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Iridoids from Vitex negundo var. heterophylla and their antioxidant activities
Phytochemistry Letters 35 (2020) 186–190
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Iridoids from Vitex negundo var. heterophylla and their antioxidant activities 1
1
Ying-Xue Niu , Dun Wang , Xin-Yue Chu, Su-Yu Gao, Dong-Xin Yang, Li-Xia Chen*, Hua Li*
T
Wuya College of Innovation, School of Pharmaceutical Engineering, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China
ARTICLE INFO
ABSTRACT
Keywords: Vitex negundo var. heterophylla Iridoids Iridoid glycosides Antioxidant activity
Two new iridoids (1-2) together with eight known iridoid glycosides (3-10) were isolated from the 70% EtOH extract of the dried leaves of Vitex negundo var. heterophylla. Their structures were elucidated by extensive spectroscopic analyses. Iridoid aglycones were found for the first time from this plant. All of these compounds were tested for their antioxidant activities. Among them, compound 9 showed moderate radical-scavenging effect on the stable free radical, 1,1-diphenyl-2-picrylhydrazyl, with IC50 values of 26.95 μM.
1. Introduction
also tested for their antioxidant activities.
The genus Vitex (Verbenaceae) consists of small trees and shrubs, with about 250 species mainly distributed in tropical and subtropical regions (Li et al., 2014; Hu et al., 2015). Vitex negundo var. heterophylla (Franch.) Rehd. as one of such species is widely distributed in the hilly areas of north, northeast, northwest, and central regions of China (Li et al., 2014; Hu et al., 2015). Various parts of V. negundo, including the leaves, roots and seeds, have been locally used as traditional folk medicines since antiquity, particularly in China to treat a wide range of ailments, such as cold, headache, migraine and ophthalmodynia (M.Y. Huang et al., 2013; Yao et al., 2016). Crude extracts and pure compounds from Vitex species have been reported to exhibit a wide array of bioactivities including anti-inflammatory (Aboul-Enein et al., 2017), antioxidant (Hajdtu et al., 2007; Huang et al., 2012), antinociceptive (Zheng et al., 2010), and anti-bacterial effects (Chang et al., 2018). Phytochemical investigations have indicated the presence of flavonoids, diterpenoids, iridoids, phenylpropanoids, triterpenoids, phytoecdysteroids, phenolic glycosides, and essential oils in Vitex plants (Suksamrarn et al., 1997; Ono et al., 2001; Li et al., 2002; Sena-Filh et al., 2008; Hu et al., 2015, 2016; Qiu et al., 2017; Bao et al., 2018). In order to find antioxidant material basis of Vitex negundo var. heterophylla, the 70% EtOH extract of the dried leaves of this plant was investigated, and two new iridoids (1-2), along with eight known iridoid glycosides (3-10) were obtained. Their structures were established on the basis of comprehensive spectroscopic analysis. Iridoid aglycones are very unusual in nature (Li et al., 2004; Liu et al., 2006; Yang et al., 2010; Jia et al., 2012; Lee et al., 2018), which were found for the first time from this plant. All of the isolated compounds were
2. Results and discussion The investigation of the constituents from the n-BuOH fraction of the dried leaves of Vitex negundo var. heterophylla led to the isolation of two new iridoid aglycones, nishindacin A (1) and isonishindacin A (2), along with eight known iridoid glycosides including nishindaside (3) (Iwagawa et al., 1993), isonishindaside (4) (Iwagawa et al., 1993), agnuside (5) (Dutta et al., 1983), 6′-O-p- hydroxybenzoylmussaenosidic acid (6) (Sehgal et al., 1983), 6′-O-p-hydroxybenzoyl-gardoside (7) (J. Huang et al., 2013), 6′-O-benzoyl-8-epi-loganic acid (8) (Harput et al., 2004), 6′-O-E-caffeoyl-mussaenosidic acid (9) (Gousiadou et al., 2012), and negundoside (10) (Roy et al., 2015) (Fig. 1). Compound 1 was isolated as yellowish oil (MeOH). The molecular