Plant extract

The Triphala capsule formulation (water extract) was prepared by the Pharmaceutical Chemistry and Natural Products Research Unit, Faculty of Pharmacy, Mahasarakham University, Thailand.  The plant materials were dried at 60 ^ºC for 48 h in the hot-air oven after thoroughly washed with tap water and remove seeds of each fruit.  All dried materials were powdered using the electric blender and passed through sieve No.14. The powdered plant material (15 g) was extracted by boiling in 1,000 ml of distilled water for 1 h, filtered through Whatman no. 1 filter paper, and freeze-dried. The Microbial contamination was controlled using gamma-irradiation. The gallic acid content of Triphala formulation was 133.80 ± 2.94 mg/g (mean+SD) of extract.

Animals

The study protocol was prepared and ethical approval of the study conduct was obtained from the Ethics Committee for Animal Research, Thammasat University, Thailand (No. 019/2559).  Males and females of Wistar rats, aged between 5-7 weeks, weighing 150 to 180 g, were used in the study (National Laboratory Animal Center of Thailand). The study was carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals (US National Research Council, 2011).  The animals were acclimatized one week before study initiation. The animals were housed in stainless-steel cages at the Laboratory Animal Center, Thammasat University under controlled light of 12 h dark/light cycle at 22±2^ºC and relative humidity 30-70%.  All were fed with the stock diet and water ad libitum. During drug administration period, all animals were observed daily for the changes in body weight, behaviors, and mortality.

Pharmacokinetic study

Animals (20 rats) were randomly allocated to two groups of 5 males and 5 females each. Triphala formulation extract was dissolved in distilled water and fed to each rat by intragastric gavage at a daily oral dose of 1,000 or 5,000 mg/kg body weight for 28 days. Blood samples (150-200 l each) were collected from the tail vein of each rat at 0, 0.5, 2, 4, 8, 12 and 24 h, respectively. Plasma samples were separated through centrifugation at 1,000 x g for 10 min and stored at -80ºC until analysis.

Determination of gallic acid concentrations in plasma

The bioactive compound, gallic acid, was used as the marker to investigate the pharmacokinetics of Triphala formulation in rats. Plasma sample (100 l) was spiked with the internal standard, apigenin (10  l of 30 ng/ml), and the resultant mixture was extracted with a mixture of 2% formic acid in acetonitrile (500  l). After centrifugation (13,800 xg, 10 min), the clear organic layer was evaporated to dryness under nitrogen flow. The dried residue was reconstituted with the mobile phase (100 l) and an aliquot (5 l) was injected onto the liquid chromatographic-mass spectrometry (LC-MS/MS) in the negative-ion mode (ESI). The system consisted of Agilent 1260 Separation Module (Agilent Technologies, CA, USA) solvent and sample delivery, an AB SCIEX QTRAP® 5500 mass-spectrometer (AB Sciex, Foster City, CA, USA) in the Scheduled Multiple Reactions Monitoring (MRM) mode as follows: -4,500V ion spray, 50 ion source gas 1 (GS1) and 50 gas 2 (GS2), 30 curtain gas (CUR), 5 collision gas (CAD), and 500 ^ºC source temperature. Optimal mass parameters, that is, declustering potential (DP), entrance potential (EP), collision energy (CE), and cell exit potential (CXP) were 50, 6, 17 and 11 psi for gallic acid and 67, 6, 42, and 8 psi for apigenin, respectively. The full-scan mass spectra of each analyze were set to monitor the transitions of the precursors to the product ions were m/z 168.9 "125.1 for gallic acid and 269"117 for apigenin. The separation was performed on a ZORBAX SB-C18 (4.6 x 50 mm, 3.5 µm particle size) (Agilent Technologies, CA, USA) with the mobile phase [a mixture of water containing 0.1 % formic acid and 100 % acetonitrile with gradient elution 0 min (60:40%), 5 min (20:80%), and 6.5 min (60:40%)] running at a flow-rate of 0.3 ml/min.

