Original Article Marker based standardization of polyherbal formulation (SJT‑DI‑02) by high performance thin layer chromatography method Bhakti J. Ladva, Vijay M. Mahida1, Urmi D. Kantaria, Rina H. Gokani
Department of Quality Assurance, S.J. Thakkar Pharmacy College, Rajkot, 1Department of Pharmacology, Anand Pharmacy College, Anand, Gujarat, India
ABSTRACT
Background: Preparation of highly standardized herbal products with respect to chemical composition and biological activity is considered to be a valuable approach in this field. SJT‑DI‑02 polyherbal formulation was successfully developed at our institute and filed for patent at Mumbai patent office. Objective: The present work was marker based standardization of patented, novel and efficacious polyherbal formulation namely SJT‑DI‑02 for the treatment of diabetes. The SJT‑DI‑02 was comprised of dried extracts of rhizomes of Acorus calamus, leaves of Aegle marmelose, fruits of Benincasa hispida, roots of Chlorophytum arendinaceum, seeds Address for correspondence: of Eugenia jambolana, leaves of Ocimum sanctum, pericarp of Punica granatum, seeds of Tamarindus indica. Prof. Bhakti J. Ladva, Selected plants were collected, dried and extracted with suitable solvents. The formulation was prepared E‑mail: bhakti_ladva@yahoo. by mixing different fractions of extracts. Materials and Methods: For successful and best standardization, com first of all selection and procurement was carried out. Selection is done on the basis of therapeutic efficacy and amount of the marker present in the particular plant part. At the time of procurement side by side phytochemical screening and estimation of phytoconstituents was carried out. After completion of preliminary screening using characterized markers, we tried to develop best TLC systems using selected solvent composition. Finally well‑developed TLC systems were applied in HPTLC. In the present study polyherbal formulation was standardized by using different four markers. TLC Densitometric methods were developed using HPTLC for the quantification of these marker compounds. Solvent systems were optimized to achieve best resolution of the marker compounds from other components of the sample extract. The identity of the bands in the sample extracts were confirmed by comparing the R and the absorption spectra by overlaying f their UV absorption spectra with those of their respective standards. The purity of the bands due to marker compounds in the sample extracts were confirmed by overlaying the absorption spectra recorded at start, middle and end position of the band in the sample tracks. After conforming all these things fingerprints were developed for all three formulations which will be act as authentification and quality control tool. Results: % w/w of asarones is 3.61, % w/w of marmelosin is 4.60, % w/w of gallic acid is 10.80 and % w/w of lupeol is 4.13.The method was validated in terms of linearity, precision, repeatability, limit of detection, limit of quantification and accuracy. In well‑developed mobile phase system linearity was found to be in the range of 0.983‑0.995, % recovery was found to be in the range of 97.48‑99.63, % RSD for intraday and interday was found to be 0.13‑ 0.70 and 0.32 ‑1.41 and LOD and LOQ was found to be in the range of 0.15‑ 0.61 and 0.45 ‑1.83 microgram per ml. Conclusion: Thus High performance thin layer chromatography (HPTLC) Received : 09‑05‑13 methods were developed and validated in terms of linearity, precision, repeatability, limit of detection, limit Review completed : 25‑09‑13 Accepted : 14‑12‑13 of quantification and accuracy. The methods were rapid, sensitive, reproducible and economical. It does not suffer any positive or negative interference due to common other component present in the formulation and would also serve as a tool Access this article online for authentication of herbal products containing marmelosin, gallic Quick Response Code: acid, lupeol and asarones. Thus this work provides standardized Website: and therapeutically active polyherbal formulations for the different www.jpbsonline.org ailments. DOI: 10.4103/0975-7406.135249
KEY WORDS: Asarones, gallic acid, HPTLC, lupeol, marmelosin, SJT‑DI‑02
How to cite this article: Ladva BJ, Mahida VM, Kantaria UD, Gokani RH. Marker based standardization of polyherbal formulation (SJT-DI-02) by high performance thin layer chromatography method. J Pharm Bioall Sci 2014;6:213-9.
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I
n India, currently 50.8 million peoples are estimated to be suffering from diabetes. Further projections indicate that India will have maximum number of diabetic patients by the year 2030.[1] Currently available drugs having plenty number of side‑effects. So in the present scenario, there is usage of safe, efficacious and cheap antidiabetic drug from the plants. Medicinal plants used in the traditional Indian system of medicine are known to produce a variety of compounds with acknowledged therapeutic properties. In the past decade, there has been a paradigm shift from single‑target drugs to multi‑target polyherbal formulation.
arendinaceum and pericarps of Punica granatum were collected from Sanjivani Ayurvedic Store Rajkot. Dried leaves of A. marmelose were collected from Bhanavad, Dist: Jamnagar and Ocimum sanctum were collected from Dhoraji, Dist: Rajkot. Dried fruit of Benincasa hispida collected from Jubeli market, Rajkot. All the plant material were identified and authenticated by Prof. Vishal Muliya, Botany Department, Christ Science College, Rajkot, Gujarat, India. The plant parts were dried, powdered to 60# and stored at 25°C in air tight containers.
Standardization encourages marketing opportunism for polyherbal formulation.[2] It enables drug companies to claim exclusive patents for processed herbs. Standardization of herbal formulation in terms of raw materials, manufacturing practices and composition is important to ensure quality and optimum level of active principles for their bio potency. Identification of major and unique compounds in herbs as markers and development of analytical methodologies for monitoring them are the key steps involved in marker based standardization.[3] High performance thin layer chromatography (HPTLC) as a preferred analytical tool for fingerprints and quantification of marker compounds in herbal drugs due to its simplicity, sensitivity, accuracy, suitability for high throughput screening etc.[4]
Extraction and preparation of polyherbal formulation
SJT‑DI‑02 polyherbal formulation comprising of dried extracts of rhizomes of Acorus calamus, leaves of Aegle marmelose, fruits of Benincasa hispida, roots of Chlorophytum arendinaceum, seeds of Eugenia jambolana, leaves of Ocimum sanctum, pericarps of Punica granatum, seeds of Tamarindus indica. The polyherbal formulation SJT‑DI‑02 was developed and evaluated for its antidiabetic activity previously. The developed formulation was safe and efficacious.[5] To makes it available to large pool of patients, it should be standardized. Thus, the present study was aimed to standardize SJT‑DI‑02 using chemical markers marmelosin, gallic acid, lupeol and asarones by HPTLC methods.
