A new isoflavone glycoside from flowers of Pueraria Montana var. lobata (Willd.) Sanjappa & Pradeep

Article abstract of DOI:10.1080/14786419.2019.1655021

A new isoflavone glycoside, named 3’-hydroxytectorigenin-7-O-β-D-xylosyl-(1→6)-β-D-glucopyranoside (1) was isolated from the flowers of Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep. The structure of compound 1 was characterised by HR-ESI-MS and NMR spectroscopic methods. In radical scavenging activity test using 2, 2-diphenyl-1-picrylhydrazyl (DPPH), compound 1 showed moderate activity with IC₅₀ value of 42 ± 4.2 μg/mL.

Full text of DOI:10.1080/14786419.2019.1655021

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
A new isoflavone glycoside from flowers of
Pueraria Montana var. lobata (Willd.) Sanjappa &
Pradeep
Yilong Ma, Yafang Shang, Zhifeng Zhong, Yingshuo Zhang, Yang Yang, Jun
Feng & Zhaojun Wei
To cite this article: Yilong Ma, Yafang Shang, Zhifeng Zhong, Yingshuo Zhang, Yang
Yang, Jun Feng & Zhaojun Wei (2019): A new isoflavone glycoside from flowers of
PuerariaꢀMontana var. lobata (Willd.) Sanjappa & Pradeep, Natural Product Research, DOI:
10.1080/14786419.2019.1655021
To link to this article: https://doi.org/10.1080/14786419.2019.1655021
View supplementary material
Published online: 21 Aug 2019.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=gnpl20

NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1655021
A new isoflavone glycoside from flowers of Pueraria
Montana var. lobata (Willd.) Sanjappa & Pradeep
ꢀ
ꢀ
ꢀ
Yilong Ma
Jun Feng and Zhaojun Wei
, Yafang Shang , Zhifeng Zhong , Yingshuo Zhang, Yang Yang,
School of Food and Biological Engineering, Hefei University of Technology, Hefei, P.R. China
ABSTRACT
ARTICLE HISTORY
Received 15 March 2019
Accepted 2 August 2019
A new isoflavone glycoside, named 3’-hydroxytectorigenin-7-O-
b-D-xylosyl-(1!6)-b-D-glucopyranoside (1) was isolated from the
flowers of Pueraria montana var. lobata (Willd.) Sanjappa &
Pradeep. The structure of compound 1 was characterised by HR-
ESI-MS and NMR spectroscopic methods. In radical scavenging
activity test using 2, 2-diphenyl-1-picrylhydrazyl (DPPH), com-
pound 1 showed moderate activity with IC50 value of 42 4.2 lg/
mL.
KEYWORDS
Pueraria montana var.
lobata; isoflavone glycoside;
3’-hydroxy -tectorigenin-7-
O-b-D-xylosyl-(1!6)-b-D-
glucopyranoside; radical
scavenging activity
1. Introduction
The flowers of Pueraria montana var. lobata (Willd.) Sanjappa
& Pradeep
(Leguminosae), known as Gehua in China and have been used in Traditional Chinese
Medicine (TCM) for thousands of years (Niiho et al. 2010; Xiong et al. 2010; Yao et al.
2010). As TCM, P. montana var. lobata flowers have been used to treat stroke (Li et al.
2017), intoxication (Zhang et al. 2012), stiffness and pain, as well as alcohol abuse and
rectal ulcers (Yu et al. 2011). Recent studies have revealed that the constituents in P.
montana var. lobata flowers are isoflavones, including tectorigenin, daidzein and genis-
tin (Yu et al. 2004; Shi et al. 2012; Cui et al. 2018). These isoflavones show various
activities towards different diseases, such as cancer (Shahana et al. 2010) and liver
injury (Lee et al. 2003). Besides isoflavones, a number of isoflavone glycosides were
isolated and identified from P. montana var. lobata flowers, including puerarin (Xia
CONTACT Yilong Ma
These authors contributed equally to this work.
yilong.ma@hfut.edu.cn; Zhaojun Wei
zjwei@hfut.edu.cn
ꢀ
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1655021.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Y. MA ET AL.
et al. 2013), kakkalide (Bai et al. 2011), tectoridin (Xiong et al. 2010) and daidzin (Shi
et al. 2012). These isoflavone glycosides were showed to possess neuroprotective and
anti-inflammatory activities (Min et al. 2011; Xing et al. 2011). Herein, a new isoflavone
glycoside, 3’-hydroxytectorigenin-7-O-b- D-xylosyl-(1!6)-b-D-glucopyranoside (1) from
flowers of this plant was isolated and characterised. In addition, the radical scavenging
activity of this compound was evaluated.
2. Results and discussion
Compound 1 was obtained as a white amorphous powder with a molecular formula
which was assigned as C27H30O16 based on the HR-ESI-MS data (m/z 609.1505, [M-H]ꢁ,
calculated for 609.1534) and NMR analyses. The characteristic UV absorption bands at
268 and 340 nm and NMR data indicated that compound 1 was an isoflavone (Huang
1
et al. 2012). From H spectrum, characteristic isoflavone signals at d 8.36 (H-2) and d
6.96 (H-8) indicated the A-ring of isoflavone was substituted at C-5, C-6 and C-7 (Park
et al. 1999), and hydroxy (d 12.97) and methoxy groups (d 3.78) could respectively be
assigned at C-5 and C-6 by the interpretation of HMBC spectrum. The proton signals
at d 7.03 (H-2’), d 6.80 (H-5’) and d 6.84 (H-6’) indicated that the B-ring of isoflavone
was substituted at C-3’ and C-4’ (Lee et al. 2014; Fu et al. 2015), and aromatic hydroxy
signals at d 9.09 and d 9.03 could be assigned at these two positions. The hydroxy
substitution at C-3’ and C-4’ has been reported in other isoflavones isolated from the
flowers of P. montana var. lobata, e.g. 3’-hydroxypuerarin (Li et al. 2017) and 3’-hydrox-
ydaidzein (Shi et al. 2012). The presence of two sugar moieties was evident from the
presence of anomeric proton signals at d 5.05 (H-1 of Glc) and d 4.19 (H-1 of Xyl), and
the sugar moieties were identified as D-glucose and D-xylose by the acid hydrolysis of
compound 1. The identity of glucose and xylose was confirmed by the presence of m/
z
317 and 302 fragments, which were deduced as [M þ H–xyl–glc]þ and
[M þ H–xyl–glc–CH3]þ, respectively (Zhang et al. 2012; Li et al. 2017). These MS frag-
ments also confirmed the presence of a methoxy group. The linkages among xylose,
glucose and isoflavone skeleton were established from HMBC spectrum, which showed
correlations between H-1 of xylose (d 4.19) and C-6 of glucose (d 69.15), as well as H-1
of glucose (d 5.05) and C-7 of isoflavone (d 156.98). From 13C NMR and DEPT spec-
trum, the presence of 27 carbon atoms was confirmed, including 10 quaternary carbon
atoms, 15 -CH (or -CH3), and 2 -CH2. Among them, 9 of 14 CH and two CH2 were
attributed to xyl-glu glycosides, while the 10 quaternary carbon atoms, 5 of 14 –CH
and one -CH3 were attributable to one isoflavone skeleton. Therefore, compound 1
was characterised as 3’-hydroxytectorigenin-7-O-b-D-xylosyl-(1!6)-b-D-glucopyrano-
side (Figure 1).
The free radical scavenging activity of compound 1 was determined against 2, 2-
diphenyl-1-picrylhydrazyl (DPPH). Compared to vitamin C (IC50 ¼ 21 2.4 lg/mL) and
tectorigenin (IC50 ¼ 297 lg/mL, one of the main isoflavones in the flower of P. mon-
tana var. lobata) (Park et al. 2004), compound 1 showed moderate activity with IC50
value of 42 4.2 lg/mL.

