Upadhyay et al., 2005). Therefore, we selected water as the solvent for the HHPE efficiency experiment to extract CGA from green coffee beans. Three variables, namely, pressure, temperature, and duration, were employed in the experiment, and nine conditions were, therefore, compared and analyzed (Table 1). The extraction was performed at 200 MPa, 400 MPa, and 600 MPa at 25 °C for 2.5 min. The results showed that higher CGA and caffeine extraction yields were obtained from HPE at 400 MPa than at 200 MPa, and this implied that a higher pressure was beneficial for improving the CGA extraction rate. However, when the pressure was increased to 600 MPa, there was no significant increase in the extraction rate compared to that at 400 MPa, as the cellular structure of the coffee beans was already fully destroyed and the CGA components had been extracted. Therefore, further experiments were conducted at 400 MPa. The use of different starting temperatures for extraction also affected the HPE efficiency. As the temperature was increased from 5 °C to 25 °C, the amounts of CGA and caffeine extracted increased from 4.32% and 3.27% to 7.48% and 5.66%, respectively. However, increasing the temperature to 50 °C provided no further improvements in the extraction rates of the crude extracts, CGA, or caffeine. Butiuk et al. (2021) observed the same trend in the extraction of yerba mate; a significant increase was observed in the CGA extraction rate when the extraction temperature was increased from 30 °C to 65 °C, but the rate reduced when the temperature was increased to 100 °C. Phenolic compounds are thermal sensitive, and certain important bioactive substances are likely to be degraded at a high temperature; therefore, the extraction temperature needs to be maintained under a certain limit (Silva et al., 2007). A comparison of the extraction durations used showed that an increase in the extraction duration increased the extraction efficiencies of CGA and caffeine, and the extraction rate was significantly higher at 2.5 min than at 1 min. However, no significant difference was observed with a 5-min extraction duration. These results suggest that the solute in the sample matrix was dissolved using high pressure, and the two target compounds in the extraction took approximately 2.5 min to reach equilibrium. Thus, it was not possible for any more CGA or caffeine to be dissolved, and the extended duration was not productive (Ahmad et al., 2021). Traditional HR extraction at 50 °C for 5 min provided 3.54% and 2.92% of CGA and caffeine, respectively, but twice the amount of CGA and caffeine was obtained using HPE extraction at 400 MPa at 25 °C within 2.5 min. Huang et al. (2013) found that HPE performed better than traditional extraction techniques (such as heat or solvent extraction) at lower temperatures, and thus, the extraction duration was reduced, the extraction efficiency was improved, thermal degradation was reduced, and fewer bioactive components were lost.
Antioxidant properties of extracts
As shown in Table 2, the TPC concentration range was 3.34–4.57 mg gallic acid equivalents/g in the coffee extracts obtained with different HHPE conditions. All three operating conditions had an impact on the amount of TPC extracted. When the initial extraction temperature was 25 °C, there was a significant increase in the amount of TPC obtained with an increase in extraction pressure and duration. However, increasing the temperature to 50 °C resulted in a slight decrease in TPC, and this was probably related to the degradation of other phenolic components at the higher temperature (Upadhyay et al., 2005). In this study, the TFC showed the same trends as those of TPC. The optimal extraction pressure of TFC was 400 MPa, and the amount of TFC increased significantly with the extraction duration and reached 2.52 mg quercetin equivalents/g at 6 min. However, temperature did not appear to have a significant impact on the amount of TFC extracted. Nevertheless, both the extraction rates of TPC and TFC obtained through HHPE were significantly higher than the 3.02 mg gallic acid equivalents/g and 1.24 mg quercetin equivalents/g obtained by heat reflux extraction, respectively. The free radical scavenging activity in the extract was measured through the percentage inhibition of DPPH free radicals (Table 2). The free radical scavenging activity of the heat reflux extract was 65.9%, while that of the HHPE extract ranged from 64.2% to 81.5%. The extract obtained at 25 °C and 600 MPa for 2.5 min had the highest scavenging activity, and the extract obtained at 50 °C and 400 MPa had the lowest scavenging capacity (with no significant difference from that of the HR extracts). Caffeine in coffee reduces oxidative stress and protects the antioxidant system in hypoxia-induced lung epithelial cells. In human skin fibroblasts, caffeine is an inhibitor of lipid peroxidation products induced by hydrogen peroxide, and it reduces lipid peroxidation and reactive oxygen species (Tiwari et al., 2014). The free radical-scavenging activity is related to the bioactive compounds, including not only CGA and its derivatives but also caffeine, theophylline, theobromine, cafestol, kahweol, tocopherols, and trigonelline. These compounds have strong antioxidant potentials, and they exhibit various health-promoting effects. For example, they have anticancer properties and are known to inhibit hyperglycemia, hyperinsulinemia, and hyperlipidemia (Jeszka‑Skowron et al., 2016). Hu et al. (2015) used HPE, HR extraction, ultrasonic extraction, and Soxhlet extraction to extract CGA from the flower buds of Lonicera japonica. The shortest extraction duration was obtained with HPE, and this process provided the highest CGA concentration. In addition, there were no significant differences between the DPPH-scavenging ability of the extract produced, and those of the other extracts, which indicated the advantages of the high extraction rate, short extraction duration, and the low energy consumption of HPE (Khan et al., 2019).
