br Abbreviations UHPLC HRMS ultra high
Abbreviations: UHPLC-HRMS, ultra high-performance liquid chromatogra-phy coupled with high resolution mass spectrometry; Cur, curcumin; DMcur, demethoxycurcumin; BMcur, bisdemethoxycurcumin; CE, curcuminoids extract containing 3 A549 cell-binding curcuminoids; TcE, turmeric crude extract; IS, internal standard; FBS, fetal bovine serum; ACN, acetonitrile; QC, quality control; PLS-DA, partial least-squares discriminant analysis; OPLS-DA, orthogonal partial least-squares discriminant analysis; VIP, Variable Importance in the Projection; ROC, receiver operating characteristic; TICs, total ion chromatograms; OSC, orthogonal signal correction.
∗ Corresponding authors. E-mail addresses: [email protected] (S.-L. Wang), [email protected] (T. Xie). 1 These authors contributed equally to this work.
Turmeric, the rhizomes of Curcuma long L. (family Zingiber-aceae), which is a tuberous herbaceous perennial plant with yellow flowers and wide leaves, has been used as condiment, dyestuff, medicine, cosmetic, flavour and food component from ancient times until now [1–3]. For instance, turmeric is an important spice and an important component of curries to which it gives the yellow color in Asian countries, and the mustard and sauces in the West . In addition, turmeric powder can also be used to add flavor and color to rice, seafood, pasta, meat and vegetable dishes, and salads. Furthermore, according to the European and Chinese Phar-macopoeias, turmeric is an official medicinal product in Europe and China for treatment of many diseases, such as stabbing pain in the
chest and the hypochondrium, chest impediment and heart pain, dysmenorrhea and amenorrhea etc [5,6].
The importance of turmeric in medical treatment primarily due to its versatile biological activities stems from the chemical components in turmeric. Two MK0683 of secondary metabo-lites, the curcuminoids and the essential oils, has been proven as the major constituents in turmeric. Among them, curcumin (Cur), demethoxycurcumin (DMcur), and bisdemethoxycurcumin (BMcur) have been listed as the key compounds of curcumi-noids, and ar-turmerone, -turmerone, and -turmerone are the major compounds in essential oils [7–9]. Reported biologic prop-erties of the chemical components or the extract of turmeric include choleretic , antioxidant , anti-inflammatory , antibacterial , antiviral , hepatoprotective , cardiopro-tective , and anti-cancer properties [17,18]. Over the past years, accumulating investigations have been performed on the major constituent-Cur, due to its high content in turmeric, particularly for its significant anti-cancer property. It has been proven that Cur has good effects or is effective in suppressing head and neck squamous cell carcinoma , gastric , colon , breast , tobacco smoke-induced liver cancer  and lung cancer cells [24,25].
As we know, the therapeutic effects of herbal medicine are expressed by multi-components which have complicated inte-gration effects of synergistic and/or antagonistic actions on multi-targets [26,27]. Thus, we have postulated that turmeric, as an herbal medicine, may also contain multiple anti-cancer components except the well-known Cur. To test this postulate, we developed a target cell extraction-chemical profiling method based on human lung adenocarcinoma cell line (A549 cells) and UHPLC-LTQ Orbitrap MS for screening of anti-lung cancer bioactive compounds from turmeric. Finally, three A549 cell-binding com-pounds were screened out and were identified as BMcur, DMcur and Cur . Compared to Cur, BMcur and DMcur are much less studied, in particular their potential in anti-lung cancer effects.
Following our previous result, the anti-A549 cells potency and mechanism of action of the three A549 cell-binding curcuminoids were studied in this paper by chemical markers’ knockout cou-pled with UHPLC-LTQ Orbitrap MS-based metabolomics. Using the chemical markers’ knockout method we developed before , four curcuminoid-containing fractions were prepared including curcuminoid extract (a mixture containing 3 cell-binding curcum-inoids, named CE), BMcur, DMcur and Cur. The cytotoxic activity on A549 cells of these fractions as well as the turmeric crude extract (TcE) were then tested by MTT assay to illustrate the major bioactive fractions or compounds. This strategy complied with the holistic thinking of herbal medicine by preparing and testing the fractions according to the natural proportion of the constituents in herbal medicine, which is totally different with that in medicinal chemistry of natural products, i.e., obtaining the pure compounds by isolation followed by activity evaluation. Meanwhile, UHPLC-LTQ Orbitrap MS-based non-targeted metabolomics was applied by analysis of the cell lysis treated with the above-mentioned frac-tions to understand the mechanism of action and synergistic and/or antagonistic actions of these fractions or compounds on metabolic pathways (see Fig. S1). To the best of our knowledge, the anti-lung cancer effects of 3 curcuminoids on A549 cells based on natural proportion and the mechanism of action and integration effects on metabolic profile has yet to be evaluated.