Tadehaginosides AâJ, Phenylpropanoid Glucosides from Tadehagi

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Tadehaginosides A−J, Phenylpropanoid Glucosides from Tadehagi triquetrum, Enhance Glucose Uptake via the Upregulation of PPARγ and GLUT‑4 in C2C12 Myotubes Xiaopo Zhang,† Changyu Chen,† Yonghui Li,† Deli Chen,‡ Lin Dong,† Wei Na,† Chongming Wu,*,‡ Junqing Zhang,*,† and Youbin Li*,† †

School of Pharmaceutical Science, Hainan Medical University, Hainan 571199, People’s Republic of China Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Ten new phenylpropanoid glucosides, tadehaginosides A−J (1−10), and the known compound tadehaginoside (11) were obtained from Tadehagi triquetrum. These phenylpropanoid glucosides were structurally characterized through extensive physical and chemical analyses. Compounds 1 and 2 represent the first set of dimeric derivatives of tadehaginoside with an unusual bicyclo[2.2.2]octene skeleton, whereas compounds 3 and 4 contain a unique cyclobutane basic core in their carbon scaffolds. The effects of these compounds on glucose uptake in C2C12 myotubes were evaluated. Compounds 3−11, particularly 4, significantly increased the basal and insulin-elicited glucose uptake. The results from molecular docking, luciferase analyses, and ELISA indicated that the increased glucose uptake may be due to increases in peroxisome proliferator-activated receptor γ (PPARγ) activity and glucose transporter-4 (GLUT-4) expression. These results indicate that the isolated phenylpropanoid glucosides, particularly compound 4, have the potential to be developed into antidiabetic compounds.

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uptake, and compound 4 exhibited the most potent activity, with an efficacy comparable to that of 100 nM insulin. In light of its unique structure and potent activity, the time- and dosedependent relationships of 4 were further evaluated. The results from molecular docking, luciferase analyses, and ELISA indicated that the increased glucose uptake may be due to increases in PPARγ activity and GLUT-4 expression. These results demonstrated that the phenylpropanoid glucosides, particularly compound 4, have the potential to be developed into antidiabetic compounds.

lants belonging to the genus Tadehagi, which are widely distributed throughout tropical and subtropical regions, have been used as traditional medicines in many countries worldwide.1 The secondary metabolites obtained from these plants, which possess interesting chemical structures and exert multiple biological effects, have attracted considerable attention from chemists and pharmacologists.2,3 Among these plants, Tadehagi triquetrum has been used for the treatment of many chronic diseases,4,5 and previous studies have shown that phenolic compounds are the main constituents of T. triquetrum. The structure of tadehaginoside, a rare phenylpropanoid glucoside isolated from this plant, consists of a glucosyl, a phloroglucinol, and a trans-p-hydroxycinnamoyl moiety, and this compound exhibits antihepatotoxic, lipid-lowering, and hypoglycemic activities.6,7 In the ongoing search for phenylpropanoid glucosides with hypoglycemic activities from this plant,8 10 new compounds, tadehaginosides A−J (1−10), and the known compound tadehaginoside (11) were obtained from its aerial parts. Tadehaginosides A−D are defined as dimeric derivatives of tadehaginoside characterized by unusual bicyclo[2.2.2]octene skeletons or cyclobutane rings in their structures. The effects of the isolated compounds on glucose uptake in C2C12 myotubes were evaluated. Compounds 3−11 significantly increased the basal and insulin-elicited glucose © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Ten new phenylpropanoid glucosides, tadehaginosides A−J (1−10), were obtained from T. triquetrum after successive separations and purifications using various column chromatography methods. Tadehaginoside A (1) possesses a molecular formula of C42H44O20, as was established by the analysis of its HRESIMS (m/z 891.2327 [M + Na]+, calculated as 891.2324 for C42H44O20Na) and 13C NMR data. The 1H NMR spectrum of 1 showed resonances attributable to four methines at δH 3.73 Received: September 11, 2015

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DOI: 10.1021/acs.jnatprod.5b00820 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Chemical structures of compounds 1−11.

observed at δH 4.86 (2H, J = 7.8 Hz, H-7″, H-7‴) indicated the presence of two β-glucopyranosyl groups. The enzymatic hydrolysis of 1 afforded D-glucose (Figure 1), as was verified by GC analysis. The 13C NMR data of 1 revealed 42 resonances, which were attributed to three aromatic rings at δC 161.0 (×2), 160.3 (×4), 157.7, 134.2, 129.7 (×2), 116.7 (×2), 98.3 (×2), and 97.1 (×4); two double bonds at δC 147.1, 141.7, 134.4, and 119.2; two glucosyl moieties at δC 102.2, 102.1, 78.1, 77.8, 75.6, 75.3, 75.0, 74.9, 71.9, 71.7, and 65.0 (×2); four methine groups at δC 59.3, 51.6, 44.8, and 37.3; one methylene group at δC 35.7; and three carbonyl groups at δC 210.7, 174.7, and 168.7. The structure of 1 was established through an analysis of its 2D NMR data. The 1H−1H COSY correlations from H-2 (δH 6.60) to H-3 (δH 3.28), H-3 to H-7′ (δH 3.67), H-7′ to H-8′ (δH 2.90), H-8′ to H-6 (δH 3.73), and H-6 to H-5 (δH 1.95,

