Simultaneous Determination of Eight Phytoconstituents in Triphala churna by HPLC–DAD.

 

Anil D. Mahajan and Nandini R. Pai*

Department of Chemistry, D. G. Ruparel College, Mahim, Mumbai - 400016, India.

ABSTRACT:

Triphala is one of the most popular herbal preparations in the world used to treat variety of diseases. A simple, precise and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous estimation of gallic acid, methyl gallate, ethyl gallate, chebulagic acid, tetra-O-galloyl-β-D-glucose, ellagic acid, chebulinic acid and penta-O-galloyl-β-D-glucose in triphala churna. The chromatographic separation was achieved on Inertsil ODS-3 column (100mm×4. 6mm, 3µm). A mixture of 0.02% triethyl amine aqueous pH 3.0 with ortho-phosphoric acid (A) and acetonitrile (B) was used as mobile phase in gradient mode at a flow rate of 1. 0 ml/min and detector wavelength at 272 nm. The validation of the proposed method was carried out for linearity, accuracy, precision, limit of detection, limit of quantification and robustness. The results from intra and inter day validation showed the method was efficient and reproducible. The advantage of the developed method is simple extraction procedure involved and short run time (27 min.). Hence this method can be used for routine quality control analysis of Triphala churna.

 

KEYWORDS: Triphala, RP-HPLC, Method validation, Chebulagic acid, Chebulinic acid, Gallic acid.

 

 

INTRODUCTION:

India has moved forward in advocating global usefulness of Ayurveda contemporary scenario of health care through global networks. As a result many foreign countries have began looking to India for understanding Ayurveda and incorporating it through education, research and practice to meet overwhelming desire of consumer to access complementary and alternative medicine. The increased use of ayurvedic medicines is mainly due to increased side effects of chemical drugs, lack of treatment for several chronic diseases, high cost of new drugs and emergence of multi-drug resistant bacteria and parasites1. Before the availability of synthetic drug, ayurvedic or herbal drugs were mainstay of treatment but the quantity and quality of the safety and efficacy data on herbal medicine are far from sufficient to meet the criteria needed to support their use worldwide2.

 

Triphala, one of the most popular herbal preparations in the world is commonly available as a finely sieved powder. According to Ayurvedic formulary of India, Triphala is prepared by mixing equal parts Amalaki (Emblica officinalis), Vibhitaka (Terminalia belerica) and Haritaki (Terminalia chebula)3-4. Triphala is used in ayurvedic medicine in the treatment of a variety of conditions and also forms part of many other formulations. Triphala is one of the powerful antioxidant and detoxifying agents5.


Triphala churna has been found very effective in treating acidity, constipation, flatulence, digestive disturbances and liver problems. It is reported to be cardio-tonic, reduce cholesterol level and control blood pressure. Triphala churana is also reported as antidiabetic, antimutagenic and radioprotective6-9.

 

Various methods have been reported for analysis of the Triphala churna such as HPLC or HPTLC methods using gallic acid, ellagic acid, chebulagic and chebulinic acid as marker10-12. Recently, a validated HPLC method has been published which quantifies gallic acid, tannic acid, syringic acid and epicatechin and ascorbic acid in Triphala churna13. These methods do not cover maximum phytoconstituents present which are responsible for therapeutic activities. Hence they cannot satisfy the requirements for studies of mechanism of action and quality control of Triphala churna. Moreover, due to multiple compounds that might be associated with the therapeutic functions, a single or a few marker compounds could not be responsible for the overall pharmacological activities of the drug. A comprehensive quality evaluation method based on analysis of all bioactive compounds is urgently needed in order to accurately reflect the quality of this herbal drug. In this study a reversed phase HPLC method for analysis with eight marker compounds as gallic acid, methyl gallate, ethyl gallate, chebulagic acid, tetra-O-galloyl-β-D-glucose, ellagic acid, chebulinic acid and penta-O-galloyl-β-D-glucose has been developed and validated. The above listed constituents are believed to be the active components in triphala churna, and could be considered as the ‘marker compounds’ for the chemical evaluation or standardization of the same.

 

MATERIALS AND METHODS:

Chemicals and materials:

The chemicals used were analytical or HPLC-grade. HPLC-grade acetonitrile ortho-phosphoric acid (AR grade) and triethyl amine (AR grade) were purchased from Merck specialty India Pvt. Ltd. Ultra pure water, generated by use of a Milli-Q System (Millipore), was used for sample preparation and preparation of mobile phases for HPLC analysis. Dried fruits of Terminalia chebula Retizus (combrataceae), Terminalia bellirica (Gaertn. ) Roxb. (combrataceae) and Emblica officinalis Gaertn. (euphorbiaceae) were procured from local market in Mumbai, India and authenticated at Agharkar Research Institute Pune, India, with a voucher specimen (F-138), (F-139), (F-140) which were deposited in the herbarium respectively. Standard compounds gallic acid (1) and ellagic acid (6) were purchased from Sigma Aldrich. Other standard compounds such as methyl gallate (2), ethyl gallate (3), chebulagic acid (4), tetra-O-Galloyl-β-D-glucose (5), chebulinic acid (7) and penta-O-Galloyl-β-D-glucose (8) were isolated from the dried fruit of the Terminalia chebula Retizus (combrataceae). (Fig-1).

