Two new glycosides from Vitex negundo

Article abstract of DOI:10.1080/14786419.2012.763126

Two new glycosides, 2-methyl pyromeconic acid 3-O-β-d-glucopyranoside- 6′-(O-4″-hydroxybenzoate) (1), 6′-O-p-hydroxybenzoyl-gardoside (2) and four known iridoid glycosides (3-6) were isolated from the whole plant of Vitex negundo. Their structures were el

Full text of DOI:10.1080/14786419.2012.763126

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Two new glycosides from Vitex
negundo
Jie Huangab, Guo-Cai Wangbc, Chun-Hua Wangab, Xiao-Jun Huangbc
& Wen-Cai Yeabc
a Department of phytochemistry, China Pharmaceutical University,
Nanjing, 210009, P.R. China
b College of Pharmacy, Institute of Traditional Chinese Medicine &
Natural Products, Jinan University, Guangzhou, 510632, P.R. China
c Guangdong Province Key Laboratory of Pharmacodynamic
Constituents of TCM and New Drugs Research, Jinan University,
Guangzhou, 510632, P.R. China
Published online: 29 Jan 2013.
To cite this article: Jie Huang, Guo-Cai Wang, Chun-Hua Wang, Xiao-Jun Huang & Wen-Cai Ye
(2013) Two new glycosides from Vitex negundo, Natural Product Research: Formerly Natural Product
Letters, 27:20, 1837-1841, DOI: 10.1080/14786419.2012.763126
To link to this article: http://dx.doi.org/10.1080/14786419.2012.763126
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Natural Product Research, 2013
Vol. 27, No. 20, 1837–1841, http://dx.doi.org/10.1080/14786419.2012.763126
Two new glycosides from Vitex negundo
Jie Huanga,b†, Guo-Cai Wangb,c†, Chun-Hua Wanga,b, Xiao-Jun Huangb,c and Wen-Cai Yea,b,c
*
aDepartment of phytochemistry, China Pharmaceutical University, Nanjing 210009, P.R. China; bCollege
of Pharmacy, Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou
510632, P.R. China; cGuangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and
New Drugs Research, Jinan University, Guangzhou 510632, P.R. China
(Received 6 August 2012; final version received 21 November 2012)
Two new glycosides, 2-methyl pyromeconic acid 3-O-b-D-glucopyranoside-60-(O-400-
hydroxybenzoate) (1), 60-O-p-hydroxybenzoyl-gardoside (2) and four known iridoid
glycosides (3–6) were isolated from the whole plant of Vitex negundo. Their structures
were elucidated on the basis of spectroscopic methods including HR-ESI-MS, 1D and
2D NMR.
Keywords: Vitex negundo; Verbenaceae; pyromeconic acid derivative; iridoid
glycoside
1. Introduction
The plant Vitex negundo Linn. is a shrub belonging to the family Verbenaceae, mainly
distributed in Southern Asia (Watt 1972). Traditionally, the extract of V. negundo is widely used
for the treatment of some female disorders (Tiwari & Tripathi 2007). Previous phytochemical
studies of this plant had led to the isolation of terpenoids (Vishnoi et al. 1983; Chawla et al. 1991,
1992a), volatile oil (Singh et al. 1999), flavonoids (Misra & Subramanian 1980; Achari et al.
1984), iridoid glycosides (Sehgal et al. 1983), stilbenes (Banerji et al. 1988) and lignans (Chawla
et al. 1992b). This paper reports the isolation and structural elucidation of two new compounds
(1–2), along with four known iridoid glycosides (3–6).
2. Results and discussion
Compound 1 was isolated as colourless needles. The molecular formula of 1 was established as
C19H20O10 by the HR-ESI-MS at m/z 431.0988 [M þ Na]þ (calcd for C19H20O10Na, 431.0954).