Assay validation

Calibration curve:  Calibration curves of gallic acid were prepared by replicate analysis as described above, of seven plasma samples (100 l each) spiked with gallic acid (50-2,000 ng/ml) and an internal standard, apigenin (30 ng/ml). Samples were analyzed as described above (CDER, 2018).

Precision:  Within-day repeatability and day-to-day variation (reproducibility) were determined by replicate analysis of six sets of samples spiked with gallic acid (400, 800, and 1,600 ng/ml). Coefficient of variation (%CV) was determined from the ratio of standard deviation (SD) and mean (CDER, 2018).

Accuracy:   The accuracy of the method was determined by replicate analysis of six sets of samples spiked with gallic acid (400, 800, and 1,600 ng/ml) and comparing the difference between spiked value and that actually found (theoretical value) (CDER, 2018).

Recovery: The analytical recovery of sample preparation procedures was estimated by comparing the peak heights obtained from the sample with that measured with post-extraction sample spiked with equivalent amounts of gallic acid (400, 800 and 1,600 ng/ml) (CDER, 2018).

Selectivity: The selectivity of the method was verified by checking for interference by commonly used drugs, that is paracetamol and dimenhydrinate (CDER, 2018).

Limit of quantification: The limit of quantification (LOQ) of the method was determined from the lowest concentration of gallic acid in spiked plasma that produced a peak height ten times of the baseline noise at - 0.2 A (CDER, 2018).

Stability: The stability of gallic acid in plasma samples was determined by storing spiked samples (400, 800 and 1,600 ng/ml, triplicate each) at -80 ºC freezer (Sanyo, Japan) for three months. Concentrations were measured on days 1, 15, and months 1 and 3 (CDER, 2018).

Quality control: Quality control (QC) samples for gallic acid were made up in plasma samples using a stock solution (400, 800 and 1,600 ng/ml, triplicate each). The samples were aliquoted and stored at -80 ºC for use with each analytical run (CDER, 2018).

Pharmacokinetic analysis

Plasma concentration-time profile of gallic acid was plotted and the pharmacokinetic parameters were determined using a model-independent analysis (Gibaldi, 2011). The maximum concentration (C_max) and time to maximum concentration (t_max) were obtained from the plasma concentration-time data. The area under the curve from zero time to twenty-four hours of dosing (AUC_0-24h) was calculated using linear trapezoidal rule (ascending data points) and log-trapezoidal rule (descending data points). The AUC from the zero time to infinity (AUC_0-∞) was determined from the sum of the AUC_0-24h and the extrapolated AUC. The terminal elimination half-life (t_1/2z) was calculated from log-linear regression of at least four of the last plasma concentration-time data. The apparent total body clearance (CL/F) and apparent volume of distribution (V_z/F) were calculated as CL/F=dose/AUC_0-∞ and V_z/F=[CL/F]/ λ_z.

Statistical analysis

Data are presented as median (interquartile range) values. Comparison of quantitative variables between the two groups was performed using Mann-Whitney U test for data not conforming to the normal distribution (SPSS version 13). Optimal sample size for the pharmacokinetic study in rats was determined based on the primary outcome pharmacokinetic parameter AUC0_0-  (Aarons and Ogungbenro, 2010).  Statistical significance level was at α < 0.05.

Assay validation

Precision: The intra-day assay (%CV) of gallic acid in plasma varied between 8.13% and 9.38 %. The inter-day assay (%CV) varied between 5.70% and 6.94% (Table 1). Results suggested acceptable precision of the assay method.

Accuracy: Good accuracy was observed from both the intra-day or inter-day assays. For intra-day assay validation, deviation of mean value from theoretical value (%DMV) varied between 2.50% and 5.46 %.  The inter-day assay (%MDV) varied between -7.17% and 5.92% (Table1).

Recovery: The mean recoveries of gallic acid at 400, 800 and 1,600 ng/ml were 105.9%, 92.8% and 95.7%, respectively (Table 1).  Results suggested high recovery of gallic acid from the spiked plasma samples.

