Chemicals and reagents
Standard camptothecin obtained from Aditya Imptex Pvt. Ltd. Mumbai, India. Methanol was supplied by OZONE International. Pvt. Ltd. Mumbai, Maharashtra. Chloroform, sodium carbonate, sulphuric acid, emulgen, and double distilled water were obtained from Unique Chemical Kolhapur. All chemicals and reagents used in the present research work were of analytical grade.
Preparation of extract
Microwave irradiation method was used for extraction of and isolation of CPT from Nothapodytes nimmoniana leaves using emulgen as surface active agent. Accurately weighted 5 g of leaf powder and placed in 250 ml conical flask along with 200 ml of emulgen solution having pH 8. Resulting solution was microwave irradiated for about 1 min for 350 V and cooled for 2 min. Resultant solution was maintained at pH 3–4 by using sulfuric acid and Mayer’s reagent added to precipitate CPT [23, 24].
Fourier transform infrared spectroscopy (FTIR) study
FTIR (Perkin Elmer FTIR model-1615) is modern analytical tool which conforms characteristic peaks present in CPT which represents functional groups present and CPT sample was scanned 4000 to 400 cm−1 range at a resolution of 4 cm−1 [25].
Mass spectroscopy
In mass spectroscopy, LC ion trap method was utilized for determination of ions which are + ve and −ve in mass spectra. For both positive and negative ion mode, capillary voltage was set to − 3800 V and 4500 V, respectively and at the end ± 500 volts plate in +ve and −ve ion mode, respectively. With help of syringe, CPT sample was injected along with micro TOF-Q detector and Apollo ESI as ion source. Structural elucidation was confirmed by comparison of isotope and mass accuracy pattern [26].
Nuclear magnetic resonance (NMR) spectroscopy
Before analysis camptothecin samples were stored at – 80 oC. For dissolved sample, solvent was evaporated by stream of nitrogen then redissolved in CDCl3/CD3OD (2:1). A high-resolution NMR spectrum of each single species was acquired on (Bruker DRX 600 MHz) NMR spectrometer equipped with TXI probe [26].
Cytotoxicity study
In a micro plate containing 96 wells, cells were seeded which are maintained overnight at 37 °C in 95% RH and CO2 5%. Various concentrations ranging from 20 to 0.625 μg/mL of samples was treated. The cells were incubated for another 48 h. Phosphate buffer used for cleaning of wells and 20 μL of the MTT was used as staining solution was poured in every well and incubated at 37 °C. In every single well, DMSO was added after 4 h which dissolve formazan and with help of micro plate reader and absorbance was measured at 570 nm [27].
$$ \%\mathrm{Surviving}\ \mathrm{cells}=\frac{\mathrm{Mean}\ \mathrm{OD}\ \mathrm{of}\ \mathrm{test}\ \mathrm{compound}}{\mathrm{Mean}\ \mathrm{OD}\ \mathrm{of}\ \mathrm{Negative}\ \mathrm{control}}\times 100 $$
Standard stock solution
Precisely weighted camptothecin 10 mg and dissolved in 10 ml of organic solvent methanol and make up the volume up to 100 ml with same solvent. The solution obtained was standard stock solution having the strength of 1000 μg/ml. From standard stock solution, 10 ml was withdrawn and 100 μg/ml concentration solution was prepared by suitable dilution which was filtered before analyzing by Whatman filter paper [28].
Calibration curve
Working solution was prepared by suitably diluting primary stock solution at room temperature. Working solution was serially diluted at different concentrations and scanned in the range of 200–400 nm (Shimadzu-UV-1900). Linearity of calibration curve was measured diluting the working solution in the range of 1–100 μg/ml.
Accuracy
Accuracy of the developed analytical method has been determined by close comparison between actual observed and standard values. Recovery was performed by addition level of 80, 100, and 120% for test solution in to fixed standard solution [29].
Precision
Both intra- and inter-day precision of developed analytical method was confirmed by observed values over a week from present day and on next 3 days at time intervals of 4 h. Observed results were calculated statistically and represented in the form of SD [30].
Repeatability
By analyzing 6 samples of CPT of 10 μg/ml the repeatability was determined and obtained results further utilized for statistical analysis [31].
Limit of detection and limit of quantification ((LOD and LOQ)
It is lowest detectible amount measured quantitatively by any analytical method. These variables were determined as per ICH guidelines by observed values along with its SD [32, 33].
LOD and LOQ were calculated using formula
LOD = 3.3 ∗ σ/s LOQ = 10 ∗ σ/s
σ- Standard deviation and S -slope.