1. INTRODUCTION:

Herbal formulations show the number of problems when quality aspect is considered. This is because of nature of the herbal ingredients and different secondary metabolites present therein. Mainly, variation in the chemical profile of the herbal due to intrinsic and extrinsic factors (growing, harvesting, storage and drying processes)^[1,3]. The subject of herbal drug standardization is massively wide and deep.

There is so much to know and so many seemingly contradictory theories on the subject of herbal medicines and their relationship with human physiology and mental function.

There is so much to know and so many seemingly contradictory theories on the subject of herbal medicines and their relationship with human physiology and mental function. For the purpose of research work on standardization of herbal formulations and neutraceuticals, a profound knowledge of the important herbs found in India and widely used in Ayurvedic formulation is of upmost importance. India can emerge as the major country and play the lead role in production of standardized, therapeutically effective Ayurvedic formulations.

Triphala is an antioxidant-rich herbal formulation, frequently used for immune system stimulation, improvement of digestion, relief of constipation, gastrointestinal tract cleansing, relief of gas, treatment of diabetes, eye disease, anemia, jaundice, asthma, fever, chronic ulcers etc. The word triphala means a mixture of three fruits and thus the preparation is a composite mixture of fruits of three medicinal herbs, Fruits of Amalaki (Emblica officinalis Linn.), Bhibitaki (Terminalia belerica Roxb.) and Haritaki (Terminalia chebula Retz.) in equal proportions^[4-6]. It is an important medicine of the ‘Rasayana’ group of Ayurveda and is believed to promote health, immunity and longevity^[7]. Since it contains enormous amount of tannins such as Ellagic acid and Gallic acid, it is required to maintain their quality and purity for safety and efficacy^[8,9].

Gallic acid is common constituent found in plants, thus it is active constituent of polyherbal formulation used as health supplements. Gallic acid (3,4,5-trihydroxybenzoic acid), a plant phenol that occurs naturally and is present in Amla, Haritaki, Bhera, tea leaves, grapes etc., in its free state and as part of the tannin molecule. Gallic acid possesses cytotoxicity against cancer cells, anti-inflammatory, antimutagenic, hepatoprotective, and neuroprotective effect, anti-tumor potential and analgesic activity^[7,10-12]. In the last two decades HPTLC and HPLC methods have emerged as important tools for the qualitative and quantitative phytochemical analysis of herbal drugs and formulations^[13].

This research paper provides qualitative determination of Gallic acid and Ellagic acid by HPTLC and quantitative estimation of Gallic acid through HPTLC and HPLC present in polyherbal formulation.

2. MATERIALS AND METHODS:

Collection of raw drugs:

All ingredients of Triphala churna were procured from the local market of Chennai and authenticated by Pharmacognosist.

Preparation of Triphala churna formulation:

Triphala churna was prepared as per Ayurvedic Formulary of India. All the ingredients were dried below 60ºC, powdered individually in a pulverizer and pass through 85# sieve and stored in air tight containers. Each ingredient were weighed separately required weight, mixed together to obtain a homogeneous blend ^[5].

2.1. Reagents and standards:

All chemicals and solvents were used analytical grade and obtained from E-Merck. Bio Marker reference standard were procured from Natural remedies, India. All other solvents and chemicals were of the highest analytical grade.

2.2. Instrumentation:

Analysis was performed on a HPLC at Natural Remedies Private Limited and CAMAG HPTLC system equipped with an Automatic TLC Sampler IV, TLC Scanner 3 linked to WIN CATS software used at CSMRADDI, Chennai.

HPTLC fingerprinting profile:

The 5µl of each test samples and standards were applied on Tracks 1- 6 on E. Merck aluminium plate pre-coated with Silica gel 60F₂₅₄ of 0.2 mm thickness using CAMAG Automatic sample applicator IV. The plate was developed in the solvent system of Toluene: Ethylacetate: Formic acid: Methanol (3: 3: 0.8: 0.2 v/v) and dried. The developed plate was observed through CAMAG TLC Visualizer under UV at 254 nm and 366 nm and photos were documented. Finally the plate was dipped in Alcoholic Ferric chloride reagent and heated in hot air oven at 105^oC untill the colour of the spots were appeared and photo was documentation under white light. Before derivatization the was scanned using CAMAG TLC Scanner with WINCATS software at a wavelength of UV 254 and 366 nm using deuterium .

2.3. Quantitative estimation of Gallic acid in Triphala Churna and its ingredients by HPLC:

1. Chromatographic system:

High Performance Liquid Chromatographic system using LC Solutions software.

2. Column:

Hibar RP-18e (5μ) Catalogue No: 1.01886, Phenomenex Luna –18 100A (5m) P/No. 00G-4041-E0, Detection at 270 nm wavelength Using Photo diode array detector column temp. at 25 ºC, Injection volume 20 ml. Flow rate 1.50 ml/min for 45 minutes. Mobile phase using gradient method is given in Table 1.