Inhibition of α-glucosidase and α-amylase
Alpha-amylase and α-glucosidase are key enzymes affecting the digestion and absorption of starch and other carbohydrates. Alpha-amylase hydrolyzes starch into dextrins and oligosaccharides by hydrolyzing the α-D-1,4-glycosidic bond, while α-glucosidase hydrolyzes dextrins and oligosaccharides into glucose, which is then transported to blood vessels; it increases postprandial blood sugar, which can result in obesity or diabetes. The inhibition of α-amylase and α-glucosidase activities reduces starch digestibility and delays starch degradation and glucose absorption (Papoutsis et al., 2019). In this study, the HPE extracts obtained using different extraction conditions showed varying α-glucosidase and α-amylase inhibition levels due to the different CGA and caffeine concentrations obtained, as shown in Fig. 2. In extracts obtained at temperatures below 25 °C for more than 2.5 min at 400 MPa, the inhibition rates of α-glucosidase and α-amylase reached a minimum of 27% and 26%, respectively, and these results were significantly higher than those of the heat reflux extracts (22% and 10%, respectively). Oboh et al. (2015) previously demonstrated the inhibitory effect of CGA on α-amylase (IC50 of 3.68 μg/mL) and α-glucosidase (4.98 μg/mL), and Zheng et al. (2020) reported the inhibitory effect of CGA on α-amylase (IC50 of 0.498 mg/mL). However, Wang et al. (2022) found that the positive effects from inhibiting phenolic acids and flavonoids on α-glucosidase and α-amylase were due to the specific functional patterns of the hydroxyl group, which formed hydrogen bonds with specific amino acids at the active site of enzymes. In this study, the coffee extract obtained at 25 °C and 600 MPa for 2.5 min had the highest inhibitory effects on α-glucosidase and α-amylase, and this result was likely related to the high TPC and TPF contents. CGA may have a low binding affinity to α-glucosidase and α-amylase, which could be the reason for the low α-glucosidase and α-amylase inhibition rates in some of the extracts that had a high CGA concentration. Therefore, it is more likely that the inhibitory effect of CGA on α-amylase and α-glucosidase is associated with non-competitive inhibition and competitive inhibition, respectively; this has been attributed to the structural differences between α-amylase and α-glucosidase (Nyambe-Silavwe et al, 2018). In this respect, Alongi et al. (2019) found that adding milk to coffee followed by homogenization at a high pressure of 150 MPa increased the bioaccessibility of CGA from nearly 25% to > 50% because the milk fat promoted the formation of CGA microencapsules, which reduced the susceptibility to degradation during digestion and also enhanced the inhibition of α-glucosidase.
