Simultaneous quantification of pharmacological markers quercetin, berberine HCl and curcumin using High-performance thin-layer chromatography (HPTLC) from polyherbal formulation
Kinjal Patel1*, Priya Shah2, Maitreyi Zaveri3
1Research scholar, K.B. Institute of Pharmaceutical Education and Research,
Kadi Sarva Vishwa Vidyalaya and Faculty at Department of Pharmacognosy,
K. B. Raval College of Pharmacy, Shertha, Gandhinagar, Gujarat, India.
2Department of Pharmacognosy, LJ Institute of Pharmacy, LJ University, Ahmedabad, Gujarat, India.
3HOD, Department of Pharmacognosy, K. B. Institute of Pharmaceutical Education and Research,
Kadi Sarva Vishwa Vidyalaya, Gandhinagar, Gujarat, India.
*Corresponding Author E-mail: kinjal9387@gmail.com
ABSTRACT:
Quercetin, curcumin and berberine HCl have anti-acne, antimicrobial, anti-inflammatory, antioxidant properties. In the present study an attempt has been made to develop a simple, precise, rapid, selective and cost-effective high-performance thin-layer chromatographic (HPTLC) method for simultaneous estimation of quercetin, curcumin and berberine HCl from polyherbal gel prepared for the treatment of Acne. The method was developed using HPTLC silica gel GF254 precoated aluminium plate as a stationary phase. The solvent system consisted of Toluene, Ethyl acetate, Methanol, Formic acid. Densitometric analysis was carried out in the absorbance mode at 254nm. This system was found to give compact spots for quercetin, curcumin and berberine HCl at specific Rf values. The linearity of quercetin, curcumin and berberine HCl were obtained between 200 and 1000 ng/spot with good correlation coefficient values. The % of quercetin, curcumin and berberine HCl from polyherbal gel was found to be 99.42±0.98, 98.67±0.76% and 99.6±1.06% respectively, which was well within the limit. It was concluded that the developed HPTLC method would be an important tool in the quality control of polyherbal formulations.
KEYWORDS: Quercetin, Curcumin, Berberine HCl, HPTLC, Polyherbal Gel, Acne.
INTRODUCTION:
Quercetin (QCN) is a flavonol, which is a subclass of flavonoids and it is present in number of fruits and vegetables such as onion (Allium cepa Linn.), apple (Malus pumila Miller), black tea (Camellia sinensis (L.) Kuntze)1-3.
Berberine(5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a] quinolizinium) is a nonbasic and quaternary benzylisoquinoline alkaloid and is yellow in color and it is present in plants such as barberry (Berberis vulgaris), tree turmeric
(Berberis aristata), Oregon grape (Mahonia aquifolium), golden seal (Hydrastis canadensis), yellow root (Xanthorhiza simplicissima), Amur cork tree (Phellodendron amurense), Chinese gold thread (Coptis chinensis), prickly poppy (Argemone mexicana) and Californian poppy (Eschscholzia californica)4-6.
Curcuminoid is yellow in color and derived from the plant Curcuma longa Linn and it is known as diferuloylmethane and its composition are curcumin7-9.
Quercetin, berberine HCl and curcumin possesses number of activities like anti-acne, antimicrobial, antioxidant, ant carcinogenic, anti-inflammatory1,4,9.
In the last two decades high performance thin layer chromatography [HPTLC] method has emerged as an important tool for the qualitative and quantitative phytochemical analysis of herbal drugs and herbal formulations that includes TLC fingerprint profiles and estimation of chemical markers and biomarkers10. The major advantage of HPTLC method is that several samples can be analyzed simultaneously using a small quantity of mobile phase.
Estimation of quercetin has been done by using HPTLC for a number of times individually and also in combination with other compounds11-15. Also a number of validated HPLC methods have been developed for the estimation of quercetin both individually and in combination with other compounds16-20. Quantification of curcumin has been done by HPTLC technique for a number of times individually and also in combination with other compounds15,21-23. Also a number of validated HPLC methods have been devised for the estimation of curcumin both individually and in combination with other compounds15,19,24,25. Estimation of berberine HCl has been done by using HPTLC for a number of times individually and also in combination with other compounds26,27. Also a number of validated HPLC methods have been devised for the estimation of berberine HCl both individually and in combination with other compounds20.