formula C18H22O7 was deduced by HRESIMS data at m/z 349.1298 [MH]- (calcd for C18H21O7, 349.1293) in combination with its NMR data (Table S1), which indicated eight degrees of unsaturation. The 1H NMR spectrum showed signals at δH 7.87 (2H, d, J =6.4 Hz) and 6.81 (2H, d, J =6.4 Hz) in combination with a series of carbon signals [δC 167.8 (C7′), 133.0 (C-2′, C-6′), 116.5 (C-3′, C-5′), 122.0 (C-1′), 164.2 (C-4′)], indicating the presence of one p-hydroxybenzoyl group. In addition, two methoxy protons at δH 3.42 (3H, s) and 3.45 (3H, s), two protons of the oxymethylene at δH 4.86 (1H, overlapped) and 4.79 (1H, d, J = 14.4 Hz), two acetal protons at δH 4.45 (1H, d, J =6.1 Hz) and 4.71 (1H, br d, J =8.5 Hz), an oxymethine proton presented at 4.65 (1H, d, J =5.5 Hz), and one olefinic proton at δH 5.78 (1H, br s) were observed in the 1H NMR spectrum. The 13C NMR spectrum of 1 revealed 18 carbon signals including one ester carbonyl carbon at δC 167.8, two
Corresponding authors. E-mail addresses: [email protected] (L.-X. Chen), [email protected] (H. Li). 1 These two authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.phytol.2019.11.018 Received 9 September 2019; Received in revised form 21 November 2019; Accepted 27 November 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 35 (2020) 186–190
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compounds 3 and 4 were immersed in dichloromethane-methanol (2:1) solution, respectively. They were continuously heated at 50℃ and detected with TLC at different time periods (6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h), and compounds 1 and 2 were not found at all the time periods. This result proved that compounds 1 and 2 are of natural origin. DPPH radical scavenging activities have been widely used for representing the total antioxidant activities of foods. The spectrophotometric technique employs the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH·), which shows a characteristic UV–vis spectrum with a maximum absorbance close to 515 nm in methanol (Brand-Williams et al., 1995). The antioxidant activities of these compounds (50 μM) are shown in Table 2. Ascorbic acid (Vitamin C) was used as the positive control with IC50 values of 6.68 μM. Among all the tested compounds, compound 9 showed moderate radical-scavenging effect on the stable free radical, with IC50 values of 26.95 μM in the DPPH scavenging assay (Fig. 4).
Fig. 1. Structures of compounds 1 and 2.
pairs of aromatic carbons at δC 116.5 × 2 and 133.0 × 2, one oxygenated aromatic carbon at δC 164.2, one oxymethylene carbon at δC 63.4, and two olefinic carbons at δC 133.1 and 143.3. All the NMR data suggested that 1 possessed the similar skeleton structure as nishindaside (Iwagawa et al., 1993), except for the downfield shift of C-1 from δC 98.7 in nishindaside to δC 103.8 in 1, and the absence of a set of sugar signals and the presence of an extra methoxy signal in 1, indicating that a methoxy group linked at C-1. The assumption was further confirmed by the HMBC experiment (Fig. 2), which showed the long-range correlations of H-1 with 1-OCH3/C-3/C-8, H-3 with C-1/C5/3-OCH3, H-7 with C-5/C-6/C-8/C-9/C-10, and H-10 with C-7/C-7′/C8. The linkages of iridoid and p-hydroxybenzoyl moieties were constructed by the key correlation from H-10 to C-7′ in the HMBC spectrum. According to the biosynthetic considerations of iridoids, H-5 and H-9 were assigned as β-oriented (Klimek, 1996; Ahmad et al., 2004; Kim, 2009; Geu-Flores et al., 2012; Piaz et al., 2013). The key NOESY correlations of H-1/H-3, H-1/H-6, H-3/H-6, and H-5/H-9, and no correlation of H-6/H-9 were observed (Fig. 2), together with the coupling constant (J =6.1 Hz) between H-1 and H-9, indicating that H-1, H-3 and H-6 were α-oriented. Based on the above spectroscopic data, combined with the biosynthetic considerations of iridoids (Geu-Flores et al., 