Materials and Methods Plant materials Dried Rhizomes of A. calamus, seeds of Eugenia jambolana, seeds of Tamarindus indica, and dried roots of Chlorophytum Table 1: Preparation of SJT‑DI‑02 polyherbal formulation Crude drug
Extract prepared
% yield
Percentage of extracts in polyherbal formulation
Acorus calamus Aegle marmelos Benincasa hispida Chlorophytum arendinaceum Eugenia jambolana Ocimum sanctum Punica granatum Tamarindus indica
Ethyl acetate Aqueous Methanol 70% ethanol
8.61 26.87 18.03 13.33
7.5 12.5 7.5 7.5
Ethanol Ethanol Methanol Aqueous
23.59 14.90 32.90 10.52
12.5 12.5 16.0 25.0
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All the crude drug powders were extracted separately using different solvents. All the crude individual herbal extracts preparations and fractions are mentioned in Table 1. The extracts were concentrated and vacuum dried. The different polyherbal formulation was prepared by ‑ mixing the dried extracts in different proportions is shown in Table 1.
Chemicals and solvents Marmelosin, gallic acid, lupeol and asarones were procured from Natural remedies, Bangalore. All the solvents and chemicals in experiments were of analytical grade (Merck, Ltd., Mumbai).
Development of solvent system by TLC study Chemical markers and formulation (SJT‑DI‑02) was subjected for TLC study using different solvent systems and optimization was carried out for each marker.[6‑9] Solvent systems were optimized in order to get maximum separation of various phytochemical and it further used for HPTLC study.
Development of HPTLC methods HPTLC instrumentation The sample solutions were spotted in the form of bands of width 6 mm with a Hamilton 100 µl syringe on pre‑coated plate G 60 F254 (20 cm × 10 cm with 250 µm thickness, E. Merck) using a Camag Linomat V applicator. The slit dimension was kept 3 mm × 0.45 mm. Each track was scanned thrice and baseline correction was carried out. The mobile phase consisted of Toluene: Ethyl acetate: Methanol: Glacial acetic acid (8:2:0.25:0.2) (for asarones and marmelosin); Toluene: Ethyl acetate: Methanol: Formic acid. (3:3:0.2:0.8) (for gallic acid) and Benzene: Ethyl acetate (9:1) (for lupeol). Linear ascending development was carried out in 20 cm × 10 cm, a Camag twin trough glass Chamber saturated with the mobile phase for each marker. The optimized chamber saturation time for mobile phase was 30 min at room temperature (25 ± 1°C). The migration distance was 60 mm. TLC plates were air dried with air dryer. Densitometric scanning was performed using Camag TLC Scanner‑III at 305 nm (marmelosin), 245 nm (gallic acid), 559 nm (lupeol) and 295 nm (asarones) operated by a Camag data evaluation 32 bit software. Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3
Ladva, et al.: Standardization of polyherbal formulation by HPTLC method
Preparation of solutions Preparation of standard solution of chemical marker compounds Accurately weighed 2.5 mg of marmelosin, gallic acid and lupeol (0.5 mg/ml) and 15 mg of asarones (3 mg/ml) were dissolved in 5 ml of methanol respectively in a volumetric flask separately.
Preparation of sample solutions
experiments were performed by adding three different amounts of standard drug, i.e., 50, 100 and 150% of the drug, to the pre‑analyzed formulation (SJT‑DI‑02) and the resultant is reanalyzed 3 times and %recovery was calculated. The specificity of the method was ascertained by matching of overlain spectra of standard and test formulation. Limits of detection (LOD) and limits of quantification (LOQ) were determined at signal‑to‑noise ratio of 3:1 and 10:1 (3.3 σ/S and 10σ/S) respectively. Determination of the signal to noise ratio was performed by comparing measured signals from samples with known low concentration of analyte can be reliably detected[4].
Accurately weighed 50 mg of SJT‑DI‑02 dissolved in 10 ml of methanol (5.0 mg/ml) in a volumetric flask.
Results and Discussion
Calibration curves of chemical marker compounds
Preliminary TLC study for solvent system optimization
From standard marmelosin, gallic acid, lupeol solutions (0.5 mg/ml), 3, 4, 5, 6 and 7 µl and for whereas standard asarones solution (3 mg/ml), 2, 3, 4, 5 and 6 µl volume were spotted separately. The plates were developed and dried. Lupeol spotted plate was derivatized by 5% aqueous H2SO4 and heated at 110°C. The marmelosin, gallic acid, lupeol and asarones spotted plates were scanned at 305, 245, 559 and 295 nm respectively. Data of peak height and peak area of each marker spots were recorded. Standard curve of peak area versus concentrations were plotted.
Solvent systems were optimized to achieve best resolution of the marker compounds from other components of the formulations through TLC study.
Estimation of chemical markers in polyherbal formulations A volume of 35 µl of test solution for marmelosin and asarones, 25 µl of test solution for gallic acid and 50 µl of test solution for lupeol were used for spotting. The plates were developed and dried. For lupeol the plate was derivatized by 5% aqueous H2SO4 and heated at 110°C. The developed plates were scanned at 305 nm (marmelosin), 245 nm (gallic acid), 559 nm (lupeol) and 295 nm (asarones). Peak areas were noted and concentration of markers in the formulation was calculated from the calibration curve.
Validation of HPTLC methods Linearity of the method was evaluated by constructing calibration curves, i.e., peak area versus concentrations, at different concentration levels (1.5‑3.5 µg/spot of pure marmelosin, gallic acid, lupeol [0.5 mg/ml] and 6‑18 µg/spot of asarones [3 mg/ml]) respectively. The results were expressed in terms of correlation co‑efficient of the linear regression analysis. Intra‑day and inter‑day precision were determined by analyzing sample solutions of analyte at three levels covering low, medium and higher concentrations of calibration curve (2‑3 µg/spot of pure marmelosin, gallic acid, lupeol (0.5 mg/ml) and 9‑15 µg/spot of pure asarones (3 mg/ml) respectively for 3 times on the same day and over a period of 3 days. The peak areas obtained were used to calculate mean and % coefficient variance (CV) values. Repeatability of measurement of peak area and sample application (by analyzing 2.5, µg/spot of pure solution of marmelosin, gallic acid, lupeol [0.5 mg/ml] and 12 µg/spot of pure solution of asarones [3 mg/ml]), Recovery Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3
Marmelosin Marmelosin showed single dark violet colored spot at Rf ‑ 0.50 in Toluene: Ethyl acetate: Glacial acetic acid: Methanol (8:2:0.2:0.25). Table 2: Estimation of marmelosin, gallic acid, lupeol and asarones in SJT‑DI‑02 Marker compounds
Mean peak area (n=3)
Average amount of marker (µg/spot)
Average % of marker compounds
% CV
Marmelosin Gallic acid Lupeol Asarones
15029.9±17.28 16697.37±65.9 13057.9±26.68 20355.77±14.2
1.61 2.70 2.06 1.62
4.60 10.80 4.13 3.61
0.11 0.39 0.20 0.11
CV: Coefficient variance
Table 3: Calibration data of standard gallic acid concentration versus mean peak area Concentration of gallic acid (μg/spot)
Mean peak area±SD (n=3)
% CV
1.5 2 2.5 3 3.5
11846.17±11.08 13761.37±15.23 16227.57±16.05 17255.4±17.88 20292.53±8.68
0.09 0.11 0.09 0.10 0.04
CV: Coefficient variance, SD: Standard deviation
Table 4: Calibration data of standard lupeol concentration versus mean peak area Concentration of lupeol (μg/spot)
Mean peak area±SD (n=3)
% CV
1.5 2 2.5 3 3.5
11962.75±14.77 12843.8±19.94 14032.2±25.32 14876.3±28.14 15739.3±30.75
0.12 0.15 0.18 0.18 0.19
CV: Coefficient variance, SD: Standard deviation 215
Ladva, et al.: Standardization of polyherbal formulation by HPTLC method
Gallic acid
Estimation of the chemical markers in SJT‑DI‑02
Gallic acid showed single dark violet colored spot at Rf ‑ 0.39 in Toluene: Ethyl acetate: Formic acid: Methanol (3:3:0.8:0.2).
