Phanerosides A–X, Phenylpropanoid Esters of Sucrose from the Rattans of Phanera championii Benth

Twenty-four new phenylpropanoid esters of sucrose, phanerosides A–X (1–24), were isolated from an EtOH extract of the rattans of Phanera championii Benth. (Fabaceae). Their structures were elucidated on the basis of comprehensive spectroscopic data analysis. A wide range of structural analogues were presented due to the different numbers and positions of acetyl substituents and the structures of phenylpropanoid moieties. Phenylpropanoid esters of sucrose were isolated from the Fabaceae family for the first time. Biologically, the inhibitory effects of compounds 6 and 21 on NO production in LPS-induced BV-2 microglial cells were better than that of the positive control, with IC50 values of 6.7 and 5.2 μM, respectively. The antioxidant activity assay showed that compounds 5, 15, 17, and 24 displayed moderate DPPH radical scavenging activity, with IC50 values ranging from 34.9 to 43.9 μM.

Fabaceae is the third largest plant family, comprising approximately 800 genera and 20,000 species worldwide [13,14]. As the largest of the several genera in the Fabaceae family, Phanera results from the reorganization of Bauhinia sensu lato. The genus Phanera encompasses about 90-100 species, mainly distributed in tropical Asia and Australasia [15]. As one of them, Phanera championii Benth. is widely distributed in Guangxi Province and was first recorded in 'Nanning Drug Chi' [16]. It is a well-known folk medicine with a rich history of being used as medication to treat rheumatoid arthritis and epigastric pain [17,18]. Phytochemical investigations of this plant have primarily resulted in the isolation of flavonoids, nitrile glucoside, dibenzofurans, and diterpenoid [16,[19][20][21]. In our ongoing research in pursuit of novel and biologically active metabolites from ethnic medicines in Guangxi, 24 new phenylpropanoid esters of sucrose (1-24) were isolated from the rattans of P. championii. Herein, the isolation, structural elucidation, anti-inflammatory, and antioxidant activities in vitro of all the isolated compounds are described.

Structural Elucidation
Compound 1 (Figure 1) was isolated as a white amorphous powder with a molecular formula of C 30 H 34 O 15 , as determined using HRESIMS (m/z 657.1798 [M + Na] + , calcd for 657.1790) and 13 C NMR data. The 1 H NMR spectrum (Table 1)  In addition, a characteristic doublet with a small coupling constant (J = 3.6 Hz) at δ H 5.60 was also shown in the 1 H NMR spectrum, which together with 12 oxygen-bearing carbon signals (including two anomers at δ C 105.7 and 90.6) in the 13 C NMR data ( Table 2) supposed the presence of a disaccharide moiety. Furthermore, detailed analysis of the 2D NMR correlations ( Figure 2) and chiral HPLC analysis of monosaccharides after acid hydrolysis of 1 revealed that the two sugars were β-D-fructose and α-D-glucose units connected via C-2→C-1 to construct a D-sucrose moiety. The 13 C NMR spectrum showed 30 carbon signals, apart from the 12 carbon signals occupied by the D-sucrose moiety; the other 18 ones were classified as 2 carbonyl carbons (δ C 169.2, 168.8) and 16 olefinic or aromatic carbons with the assistance of the HSQC data. Two discrete spin systems, H-7 /H-8 and H-7 /H-8 , in the 1 H-1 H COSY spectrum, and the correlations from H-7 to C-1 /C-2 /C-6 /C-9 and H-7 to C-1 /C-2 /C-6 /C-9 in the HMBC spectrum ( Figure 2), established two trans-p-coumaroyl moieties. Subsequently, the key HMBC correlations from H-2 to C-9 as well as from H 2 -6 to C-9 suggested that the two trans-p-coumaroyl moieties were located at C-2 and C-6 . Thus, the structure of 1 was identified and named phaneroside A.