MATERIAL AND METHODS:
Solvents and chemicals:
Quercetin, berberine HCl and curcumin standard were procured from Yucca Enterprises, Mumbai. Silica gel 60F254 TLC plates (20×10 cm, layer thickness 0.2 mm, E. Merck, Germany) were used as a stationary phase. All chemicals and reagents were of analytical grade and they were obtained from SD Fine Chem. Ltd., India. Prepared formulation was used for analysis.
Instrumentation:
The HPTLC system (Camag, Muttenz, Switzerland) consisted of Linomat V applicator connected to a nitrogen cylinder, a twin trough chamber (10 × 10 cm) and precoated silica gel 60 F254 TLC plates (10 × 10 cm, layer thickness 0.2 mm, E. Merck, Darmstadt, Germany) were used as stationary phase. TLC plates were prewashed twice with 10 ml of methanol and activated at 105 °C for 20 min prior to sample application. Analysis was carried out using a TLC scanner IV with win CATS software Version 2.5.18072.1 (Camag, Switzerland).
Preparation of Standard Solution:
10 mg of quercetin, berberine HCl and curcumin were weighed and transferred into 100 ml volumetric flask. These drugs were dissolved in 50 ml methanol by vigorous shaking and then volume was made up to mark with methanol to obtain a final concentration of 100 µg/ml.
HPTLC method development and chromatographic conditions:
The sample solutions were spotted on precoated silica gel aluminium plate 60F254 (10 cm × 10 cm) with 250 μm thickness (E. Merck, Darmstadt, Germany) in the form of bands of 8 mm width with a Hamilton syringe (100 μL) using a Camag Linomat V (Switzerland) sample applicator. The slit dimension was kept at 6 mm × 0.45 mm and 20 mm/s scanning speed was employed. Linear ascending development was carried out in 10 cm x 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) previously saturated with the mobile phase. Plates were then developed, at a constant temperature with 20 ml mobile phase consisting of Toluene: Ethyl acetate: Methanol: Formic acid (4:4:1.5:0.5 v/v/v/v). The optimized chamber saturation time for mobile phase was 20 min at room temperature (25±2 °C) at a relative humidity of 60±5%. Densitometric scanning was performed on Camag TLC scanner IV in the absorbance mode at 254nm after development for all measurement and operated by win CATS software Version 2.5.18072.1 (Camag, Switzerland).
Validation of the method:28
The developed method was validated for linearity, accuracy, precision, and repeatability, limit of detection and limit of quantification. To check the accuracy of the proposed method recovery studies were carried out by spiking 80%, 100% and 120% of standard concentration. The intra-day and inter-day precision studies were evaluated by estimating the responses of three quality control (QC) standards in triplicates under same experimental conditions three times on the same day and on three different days and precision was expressed in terms of %RSD.
Estimation of quercetin, berberine HCl and curcumin in the formulation:
A 1g polyherbal gel formulation (7.5mg quercetin, 0.5mg berberine HCl, 20mg curcumin) was weighed and transferred into volumetric flask containing 100ml of methanol and kept in the ultrasonicator for 15 min for extraction of drugs from formulation. The amount present in the formulation was calculated from the regression equation obtained from the calibration curve.
RESULTS AND DISCUSSION:
Development of the Optimum Mobile Phase:
The method was developed using HPTLC silica gel GF254 precoated aluminium plate (10×10 cm, layer thickness 0.2 mm, E. Merck, Germany) as a stationary phase. The mobile phase Toluene: Ethyl acetate: Methanol: Formic acid (4:4:1.5:0.5 v/v/v/v) gave good resolution with Rf values 0.40, 0.63 and 0.74 for berberine HCl, quercetin and curcumin, respectively. Densitometric analysis was carried out in the absorbance mode at 254nm.
Validation of the proposed method:
The linearity regression data for calibration curve showed good linear relationship over the range of 200-1000ng/spot for quercetin, berberine HCl and curcumin, respectively (Figure 3,4,5). The correlation coefficient was 0.999, 0.9997 and 0.9991 for quercetin, berberine HCl and curcumin, respectively. %RSD values were found to be less than 2% for quercetin, berberine HCl and curcumin, respectively for intra-day precision, inter-day precision and repeatability, which suggested high level of precision and reproducibility of the given method (Table 1,2). The percentage recovery was found to be in the range of 98.44– 99.58%, 98.5- 99.63% and 98.18- 99.26% for quercetin, berberine HCl and curcumin, respectively (Table 3). The results for method validation are shown in (Table 4).
