Isolation and identification of phenolic compounds from Gynura divaricata leaves

Background: Phenolic constituents were the principle bioactivity compounds exist in Gynura divaricata, little phenolic compounds were reported from the plant previously. Materials and Methods: 60% ethanol extract from the leaves of Gynura divaricata were isolated and purified by column chromatography of Silica gel, ODS and Sephadex LH-20, the structures of the isolated compounds were identified by UV, 1H-NMR, 13C-NMR and MS spectroscopic techniques. Additionally, a high-performance liquid chromatography-diode array detector-electrospray ionization-mass (HPLC-DAD-ESI-MS) analytical method was developed to identify some minor constituents in the n-butanol fraction of the ethanol extract of Gynura divaricata. Results: Six flavonols and one Dicaffeoylquinic acid were isolated from the leaves of Gynura divaricata, and these compounds were identified as follows: quercetin (1), kaempferol (2), kaempferol-3-O-β-D-glucopyranoside (3), quercetin-3-O-rutinoside (4), kaempferol-3,7-di-O-β-D-glucopyranoside (5), kaempferol-3-O-rutinoside-7-O-β-D-glucopyranoside (6), and 3,5-dicaffeoylquinic acid (7). A total of 13 compounds, including 9 flavonol glycosides and 4 phenolic acids, were tentatively identified by comparing their retention time (RT), UV, and MS spectrum values with those that had been identified and the published data. Conclusion: This was the first time to use the HPLC-DAD-ESI-MS method to identify the phytochemicals of the genera Gynura. Moreover, compounds (6) and (7) have been isolated for the first time from the genus Gynura.


INTRODUCTION
Gynura genus belongs to the family Asteraceae, consisting of 12 species in China. [1] Many species are edible medicinal plants and the leaves are also used as a vegetable by the locals in Southwestern China. [2] G. divaricata is a traditional Chinese medicinal plant, which is called "Bai Bei San Qi" in Chinese. It has a long history of use for treatment of diabetes in the folk medicine. The ethanol extract of aerial parts of G. divaricata was reported to demonstrate hypoglycemic activity in vivo, the flavonoid compounds were the active constituents. [3,4] It also has been reported that many constituents with antiproliferation activity exist in G. divaricata. [5,6] The chemical constituents of G. divaricata include flavonols, phenolic acids, cerebrosides, polysaccharides, alkaloids, terpenoids, and sterols. [5][6][7][8][9][10] Flavonols were the principal constituents of the plant, 4 flavonol compounds, including quercetin, isoquercitrin, rutin, and kaempferol-3-O-rutinoside, have been isolated and identified from the aerial parts of the plant. [9] This article herein describes the isolation and structure elucidation of the flavonol and phenolic acid compounds from the ethanol extract of G. divaricata DC. leaves by NMR and high-performance liquid chromatography-diode array detector-electrospray ionization-mass spectrometry (HPLC-DAD-ESI-MS) methods.

General
The 1 H-NMR and 13

Plant material
The Gynura divaricata plant was obtained in 2009 from Guangdong province, China. A voucher specimen (201001) was deposited at the Department of Chemistry, Nanchang University. The leaves of G. divaricata were dried at 40°C in an air oven and finely powdered.

Extraction and isolation
The weighed portion of the crude drug 5 kg was extracted twice with 60% ethanol (v/v) under reflux at 90°C. The extract was evaporated to dryness in vacuo. Extract yield with respect to the dried herb was 25%. The dry extract was suspended in water and subjected to sequential liquid-liquid extraction with chloroform, ethyl acetate (EA), and n-butanol, the yield of those 3 extracts were 31.2, 56.5, and 89.5 g, respectively. The EA fraction was chromatographed using flash column on a Silica gel eluted with chloroform-methanol step-gradient (starting with 100:0 to 4:1), eluted fractions were combined on their TLC pattern to yield 8 fractions. The chloroform-methanol fraction (10:1) was chromatographed on a Sephadex LH-20 column eluted with chloroform-methanol (1:1) to yield compounds 1 and 2. The chloroform-methanol fraction (6:1) chromatographed on a Sephadex LH-20 column eluted with methanol and further chromatographed on an RP-ODS column gradient eluted with methanolwater (40%-60%, v/v) gave compounds 3 and 7. The chloroform-methanol fraction (4:1) chromatographed on a Sephadex LH-20 column eluted with methanol yields compound 4 [ Figure 1].
The n-butanol fraction was chromatographed using flash RP-ODS column gradient eluted with methanol-water (10%-50%, v/v), and the eluted fractions were combined on their HPLC pattern to yield 4 fractions. The methanolwater fraction (25%, v/v) was further chromatographed using flash RP-ODS column and isocratic eluted with methanol-water (18%, v/v) gave compounds 5 and 6. The other minor constituents of n-butanol extracts were separated and identified by HPLC-DAD-ESI-MS method.