2012), compound 1 was determined and named nishindacin A. As for 2, its UV, IR, 1H NMR, 13C NMR, and HRESIMS data were similar to those of 1. By comparison of its 13C NMR signals with those of 1, the signal assigned to C-1 was shifted upfield by 3.2 ppm, suggesting that 2 was an epimer of 1 at C-3. In the NOESY spectrum of 2, the crosspeaks of H-5/H-9 with H-3, while no correlations between H-5/H-9 and H-1/H-6 indicated that H-3 was β-oriented, H-1 and H-6 were α-oriented (Fig. 3). Therefore, 2 was elucidated as 3R-diastereomer of 1, and named isonishindacin A. Specific experiments were carried out to exclude the possibility of compounds 1 and 2 as artificial products. In the isolation process, the fraction containing 1 and 2 was divided on silica gel (a possible catalyst) column chromatography (CC) and eluted with MeOH/CH2Cl2 (always with trace of acid), and possible equilibrium between two acetals (a glucoside and a methyl acetal) may occur. Therefore,
3. Experimental 3.1. General experimental procedures Bruker AV-400 and AV-600 spectrometers were used in the NMR experiments. Chemical shift values are expressed in δ (ppm) using the peak signals of the solvent methanol-d4 (δH 4.87, 3.31 and δC 49.15) as references, and coupling constants (J in Hz) are given in parentheses. Optical rotations were recorded on a Perkin-Elmer 241 polarimeter (PerkinElmer, Waltham, MA, USA). UV spectra were measured on a Shimadzu UV 2201 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). HRESIMS data were acquired on an Agilent 6210 TOF mass spectrometer (Palo Alto, CA, USA). Silica gel GF254 prepared for TLC was purchased from Qingdao Marine Chemical Factory (Qingdao, China). Silica gel (200 − 300 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20 (Pharmacia, USA), and octadecyl silica gel (Merck Chemical Company Ltd., Germany) were used for column chromatography (CC). RP-HPLC was equipped with an LC-6CE liquid chromatograph, SPD-20A UV detector (Shimadzu, Kyoto, Japan), and RP-C18 column (250 × 20 mm, 120 Å, 5 μm, YMC Co. Ltd.). All HPLC or analytical grade reagents were purchased from Tianjin Damao Chemical Company (Tianjin, China). Trace of samples on silica gel plates were detected under UV light and heating after spraying with anisaldehyde-H2SO4 reagent. DPPH were purchased from Sigma–Aldrich (USA). Methanol was purchased from Friendemann Schmidt (Germany). All chemicals were of analytical reagent grade.
Fig. 2. Key HMBC and NOESY correlations of compounds 1.
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Fig. 3. Key HMBC and NOESY correlations of compound 2.
Fig. 4. The IC50 of compound 9 and Vitamin C. Table 1 The 1H (600 MHz) and ppm). No.
13
1a
2a
δC
δH (J in Hz)
δC
δH (J in Hz)
1 3 4
103.8 100.2 30.6
4.45 (1H, d, 6.1) 4.71 (1H, br d, 8.5) 1.98 (1H, dd, 14.4, 3.0) 1.86 (1H, m)
100.6 100.6 30.5
5 6 7 8 9 10
46.2 80.7 133.1 143.3 49.8 63.4
45.6 81.1 132.2 143.8 50.0 63.4
1′ 2′ 3′ 4′ 5′ 6′ 7′ 1-OCH3 3-OCH3
122.0 133.0 116.5 164.2 116.5 133.0 167.8 56.4 56.2
2.40 (1H, m) 4.65 (1H, d, 5.5) 5.78 (1H, br s) – 2.76 (1H, br t-like, 6.1) 4.86 (1H, overlapped) 4.79 (1H, d, 14.4) – 7.87 (1H, d, 6.4) 6.81 (1H, d, 6.4) – 6.81 (1H, d, 6.4) 7.87 (1H, d, 6.4) – 3.42 (3H, s) 3.45 (3H, s)
4.53 (1H, d, 7.5) 4.86 (1H, d, 5.1) 1.92 (1H, ddd, 13.2, 11.4, 5.5) 1.62 (1H, ddd, 13.2, 7.9, 5.5) 2.29 (1H, m) 4.57 (1H, m) 5.82 (1H, br s) – 2.80 (1H, br t, 7.6) 4.84 (1H, overlapped) 4.77 (1H, br d, 14.8) – 7.88 (1H, d, 6.4) 6.81 (1H, d, 6.4) – 6.81 (1H, d, 6.4) 7.88 (1H, d, 6.4) – 3.42 (3H, s) 3.39 (3H, s)
a
Table 2 DPPH radical-scavenging activity of compounds 1–10.
C (150 MHz) NMR Data of Compounds 1 and 2 (δ in
122.0 133.0 116.5 164.2 116.5 133.0 167.8 56.7 55.7
compounds
Scavenging activity (%)
compounds
Scavenging activity (%)
1 2 3 4 5 Vitamin Ca
27.14% 25.80% 26.07% 24.46% 21.25% 92.5%
6 7 8 9 10
19.10% 21.51% 23.12% 88.48% 22.13%
a
positive control.
