E Sharma and B S Arora
N. foetida , LC-MS, camptothecin, camptothecinoids, h yphenated technique, quantitative analysis
|PUBLISHED DATE||September 2015|
|PUBLISHER||The Author(s) 2015. This article is published with open access at www.chitkara.edu.in/publications|
In the present study, hyphenated technique using dyad system of high performance liquid chromatography with diode array detection directly coupled with electrospray ionization mass spectrometry (LC-ESI-MS) has been employed for identification of camptothecines and some minor constituents of methanol extract from different parts of Nothapodytes foetida on the basis of systematic mass spectrum analysis without isolation and purification of these components. Also, a simple, precise, sensitive and reproducible LC-MS method has been developed for quantification of camptothecin (CPT), a potent cytotoxic isoquinoline alkaloid, in methanol extract from these different parts of Nothapodytes foetida using the same technique.
Nothapodytes foetida (Wt.) Sleumer, formerly known as Mappia foetida Miers, is a small, spreading, sub-canopy evergreen tree of family Icacinaceae naturally distributed in in the Indo-Malaysian region and China. In India, it is distributed in the wild forests of Western Ghats, some parts of west Bengal and Assam. This plant species has gained substantial commercial interest in recent years, as it is a prospective source of an expensive cytotoxic monoterpenoid isoquinoline alkaloid, camptothecin (CPT), used as an antitumor drug in thetreatment of colon, head, breast and bladder cancers acting by stabilizing the DNA-protein complex by forming topoisomerase I-DNA adduct , . A number of reports have indicated its therapeutic potential against colon cancer , ovarian cancer , HIV-1 , HSV-2 , parasitic trypanosomas & leishmania  and malaria . CPT is believed to be the third most important alkaloid sought after by the pharmaceutical companies around the world. Various semi-synthetic analogs of CPT including topotecan and irinotecan have been synthesized as potential therapeutic agents against different types of cancer , . Nothapodytes foetida has been reported for the highest concentration of CPT amongst the other known botanical sources . This has lead to the large scale exploitation resulting serious threat of extinction of the species from its wild habitat. Phytochemical study of this plant species utilizing simple and reproducible analytical methods is of great significance.
Different analytical methods like high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC), proton nuclear magnetic resonance spectroscopy (1H-NMR) and desorption electrospray ionization mass spectrometry (DESI-MS) have been reported for quantification of CPT from N. foetida. [12-18]. Furthermore, HPLC profiles have been developed for qualitative and quantitative analysis of active constituents in different parts of this plant, . In recent years, hyphenated techniques involving coupling of a separation technique and an spectroscopic detection technology have received much attraction as the principal means to obtain structural information leading to the identification of phytochemicals present in a crude samples . Aim of the present study was to evaluate the percentage of camptothecin and identify camptothecin-related alkaloids and other minor components in the methanol extracts from stem, young leaves, stem bark, seeds, fruits and roots of Nothapodytes foetida using reverse-phase high performance liquid chromatography coupled with on-line photodiode array detection and electrospray-ionization tandem mass spectrometry without isolation and purification of these phytochemicals.
|ISSN||Print : 2349-7564, Online : 2349-7769|
The quantitative analysis of CPT (1) was carried out by LC-ESI-MS. Under the LC-MS conditions mentioned in experimental section, purified CPT was eluted at retention time (Rt) of 20.1 min. (Figure 2) and by applying these condition in positive ion mode of ESI-MS, the same peak exhibited a molecular adduct at m/z 349.1 (M+H)+.
Quantitation of CPT in the extracts prepared from different parts of the plant (stem, stem bark, seeds, roots, fruits and young leaves) was done on the basis of the calibration curve established by injecting five different concentrations of CPT standard in the concentration range of 1μg/μL to 10μg/μL each time before sample analysis. Quantitation of CPT was carried out using selective ion monitoring (SIM) detection of the molecular ion peak at m/z 349.1 [M+H]+. Linear calibration curve of CPT within the concentration range of 1μg to 10μg (R2 = curve coefficient 0.9999) was obtained (Figure 3). Validation of the method was carried out by spiking 10μg of standard CPT to 10 mg of the dried plant extract. The spiked sample was extracted with methanol and analyzed by the proposed method. The recovery was within the range of 92% to 99%. The precision of the method was assessed by adding different concentrations of CPT standard to the samples and comparing the amounts determined from their chromatograms with the actually added amounts. Total CPT concentration in plant tissues has been expressed on a dry weight basis (Table 1).
LC-MS analysis of stem, young leaves, stems bark, seeds, fruits and root bark tissues of Nothapodytes foetida led to the identification of different phytochemicals (Table 2) and a number of as yet unidentified camptothecines. A number of compounds having different retention time but the same molecular weight were identified from these different plant tissues. These compounds were isomeric entities of camptothecinoids detected in the accessions of N. foetida.
In the present study, hyphenated technique involving dyad system of reverse-phase high performance liquid chromatography and electrosprayionization tandem mass spectrometry (LC-ESI-MS) has been applied for the phytochemical study of methanol extracts from stem, young leaves, stem bark, seeds, fruits and roots of Nothapodytes foetida leading to the unanimous identification of some camptothecins and other minor components on the basis of systematic mass spectrum analysis including fragmentation patterns and comparison with literature data, without involving the time consuming and hectic separation process of these individual constituents. There were a number of other components that could not be identified only by LC-MS. Also, an LC-MS method has been developed for the quantitative analysis of CPT in crude methanolic extract from these different parts of the plant using the same technique.