Structural Elucidation
Compound 1 (Figure 1) was isolated as a white amorphous powder with a molecular formula of C30H34O15, as determined using HRESIMS (m/z 657.1798 [M + Na] + , calcd for 657.1790) and 13 C NMR data. The 1 H NMR spectrum (Table 1)  In addition, a characteristic doublet with a small coupling constant (J = 3.6 Hz) at δH 5.60 was also shown in the 1 H NMR spectrum, which together with 12 oxygen-bearing carbon signals (including two anomers at δC 105.7 and 90.6) in the 13 C NMR data ( Table 2) supposed the presence of a disaccharide moiety. Furthermore, detailed analysis of the 2D NMR correlations ( Figure  2) and chiral HPLC analysis of monosaccharides after acid hydrolysis of 1 revealed that the two sugars were β-D-fructose and α-D-glucose units connected via C-2→C-1′ to construct a D-sucrose moiety. The 13 C NMR spectrum showed 30 carbon signals, apart from the 12 carbon signals occupied by the D-sucrose moiety; the other 18 ones were classified as 2 carbonyl carbons (δC 169.2, 168.8) and 16 olefinic or aromatic carbons with the assistance of the HSQC data. Two discrete spin systems, H-7″/H-8″ and H-7‴/H-8‴, in the 1 H-1 H COSY spectrum, and the correlations from H-7″ to C-1″/C-2″/C-6″/C-9″ and H-7‴ to C-1‴/C-2‴/C-6‴/C-9‴ in the HMBC spectrum ( Figure 2), established two trans-p-coumaroyl moieties. Subsequently, the key HMBC correlations from H-2′ to C-9″ as well as from H2-6′ to C-9‴ suggested that the two trans-p-coumaroyl moieties were located at C-2′ and C-6′. Thus, the structure of 1 was identified and named phaneroside A.    Compounds 2-24 were determined to be structurally related to 1 according to their extremely similar physicochemical properties and NMR data. The latter all showed the characteristics of a core D-sucrose unit, while the variation of substituents and their positions obtained different structures. The structural elucidation of 2-24 was as follows.
The molecular formula of compound 2 was assigned as C 30 H 34 O 14 based on its HRES-IMS (m/z 641.1847 [M + Na] + , calcd for 641.1841) and 13 C NMR data, with 16 mass units less than 1. The analogous 1 H NMR data (Table 1) of 1 and 2 indicated that they were structural analogues, excepting of the presence of a monosubstituted aromatic moiety [δ H 7.65 (1H, m, H-2 /6 ), 7.41 (3H, overlapped, H-3 /4 /5 )] and the absence of an AA BB aromatic unit in 2. The long-range correlations from phenyl protons to olefinic carbons and correlations from olefinic protons to ester carbonyl carbons in the HMBC spectrum indicated that a trans-p-coumaroyl group and a trans-cinnamoyl group were presented ( Figure 3). The HMBC correlations from H-2 (δ H 4.73) to C-9 and H 2 -6 (δ H 4.56, 4.34) to C-9 confirmed the structure of 2 as shown, and this compound was named phaneroside B.      (Table 1) (Tables 1 and 2) indicated the presence of two trans-feruloyl moieties and a D-sucrose unit. The key HMBC crosspeaks from H-2 (δ H 4.73) to C-9 and H 2 -6 (δ H 4.54, 4.31) to C-9 suggested that the two trans-feruloyl moieties were linked to C-2 and C-6 of glucopyranose unit ( Figure 3). Thus, the structure of compound 5 was identified and named phaneroside E.
Compounds 6-8 shared the same molecular formula of C 32 H 36 O 16 , as obtained from their respective HRESIMS data. Their molecular masses were 42 mass units more than that of 1, which combined with the 1D NMR data of 6-8 (Tables 3 and 5) 4.11, 4.06) to carbonyl (δ C 172.0) in 6, from H-3 (δ H 5.43) to carbonyl (δ C 172.2) in 7, and from H-4 (δ H 5.30) to carbonyl (δ C 172.4) in 8 indicated that the acetyl group was attached to C-1, C-3, and C-4 of the fructofuranose unit in compounds 6, 7, and 8, respectively ( Figure 4). In addition, the chemical shifts of the protons linked to the acetyl group in 6-8 were obviously shifted downfield compared to those of 1, which also supported the above description. Thus, the structures of 6, 7, and 8 were established and named phanerosides F, G, and H, respectively.   Compounds 9-11 were assigned the same molecular formula, C33H38O17, according to their HRESIMS and 13 C NMR data. Compounds 9-11 were determined to be acetylated derivatives of 4, as their molecular masses were 42 mass units more than that of 4. Their 1 H and 13    In their HMBC spectra, the correlations from H 2 -1 (δ H 4.14, 4.03) to carbonyl (δ C 172.0) in 12, from H-3 (δ H 5.43) to carbonyl (δ C 172.2) in 13, and from H-4 (δ H 5.31) to carbonyl (δ C 172.3) in 14 suggested that the acetyl group was linked to C-1, C-3, and C-4 in 12, 13, and 14, respectively. Therefore, the structures of compounds 12-14 (phanerosides L-N) were established as shown.
Compounds 15-17 were determined to have the same molecular formula, C 34 H 40 O 18 , on the basis of their HRESIMS and 13 C NMR data, displaying 42 mass units more than that of 5. Compounds 15-17 were suggested to be acetylated derivatives of 5 after an analysis of their 1D NMR data (Tables 4 and 5). The acetyl group was, respectively, located at C-1, C-3, and C-4 of the fructofuranose unit in 15, 16, and 17, which was confirmed using the key HMBC correlations from the protons of the sugar unit to corresponding carbonyl carbons. Accordingly, the structure of compounds 15-17 (phanerosides O-Q) were determined as shown.