3.2. Plant material The dried leaves of Vitex negundo var. heterophylla (Franch.) Rehd. were collected from Chaoyang, Liaoning Province, China, and were identified by Prof. Jing-Ming Jia, Department of Pharmaceutical Botany, Shenyang Pharmaceutical University. A voucher specimen (VN. 20,140,915) was deposited in the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University. 3.3. Extraction and isolation The dried leaves of Vitex negundo var. heterophylla. (6.0 kg) were extracted with 70% EtOH (3 × 2 h × 76 L) to afford the crude extract after solvent removal in vacuo. The crude extract (406.5 g) was
Spectra of 1 and 2 were tested in methanol-d4. 188
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suspended in water and partitioned successively with petroleum ether (PE), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) in the same volume for three times. The n-BuOH extracts (200.5 g) were subjected to D101 Macroporous adsorption resin eluted with EtOH-H2O (0:100−100:0, v/v) to afford six fractions (D1−D6). Fraction D1 (52.0 g) was divided on silica gel CC and eluted by CH2Cl2/MeOH (100:1 to 1:1) into six fractions (D11-D16) according to TLC analysis. Subfraction D13 was subjected to an ODS column and eluted with CH3OH/H2O (1:9 to 1:0) to yield D131-D137. Then fraction D136 was separated by HPLC eluted with 50% CH3OH/H2O to give compound 1 (12.3 mg, tR =20.1 min) and compound 2 (15.4 mg, tR =23.1 min). Subfraction D15 was separated on ODS CC and eluted with MeOH/H2O (1:9 to 1:0) to obtain compound 3 (23.3 mg) and six subfractions (D151-D156), D155 was separated by preparative HPLC (40% MeOH/ H2O, flow rate 8.0 mL/min) to compound 4 (38.3 mg, tR =22.7 min), compound 5 (395.8 mg, tR =19.1 min) and compound 6 (304.7 mg, tR =29.2 min). Fraction D16 was chromatographed over an ODS column, MeOH/H2O (1:9 to 1:0) as solvent, yielding compounds 7 (43.7 mg), 10 (24.2 mg) and eight subfractions (D161-168). Purifcation of subfraction D167 by HPLC (CH3OH-0.0001trifluoroacetic acid-H2O = 3:7, 8.0 mL/ min) led to the isolation of compounds 8 (106.3 mg, tR =25.4 min) and 9 (80.1 mg, tR =36.8 min).
Acknowledgments This work was supported by the National Natural Science Foundation of China (NO. 81773594), Liaoning Revitalization Talents Program (NO. XLYC1807182), and Shenyang Planning Project of Science and Technology (NO. 18-013-0-46). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.11.018. References Aboul-Enein, H.Y., Cheriti, A., Belboukhari, N., Sekkoum, K., Habbab, A., 2017. Analgesic and anti-inflammatory effects of essential oils of Vitex agnuscastus L. From south-west of Algeria. Curr. Bioact. 13, 165–169. Ahmad, I., Afza, N., Anis, I., Malik, A., Tareen, R.B., 2004. Iridoid galactosides and a benzofuran type sesquiterpene from Buddleja crispa. Heterocycles 35, 1875–1881. Bao, F.Y., Tang, R.T., Cheng, L., Zhang, C.Y., Qiu, C.Y., Yuan, T., Zhu, L.H., Li, H., Chen, L.X., 2018. Terpenoids from Vitex trifolia and their anti-infammatory activities. J. Nat. 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3.4. Physical and spectroscopic data of new compounds 3.4.1. Nishindacin A (1) Yellowish oil (MeOH); [α]eq \o(\s\up 7(20),\s\do 3(D))-23.0 (c = 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (5.1) nm; IR (KBr) vmax 3395, 2923, 2850, 1651, 1515, 1448, 1396, 1165, 618 cm−1; 1H-NMR (600 MHz, CD3OD) and 13C-NMR (150 MHz, CD3OD) data, see Table 1; HRESIMS (negative): m/z 349.1298 [M-H]- (calcd for C18H21O7, 349.1293). 3.4.2. Isonishindacin A (2) Yellowish oil (MeOH); [α]eq \o(\s\up 7(20),\s\do 3(D))-115.0 (c = 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (5.1) nm; IR (KBr) vmax 3396, 2924, 2850, 1652, 1515, 1447, 1396, 1165, 617 cm−1; 1H-NMR (600 MHz, CD3OD) and 13C-NMR (150 MHz, CD3OD) data, see Table 1; HRESIMS (negative): m/z 349.1294 [M-H]- (calcd for C18H21O7, 349.1293). 3.5. DPPH radical-scavenging assay The compounds (50 μM, 200 μL each) were added to 1.8 mL of 0.1 mM DPPH and mixed vigorously. The mixture was shaken and then incubated for 30 min in the dark, then the absorbance was measured at 515 nm using a spectrophotometer (UV-2000, Unico, China) (BrandWilliams et al., 1995). The absorbance of the control was obtained by replacing the sample with ethanol. The radical scavenging activity was determined using the following equation: Scavenging activity(%) = (Acontrol−Asample)/Acontrol × 100% Here, Asample is the absorbance of the sample, and Acontrol is the absorbance of the control containing all of the reaction reagents except the sample. Supplementary material Supplementary material relating to this article is available online, including Figures S1-S16. Declaration of Competing Interest No potential conflict of interest was reported by the authors. 189
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Iridoids from Vitex negundo var. heterophylla and their antioxidant activities 1
1
Ying-Xue Niu , Dun Wang , Xin-Yue Chu, Su-Yu Gao, Dong-Xin Yang, Li-Xia Chen*, Hua Li*
T
Wuya College of Innovation, School of Pharmaceutical Engineering, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China
ARTICLE INFO
ABSTRACT
Keywords: Vitex negundo var. heterophylla Iridoids Iridoid glycosides Antioxidant activity
Two new iridoids (1-2) together with eight known iridoid glycosides (3-10) were isolated from the 70% EtOH extract of the dried leaves of Vitex negundo var. heterophylla. Their structures were elucidated by extensive spectroscopic analyses. Iridoid aglycones were found for the first time from this plant. All of these compounds were tested for their antioxidant activities. Among them, compound 9 showed moderate radical-scavenging effect on the stable free radical, 1,1-diphenyl-2-picrylhydrazyl, with IC50 values of 26.95 μM.