The amount of marmelosin, gallic acid, lupeol and asarones were computed from the calibration curves as shown in Table 2. 4.60%, 10.80, 4.13 and 3.61% w/w were found to be present in SJT‑DI‑02.
Lupeol Lupeol showed single dark brown colored spot at Rf ‑ 0.34 in Benzene: Ethyl acetate (9:1) and derivatization with 5% sulfuric acid.
Calibration curve data for marmelosin, gallic acid, lupeol and asarones are shown in Tables 3-6 respectively.
Validation of HPTLC method
Asarones
Linearity
Asarones showed single dark violet colored spot at Rf ‑ 0.56 in Toluene: Ethyl acetate: Formic acid: Methanol (3:3:0.8:0.2).
Linearity were obtained for marmelosin, gallic acid, lupeol and asarones in the range of 1.5‑3.5, 1.5‑3.5, 1.5‑3.5 and 6‑18 µg/spot with correlation coefficient of 0.988, 0.983, 0.995 and 0.984 respectively.
The purity of the spot of standard and test was confirmed by same Rf value, same colored spot was found in SJT‑DI‑02 formulation and it was further confirmed by matching of ultraviolet absorption spectra. Table 5: Calibration data of standard asarones concentration versus mean peak area Concentration of asarones (μg/spot)
Mean peak area±SD (n=3)
% CV
6 9 12 15 18
25238.3±37.70 27716.4±32.15 31739.63±35.74 33468.57±62.27 35925.31±36.68
0.14 0.11 0.11 0.18 0.10
CV: Coefficient variance, SD: Standard deviation
Table 6: Calibration data of standard marmelosin concentration versus mean peak area Concentration of marmelosin (µg/spot)
Mean peak area±SD (n=3)
% CV
1.5 2 2.5 3 3.5
14887.6±58.37 15715.63±36.15 17535±54.84 19030.03±20.10 20080.5±57.59
0.39 0.23 0.31 0.10 0.28
CV: Coefficient variance, SD: Standard deviation
Precision The intra‑day and inter‑day (%CV) for marmelosin (0.15‑0.19 and 0.51‑0.62%), gallic acid (0.13‑0.15 and 0.43‑0.72%), lupeol (0.12‑0.17 and 0.32‑0.65%) and asarones (0.55‑0.70 and 1.13‑1.41%) as shown in Table 7 were found less than 1.5% CV and suggested that all the developed methods were precise. Repeatability CV for repeatability of measurement of peak area of 7 times measurement of the same spots of marmelosin, gallic acid, lupeol and asarones were found to be 0.31, 0.16, 0.18, and 0.13 respectively. The %CV of repeatability of sample application of marmelosin, gallic acid, lupeol and asarones were found to be 0.74, 0.52, 0.35 and 0.72 respectively. The low values of %CV indicate the precision of the method. Accuracy (%recovery) The %recovery of marmelosin, gallic acid, lupeol and asarones were found to be 97.48‑98.21, 98.13‑99.49, 98.23‑99.63 and 97.76‑99.30 respectively in SJT‑DI‑02 which was satisfactory as shown in Table 8.
Table 7: Data for intra‑day and inter‑day precision Marker compounds
Concentration (µg/spot)
Peak area (mean±SD)
% CV
Peak area (mean±SD)
% CV
Marmelosin
2 2.5 3 2 2.5 3 2 2.5 3 9 12 15
15678.23±24.45 17932.12±30.59 19056.46±36.65 13749.50±21.12 16234.63±21.68 17253.46±27.06 12833.57±22.55 14143.03±25.35 14829.03±18.90 27709.03±164.2 31719.0 6±222.1 33589.56±186.2
0.15 0.17 0.19 0.15 0.13 0.15 0.17 0.17 0.12 0.59 0.70 0.55
15599.6±96.75 17834.7±90.62 19122.4±98.13 13653.3±98.55 16185.5±70.30 17178.7±78.97 12753.3±83.28 14189.1±68.68 14785.9±48.61 27363.2±283.8 31253.3±442.1 33040.7±375.6
0.62 0.50 0.51 0.72 0.43 0.45 0.65 0.48 0.32 1.03 1.41 1.13
Gallic acid Lupeol Asarones
Intra‑day precision (n=3)
Inter‑day precision (n=5)
CV: Coefficient variance, SD: Standard deviation
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Table 8: Data of accuracy in SJT-DI‑02 Marker compounds
Concentration of marker compounds (µg/spot)
Marmelosin Gallic acid Lupeol Asarones
Added
Amount of marker compounds found mean±SD (n=3)
% recovery (n=3)
Taken 2.3 2.3 2.3 2.7 2.7 2.7 2.06 2.06 2.06 2.34 2.34 2.34
1.15 2.3 3.45 1.35 2.7 4.05 1.03 2.06 3.09 1.17 2.34 3.51
19883.9±28.89 22956.9±45.32 25941.8±17.45 21896.6±49.81 27588.6±94.47 32688.2±35.92 14922.6±66.04 16980.8±55.75 18866.1±67.35 23118.6±19.00 24151.2±48.62 25138.2±54.80
98.21 98.10 97.48 98.19 99.49 98.13 98.23 99.63 98.72 99.30 98.87 97.76
SD: Standard deviation
Table 9: Summary of the validation parameters of the developed HPTLC methods Marker compounds
Regression coefficient
Precision % RSD (intra and inter day)
Repeatability of measurement
Repeatability sample application
Accuracy
LOD µg/spot
Limit of quantification µg/spot
Marmelosin
0.98
0.31
0.74
97.48‑98.21
0.15
0.45
Gallic acid
0.983
0.16
0.52
98.13‑99.49
0.15
0.45
Lupeol
0.995
0.18
0.35
98.23‑99.63
0.15
0.46
Asarones
0.984
0.15‑0.19 0.50‑0.62 0.13‑0.15 0.43‑0.72 0.12‑0.17 0.32‑0.65 0.55‑0.70 1.03‑1.41
0.13
0.72
97.76‑99.30
0.613
1.83
HPTLC: High performance thin layer chromatography, RSD: Relative standard deviation, LOD: Limit of detection, LOQ: Limit of quantification
Specificity The spectra of spot of standard markers were matched with the spots found in the formulation suggested that there were no integer with the peak of the markers. Therefore, all the developed methods were specific.
