Simultaneous estimation of Gallic acid and rutin in Kaishore guggulu and Vatari guggulu by HPTLC

 

K.V.V.S. Krishna*, K. Gouri Sankar, M. Syam Vardhan, S. Rama Krishna, T. Tamizhmani

Department Of Pharmacognosy, Bharathi College  Of Pharmacy, Bharathi Nagara, Maddur Tq., Mandya Dt., Karnataka, India-571422.

*Corresponding Author E-mail: kvvskrishna@rediffmail.com

ABSTRACT:

A simple, rapid, robust and reliable HPTLC method has been developed for simultaneous estimation of gallic acid and rutin in Kaishore Guggulu and Vatari Guggulu. Identification and quantification were performed on 20cm x 10cm, layer thickness 0.2mm, aluminium-backed silica gel 60 F254 HPTLC plates previously washed with methanol. Toluene: Acetone: Ethyl Acetate: Formic Acid: Water (2: 3: 2: 1: 2, v/v), was used as a mobile phase. Calibration plots were established showing the dependence of response on the amount chromatographed. The validated calibration range was 400-800ng per spot (R2 = 0.998; 0.996). The spots were scanned at λ = 312 nm. The suitability of this HPTLC method for simultaneous estimation of the marker constituents was proved by validation in accordance with ICH Guidelines. Determination of method accuracy by the standard addition method at three concentration levels returned a mean recovery of 98.92 ± 0.16 - 101.61 ± 0.24. The developed method has the advantage of being rapid, robust and easy. Hence, it can be applied for routine quality control analysis of gallic acid and rutin in those Ayurvedic formulations.

 

KEYWORDS: Ayurvedic formulations, Kaishore Guggulu, Vatari Guggulu, HPTLC, gallic acid and rutin.

 

 


INTRODUCTION:

Bahera (Terminalia belerica, Family-Combretaceae)[1] is a proven anti-atherogenic agent that reduces cholesterol and good for eyes, hair and voice. The fruits of Terminalia bellerica are commonly used in the treatment of dyspepsia and diarrhea. The fruits contain about 20-30% of tannins[2] of which gallic acid, ellagic acid, phyllemblin, ethyl gallate and galloglucose are major hydrolysable tannins and a flavonoid rutin. Therefore, estimation of gallic acid and rutin would be an important parameter for quality control of Bahera containing Ayurvedic formulations.

 

Gallic acid[3] is an organic acid also known as 3, 4, 5 trihydroxybenzoicacid. It is commonly used in pharmaceutical industry as a standard for determining the phenol content of various analytes by the Folin-ciocalteau assay; results are reported in gallic acid equivalents. It seems to have antifungal and antiviral properties. It acts as an antioxidant[4] and helps to protect our cells against oxidative damage, it was found to show cytotoxic action against cancer cells, without harming healthy cells. It is used as a remote astringent in cases of internal hemorrhage. It is also used to treat albuminuria and diabetes. Some ointments to treat psoriasis and external hemorrhoids contain gallic acid.

 

Rutin[5] is a citrus flavonoid glycoside found in buckwheat the leaves and petioles of rheum species and asparagus. It inhibits platelet aggregation as well as decreasing capillary permeability, making the blood thinner and improves circulation.

Extensive Literature survey revealed HPTLC and HPLC [6] methods for the determination of gallic acid and rutin. But, no method is so far reported for the simultaneous estimation of gallic acid and rutin in Kaishore Guggulu and Vatari Guggulu. The aim of the present work is to develop a simple, precise, rapid and cost effective HPTLC method for the simultaneous estimation of gallic acid and rutin in the marketed Ayurvedic formulations.

 

MATERIALS AND METHODS:

All chemicals and reagents used throughout the experiment including Ethyl Acetate, Formic Acid, Methanol, Toluene and Alcoholic FeCl3 were of analytical grade. Analytically pure samples of gallic acid and rutin were procured as gift samples from M/s Natural Remedies Pvt. Ltd., Bangalore, India. The Ayurvedic formulations i.e., Kaishore Guggulu and Vatari Guggulu, used for present study were purchased from Gururaja Pharmacy, Bangalore.