(br s, H-6), 3.67 (t, J = 6.6 Hz, H-7′), 3.28 (br d, J = 6.6 Hz, H3), and 2.90 (br d, J = 6.6 Hz, H-8′). Protons resonating at δH 1.95 (d, J = 18.0 Hz, H-5a) and 2.40 (d, J = 18.0 Hz, H-5b) indicated the presence of an isolated methylene group. The two low-field doublets observed at δH 6.23 (d, J = 15.6 Hz, H-8) and 7.39 (d, J = 15.6 Hz, H-7) were assigned as the α and β protons of a trans-enoyl group.9 Additionally, the signal at δH 6.60 (d, J = 6.0 Hz, H-2) was characteristic of an olefinic methine proton. Proton signals at δH 6.97 (2H, d, J = 8.4 Hz, H-2′, H-6′) and δH 6.68 (2H, d, J = 8.4 Hz, H-3′, H-5′) indicated the presence of a p-substituted aromatic moiety.10 Signals for two 1,3,5-trisubstituted aromatic rings were observed at δH 6.07 (2H, d, J = 1.8 Hz, H-2″, H-6″), 6.06 (2H, d, J = 1.8 Hz, H-2‴, H-6‴), and 5.95 (2H, br s, H-4″, H4‴).11 The resonances corresponding to anomeric protons B



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Figure 2. Key 1H−1H COSY () and HMBC (→) connections of compounds 1, 3, 5, 6, 7, and 9.

H-8′ (δH 2.90)/H-2′ and H-6′ (δH 6.97) indicated the endo position of the p-hydroxyphenyl group.12 Additionally, the correlation between H-6 (δH 3.73) and H-8 (δH 6.23) together with the large coupling constant (J7, 8 = 15.6 Hz) suggested the E configuration of the Δ7(8) double bond, as shown in Figure 3. Because empirical rules for elucidating structures of compounds with a bicyclo[2.2.2]octene skeleton had been

2.40) revealed the presence of an isolated spin system. In the HMBC spectrum, the correlations from H-2 (δH 6.60) to C-3 (δC 59.3) and C-4 (δC 210.7); H-7′ (δH 3.67) to C-3 (δC 59.3), C-8′ (51.6), C-1′ (δC 134.2), C-2′ (δC 129.7), and C-6′ (δC 129.7); and H-7 (δH 7.39) to C-6 (δC 37.3) revealed the bicyclo[2.2.2]octene framework of 1. The correlations from H7′ (δH 3.67) to C-1′ (δC 134.2), C-2′ (δC 129.7), and C-6′ (δC 129.7) indicated that the 1,4-disubstituted phenyl ring was linked to the bicyclo[2.2.2]octene skeleton from C-1′ to C-7′. The locations of the carbonyls at C-9 and C-9′ were determined by the correlations from H-7′ (δH 3.67) to C-9′ (δC 174.7) and from H-7 (δH 7.39) and H-8 (δH 6.23) to C-1 (δC 147.1), C-2 (δC 134.4), and C-9 (δC 168.7). The connections between the phloroglucinol and glucosyl moieties were established based on the HMBC correlations from H-7″ (δH 4.86) to C-1″ (δC 161.0) and from H-7‴ (δH 4.86) to C-1‴ (δC 161.0). The correlations from H-12″ (δH 4.55, 4.34) to C-9 and from H-12‴ (δH 4.49, 4.36) to C-9′ established the linkages between the glucosyl (C-12″ and C-12‴) and carbonyl (C-9 and C-9′) moieties, as shown in Figure 2. The relative configuration of 1 was determined through a NOESY experiment. The correlations between H-3 (δH 3.28) and H-8′ (δH 2.90) indicated that C-9′ occupied an exo position. The correlations of H-6 (δH 3.73)/H-7′ (δH 3.67) and

Figure 3. Key NOESY interactions of compounds 1, 3, and 4. C



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7′), 3.81 (1H, dd, J = 9.6, 7.2 Hz, H-8), and 3.76 (1H, dd, J = 9.6, 6.6 Hz, H-8′); δC 44.1, 43.8, 43.0, and 42.1]. These NMR signals resonated in pairs, which unequivocally indicated that 3 was a dimeric compound. On the basis of the 1H−1H COSY spectrum, the four methine groups (C-7, C-8, C-7′, and C-8′) of 3 were determined to be covalently connected. Thus, compound 3 was identified as a new dimeric derivative of tadehaginoside, and its dimerization occurred through a [2+2] cycloaddition reaction involving the α,β-unsaturated carbonyl moiety of the p-hydroxycinnamoyl unit. This result was confirmed by a comparison of its chemical shift values with those of similar compounds.15,16 The NMR data of 3 were fully assigned through an analysis of its 1H−1H COSY, HSQC, and HMBC data (Figure 2). The relative configuration of 3 was defined via a NOESY experiment, which revealed correlations between H-6 (δH 6.76) and H-7′ (δH 4.00), H-6 and H-8′ (δH 3.76), H-6′ (δH 6.69) and H-7 (δH 4.07), and H-6′ and H-8 (δH 3.81). Similar NOE associations between H-6′ and H-12‴a (δH 4.26), H-12’″a and H-8 (δH 3.81), H-8′ (δH 3.76) and H-12″a (δH 4.47), and H12″a and H-6 (δH 6.76) were observed. These NOESY crosspeaks (Figure 3) suggested that the substituents on the fourmembered ring had a μ-truxinate arrangement.17 The Dconfigurations of the glucosyl moieties were also established through a hydrolysis and GC analysis. Therefore, the structure of 3, tadehaginoside C, was unequivocally established as shown in Figure 1. Tadehaginoside D (4) had the same molecular formula as 3, as determined through its HRESIMS and 13C NMR data. Its NMR data (Table 1) resembled those of 3, indicating that 4 was also a dimeric derivative of tadehaginoside with a truxillyltype structure. Because of the different arrangement of the substituents, their cyclobutyl protons showed different chemical shifts and coupling constants (Table 1). The 13C NMR chemical shifts of the cyclobutane moiety, C-7 (δC 40.2), and C-7′ (δC 40.1) were shielded, whereas those of C-8 (δC 46.1) and C-8′ (δC 45.8) were relatively deshielded. These data clearly indicated that the connectivity of the two phydroxyphenyl and two acyl groups of the cyclobutane moiety was of the α-truxillyl and not the μ-truxinyl type.18 This result was further confirmed by the diagnostic NOESY correlations from H-2 to H-6 (δH 7.10) and H-7′ (δH 3.84) and from H-8′ (δH 4.18) to H-2′, H-6′ (δH 7.01), H-7 (δH 3.88), and H-8 (δH 4.25), as shown in Figure 3. The absolute configurations of the glucosyl moieties were determined using the same method as was used for 3. Therefore, the structure of 4 was established as shown in Figure 1. Tadehaginoside E (5) was obtained as an amorphous powder. Its molecular formula, C21H22O10, was established by an analysis of its HRESIMS (m/z 457.1104 [M + Na]+, calcd 457.1111 for C21H22O10Na) and 13C NMR data. The NMR data of 5 were similar to those of tadehaginoside, with the exception of its glucosyl unit. A comparison of the 13C NMR data of 5 with those of tadehaginoside revealed changes in the chemical shifts (Table 2) of C-4″ (+0.5 ppm), C-3″(−2.1 ppm), and C-6″ (−2.3 ppm), indicating that esterification occurred at C-4″ rather than at C-6″ of the glucosyl moiety.19,20 The HMBC correlation from H-4″ (δH 4.95) to C-9 (δC 168.8) further confirmed such a connectivity, as shown in Figure 2. Therefore, the structure of 5, tadehaginoside E, was established as shown in Figure 1.

