Three major compounds showing significant antioxidative, α-glucosidase inhibition, and antiglycation activities in Robusta coffee brew

Figures & data

Figure 1. Flow chart of the separation and isolation of the antioxidant, α-glucosidase inhibitor, and glycation inhibitor from Robusta coffee brew. The separation and isolation were conducted in triplicate (n =3). The concentration of the coffee brew, coffee fractions, and single compounds were equivalent with 5 g ground coffee/100 mL coffee brew (5 g eq./100 mL). P.1.1-P.1.8*: P.1.1, P.1.2, P.1.4, P.1.5, P.1.7, and P.1.8 were the peak groups separated from the water fraction. P.2.1-P.2.12**: P.2.1, P.2.2, P.2.3, P.2.4, P.2.6, P.2.8, P.2.9, P.2.10, P.2.11, and P.2.12 were the peak groups separated from the 20% MeOH fraction

Figure 2. Different activities of coffee brew fractions separated in the ODS Sep-Pak as determined by the DPPH assay (a), H-ORAC assay (b), and anti-α-glucosidase assay (c). The concentration of coffee fractions was 5 g eq./100 mL. The values were expressed as the mean ± SD (n= 3). Different superscripts indicate significant differences in the Duncan test (p< .05)

Table 1. Antiglycation activities of the Robusta coffee brew fractions separated in the ODS Sep-Pak

Fractions	IC50 (mg mL^−1)
	24 h	48 h	72 h
Aminoguanidine (positive control)	2.43 0.38^a	3.87 ± 0.55^a	4.22 ± 0.50^a
Mixed fractions (water + 20% MeOH + 40% MeOH + MeOH + EtOH)	5.59 ± 0.45^b	5.48 ± 0.28^b	5.93 ± 0.28^b
Mixed fractions without water fraction	6.33 ± 0.20^cd	6.40 ± 0.03^c	6.94 ± 0.09^c
Mixed fractions without 20% MeOH fraction	8.19 ± 0.23^e	8.23 ± 0.44^d	9.15 ± 0.74^d
Mixed fractions without 40% MeOH fraction	6.48 ± 0.24^d	6.60 ± 0.20^c	7.12 ± 0.44^c
Mixed fractions without MeOH fraction	6.12 ± 0.41^bc	5.61 ± 0.39^b	5.96 ± 0.44^b
Mixed fractions without EtOH fraction	5.96 ± 0.48^bc	5.49 ± 0.37^b	5.76 ± 0.58^b

Values with different superscripts in the same column are significantly different in the Duncan test (p< 0.05). Average of three data samples ± SD.

Figure 3. Different activities of the subfractions separated from the water, 20% methanol, and 40% methanol fractions in the HPLC-5C18-MS-II column: DPPH (a), H-ORAC (b), and anti-α-glucosidase (c). The concentrations of coffee subfractions were 5 g eq./100 mL. The values were expressed as the mean ± SD (n =3). Different superscripts indicate significant differences in the Duncan test (p< .05)

Figure 4. HPLC chromatograms of water (a) and 20% MeOH (b) fractions separated in the HPLC-ODS 4 column

Figure 5. Different activities of peaks and compounds of the water and 20% MeOH fractions separated in the HPLC-ODS 4 column: DPPH (a), H-ORAC (b) and anti-α-glucosidase (c). The concentrations of the peaks or compounds were 5 g eq./100 mL. All values were expressed as the mean ± SD (n = 3). Different superscripts indicate significant differences in the Duncan test (p < .05)

Table 2. LC-MS molecular ion; ¹³C NMR and ¹H NMR (500 MHz, D₂O) chemical shifts; and quantitative data of 5-CQA, 4-CQA, and 3-CQA isolated from coffee brew

Analysis	5-CQA (compound 1.6)		4-CQA (compound 2.5)		3-CQA (compound 1.3)
LC-MS
Molecular ion	[M + H]^+ (m/z 355)		[M-H]^− (m/z 353)		[M-H]^− (m/z 353)
^13C NMR and ^1H NMR
Carbon atom number	^13C (δ/ppm)	^1H(δ/ppm)	^13C (δ/ppm)	^1H(δ/ppm)	^13C (δ/ppm)	^1H(δ/ppm)
Quinic moiety
1	75.65 (C)		77.91 (C)		76.33 (C)
2	37.10 (CH2)	2.10 br t (2H)	37.86 (CH2)	1.92 t (2H)	35.55 (CH2)	2.16 d (2H)
3	72.00 (CH)	4.12 d(H)	68.31 (CH)	4.21 br m (H)	72.80 (CH)	5.28 t (H)
4	69.81 (CH)	3.77 t(H)	78.25 (CH)	4.75 m(H)	73.60 (CH)	3.64 t (H)
5	71.05 (CH)	5.15 br m (H)	65.00 (CH)	4.18 t(H)	66.85 (CH)	4.08 t (H)
6	36.74 (CH2)	1.96 br d(2H)	41.02 (CH2)	2.06 br d (2H)	40.48 (CH2)	1.84 t (2H)
7	178.39 (C)		179.98 (C)		180.65 (C)
Caffeoyl moiety
1′	127.20 (C)		127.78 (C)		127.16 (C)
2′	115.36 (CH)	6.24 d (H)	114.87 (CH)	7.07 d (H)	114.99 (CH)	7.09 s (H)
3′	144.47 (C)		145.04 (C)		144.33 (C)
4′	147.31 (C)		147.90 (C)		147.10 (C)
5′	116.45 (CH)	6.81 d(H)	116.69 (CH)	6.79 m(H)	116.33 (CH)	6.32 d (H)
6′	122.93 (CH)	6.94 dd (H)	123.18 (CH)	7.00 dd (H)	122.74 (CH)	7.09 dd (H)
7′	146.39 (CH)	7.46 d (H)	147.20 (CH)	7.54 s (H)	146.20 (CH)	7.55 d (H)
8′	114.78 (CH)	7.04 s(H)	115.60 (CH)	6.30 d(H)	115.21 (CH)	6.30 d (H)
9′	169.03 (C)		169.98 (C)		169.19 (C)
Weighing
Yield from 4 mL solution (mg)*	2.9 ± 0.1		<1		1.0 ± 0.0
HPLC
Concentration (mg mL^−1)**	0.7 ± 0.0		0.2 ± 0.0		0.3 ± 0.0

D₂O: Deuterium oxide. 5-CQA: 5-O-Caffeoylquinic acid, 4-CQA: 4-O-Caffeoylquinic acid, 3-CQA: 3-O-Caffeoylquinic acid. s: singlet, d: duplet, dd: duplet-duplet, t: triplet, br: broad, m: multiplet. *Yields were mean values ± SD (n= 3). **The concentrations were quantified as mean values in 5-CQA equivalent ± SD (n= 3).

Figure 6. The chemical structure of 5-O-caffeoylquinic acid (a), 4-O-caffeoylquinic acid (b), and 3-O-caffeoylquinic acid (c)