Isolation, Cytotoxicity Evaluation and HPLC-Quantification of the Chemical Constituents from Prangos pabularia Saleem Farooq 1 *, Shakeel-u-Rehman 2,3 , Nisar Ahmad Dangroo 1 , Dev Priya 4 , Javid Ahmad Banday 3 , Pyare Lal Sangwan 1 , Mushtaq Ahmad Qurishi 3 , Surrinder Koul 1 *, Ajit Kumar Saxena 4 1 Bio-organic Chemistry Section, CSIR-Indian Institute of Integrative Medicine, Jammu, India, 2 Bio-organic Chemistry Section, CSIR-Indian Institute of Integrative Medicine, Srinagar, India, 3 Department of Chemistry, University of Kashmir, Srinagar, India, 4 Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India Abstract Phytochemical analysis of the dichloromethane:methanol (1:1) extract of root parts of Prangos pabularia led to the isolation of twelve cytotoxic constituents, viz., 6-hydroxycoumarin (1), 7-hydroxycoumarin (2), heraclenol-glycoside (3), xanthotoxol (4), heraclenol (5), oxypeucedanin hydrate (6), 8-((3,3-dimethyloxiran-2-yl)methyl)-7-methoxy-2H-chromen-2-one (7), oxypeucedanin hydrate monoacetate (8), xanthotoxin (9), 4-((2-hydroxy-3-methylbut-3-en-1-yl)oxy)-7H-furo[3,2-g]chro- men-7-one (10), imperatorin (11) and osthol (12). The isolates were identified using spectral techniques in the light of literature. 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity screening of the isolated constituents was carried out against six human cancer cell lines including lung (A549 and NCI-H322), epidermoid carcinoma (A431), melanoma (A375), prostate (PC-3) and Colon (HCT-116) cell lines. Osthol (12) exhibited the highest cytotoxicity with IC 50 values of 3.2, 6.2, 10.9, 14.5, 24.8, and 30.2 mM against epidermoid carcinoma (A431), melanoma (A375), lung (NCI-H322), lung (A549), prostate (PC-3) and colon (HCT-116) cell lines respectively. Epidermoid carcinoma cell line A431 was sensitive to most of the compounds followed by lung (A549) cancer cell line. Finally a simple and reliable HPLC method was developed (RP-HPLC-DAD) and validated for the simultaneous quantification of these cytotoxic constituents in Prangos pabularia. The extract was analyzed using a reversed-phase Agilent ZORBAX eclipse plus column C 18 (4.6 6 250 mm, 5 mm) at 250 nm wavelength using a gradient water-methanol solvent system at a flow rate of 0.8 ml/min. The RP-HPLC method is validated in terms of recovery, linearity, accuracy and precision (intra and inter-day validation). This method, because of shorter analysis time, makes it valuable for the commercial quality control of Prangos pabularia extracts and its future pharmaceutical preparations. Citation: Farooq S, Shakeel-u-Rehman, Dangroo NA, Priya D, Banday JA, et al. (2014) Isolation, Cytotoxicity Evaluation and HPLC-Quantification of the Chemical Constituents from Prangos pabularia. PLoS ONE 9(10): e108713. doi:10.1371/journal.pone.0108713 Editor: Manuel Reigosa, University of Vigo, Spain Received April 8, 2014; Accepted September 1, 2014; Published October 14, 2014 Copyright: ß 2014 Farooq et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The authors have received fellowship/funding from CSIR India. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: farooqprince100@yahoo.in (SF); skoul@iiim.ac.in (SK) Introduction Prangos, one of the largest and most widely distributed genus of family Umbelliferae consists of around 30–40 species. Most of the Prangos species are reported to possess diverse pharmacological activities viz. anti-microbial [1], anti-oxidant [2], cytotoxic [3,4], anti-helmintic and aphrodisiac [5], besides, their use in the treatment of haemorrhoids, wounds and leukoplakia [6]. In Central Asia, the extracts of Prangos species have been used to stop bleeding and heal scars [4]. Prangos pabularia commonly known as ‘‘Komal’’ in Hindi and ‘‘Kurangas’’ locally (Kashmir), occurs in stony slopes of Ladakh (Jammu and Kashmir, India). It is the only species of the genus found in India. In Indian traditional system of medicine, its roots and fruits have been used as diuretic, carmative, laxative, stimulant and liver tonic [7]. It is also used for treatment of itches, and as a promoter for the expulsion of foetus [7]. An infusion of the roots is useful in indigestion, flatulence and regularization of menstrual cycle in females [8]. Previous phytochemical investigations on the fruits and roots of p. pabularia revealed the presence of various chemical constitu- ents, consisting of coumarins of diversified structures, terpenoids and glycosides [9–11]. It may be pertinent to say that coumarins form an important class of compounds known to possess various pharmacological activities viz. anti-inflammatory, anti-pyretic [12], anti-oxidant [13], bronchodilator [14], vasodilator [15], anti-amoebic [16], anti-bacterial [17] and anti-fungal [18]. Physiological, bacteriostatic and antitumor activity make these compounds attractive for further backbone derivatization and screening as novel therapeutic agent/s [19]. Weber and co- workers have shown that coumarin and its metabolite 7- hydroxycoumarin exhibit antitumor activity against several human tumour cell lines. In addition, it has been shown that PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e108713