New Caffeoylquinic Acid Derivatives and Flavanone Glycoside from the Flowers of Chrysanthemum morifolium and Their Bioactivities

The Chrysanthemum morifolium flower is widely used in China and Japan as a food, beverage, and medicine for many diseases. In our work, two new caffeoylquinic acid derivatives (1, 2), a new flavanone glycoside (3), and six reported flavanones (4–9) were isolated and identified from the flowers of C. morifolium. The chemical structures of all isolates were elucidated by the analysis of comprehensive spectroscopic data as well as by comparison with previously reported data. The isolated constituents 1–8 were evaluated for their neuroprotective activity, and compounds 3 and 4 displayed neuroprotective effects against hydrogen peroxide-induced neurotoxicity in human neuroblastoma SH-SY5Y cells.


Introduction
The flowers of Chrysanthemum morifolium Ramat., Flos Chrysanthemi, which are cultivated in Zhejiang Province as 'Hangbaiju (HJ), have been widely and traditionally consumed in China as a medicinal and edible cognate for about 2000 years. As a well-known herb medicine, HJ is used for its efficacy in dispersing cold, eliminating heat, and improving liver function [1]. Simultaneously, tea made from HJ with hot or boiling water is a popular beverage that cools fever and heightens eyesight [2]. The biological activities of HJ have been exhaustively reported and include cardiovascular protection [3], anti-oxidation [4][5][6], anti-human immune deficiency viruses activity [7,8], anti-inflammatory activity [9], vasorelaxant activity [10], neuroprotective activity [11], anti-cancer activity [12,13], hepatoprotective effects [14,15], aldose reductase inhibition [16,17], and anti-mutagenesis activity [18]. Because of its diversiform and appealing pharmacological activities, HJ has been the subject of intense investigation. Previous research hasshown that HJ containes a wide variety of chemical compounds, including flavonoids and their corresponding glucosides [6][7][8], caffeoylquinic acids [4,5], sesquiterpenes [19], triterpenes [9], and unsaturated fatty acids [20]. Among them, caffeoylquinic acids, flavonoids, and their corresponding glycosides were considered to be the compounds from this flower. We herewith report the isolation and elucidation of two new caffeoylquinic acid derivatives (1, 2), a new flavanone glycoside (3), and six reported flavanones (4-9) (Figure 1). The structures of the new compounds were identified by analyzing spectroscopic data (1D and 2D NMR, MS, IR, ECD, ORD, and UV). The isolated compounds 1-8 were also examined for their neuroprotective and hepatoprotective properties. The isolation, structure determination, and bioactivity of these compounds are described in this paper.
Compound 1, a white, amorphous power, possesses a molecular formula of C 29 H 32 O 14 , which was determined by HRESIMS (m/z 627.1704 [M + Na] + , calculated for C 29 H 32 NaO 14 , 627.1684) and 13 C-NMR data, corresponding to 14 degrees of unsaturation. The IR spectrum displayed characteristic absorptions of hydroxy (3365 cm −1 ), carbonyl (1711 cm −1 ), and aromatic ring (1522 and 1445 cm −1 ) groups. The 1 H-NMR data (Table 1) (Figure 2a) spectra was observed, in addition to HMBC correlations ( Figure 2a) from H-7 to C-1 , C-2 , C-6 , and C-9 and from H-8 to C-1 and C-9 . Taken together, the above NMR data suggested the existence of a caffeoyl motif in the structure. A quinic acid moiety was revealed via 1 H NMR resonances of three oxymethine protons at δ H 5.44 (m, H-5), 4.90 (m, H-4), and 4.28 (m, H-3), together with two sets of sp 3 methylene protons at δ H 2.06 (m, 2H) and δ H 1.70 (2H, m) for H 2 -2 and H 2 -6, respectively, as shown in Table 1. In combination with the HSQC correlations, these resonances corresponded to three oxygenated methine carbons at δ C 69.4 (C-5), 76.3 (C-4), 70.3 (C-3), as well as to two sp 3 methylene carbons at δ C 40.2 (C-6) and 38.2 (C-2). In addition, there were an oxygenated tertiary carbon at δ C 75.2 (C-1) and a carbonyl at δ C 175.9 (C-7) in the 13 C-NMR spectrum, which are characteristic of a quinic acid unit. The assignments for the quinic acid nucleus were confirmed by analysis of the 1 H-1 H COSY cross peaks H-2ax/H-3/H-4/H-5/H-6ax and HMBC correlation from H-2ax to C-7 (175.9). The HMBC correlation between δ H 4.90 (H-4) and 168.2 (C-9 ) suggested the caffeoyl group was esterified to 4-OH of the quinic acid. Besides the signals of a caffeoyl group and a quinic acid, additional 