HPLC-MS instrument and conditions
The HPLC-DAD-ESI-MS system consists of a Waters 2995 Series LC and ZQ-4000 Mass spectrometer (Waters, USA), equipped with a vacuum degasser, a quaternary pump, an autosampler, a thermostatted column compartment, a diode array detector (DAD), and an ion-trap mass spectrometer with electrospray ionization interface, controlled by Waters 2995 Series LC/ZQ-4000 Trap Software. Shimadzu shimpack VP-ODS (150 mm × 4.6 mm i.d., 5 μm particle size) was used for separation. Solvents for the mobile phase were water-0.1% acetic acid (A) and acetonitrile (B). The gradient elution was 0-30 min, linear gradient 10%-30% B; 30-40 min, linear gradient 30%-100% B. The flow rate was 0.8 mL/min and the column was operated at 30°C. Peaks were detected with the DAD at 254 nm. The ESI negative and positive ionization (NI and PI) total ion current (TIC) modes were used for MS detection. The m/z values of the monitored ions were from 100 to 800. The other parameters were as follows: capillary voltage, 3.5 kV; cone voltage, 30 V; extraction voltage, 5 V; RF voltage, 0.5 V; source temperature, 90°C; nitrogen gas flow for desolvation, 300 L/h; and temperature of the nitrogen gas for desolvation, 350°C. Samples for assay were dissolved in 45% MeOH as 3 mg/mL solutions and centrifuged at 12,000 rpm (Beckman, USA) for 15 min to remove particles before injection.
Compound 6 was obtained as a faint yellow powder, the molecular formula C 33 H 40 O 20 was suggested by a mass spectrum with a quasi-molecular ion peak [M-H] − at m/z 755. The UV and 1 H-NMR spectrum of compound 6 was similar to that of 5, suggesting that compound 6 also was a kaempferol glycoside derivative, the only difference being the presence of a methyl signal (δ 0.99) in the highfield region, which was assigned to rhamnose, further confirmed by the doublet proton at δ 4.44, was assigned to the anomeric proton of rhamnose with a coupling constant (J = 1.6 Hz) characteristic for α-linked rhamnose. The 13 C-NMR spectrum of 6 confirms that compound 6 is a triglycoside of kaempferol [ Table 1]. Careful examination of the 13 C-NMR spectrum of 6 showed that the signal assigned to the glucose C-6 [ Table 1] was shifted downfield by appropriately 6 ppm (from 61.3 to 67.3) confirming that the rhamnose moiety linkage to the glucose C-6. [17] From the above data, compound 6 was identified as kaempferol-  2H, m, H-2). The 1 H-NMR data were in agreement with the literature [18] and compound 7 was identified as 3,5-Dicaffeoylquinic acid.
An HPLC-DAD-ESI-MS method was developed to  identify the minor phytochemical constituents of n-butanol fraction of G. divaricata extract. The chromatogram of MS TIC in negative mode is shown in Figure 2a. As shown in Figure 2b, 13 major peaks were detected under the HPLC conditions with DAD detection at 254 nm. Peaks of 2, 3, and 11, 12 were co-eluted in the present conditions and unequivocally determined to be kaempferol-3,7di-O-β-d-glucopyranoside, kaempferol-3-O-rutinoside-7-O-d-glucopyranoside, 3,5-Dicaffeoylquinic acid, and kaempferol-3-O-β-d-glucopyranoside, respectively. And peak 5 was identified as quercetin-3-O-rutinoside. All of those 5 peaks were identified by comparing the retention time (RT), UV [ Figure 3], and ESI-MS values with isolation compounds. The other compounds were tentatively identified based on the UV adsorption value, m/z value, and elution order compared with the published data.
The flavonoid and phenolic acid compounds were affected by the concentration of extraction ethanol. The singlefactor experiment showed that 60% ethanol was suitable to extract the phenolic constituents from the plant. The levels of phenolic contents were decreased as the concentration of ethanol increased. Chloroform was used to remove the nonpolar constituents, while little extracts were obtained using diethyl ether and petroleum ether. The ethyl acetate extracts showed powerful antioxidant activity and highest total phenolic content. HPLC analysis showed that ethyl acetate extracts only shared 3 principal peaks, and the kaempferol-3-O-β-d-glucopyranoside was the major constituent. However, n-butanol extract shared numerous flavonoid compounds, while the total phenolic was lower.
In order to fully elaborate the phenolic compounds of the extract from G. divaricata, the extracts of ethyl acetate and n-butanol were isolated using chromatograph column and HPLC-DAD-ESI-MS method. To our best knowledge, the present study is the first report of the isolation and identification of triglycoside of kaempferol and Dicaffeoylquinic acid from the leaves of G. divaricata.