Abstract

The gallic acid and chebulinic acid contents in Phyllanthus emblica Linn., Terminalia bellirica Roxb., Terminalia chebula Retz. and Triphala products from Chae Son district, Lampang, Thailand at 2017 and 2018 seasons were studied. The HPLC separation was validated for analysis the hexane, ethyl acetate and methanol herb extracts. The gallic acid contents in the methanol extracts of P. emblica and T. chebula collected in 2018 were amounted to be double those of the same plants collected in 2017. The chebulinic content in the methanol extract of P. emblica collected in 2018 was almost double that of the same plant collected in 2017. The gallic acid contents of two Triphala products (with sugar and sugar-free) were not found to be different while the chebulinic contents of the two Triphala products for 2018 were 50% higher than those of 2017. The gallic acid and chebulinic acid contents variation form year to year should be taken into consideration for the production of Triphala products.

1. Introduction

Triphala is a plant product which has a long history of usage in India. Triphala is composed of P. emblica, T. bellerica and T. chebula usually in the 1:1:1 composition ¹. It is believed that regular consumption of Triphala leads to good health and longevity. There have been reports on the biological activities of Triphala including anti-cancer, anti-inflammatory, anti-diabetic, muscle-relaxing, anti-microbial, anti-depression, immune-enhancing and ulcer-healing activities ².

The main constituents of Triphala products were found to be gallic acid, ellagic acid, chebulinic acid, ascorbic acid, syringic acid, tannic acid and other phenolic compounds ^{1, 3}. In Thailand Triphala products enjoy increasing popularity and there are a number of formulations (with sugar and sugar-free) with different proportions of the three plants. There have been reports that four formulations of Triphala are effective in enhancing the immune system. It has been suggested that formulations with equal proportion of the three plants are recommended for daily consumption while those with higher proportions of T. bellerica are suitable for relieving allergic symptoms. There were reports on the uric acid-lowering, anti-inflammability, blood pressure-reducing and blood lipid-lowering activities. For Triphala products to be effective, the exact composition of the three plants is of vital importance ^{4, 5, 6}. To date, there have been no studies on the seasonal variation of the active constituents of the three plants, leading to uncertain composition of the Triphala products.

Therefore, it was decided to investigate the seasonal variation of the active constituents of the three plants (collected in 2017 and 2018 seasons) by studying the gallic acid and chebulinic acid contents of the hexane, ethyl acetate and methanol extracts. The gallic acid and chebulinic acid (Figure 1) contents in the methanol extract of Triphala products (2:1:1; with sugar and sugar-free) for 2017 and 2018 were also studied to establish the correlation of the contents of these two acids in the plants and in the Triphala products in the same period. It was anticipated that the study will be useful for the future production of Triphala products with desired compositions of the two acids.

2. Materials and Methods

P. emblica (BKF 113645), T. bellirica (BKF 188656) and T. chebula (BKF 170476) were collected from Chae Son National Park (1845′12″N, 9924′32″E.) , Lampang, Thailand in January, 2017 and 2018 by Mr. Narong Nuntasaen. The plants were identified and deposited in the Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of National Resources and Environment, Bangkok, Thailand. Gallic acid monohydrate and chebulinic acid were purchased from Sigma-Aldrich Co.

The dried fruits (collected in 2017 and 2018) (2 kg) of P. emblica, T. bellirica and T. chebula (Figure 2) ⁷ were ground into powder and then separately successively extracted with hexane, ethyl acetate and methanol (5×7 L for each solvent). Removal of the solvents from each extract under reduced pressure gave the crude extracts of P. emblica, T. bellirica and T. chebula. Dry Triphala (2017 and 2018) in 2:1:1 (P. emblica, T. bellirica and T. chebula) proportions was extracted only with methanol to give the crude methanol extract.

The weights of all extracts were 20.0-21.0 ± 0.9 mg. They were separately extracted with methanol (1 mL) under vortex mixture for 3 minutes, and filtered through nylon membrane. The filtrates were stored under 25 °C and the gallic acid and chebulinic acid contents were determined using HPLC.

The two main types of Triphala drinks available in the market are sugar and without sugar which are totally different in the ratio of ingredients. The type of Triphala drink on 2017 and 2018 years are shown in Table 1.

HPLC analysis was carried out on a HITACHI Chromaster equipped with a 5110 pump, a 5310 column oven, a 5430 diode array detector and a 5210 auto sampler. Separation was undertaken on a HITACHI LaChrom C₁₈ column (250 4.6 mm, 5 µm) at 25 °C. The mobile phase flow rate was 1 mL/min. The injection volume was 10 μL. The quantitation wavelength was set at 270 nm. Identification of gallic acid and chebulinic acid was accomplished by comparing the retention times and absorption spectra of relevant peaks to those of standard compounds. The separation condition for HPLC analysis was modified according to previous papers ⁸. The mobile phase consisted of 1% acetic acid in water (A), pH 2.65 and methanol (B). The program for gradient elution started at 90% solvent A and 10% solvent B, linearly increased to 50% solvent A and 50% solvent B in 25 min, followed by washing for 25 min (Table 2).