established, ECD spectroscopic data were used to determine the absolute configuration of 1.13 By applying the octant rule and comparing the ECD curves of 1 (Cotton effects at −268.5 and +310.5 nm) with those of similar compounds,14 as shown in Figure 4, the absolute configuration of 1 was defined as 3S,

Figure 4. ECD spectroscopic data of compounds 1 and 2.

6S, 7′S, 8′S. Therefore, the structure of 1 was established as a dimeric derivative of tadehaginoside and named tadehaginoside A. Tadehaginoside B (2) had the same molecular formula as 1, as determined through HRESIMS and 13C NMR data (Table 1). The NMR data showed that 2 was also a dimeric derivative of tadehaginoside possessing a bicyclo[2.2.2]octene skeleton. The NMR spectroscopic data (Table 1) attributed to the bicyclo[2.2.2]octene skeleton of 2 were identical to those of 1. However, the ECD spectroscopic data of the two compounds exhibited similar Cotton effects but of opposite signs (Figure 4), suggesting the enantiomeric relationship of the bicyclo[2.2.2]octene skeletons of 1 and 2, as shown in Figure 1. Although the proton signals of the glucosyl moieties of 1 and 2 exhibited differences, the D-configurations of the glucosyl groups were determined by enzymatic hydrolysis and GC analysis. Accordingly, the structure of 2, tadehaginoside B, was established as shown in Figure 1. Tadehaginoside C (3) was determined to have the molecular formula C42H44O20 based on its HRESIMS (m/z 891.2322 [M + Na]+, calculated as 891.2324) and 13C NMR data. The NMR data revealed that 3 possessed two 1,4-disubstituted aromatic rings [δH 6.76 (2H, d, J = 8.4 Hz, H-2, 6), 6.47 (2H, d, J = 8.4 Hz, H-3, H-5), 6.69 (2H, d, J = 8.4 Hz, H-2′, 6′), and 6.41 (2H, d, J = 8.4 Hz, H-3′, 5′); δC 155.3, 155.2, 129.3, 129.1, 129.0 (×2), 128.8 (×2), 114.7 (×2), and 114.6 (×2)], two 1,3,5trisubstituted benzene rings [δH 5.93 (2H, s, H-4″, 4‴) and 5.91 (4H, s, H-2″, 6″, 2‴, 6‴); δC 158.9 (×4), 159.2, 159.1, 96.8, 96.7, 95.1 (×2), and 94.9 (×2)], two glucosyl moieties [δH 4.73 (1H, J = 7.8 Hz, H-7″), 4.71 (1H, J = 7.8 Hz, H-7‴), 4.47 (1H, dd, J = 12.0, 2.4 Hz, H-12″a), 4.20 (1H, dd, J = 12.0, 7.2 Hz, H-12″b), 4.26 (1H, dd, J = 12.0, 2.4 Hz, H-12‴a), 4.00 (1H, dd, J = 12.0, 7.2 Hz, H-12‴b), and 3.12−3.52 (8H, overlapped); δC 100.4, 100.2, 76.3, 76.2, 73.7, 73.6, 73.1, 73.0, 70.1, 69.8, and 63.7 (×2)], and four methine groups [δH 4.07 (1H, dd, J = 9.6, 7.2 Hz, H-7), 4.00 (1H, dd, J = 9.6, 6.6 Hz, HD



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Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1−4 1a position

a

δC, type

1 2 3 4 5

147.1, 134.4, 59.3, 210.7, 35.7,

C CH CH C CH2

6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 11″ 12″

37.3, 141.7, 119.2, 168.7, 134.2, 129.7, 116.7, 157.7, 116.7, 129.7, 44.8, 51.6, 174.7, 161.0, 97.1, 160.3, 98.3, 160.3, 97.1, 102.2, 74.9, 78.1, 71.9, 75.6, 65.0,