13 carbon resonances were observed. After analyzing the 1 H, 13 C, and HSQC data, the remaining signals were attributed to one carbonyl groups (δ C 171.8), six aromatic carbons (one ABX system, δ C 131.7, 115.2, 146.6, 146.7, 116.0, and 119.8), three oxygenated methane groups (δ C 84.3, 75.4, 100.2), one methylene group (δ C 57.2), and two secondary methyls (δ C 20.3, 21.2). The assignments of substitution pattern and planar structure of compound 1 were further confirmed by analysis of the 1 H-1 H COSY and HMBC correlations. The 1 H-1 H COSY cross peaks H-7 /H-8 /H-10 / H 3 -11 together with the HMBC correlations from H-7 , H-8 , and C-9 established the structural fragment C-7 -C-8 -(C-9 )-C-10 −C-11 . The 1 H-1 H COSY cross peaks H-12 /H 3 -13 and HMBC correlations from H-12 to C-7 and C-10 enabled the establishment of a 2,6-dimethyl-5-carboxyl-1,3-dioxane ring. The HMBC correlations from H-7 to C-1 , C-2 , and C-6 and from H-8 to C-1 indicated the connectivity C-7 -C-1 . Moreover, the HMBC correlations from H-5 to C-9 confirmed C-9 is connected with C-3 via an ester bond. Thus, the planar structure of 1 is shown in Figure 1 The UV spectra (λmax 330, 288 nm) was highly similar to that of 1 (λmax 331, 288 nm), which indicated that compound 2 may be a caffeoylquinic acid derivative as well. The detailed analyses of 1D NMR (Table 1) and HSQC spectra suggested the presence of 3 carbonyls, 12 aromatic carbons, 2 olefinic carbons, 1 secondary methyl, 2 methylenes, 6 oxygenated methines, and 1 oxygenated quaternary carbon. Compared with those of 1, these characteristic signals imply that one quinic moiety and one caffeoyl group exist in the structure of 2. The assignments of substitution pattern and planar structure of compound 2 were further confirmed by analysis of the 1 H-1 H COSY and HMBC correlations ( Figure 3a). The 1 H-1 H COSY cross peaks H-7 /H-8 /H-10 / H3-11 together with the HMBC correlations from H-7 , H-8 , and H-10 established the structural fragment C-7 -C-8 -(C-9 )-C-10 −C-11 . The HMBC correlations from H-7 to C-1 , C-2 , and C-6 and from H-8 to C-1 indicated the connectivity C-7 -C-1 . The HMBC correlations from H-2 to C-7 and from C-4 and H-6 to C-4 along with that from H-5 to C-3 suggested the presence of a 1, 3, 4-trisubstituted phenyl unit. Moreover, the HMBC correlations from H-3 to C-9 confirmed C-9 is connected with C-3 via an ester bond, whereas the HMBC cross-peak of H-4 with its ester carbonyl carbon C-9 indicated the attachment of the caffeoyl group at C-4. On the basis of these results, the planar structure of 2 was assigned as shown in Figure      , respectively, were found in the high-field region. The 13 C-NMR spectrum exhibited 27 carbon signals, corresponding to a flavanone skeleton bearing two hydroxyl units and two sugar moieties. The locations of two glucose units were determined at C-7 and C-3′ on the basis of the cross-peak from the anomeric protons H-1″ to C-7, and H-1″′ to C-3′ in the HMBC spectra ( Figure 4). The coupling constant (J = 7.5 Hz, H-1′′ and H-1′′′) of two anomeric protons indicated two β-glycosidic linkages. The D-configuration of the glucose moieties was determined via GC analysis of its chiral derivatives after acid hydrolysis. Moreover, the stereochemistry at C-2 was established as S due to the presence of a negative Cotton effect at 287 nm and a positive Cotton effect at 342 nm in the circular dichroism (CD) spectrum (see Supplementary Materials) [27]. Accordingly, the structure of 3 was identified as (2S)-eriodictyol 7,3′-di-O-β-D-glucopyranoside.  , respectively, were found in the high-field region. The 13 C-NMR spectrum exhibited 27 carbon signals, corresponding to a flavanone skeleton bearing two hydroxyl units and two sugar moieties. The locations of two glucose units were determined at C-7 and C-3 on the basis of the cross-peak from the anomeric protons H-1" to C-7, and H-1" to C-3 in the HMBC spectra ( Figure 4). The coupling constant (J = 7.5 Hz, H-1 and H-1 ) of two anomeric protons indicated two β-glycosidic linkages. The D-configuration of the glucose moieties was determined via GC analysis of its chiral derivatives after acid hydrolysis. Moreover, the stereochemistry at C-2 was established as S due to the presence of a negative Cotton effect at 287 nm and a positive Cotton effect at 342 nm in the circular dichroism (CD) spectrum (see Supplementary Materials) [27]. Accordingly, the structure of 3 was identified as (2S)-eriodictyol 7,3 -di-O-β-D-glucopyranoside.