 

Instrumentation:

Analysis was performed on 10cm x 10cm plates cut from 20cm x 20cm aluminium-backed silica gel 60 F254 plates (E. Merck). Samples were applied to the plates by means of a Linomat-V automatic spotter with the aid of Hamilton 100μl syringe. TLC plates were developed in flat bottom twin trough chamber. Densitometry was performed with a TLC scanner-3 with Win-CATS 4 software resident in a Pentium IV computer. 

 

Preparation of standard solution

Accurately weighed 10mg of gallic acid standard was dissolved in 10ml of methanol in a volumetric flask (A). 2ml of this solution was diluted to 10ml with methanol (B). Accurately weighed 10mg of rutin standard was dissolved in 10ml of methanol in a volumetric flask (C). 2ml of this solution was diluted to 10ml with methanol (D). Working standard solution (E) was prepared by mixing 5ml of B and 5ml of D. Solution E was used for the HPTLC analysis. A stock solution containing 200mcg/ml gallic acid (B) and rutin (D) were prepared in methanol. Calibration solutions were prepared by diluting the stock solution so that application of 4-8µl volumes gave a series of spots covering the range 400 to 800ng of gallic acid and rutin respectively.

 

Preparation of sample solution:

Amount equivalent to the contents of the formulation was extracted twice with 10ml of methanol by boiling for 10 minutes. Extract obtained was filtered using Whatman filter paper, concentrated to less than 10ml and transferred to a 10ml volumetric flask and volume was made up with methanol. Kaishore Guggulu and Vatari Guggulu were weighed in amounts of 0.40gm and 0.46gm respectively.

 

 

Simultaneous estimation of markers in Kaishore Guggulu and Vatari Guggulu:

From the sample solutions, 5.0μl was applied on the pre-coated silica gel plate and process was repeated to develop and the plate was scanned at λ=312nm. The amount of gallic acid and rutin in the samples was calculated from the calibration equation generated and by using the average area of triplicate sample aliquots.

 

Validation of the method:

After the development of HPTLC method for the simultaneous estimation of the poly herbal formulations, Validation of the method was carried out according to the ICH guidelines with respect to Linearity, Accuracy, Precision, Limit of Detection and Limit of Quantification, Robustness. [7-9]

 

RESULTS AND DISCUSSION:

A wavelength of 312nm was chosen for quantification. The Rf value of gallic acid and rutin after development with the mobile phase Toluene: Acetone: Ethyl Acetate: Formic Acid: Water (2: 3: 2: 1: 2, v/v) was 0.43 and 0.05 respectively. When the concentrations of gallic acid and rutin and their respective peak areas were subjected to regression analysis by least squares method, a good linear relationship (R2=0.998; 0.996) was observed between the concentrations of gallic acid and rutin and the respective peak areas in the range 400-800 ng/spot. The regression of gallic acid and rutin was found to be Y=4.482 X – 299.9 and Y= 10.75 X + 9718 respectively, where ‘Y’ is the peak area and ‘X’ is the concentration of gallic acid and rutin respectively were shown in Table 1.

 

Table 1: Characteristic parameters for the proposed HPTLC method

Parameters

HPTLC

Gallic Acid

Rutin

Calibration range (ng /spot)

400-800

400-800

Detection wavelength

312nm

312nm

Mobile phase ( Toluene : Acetone : Ethylacetate : Formic acid : Water)

2 : 3 : 2 : 1 : 2

2 : 3 : 2 : 1 : 2

Rf value

0.43

0.05

Regression equation (y*)

Y = 4.482x – 299.9

Y=10.75x + 9718.6

Slope (b)

4.4821

10.7521

Intercept (a)

-299.94

9718.6

Correlation coefficient (R2)

0.998

0.996

 

The regression equations were used to estimate the amounts of gallic acid and rutin in tablet Ayurvedic formulations or in validation study (precision and accuracy). The content of gallic acid and rutin present in Ayurvedic formulations were shown in Table 2.