1. Introduction
also tested for their antioxidant activities.
The genus Vitex (Verbenaceae) consists of small trees and shrubs, with about 250 species mainly distributed in tropical and subtropical regions (Li et al., 2014; Hu et al., 2015). Vitex negundo var. heterophylla (Franch.) Rehd. as one of such species is widely distributed in the hilly areas of north, northeast, northwest, and central regions of China (Li et al., 2014; Hu et al., 2015). Various parts of V. negundo, including the leaves, roots and seeds, have been locally used as traditional folk medicines since antiquity, particularly in China to treat a wide range of ailments, such as cold, headache, migraine and ophthalmodynia (M.Y. Huang et al., 2013; Yao et al., 2016). Crude extracts and pure compounds from Vitex species have been reported to exhibit a wide array of bioactivities including anti-inflammatory (Aboul-Enein et al., 2017), antioxidant (Hajdtu et al., 2007; Huang et al., 2012), antinociceptive (Zheng et al., 2010), and anti-bacterial effects (Chang et al., 2018). Phytochemical investigations have indicated the presence of flavonoids, diterpenoids, iridoids, phenylpropanoids, triterpenoids, phytoecdysteroids, phenolic glycosides, and essential oils in Vitex plants (Suksamrarn et al., 1997; Ono et al., 2001; Li et al., 2002; Sena-Filh et al., 2008; Hu et al., 2015, 2016; Qiu et al., 2017; Bao et al., 2018). In order to find antioxidant material basis of Vitex negundo var. heterophylla, the 70% EtOH extract of the dried leaves of this plant was investigated, and two new iridoids (1-2), along with eight known iridoid glycosides (3-10) were obtained. Their structures were established on the basis of comprehensive spectroscopic analysis. Iridoid aglycones are very unusual in nature (Li et al., 2004; Liu et al., 2006; Yang et al., 2010; Jia et al., 2012; Lee et al., 2018), which were found for the first time from this plant. All of the isolated compounds were
2. Results and discussion The investigation of the constituents from the n-BuOH fraction of the dried leaves of Vitex negundo var. heterophylla led to the isolation of two new iridoid aglycones, nishindacin A (1) and isonishindacin A (2), along with eight known iridoid glycosides including nishindaside (3) (Iwagawa et al., 1993), isonishindaside (4) (Iwagawa et al., 1993), agnuside (5) (Dutta et al., 1983), 6′-O-p- hydroxybenzoylmussaenosidic acid (6) (Sehgal et al., 1983), 6′-O-p-hydroxybenzoyl-gardoside (7) (J. Huang et al., 2013), 6′-O-benzoyl-8-epi-loganic acid (8) (Harput et al., 2004), 6′-O-E-caffeoyl-mussaenosidic acid (9) (Gousiadou et al., 2012), and negundoside (10) (Roy et al., 2015) (Fig. 1). Compound 1 was isolated as yellowish oil (MeOH). The molecular formula C18H22O7 was deduced by HRESIMS data at m/z 349.1298 [MH]- (calcd for C18H21O7, 349.1293) in combination with its NMR data (Table S1), which indicated eight degrees of unsaturation. The 1H NMR spectrum showed signals at δH 7.87 (2H, d, J =6.4 Hz) and 6.81 (2H, d, J =6.4 Hz) in combination with a series of carbon signals [δC 167.8 (C7′), 133.0 (C-2′, C-6′), 116.5 (C-3′, C-5′), 122.0 (C-1′), 164.2 (C-4′)], indicating the presence of one p-hydroxybenzoyl group. In addition, two methoxy protons at δH 3.42 (3H, s) and 3.45 (3H, s), two protons of the oxymethylene at δH 4.86 (1H, overlapped) and 4.79 (1H, d, J = 14.4 Hz), two acetal protons at δH 4.45 (1H, d, J =6.1 Hz) and 4.71 (1H, br d, J =8.5 Hz), an oxymethine proton presented at 4.65 (1H, d, J =5.5 Hz), and one olefinic proton at δH 5.78 (1H, br s) were observed in the 1H NMR spectrum. The 13C NMR spectrum of 1 revealed 18 carbon signals including one ester carbonyl carbon at δC 167.8, two
Corresponding authors. E-mail addresses: [email protected] (L.-X. Chen), [email protected] (H. Li). 1 These two authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.phytol.2019.11.018 Received 9 September 2019; Received in revised form 21 November 2019; Accepted 27 November 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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compounds 3 and 4 were immersed in dichloromethane-methanol (2:1) solution, respectively. They were continuously heated at 50℃ and detected with TLC at different time periods (6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h), and compounds 1 and 2 were not found at all the time periods. This result proved that compounds 1 and 2 are of natural origin. DPPH radical scavenging activities have been widely used for representing the total antioxidant activities of foods. The spectrophotometric technique employs the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH·), which shows a characteristic UV–vis spectrum with a maximum absorbance close to 515 nm in methanol (Brand-Williams et al., 1995). The antioxidant activities of these compounds (50 μM) are shown in Table 2. Ascorbic acid (Vitamin C) was used as the positive control with IC50 values of 6.68 μM. Among all the tested compounds, compound 9 showed moderate radical-scavenging effect on the stable free radical, with IC50 values of 26.95 μM in the DPPH scavenging assay (Fig. 4).