CH CH CH C C CH CH C CH CH CH CH C C CH C CH C CH CH CH CH CH CH CH2

1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 10‴ 11‴ 12‴

161.0, 97.1, 160.3, 98.3, 160.3, 97.1, 102.1, 75.0, 77.8, 71.7, 75.3, 65.0,

C CH C CH C CH CH CH CH CH CH CH2

2a δH (J in Hz)

6.60 d (6.0) 3.28 br d (6.6) 1.95 2.40 3.73 7.39 6.23

d (18.0) d (18.0) br s d (15.6) d (15.6)

6.97 d (8.4) 6.68 d (8.4) 6.68 6.97 3.67 2.90

d (8.4) d (8.4) t (6.6) br d (6.6)

6.07 d (1.8) 5.95 br s 6.07 4.86 3.42 3.44 3.42 3.70 4.55 4.34

d (1.8) d (7.8) mc mc mc m dd (12.0, 2.4) dd (12.0, 7.2)

6.06 d (1.8) 5.95 br s 6.06 4.86 3.42 3.44 3.34 3.66 4.49 4.36

d (1.8) d (7.8) mc mc mc m dd (12.0, 2.4) dd (12.0, 7.2)

δC, type 147.1, 134.4, 59.3, 210.7, 35.7,

C CH CH C CH2

37.3, 141.7, 119.2, 168.7, 134.2, 129.7, 116.7, 157.7, 116.7, 129.7, 44.9, 51.6, 174.7, 161.0, 97.1, 160.3, 98.3, 160.3, 97.1, 102.2, 74.9, 78.1, 71.9, 75.6, 65.0,


161.0, 97.1, 160.3, 98.3, 160.3, 97.1, 102.1, 75.0, 77.8, 71.7, 75.3, 65.1,


3b δH (J in Hz)

δC, type

6.60 d (6.0) 3.28 br d (6.6) 2.03 2.41 3.78 7.39 6.24

d (18.0) d (18.0) br s d (15.6) d (15.6)

6.94 d (8.4) 6.65 d (8.4) 6.65 6.94 3.67 2.87

d (8.4) d (8.4) t (6.6) br d (6.6)

6.07 d (1.8) 5.95 br s 6.07 4.86 3.40 3.46 3.43 3.67 4.63 4.23

d (1.8) d (7.8) mc mc mc m dd (12.0, 2.4) dd (12.0, 7.2)

6.06 d (1.8) 5.95 br s 6.06 4.86 3.44 3.46 3.38 3.64 4.55 4.30

d (1.8) d (7.8) mc mc mc m dd (12.0, 2.4) dd (12.0, 7.2)

129.3, 129.0, 114.7, 155.3, 114.7,

C CH CH C CH

129.0, 44.1, 43.0, 172.4, 129.1, 128.8, 114.6, 155.2, 114.6, 128.8, 43.8, 42.1, 172.3, 159.2, 95.1, 158.9, 96.8, 158.9, 95.1, 100.4, 73.1, 76.3, 70.1, 73.7, 63.7,


159.1, 94.9, 158.9, 96.7, 158.9, 94.9, 100.2, 73.0, 76.2, 69.8, 73.6, 63.7,


4b δH (J in Hz)

6.76 d (8.4) 6.47 d (8.4) 6.47 d (8.4) 6.76 d (8.4) 4.07 dd (9.6, 7.2) 3.81 dd (9.6, 7.2)

6.69 (d, 8.4) 6.41 (d, 8.4) 6.41 6.69 4.00 3.76

(d, 8.4) (d, 8.4) dd (9.6, 6.6) dd (9.6, 6.6)

5.91 s 5.93 s 5.91 4.73 3.17 3.27 3.12 3.52 4.47 4.20

s d (7.8) mc mc mc mc dd (12.0, 2.4) dd (12.0, 7.2)

5.91 s 5.93 s 5.91 4.71 3.16 3.27 3.12 3.52 4.26 4.00

s d (7.8) mc mc mc mc dd (12.0, 2.4) dd (12.0, 7.2)

δC, type 128.7, 128.6, 115.1, 156.4, 115.1,

C CH CH C CH

128.7, 40.2, 46.1, 171.6, 128.4, 128.5, 115.1, 156.0, 115.1, 128.5, 40.1, 45.8, 171.4, 159.3, 95.2, 159.0, 96.8, 159.0, 95.2, 100.5, 73.1, 76.4, 70.1, 73.8, 63.9,


159.2, 95.1, 159.0, 96.7, 159.0, 95.1, 100.2, 73.1, 76.4, 70.0, 73.8, 63.5,


δH (J in Hz) 7.10 d (7.2) 6.64 d (7.2) 6.64 d (7.2) 7.10 d (7.2) 3.88 br d (9.6) 4.25 br d (9.6)

7.01 d (7.2) 6.55 d (7.2) 6.55 7.01 3.84 4.18

d (7.2) d (7.2) br d (8.4) br d (8.4)

5.94 s 5.96 s 5.94 4.67 3.16 3.22 3.00 3.31 3.99 3.76

s d (7.8) mc m m mc m m

5.94 s 5.96 s 5.94 4.56 3.16 3.18 2.98 3.31 4.23 3.34

s d (7.8) mc m m mc m m

Detected in methanol-d4. bDetected in DMSO-d6. cSignals overlapped.

