Quantitation of Bioactive Phytochemicals in Ocimum Species and Its Marketed Herbal Formulations Using UHPLC-MS
Ocimum sanctum L., with phenolic acids, flavonoids, propenyl phenols and terpenoids as active pharmacological constituents, is a prominent therapeutic herb. It is used as an ingredient in many herbal formulations. Therefore, development of an analytical method for simultaneous determination of bioactive compounds of O. sanctum is required for quality control of herbal formulations. The aim of this study was to develop and validate a new, rapid, sensitive and selective ultra high performance liquid chromatograhy electrospray tandem mass spectrometry (UHPLC-ESI-MS/MS) strategy for simultaneous determination of bioactive markers including phenolic acids, flavonoids, propenyl phenol and terpenoid in the leaf extract and marketed herbal formulations of O. sanctum. The simultaneous determination was carried out using negative electrospray ionization with multiple reactions monitoring (MRM) mode. Chromatographic separation was achieved on an Acquity UHPLC BEH C,g column using gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Principal component analysis (PCA) was carried out to correlate and discriminate eight geographical collections of O. sanctum based on quantitative data of analytes. The developed method was validated as per International Conference on Harmonization (ICH, 2005) guidelines and found to be accurate with overall recovery in the range from 95.09% to 104.84% (RSD < 1.85%), precise (RSD < 1.98%) and linear (r2 > 0.9971) over the concentration range of 0.5-1,000 ng/mL. Ursolic acid was found to be the most abundant marker in all the investigated samples except for the marketed tablet. The established method is simple, rapid and sensitive and hence could be reliably utilized for the quality control of O. sanctum and derived herbal formulations.
Plant Extracts and Marketed Formulations
The leaves of O. sanctum (Figure 3.1) were collected from various geographical regions of India such as Nauni, Solan (Himachal Pradesh), Lucknow (Uttar Pradesh), Jabalpur (Madhya Pradesh) and Kolkata (West Bengal). The voucher specimens of O. sanctum from Uttar Pradesh (U.P.), Madhya Pradesh (M. P.) and West Bengal (W. B.) have been deposited in the Botany Department of CSIR-Central Drug Research Institute, Lucknow, India. The voucher specimen of O. sanctum from Himachal Pradesh (H. P.) has been deposited in the Department of Forest Products, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India. The details of plant samples of six Ocimum species are given in Section 2.1.
FIGURE 3.1 Pictures of 0. sanctum and its herbal formulations.