Compounds 18 and 19 had the same molecular formula, C 34 H 40 O 18 , as 17 according to their HRESIMS and 13 C NMR data. The 1D NMR data (Tables 4 and 5) closely resembled those of 17, with the exception that one of the two trans-feruloyl groups was replaced by a cis-feruloyl group according to the smaller coupling constants of 3 J 7 ,8 (12.6 Hz) in 18 and 3 J 7 ,8 (13.2 Hz) in 19. The cis-feruloyl group was linked to C-2 and C-6 of the glucopyranose unit in 18 and 19, respectively, which was proved by the key HMBC cross-peaks from H-2 /H-7 to C-9 in 18 and from H-6 /H-7 to C-9 in 19 ( Figure 5). Thus, the structures of compounds 18 and 19 were identified and named phanerosides R and S, respectively.  Compounds 18 and 19 had the same molecular formula, C34H40O18, as 17 according to their HRESIMS and 13 C NMR data. The 1D NMR data (Tables 4 and 5) closely resembled those of 17, with the exception that one of the two trans-feruloyl groups was replaced by a cis-feruloyl group according to the smaller coupling constants of 3 J7″,8″ (12.6 Hz) in 18 and 3 J7 ‴ ,8 ‴ (13.2 Hz) in 19. The cis-feruloyl group was linked to C-2′ and C-6′ of the glucopyranose unit in 18 and 19, respectively, which was proved by the key HMBC cross-peaks from H-2′/H-7″ to C-9″ in 18 and from H-6′/H-7‴ to C-9‴ in 19 ( Figure 5). Thus, the structures of compounds 18 and 19 were identified and named phanerosides R and S, respectively.   Figure 6). Thus, the structures of compounds 20 and 21 were characterized and named phanerosides T and U, respectively.    Figure 6). Thus, the structures of compounds 20 and 21 were characterized and named phanerosides T and U, respectively.    Compounds 22 and 23 showed the same molecular formula, C35H40O18, as determined by their 13 C NMR data and respective HRESIMS ion peaks at m/z 771.2093 and 771.2112 ([M + Na] + , calcd for 771.2107). Analysis of the NMR data (Figures S194-S199,S203-S208) of 22 and 23 proclaimed that the sugar moieties in both compounds were acylated by a trans-ferulic acid, a trans-p-coumaric acid, and two acetic acids. The trans-feruloyl and trans-p-coumaroyl units were, respectively, located at C-2′ and C-6′ in 22 based on the key HMBC correlations from H-2′ to C-9″ and H2-6′ to C-9‴, while the locations of these two substituents in 23 were the opposite. The key HMBC correlations from H-3 (δH 5.65) to carbonyl (δC 172.0) and H-4 (δH 5.50) to carbonyl (δC 171.8) in 22, and from H2-1 (δH 4.16, 3.99) to carbonyl (δC 171.9) and H-3 (δH 5.30) to carbonyl (δC 172.1) in 23, indicated that the two acetyl groups were placed at C-3 and C-4 in 22 and C-1 and C-3 in 23. Accordingly, the structures of compounds 22 and 23 (phanerosides V and W) were defined as shown.
The molecular formula of compound 24 was assigned as C36H42O19 based on the HRESIMS (m/z 801.2226 [M + Na] + , calcd for 801.2213) and 13 C NMR data. The NMR data ( Figures S212-S217) indicated that it possessed two trans-feruloyl moieties, D-sucrose, and two acetyl groups. The two trans-feruloyl moieties were linked to C-2′ and C-6′ of the glucopyranose ring, and two acetyl groups were attached to C-3 and C-4 of the fructofuranose ring, according to the key HMBC correlations as shown in Figure 6. Thus, the structure of compound 24 was identified and named phaneroside X.
a. In Vitro Anti-inflammatory Effects of Compounds 1-24 Nitric oxide (NO) is one of the major inflammatory mediators, and phenylpropanoid Analysis of the NMR data (Figures S194-S199 and S203-S208) of 22 and 23 proclaimed that the sugar moieties in both compounds were acylated by a trans-ferulic acid, a trans-p-coumaric acid, and two acetic acids. The trans-feruloyl and trans-p-coumaroyl units were, respectively, located at C-2 and C-6 in 22 based on the key HMBC correlations from H-2 to C-9 and H 2 -6 to C-9 , while the locations of these two substituents in 23 were the opposite. The key HMBC correlations from H-3 (δ H 5.65) to carbonyl (δ C 172.0) and H-4 (δ H 5.50) to carbonyl (δ C 171.8) in 22, and from H 2 -1 (δ H 4.16, 3.99) to carbonyl (δ C 171.9) and H-3 (δ H 5.30) to carbonyl (δ C 172.1) in 23, indicated that the two acetyl groups were placed at C-3 and C-4 in 22 and C-1 and C-3 in 23. Accordingly, the structures of compounds 22 and 23 (phanerosides V and W) were defined as shown.
The molecular formula of compound 24 was assigned as C 36 H 42 O 19 based on the HRESIMS (m/z 801.2226 [M + Na] + , calcd for 801.2213) and 13 C NMR data. The NMR data ( Figures S212-S217) indicated that it possessed two trans-feruloyl moieties, D-sucrose, and two acetyl groups. The two trans-feruloyl moieties were linked to C-2 and C-6 of the glucopyranose ring, and two acetyl groups were attached to C-3 and C-4 of the fructofuranose ring, according to the key HMBC correlations as shown in Figure 6. Thus, the structure of compound 24 was identified and named phaneroside X.