LOD The minimum detectable limit was found to be 0.15, 0.15, 0.15 and 0.61 µg/spot for marmelosin, gallic acid, lupeol and asarones respectively.
LOQ The minimum quantification limit was found to be 0.45, 0.45, 0.46 and 1.83 µg/spot for marmelosin, gallic acid, lupeol and asarones respectively. Thus the developed HPTLC methods were found to simple, precise, sensitive and accurate and can be used for the quantification of the marmelosin, gallic acid, lupeol and asarones in SJT‑DI‑02 and also in routine quality control of the raw materials as well as formulations containing any of these compounds. In Table 9, summary of the validation parameters of the developed HPTLC methods are shown. Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3
Figure 1a-e shows Co‑chromatography of marmelosin, Densotometric chromatogram of calibration curve of marmelosin, Calibration curve of marmelosin concentration versus mean peak area, Overlain ultraviolet spectra of marmelosin with SJT‑DI‑02 and Densitometric chromatogram of marmelosin with SJT‑DI‑02 respectively. Figure 2a-e shows Co‑chromatography of gallic acid, Densitometric chromatogram of calibration curve of gallic acid, Calibration curve of gallic acid concentration versus mean peak area, Overlain ultraviolet spectra of gallic acid with SJT‑DI‑02 and Densitometric chromatogram of gallic acid with SJT‑DI‑02 respectively. Figure 3a-e shows Co‑chromatography of lupeol, Densitometric chromatogram of calibration curve of lupeol, Calibration curve of lupeol concentration versus mean peak area, Overlain ultraviolet spectra of lupeol with SJT‑DI‑02 and Densitometric chromatogram of lupeol with SJT‑DI‑02 respectively. Figure 4a-e shows Co‑chromatography of asarones, Densitometric chromatogram of calibration curve of asarones, Calibration curve of asarones concentration versus mean peak area, Overlain ultraviolet spectra of asarones with SJT‑DI‑02 and Densitometric chromatogram of asarones with SJT‑DI‑02 respectively.
Conclusion We have established simple, precise, specific and economical HPTLC Densitometric methods for the quantification of the marmelosin, gallic acid, lupeol and asarones bio active markers found in SJT‑DI‑02. The methods were validated to trace the 217
Ladva, et al.: Standardization of polyherbal formulation by HPTLC method
a
b
(B) Densotometric chromatogram of calibration curve of marmelosin scanned at 305nm
(a) 35 μg/spot of SJT-DI-02 (b) 1.5-3.5 μg/spot of marmelosin
a2
(B) Densotometric chromatogram of calibrationcurve of gallic acid scanned at 245nm
b
(b) 1.5-3.5 μg/spot of gallic acid
(A) Co chromatography of gallic acid in SJT-DI-02
25000
y = 2740x + 10600 R² = 0.988
20000 15000
a – Marmelosin
25000
b – SJT-DI-02
20000
10000
Peak area
Peak area
a1
(a1, a2) 35, 50 μg/spot of SJT-DI-02
(A) Co chromatography of marmelosinin SJT-DI-02
5000 a
0 0
1 2 3 Concentration (µg/spot)
y = 4077.4x + 5683.2 R² = 0.9839 a - Gallic acid
15000
b – SJT-DI-02
10000
b
5000
4
ab
0 0
(C) Calibration curve of marmelosin concentration Vs mean peak area
1 2 3 Concentration (µg/spot )
4
(D) Overlain UV spectra of marmelosin with SJT-DI-02
(C) Calibration curve of gallic acidconcentration Vs mean peak area.
(D) Overlain UV spectra of gallic acid with SJT-DI-02
a - Marmelosin
a - Gallic acid
b –SJT-DI-02
b - SJT-DI-02
a
a b
b
(E) Densotometric chromatogram of Marmelosin with SJT-DI-02
(E) Densotometric chromatogram of gallic acid with SJT-DI-02
Figure 1: Estimation of Marmelosin by HPTLC Method
Figure 2: Estimation of Gallic acid by HPTLC Method
ab
a
(a) 1.5-3.5 μg/spot of lupeol
(B) Densotometric chromatogram of calibration curve of lupeol scanned at 559 nm
(b) 50 μg/spot of SJT-DI-02
Peak area
Peak area
a b
a - Lupeol
y = 1924.66x + 9080.38 R² = 0.995 0
1 2 3 concentration(µg/spot)
(B) Densotometric chromatogram of calibration curve of asarones scanned at 295 nm
(A) Co chromatography of asarones in SJT-DI-02
(A) Co chromatography of lupeolSJT-DI-02 18000 16000 14000 12000 10000 8000 6000 4000 2000 0
b
(b) 1.5-3.5 μg/spot of asarones
(a) 35 μg/spot of SJT-DI-02
b - SJT-DI-02
40000 35000 30000 25000 20000 15000 10000 5000 0
a – Asarones b - SJT-DI-02
y = 904.21x + 19967 R² = 0.9849 a b
0
5
10
15
20
Concentration (µg/spot)
4
(C) Calibration curve of asarones concentration Vs mean peak area
(C) Calibration curve of lupeol concentration Vs mean peak area
(D) Overlain UV spectra of asarones with SJT-DI-02
(D) Overlain UV spectra of lupeol with SJT-DI-02
a -Asarones
a - Lupeol
b - SJT-DI-02
ab
a b
(E) Densotometric chromatogram of lupeol with SJT-DI-02
Figure 3: Estimation of Lupeol by HPTLC Method
218
(E) Densitometric chromatogram of asarones with SJT-DI-02
Figure 4: Estimation of Asarones by HPTLC Method Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3
Ladva, et al.: Standardization of polyherbal formulation by HPTLC method
markers in complex mixtures of herbal drugs used in SJT‑DI‑02. Developed methods can also be used in routine quality control of herbal raw material as well as formulation containing the same markers.
Acknowledgment
4. 5. 6.
The authors are thankful to Anchorm Pvt. Ltd., Mumbai and Saurashtra University Rajkot for providing guidance and facilities.
7.