separation of the two compounds using any of the methods applied in this study was unsuccessful. However, their NMR spectroscopic data permitted the elucidation of their structures. Compounds 7 and 8 possessed the same molecular formula and similar NMR data as tadehaginoside. The only difference between these two compounds and tadehaginoside was the esterification that occurred at C-2″ of the glucosyl moiety. The changes in the chemical shifts of C-1″, C-2″, and C-3″ and the HMBC correlation from H-2″ to C-9 supported this structural elucidation, as shown in Figure 2. The coupling constants (Table 2) demonstrated that the Δ7(8) double bonds of 7 and 8 were in a trans and cis configuration, respectively. Therefore,

Tadehaginoside F (6) was determined to have a molecular formula of C21H22O10 according to its HRESIMS and 13C NMR data. The structure of 6 was similar to that of tadehaginoside with the exception that esterification occurred at C-3″ rather than at C-6″ of the glucosyl group. This finding was supported by the changes in the chemical shifts of C-3″ (+0.9 ppm), C-2″(−1.4 ppm), and C-4″ (−2.2 ppm) and the HMBC correlation between H-3″ (δH 5.13) and C-9 (δC 169.2). Therefore, the structure of 6, tadehaginoside F, was established as shown in Figure 1. Tadehaginosides G and H (7 and 8) were obtained as a mixture with an approximate ratio of 4:1. However, further E



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Table 2. 1H and 13C NMR Spectroscopic Data for Compounds 5−8 in Methanol-d4 5 position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ a

δC, type 127.4, 131.5, 117.1, 161.6, 117.1, 131.5, 147.5, 115.0, 168.8, 161.0, 97.1, 160.3, 98.4, 160.3, 97.1, 102.3, 75.2, 76.0, 72.4, 76.4, 62.5,

C CH CH C CH CH CH CH C C CH C CH C CH CH CH CH CH CH CH2

6 δH (J in Hz)

δC, type

7.47 d (8.4) 6.81 d (8.4) 6.81 7.47 7.68 6.38

d d d d

(8.4) (8.4) (15.6) (15.6)

6.10 d (1.8) 5.97 t (1.8) 6.10 4.91 3.53 3.73 4.95 3.67 3.67 3.58

d (1.8) d (7.8) dd (9.6, 7.8) t (9.6) t (9.6) m dd (12.0, 2.4) dd (12.0, 6.6)

127.5, C 131.4, CH 117.0, CH 161.5, C 117.0, CH 131.4, CH 146.8, CH 116.0, CH 169.2, C 161.0, C 96.9, CH 160.4, C 98.3, CH 160.4, C 96.9,CH 102.3, CH 73.5, CH 79.0, CH 69.7, CH 78.1, CH 62.4, CH2

7 δH (J in Hz)

7.47 d (8.4) 6.81 d (8.4) 6.81 7.47 7.68 6.42

d d d d

(8.4) (8.4) (15.6) (15.6)

6.09 br s 5.95 br s 6.09 4.95 3.60 5.13 3.56 3.64 3.92 3.75

br s d (7.8) dd (9.0, 7.8) t (9.6) m m dd (12.0, 2.4) dd (12.0, 7.2)

δC, type 127.4, 131.4, 117.0, 161.5, 117.0, 131.4, 147.2, 115.2, 168.6, 160.9, 96.9, 160.3, 98.5, 160.3, 96.9, 101.0, 75.2, 76.3, 71.6, 78.5, 62.6,


8 δH (J in Hz)

δC, type

7.46 d (8.4) 6.79 d (8.4) 6.79 7.46 7.67 6.40

d d d d

(8.4) (8.4) (15.6) (15.6)

5.98 d (1.8) 5.93 d (1.8) 5.98 5.03 5.02 3.68 3.50 3.50 3.94 3.76

d (1.8) d (7.8) m t (9.0) ma ma dd (12.0, 2.4) dd (12.0, 7.2)

127.8, 133.8, 116.0, 161.5, 116.0, 133.8, 145.6, 116.6, 167.5, 160.8, 96.8, 160.4, 98.5, 160.4, 96.8, 100.5, 75.2, 76.3, 71.6, 78.4, 62.5,

δH (J in Hz)


7.58 d (8.4) 6.73 d (8.4) 6.73 7.58 6.89 5.80

d d d d

(8.4) (8.4) (12.6) (12.6)

6.00 d (1.8) 5.96 d (1.8) 6.00 5.01 4.98 3.60 3.50 3.50 3.92 3.75

d (1.8) d (7.8) dd (9.0, 7.8) t (9.0) ma ma dd (12.0, 2.4) dd (12.0, 7.2)

Signals overlapped.

Table 3. 1H and 13C NMR Spectroscopic Data for Compounds 9−11 in Methanol-d4 9 position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″ 3″ 4″ 5″ 6″

δC, type 127.5, 131.4, 117.0, 161.4, 117.0, 131.4, 147.1, 115.2, 169.3, 162.8, 96.1, 166.5, 98.5, 167.8, 107.1, 205.0, 33.6, 102.4, 75.0, 78.7, 71.8, 76.0, 64.8,

C CH CH C CH CH CH CH C C CH C CH C C C CH3 CH CH CH CH CH CH2

10 δH (J in Hz)

7.47 d (8.4) 6.81 d (8.4) 6.81 7.47 7.62 6.38

d d d d

(8.4) (8.4) (15.6) (15.6)

6.21 d (1.8) 5.96 d (1.8)

2.69 5.04 3.57 3.50 3.43 3.74 4.57 4.29

s d (7.8) dd (9.0, 7.8) t (9.0) t (9.0) ddd (9.0, 7.2, 2.4) dd (12.0, 2.4) dd (12.0, 7.2)

δC, type 127.5, 133.9, 116.0, 161.0, 116.0, 133.9, 145.8, 116.5, 167.8, 162.7, 96.1, 166.4, 98.5, 167.8, 107.1, 205.0, 33.6, 102.3, 74.9, 78.6, 71.7, 75.9, 64.3,