 

Table 2: Content of gallic acid and rutin in Kaishore Guggulu and Vatari Guggulu

S.No.

Samples

Gallic acid (%)

Rutin (%)

1.

Kaishore Guggulu

0.2414

ND

2.

Vatari Guggulu

0.5385

0.00494

The chromatograms containing peaks of gallic acid and rutin in Ayurvedic formulations are shown in Figure 1 and 2 respectively.


 

Figure 1: Typical HPTLC Chromatogram of Kaishore Guggulu by HPTLC method

 



Figure 2: Typical HPTLC Chromatogram of Vatari Guggulu by HPTLC method

 


Precision:

The precision of the method in terms of intra - day precision (% RSD) was determined by analyzing gallic acid and rutin standard solutions in the range (400 - 800ng/spot) three times on the same day. Inter - day precision (% RSD) was assessed by analyzing these solutions (400 - 800ng/spot) on three different days over a period of one week. The results of the precision studies are shown in Table 3 and 4.

 

Table 3: Precision of Gallic acid by HPTLC method

S. No.

Concentration

(ng/spot)

Intraday precision (Area)

Interday precision (Area)

1.

600

2380.7

2452.8

2.

600

2390.8

2491.3

3.

600

2415.7

2501.8

4.

600

2438.5

2520.4

5.

600

2445.9

2544.8

6

600

2456.8

2589.7

Mean

 

2421.4

2516.8

Std.Dev

 

30.890

47.079

%RSD

 

1.275

1.870

 

Table 4: Precision of Rutin by HPTLC method

S. No.

Concentration (ng/spot)

Intraday precision (Area)

Interday precision (Area)

1.

600

16112.1

15914.1

2.

600

16114.1

15863.1

3.

600

16113.1

15962.1

4.

600

15962.1

16063.1

5.

600

15863.1

15564.1

6.

600

15914.1

15712.1

Mean

 

16013.1

15846.4

Std.Dev

 

113.93

180.52

%RSD

 

0.71

1.13

 

Accuracy:

Determination of method accuracy by the standard addition method at three concentration levels returned a mean recovery of 98.92 ± 0.16 - 101.61 ± 0.24 is given in Table 5 and 6.

 

 

 

 

 


 

Table 5: Recovery studies of Gallic acid by HPTLC method

S. No.

Sample

 

Initial amount

(ng/spot)

Amount added

(ng/spot)

Amount recovered*

(ng/spot)

Recovery ± SD* (%)

% RSD

1.

Kaishore guggulu

600

300(50%)

600(100%)

900(150%)

98.98

101.51

99.39

98.98±0.21

101.51±0.31

99.39±0.12

0.213

0.312

0.127

2.

Vatari guggulu

600

300(50%)

600(100%)

900(150%)

99.04

101.43

99.42

99.04±0.20

101.43±0.31

99.42±0.12

0.211

0.309

0.126

Mean* ± S.D. from six determinations

 

Table 6: Recovery studies of Rutin by HPTLC method

S. No.

Sample

Initial amount

(ng/spot)

Amount added

(ng/spot)

Amount recovered* (ng/spot)

Recovery ± SD* (%)

% RSD

1

Kaishore guggulu

600

300(50%)

600(100%)

900(150%)

101.25

100.14

100.31

101.25±0.60

100.14±0.12

100.31±0.19

0.603

0.123

0.195

2

Vatari guggulu

600

300(50%)

600(100%)

900(150%)

99.32

101.57

100.11

99.32±0.16

101.57±0.24

100.11±0.98

0.164

0.241

0.983

Mean* ± S.D. from six determinations

 


Linearity:

The linearity was found in the concentration range of 400 - 800ng/spot. The correlation coefficient was found to be 0.998 and 0.996 for gallic acid and rutin respectively. The results are presented in Table 7 and 8 and Figure 3 and 4 respectively.

 

 

Figure 3: Calibration curve of Gallic acid by HPTLC method

 

Table 7: Calibration data of Gallic acid by HPTLC method

S. No.

Amount in ng/Spot

Rf values

Peak area

1.