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2007. Available from: http://www.pharmainfo.net/reviews/ herbal‑medicine‑and‑its‑standardization. [Accessed on 2011 Dec 25]. Srivastava M. High‑Performance Thin‑Layer Chromatography (HPTLC). 5th ed. Heidelberg, Dordrecht, London, New York: Springer; 1965. p. 28‑34. Meghanathi MB. Thesis‑Evaluation of polyherbal formulation for the treatment of diabetes. Rajkot (Gujarat): S.J. Thakkar Pharmacy College; 2011. Widmer V, Schibli A, Reich E. Quantitative determination of beta‑asarone in calamus by high‑performance thin‑layer chromatography. J AOAC Int 2005;88:1562‑7. Patel NV, Telange DR. Qualitative and quantitative estimation of gallic acid and ascorbic acid in polyherbal tablets. Int J Pharm Sci Res 2011;2:2394‑8. Rachchh MA, Jain SM. Gastroprotective effect of Benincasa hispida fruit extract. Indian J Pharmacol 2008;40:271‑5. Shailajan S, Menon S, Hande H. Method validation of marmelosin from fruit pulp of Aegle marmelose Correa using HPTLC technique. J Pharm Res 2011;4:1353‑5. Source of Support: Nil, Conflict of Interest: None declared.
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Department of Quality Assurance, S.J. Thakkar Pharmacy College, Rajkot, 1Department of Pharmacology, Anand Pharmacy College, Anand, Gujarat, India
ABSTRACT
Background: Preparation of highly standardized herbal products with respect to chemical composition and biological activity is considered to be a valuable approach in this field. SJT‑DI‑02 polyherbal formulation was successfully developed at our institute and filed for patent at Mumbai patent office. Objective: The present work was marker based standardization of patented, novel and efficacious polyherbal formulation namely SJT‑DI‑02 for the treatment of diabetes. The SJT‑DI‑02 was comprised of dried extracts of rhizomes of Acorus calamus, leaves of Aegle marmelose, fruits of Benincasa hispida, roots of Chlorophytum arendinaceum, seeds Address for correspondence: of Eugenia jambolana, leaves of Ocimum sanctum, pericarp of Punica granatum, seeds of Tamarindus indica. Prof. Bhakti J. Ladva, Selected plants were collected, dried and extracted with suitable solvents. The formulation was prepared E‑mail: bhakti_ladva@yahoo. by mixing different fractions of extracts. Materials and Methods: For successful and best standardization, com first of all selection and procurement was carried out. Selection is done on the basis of therapeutic efficacy and amount of the marker present in the particular plant part. At the time of procurement side by side phytochemical screening and estimation of phytoconstituents was carried out. After completion of preliminary screening using characterized markers, we tried to develop best TLC systems using selected solvent composition. Finally well‑developed TLC systems were applied in HPTLC. In the present study polyherbal formulation was standardized by using different four markers. TLC Densitometric methods were developed using HPTLC for the quantification of these marker compounds. Solvent systems were optimized to achieve best resolution of the marker compounds from other components of the sample extract. The identity of the bands in the sample extracts were confirmed by comparing the R and the absorption spectra by overlaying f their UV absorption spectra with those of their respective standards. The purity of the bands due to marker compounds in the sample extracts were confirmed by overlaying the absorption spectra recorded at start, middle and end position of the band in the sample tracks. After conforming all these things fingerprints were developed for all three formulations which will be act as authentification and quality control tool. Results: % w/w of asarones is 3.61, % w/w of marmelosin is 4.60, % w/w of gallic acid is 10.80 and % w/w of lupeol is 4.13.The method was validated in terms of linearity, precision, repeatability, limit of detection, limit of quantification and accuracy. In well‑developed mobile phase system linearity was found to be in the range of 0.983‑0.995, % recovery was found to be in the range of 97.48‑99.63, % RSD for intraday and interday was found to be 0.13‑ 0.70 and 0.32 ‑1.41 and LOD and LOQ was found to be in the range of 0.15‑ 0.61 and 0.45 ‑1.83 microgram per ml. Conclusion: Thus High performance thin layer chromatography (HPTLC) Received : 09‑05‑13 methods were developed and validated in terms of linearity, precision, repeatability, limit of detection, limit Review completed : 25‑09‑13 Accepted : 14‑12‑13 of quantification and accuracy. The methods were rapid, sensitive, reproducible and economical. It does not suffer any positive or negative interference due to common other component present in the formulation and would also serve as a tool Access this article online for authentication of herbal products containing marmelosin, gallic Quick Response Code: acid, lupeol and asarones. Thus this work provides standardized Website: and therapeutically active polyherbal formulations for the different www.jpbsonline.org ailments. DOI: 10.4103/0975-7406.135249
KEY WORDS: Asarones, gallic acid, HPTLC, lupeol, marmelosin, SJT‑DI‑02
How to cite this article: Ladva BJ, Mahida VM, Kantaria UD, Gokani RH. Marker based standardization of polyherbal formulation (SJT-DI-02) by high performance thin layer chromatography method. J Pharm Bioall Sci 2014;6:213-9.
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Ladva, et al.: Standardization of polyherbal formulation by HPTLC method
I
n India, currently 50.8 million peoples are estimated to be suffering from diabetes. Further projections indicate that India will have maximum number of diabetic patients by the year 2030.[1] Currently available drugs having plenty number of side‑effects. So in the present scenario, there is usage of safe, efficacious and cheap antidiabetic drug from the plants. Medicinal plants used in the traditional Indian system of medicine are known to produce a variety of compounds with acknowledged therapeutic properties. In the past decade, there has been a paradigm shift from single‑target drugs to multi‑target polyherbal formulation.
arendinaceum and pericarps of Punica granatum were collected from Sanjivani Ayurvedic Store Rajkot. Dried leaves of A. marmelose were collected from Bhanavad, Dist: Jamnagar and Ocimum sanctum were collected from Dhoraji, Dist: Rajkot. Dried fruit of Benincasa hispida collected from Jubeli market, Rajkot. All the plant material were identified and authenticated by Prof. Vishal Muliya, Botany Department, Christ Science College, Rajkot, Gujarat, India. The plant parts were dried, powdered to 60# and stored at 25°C in air tight containers.
Standardization encourages marketing opportunism for polyherbal formulation.[2] It enables drug companies to claim exclusive patents for processed herbs. Standardization of herbal formulation in terms of raw materials, manufacturing practices and composition is important to ensure quality and optimum level of active principles for their bio potency. Identification of major and unique compounds in herbs as markers and development of analytical methodologies for monitoring them are the key steps involved in marker based standardization.[3] High performance thin layer chromatography (HPTLC) as a preferred analytical tool for fingerprints and quantification of marker compounds in herbal drugs due to its simplicity, sensitivity, accuracy, suitability for high throughput screening etc.[4]
Extraction and preparation of polyherbal formulation
SJT‑DI‑02 polyherbal formulation comprising of dried extracts of rhizomes of Acorus calamus, leaves of Aegle marmelose, fruits of Benincasa hispida, roots of Chlorophytum arendinaceum, seeds of Eugenia jambolana, leaves of Ocimum sanctum, pericarps of Punica granatum, seeds of Tamarindus indica. The polyherbal formulation SJT‑DI‑02 was developed and evaluated for its antidiabetic activity previously. The developed formulation was safe and efficacious.[5] To makes it available to large pool of patients, it should be standardized. Thus, the present study was aimed to standardize SJT‑DI‑02 using chemical markers marmelosin, gallic acid, lupeol and asarones by HPTLC methods.