Fig. 1. Structures of compounds 1 and 2.
pairs of aromatic carbons at δC 116.5 × 2 and 133.0 × 2, one oxygenated aromatic carbon at δC 164.2, one oxymethylene carbon at δC 63.4, and two olefinic carbons at δC 133.1 and 143.3. All the NMR data suggested that 1 possessed the similar skeleton structure as nishindaside (Iwagawa et al., 1993), except for the downfield shift of C-1 from δC 98.7 in nishindaside to δC 103.8 in 1, and the absence of a set of sugar signals and the presence of an extra methoxy signal in 1, indicating that a methoxy group linked at C-1. The assumption was further confirmed by the HMBC experiment (Fig. 2), which showed the long-range correlations of H-1 with 1-OCH3/C-3/C-8, H-3 with C-1/C5/3-OCH3, H-7 with C-5/C-6/C-8/C-9/C-10, and H-10 with C-7/C-7′/C8. The linkages of iridoid and p-hydroxybenzoyl moieties were constructed by the key correlation from H-10 to C-7′ in the HMBC spectrum. According to the biosynthetic considerations of iridoids, H-5 and H-9 were assigned as β-oriented (Klimek, 1996; Ahmad et al., 2004; Kim, 2009; Geu-Flores et al., 2012; Piaz et al., 2013). The key NOESY correlations of H-1/H-3, H-1/H-6, H-3/H-6, and H-5/H-9, and no correlation of H-6/H-9 were observed (Fig. 2), together with the coupling constant (J =6.1 Hz) between H-1 and H-9, indicating that H-1, H-3 and H-6 were α-oriented. Based on the above spectroscopic data, combined with the biosynthetic considerations of iridoids (Geu-Flores et al., 2012), compound 1 was determined and named nishindacin A. As for 2, its UV, IR, 1H NMR, 13C NMR, and HRESIMS data were similar to those of 1. By comparison of its 13C NMR signals with those of 1, the signal assigned to C-1 was shifted upfield by 3.2 ppm, suggesting that 2 was an epimer of 1 at C-3. In the NOESY spectrum of 2, the crosspeaks of H-5/H-9 with H-3, while no correlations between H-5/H-9 and H-1/H-6 indicated that H-3 was β-oriented, H-1 and H-6 were α-oriented (Fig. 3). Therefore, 2 was elucidated as 3R-diastereomer of 1, and named isonishindacin A. Specific experiments were carried out to exclude the possibility of compounds 1 and 2 as artificial products. In the isolation process, the fraction containing 1 and 2 was divided on silica gel (a possible catalyst) column chromatography (CC) and eluted with MeOH/CH2Cl2 (always with trace of acid), and possible equilibrium between two acetals (a glucoside and a methyl acetal) may occur. Therefore,
3. Experimental 3.1. General experimental procedures Bruker AV-400 and AV-600 spectrometers were used in the NMR experiments. Chemical shift values are expressed in δ (ppm) using the peak signals of the solvent methanol-d4 (δH 4.87, 3.31 and δC 49.15) as references, and coupling constants (J in Hz) are given in parentheses. Optical rotations were recorded on a Perkin-Elmer 241 polarimeter (PerkinElmer, Waltham, MA, USA). UV spectra were measured on a Shimadzu UV 2201 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). HRESIMS data were acquired on an Agilent 6210 TOF mass spectrometer (Palo Alto, CA, USA). Silica gel GF254 prepared for TLC was purchased from Qingdao Marine Chemical Factory (Qingdao, China). Silica gel (200 − 300 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20 (Pharmacia, USA), and octadecyl silica gel (Merck Chemical Company Ltd., Germany) were used for column chromatography (CC). RP-HPLC was equipped with an LC-6CE liquid chromatograph, SPD-20A UV detector (Shimadzu, Kyoto, Japan), and RP-C18 column (250 × 20 mm, 120 Å, 5 μm, YMC Co. Ltd.). All HPLC or analytical grade reagents were purchased from Tianjin Damao Chemical Company (Tianjin, China). Trace of samples on silica gel plates were detected under UV light and heating after spraying with anisaldehyde-H2SO4 reagent. DPPH were purchased from Sigma–Aldrich (USA). Methanol was purchased from Friendemann Schmidt (Germany). All chemicals were of analytical reagent grade.
