Nitidine, Chelerythrine and Sanguinarine, all these three alkaloids are benzophenanthridine alkaloids. Nitidine was used as an anti-HIV, anti-malarial and anti-cancer. Chelerythrine had anti-cancer and anti-inflammatory activities. Sanguinarine was widely used as an anti-plaquestic and anti-cancer. High performance thin layer chromatography (HPTLC) method was used for simultaneous quantification of Nitidine, Chelerythrine and Sanguinarine in callus extract of Zanthoxylum rhetsa by using Silica gel 60 F 254 as stationary phase and ethyl acetate:methanol:water:diethylamine (30:5:2:0.5 v/v) as mobile phase at 280 nm. The linearity concentration range was 5 - 160 μg/band of each alkaloid. The R f values of Nitidine, Chelerythrine and Sanguinarine were found to be 0.28, 0.49 and 0.73. The limit of detection and limit of quantification were found to be 0.026, 0.088 μg/spot and 0.010 and 0.033 μg/spot, 0.0104 and 0.035 μg/spot respectively for Nitidine, Chelerythrine and Sanguinarine. HPTLC method was developed and validated according to ICH guidelines for simultaneous estimation of Nitidine, Chelerythrine and Sanguinarine and proved to be simple, specific, accurate, robust and rapid.
Benzophenanthridine alkaloids are one of the most important sub-classes of isoquinoline alkaloids, which are the major group of pharmacologically useful compounds, such as nitidine, chelerythrine, sanguinarine, arborine, chelirubine, angoline, chelidonine, chelilutine, corynoline, marcapine, fagaridine, decarine, sanguilutine, sanguirubine and aricine [
The present paper deals with simultaneous HPTLC quantification of three benzophenanthridine alkaloids namely nitidine, chelerythrine and sanguinarine. Nitidine was reported to be used as an anti-cancer [
Praveena and Veeresham (2014 and 2015) were reported the HPTLC quantification of nitidine from Toddalia asiatica roots and callus cultures [
Z. rhetsa plants were identified and collected from Medicinal plants garden of Kerala Forest Research Institute (KFRI) of Peechi, Kerala, India and it was authenticated by Prof. T. Christopher, Taxonomist, department of Botany, Kakatiya University, Warangal, Telangana, India. Voucher specimen of the plant was deposited in the author laboratories.
All solvents and reagents used were purchased from Merck, Mumbai, India. Standard drugs nitidine (≥97%), chelerythrine (≥95%) and sanguinarine (≥98%) were purchased from Sigma, Mumbai, India.
1 mg/ml stock solutions of nitdine, chelerythrine and sanguinarine were prepared by dissolving an accurately weighed 10 mg of each standard in 10 ml of 70% methanol in volumetric flask. Further dilutions were made from this stock.
5 gms of dried leafy callus of Z. rhetsa, was taken and extracted with 10 ml of 70% v/v methanol by refluxing for 30 minutes, and then concentrated to dryness by vacuum. Dried extract was re-dissolved in 70% v/v methanol to get sample stock solution.
The method was developed on Camag HPTLC system, consisting of Linomat V 10 AT semi automatic applicator (Muttenz, Switzerland), Camag twin trough chamber (20 cm × 20 cm) for TLC plate development and Camag TLC scanner 3 20AT, equipped with software (version 1.4.3) win CATS and 100 µl capacity Camag Syringe. HPTLC analysis was performed by application of 10 µl of each standard drug on 10 cm × 10 cm, 0.2 mm layer thickness silica gel 60F254 (Merck, Germany) pre-coated aluminum plates as 8 mm band width with the help of semi automatic applicator under pressure of nitrogen gas. The space between each band is 6 mm, 15 mm from side and 8 mm from bottom. Development was done through twin trough chamber by linear ascending mechanism. The chamber is pre saturated with mobile phase i.e., ethyl acetate: methanol: water: di ethyl amine (30:5:2:0.5 v/v) for 20 minutes at room temperature in prior to insertion of plate into solvent system. The development distance was 80 mm. After this process the plates were dried. Densitometric scanning at 280 nm was selected the maximum absorption of band, performed with Camag TLC scanner in reflection absorbance made by using a slit width 6 mm × 0.3 mm, data resolution 100 mm∙sec−1, 20 mm∙sec−1 scanning speed. For continuous radiation purpose deuterium lamp was used for UV-Visible region 190 - 800 nm.
An optimized HPTLC densitometry method was validated by following parameters.
5, 10, 20, 40, 80, 160 µg/spot concentrations of standards were loaded on to TLC plate by using semi automatic applicator, which were prepared from standard solutions. Each different concentration was loaded for 3 times on the plate. The plate was developed by using mobile phase and plotted the peak areas of each spot against concentration to obtain the calibration curve.
Slope and standard deviation of the calibration curve were used for calculation of LOD and LOQ.
LOD = 3.3 σ/S
where σ is the standard deviation of the response and S is the slope of the calibration curve.
LOQ = 10 σ/S
Specificity of the method was analyzed by comparing the callus extracts and standards. The spot for nitidine, chelerythrine and sanguinarine was confirmed by comparing their Rf values with standard compounds.