Antibacterial activity of extracts
The results of the zone of inhibition test showed that coffee extracts obtained under high-pressure inhibited food pathogens (Table 3). The MICs of coffee extracts obtained at different HHPE conditions against the gram-positive bacteria S. aureus and L. innocua were 27.5–37.3 mg/mL and 30.5–43.3 mg/mL, respectively, while the MICs of the HR extract against S. aureus and L. innocua were 39.2 mg/mL and 44.5 mg/mL, respectively, which were significantly higher than those of all the HPE extracts. However, the MICs of the HPE extracts against gram-negative bacteria were significantly higher than those against gram-positive bacteria. The MICs for E. coli and S. enterica ranged between 35.5 mg/mL and 45.6 mg/mL and between 41.9 mg/mL and 49.7 mg/mL, respectively, although these were lower than MICs of the HR extracts (45.6 mg/mL and 51.7 mg/mL, respectively). This result suggested that the extraction temperature had no effect on antibacterial activity of the coffee extract and that the MIC was reduced when the extraction duration and pressure were increased; this implication agrees with those of our previous reports. Duangjai et al. (2016) found that CGA and caffeine were the main components of the water extract of coffee pulp, and these showed antibacterial activity against both gram-positive bacteria (S. aureus and S. epidermidis) and gram-negative bacteria (Pseudomonas aeruginosa and E. coli). In particular, the MIC of S. epidermidis was the lowest at only 4.69 mg/mL. Tasew et al. (2020) obtained different antibacterial activity results from green coffee bean extracts, with an MIC against S. typhimurium of 15.62 mg/mL, which was lower than that of E. coli (31.25 mg/mL) and S. aureus (31.25 mg/mL). Due to the different levels of antibacterial activity of phenolic acids, CGA, and caffeine in coffee and the differences in the extraction conditions, the mechanisms used to destroy the biological structure of bacteria were also different, which resulted in the coffee extracts having different inhibition efficiencies against each type of bacteria. In general, the antibacterial activity is mediated through the inactivation of the cytoplasmic membrane and the inhibition of intracellular and extracellular enzymes. However, it is difficult for hydrophobic compounds to absorb the outer membrane of gram-negative bacteria, which is composed of phospholipids; this results in a poorer antibacterial activity against gram-negative bacteria (Runti et al., 2015). The data obtained in this study show that the high-pressure extracts with higher total phenol, CGA, and caffeine contents provided better antibacterial activity against gram-positive bacteria.
Microstructure changes of green coffee bean
To understand the effect of HPE on the microstructure of coffee beans, a scanning electron microscope was used to observe the cellular structure of unextracted coffee beans and that of coffee beans extracted by both hot reflux extraction and HHPE. Figure 3 (a), (b), and (c) show the morphology of the unpressurized coffee beans observed via SEM (magnification 300–1000 times). The cells were relatively closely connected in an orderly manner, with obvious intracellular organelle spacing. Figure 3 (d), (e), and (f) show coffee beans extracted via hot reflux extraction. The intercellular compartments were partially damaged and showed evidence of irregular and sudden shrinkage. However, the patterns of most of the cell compartments were similar to those of the control group. Figure 3 (g), (h), and (i) show coffee beans obtained via HPE. Although the basic cellular structure was visually distinguishable, the intracellular pores were significantly compressed following high pressure treatment, and the volume was noticeably reduced. The cell pores were irregular with significant intracellular tissue ruptures and were squeezed into irregular or loose porous floccules. According to the Le Chatelier’s principle, the volume of the system tended to decrease during pressurization, as the extraction solvent entered the cells and made contact with bioactive components. These components were then dissolved in the solvent and extracted out of the cell. During HPE, the pressurized cells also exhibited increased solvent permeability. The higher the water pressure, the greater was the amount of solvent that entered the cell pores and the greater were the amounts of biological compounds dissolved in the solvent; this suggests that there was an increase in the dissolved functional components of cells, which improved the extraction efficiency. An equilibrium concentration of the solvent between the inside and outside of the cell was established when the pressure is maintained. When the pressure was suddenly reduced, the cell wall was disrupted to release an extract that contained high concentrations of solutes (Xi et al., 2017). In HPE studies of broccoli seeds (Xing et al., 2019) and Lonicera japonica (Hu et al., 2015), the extraction efficiencies showed increasing trends because of the damage conferred to the sample cell structure, owing to the pressure applied, as observed via SEM.