Fig. 2. Key HMBC and NOESY correlations of compounds 1.
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Fig. 3. Key HMBC and NOESY correlations of compound 2.
Fig. 4. The IC50 of compound 9 and Vitamin C. Table 1 The 1H (600 MHz) and ppm). No.
13
1a
2a
δC
δH (J in Hz)
δC
δH (J in Hz)
1 3 4
103.8 100.2 30.6
4.45 (1H, d, 6.1) 4.71 (1H, br d, 8.5) 1.98 (1H, dd, 14.4, 3.0) 1.86 (1H, m)
100.6 100.6 30.5
5 6 7 8 9 10
46.2 80.7 133.1 143.3 49.8 63.4
45.6 81.1 132.2 143.8 50.0 63.4
1′ 2′ 3′ 4′ 5′ 6′ 7′ 1-OCH3 3-OCH3
122.0 133.0 116.5 164.2 116.5 133.0 167.8 56.4 56.2
2.40 (1H, m) 4.65 (1H, d, 5.5) 5.78 (1H, br s) – 2.76 (1H, br t-like, 6.1) 4.86 (1H, overlapped) 4.79 (1H, d, 14.4) – 7.87 (1H, d, 6.4) 6.81 (1H, d, 6.4) – 6.81 (1H, d, 6.4) 7.87 (1H, d, 6.4) – 3.42 (3H, s) 3.45 (3H, s)
4.53 (1H, d, 7.5) 4.86 (1H, d, 5.1) 1.92 (1H, ddd, 13.2, 11.4, 5.5) 1.62 (1H, ddd, 13.2, 7.9, 5.5) 2.29 (1H, m) 4.57 (1H, m) 5.82 (1H, br s) – 2.80 (1H, br t, 7.6) 4.84 (1H, overlapped) 4.77 (1H, br d, 14.8) – 7.88 (1H, d, 6.4) 6.81 (1H, d, 6.4) – 6.81 (1H, d, 6.4) 7.88 (1H, d, 6.4) – 3.42 (3H, s) 3.39 (3H, s)
a
Table 2 DPPH radical-scavenging activity of compounds 1–10.
C (150 MHz) NMR Data of Compounds 1 and 2 (δ in
122.0 133.0 116.5 164.2 116.5 133.0 167.8 56.7 55.7
compounds
Scavenging activity (%)
compounds
Scavenging activity (%)
1 2 3 4 5 Vitamin Ca
27.14% 25.80% 26.07% 24.46% 21.25% 92.5%
6 7 8 9 10
19.10% 21.51% 23.12% 88.48% 22.13%
a
positive control.