Hepatoprotective Activity
Compounds 1-8 were tested for their hepatoprotective activities against N-acetyl-p-

Hepatoprotective Activity
Compounds 1-8 were tested for their hepatoprotective activities against N-acetyl-p-aminophenol (APAP)-induced injury in HepG2 cells using bicyclol as a positive control in the MTT method. All compounds were inactive at a concentration of 10 µM ( Figure 5).

Hepatoprotective Activity
Compounds 1-8 were tested for their hepatoprotective activities against N-acetyl-paminophenol (APAP)-induced injury in HepG2 cells using bicyclol as a positive control in the MTT method. All compounds were inactive at a concentration of 10 μM ( Figure 5).

Neuroprotective Activity
The isolated compounds 1-8 were tested for their neuroprotective effect against H 2 O 2 -induced cell toxicity in SH-SY5Y cells. Compounds 3 and 4 exhibited a moderate neuroprotective effect at a concentration of 10 µM against SH-SY5Y cell damage with cell viability of 65.08% and 62.24%, respectively, compared with that measured in the presence of L-NBP (L-3-n-butylphthalide) corresponding to 59.74%. Other compounds displayed mild activities, ranging from 57.19% to 59.57% in cell viability at 10 µM ( Figure 6).
The isolated compounds 1-8 were tested for their neuroprotective effect against H2O2-induced cell toxicity in SH-SY5Y cells. Compounds 3 and 4 exhibited a moderate neuroprotective effect at a concentration of 10 μM against SH-SY5Y cell damage with cell viability of 65.08% and 62.24%, respectively, compared with that measured in the presence of L-NBP (L-3-n-butylphthalide) corresponding to 59.74%. Other compounds displayed mild activities, ranging from 57.19% to 59.57% in cell viability at 10 μM ( Figure 6).

Plant Materials
The dried flower of C. morifolium Ramat. was collected in Tongxiang, Zhejiang province, China, in September 2014. The plant was identified by Professor Lin Ma Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing.

Extraction and Isolation
Dried flowers of C. morifolium Ramat. (100 kg) were extracted three times with 80% EtOH (3 × 150 L) under reflux for 3 h. The EtOH solution was evaporated under reduced pressure, and then the

Plant Materials
The dried flower of C. morifolium Ramat. was collected in Tongxiang, Zhejiang province, China, in September 2014. The plant was identified by Professor Lin Ma Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing.

Hydrolysis of Compound 3 and GC Analysis
Compounds (4 mg) was hydrolyzed with 2 M HCl (3 mL) at 80 • C for 5 h. The sugars gained from the hydrolysates were analyzed by GC on the basis of the reported method [28].

Hepatoprotective Activity Assay
HepG2 cells were cultured in DMEM with 10% FCS, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 • C (5% CO 2 , 100% relative humidity). These cells were digested using 0.25% trypsin and then seeded into 96-well plates. After incubation for 24 h, the cells were treated with the isolated compounds (10 µM) and APAP (8 mM) and incubated for 48 h. A volume of 100 µL of MTT solution (0.5 mg/mL) was then added into each well. After incubation for 4 h at 37°C, the cells were finally lysed with 150 µL DMSO. Finally, the absorbance was measured at 570 nm with a microplate reader, using bicyclol as the positive control. The results were expressed as percentage of cell viability (%), hypothesizing the viability of control cells as 100%.

Neuroprotective Activity Assay
Human neuroblastoma SH-SY5Y cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C under a water-saturated atmosphere of 5% CO 2 . The cells were seeded in 96-well culture plates (1 × 10 4 cells/well) in 100 µL for 18 h, then incubated with the isolated compounds (10 µL, 10 µM) for 4 h. In order to induce an oxidative stress, 100 µL freshly prepared culture medium with 200 µM H 2 O 2 (IC 50 = 128.5 µM) was added to the cells and incubated with the compounds at 37 • C for 24 h. Then, 10 µL MTT solution (5 mg/mL) was added into each well. After incubation for 4 h at 37 • C, the cells were finally lysed with 150 µL DMSO. The absorbance was measured at 570 nm, using l-NBP (l-3-n-butylphthalide) as the positive control. The results were expressed as percentage of cell viability (%).

Conclusions
In summary, two new caffeoylquinic acid derivatives (1,2), a new flavanone glycoside (3), and six reported flavanone glycosides (4-9), were isolated and characterized from the dried flowering head of C. morifolium Ramat. The in vitro evaluation of neuroprotective activity suggested that compounds 3 and 4 could improve cell viability at a concentration of 10 µM. This paper not only enriches the chemical diversity of C. morifolium compounds but also provides useful clues on the neuroprotective agents of C. morifolium Ramat cv. "HJ", which will contribute to the development and application of this functional food. Further investigations should be implemented to examine the neuroprotective activity in vivo and the required isolates' concentrations and provide real biological significance in the future.