400

0.45

1463.3

2.

500

0.44

1979.8

3.

600

0.43

2380.8

4.

700

0.43

2856.9

5.

800

0.42

3265.8

 

Table 8: Calibration data of Rutin by HPTLC method

S. No.

Amount in ng/Spot

Rf values

Peak area

1.

400

0.05

13989.8

2.

500

0.05

15214.1

3.

600

0.05

16112.1

4.

700

0.05

17121.2

5.

800

0.05

18412.3

 

 

 

Figure 4: Calibration curve of Rutin by HPTLC method

 

Limit of Detection and Limit of Quantitation:

The LOD and LOQ of gallic acid were found to be 9.504 and 28.802 respectively. The LOD and LOQ of rutin were found to be 0.306 and 0.930 respectively. The LOD and LOQ of gallic acid and rutin were reported in Table 9.

 

Table 9: Validation parameters for the proposed HPTLC method

Parameters

HPTLC

Gallic Acid

Rutin

Limit of detection (ng/spot)

9.504

0.306

Limit of quantitation (ng/spot)

28.802

0.930

*y = b x + a, where x is the concentration of Gallic acid and Rutin in ng/spot and y is the peak area at respective wavelength. 

Mean** = Average of three linearity curves 

 

The drug peak-area was calculated for each concentration level and a graph was plotted of drug concentration against the peak area and shown in Figure 5.

 

 

Figure 5: Typical HPTLC Chromatogram of gallic acid and rutin by HPTLC method

 

Robustness:

The robustness of the method was studied, during method development, by determining the effect of small variation, of mobile phase composition (±2%), chamber saturation period, development distance, derivatization time, and scanning time (10% variation of each). No significant change of Rf values was observed, indicating the robustness of the method.

 

CONCLUSION:

In the present study, on the simultaneous estimation of gallic acid and rutin in Kaishore Guggulu and Vatari Guggulu by HPTLC, wide variations in the content of gallic acid and rutin were observed in the marketed Ayurvedic formulations. This leads to marked differences in the therapeutic efficacy of those formulations when administered. Hence, the newly developed method can be utilized to standardize any such Ayurvedic formulation, thereby ensuring their desired therapeutic efficacy. This would also minimize or avoid batch-to-batch variations of such Ayurvedic formulations. The newly developed HPTLC method is simple, sensitive, precise, accurate, reproducible, robust, selective and economic and can be used for routine quality control analysis of the marker constituents in Ayurvedic and polyherbal formulations in Herbal Drug Industries, Research institutions and Quality Control departments.

 

REFERENCES:

1.      Marker Compounds of Ayurvedic Drugs, WHO Country Office for India, New Delhi, July 2009.

2.      Kokate CK, Purohit AP and Gokhale SB. Text book of Pharmacognosy. Jan2007; p.258-59.

3.      URL: http://en.wikipedia.org/wiki/File:Gallic_acid.svg    

4.      Natvarbhai M Petal, Vineet C Tain, Anurag Bhargav and Dhiren Pshah. HPTLC method for Quantification of Gallic acid in root powder of Alangium salvifolium wang. Int J Pharm Research. April-June2010; 2(3):40-44.

5.      URL: http://en.wikipedia.org/wiki/File:Rutin_structure.svg

6.      Ashok kumar BS, Lakshman K, Jayaveera KN and Satish KV. Estimation of Rutin and Quercetin in Terminalia chebula by HPLC. The Internet J Aesthetic & Antiaging medicine. 2009.

7.      International Conference on Harmonization (ICH), Validation of Analytical Procedures: Text on Validation of Analytical Procedures Q2A, 1994.

8.      International Conference on Harmonization (ICH), Validation of Analytical Procedures: Methodology Q2B, 1996.

9.      International Conference on Harmonization (ICH), Validation of Analytical Procedures: Text and Methodology Q2 (R1), 2005.

 

 

 

 

Received on 25.01.2018                                Modified on 23.02.2018

Accepted on 13.04.2018       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2018; 10(3): 221-225.

DOI:  10.5958/0975-4385.2018.00036.5