The IR spectrum indicated the presence of hydroxyl (3317 cm21), carboxyl (1696 cm21) and
1
aromatic ring (1608 and 1516 cm21). The H NMR spectrum showed the presence of two
olefinic protons at dH 7.86 (1H, d, J ¼ 5.6 Hz) and 6.34 (1H, d, J ¼ 5.6 Hz), a 1,4-disubstituted
benzene ring at dH 7.82 (2H, d, J ¼ 7.8 Hz) and 6.81 (2H, d, J ¼ 7.8 Hz), an anomeric proton of
sugar unit at dH 4.89 (1H, d, J ¼ 7.3 Hz) and a methyl at dH 2.29 (3H, s). The 13C NMR and
DEPT spectra displayed the signals of a benzene ring at dC 163.6, 132.8 £ 2, 122.2 and 116.4
£ 2; a pyranone moiety at dC 176.9, 164.5, 156.9, 143.1 and 117.2; a glucopyranosyl unit at dC
104.6, 77.9, 76.0, 75.4, 71.8 and 64.4 and a methyl at dC 15.6.
Comparison of 1H, 13C NMR data of 1 with those of pyromeconic acid 3-O-b-D-
glucopyranoside-60-(O-400-hydroxybenzoate) (Ahmed & Mohamed 2002) revealed that most
*Corresponding author. Email: chywc@yahoo.com.cn
†These authors contributed equally to this work.
q 2013 Taylor & Francis

1838
J. Huang et al.
signals of the two compounds were similar except that 1 had the signal of an extra methyl at dH
2.29 (3H, s)/dC 15.6, and the downfield shift of C-3 from dC 146.5 to dC 164.5, as well as the
upfield shift of C-2 from dC 147.8 to dC 143.1. All the above data suggested that the methyl was
located at C-2 position, which was confirmed by the HMBC correlations between H-7 (dH 2.27)
and C-2 (dC 143.1)/C-3 (dC 164.5) (Figure 1). Acid hydrolysis of 1 afforded D-glucose, which
was identified by GC analysis. The b-configuration of D-glucose was determined by the coupling
constant (J ¼ 7.3 Hz) of the anomeric proton. Consequently, compound 1 was established as 2-
methyl pyromeconic acid 3-O-b-D-glucopyranoside-60-(O-400-hydroxybenzoate).
Compound 2 was obtained as amorphous powder. The molecular formula of 2 was
determined as C23H26O12 by its HR-ESI-MS at m/z 493.1332 [M 2 H]þ (calcd for C23H25O12,
493.1346). Acid hydrolysis of 2 also afforded D-glucose. The IR spectrum showed the presence
1
of hydroxyl (3378 cm21), carboxyl (1697 cm21) and aromatic ring (1608, 1516 cm21). The H
NMR spectrum showed two olefinic protons at dH 5.23 (1H, t, J ¼ 1.8 Hz) and 5.15 (1H, br s),
suggesting the presence of an exo-methylene group. The protons of a 1,4-disubstituted benzene
ring at dH 7.87 (2H, d, J ¼ 7.8 Hz) and 6.81 (2H, d, J ¼ 7.8 Hz), as well as an anomeric proton of
sugar unit at dH 4.69 (1H, d, J ¼ 7.9 Hz), were also displayed in the 1H NMR spectrum. The 13
C
NMR spectrum showed 23 carbon signals including a glucopyranosyl unit at dC 100.2, 77.9,
75.6, 74.7, 71.9 and 64.6; a p-hydroxybenzoyl moiety at dC 167.9, 163.8, 132.8 £ 2, 122.2 and
116.2 £ 2 and an iridoid aglycone at dC 172.1, 152.8, 152.0, 113.5, 113.2, 96.8, 73.9, 44.7, 41.1
and 32.5. All the above data indicated that 2 was an iridoid glycoside with a benzoyl unit.
The NMR data of 2 were very similar to those of the known compound 60-O-benzoyl-
gardoside (Harput et al. 2004). The difference was the presence of a p-hydroxybenzoyl (dC
167.9, 163.8, 132.8 £ 2, 122.2, 116.2 £ 2) in 2 instead of a benzoyl (dC 167.8, 134.4, 131.4,
130.6 £ 2, 129.7 £ 2) in 60-O-benzoyl-gardoside. The HMBC correlation between H-60 (dH
4.58) and C-700 (dC 167.9) confirmed that the p-hydroxybenzoyl group was connected to C-60
position of glucose. Thus, Compound 2 was elucidated as 60-O-p-hydroxybenzoyl-gardoside.
