Pharmacokinetics of gallic acid and protocatechuic acid in humans after dosing with Relinqing (RLQ) and the potential for RLQ-perpetrated drug–drug interactions on organic anion transporter (OAT) 1/3

Figures & data

Figure 1. Chemical structures of (A) gallic acid, (B) protocatechuic acid, (C) vanillic acid, (D) quercitrin, (E) quercetin and (F) kaempferol.

Figure 2. Typical chromatograms of (1) gallic acid, (2) protocatechuic acid, (3) quercitrin, (4) quercetin and (5) kaempferol obtained from an analysis of standard solution (A) and RLQ solution (B).

Figure 3. Multiple reaction monitoring chromatograms of (A) blank plasma; (B) blank plasma spiked with a standard mixture of gallic acid (GA, 1) and protocatechuic acid (PCA, 2) at the LLOQ level with internal standard (IS, 3); (C) human plasma sample taken 2 h following the oral administration of RLQ to human subjects.

Table 1. Accuracy, precision, recovery and matrix effect for analysis of gallic acid (GA) and protocatechuic acid (PCA) in quality control samples (n = 6).

Analyte	Spiked concentration (μg/L)	Intra-day		Inter-day		Recovery (%, means ± SD)	Matrix effect (%, means ± SD)
		Accuracy (%)	Precision (%)	Accuracy (%)	Precision (%)
GA	1.00	−5.13	7.04	−3.14	9.36	–	–
	3.00	−3.62	1.53	−3.98	2.62	99.22 ± 3.62	109.27 ± 5.77
	60.0	−2.98	2.44	−3.38	3.13	92.49 ± 2.01	103.11 ± 0.51
	300.0	−7.41	0.95	−6.87	1.93	91.08 ± 2.37	102.36 ± 0.47
PCA	0.50	−4.29	9.06	−8.54	9.98	–	–
	1.50	−6.93	2.46	−7.01	4.01	99.79 ± 3.34	111.65 ± 8.18
	30.0	−5.56	2.07	−5.03	2.63	92.49 ± 2.45	101.39 ± 0.51
	150.0	−5.54	1.42	−6.39	1.74	92.26 ± 2.21	101.40 ± 0.74

Table 2. Stability of gallic acid (GA) and protocatechuic acid (PCA) in quality control samples (n = 3).

Analyte	Spiked concentration (μg/L)	Post-preparative		Bench-top		Long-term		Three freeze-thaw cycles
		Stability (%)	RSD (%)	Stability (%)	RSD (%)	Stability (%)	RSD (%)	Stability (%)	RSD (%)
GA	3.00	88.48	2.15	98.11	6.99	99.70	2.30	85.89	2.76
	300.0	94.37	5.00	94.81	8.21	93.68	2.33	93.17	3.83
PCA	1.50	90.60	3.85	97.69	2.96	99.04	2.15	87.22	4.42
	150.0	96.93	5.21	95.44	7.48	93.67	2.35	94.22	3.79

Table 3. Demographic characteristics of healthy subjects in this study (n = 12).

Characteristic	Mean ± SD (range)
Male/female	6/6
Age (years)	27.0 ± 1.94 (24–30)
Height (m)	1.65 ± 0.08 (1.57–1.81)
Weight (kg)	58.6 ± 6.27 (50.2–70.1)
BMI (kg/m^2)	21.5 ± 1.37 (19.5–23.3)

Figure 4. Plasma concentration-time and semilogarithmic scale profiles of gallic acid (A and B) and protocatechuic acid (C and D) after oral administration of single (▲) and multiple (▼) doses of Relinqing granules (RLQ, 8 g) in human subjects (means ± SD, n = 12).

Table 4. Pharmacokinetic parameters of gallic acid and protocatechuic acid after oral administration of single and multiple doses of Relinqing to human subjects (means ± SD, n = 6).

Variable	Gallic acid		Protocatechuic acid
	Single dose	Multiple dose	Single dose	Multiple dose
Cmax (μg/L)	139 ± 49.4	118 ± 39.5	6.06 ± 2.28	5.45 ± 1.58
AUC0–t (h·μg/L)	251 ± 74.4	205 ± 62.8	3.20 ± 1.17	2.81 ± 0.81
AUC0–∞ (h·μg/L)	256 ± 74.9	209 ± 63.5	3.64 ± 1.35	3.09 ± 0.90
tmax (h)^a	0.75 (0.5, 1)	0.75 (0.75, 1)	0.33 (0.17, 0.5)	0.33 (0.17, 0.5)
ke (h^−1)	0.53 ± 0.11	0.54 ± 0.12	2.42 ± 0.87	2.84 ± 0.72
t1/2 (h)	1.37 ± 0.37	1.34 ± 0.32	0.33 ± 0.14	0.26 ± 0.08
Vz/F (L)	241 ± 82.7	292 ± 113	158 ± 101	139 ± 46.1
CL/F (L/h)	124 ± 42.0	152 ± 51.6	337 ± 135	379 ± 119
Fu (%)	8.55 ± 4.62	10.7 ± 7.87	4.24 ± 3.72	5.41 ± 3.40

^aMedian (range).

AUC_0–t: area under the plasma concentration-time curve from time zero to the last measurable concentration; AUC_0–∞: area under the plasma concentration–time curve from time 0 to infinity; C_max: maximum plasma concentration after administration; CL/F: total body clearance; t_1/2: terminal elimination half-life; t_max: time of C_max; F_u: fraction of administered dose excreted in the urine; V_z/F: oral volume of distribution

No statistically significant differences were observed between single and multiple doses of RLQ in human using a two-tailed unpaired t- test (p > 0.05).

Figure 5. Urinary concentration-time profiles of gallic acid (GA, A) and protocatechuic acid (PCA, B), and urinary recovery-time curves of GA (C) and PCA (D) after oral administration of single (▲) and multiple (▼) doses of Relinqing granules (8 g) to human subjects, as well as the cumulative urinary recoveries of GA (E) and PCA (F) in urine (means ± SD, n = 12).

Figure 6. Gender difference on the main pharmacokinetic parameters C_max (A) and AUC_0–t (B) of gallic acid, and C_max (C) and AUC_0–t (D) of protocatechuic acid after single and multiple doses of Relinqing granules (8 g) in human subjects (means ± SD, n = 6).

Figure 7. Inhibitory effects of gallic acid (GA) and protocatechuic acid (PCA) on the uptake of ¹⁴C-amino hippuric acid (¹⁴C-PAH, A), ³H-estrone sulphate-ammonium salt (³H-ES, B),¹⁴C-tetraethylammonium bromide (¹⁴C-TEA, C) and ³H-ES (D) into MDCK-hOAT1, S2-hOAT3, S2-hOCT2 and HEK293-hOATP1B1 cells, respectively.

Figure 8. Inhibitory effects of gallic acid (GA) and protocatechuic acid (PCA) on the efflux of rhodamine 123 (A) and lucifer yellow (B) across MDCK-hMDR1 and MDCK-hBCRP cells, respectively.