Materials and Methods Plant materials Dried Rhizomes of A. calamus, seeds of Eugenia jambolana, seeds of Tamarindus indica, and dried roots of Chlorophytum Table 1: Preparation of SJT‑DI‑02 polyherbal formulation Crude drug
Extract prepared
% yield
Percentage of extracts in polyherbal formulation
Acorus calamus Aegle marmelos Benincasa hispida Chlorophytum arendinaceum Eugenia jambolana Ocimum sanctum Punica granatum Tamarindus indica
Ethyl acetate Aqueous Methanol 70% ethanol
8.61 26.87 18.03 13.33
7.5 12.5 7.5 7.5
Ethanol Ethanol Methanol Aqueous
23.59 14.90 32.90 10.52
12.5 12.5 16.0 25.0
214
All the crude drug powders were extracted separately using different solvents. All the crude individual herbal extracts preparations and fractions are mentioned in Table 1. The extracts were concentrated and vacuum dried. The different polyherbal formulation was prepared by ‑ mixing the dried extracts in different proportions is shown in Table 1.
Chemicals and solvents Marmelosin, gallic acid, lupeol and asarones were procured from Natural remedies, Bangalore. All the solvents and chemicals in experiments were of analytical grade (Merck, Ltd., Mumbai).
Development of solvent system by TLC study Chemical markers and formulation (SJT‑DI‑02) was subjected for TLC study using different solvent systems and optimization was carried out for each marker.[6‑9] Solvent systems were optimized in order to get maximum separation of various phytochemical and it further used for HPTLC study.
Development of HPTLC methods HPTLC instrumentation The sample solutions were spotted in the form of bands of width 6 mm with a Hamilton 100 µl syringe on pre‑coated plate G 60 F254 (20 cm × 10 cm with 250 µm thickness, E. Merck) using a Camag Linomat V applicator. The slit dimension was kept 3 mm × 0.45 mm. Each track was scanned thrice and baseline correction was carried out. The mobile phase consisted of Toluene: Ethyl acetate: Methanol: Glacial acetic acid (8:2:0.25:0.2) (for asarones and marmelosin); Toluene: Ethyl acetate: Methanol: Formic acid. (3:3:0.2:0.8) (for gallic acid) and Benzene: Ethyl acetate (9:1) (for lupeol). Linear ascending development was carried out in 20 cm × 10 cm, a Camag twin trough glass Chamber saturated with the mobile phase for each marker. The optimized chamber saturation time for mobile phase was 30 min at room temperature (25 ± 1°C). The migration distance was 60 mm. TLC plates were air dried with air dryer. Densitometric scanning was performed using Camag TLC Scanner‑III at 305 nm (marmelosin), 245 nm (gallic acid), 559 nm (lupeol) and 295 nm (asarones) operated by a Camag data evaluation 32 bit software. Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3
Ladva, et al.: Standardization of polyherbal formulation by HPTLC method
Preparation of solutions Preparation of standard solution of chemical marker compounds Accurately weighed 2.5 mg of marmelosin, gallic acid and lupeol (0.5 mg/ml) and 15 mg of asarones (3 mg/ml) were dissolved in 5 ml of methanol respectively in a volumetric flask separately.
Preparation of sample solutions
experiments were performed by adding three different amounts of standard drug, i.e., 50, 100 and 150% of the drug, to the pre‑analyzed formulation (SJT‑DI‑02) and the resultant is reanalyzed 3 times and %recovery was calculated. The specificity of the method was ascertained by matching of overlain spectra of standard and test formulation. Limits of detection (LOD) and limits of quantification (LOQ) were determined at signal‑to‑noise ratio of 3:1 and 10:1 (3.3 σ/S and 10σ/S) respectively. Determination of the signal to noise ratio was performed by comparing measured signals from samples with known low concentration of analyte can be reliably detected[4].
Accurately weighed 50 mg of SJT‑DI‑02 dissolved in 10 ml of methanol (5.0 mg/ml) in a volumetric flask.
Results and Discussion
Calibration curves of chemical marker compounds
Preliminary TLC study for solvent system optimization
From standard marmelosin, gallic acid, lupeol solutions (0.5 mg/ml), 3, 4, 5, 6 and 7 µl and for whereas standard asarones solution (3 mg/ml), 2, 3, 4, 5 and 6 µl volume were spotted separately. The plates were developed and dried. Lupeol spotted plate was derivatized by 5% aqueous H2SO4 and heated at 110°C. The marmelosin, gallic acid, lupeol and asarones spotted plates were scanned at 305, 245, 559 and 295 nm respectively. Data of peak height and peak area of each marker spots were recorded. Standard curve of peak area versus concentrations were plotted.
Solvent systems were optimized to achieve best resolution of the marker compounds from other components of the formulations through TLC study.
Estimation of chemical markers in polyherbal formulations A volume of 35 µl of test solution for marmelosin and asarones, 25 µl of test solution for gallic acid and 50 µl of test solution for lupeol were used for spotting. The plates were developed and dried. For lupeol the plate was derivatized by 5% aqueous H2SO4 and heated at 110°C. The developed plates were scanned at 305 nm (marmelosin), 245 nm (gallic acid), 559 nm (lupeol) and 295 nm (asarones). Peak areas were noted and concentration of markers in the formulation was calculated from the calibration curve.
Validation of HPTLC methods Linearity of the method was evaluated by constructing calibration curves, i.e., peak area versus concentrations, at different concentration levels (1.5‑3.5 µg/spot of pure marmelosin, gallic acid, lupeol [0.5 mg/ml] and 6‑18 µg/spot of asarones [3 mg/ml]) respectively. The results were expressed in terms of correlation co‑efficient of the linear regression analysis. Intra‑day and inter‑day precision were determined by analyzing sample solutions of analyte at three levels covering low, medium and higher concentrations of calibration curve (2‑3 µg/spot of pure marmelosin, gallic acid, lupeol (0.5 mg/ml) and 9‑15 µg/spot of pure asarones (3 mg/ml) respectively for 3 times on the same day and over a period of 3 days. The peak areas obtained were used to calculate mean and % coefficient variance (CV) values. Repeatability of measurement of peak area and sample application (by analyzing 2.5, µg/spot of pure solution of marmelosin, gallic acid, lupeol [0.5 mg/ml] and 12 µg/spot of pure solution of asarones [3 mg/ml]), Recovery Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3
Marmelosin Marmelosin showed single dark violet colored spot at Rf ‑ 0.50 in Toluene: Ethyl acetate: Glacial acetic acid: Methanol (8:2:0.2:0.25). Table 2: Estimation of marmelosin, gallic acid, lupeol and asarones in SJT‑DI‑02 Marker compounds
Mean peak area (n=3)
Average amount of marker (µg/spot)
Average % of marker compounds
% CV
Marmelosin Gallic acid Lupeol Asarones
15029.9±17.28 16697.37±65.9 13057.9±26.68 20355.77±14.2
1.61 2.70 2.06 1.62
4.60 10.80 4.13 3.61
0.11 0.39 0.20 0.11
CV: Coefficient variance
Table 3: Calibration data of standard gallic acid concentration versus mean peak area Concentration of gallic acid (μg/spot)
Mean peak area±SD (n=3)
% CV
1.5 2 2.5 3 3.5
11846.17±11.08 13761.37±15.23 16227.57±16.05 17255.4±17.88 20292.53±8.68
0.09 0.11 0.09 0.10 0.04
CV: Coefficient variance, SD: Standard deviation
Table 4: Calibration data of standard lupeol concentration versus mean peak area Concentration of lupeol (μg/spot)
Mean peak area±SD (n=3)
% CV
1.5 2 2.5 3 3.5
11962.75±14.77 12843.8±19.94 14032.2±25.32 14876.3±28.14 15739.3±30.75
0.12 0.15 0.18 0.18 0.19
CV: Coefficient variance, SD: Standard deviation 215
Ladva, et al.: Standardization of polyherbal formulation by HPTLC method
Gallic acid
Estimation of the chemical markers in SJT‑DI‑02
Gallic acid showed single dark violet colored spot at Rf ‑ 0.39 in Toluene: Ethyl acetate: Formic acid: Methanol (3:3:0.8:0.2).