a. In Vitro Anti-inflammatory Effects of Compounds 1-24
Nitric oxide (NO) is one of the major inflammatory mediators, and phenylpropanoid esters of sucrose have been previously reported to possess potent anti-inflammatory activity [3,7,22]. Therefore, all the isolates were evaluated in vitro for their anti-inflammatory potential via the Griess reaction in LPS-induced BV-2 microglial cells (Figure 7) [23]. Especially, compounds 6 and 21 exhibited potent inhibitory activities on NO production, with IC 50 values of 6.7 ± 1.7 and 5.2 ± 3.5 µM, which were better than the positive control, L-NMMA (IC 50 = 7.0 ± 2.7 µM) . Compounds 10, 14, and 19 showed moderate inhibitory effects on NO production, with IC 50 values of 72.7, 46.0, and 57.7 µM, respectively. These results suggested that the anti-inflammatory activities of these compounds were not determined by a single variable, while the type, number, and position of the substituents may all affect their inhibitory activities.

b. Antioxidant Effects of Compounds 1-24
Many isolated phenylpropanoid esters of sucrose are thought to act as potential antioxidants [3]. Consequently, their antioxidant activities were also tested using the DPPH radical scavenging assay [24]. Compounds 5, 15, 17, and 24 exhibited moderate inhibitory effects with EC50 values of 43.9 ± 0.2, 43.8 ± 0.1, 34.9 ± 0.1, 39.4 ± 0.3 µM, respectively. As it stands, the compounds whose C-2′ and C-6′ of the glucopyranose ring were both substituted by trans-feruloyl groups showed a more positive impact on their antioxidant effects.

