2.1. Identification and Quantification of Major Bioactive Compounds in WCC, WCCE1, and WCCE2
Our previous reports and studies from other laboratories have shown that coffee cherries are a rich source of polyphenolic compounds [
13,
14,
15,
16]. Various extraction and analytical methods have been used to identify several phenolic compounds, including phenolic acids, flavonoids, alkaloids, and organic acids. In the current study, we quantified the major components of the whole coffee cherry (WCC) and its two commercially available extracts WCCE1 and WCCE2 using HPLC. The UV-HPLC profiles of WCC and WCCE1 are shown in
Figure 1A,B. LC-MS/MS analysis of the major chromatographic peaks identified a total of eight chlorogenic acids. Peaks at retention times of 11.4–11.5, 14.2, and 15.1 min were identified as 3-CQA, 5-CQA, and 4-CQA, respectively. Each CQA isoform exhibited unique fragmentation patterns (
Figure 2A–C). Dissociation of 3-CQA produced three major fragment ions at
m/
z 135, 179, and 191, representing the molecular ions [
caffeic acid-H-H
2O]
−, [
caffeic acid-H]
−;, and [
quinic acid-H]
−, respectively. Dissociation of 5-CQA generated a major fragment ion at
m/
z 191, corresponding to [
quinic acid-H]
−;. 4-CQA generated more fragment ions upon dissociation—the major fragments include
m/
z 135, 173, 179, and 191, representing the molecular ions [
caffeic acid-H-H
2O]
−, [
quinic acid-H-H
2O]
−, [
caffeic acid-H]
−, and [
quinic acid-H]
−, respectively. Following a similar approach, we identified two FQA isoforms: 5-FQA (
m/
z 367) and 4-FQA (
m/
z 367) corresponding to the UV-HPLC peaks at RT 18.2/18.3, and 18.4 min, respectively; and three diCQA isoforms: 3,4-diCQA (
m/
z 515), 3,5-diCQA (
m/
z 515) and 3,4-diCQA (
m/
z 515) corresponding to the UV-HPLC peaks at RT 21.9/22.0, 22.5, and 23.1 min, respectively. Moreover, the major peak at RT 14.5 min in the WCCE2 UV-HPLC chromatogram (
Figure 1C) was identified as caffeine by positive ion mode MS/MS analysis. Dissociation of caffeine produced a fragment ion at
m/
z 138 (
Figure 2D), corresponding to the neutral loss of methyl isocyanate, [caffeine + H–O=C=NCH
3]
+. The major MS/MS peak at
m/
z 195 corresponded to the undissociated caffeine molecular ion [caffeine + H]
+.
Quantitative data for the major identified compounds in WCC, WCCE1, and WCCE2 (expressed as %
w/
w) are shown in
Table 1. The total amounts of CGAs in WCC, WCCE1, and WCCE2 were 6.76 ± 1.34, 46.46 ± 0.93, and 6.1 ± 0.01 %
w/
w, respectively. 5-CQA was the major CGA found in all samples. The caffeine level in WCCE2 was 73.60 ± 0.65 %
w/
w The caffeine levels were very low in WCC and WCCE1 (
Table 1). The amount of trigonelline was found to be much higher in WCCE1 than in WCC and WCCE2 (
Table 1). This result suggests that the extraction and purification processes involved in the preparation of WCCE1 indirectly favor trigonelline, whereas the preparation of the caffeine-rich sample, WCCE2, disfavors trigonelline.
2.3. Inhibition of α-Glucosidase Activity, α-Amylase, and AChE Activities
Alpha glucosidase, located at the brush-border surface membrane of intestinal cells, is the key enzyme that catalyzes the final step in the hydrolysis of carbohydrates and releases glucose from disaccharides and oligosaccharides. Controlling the activities of this enzyme plays a crucial role in the regulation of blood glucose. These activities were monitored by in vitro assays using different combinations of enzyme sources and substrates.