In addition, four known compounds (3–6) (Figure 2) were elucidated as 60-p-
hydroxybenzoylmussaenosidic acid (3) (Sehgal et al. 1983), 20-p-hydroxybenzoylmussaenosidic
acid (4) (Sehgal et al. 1982), 20-O-trans-p-coumaroylloganic acid (5) (Wu et al. 2009) and 20-O-
trans-p-hydroxybenzoyl-8-epiloganic acid (6) (Sridhar & Subbaraju 2004), respectively, by
comparison of their spectral data with those of the literatures.
3. Experimental
3.1. General methods
Melting points were obtained on an X-5 micro-melting point detector (uncorrected). Optical
rotation values were measured on a JASCO P-1020 polarimeter. UV spectra were recorded by a
JASCO V-550 UV/Vis spectrophotometer. IR spectra were measured on a JASCO FT/IR-480 plus
FT-IR spectrometer. HR-ESI-MS data were carried out on an Agilent 6210 ESI/TOF mass
Figure 1. The chemical structure and key HMBC of compounds 1 and 2.

Natural Product Research
1839
Figure 2. The chemical structure of compounds 3–6.
spectrometer. 1D and 2D NMR spectra were recorded on Bruker AV-400 spectrometer using
tetramethylsilane (TMS) as internal standard. GC was carried out on a Shimadzu GCMS-QP2010
plus gas chromatograph–mass spectrometer using HP-1701 column (0.25 mm £ 30 m). A Waters
1525 pump and a 2487 Dual l absorbance detector for analytical HPLC using Cosmosil 5C18-
MS-II waters column (4.6 mm £ 250 mm). A Gilson 360 pump and a UV/Vis-152 detector for
preparative HPLC using Cosmosil 5C18-MS-II waters column (20 mm £ 250 mm). Column
chromatographies were carried out on macroporous resin such as Diaion HP-20 (Mitsubishi
Chemical Corporation, Japan), silica gel (200–300 mesh; Qingdao Marine Chemical Group Co.
˚
Ltd, Qingdao, China), ODS (50 mm, 120 A; YMC) and Sephadex LH-20 (Pharmacia Biotech AB,
Uppsala, Sweden). TLC was performed using precoated silica gel GF254 plates (Yantai Chemical
Industry Research Institute, Yantai, China) or RP-18 F254 plates (Merck, Darmstadt, Germany).
D- and L-glucose were purchased from Sigma (St Louis, MO, USA).
3.2. Plant material
The whole plant of V. negundo was collected in Guangzhou city, Guangdong Province of P.R.
China, and authenticated by Prof. Guang-Xiong Zhou (College of Pharmacy, Jinan University).
A voucher specimen (No. 20110910) was deposited in the Institute of Traditional Chinese
Medicine & Natural Products, Jinan University, Guangzhou, P.R. China.
3.3. Extraction and isolation
The dried and powdered whole plant of V. negundo (9.0 kg) was extracted with 95% EtOH at
room temperature for three times (3 £ 20 L, each 10 h). The EtOH extract was concentrated in
vacuum to yield a residue (450 g), which was suspended in water and partitioned using
petroleum ether, EtOAc and n-BuOH, respectively. The n-BuOH soluble fraction (200 g) was
subjected to silica gel column (10 £ 150 cm, 2000 g) eluting with CHCl3 –MeOH
(100:0 ! 0:100) to afford six fractions (A–F). Fraction C (16 g) was separated by Sephadex
LH-20 column (2.5 £ 150 cm, CHCl3–MeOH, 50:50) to afford three subfractions (I–III).
Subfraction II was subjected to ODS column (3 £ 60 cm, MeOH–H2O) and preparative HPLC
(MeOH–H2O, 40:60) to afford 1 (6.0 mg) and 2 (4.0 mg). Subfraction III was also subjected to

1840
J. Huang et al.
an ODS column and preparative HPLC (MeOH–H2O, 30:70) to afford 3 (3.4 mg) and
6 (2.9 mg). Fraction E (7 g) was separated by Sephadex LH-20 column (3.0 £ 80 cm, CHCl3–
MeOH, 50:50) to afford 4 (4.1 mg) and 5 (3.2 mg).