General Experimental Procedures
Optical rotations were obtained on a JASCO P-2000 polarimeter. UV absorption spectra were determined on a PerkinElmer 650 spectrophotometer. NMR spectra were acquired on a 400 or 600 MHz Bruker AVANCE apparatus. Chemical shifts are expressed in δ (ppm) and referenced to the solvent residual peak. HRESIMS data were obtained on an Agilent 6545 Q-TOF LC-MS spectrometer. The other instruments and materials serving for the isolation and purification of compounds were coincident with previous papers [25,26]

b. Extraction and Isolation
The dried rattans of P. championii (21.0 kg) were soaked for 12 h in 95% aqueous EtOH (100 L) at room temperature, and then extracted three times with 95% aqueous EtOH (3 ×

b. Antioxidant Effects of Compounds 1-24
Many isolated phenylpropanoid esters of sucrose are thought to act as potential antioxidants [3]. Consequently, their antioxidant activities were also tested using the DPPH radical scavenging assay [24]. Compounds 5, 15, 17, and 24 exhibited moderate inhibitory effects with EC 50 values of 43.9 ± 0.2, 43.8 ± 0.1, 34.9 ± 0.1, 39.4 ± 0.3 µM, respectively. As it stands, the compounds whose C-2 and C-6 of the glucopyranose ring were both substituted by trans-feruloyl groups showed a more positive impact on their antioxidant effects.

General Experimental Procedures
Optical rotations were obtained on a JASCO P-2000 polarimeter. UV absorption spectra were determined on a PerkinElmer 650 spectrophotometer. NMR spectra were acquired on a 400 or 600 MHz Bruker AVANCE apparatus. Chemical shifts are expressed in δ (ppm) and referenced to the solvent residual peak. HRESIMS data were obtained on an Agilent 6545 Q-TOF LC-MS spectrometer. The other instruments and materials serving for the isolation and purification of compounds were coincident with previous papers [25,26].  Tables 3 and 5. All significant data are presented in Supplementary Materials ( Figures S47-S55).
Phaneroside G (7) Compound 1 (4.0 mg) was added to 5.0 mL of 9% aqueous HCl in a sealed flask, which was refluxed at 80 • C for 5 h. The acidic aqueous mixture was dried, H 2 O (2 mL) was added, and the mixture was extracted with EtOAc (3 × 2 mL). The aqueous layer was concentrated to obtain the sugar fraction, which was dissolved with MeOH and analyzed using chiral-phase HPLC equipped with a Daicel Chiralpak AD-H column (250 × 4.6 mm, 5 µm) and an evaporative light-scattering detector (ELSD) using n-hexane:EtOH (82:18) as the mobile phase (0.7 mL/min) [27]. The sugars were confirmed to be D-glucose and D-fructose by comparing their retention times with those of D-glucose (17.4 min), L-glucose (18.2 min), D-fructose (25.6 min), and L-fructose (26.4 min) ( Figure S10).

e. NO Production Measurements and Cell Viability Assays
The inhibitory effects of the isolated compounds on LPS-stimulated NO production were evaluated using the Griess reaction, and the cytotoxicities of compounds on BV-2 microglial cells were evaluated using MTT assays, as described in our previous report [23]. The result is shown in Figure 7.

f. Antioxidant Activity Assay
The antioxidant activity of the isolated compounds was tested using a DPPH radical scavenging assay as previously described, and vitamin C was used as the positive control [24].

Conclusions
In conclusion, 24 new phenylpropanoid esters of sucrose were isolated from the rattans of P. championii. Their structures were determined via extensive spectroscopic methods. The configuration of sugar moiety was determined via chiral-phase HPLC equipped with an evaporative light-scattering detector (ELSD) after acid hydrolysis of compound 1. This is the first report of phenylpropanoid esters of sucrose isolated from the family Fabaceae. Structurally, these compounds revealed a huge structural diversity in terms of the number and position of phenylpropanoid and acetyl substituents. Biologically, all the isolated compounds were evaluated for their anti-inflammatory and antioxidant activities, and several compounds showed potent or moderate effects. Additionally, the structure-activity relationship was briefly discussed. These compounds may serve as potential leads for the development of anti-inflammatory agents.