In the present study, we tested the potential inhibitory effects of α-glucosidase using pNPG as substrates with and without WCC, WCCE1, and WCCE2. WCCE1 inhibited α-glucosidase activity in a dose-dependent manner (
p < 0.05) (
Figure 4A). WCCE2 did not show any inhibitory against α-glucosidase effects in our assays within the tested concentration range. WCC showed an inhibitory effect at higher concentrations compared to WCC1 (
Table 4). The minimal 50% inhibition of glucosidase for WCCE1 was 2.42 mg/mL, which is 12 times lower than that of WCC (
Table 4). As mentioned before, WCCE1 is enriched with chlorogenic acid and the major phenolic compound in this extract is 5-CQA; we were interested in determining the inhibitory effect of 5-CQA and found an IC
50 value of 2.1 mg/mL. The comparable IC
50 of CGAs and WCCE1 (with 48% of CGAs) indicated that other chlorogenic acids also play an additive role in the inhibition of α-glucosidase.
Chlorogenic acids and their derivatives isolated from coffee and other natural products have been investigated in many studies because of their potential role in the inhibition of α-glucosidase activity. Xu et al. [
28] isolated a polyphenol extract from
IIex kudingcha that contained 74.3% chlorogenic acid derivatives, comprising of 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA, and reported the IC
50 as 0.42 mg/mL from the inhibitory effects on the activities of α-glucosidase. The purified fractions of the CGA derivatives had a range 0.16–0.39 mg/mL, where 5-CGA had an IC
50 of 0.30 mg/mL. The chlorogenic acid-rich extract obtained from
Echinacae purpurea flower extract exhibited a strong inhibitory effect, where the IC
50 of chlorogenic acid was reported be 0.90 mg/mL for glucosidase [
29]. Alongi et al. [
30] reported the IC
50 as 2.41–3.24 mg/mL in coffee brews prepared from green coffee beans,
C. canephora var. robusta. In another report, they tested α-glucosidase inhibition activity from the brews prepared from roasted and unroasted coffee and found an IC
50 of 0.50–0.56 mg/mL [
31]. The IC
50 for acarbose was 0.09 mg/mL. CGAs and melanoidins were described as the major inhibitors of this enzyme activity, in support of the antidiabetic potential of coffee consumption [
31]. The α-glucosidase inhibitory capacity resulted from the composition of the coffee samples with different degrees of roasting. CGAs represented 77% of phenolic compounds in the sample, followed by di-chlorogenic acids (di-CGA, 22%), which were (both) responsible for glucosidase inhibition activity [
31]. In another study, Duangjai et al. [
32] reported that 5 mg/mL coffee cherry extract inhibited up to 28.85% of the glucosidase activity. The active compounds—namely ferulic acid, maleic acid, citric acid, caffeic acid, and chlorogenic acid—inhibited glucosidase activity by up to 46.57% at a concentration of 5 mM (1.78 mg/mL). Chlorogenic acid and its synthetic acetyl derivative showed significant inhibition of glucosidase activity at 2.8 mM (1 mg/mL) [
33]. Herawati et al. [
34] investigated the α-glucosidase inhibitory activity in
C. robusta coffee beans and reported that the coffee brew extract showed a reduction in glucosidase activity of 69.41% at a concentration of 12.5 g/100 mL concentration [
34,
35]. The bioactive compounds responsible for antioxidant and anti-α-glucosidase activities were proposed to be phenolic acids.
Alpha amylase (1,4-α-d-glucan-glucanohydrolase, EC 3.2.1.1) is another primary digestive enzyme that catalyzes the hydrolysis of α-1-4-glycosidic linkages of starch and converts starch to its oligosaccharides. Inhibition of α-amylase can be considered a strategy for the treatment of disorders of carbohydrate metabolism by controlling their rate of release. Using human salivary and pancreatic α-amylase and various substrates such as starch and p-nitrophenol derivatives, inhibitors have been screened. We tested the potential anti-amylase activity of coffee cherries and their extracts using human salivary α-amylase. WCCE1 and WCC effectively inhibited amylase activity in a dose-dependent manner (p < 0.05). WCCE1 inhibited amylase activity with an IC50 of 1.7 mg/mL and WCC had an almost eight times higher IC50 (similar α-glucosidase activity) in the extracts. WCCE2 was not an effective inhibitor of amylase activity in our assay.