Quantification estimation of gallic acid and chebulinic acid were carried out based on the calibration curves of standard gallic acid and chebulinic acid. Solutions of 15.625, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL of gallic acid and chebulinic acid were prepared. The standard curves were plotted between the peak areas and concentrations. Peak areas of gallic acid monohydrate were compared to the standard curve and calculated to give their contents. The solutions were filtered through a nylon membrane (0.45 mm) syringe filter and analyses were carried out in triplicate. Peak area of standard gallic acid and chibulinic acid are shown in Table 3 and Table 4.

3. Results and Discussion

The total weights of the relevant extracts are shown in the Table 5.

From Table 5, it could be concluded that the weights of the extracts increased with increasing polarity of the solvents. This is consistent with previous reports of the occurrence of ascorbic acid, gallic acid, amalic acid, chebulic acid, chebulinic acid and quercitin in P.emblica ⁹ T. bellirica fruit was found to contain ellargic acid, gallic acid, chebulaginic acid, bellericanin and chebulinic acid ¹⁰ while T. chebula fruit was found to contain gallic acid, tannin, chebulic acid and chebulinic acid ¹¹. According to solvent extraction, researches on Triphala indicated that the methanol extracted reviewed the highest bioactivities and the higher amount of phenolic compounds including gallic and chebulinic acid than the others. Then, only Triphala (2:1:1) was extracted by methanol. ^{12, 13, 14, 15}

Gallic acid and chebulinic acid standards were used as references to construct the standard curves for the calculation of sensitivity, stability, precision, repeatability, and recovery rate; the method was validated in terms of all these parameters. The parameters related to the standard curves, such as regression equation, correlation coefficient, and linear range are shown in Table 6. The calibration curves of gallic acid and chebulinic acid showed good linearity (R² ≥ 0.9998) within the test range, and their content could be accurately determined using the regression equation. (Figure 3 and Figure 4)

Retention time values of gallic acid and chebulinic acid were found to be 7.56 and 22.8 minutes respectively, consistent with previous reports ⁸ (Figure 5). HPLC chromatogram of P. emblica, T. bellirica, T. chebula, Triphala(2:1:1), Triphala herbal drink (sugar-free) and Triphala herbal drink (with sugar) in 2017 and 2018 were shown in Figure 5– Figure 15 and 16-27. (Chromatogram which appear after the 26^th minute would not be considered, were column washing period.)

The contents of gallic acid, chebulinic acid in different extracts of Triphala herb and Triphala herbal drinks are listed in Table 7, Table 8, Table 9 and Table 10.

The crude methanol extracts of the three plants possessed the highest contents of gallic acid, followed by the ethyl acetate extracts while gallic acid was not found in the hexane extracts. The contents of gallic acid in the three herbs ranged from 0.15 g/100 g in the ethyl acetate extracts of T. bellirica to 0.46 g/100 g in the methanol extracts of P. emblica for 2017. For 2018, the gallic acid contents in the methanol extracts ranged from 1.06 g/100 g for P. emblica to 0.12 g/100 g for T. bellirica. In the case of Triphala (2: 1: 1) products, the gallic acid content for 2018 was found to be 28% higher than that for 2017.

The results of the determination of gallic acid content in both formulas in 2017 and 2018 showed that the amount of gallic acid ranged from 3.56-4.26 g /100 mL. The contents of chebulinic acid in different extracts and herb samples are listed in Table 9.

In the methanol extracts, the contents of chebulinic acid in the three herbs ranged from 0.06 g/100 g for P. emblica to 5.83 g/100 g for T. bellirica. For 2017, the contents of chebulinic acid in other extracts for the 2 years did not show a clear trend. The content of chebulinic acid in both Triphala formulas are shown in the Table 10.

The chebulinic acid contents in both formulas for 2017 and 2018 ranged from 0.10-0.60 g /100 mL. In P. emblica, the contents of gallic acid were 0.17-1.06% and those of chebulinic acid were 0.01-1.71%. In T. bellirica. The gallic acid contents were 0.12-0.57% and those of chebulinic acid were 0.34-7.71%. In T. chebula, the gallic acid contents were 0.13-0.70% and those of chebulinic acid were 0.01-4.37%. In Triphala (2:1:1) the gallic acid contents were 1.47-2.05% and those of chebulinic acid were 2.08-7.21%. In sugar-free Triphala herbal drinks, the gallic acid contents were 3.61-3.64% and those of chebulinic acid were 0.10-0.26%. In Triphala with sugar herbal drinks, the gallic acid contents were 3.56-4.26% and those of chebulinic acid were 0.58-0.60%.

4. Conclusion

Determination of the gallic acid and chebulinic acid contents of P. emblica, T. bellirica and T. bellirica as well as Triphala products for 2017 and 2018 revealed that there were seasonal variations of these constituents which may have a significant impact on the quality of these Triphala products. In order to offer a consistent and desirable quality in these Triphala products, determination of the gallic acid and chebulinic acid contents prior to production is an important necessity.

References