3.3.1. 2-Methyl pyromeconic acid 3-O-b-D-glucopyranoside-60-(O-400-hydroxybenzoate) (1)
25
Colourless needles, mp 205–2078C. ½aꢀD 243 (c 0.5, MeOH). UV (MeOH) lmax (log 1): 208
(1.97), 258 (1.92) nm. IR (KBr) nmax: 3317, 1696, 1638, 1602, 1557, 1455, 1281, 1167, 1125,
1081, 848, 773, 618 cm21. HR-ESI-MS m/z: 431.0988 [M þ Na]þ (calcd for C19H20O10Na,
431.0954). 1H NMR (CD3OD, 400 MHz) d: 6.34 (1H, d, J ¼ 5.6 Hz, H-5), 7.86 (1H, d,
J ¼ 5.6 Hz, H-6), 2.29 (3H, s, H-7), 4.89 (1H, d, J ¼ 7.3 Hz, H-10), 3.43 (1H, m, H-20), 3.41 (1H,
m, H-30), 3.40 (1H, m, H-40), 3.35 (1H, m, H-50), 4.45 (1H, dd, J ¼ 11.8, 6.7 Hz, H-60a), 4.55
(1H, dd, J ¼ 11.8, 2.4 Hz, H-60b), 7.82 (2H, d, J ¼ 7.8 Hz, H-200, H-600), 6.81 (2H, d, J ¼ 7.8 Hz,
H-300, H-500). 13C NMR (CD3OD, 100 MHz) d: 143.1 (C-2), 164.5 (C-3), 176.9 (C-4), 117.2 (C-
5), 156.9 (C-6), 15.6 (C-7), 104.6 (C-10), 77.9 (C-20), 75.4 (C-30), 71.8 (C-40), 76.0 (C-50), 64.4
(C-60), 122.2 (C-100), 132.8 (C-200, C-600), 116.4 (C-300, C-500), 163.6 (C-400), 167.7 (C-700).
3.3.2. 60-O-p-Hydroxybenzoyl-gardoside (2)
25
Amorphous powder, mp 178–1808C. ½aꢀD 254 (c 0.5, MeOH). UV (MeOH) lmax (log 1): 207
(1.82), 255 (1.90) nm. IR (KBr) nmax: 3378, 1697, 1608, 1516, 1282, 1169, 1078, 1013,
1
771 cm21. HR-ESI-MS m/z: 493.1332 [M 2 H]þ (calcd for C23H25O12, 493.1346). H NMR
(CD3OD, 400 MHz) d: 5.14 (1H, d, J ¼ 5.2 Hz, H-1), 7.34 (1H, br s, H-3), 3.13 (1H, dt, J ¼ 7.6,
7.0 Hz, H-5), 1.93 (1H, m, H-6a), 1.80 (1H, dt, J ¼ 6.4, 12.8 Hz, H-6b), 4.28 (1H, m, H-7), 2.88
(1H, m, H-9), 5.15 (1H, br s, H-10a), 5.23 (1H, t, J ¼ 1.8 Hz, H-10b), 4.69 (1H, d, J ¼ 7.9 Hz, H-
10), 3.25 (1H, m, H-20), 3.41 (1H, m, H-30), 3.40 (1H, m, H-40), 3.60 (1H, m, H-50), 4.42 (1H, dd,
J ¼ 11.8, 6.7 Hz, H-60a), 4.58 (1H, dd, J ¼ 11.8, 2.4 Hz, H -60b), 7.87 (2H, d, J ¼ 7.8 Hz, H-200,
H-600), 6.81 (2H, d, J ¼ 7.8 Hz, H-300, H-500). 13C NMR (CD3OD, 100 MHz) d: 96.8 (C-1), 152.0
(C-3), 113.5 (C-4), 32.5 (C-5), 41.1 (C-6), 73.9 (C-7), 152.8 (C-8), 44.7 (C-9), 113.2 (C-10),
172.1 (C-11), 100.2 (C-10), 74.7 (C-20), 77.9 (C-30), 71.9 (C-40), 75.6 (C-50), 64.6 (C-60), 122.2
(C-100), 132.8 (C-200, C-600), 116.2 (C-300, C-500), 163.8 (C-400), 167.9 (C-700).
3.4. Acid hydrolysis and GC analysis of 1 and 2
Each solution of compounds 1 and 2 (each 1.5 mg) was hydrolysed with 1.5 mL of 2 N HCl
(CH3OH) for 3 h at 808C. The reaction mixture was dissolved in H2O and extracted with CHCl3.