NATURAL PRODUCT RESEARCH
3
Figure 1. Structure of compound 1.
3. Experimental
3.1. General experimental procedures
The crude sample of compound 1 was obtained from the extract of P. montana var.
lobata flowers by zirconia absorption, using a procedure similar to that reported previ-
ously (Ma et al. 2018). Further purification of compound 1 was achieved by prepara-
tive high performance liquid chromatography (HPLC, Shimadzu, Suzhou, China), which
was equipped with a SinoChrom ODS-BP column (250 ꢂ 10 mm, 10 lm, Dalian Elite).
The analytical HPLC was carried out using a reverse-phase HPLC (RP-HPLC) system
(Dalian Elite, ichrom M5100), which was equipped with an HC-C18 reverse-phase col-
umn (250 ꢂ 4.6 mm, 5 lm, Dalian Elite). LC-MS analysis was performed with 1260/6460
LC/MSD system (Agilent Technologies, Santa Clara, CA, USA). The NMR spectra were
recorded on a Bruker AVANCEIII 400 MHz spectrometer (Bruker, Rheinstetten, Germany)
in DMSO as a solvent. Chemical shifts are expressed in d (ppm) and are referenced to
the residual solvent signals. All other chemicals and solvents were of analytical grade
and were used without further purification.
3.2. Plant material
The flowers of P. montana var. lobata were collected in June of 2017 from Bozhou
(Anhui, China) and the plant was identified as Pueraria montana var. lobata (Willd.)
Sanjappa & Pradeep by Dr. Xiangli Niu, professor of Hefei University of Technology
(Hefei, China). In addition, the plant was also known as Pueraria lobata (Willd.) Ohwi,
which is a synonym of Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep (Cui
et al. 2018). A voucher specimen (FPL-2017-001) was deposited in the herbarium of
School of Food and Biological Engineering, Hefei University of Technology,
Hefei, China.
3.3. Extraction and isolation
The dried P. montana var. lobata flowers were ground into 40 mesh size and extracted
with water for 1 hour by an ultrasonic cleaner (45 kHz, 240 W). In a typical extraction,
the flowers (1 g) were mixed with water (10 mL) and sonicated for 1 hour, and the
extract was separated by a 0.45 lm filter and further analysed by HPLC and LC-MS.
The filtrate was then mixed with zirconia, this adsorbent was prepared using the

4
Y. MA ET AL.
procedure described previously (Ma et al. 2018). After 24 h adsorption using a shaker,
the adsorbent was separated by centrifugation and washed with water; the adsorbent
was then suspended in 70% acetic acid (v/v) and shaken for 24 h, the desorption solu-
tion was separated by centrifugation to give crude compound 1 with HPLC purity of
90%, which was lyophilized and further purified by preparative HPLC. In a typical puri-
fication, 2 g flower gave 6.3 mg compound 1 with a purity of 96%, and the corre-
sponding yield of 0.3%.
Compound 1: white amorphous powder; HR-ESI-MS m/z [M-H]ꢁ ¼ 609.1505 (calcu-
1
lated [M-H]ꢁ for 609.1534); H NMR (DMSO-d6, 400 MHz): d 12.97 (s, 5-OH), 9.09 (brs,
3’-OH), 9.03 (brs, 4’-OH), 8.36 (s, H-2), 7.03 (d, J ¼ 1.9 Hz, 2’-H), 6.96 (s, 8-H), 6.80 (m, 5’-
H), 6.84 (m, 6’-H), 5.05 (d, J ¼ 7.2 Hz, Glc-1), 4.19 (d, J ¼ 7.4 Hz, Xyl-1), 3.94 (d, J ¼ 10 Hz,
Glc-6b), 3.78 (s, 6-OMe), 3.71 (m, Xyl-5b), 3.66 (m, Glc-6a), 3.63 (m, Glc-5), 3.35 (m, Glc-
2), 3.33 (m, Glc-3), 3.31 (m, Xyl-4), 3.21 (m, Glc-4), 3.09 (m, Xyl-3), 3.01 (m, Xyl-2), 2.98
(m, Xyl-5a); 13C NMR (DMSO-d6, 100 MHz) d 181.30 (C-4), 156.98 (C-7), 155.08 (C-2),
153.34 (C-5), 152.95 (C-9), 146.06 (C-4’), 145.42 (C-3’), 132.88 (C-6), 122.64 (C-3), 121.97
(C-1’), 120.45 (C-6’), 116.99 (C-2’), 115.89 (C-5’), 107.01 (C-10), 104.64 (Xyl-1), 100.65
(Glc-1), 94.67 (C-8), 77.10 (Glc-3), 77.00 (Xyl-3), 76.23 (Glc-5), 73.90 (Xyl-2), 73.54 (Glc-2),
70.24 (Glc-4), 69.93 (Xyl-4), 69.15 (Glc-6), 66.11 (Xyl-5), 60.79 (6-OMe). The spectra of
1D and 2D NMR, HR-ESI-MS(/MS) and HPLC profiles are given in supplemental materi-
als as Figure S1–S10.
3.4. Acid hydrolysis
Compound 1 (2 mg) was hydrolyzed with HCl (2 N, 5 mL) at 80 ꢃC for 3 h. The solvent
was removed by freeze-drying, after which 1 mL of pyridine and 2 mg of L-cysteine
methyl ester hydrochloride were added to the residue, and the mixture was heated at
60 ꢃC for 1 h. After cooling the solution to room temperature, O-tolyl isothiocyanate
(5 mL) was added to the mixture and heated at 60 ꢃC for an additional 1 h. The reac-
tion mixture was then analysed by HPLC and detected at 250 nm. Analytical HPLC was
performed on a HC-C18 reverse-phase column (250 ꢂ 4.6 mm, 5 lm, Agilent) at 25 ꢃC
using acetonitrile and H2O (containing 0.05% acetic acid) (25:75, 1.0 mL/min) as the
mobile phase. D-glucose and D-xylose were identified as the sugar units of compound
1 by comparison with authentic samples of D-glucose and D-xylose (Tanaka
et al. 2007).
3.5. Radical scavenging activity
The DPPH radical scavenging activity was performed by using previous methods with
some modifications (Ma et al. 2018). Briefly, compound 1 was dissolved in ultrapure
water with series of concentrations from 0 to 1.0 mg/mL. The resulting sample solu-
tions (100 lL) were mixed with freshly prepared DPPH methanolic solution (125 lL,
0.1 mM) followed by incubation for 30 min and absorbance reading at 517 nm.
Methanol was used as a blank control and vitamin C (Vc) served as a positive control;
the DPPH radical scavenging activity was calculated using the following formula:
DPPH radical scavenging activity ð%Þ ¼ ðA0 ꢁ A1 þ A2Þ=A0 ꢂ 100%

