Identification of Phytoconstituents in Leea indica (Burm. F.) Merr. Leaves by High Performance Liquid Chromatography Micro Time-of-Flight Mass Spectrometry

Leea indica (Vitaceae) is a Southeast Asian medicinal plant. In this study, an ethyl acetate fraction of L. indica leaves was studied for its phytoconstituents using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-microTOF-Q-MS/MS) analysis. A total of 31 compounds of different classes, including benzoic acid derivatives, phenolics, flavonoids, catechins, dihydrochalcones, coumarins, megastigmanes, and oxylipins were identified using LC-MS/MS. Among them, six compounds including gallic acid, methyl gallate, (−)-epigallocatechin-3-O-gallate, myricetin-3-O-rhamnoside, quercetin-3-O-rhamnoside, and 4′,6′-dihydroxy-4-methoxydihydrochalcone 2′-O-β-d-glucopyranoside were isolated and identified by NMR analysis. The LC-MS/MS analysis led to the tentative identification of three novel dihydrochalcones namely 4′,6′-dihydroxy-4-methoxydihydrochalcone 2′-O-rutinoside, 4′,6′-dihydroxy-4-methoxydihydrochalcone 2′-O-glucosylpentoside and 4′,6′-dihydroxy-4-methoxydihydrochalcone 2′-O-(3″-O-galloyl)-β-d-glucopyranoside. The structural identification of novel dihydrochalcones was based on the basic skeleton of the isolated dihydrochalcone, 4′,6′-dihydroxy-4-methoxydihydrochalcone 2′-O-β-d-glucopyranoside and characteristic LC-MS/MS fragmentation patterns. This is the first comprehensive analysis for the identification of compounds from L. indica using LC-MS. A total 24 compounds including three new dihydrochalcones were identified for the first time from the genus Leea.


Identification of Dihydrochalcones by LC-ESI-MS/MS Analysis
The ethyl acetate fraction of L. indica leaves was analyzed by the LC-ESI-MS/MS method. Figure 1 shows the base peak chromatogram (BPC) of the ethyl acetate fraction of L. indica leaves at 254 nm. Figure 2 shows the structures of the 31 compounds identified. In total, 31 compounds were identified of which ten compounds ( While dihydrochalcone phloridzin has been previously reported in L. indica [13], the other six dihydrochalcone derivatives have not been previously reported in the same plant species. The observed MS peaks including retention time, observed mass, calculated mass, molecular formula, ppm error, and MS/MS data are presented in Table 1.     The structural identification of three new dihydrochalcones 25, 26 and 29 was based on the relevance of the LC-MS/MS fragmentation patterns with the isolated compound 4',6'-dihydroxy-4methoxy dihydrochalcone 2'-O-β-D-glucopyranoside 27. The MS/MS spectra of compounds 25, 26, 27 and 29, showed a common base ion peak at m/z 287 for 2′,4′,6′-trihydroxy-4methoxydihydrochalcone, which is a characteristic ion formed by the loss of glycoside(s) and/or galloyl glycoside moieties.      Figure S1). In the MS/MS spectrum, a characteristic fragment ion at m/z 287 as base peak suggested that this compound corresponded to a 2 ,4 ,6 -trihydroxy-4-methoxydihydrochalcone linked to a rutinose moiety, where the neutral loss of 308 Da is characteristic of the loss of a rutinose moiety [21].  Figure S1). In the MS/MS spectrum, a characteristic fragment ion at m/z 287 as base peak suggested that this compound corresponded to a 2′,4′,6′-trihydroxy-4methoxydihydrochalcone linked to a rutinose moiety, where the neutral loss of 308 Da is characteristic of the loss of a rutinose moiety [21].    The LC-MS fragmentation patterns of the three novel dihydrochalcones (25, 26 and 29) were compared to the isolated dihydrochalcone (27), and we noted that the observed HR-MS data were in good agreement with the calculated masses. Further isolation of the peaks 25, 26 and 29 and spectroscopic analyses would be required to unambiguously confirm the proposed structures of these The LC-MS fragmentation patterns of the three novel dihydrochalcones (25, 26 and 29) were compared to the isolated dihydrochalcone (27), and we noted that the observed HR-MS data were in good agreement with the calculated masses. Further isolation of the peaks 25, 26 and 29 and spectroscopic analyses would be required to unambiguously confirm the proposed structures of these dihydrochalcones.

Plant Materials
Fresh ground leaves of L. indica were collected in Singapore. A voucher specimen (no. LI-0109) was deposited at the herbarium of the National University of Singapore (NUS) Medicinal Plant Research Group.

Extraction and Isolation
The fresh ground leaves of L. indica (2.8 kg) were macerated with 70% v/v MeOH at room temperature. The extract was filtered and concentrated under vacuum, yielding a crude methanolic extract. The dried methanolic extract was dissolved in water and partitioned with different solvents, concentrated under vacuum to give hexane (0.005%), dichloromethane (0.027%) and ethyl acetate (1.32%) fractions.

General Information
NMR spectra were recorded on a Bruker Avance-400 Spectrometer (Fallanden, Switzerland), 1 H at 400 MHz and 13 C at 100 MHz in deuterated solvents using tetramethylsilane (TMS) as an internal reference. Deuterated solvents, methanol-d 4 and dimethyl sulfoxide-d 6 for NMR were purchased from Sigma-Aldrich (USA).
The LC-MS analysis was carried out using a Dionex Ultimate 3000 VWD system coupled with a VWD and a micro-TOF-Q mass detector (Bruker Daltonics Inc., Billerica, MA, USA). Chromatographic separation was performed on an RP-C 18 column (3.0 × 150 mm; particle size 2.7 µM; Agilent Poroshell 120, New Castle, DE, USA), operated at 25 • C. Analysis was carried out using a gradient elution program of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) as a mobile phase at a flow rate of 0.5 mL/min. The following gradient system was used: 0-45 min, 5-30% B; 45-60 min 30-100% B and 60-65 min 100% B. UV detection was performed by scanning the samples at 210, 254, 280, and 360 nm. Electrospray ionization mass spectra (ESI-MS) were recorded in negative ionization mode. The mass range of m/z 50-2000 was scanned. For MS/MS analysis, collision energies were set automatically.