2. Chromatographic Conditions:

Solution A (Buffer): Dissolve 0.136 g of anhydrous potassium dihydrogen orthophosphate (KH₂PO₄) in 900 ml of HPLC grade water. Add 0.5ml of Orthophosphoric acid. Make upto 1000 mL with water, filter through 0.45 m membrane and degas in a sonicator for 3 minutes.

Solution B: Acetonitrile

Table 1. Mobile phase using gradient method

Time	Buffer concentration (%)	Acetonitrile concentration (%)
0.01	95.0	5.0
18.0	80.0	20.0
25.0	65.0	35.0
28.0	65.0	35.0
35.0	80.0	20.0
40.0	95.0	5.0
45.0	95.0	5.0

Standard preparation:

Weigh accurately 10 mg of Gallic acid in to 100 ml volumetric flask, dissolve in 50 ml of hot water by sonication, cool and make up to 100 ml with water.

Raw material preparation:

Weigh about 0.250 g of finely powdered sample raw material into a 100 ml volumetric flask, dissolve in 50ml of hot water and sonicate for 10 minutes, cool and make up to 100 ml with water, Filter through 0.45 microns (PES filter papers only) membrane filter paper.

Procedure:

Inject three times the standard preparation and calculate the mean area and the RSD. The RSD should not be more than 2%. Inject 20 μl of sample preparation and record the chromatogram at 270nm.

Quantitative estimation of Gallic acid in Triphala Churna and its ingredients by HPTLC:

Standard preparation:

2 mg of Gallic acid was dissolved in ethanol and made up to 10 ml volumetric flask.

Raw material preparation:

5 g of finely powdered samples was taken in a soxhlet apparatus for 24 hrs, the extract was concentrated, filtered and made upto 25 ml of volumetric flask.

Procedure:

All the test solutions (1 µl & 2 μl) and the standard solution of different concentrations (8 μl, 10 μl, 12 μl & 14 μl ) were applied on respective Tracks on E. Merck aluminium plate pre-coated with Silica gel 60F₂₅₄ of 0.2 mm thickness using CAMAG Automatic sample applicator IV. The plate was developed in the solvent system of Toluene: Ethylacetate: Formic acid (3: 3.5: 0.5 v/v) and dried. The developed plate was observed through CAMAG TLC Visualizer under UV at 254 nm and 366 nm and photos were taken. The plate was scanned using CAMAG TLC Scanner 3 with WINCATS software at a wavelength of UV 254 nm using deuterium lamp.

3. RESULTS AND DISCUSSION:

Standardization promises constant composition of all herbals including analytical operations for identification, markers and assay of bioactive constituents. In the current study quantitative estimation of specific biologically active Gallic acid component was conducted in the Ayurvedic Polyherbal formulation and its ingredients using HPLC and HPTLC. The quantitative estimation of Gallic acid is using calibration curve of standard Gallic acid. The chromatograms of the components of Triphala churna and its ingredients were also quantified with respect to the standards. The chromatograms of the individual components of Triphala churna and its ingredients were also quantified with respect to the standards given in Figure 1 to 4. The results obtained are shown in Table 2.

HPTLC method is an accurate, simple and specific method for quantitative estimation of biomarkers. For optimization of different mobile phase compositions were employed to achieve good separation. Among the various solvent systems tried the solvent system containing Toluene: Ethyl acetate: Formic acid in the volume ratio of (3: 3.5: 0.5 v/v) resulted in good separation of the Gallic acid and Ellagic acid (Figure 5). TLC plate was observed under UV light for the presence of Gallic acid and Ellagic acid, which was detected by prominent Green spot in UV 254 nm, dark blue spots in UV 366 and appeared in alcoholic ferric chloride reagent. The R_f value of Ellagic acid 0.47 and Gallic acid is 0.56 in all the samples and reference standards were found comparable under UV light at 254 nm and 366 nm. The quantity of Gallic acid was estimated by comparing the peak area of the standard with that of the sample extracts (Figure 6-7).

Figure 1. HPLC Chromatogram and Peak Table of Amalaki (Emblica officinalis) Fruit

Figure 2. HPLC Chromatogram and Peak Table of Bibhitaki (Terminalia belerica) Fruit

Figure 3. HPLC Chromatogram and Peak Table of Haritaki (Terminalia chebula) Fruit

Figure 4. HPLC Chromatogram and Peak Table of Triphala Churna


1 2 3 4 5 6	1 2 3 4 5 6	1 2 3 4 5 6

Figure 5. HPTLC profile of Ethanol extract of Triphala Churna and its Ingredients at UV 254 nm, UV 366 nm and after derivatization.

