Yeast extract and salicylic acid were used as the elicitors at final concentrations of 100, 250, and 500mgL?1, whereas Cabozantinib clinical trial tryptophan and loganin were used as the exogenous precursors at final concentration of 3, 6, and 9��M. These compounds were added to the M. speciosa suspension culture to test mitragynine production. Suspension culture medium that lacked elicitors and precursors was used as the control in this experiment. The cells were harvested six days and twelve days after treated with elicitor and exogenous precursor to analyze the mitragynine content.2.6. ExtractionTo extract the alkaloids, the cells were separated from the liquid media and ground with liquid nitrogen. Approximately 8g was collected before it was extracted with 50mL of methanol for three days.
The methanol extract was filtered, and new methanol was added to the remaining cells; this process was repeated three times. The methanol filtrates were combined and evaporated under reduced pressure. The crude methanol extract was dissolved in a 10% acetic acid solution; it was shaken well and allowed to stand overnight. The acidic filtrate was adjusted to pH 11 with sodium carbonate and extracted with chloroform. The chloroform extract then was washed, dried over anhydrous sodium sulfate, and evaporated to yield a dry, crude alkaloid extract.2.7. HPLC Quantification of MitragynineThe presence of mitragynine in the samples was verified by the comparison of the Rt and UV spectral peaks of the sample with the standard peak. The HPLC system consisted of a Waters 2707 autosampler, a Waters 600 controller, and a Waters 2998 photodiode array detector.
The data were collected and processed using the Empower Software System.The analytical method was performed using Column ��Bridge C18 (size 5��m, 4.6 �� 250mm) from Waters. The mobile phase was a methanol-water mixture (80:20, v/v) and was filtered separately before it was mixed through a 0.22��m nylon membrane filter. The flow rate was 0.6mL/min, and the autosampler vials were at an ambient temperature of 25 �� 1��C. A sampler volume of 10��L was injected, and the detector was set at 254nm. A standard calibration curve was constructed by injecting different concentrations of mitragynine standard. The calibration curve for the mitragynine standard was linear (1mgL?1�C5mgL?1) with a regression factor of 0.989240.2.8.
Statistical AnalysisAnalysis of variance (ANOVA) was performed using the Statistical Analysis System (SAS) Version 9.0 software [24] to determine the significance of the treatment effects for each experiment. A P value of < 0.05 was considered to be significant.3. Results3.1. Plant Growth Regulator and Explants Effects on Callus InductionsThe petiole and leaf explants were placed on WPM that was supplemented with different concentrations of 2,4-D Batimastat (2, 4, 6, 8, and 10mgL?1) for callus induction.