Few studies have demonstrated that natural CGA-containing extracts strongly inhibit α-amylase activity. Narita and Inouye isolated nine different isomers of CGAs from green coffee beans and investigated their effects on the inhibition of amylase [
36]. The IC
50 values ranged from 0.02 mM to 26.5 mM, where di-caffeoylquinic acid was found to be a stronger inhibitor. The IC
50 values for 5, 3, 4-CQA were 0.08 mM, 0.23, and 0.12 mM, respectively. In another study, Narita et al. [
37] reported that 5-CQA, caffeic acid, and quinic acid inhibited porcine pancreas α-amylase with IC
50 values of 0.08 mM, 0.40 mM, and 26.5 mM, respectively [
36]. The higher inhibitory effect of di-CQA than CQA on α-amylase activity suggests that inhibitory properties increase with an increasing number of caffeic acid (CA) sub-structures [
36,
37]. Moreover, the inhibitory activities of CA derivatives were always higher than those of ferulic acid derivatives, suggesting that the two neighboring hydroxyl groups on the catechol ring were effective in the inhibition. Zheng et al. [
38] examined the inhibitory effect of chlorogenic acid against porcine pancreatic amylase and potato starch as substrates and obtained an IC
50 of 0.498 mg/mL and acarbose of 2.28 mg/mL. Different results were observed with inhibition assays because of the physicochemical properties of the substrates, which could affect the affinity to the active site of the enzymes [
39]. It is now well established that the use of different enzyme sources for inhibition assays and different substrates can yield very different results [
40,
41]. The chlorogenic acid-rich extract obtained from
Echinaca purpurea flower extract exhibited a strong inhibitory effect, with an IC
50 of 1.71 mg/mL for amylase [
29]. Coffee brew extract at 5 mg/mL inhibited up to 49.32% of α-amylase activity and its constituents; caffeic acid and chlorogenic acid inhibited 93.35% and 50%, respectively [
32]. Nyambe-Silavwe et al. [
40] reported that chlorogenic acid and phenolic acids are weak inhibitors of human salivary amylase, as they showed 20% inhibition at 5 mM.
AChE catalyzes the hydrolysis of the neurotransmitter acetylcholine into choline and acetate. The rate of hydrolysis of AChE in E. electricus with DTNB produces thiocholine, which was used to screen the potential anticholinesterase activity of WCC, WCCE1, and WCCE2.
In our assay, WCCE2 inhibited AChE activity in a dose-dependent manner in the tested range (0–125 µg/mL) (
p < 0.05). WCCE1 and WCC did not inhibit AChE activity at the concentrations used in the assay.
Figure 4C shows the inhibitory pattern of WCCE2, and the IC
50 was determined to be 90 µg/mL (
Table 3). Physostigmine is a strong commercial inhibitor of AChE that showed nearly complete inhibition at 5 µg/mL (data not shown). We quantified the level of caffeine and found it to be 73%, which demonstrates that the inhibition of AChE occurred due to the presence of caffeine in WCCE2. Synthetic pure caffeine supported this hypothesis, which showed an IC
50 of 65 µg/mL in our assay. In their study, Pohanka et al. [
42] reported caffeine to be a moderate inhibitor of AChE activity, with an IC
50 of 87 µM. Caffeine inhibits AChE in a noncompetitive manner. The differences in the results from various studies have been found to be due to the sources of AChE and the assay used [
43]. Small structural alterations in AChE from different organisms can be responsible for the difference in results [
43]. In one study [
44], chlorogenic acid showed a moderate inhibitory effect on AChE activity. Although CGAs are a major component of WCCE1, the concentrations used in our assay may not be sufficient to inhibit the activity of AChE.
In the present study, we found a positive correlation between chlorogenic acid content and antioxidant, α-amylase inhibition, and α-glucosidase inhibition, whereas caffeine had a strong positive correlation with AChE inhibition.