Limit of quantification: The LOQ of gallic acid in plasma was accepted as 2.5 ng/ml using 100 l blood.

Stability:  The analysis of gallic in plasma samples was found to be stable following storage at -80 ^?C for a minimum of three months without significant decomposition of the drug.

Pharmacokinetics of gallic acid

Triphala formulation was well tolerated in all rats receiving both dose levels of Triphala formulation with no abnormal signs and symptoms and body weight changes.  Gallic acid in both groups of Triphala formulation was rapidly absorbed after oral dose administration with a median t_max of 0.5-1 h that received a dose of 5,000 mg/kg body weight and 1,000 mg of Triphala formulation, respectively (Figure 2). It disappeared after that from systemic circulation within 12 h. The median C_max increased from 194.7 ng/ml to 866.2 ng/ml and the AUC_0-24h and AUC_0-  increased from 383.2 ng*h/ml to 1,910.6 ng*h/ml and 421.8 ng*h/ml to 1,917.9 ng*h/ml, respectively. It was found that almost 5-times in the group receiving 1,000 mg/kg body weight compared with 5,000 mg/kg body weight, while other pharmacokinetic parameters (CL/F, V_z/F, and t_1/2z) were comparable (Table 2).

The pharmacokinetics of gallic acid has previously been reported in rats following oral administration of gallic acid or other herbal extracts, but not Triphala formulation (Ferruzzi et al., 2009; Konishi et al., 2004; Ma et al., 2015; Song et al., 2010). Characterization of the pharmacokinetic profile of gallic acid in animals following the clinically used formulation (Triphala) is essential to provide preliminary information for human study. In the current study, the pharmacokinetics of gallic acid was investigated in rats following 1,000 and 5,000 mg/kg body weight Triphala formulation, which corresponds to the minimally and maximally safe dose levels of Triphala formulation reported in rats (Phetkate et al., 2019). Gallic acid from both dose levels of Triphala formulation was rapidly absorbed, reaching its maximum plasma concentration within 1 h. The compound was rapidly cleared from blood circulation within 12 h of administration. The pharmacokinetics of gallic acid was linear with about 5-fold increase in C_max and systemic exposure AUC_0-  when the dose was increased from 1,000 to 5,000 mg/kg body weight. This pharmacokinetic profile was in general, in agreement with those reported following the administration of gallic acid and other herbal extracts (Ferruzzi et al., 2009; Konishi et al., 2004; Ma et al., 2015; Song et al., 2010). Gallic acid at the dose of 100 μmol/kg body weight (17 mg/kg) was shown to be rapidly absorbed with C_max of 0.71 μmol/l (120.78 ng/ml) and AUC of 42.6 μmol*min/l) (120.78 ng*h/ml) (Konishi et al., 2004). In another study, after oral Polygonum capitatum extract containing 4-24 mg/kg gallic acid, C_max of gallic acid was 246-806 ng/ml. AUC_0-t and AUC_0- were 695-1,832 and 896-3,403 ng/ml*h, respectively (Ma et al., 2015). Following the administration of 50, 100, and 150 mg grape seed polyphenolic extract, C_max and AUC_0-8h were 239-323 ng/ml and 512-707 ng*h/ml (Ferruzzi et al., 2009). The variation of the reported C_max and AUC of gallic acid is explained by the variability of sources of gallic acid and species of plants under investigation.

The pharmacokinetics of gallic acid obtained from the present study in rats provides preliminary information for designing proper pharmacokinetic studies in humans for further dose optimization of appropriate dosage regimens of Triphala formulation for treatment of various diseases or health conditions.

The authors have not declared any conflicts of interests.

The study was funded by Thammasat University (no. 2018010) and Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma.

The authors gratefully acknowledge Assistant Professor Prasob-orn Rinthong, Mahasarakham University for her kindness in supporting the aqueous extract of Triphala and would also like to thank the staff of Drug Discovery and Development Center and Laboratory Animal Center, Thammasat University for their technical assistance during the study.