NATURAL PRODUCT RESEARCH
5
where, A0 is the absorbance of the DPPH solution without any sample, A1 and A2
being the absorbance of the solution with different concentrations of samples and
samples without DPPH solution, respectively.
4. Conclusion
A new isoflavone glycoside, 3’-hydroxytectorigenin-7-O-b-D-xylosyl-(1!6)-b-D-gluco-
pyranoside (1) was isolated from the flower of P. montana var. lobata through a com-
bined method consisting of zirconia adsorption and preparative HPLC. The structure of
compound 1 was elucidated by HR-ESI-MS, 1D and 2D NMR, and the radical scaveng-
ing activity was also evaluated. The new isoflavone showed moderate radical scaveng-
ing activity against DPPH radical.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the Fundamental Research Funds for the Central
Universities [grant number: JZ2019HGTB0061, JZ2016HGTA0680], the Major Projects of Science
and Technology in Anhui Province [grant numbers: 18030701144, 18030701142, 17030701058,
17030701024 and 17030701028], and Natural Science Foundation of Anhui Province [grant num-
ber 1608085QH178].
ORCID
Yilong Ma
Zhaojun Wei
http://orcid.org/0000-0001-5912-3943
http://orcid.org/0000-0003-1729-209X
References
Bai X, Qu J, Lu J, Kano Y, Yuan D. 2011. Pharmacokinetics of kakkalide and its main metabolites
in rat plasma determined by HPLC-DAD and LC-MSn. J Chromatogr B Analyt Technol Biomed
Life Sci. 879(5–6):395–402.
Cui T, Tang S, Liu C, Li Z, Zhu Q, You J, Si X, Zhang F, He P, Liu Z, et al. 2018. Three new isofla-
vones from the Pueraria montana var. lobata (Willd.) and their bioactivities. Nat Prod Res.
32(23):2817–2824.
Fu J, Jing W, Wang W, Chen S, Zhang J, Liu A. 2015. A novel and effective chromatographic
approach to the separation of isoflavone derivatives from Pueraria lobata. Molecules. 20(3):
4238–4253.
Huang Z, Yang J, She Z, Lin Y. 2012. A new isoflavone from the mangrove endophytic fungus
Fusarium sp. (ZZF60). Nat Prod Res. 26(1):11–15.
Lee H, Choo M, Bae E, Kim D. 2003. b-Glucuronidase inhibitor tectorigenin isolated from the
flower of Pueraria thunbergiana protects carbon tetrachloride-induced liver injury. Liver Int.
23(4):221–226.
Lee H, Kim B, Ahn J. 2014. Production of bioactive hydroxyflavones by using monooxygenase
from Saccharothrix espanaensis. J Biotechnol. 176:11–17.