 

 

Sample preparation:

The samples were powdered, and passed through a 60-mesh sieve. The accurately weighed powder (0.2 g) was transferred in 50ml volumetric flask contains 30ml extraction solvent (methanol–water (70:30, v/v) and sonicated for 20 min at 27±3°C in ultra sonicator water bath and diluted up to mark. The solution was filtered through a 0.45-µm membrane prior to injection into the HPLC system.

 

Chromatography:

Waters alliance liquid chromatography system consisting Waters 2695 separation module (quaternary pump) and a photodiode array detector(Waters 2998) coupled with waters Empower Pro software was used. Separations were carried out with Inertsil ODS-3 reversed-phase column (100mm×4. 6mm, 3µm) (GL Sciences, Japan). The mobile phase was consisted of 0.02% triethyl amine aqueous pH 3.0 with ortho-phosphoric acid (A) and acetonitrile (B). The gradient program was as follows: 0–15 min, linear gradient 10–33% B; 15–20 min, linear 33–33% B; 20–21 min linear gradient 33–10% B; 22–27 min linear gradient 10–10% B. The flow rate was 1. 0 ml/min and aliquots of 10µl were injected. The UV detection wavelength was set at 272 nm. Absorption spectra of compounds were recorded between 200 and 400 nm. The compounds were identified by comparing their retention times and UV spectra with those of the markers.

 

RESULTS AND DISCUSSION:

Extraction procedure:

Various extraction methods, solvents and times were evaluated to obtain the best extraction efficiency. The results revealed that ultrasonic bath extraction was better than other extraction methods, so the further experiments were carried out with ultrasonic bath extraction. Various solvents including water, methanol–water (50:50 v/v; 60:40 v/v; 70:30 v/v; 80:20 v/v) and methanol were screened. Methanol–water (70:30 v/v) exhibited complete extraction of all the major constituents. No second extraction step was found necessary.

                

1. Gallic acid                                            2. Methyl gallate

 

3. Ethyl gallate                                 4. Chebulagic acid

 

5. Tetra-O-galloyl-β-D-glucose

 

6. Ellagic acid

 

7. Chebulinic acid

 

 

8. Penta-O-Galloyl-β-D-glucose

Fig. 1. Structures of phytoconstituents in Triphala Churna

 

Optimization of chromatographic conditions:

Various compositions of mobile phase with different organic modifiers were tried to obtain chromatograms with good resolution of adjacent peaks. 0.02% triethyl amine aqueous pH 3. 0 with ortho-phosphoric acid and acetonitrile in the gradient mode were chosen to give the desired separation and acceptable tailing factor within the running time of 27 min. As the maximum types of phytoconstituents show UV maxima between 270-278nm UV detector was set at 272nm to provide sufficient sensitivity for each analyte.

 

Sample analysis:

The method was applied to simultaneous determination of phytoconstituents (1-8) in three samples of Triphala churna (TC2, TC3, TC4) collected from market to compare with siddha Triphala churna sampleTC1). Representative chromatograms are shown in Figure. 2. The contents of the eight compounds in the samples were quantified and the results are shown in Table 3 with the mean values of three replicate injections. Variations of the eight compounds content in three market samples (TC2, TC3, TC4)and sidhha sample (TC1) are shown in histogram Figure 3

 

Fig. 2. HPLC chromatograms of

 

Fig. 2. (A) standard mixture: gallic acid (1), methyl gallate (2), ethyl gallate (3), chebulagic acid (4), tetra-O-galloyl-β-D-glucose (5), ellagic acid (6), chebulinic acid (7), penta-O-galloyl-β-D-glucose (8)

 

Fig. 2. (B) Triphala churna 1 (TC1)

 

Fig. 2. (C) Triphala churna 2 (TC2)

 

Fig. 2. (D) Triphala churna 3 (TC3)

 

Fig. 2. (E) Triphala churna 4 (TC4)

 

Fig. 3. Histogram presenting phytoconstituents contents in Triphala samples.

 

Accuracy and precision:

Recovery tests were carried out to further investigate the accuracy of the method by adding three concentration levels of the mixed standard solutions to known amounts of Triphala churna samples prior to extraction. The resultant samples were then extracted and analyzed with the described method. The average percentage recoveries were evaluated by calculating the ratio of detected amount versus added amount. The recovery of the method was in the range of 97. 5–102. 2%, as shown in Table 2. Considering the results, the method was deemed to be accurate.