11 δH (J in Hz)

C CH CH C CH CH CH CH C C CH C CH C C C CH3 CH CH CH CH CH CH2

7.60 d (8.4) 6.70 d (8.4) 6.70 7.60 6.85 5.83

d d d d

(8.4) (8.4) (12.6) (12.6)

6.14 d (1.8) 5.94 d (1.8)

2.68 5.01 3.54 3.48 3.41 3.68 4.47 4.34

s d (7.8) dd (9.0, 7.8) t (9.0) t (9.0) ddd (9.0, 7.2, 2.4) dd (12.0, 2.4) dd (12.0, 7.2)

δC, type 127.4, 131.4, 116.9, 160.9, 116.9, 131.4, 147.0, 115.1, 169.4, 161.3, 97.0, 160.2, 98.2, 160.2, 97.0,

C CH CH C CH CH CH CH C C CH C CH C CH

102.2, 74.9, 78.1, 71.9, 75.6, 64.8,

CH CH CH CH CH CH2

δH (J in Hz) 7.47 d (8.4) 6.80 d (8.4) 6.80 7.47 7.62 6.40

d d d d

(8.4) (8.4) (15.6) (15.6)

6.10 d (1.8) 5.97 t (1.8) 6.10 d (1.8)

4.88 3.58 3.46 3.42 3.67 4.55 4.28

d (7.8) dd (9.0, 7.8) t (9.0) t (9.0) m dd (12.0, 2.4) dd (12.0, 6.6)

that these compounds have the same carbon skeleton as tadehaginoside with the exception of the presence of an acetyl group at C-6′ of the phloroglucinol groups. The coupling constants (Table 3) demonstrated that the Δ7(8) double bonds of 9 and 10 were in a trans and cis configuration, respectively.

the structures of 7 and 8, tadehaginosides G and H, were assigned as shown in Figure 1. Tadehaginosides I and J (9 and 10) were also obtained as a mixture. These two compounds were determined to have the same molecular formula, C23H24O11, through an analysis of their HRESIMS and 13C NMR data. Their NMR data revealed F



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Figure 5. Effects of the phenylpropanoid glucosides on glucose uptake in C2C12 myotubes. (A) Effects of the phenylpropanoid glucosides (10 μM) on glucose uptake. (B) Time course of the effect of compound 4 (10 μM) on glucose uptake. (C) Dose-dependent synergistic effects of compound 4 and insulin on glucose uptake. (D) Cytotoxicity of compound 4 against C2C12 myotubes.

Figure 6. Compound 4 enhanced the transcriptional activity of PPARγ and increased the GLUT-4 protein level in C2C12 myotubes. (A) Key interaction patterns between compound 4 and PPARγ. Hydrogen bonds are presented as dotted lines. (B) Binding scores of compounds 3−11 with PPARγ. (C) Effects of compound 4 and rosiglitazone (10 μM) on the transcriptional activity of PPARγ. (D) Effects of compound 4 and rosiglitazone (10 μM) on the GLUT-4 protein level. G



Article

from Sigma-Aldrich, Inc. (St. Louis, MO, USA), and a GLUT-4 ELISA kit and a PPARγ luciferase assay kit were purchased from eBioscience (San Diego, CA, USA) and Promega (Beijing, China), respectively. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (NY, USA) and were used for the culture and differentiation of the C2C12 mouse muscle myoblasts. The organic solvents (analytical grade) used for isolation, purification, and biological evaluations were purchased from Tianjin DaMao Chemical Reagent (Tianjin, China). Plant Material. Samples of T. triquetrum were collected in August 2014 from the town of Changliu in Haikou, China. The plant samples were authenticated by Prof. Naikai Zeng (School of Pharmaceutical Science, Hainan Medical University), and a voucher specimen (No. TT201408) was deposited. Extraction and Isolation. Air-dried and pulverized aerial parts of T. triquetrum (5.0 kg) were extracted with an EtOH−H2O (70:30) mixture (3 × 50.0 L) at room temperature. After filtration and evaporation of the solvent, the extract (500 g) was dissolved and partitioned with EtOAc (3× 3.0 L) and n-BuOH (3× 4.0 L). The nBuOH extract (180.5 g) was subjected to a silica gel column, eluting with different ratios of CH2Cl2−MeOH, and seven fractions (A−G) were collected. Fraction B was purified by HPLC, eluting with MeOH−H2O (52:48), to afford a mixture of 9 and 10 (8.0 mg). Fraction C (12.5 g) was further separated to afford subfractions 1−5. Compounds 5 (3.5 mg) and 11 (25.0 mg) were obtained from fraction C using different column chromatography methods, particularly HPLC eluting with MeOH−H2O (45:55). Subfraction 4 was further fractionated by HPLC, eluting with MeOH−H2O (40:60), to afford 6 (3.0 mg) and a mixture of 7 and 8 (5.0 mg). Compounds 1 (15.0 mg) and 2 (22.0 mg) were isolated from fraction F (1.3 g) using a Sephadex LH-20 column and HPLC eluting with MeOH−H2O (38:62). Fraction G (1.3 g) was further purified using a Sephadex LH20 column and HPLC, eluting with MeOH−H2O (35:65), to afford 3 (12.0 mg) and 4 (8.0 mg). Tadehaginoside A (1): amorphous powder (MeOH); [α]25 D −28 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 270 (2.81), 307 (0.15) nm; ECD (0.008 M, MeOH) 268.5 (Δε = −28.2), 310.5 (Δε = +12.3) nm; IR (film) νmax 3405, 3392, 1717, 1709, 1612, 1607, 1515, 1167, 1074, 828 cm−1; 1H and 13C NMR (methanol-d4) data, see Table 1; (+)-HRESIMS m/z 891.2327 [M + Na]+ (calcd for C42H44O20Na, 891.2324). Tadehaginoside B (2): Aamorphous powder (MeOH); [α]25 D −30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (2.81), 308 (0.15) nm; ECD (0.008 M, MeOH) 268.0 (Δε = +26.3), 310.5 (Δε = −13.5) nm; IR (film) νmax 3428, 1718, 1620, 1514, 1189, 1070, 830 cm−1; 1H and 13 C NMR (methanol-d4) data, see Table 1; (+)-HRESIMS m/z 891.2318 [M + Na]+ (calcd for C42H44O20Na, 891.2324). Tadehaginoside C (3): amorphous powder (MeOH); [α]25 D −108 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 225 (1.02), 277 (0.15) nm; IR (film) νmax 3356, 1723, 1715, 1609, 1516, 1072, 827 cm−1; 1H and 13C NMR (DMSO-d6) data, see Table 1; (+)-HRESIMS m/z 891.2322 [M + Na]+ (calcd for C42H44O20Na, 891.2324). Tadehaginoside D (4): amorphous powder (MeOH); [α]25 D −58 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 226 (1.02), 277 (0.15) nm; IR (film) νmax 3407, 1717, 1612, 1515, 1072, 828 cm−1; 1H and 13C NMR (DMSO-d6) data, see Table 1; (+)-HRESIMS m/z 891.2321 [M + Na]+ (calcd for C42H44O20Na, 891.2324). Tadehaginoside E (5): amorphous powder (MeOH); [α]25 D −30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (2.81), 226 (1.02), 277 (0.15) nm; IR (film) νmax 3356, 1690, 1612, 1560, 1072, 828 cm−1; 1H and 13C NMR (methanol-d4) data, see Table 2; (+)-HRESIMS m/z 457.1104 [M + Na]+ (calcd for C21H22O10Na, 457.1111). Tadehaginoside F (6): amorphous powder (MeOH); [α]25 D −22 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (2.62), 227 (1.34), 275 (0.18) nm; IR (film) νmax 3356, 1691, 1614, 1562, 1070, 827 cm−1; 1H and 13C NMR (methanol-d4) data, see Table 2; (+)-HRESIMS m/z 457.1112 [M + Na]+ (calcd for C21H22O10Na, 457.1111). Tadehaginosides G and H (7 and 8): amorphous powder (MeOH); 1H and 13C NMR (methanol-d4) data, see Table 2;