Accuracy of the method was established by performing recovery experiments using the standard addition method. To the pre analyzed samples of callus extract, standard nitidine, chelerythrine and sanguinarine solution was added by spiking at 100 µg level and the mixture was analyzed by the proposed HPTLC method.
Random errors were identified by precision. Results were expressed in relative standard deviation (% RSD). Standard solution of nitidine, chelerythrine and sanguinarine (5, 20, 80 µg/band) were applied. Inter day precision was evaluated by applying each concentration for 3 times on three different days with an interval of 24 hrs. Intraday precision was evaluated by applying each concentration three times within the day.
To test the robustness of the method, deliberately small changes were made in the chromatographic parameters that may affect the performance of the method, i.e., mobile phase composition, mobile phase value. The RSD of the peak areas was calculated for each parameter.
System suitability was carried out to check the reproducibility and resolution of the method. After development, the plates were scanned and peak area of each spot and their Rf values were calculated.
The present study deals with simultaneous quantification of three benzophenanthridine alkaloids namely nitidine, chelerythrine and sanguinarine by using densitometric HPTLC method.
The Present paper aimed to establish optimum mobile phase for TLC analysis, which would shows clear separation of nitidine, chelerythrine and sanguinarine. A number of TLC analysis as preliminary tests to separate above said alkaloids were performed by using different combinations of solvents and modifications of mobile phases. Different methods which were proposed by earlier authors for HPTLC individual quantification of nitidine (Praveena and Veeresham in 2014 and 2015 [
Linearity was achieved with concentration range from 5 - 160 µg/band for all the
three compounds nitidine, chelerythrine and sanguinarine (Figures 2(a)-(c)). The Correlation coefficient, intercept and the slope were 0.998, 1447 and 65.36 for nitidine, 0.997, 14581 and 688.7 for chelerythrine and 0.997, 337.2 and 87.72 for sanguinarine respectively (
The values of LOD and LOQ (µg/band) of nitidine, chelerythrine and sanguinarine are 0.026, 0.088, 0.010 and 0.033 and 0.0104, 0.035 respectively and are summarized in (
From the results of repeatability and intermediate precision experiments (
Specificity of the method was ascertained by comparing Rf values and the spectras of sample with that of standards nitidine, chelerythrine and sanguinarine (
The results of recovery studies of leafy callus extracts are listed in (
Parameter | Nitidine | Chelerythrine | Sanguinarine |
---|---|---|---|
Linearity range (µg/band) | 5 - 160 | 5 - 160 | 5 - 160 |
Correlation coefficient (r2) | 0.998 | 0.997 | 0.997 |
Slope | 65.36 | 688.7 | 87.72 |
Intercept | 1447 | 14581 | 337.4 |
LOD [µg/band] | 0.026 | 0.010 | 0.0104 |
LOQ [µg/band] | 0.088 | 0.0335 | 0.0347 |
Intraday precision [%RSD, n = 3] | 0.0975 - 1.906991 | 0.670 - 1.414 | 0.21 - 1.8404 |
Inter day precision [%RSD, n = 3] | 0.460 - 1.795533 | 0.323 - 1.8136 | 0.069 - 1.79292 |
Drug name | Nitidine (µg) | Chelerythrine (µg) | Sanguinarine (µg) |
---|---|---|---|
Amount present | 42.78 | 22 | 2.7 |
Amount added (n =3) | 100 | 100 | 100 |
Amount recovered (n = 3) (mean ± sd) RSD | 141.76 ± 0.778 (0.54) | 121.5 ± 0.7 (0.57) | 102.2 ± 0.57 (0.56) |
Overall Recovery (n = 3) (%) (mean ± sd) RSD | 99.46 ± 0.394 (0.395) | 99.59 ± 0.69 (0.46) | 99.48 ± 0.56 (0.565) |
Parameter | RSD (%) |
---|---|
Mobile phase composition (Ethyl acetate: methanol: water: di ethylamine―28:6:3:1) | 1.21 |
Duration of chamber saturation (30 min) | 0.87 |
Mobile phase volume (25 ml) | 0.58 |
alkaloids, which are much better than previous reports.
The low values of the % RSD (less than 2%) for introduction of small changes in mobile phase composition, mobile phase volume and duration of mobile phase saturation time indicated the robustness of the method (
The present study was taken into consideration for the development and validation of HPTLC densitometric method for the simultaneous quantitative estimation of nitidine, chelerythrine and sanguinarine in the callus of Zanthoxylum rhetsa. HPTLC method was developed and validated according to the ICH guidelines. The technique was proved to be simple, specific, accurate, robust and rapid.
One of the author (P. Kavitha) is thankful to AICTE, New Delhi for granting QIP fellowship.
The authors declare no conflicts of interest regarding the publication of this paper.
Perala, K. and Ciddi, V. (2018) Simultaneous Quantitative Determination of Nitidine, Chelerythrine and Sanguinarine Using HPTLC from Callus Extract of Zanthoxylum rhetsa. American Journal of Analytical Chemistry, 9, 386-396. https://doi.org/10.4236/ajac.2018.98030