Track 1- Emblica officinalis – 5µl; Track 2- Terminalia belarica -5µl; Track 3- Gallic acid- 5 µl; Track 4 – Ellagic acid- 5µl; Track 5- Terminalia chebula- 5 µl; Track 6- Triphala curna- 5 µl; Solvent system: Toluene: Ethylacetate: Formic acid: Methanol (3: 3: 0.8: 0.2 v/v)

Figure 6. Linearity curve for Gallic acid and Superimposed spectra of Gallic acid with test solutions







Rf values and HPTLC Finger print profile of test samples and standards at UV 366 nm:

Rf values and HPTLC Finger print profile of test samples and standards after derivatized with Ferric Chloride:

Figure 7. R_f values and HPTLC Finger print profile of test samples and standards at UV 254nm.

Track 1: Phyllanthus emblica Linn.; Track 2 :Terminalia belerica Roxb.; Track 3:Gallic acid; Track 4: Ellagic acid; Track 5: Terminalia chebula Retz.; Track 6: Triphala churna

CONCLUSION:

All the ingredients of triphala curna having more gallic acid and number of peaks were observed in formulation, which can be used for routine Polyherbal drug analysis and for quality assurance. In this study, qualitative estimation of biologically active Gallic acid and Ellagic acid and quantitative estimation of Gallic acid was conducted in the polyherbal formulation and ingredients using HPLC and HPTLC. The R_f value of ellagic acid 0.47 and Gallic acid is 0.56 in all the samples and reference standard was found comparable under UV light at 254 nm and 366 nm. The percentage of free Gallic acid is prominent in Emblica officinalis and lower in Terminalia chebula of Triphala ingredients.

ACKNOWLEDGEMENT:

The authors are very grateful to Director General, CCRAS, Ministry of AYUSH, New Delhi for providing encouragement and facilities for carrying out this work.

REFERENCES:

1. Gilani A H and Rahman A. Trends in ethnopharmacology, Journal of Ethanopharmacology, 2005, 100(1-2), 43-49.

2. WHO. Traditional Medicine Strategy 2002-2005.World Health Organization; Geneva, 2002.

3. Zidorn C, Schubert B, Stuppner H. Altitudinal differences in the contents of phenolics in flowering heads of three members of the tribe Lactuceae (Asteraceae) occurring as introduced species in New Zealand, Biochem Sys and Eco, 2005,33, 855.

4. Gajendra Singh, Pushkar Choudhary, Syed Asif Yaqoob, Rajveer Singh Rawat and Bhanwar Lal Jat Antibacterial and antioxidant activity of triphala extracts. International Journal of Current Research, 2016. 8(9) 38335-38348.

5. Anonymous, The Ayurvedic Formulary of India. Part I, 2^nd edition, Government of India, New Delhi, 2003,113.

6. Biradar YS, Jagatap Sh, Khandelwal KR and Singhania SS. Exploring of antimicrobial activity of Triphala Mashi- an ayurvedic formulation. Evid. Based Complement. Alternat. Med. 2008, 5: 107-113.

7. Jagetia GC, Baliga MS, Malagi KJ and Kamath MS. The evaluation of the radioprotective effect of Triphala (an Ayurvedic rejuvenating drug) in the mice exposed to radiation. Phytomedicine. 2002, 9: 99–108.

8. Patel Madhavi G, Patel Vishal R, Patel Rakesh K. Development and Validation of Improved RP-HPLC method for Identification and Estimation of Ellagic and Gallic acid in Triphala churna , International Journal of Chem Tech Research, 2010(2)1486.

9. Singh DP, Govindarajan R. and Rawat AKS. High-performance Liquid Chromatography as a Tool for the Chemical Standardization of Triphala an Ayurvedic Formulation, Phytochem. Anal. 2007, 10: 1002.

10. Sajeeth C, Manna PK, Manavalan R, Jolly C. Quantitative estimation of Gallic Acid, Rutin and Quercetin in certain herbal plants by HPTLC method. Der Chemica Sinica 2010, 1(2): 80-85.

11. Tatiya RP, Sutar MP, Shirkhedkar AA, Surana SJ. Determination of Phyllanthin and Gallic Acid in Herbal Hepatoprotective Formulation by TLC-Densitometry Analysis. Pharmacognosy Journal 2011, 3(26): 39-43.

12. Kuamwat RS, Mruthunjaya K, Gupta MK. Preparation, Characterization and Antioxidant Activities of Gallic Acid- Phospholipids Complex. IJRPS, 2012, 2(1): 138-148.

13. Ranjana, Anurag Mishra, Ashutosh Mishra and Rajiv Gupta. Determination of Gallic Acid and β - Sitosterol in Poly-Herbal Formulation by HPTLC. Pharm Pharmacol Int J 2016, 4(4): 00081.

Received on 22.06.2018 Modified on 12.07.2018

Research J. Pharm. and Tech 2018; 11(8): 3243-3249.

DOI: 10.5958/0974-360X.2018.00596.6

Parameter	Triphala Churna	Amalaki *(Emblica officinalis) Fruit*	Bibhitaki *(Terminalia belerica) Fruit*	Haritaki (Terminalia chebula) Fruit
Parameter	Assay by HPLC
Gallic acid (%w/w)	3.27	4.16	4.16	1.26
	Assay by HPTLC
	3.11	5.5	3.17	1.82