Therefore, the structures of 9 and 10, tadehaginosides I and J, were defined as shown in Figure 1. It has been well documented that Z/E isomeric compounds commonly coexist in nature and are difficult to separate.21 From a biosynthesis perspective, tadehaginosides A−D may have formed from tadehaginoside via different phytochemical cyclization processes. Compounds with bicyclo[2.2.2]octene or cyclobutane frameworks are known natural products.22,23 The abilities of the isolated compounds to stimulate glucose uptake in C2C12 mouse skeletal muscle myotubes, which have frequently been used to evaluate the hypoglycemic activities of natural agents, were tested.24,25 The C2C12 myotubes were treated with the isolated compounds (10 μM) for 6 h. Compounds 3−11 significantly increased the glucose uptake of the C2C12 myotubes, and 4 exhibited the most potent effect, with an efficacy comparable to that of 100 nM insulin (Figure 5A). Because of its interesting structure and potent activity, the time- and dose-dependent effects of 4 were subsequently evaluated in detail. The C2C12 myotubes were treated with 4 (10 μM) at the indicated time points. Compound 4 significantly increased glucose uptake from 2 to 8 h with a maximum enhancement at 4 h, as shown in Figure 5B. Thus, the dose-dependent effect of 4 was evaluated at 4 h, and the results revealed that 4 at concentrations of 5−20 μM increased the basal and insulin-elicited glucose uptake of C2C12 myotubes. Similar effects were observed at concentrations of 10 and 20 μM (Figure 5C). The effect of 4 (10 and 20 μM) on glucose uptake was comparable to that of 20 μM rosiglitazone. Moreover, the MTT assay revealed that 4 (1−20 μM) was not cytotoxic toward C2C12 myotubes after a 4 h incubation period (Figure 5D). Molecular docking experiments demonstrated that 3 and 4 bind tightly to PPARγ, a key regulator of glucose homeostasis that regulates the expression of GLUT-4. Ile281, His323, and Tyr473 were identified as the key residues involved in the interactions between 4 and PPARγ, as shown in Figure 6A. Luciferase assays and enzyme-linked immunosorbent assays (ELISAs) demonstrated that 4 significantly enhanced the transcriptional activity of PPARγ (Figure 6C) and increased the GLUT-4 protein level (Figure 6D). Collectively, these results suggested that 4 could stimulate basal and insulinelicited glucose uptake through upregulation of PPARγ and GLUT-4.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations and UV spectrometric data were recorded using a PerkinElmer 341 (San Diego, CA, USA) and a Shimadzu UV2550 (Kyoto, Japan) spectrometer, respectively. ECD spectroscopic data were measured on a JASCO J-815 instrument (Kyoto, Japan). IR data were recorded using a Shimadzu FTIR-8400S spectrometer (Kyoto, Japan). NMR experiments were performed using a Bruker AV III 600 spectrometer (Bruker BioSpin, Germany). HRESIMS data were obtained using an LTQ-Orbitrap (Thermo Scientific, MA, USA) and a Q-TOF (Agilent 6540, CA, USA) instrument. A silica gel (200−300 mesh, Qingdao Marine Chemistry Co. Ltd., Qingdao, China) column was used to isolate the n-BuOH extract, and a Sephadex LH-20 (GE Healthcare Biosciences AB, Uppsala, Sweden) column was used to purify the compounds. HPLC was conducted using a Shimadzu LC-6AD system equipped with an SPD-10A detector (Kyoto, Japan). Molecular Operating Environment (MOE) 2014.09 (Chem Comp Group, Canada) was applied for the molecular docking studies. Fluorescence intensities were determined using an M1000 PRO apparatus (Tecan, Switzerland). Centrifugation was performed using a SIGMA 3-18K centrifuge (Sigma, Germany). GC data were recorded on a Shimadzu GC-14C instrument (Kyoto, Japan). Rosiglitazone was purchased H



