|Year : 2016 | Volume
| Issue : 1 | Page : 51-54
Quantification of vasaka alkaloids in in vitro cultures and in natural leaves from Indian subcontinents by reversed phase- high performance liquid chromatography
Madhukar Garg, Ennus Tajuddin Tamboli, YT Kamal, SH Ansari, Sayeed Ahmad
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Bioactive Natural Product Laboratory, Jamia Hamdard, Hamdard University, New Delhi, India
|Date of Web Publication||13-Apr-2016|
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Bioactive Natural Product Laboratory, Jamia Hamdard, Hamdard University, New Delhi
Source of Support: None, Conflict of Interest: None
Background: The present study was designed to develop an efficient protocol for studying the enhancement of in vitro production of secondary metabolites in Adhatoda vasica leaves collected from the varied geographical locations of Indian sub-continents by a validated simultaneous high-performance liquid chromatography gradient method for the quantification of vasicine and vasicinone in the developed calli and crude samples. Materials and Methods: The analysis was carried out on a reverse phase C18column using 0.1% of orthophosphoric acid: Acetonitrile in gradient manner and carried out the detection at 280 nm wavelength keeping the flow rate of 1.0 mL/min. Results: The simultaneous method was found linear with regression coefficient r2 = 0.991 in a wide range (100–500 μg/mL) precise, accurate, and robust for quinazoline alkaloids in samples. Results clearly showed a significant increase in the concentration of alkaloids in in vitro cultures as compared to natural ones. The proposed method was validated as per the International Conference on Harmonization guidelines for accuracy, precision, robustness, limit of detection, and limit of quantitation. Conclusion: The developed method was found suitable for quality control of A. vasica and for the analysis of vasicine and vasicinone in any type of sample.
Keywords: Adhatoda vasica (L.) nees, callus cultures, high-performance liquid chromatography, validation, vasicine, vasicinone
|How to cite this article:|
Garg M, Tamboli ET, Kamal Y T, Ansari S H, Ahmad S. Quantification of vasaka alkaloids in in vitro cultures and in natural leaves from Indian subcontinents by reversed phase- high performance liquid chromatography. Drug Dev Ther 2016;7:51-4
|How to cite this URL:|
Garg M, Tamboli ET, Kamal Y T, Ansari S H, Ahmad S. Quantification of vasaka alkaloids in in vitro cultures and in natural leaves from Indian subcontinents by reversed phase- high performance liquid chromatography. Drug Dev Ther [serial online] 2016 [cited 2017 May 23];7:51-4. Available from: http://www.ddtjournal.org/text.asp?2016/7/1/51/180165
| Introduction|| |
Adhatoda vasica commonly known as vasaka; Malabar nut tree in English and arusa in local Hindi  has been used in indigenous systems over last 2000 years. Leaves are administered to clear respiratory passages in the preparations such as cough syrups  containing pyrroquinazoline alkaloids mainly vasicine, vasicinone up to 1.3% possessing number of biological activities that is antibacterial, wound healing, abortifacient/oxytocic, antitussive, anti-inflammatory, anti-ulcer, hepatoprotective, and anti-tubercular.
As the large-scale destruction of the natural habitat by it is over exploitation made it endangered hence Food and Agriculture Organization recommended the need for its conservation via in vitro production of metabolites. Literature survey revealed that the previously reported methods for the quantitative estimation of vasicine and vasicinone in A. vasica and Adhatoda zeylanica have poor range of linearity and improper separations in an isocratic mode. Therefore, this study was designed for the development of in vitro cultures and compared the same to natural ones.
| Materials and Methods|| |
Chemicals, standards, samples
Chemicals utilized viz., growth hormones and constituents of Murashige and Skoog  were of analytical grade obtained from Merck (India). Vasicine (C11H12N2O; assigned purity >99.5%; melting point 212°C) was obtained from Sami Labs Ltd. (Bengaluru, India) as a gift sample and vasicinone from Chromadex, Bengaluru. Methanol, acetonitrile, orthophosphoric acid, and water used were of high performance liquid chromatography (HPLC) grade. The solvents were freshly prepared and degassed before use.
The samples collected in the month of March from the different eco-geographical locations that is Banaras (Uttar Pradesh), Chandigarh, Patiala, Mohali (Punjab), Solan, Nahan, Dalhousie (Himachal Pradesh), Dehradun (Uttrakhand), Hisar, Kurukshetra (Haryana), and Delhi were authenticated by a botanist Dr. Altaf Ahmad, Jamia Hamdard and deposited the voucher specimen in herbarium of BNPL (JH/BNPL/AV1-AV11/2010) Jamia Hamdard, New Delhi, India.
High-performance liquid chromatography instrumentation and conditions
It comprised system quaternary YL9100 pumps (South Korea), a variable wavelength programmable YL9120 ultraviolet (UV) detector and YL9130 column oven, controlled by the YL-Clarity software (Younglin Lin Instrument Co. Ltd, Korea). Standard and sample were filtered through 0.22 µm syringe assisted through rheodyne injector fitted with 0.20 µl fixed loop. The separation was achieved by acetonitrile and 0.1% orthophosphoric acid in a gradient at the flow rate of 1.0 mL/min passing through a C18 reverse phase column (Luna) followed by detection at 280 nm under UV. Whole of the analyses were performed in triplicate. Individual peaks were identified from their retention times and calculated the peak areas to obtain the respective concentrations for appropriate solutions by employing the regression equations as obtained from the calibration plots.