 

 

The intra and inter-day precisions were determined by analyzing known concentrations of the eight analytes in six replicates during a single day and by duplicating the experiments on two successive days. In order to confirm the repeatability, six different working solutions prepared from the same sample obtained from different manufacturers were analyzed. The relative standard deviation (R. S. D.) was taken as a measure of precision and repeatability. The results are shown in Table 2, indicating that the intra-, inter-day and repeatability R. S. D. values of the eight compounds were all less than 3. 5%, which showed good reproducibility of the developed method.

 

Linearity:

Standard stock solutions containing gallic acid, methyl gallate, ethyl gallate, chebulagic acid, tetra-O-galloyl-β-D-glucose, ellagic acid, chebulinic acid and penta-O-galloyl-β-D-glucose were prepared and diluted to appropriate concentrations for plotting the calibration curves. At least six concentrations of the analyte solutions were analyzed in triplicate, and then the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The calculated results are given in Table 1. All the analytes showed good linearity (r2 > 0.998) in a relatively wide concentration range.

 


 

Table. 1 Method validation data (Linearity, LOD and LOQ)

Phytoconstituent

RT

(min)

Regression

equation

R2

Linear range

(µg/ mL)

LOD

(µg/ mL)

LOQ

(µg/ mL)

Gallic acid

2.36

y = 32143x - 343. 2

0.999

2-140

0.6

1.998

Methyl gallate

6.6

y = 33724x - 781. 7

0.999

2-30

0.5

1.665

Ethyl gallate

12.05

y = 30948x + 477. 6

0.999

2-30

0.5

1.665

Chebulagic acid

13.48

y = 8771. x + 29500

0.999

2-350

0.5

1.665

TGG

14.56

y = 48100x + 1338.

0.999

2-80

0.55

1.8315

Ellagic acid

15.18

y = 48100x + 1342.

0.999

2-80

0.6

1.998

Chebulinic acid

17.11

y = 11408x + 2102.

0.999

2-45

0.55

1.8315

PGG

17.38

y = 11600x + 5747.

0.999

2-45

0.5

1.665

 


 

Table. 2 Method validation data (Recovery and precision)

Phytoconstituent

Recovery a

Precision (RSD%)

 

(%)

Intra-day b

inter-day c

Gallic acid

98.8

1.2

1.3

Methyl gallate

102.2

2.2

2 4

Ethyl gallate

97.5

2.7

2.5

Chebulagic acid

100.8

2.8

2.7

TGG

97.7

3.1

2.8

Ellagic acid

99.7

2.5

2 6

Chebulinic acid

99.0

3.2

2.9

PGG

97 6

3.5

3.4

a mean of triplicate analysis of three different concentrations

b mean of samples were analyzed on same day n=6

c mean of samples were analyzed on two consecutive day n=6

 

Limits of detection and quantification:

The working solutions of the analytes were further diluted with methanol to yield a series of appropriate concentrations. Limit of detection (LOD) and limit of quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solutions using the developed RP-HPLC method. The LOD and LOQ for each investigated compounds were calculated at signal to noise ratio of 3:1 and 10:1 respectively as shown in Table. 1.

 

Table. 3 Quantification of Phytoconstituents in Triphala samples

Phytoconstituent (%)

TC1

TC2

TC3

TC4

Gallic acid

2. 00

3. 05

2. 17

1. 08

Methyl gallate

0. 08

0. 04

0. 15

0. 17

Ethyl gallate

0. 18

0. 36

0. 23

0. 21

Chebulagic acid

3. 44

5. 74

6. 98

5. 96

TGG

0. 39

0. 36

0. 51

0. 13

Ellagic acid

1. 17

1. 62

1. 49

1. 67

Chebulinic acid

1. 43

5. 91

6. 28

4. 56

PGG

0.81

0.50

0.97

0.58

 

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Robustness of the method was investigated under a variety of conditions including changes of pH of the mobile phase, flow rate and gradient variation.  The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters has proven that the method is robust. The ruggedness of the method was determined by repeating the experiments on Jasco HPLC system by different operators in addition to Waters HPLC system.

 

CONCLUSION:

In this study, an HPLC–DAD method for the qualification and quantification of phtyoconstituents in Triphala churna has been developed and successfully applied for comparison of three marketed samples (TC2, TC3, TC4) with siddha Triphala churna sample (TC1). Significant variation in phytochemical composition of these samples observed. The possible reasons for these variations may be due to quality of the source materials and how elements are handled in production processes i. e. improper and inadequate mixing, variation in particle size of the each myrobalan and demixing during transportation. This method is validated for good accuracy, repeatability and precision, and can be used to evaluate the quality of the drug. This multi-phytoconstituents assay method will be helpful to quality control and stability studies of Triphala churna.

 

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Received on 11. 11. 2010

Accepted on 03. 12. 2010   

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 3(2): March- April 2011, 62-66