Article

54 column. The selected temperature for the injector was 230 °C, and the detector temperature was 270 °C. The oven temperature was first set to 170 °C for 5 min and then increased to 250 °C over the course of 45 min. The chromatographic behaviors of the sugar residues were determined to be the same as those of D-glucose.

(+)-HRESIMS m/z 457.1108 [M + Na]+ (calcd for C21H22O10Na, 457.1111). Tadehaginosides I and J (9 and 10): amorphous powder (MeOH); 1H and 13C NMR (methanol-d4) data, see Table 3; (+)-HRESIMS m/z 499.1219 [M + Na]+ (calcd for C23H24O11Na, 499.1216). Cell Culture and Differentiation. The C2C12 mouse muscle myoblasts were cultured as previously described.7,26 The confluent myoblasts were differentiated into myotubes by changing the culture medium to DMEM containing 1% FBS daily for 4 days. The isolated phenylpropanoid glucosides were then dissolved in DMSO for further studies. Glucose Uptake Assay. The assay was performed as previously reported with minor modifications.26 Briefly, the differentiated C2C12 myotubes were cultured in serum-free DMEM. 2-Deoxy-2-[(7-nitro2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) (10 μM) and the phenylpropanoid glucosides were added to the culture medium, and the cells were incubated for the indicated incubation time. After the incubation, the culture medium was discarded, and the C2C12 myotubes were washed twice with phosphate-buffered saline (PBS) and scraped with ice-cold PBS. The biological activity was evaluated by determining the 2-NBDG amount consumed by the cells based on their fluorescence intensities (Ex/Em = 475 nm/550 nm). Cell Viability Assay. The cell viability was evaluated using the MTT method. After the differentiated C2C12 myotubes were seeded into 96-well plates, the cells were treated with various concentrations of compound 4 or 100 μg/mL digitonin, which was used as a positive control. After incubation for the indicated time, each well was treated with the MTT reagent. DMSO was added to the cells, and the absorbance (565 nm), which reflected the cell viability, was measured using a microplate reader. Molecular Docking. Molecular docking studies between the small molecules and the 3D PPARγ receptor binding sites (PDBID: 1k74) were performed using the MOE software package. To determine the proper conformations and the minimum-energy structures of the ligands, the rigid-receptor/flexible-ligand approach was employed. The binding affinities (E-place values) in MOE were used to evaluate the interactions between PPARγ and the ligands. The scores (binding affinities) were obtained based on the virtual calculation of various interactions of the ligands with the targeted receptor. The computational results demonstrated that these new molecules exhibited potential agonist activities, with tadehaginosides C and D exhibiting higher activities than rosiglitazone. Figure 6B shows the recorded docking scores for the isolated compounds compared with the active hit molecule. Measuring the PPARγ Promoter Activity. A transactivation reporter assay was performed with the C2C12 myotubes as previously described.26 In brief, vectors for a DR-1 luciferase reporter and for PPARγ expression were transiently transfected into C2C12 myotubes. Six hours after transfection, the culture medium was replaced with fresh medium containing a suitable agonist. After 24 h, luciferase assays were performed according to the manufacturer’s instructions. Measuring the GLUT-4 Levels. The cells were cultured in medium (serum-free DMEM) containing tadehaginoside D at different concentrations (1 and 10 μM) or 10 μM rosiglitazone for 6 h. The cells were washed with PBS and lysed using the same lysis buffer, as previously reported.26 The cell lysates were centrifuged for 20 min (13000g, 4 °C) to obtain the supernatants, which were collected for quantification of GLUT-4 using an ultrasensitive mouse GLUT-4 ELISA kit. Enzymatic Hydrolysis. Each new isolate (2.0 mg) was added to a test tube containing H2O (1.0 mL) and β-D-glucosidase (10 mg). After allowing the mixtures to react for 24 h, the mixtures were filtrated and washed to obtain a residue. The residue was further extracted with CHCl3 to afford the organic and aqueous layers. The aqueous layer and authentic samples of D-glucose and L-glucose were dissolved in pyridine (anhydrous, 0.2 mL) and L-cysteine methyl ester hydrochloride and then heated. The aqueous layer was treated with trimethylsilylimidazole and then warmed.27 After extraction with nhexane and H2O, the n-hexane layer was subjected to GC using an SE-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00820. IR, NMR, and HRESIMS data as well as CD spectra (Figures S1−S52) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: 86-10-57833285. E-mail: [email protected] (C.M. Wu). *Tel/Fax: 86-898-66893460. E-mail: [email protected] (J.-Q. Zhang). *Tel/Fax: 86-898-66895337. E-mail: [email protected] (Y.B. Li). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial assistance provided by the National Natural Science Foundation of China (Nos. 81560696 and 81202994).

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REFERENCES

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Tadehaginosides AâJ, Phenylpropanoid Glucosides from Tadehagi

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