In vitro development of static and suspension cultures
The immature leaf explants collected from herbal garden of Jamia Hamdard were subjected for in vitro studies. The developed calli showing the best results were further elicited by varying concentration of potassium nitrate (20–35 mM) and NaCl (25–200 mM)., The concentrations having the highest potential for production were chosen as optimized medium to get maximum amount of vasicine [Figure 1].
|Figure 1: (a) Field growing Adhatoda vasica (b) 120 days old callus on Murashige and Skoog + 2,4-D + 6-benzyladenine + indole acetic acid (1 ppm each) (c) 120 days old callus on Murashige and Skoog + 6-benzyladenine + indole butyric acid (1 ppm each) (d) 120 days old callus on Murashige and Skoog + 6-benzyladenine (0.5 ppm) + indole butyric acid (1 ppm) (e) 120 days old callus on Murashige and Skoog + 2,4-D + 6-benzyladenine + indole acetic acid (1 ppm each) treated with 28 mM KNO3 (f) 120 days old callus on Murashige and Skoog + 2,4-D + 6-benzyladenine + indole acetic acid (1 ppm each) treated with 28 mM KNO3+100 mM NaCl|
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Calibration and validation of analytical method
Working standards of 500 µg/mL were prepared for vasicine and vasicinone in methanol and obtained the concentrations of 10–500 µg/mL for vasicine and 1–500 µg/mL for vasicinone by appropriate dilutions. The proposed method was validated as per the International Conference on Harmonization guidelines  for linearity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantitation (LOQ).
Preparation of samples
Dried and powdered samples were refluxed with methanol for 2 h. The aliquots obtained were filtered and evaporated to dryness and reconstituted methanol and made up the volume of 10 ml. A similar method was adopted for 1.0 g of dried calli and 0.5 g of suspension cells and adjusted the volume after the filtration with syringe filter of 0.45 µm. The 10 µl of each sample was injected for HPLC analysis.
| Results and Discussion|| |
Selection of mobile phase and chromatographic conditions
Number of trials was conducted with different combinations of solvents that are acetonitrile, water, methanol, orthophosphoric acid, and phosphate buffer in the various ratios. Among all acetonitrile and 0.1% orthophosphoric acid in the ratio of 70:30 (v/v) in gradient mode resulting in sharp peaks with good resolution between them.
The calibration showed good linearity over a wide range of 10–500 and 1–500 µg/mL for vasicine and vasicinone with r2 = 0.99. The accuracy of the method was evaluated as recovery by standard addition method. The preanalyzed samples were spiked with standard at three different concentration levels and got re-analyzed by the proposed method and mean value was expressed in [Table 1].
The precision of the methods was determined by injecting six different injections of the same standard (three concentrations) for repeatability experiment and calculated the assay, relative standard deviation (%RSD) and Rf values.
Robustness was determined by changing the detecting wavelengths and by analyzing the temperature. The %RSD of the experiment was calculated so as to assess the robustness of the method at 10 µg/mL.
The LOD was determined on the basis of signal to noise ratio and found to be 3.0 µg/mL for vasicine and 0.53 µg/mL for vasicinone whereas LOQ as 10 µg/mL for vasicine and 1.0 µg/mL for vasicinone, respectively. The results obtained were summarized in [Table 1].
Determination of vasicine and vasicinone in all the samples
The content was analyzed by injecting the samples in triplicates and calculated the peak area by regression equation. The developed mobile phase resulted in the optimal separation of components with well-defined and resolved sharp peaks in both standard and samples [Figure 2].
|Figure 2: A typical high-performance liquid chromatography chromatogram of standard vasicine (1) and vasicinone (2) eluting at the Rt 7.81 and 9.3 min|
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Comparative analysis of vasicine content in different sample of natural leaves
The analysis of samples showed the presence of vasicine ranging from 0.64% to 0.88% and vasicinone from 0.56% to 0.43% containing highest amounts in Delhi ones, which in turn was not a significant variation among the different collections [Figure 3].
|Figure 3: Content of vasicine (a) and vasicinone (b) (mg/g) in the samples of different locations/in vitro cultures by high-performance liquid chromatography|
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Development of in vitro cultures and analysis of vasicine content
The Delhi sample [Figure 1] containing highest content of vasicine was subjected to tissue culture studies as the leaves of plant were available throughout the year. The analysis of all the cultures showed significant content of vasicine, whereas cultures treated with 28 mM KNO3 and 100 mM NaCl showed significant increase as compared to control [Figure 3].
| Conclusion|| |
The content of vasicine analyzed in optimized suspension cultures showed manifold increase in comparison to natural leaf led to conclude that it possess a great scope for the production of pharmaceutically important quinazoline alkaloids using bioreactors at industrial level.
Tissue culture of the highest yielding explants showed a higher increase whereas suspension cultures showed the better ability for their production. The method developed was found to be accurate, reproducible, specific, and precise and well enough to separate the desired components from the immediate constituents present in the samples. The method is suitable for the routine analysis of the components in crude as well as in vitro samples.
The authors are thankful to the DST for financial support and UGC-SAP for providing the facilities in Bioactive Natural Product Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard to carry out the present research work.
Financial support and sponsorship
DST for financial support and UGC-SAP for providing the facilities in Bioactive Natural Product Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard to carry out the present research work.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]