6
Y. MA ET AL.
Li S, Li S, Liu C, Liu C, Zhang Y. 2017. Extraction and isolation of potential anti-stroke com-
pounds from flowers of Pueraria lobata guided by in vitro PC12 cell model. J Chromatogr B.
1048:111–120.
Ma Y, Zhu D, Thakur K, Wang C, Wang H, Ren Y, Zhang J, Wei Z. 2018. Antioxidant and antibac-
terial evaluation of polysaccharides sequentially extracted from onion (Allium cepa L.). Int J
Biol Macromol. 111:92–101.
Ma Y, Shang Y, Liu F, Zhang W, Wang C, Zhu D. 2018. Convenient isolation of strictinin-rich tea
polyphenol from Chinese green tea extract by zirconium phosphate. J Food Drug Anal. 26(1):
100–106.
Min S, Park Y, Kim D. 2011. Kakkalide and its metabolite irisolidone ameliorate carrageenan-
induced inflammation in mice by inhibiting NF-jB pathway. Inflammation. 34(5):344–351.
Niiho Y, Nakajima Y, Yamazaki T, Okamoto M, Tsuchihashi R, Kodera M, Kinjo J, Nohara T. 2010.
Simultaneous analysis of isoflavones and saponins in Pueraria flowers using HPLC coupled to
an evaporative light scattering detector and isolation of a new isoflavone diglucoside. J Nat
Med. 64(3):313–320.
Park E, Shin Y, Lee H, Lee C, Kim D. 2004. Passive cutaneous anaphylaxis-inhibitory action of tec-
torigenin, a metabolite of tectoridin by intestinal microflora. Biol Pharm Bull. 27(7):1099–1102.
Park H, Park J, Moon J, Lee K, Jung W, Oh S, Lee H. 1999. Isoflavone glycosides from the flowers
of Pueraria thunbergiana. Phytochemistry. 51(1):147–151.
Shahana M, ADA, Varahram S, Hiroshi H, Ardalan A, Sharanjot S, Yuichiro T, VD, Rajvir D. 2010.
Genistein reverses hypermethylation and induces active histone modifications in tumor sup-
pressor gene B-Cell translocation gene 3 in prostate cancer. Cancer. 116:66–76.
Shi S, Ma Y, Zhang Y, Liu L, Liu Q, Peng M, Xiong X. 2012. Systematic separation and purification
of 18 antioxidants from Pueraria lobata flower using HSCCC target-guided by DPPH–HPLC
experiment. Sep Purif Technol. 89:225–233.
Tanaka T, Nakashima T, Ueda T, Tomii K, Kouno I. 2007. Facile Discrimination of Aldose
Enantiomers by Reversed-Phase HPLC. Chem Pharm Bull. 55(6):899–901.
Xia D, Zhang P, Fu Y, Yu W, Ju M. 2013. Hepatoprotective activity of puerarin against carbon
tetrachloride-induced injuries in rats: A randomized controlled trial. Food Chem Toxicol. 59:
90–95.
Xing G, Dong M, Li X, Zou Y, Fan L, Wang X, Cai D, Li C, Zhou L, Liu J, Niu Y. 2011.
Neuroprotective effects of puerarin against beta-amyloid-induced neurotoxicity in PC12 cells
via a PI3K-dependent signaling pathway. Brain Res Bull. 85(3–4):212–218.
Xiong Y, Yang Y, Yang J, Chai H, Li Y, Yang J, Jia Z, Wang Z. 2010. Tectoridin, an isoflavone
glycoside from the flower of Pueraria lobata, prevents acute ethanol-induced liver steatosis in
mice. Toxicology. 276(1):64–72.
Yao M, Liao Y, Li G, Law F, Tang Y. 2010. Quantitative analysis of two isoflavones in Pueraria
lobata flowers from eleven Chinese provinces using high performance liquid chromatography.
Chin Med. 5(1):14–17.
Yu D, Ying C, Liang C, Ching L. 2004. Flavonoids from the Flowers of Pueraria lobata. J Chin
Chem Soc. 51:1425–1428.
Yu Y, Liao Y, Li X, Ye Y, Ke C, Li X, Yang X, Yao M. 2011. Isoflavonoid glycosides from the flowers
of Pueraria lobata. J Asian Nat Prod Res. 13(4):284–289.
Zhang Y, Shi S, Xiong X, Chen X, Peng M. 2012. Comparative evaluation of three methods based
on high-performance liquid chromatography analysis combined with a 2,2’-diphenyl-1-picryl-
hydrazyl assay for the rapid screening of antioxidants from Pueraria lobata flowers. Anal
Bioanal Chem. 402(9):2965–2976.