The amount of marmelosin, gallic acid, lupeol and asarones were computed from the calibration curves as shown in Table 2. 4.60%, 10.80, 4.13 and 3.61% w/w were found to be present in SJT‑DI‑02.
Lupeol Lupeol showed single dark brown colored spot at Rf ‑ 0.34 in Benzene: Ethyl acetate (9:1) and derivatization with 5% sulfuric acid.
Calibration curve data for marmelosin, gallic acid, lupeol and asarones are shown in Tables 3-6 respectively.
Validation of HPTLC method
Asarones
Linearity
Asarones showed single dark violet colored spot at Rf ‑ 0.56 in Toluene: Ethyl acetate: Formic acid: Methanol (3:3:0.8:0.2).
Linearity were obtained for marmelosin, gallic acid, lupeol and asarones in the range of 1.5‑3.5, 1.5‑3.5, 1.5‑3.5 and 6‑18 µg/spot with correlation coefficient of 0.988, 0.983, 0.995 and 0.984 respectively.
The purity of the spot of standard and test was confirmed by same Rf value, same colored spot was found in SJT‑DI‑02 formulation and it was further confirmed by matching of ultraviolet absorption spectra. Table 5: Calibration data of standard asarones concentration versus mean peak area Concentration of asarones (μg/spot)
Mean peak area±SD (n=3)
% CV
6 9 12 15 18
25238.3±37.70 27716.4±32.15 31739.63±35.74 33468.57±62.27 35925.31±36.68
0.14 0.11 0.11 0.18 0.10
CV: Coefficient variance, SD: Standard deviation
Table 6: Calibration data of standard marmelosin concentration versus mean peak area Concentration of marmelosin (µg/spot)
Mean peak area±SD (n=3)
% CV
1.5 2 2.5 3 3.5
14887.6±58.37 15715.63±36.15 17535±54.84 19030.03±20.10 20080.5±57.59
0.39 0.23 0.31 0.10 0.28
CV: Coefficient variance, SD: Standard deviation
Precision The intra‑day and inter‑day (%CV) for marmelosin (0.15‑0.19 and 0.51‑0.62%), gallic acid (0.13‑0.15 and 0.43‑0.72%), lupeol (0.12‑0.17 and 0.32‑0.65%) and asarones (0.55‑0.70 and 1.13‑1.41%) as shown in Table 7 were found less than 1.5% CV and suggested that all the developed methods were precise. Repeatability CV for repeatability of measurement of peak area of 7 times measurement of the same spots of marmelosin, gallic acid, lupeol and asarones were found to be 0.31, 0.16, 0.18, and 0.13 respectively. The %CV of repeatability of sample application of marmelosin, gallic acid, lupeol and asarones were found to be 0.74, 0.52, 0.35 and 0.72 respectively. The low values of %CV indicate the precision of the method. Accuracy (%recovery) The %recovery of marmelosin, gallic acid, lupeol and asarones were found to be 97.48‑98.21, 98.13‑99.49, 98.23‑99.63 and 97.76‑99.30 respectively in SJT‑DI‑02 which was satisfactory as shown in Table 8.
Table 7: Data for intra‑day and inter‑day precision Marker compounds
Concentration (µg/spot)
Peak area (mean±SD)
% CV
Peak area (mean±SD)
% CV
Marmelosin
2 2.5 3 2 2.5 3 2 2.5 3 9 12 15
15678.23±24.45 17932.12±30.59 19056.46±36.65 13749.50±21.12 16234.63±21.68 17253.46±27.06 12833.57±22.55 14143.03±25.35 14829.03±18.90 27709.03±164.2 31719.0 6±222.1 33589.56±186.2
0.15 0.17 0.19 0.15 0.13 0.15 0.17 0.17 0.12 0.59 0.70 0.55
15599.6±96.75 17834.7±90.62 19122.4±98.13 13653.3±98.55 16185.5±70.30 17178.7±78.97 12753.3±83.28 14189.1±68.68 14785.9±48.61 27363.2±283.8 31253.3±442.1 33040.7±375.6
0.62 0.50 0.51 0.72 0.43 0.45 0.65 0.48 0.32 1.03 1.41 1.13
Gallic acid Lupeol Asarones
Intra‑day precision (n=3)
Inter‑day precision (n=5)
CV: Coefficient variance, SD: Standard deviation
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Table 8: Data of accuracy in SJT-DI‑02 Marker compounds
Concentration of marker compounds (µg/spot)
Marmelosin Gallic acid Lupeol Asarones
Added
Amount of marker compounds found mean±SD (n=3)
% recovery (n=3)
Taken 2.3 2.3 2.3 2.7 2.7 2.7 2.06 2.06 2.06 2.34 2.34 2.34
1.15 2.3 3.45 1.35 2.7 4.05 1.03 2.06 3.09 1.17 2.34 3.51
19883.9±28.89 22956.9±45.32 25941.8±17.45 21896.6±49.81 27588.6±94.47 32688.2±35.92 14922.6±66.04 16980.8±55.75 18866.1±67.35 23118.6±19.00 24151.2±48.62 25138.2±54.80
98.21 98.10 97.48 98.19 99.49 98.13 98.23 99.63 98.72 99.30 98.87 97.76
SD: Standard deviation
Table 9: Summary of the validation parameters of the developed HPTLC methods Marker compounds
Regression coefficient
Precision % RSD (intra and inter day)
Repeatability of measurement
Repeatability sample application
Accuracy
LOD µg/spot
Limit of quantification µg/spot
Marmelosin
0.98
0.31
0.74
97.48‑98.21
0.15
0.45
Gallic acid
0.983
0.16
0.52
98.13‑99.49
0.15
0.45
Lupeol
0.995
0.18
0.35
98.23‑99.63
0.15
0.46
Asarones
0.984
0.15‑0.19 0.50‑0.62 0.13‑0.15 0.43‑0.72 0.12‑0.17 0.32‑0.65 0.55‑0.70 1.03‑1.41
0.13
0.72
97.76‑99.30
0.613
1.83
HPTLC: High performance thin layer chromatography, RSD: Relative standard deviation, LOD: Limit of detection, LOQ: Limit of quantification
Specificity The spectra of spot of standard markers were matched with the spots found in the formulation suggested that there were no integer with the peak of the markers. Therefore, all the developed methods were specific.