3.2. Plant material The dried leaves of Vitex negundo var. heterophylla (Franch.) Rehd. were collected from Chaoyang, Liaoning Province, China, and were identified by Prof. Jing-Ming Jia, Department of Pharmaceutical Botany, Shenyang Pharmaceutical University. A voucher specimen (VN. 20,140,915) was deposited in the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University. 3.3. Extraction and isolation The dried leaves of Vitex negundo var. heterophylla. (6.0 kg) were extracted with 70% EtOH (3 × 2 h × 76 L) to afford the crude extract after solvent removal in vacuo. The crude extract (406.5 g) was
Spectra of 1 and 2 were tested in methanol-d4. 188
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suspended in water and partitioned successively with petroleum ether (PE), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) in the same volume for three times. The n-BuOH extracts (200.5 g) were subjected to D101 Macroporous adsorption resin eluted with EtOH-H2O (0:100−100:0, v/v) to afford six fractions (D1−D6). Fraction D1 (52.0 g) was divided on silica gel CC and eluted by CH2Cl2/MeOH (100:1 to 1:1) into six fractions (D11-D16) according to TLC analysis. Subfraction D13 was subjected to an ODS column and eluted with CH3OH/H2O (1:9 to 1:0) to yield D131-D137. Then fraction D136 was separated by HPLC eluted with 50% CH3OH/H2O to give compound 1 (12.3 mg, tR =20.1 min) and compound 2 (15.4 mg, tR =23.1 min). Subfraction D15 was separated on ODS CC and eluted with MeOH/H2O (1:9 to 1:0) to obtain compound 3 (23.3 mg) and six subfractions (D151-D156), D155 was separated by preparative HPLC (40% MeOH/ H2O, flow rate 8.0 mL/min) to compound 4 (38.3 mg, tR =22.7 min), compound 5 (395.8 mg, tR =19.1 min) and compound 6 (304.7 mg, tR =29.2 min). Fraction D16 was chromatographed over an ODS column, MeOH/H2O (1:9 to 1:0) as solvent, yielding compounds 7 (43.7 mg), 10 (24.2 mg) and eight subfractions (D161-168). Purifcation of subfraction D167 by HPLC (CH3OH-0.0001trifluoroacetic acid-H2O = 3:7, 8.0 mL/ min) led to the isolation of compounds 8 (106.3 mg, tR =25.4 min) and 9 (80.1 mg, tR =36.8 min).
Acknowledgments This work was supported by the National Natural Science Foundation of China (NO. 81773594), Liaoning Revitalization Talents Program (NO. XLYC1807182), and Shenyang Planning Project of Science and Technology (NO. 18-013-0-46). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.11.018. References Aboul-Enein, H.Y., Cheriti, A., Belboukhari, N., Sekkoum, K., Habbab, A., 2017. Analgesic and anti-inflammatory effects of essential oils of Vitex agnuscastus L. From south-west of Algeria. Curr. Bioact. 13, 165–169. Ahmad, I., Afza, N., Anis, I., Malik, A., Tareen, R.B., 2004. Iridoid galactosides and a benzofuran type sesquiterpene from Buddleja crispa. Heterocycles 35, 1875–1881. Bao, F.Y., Tang, R.T., Cheng, L., Zhang, C.Y., Qiu, C.Y., Yuan, T., Zhu, L.H., Li, H., Chen, L.X., 2018. Terpenoids from Vitex trifolia and their anti-infammatory activities. J. Nat. 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3.4. Physical and spectroscopic data of new compounds 3.4.1. Nishindacin A (1) Yellowish oil (MeOH); [α]eq \o(\s\up 7(20),\s\do 3(D))-23.0 (c = 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (5.1) nm; IR (KBr) vmax 3395, 2923, 2850, 1651, 1515, 1448, 1396, 1165, 618 cm−1; 1H-NMR (600 MHz, CD3OD) and 13C-NMR (150 MHz, CD3OD) data, see Table 1; HRESIMS (negative): m/z 349.1298 [M-H]- (calcd for C18H21O7, 349.1293). 3.4.2. Isonishindacin A (2) Yellowish oil (MeOH); [α]eq \o(\s\up 7(20),\s\do 3(D))-115.0 (c = 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (5.1) nm; IR (KBr) vmax 3396, 2924, 2850, 1652, 1515, 1447, 1396, 1165, 617 cm−1; 1H-NMR (600 MHz, CD3OD) and 13C-NMR (150 MHz, CD3OD) data, see Table 1; HRESIMS (negative): m/z 349.1294 [M-H]- (calcd for C18H21O7, 349.1293). 3.5. DPPH radical-scavenging assay The compounds (50 μM, 200 μL each) were added to 1.8 mL of 0.1 mM DPPH and mixed vigorously. The mixture was shaken and then incubated for 30 min in the dark, then the absorbance was measured at 515 nm using a spectrophotometer (UV-2000, Unico, China) (BrandWilliams et al., 1995). The absorbance of the control was obtained by replacing the sample with ethanol. The radical scavenging activity was determined using the following equation: Scavenging activity(%) = (Acontrol−Asample)/Acontrol × 100% Here, Asample is the absorbance of the sample, and Acontrol is the absorbance of the control containing all of the reaction reagents except the sample. Supplementary material Supplementary material relating to this article is available online, including Figures S1-S16. Declaration of Competing Interest No potential conflict of interest was reported by the authors. 189
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