Figure 1. HPTLC of berberine HCl, quercetin and curcumin at 254 nm
Figure 2. Overlay spectra of berberine HCl, quercetin and curcumin at 254 nm
Figure 3. Calibration curve of quercetin Figure 4. Calibration curve of berberine HCl
Figure 5. Calibration curve of curcumin
Table 1. Precision studies
Analyte |
Concentration (ng/spot) |
Intraday Precision (n=3) |
Interday Precision (n=3) |
||
Concentration found±SD |
%RSD |
Concentration found±SD |
%RSD |
||
Quercetin |
400 |
396.88±3.32 |
0.84 |
398.33±1.25 |
0.31 |
600 |
597.61±2.18 |
0.36 |
599.06±1.25 |
0.21 |
|
800 |
802.68±3.32 |
0.41 |
801.23±4.52 |
0.56 |
|
Berberine HCl |
400 |
397.67±1.15 |
0.29 |
398.33±1.53 |
0.38 |
600 |
598.67±0.58 |
0.10 |
598±1 |
0.17 |
|
800 |
795.67±2.08 |
0.26 |
797.33±2.08 |
0.26 |
|
Curcumin |
400 |
398±2 |
0.50 |
398.67±2.31 |
0.58 |
600 |
598.67±1.15 |
0.19 |
599.33±1.15 |
0.19 |
|
800 |
798.67±3.06 |
0.38 |
798±2 |
0.25 |
Table 2. Repeatability studies
Analyte |
Concentration (ng/spot) (n=6) |
Concentration found±SD |
%RSD |
Quercetin |
600 |
598.33±1.78 |
0.3 |
Berberine HCl |
600 |
598±0.89 |
0.15 |
Curcumin |
600 |
599.33±1.03 |
0.17 |
Table 3. Accuracy studies
Analyte |
Concentration of sample taken(µg/ml) (n=3) |
Concentration of standard added (µg/ml) (n=3) |
Total Conc. (μg/ml) |
Amount Recovered Mean((μg/ml) |
%Recovery Mean± SD |
Quercetin |
160 |
200 |
360 |
358.48 |
99.58± 0.61 |
200 |
200 |
400 |
396.88 |
99.22± 0.84 |
|
240 |
200 |
440 |
433.12 |
98.44± 0.77 |
|
Berberine HCl |
160 |
200 |
360 |
358.67 |
99.63±1.25 |
200 |
200 |
400 |
394 |
98.5±0.50 |
|
240 |
200 |
440 |
433.67 |
98.56±0.95 |
|
Curcumin |
160 |
200 |
360 |
357.33 |
99.26±1.40 |
200 |
200 |
400 |
394.67 |
98.67±0.76 |
|
240 |
200 |
440 |
432 |
98.18±0.46 |
Table 4. Summary of validation parameters for quercetin, berberine HCl and curcumin
Sr. No. |
Validation Parameters |
Result obtained |
||
Result for Quercetin |
Result for Berberine HCl |
Result for Curcumin |
||
1. |
Linearity range (ng/spot) |
200-1000 |
200-1000 |
200-1000 |
2. |
Correlation coefficient |
0.999 |
0.9997 |
0.9991 |
3. |
Repeatability (n=6) (%RSD) |
0.3% |
0.15% |
0.17% |
4. |
Precision (%RSD) Intraday (n= 3) Interday (n= 3) |
0.36%-0.84% 0.21%-0.56% |
0.10%-0.29% 0.17%-0.38% |
0.19%-0.50% 0.19%-0.58% |
5. |
Accuracy (% Recovery) |
98.44– 99.58% |
98.5- 99.63% |
98.18- 99.26% |
6. |
Limit of detection(ng) |
38.21 |
19.98 |
36.31 |
7. |
Limit of quantification (ng) |
115.78 |
60.55 |
110.03 |
Table 5. Estimation of quercetin, berberine HCl and curcumin in the Polyherbal gel formulation
Drugs |
% Drug content |
Quercetin |
99.42±0.98 |
Berberine HCl |
99.6±1.06 |
Curcumin |
98.67±0.76 |
CONCLUSION:
The developed HPTLC method was found to be accurate and precise for determination of quercetin, berberine HCl and curcumin in polyherbal gel formulation and can be used for its quantification in plant materials and also in routine quality control of the raw materials as well as formulations containing these Phytoconstituents.
AKNOWLEDGEMENT:
The authors are thankful to K. B. Raval college of Pharmacy, Kasturinagar, Gandhinagar for providing the research facilities.
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Received on 17.07.2021 Modified on 05.11.2021
Accepted on 16.02.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2022; 15(11):4919-4923.
DOI: 10.52711/0974-360X.2022.00826