LOD The minimum detectable limit was found to be 0.15, 0.15, 0.15 and 0.61 µg/spot for marmelosin, gallic acid, lupeol and asarones respectively.
LOQ The minimum quantification limit was found to be 0.45, 0.45, 0.46 and 1.83 µg/spot for marmelosin, gallic acid, lupeol and asarones respectively. Thus the developed HPTLC methods were found to simple, precise, sensitive and accurate and can be used for the quantification of the marmelosin, gallic acid, lupeol and asarones in SJT‑DI‑02 and also in routine quality control of the raw materials as well as formulations containing any of these compounds. In Table 9, summary of the validation parameters of the developed HPTLC methods are shown. Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3
Figure 1a-e shows Co‑chromatography of marmelosin, Densotometric chromatogram of calibration curve of marmelosin, Calibration curve of marmelosin concentration versus mean peak area, Overlain ultraviolet spectra of marmelosin with SJT‑DI‑02 and Densitometric chromatogram of marmelosin with SJT‑DI‑02 respectively. Figure 2a-e shows Co‑chromatography of gallic acid, Densitometric chromatogram of calibration curve of gallic acid, Calibration curve of gallic acid concentration versus mean peak area, Overlain ultraviolet spectra of gallic acid with SJT‑DI‑02 and Densitometric chromatogram of gallic acid with SJT‑DI‑02 respectively. Figure 3a-e shows Co‑chromatography of lupeol, Densitometric chromatogram of calibration curve of lupeol, Calibration curve of lupeol concentration versus mean peak area, Overlain ultraviolet spectra of lupeol with SJT‑DI‑02 and Densitometric chromatogram of lupeol with SJT‑DI‑02 respectively. Figure 4a-e shows Co‑chromatography of asarones, Densitometric chromatogram of calibration curve of asarones, Calibration curve of asarones concentration versus mean peak area, Overlain ultraviolet spectra of asarones with SJT‑DI‑02 and Densitometric chromatogram of asarones with SJT‑DI‑02 respectively.
Conclusion We have established simple, precise, specific and economical HPTLC Densitometric methods for the quantification of the marmelosin, gallic acid, lupeol and asarones bio active markers found in SJT‑DI‑02. The methods were validated to trace the 217
Ladva, et al.: Standardization of polyherbal formulation by HPTLC method
a
b
(B) Densotometric chromatogram of calibration curve of marmelosin scanned at 305nm
(a) 35 μg/spot of SJT-DI-02 (b) 1.5-3.5 μg/spot of marmelosin
a2
(B) Densotometric chromatogram of calibrationcurve of gallic acid scanned at 245nm
b
(b) 1.5-3.5 μg/spot of gallic acid
(A) Co chromatography of gallic acid in SJT-DI-02
25000
y = 2740x + 10600 R² = 0.988
20000 15000
a – Marmelosin
25000
b – SJT-DI-02
20000
10000
Peak area
Peak area
a1
(a1, a2) 35, 50 μg/spot of SJT-DI-02
(A) Co chromatography of marmelosinin SJT-DI-02
5000 a
0 0
1 2 3 Concentration (µg/spot)
y = 4077.4x + 5683.2 R² = 0.9839 a - Gallic acid
15000
b – SJT-DI-02
10000
b
5000
4
ab
0 0
(C) Calibration curve of marmelosin concentration Vs mean peak area
1 2 3 Concentration (µg/spot )
4
(D) Overlain UV spectra of marmelosin with SJT-DI-02
(C) Calibration curve of gallic acidconcentration Vs mean peak area.
(D) Overlain UV spectra of gallic acid with SJT-DI-02
a - Marmelosin
a - Gallic acid
b –SJT-DI-02
b - SJT-DI-02
a
a b
b
(E) Densotometric chromatogram of Marmelosin with SJT-DI-02
(E) Densotometric chromatogram of gallic acid with SJT-DI-02
Figure 1: Estimation of Marmelosin by HPTLC Method
Figure 2: Estimation of Gallic acid by HPTLC Method
ab
a
(a) 1.5-3.5 μg/spot of lupeol
(B) Densotometric chromatogram of calibration curve of lupeol scanned at 559 nm
(b) 50 μg/spot of SJT-DI-02
Peak area
Peak area
a b
a - Lupeol
y = 1924.66x + 9080.38 R² = 0.995 0
1 2 3 concentration(µg/spot)
(B) Densotometric chromatogram of calibration curve of asarones scanned at 295 nm
(A) Co chromatography of asarones in SJT-DI-02
(A) Co chromatography of lupeolSJT-DI-02 18000 16000 14000 12000 10000 8000 6000 4000 2000 0
b
(b) 1.5-3.5 μg/spot of asarones
(a) 35 μg/spot of SJT-DI-02
b - SJT-DI-02
40000 35000 30000 25000 20000 15000 10000 5000 0
a – Asarones b - SJT-DI-02
y = 904.21x + 19967 R² = 0.9849 a b
0
5
10
15
20
Concentration (µg/spot)
4
(C) Calibration curve of asarones concentration Vs mean peak area
(C) Calibration curve of lupeol concentration Vs mean peak area
(D) Overlain UV spectra of asarones with SJT-DI-02
(D) Overlain UV spectra of lupeol with SJT-DI-02
a -Asarones
a - Lupeol
b - SJT-DI-02
ab
a b
(E) Densotometric chromatogram of lupeol with SJT-DI-02
Figure 3: Estimation of Lupeol by HPTLC Method
218
(E) Densitometric chromatogram of asarones with SJT-DI-02
Figure 4: Estimation of Asarones by HPTLC Method Journal of Pharmacy and Bioallied Sciences July-September 2014 Vol 6 Issue 3
Ladva, et al.: Standardization of polyherbal formulation by HPTLC method
markers in complex mixtures of herbal drugs used in SJT‑DI‑02. Developed methods can also be used in routine quality control of herbal raw material as well as formulation containing the same markers.
Acknowledgment
4. 5. 6.
The authors are thankful to Anchorm Pvt. Ltd., Mumbai and Saurashtra University Rajkot for providing guidance and facilities.
7.
References
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Author Institution Mapping (AIM)
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