The aqueous layer was concentrated and dried by N2. Then, 1 mL of dried pyridine and 2 mg of
L-cysteine methylester hydrochloride were added to the residue. The mixture was heated at 608C
for 2 h and concentrated to dryness with N2. N-(Trimethylsilyl) imidazole (0.2 mL) was added
into the mixture, and then kept at 608C for 1 h. At last, the solution was diluted with H2O (1 mL)
and extracted with hexane (1 mL). The organic layer was analysed under the following
conditions: HP-1701 (0.25 mm £ 30 m), detector: FID, column temperature: 200–2508C (58C/
min), detector temperature: 2808C, injector temperature: 2508C and carried gas: N2. The
standard D-glucose and L-glucose were subjected to the same reaction and GC analysis under
the above conditions [tR (min): 30.63 (D-glucose), tR (min): 33.23 (L-glucose)]. As a result,
D-glucose [tR (min): 30.68–30.79] was detected from the hydrolysates of 1 and 2, respectively.

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Acknowledgements
This work was supported by grants from the Programme for Changjiang Scholars and Innovative Research
Team in University (IRT0965) and Research Team Programme of Natural Science Foundation of
Guangdong Province (No. 8351063201000003).
References
Achari, B, Chowdhury, US, Dutta, PK, & Pakrashi, SC. 1984. Two isomeric flavanones from Vitex negundo.
Phytochemistry 23: 703–704.
Ahmed, AA, & Mohamed, AEH. 2002. Three pyrone glucosidic derivatives from Conyza albida. Planta Med 68:
664–666.
Banerji, J, Das, B, & Chakrabarty, R. 1988. Isolation of 4,40-dimethoxy-trans-stilbene & flavonoids from leaves & twigs
of Vitex negundo Linn. Indian J Chem 27B: 597–599.
Chawla, AS, Sharma, AK, Handa, SS, & Dhar, KL. 1991. Chemical investigation and anti-inflammatory activity of Vitex
negundo seeds, part I. Indian J Chem 30B, 773–776.
Chawla, AS, Sharma, AK, Handa, SS, & Dhar, KL. 1992a. Chemical investigation and anti-inflammatory activity of
Vitex negundo seeds. J Nat Prod 55: 163–167.
Chawla, AS, Sharma, AK, Handa, SS, & Dhar, KL. 1992b. A lignan from Vitex negundo seeds. Phytochemistry 31:
4378–4379.
Harput, US, Varel, M, & Nagatsu, A. 2004. Acylated iridoid glucosides from Veronica anagallis-aquatica.
Phytochemistry 65: 2135–2149.
Misra, GS, & Subramanian, PM. 1980. Three new flavone glycosides from Vitex negundo. Planta Med 38: 155–160.
Sehgal, CK, Taneja, SC, Dhar, KL, & Atal, CK. 1982. 20-p-Hydroxybenzoyl mussaenosidic acid, a new iridoid glucoside
from Vitex negundo. Phytochemistry 21: 363–366.
Sehgal, CK, Taneja, SC, Dhar, KL, & Atal, CK. 1983. 60-p-Hydroxybenzoylmussaenosidic acid – an iridoid glucoside
from Vitex negundo. Phytochemistry 22: 1036–1037.
Singh, V, Dayal, R, & Bartley, JP. 1999. Volatile constituents of Vitex negundo leaves. Planta Med 65: 580–582.
Sridhar, C, & Subbaraju, GV. 2004. New acylated iridoid glucosides from Vitex altissima. J Nat Prod 67: 2012–2016.
Tiwari, OP, & Tripathi, YB. 2007. Antioxidant properties of different fractions of Vitex negundo Linn. Food Chem 100:
1170–1171.
Vishnoi, SP, Shoeb, A, Kapil, RS, & Popli, SP. 1983. A furanoeremophilane from Vitex negundo. Phytochemistry 22:
597–598.
Watt, SG. 1972. A dictionary of the economic products of India. Delhi: Cosmo Publications. p. 248–249.
Wu, M, Wu, P, Liu, MF, & Xie, HH. 2009. Iridoids from Gentiana loureirii. Phytochemistry 70: 747–748.