Caragiside D, a new isoflavone glucoside from caragana conferta

Article abstract of DOI:10.1007/s10600-014-0981-1

Caragiside D (1), a new isoflavone glucoside, has been isolated from the n-BuOH soluble subfraction of the MeOH soluble extract of the whole plant of Caragana conferta along with one known isoflavone glucoside, caragiside A (2), and two lignan glucosides, pinoresinol 4-O-glucoside (3) and syringaresinol 4-O-glucoside (4). The structures of these compounds were elucidated through spectroscopic techniques including MS and 2D NMR. The purity of compound 1 was confirmed by HPLC.

Full text of DOI:10.1007/s10600-014-0981-1

Chemistry of Natural Compounds, Vol. 50, No. 3, July, 2014 [Russian original No. 3, May–June, 2014]
CARAGISIDE D, A NEW ISOFLAVONE GLUCOSIDE
FROM Caragana conferta
1*
1
Shagufta Perveen, Areej Mohammad Al-Taweel,
2
1,3
4
Afsar Khan, Ghada Ahmed Fawzy, and Abdul Malik
Caragiside D (1), a new isoflavone glucoside, has been isolated from the n-BuOH soluble subfraction of the
MeOH soluble extract of the whole plant of Caragana conferta along with one known isoflavone glucoside,
caragiside A (2), and two lignan glucosides, pinoresinol 4-O-glucoside (3) and syringaresinol 4-O-glucoside
(4). The structures of these compounds were elucidated through spectroscopic techniques including MS and
2D NMR. The purity of compound 1 was confirmed by HPLC.
Keywords: Caragana conferta, caragiside D, isoflavone glucoside.
The genus Caragana comprises over 80 species, of which 10 species have so far been identified in Pakistan [1]. Some
of these species are used for the treatment of fever, hypertension, irregular menstruation, and fatigue [2, 3]. Caragana conferta
Benth. ex Bake (Fabaceae) is a shrub that grows in Asia, Africa, and Southeast Europe. In Pakistan it is mainly found in the
Gilgit and Kashmir Valleys [1]. The whole plant is used as folk medicine in China and Korea for the treatment of neuralgia,
rheumatism, arthritis, and hypertension [4]. Literature survey revealed that many compounds have so far been reported from
this species [5–10]. The ethnopharmacological and chemotaxanomic importance of this species prompted us to carry out
further studies on C. conferta. Isolation studies on the n-BuOH-soluble subfraction resulted in one new isoflavone glucoside,
named caragiside D (1), along with one known isoflavone glucoside named caragiside A (2) [5] and two lignan glucosides,
pinoresinol 4-O-glucoside (3) [11] and syringaresinol 4-O-glucoside (4) [12]. The MeOH extract of the aerial parts of
C. conferta was divided into subfractions soluble in n-hexane, CHCl , EtOAc, and n-BuOH. A part of the n-BuOH soluble
3
subfraction was subjected to a series of column chromatographic techniques to afford compounds 1–4. Their structures were
established through spectroscopic techniques.
25
Caragiside D (1) was obtained as a yellow gummy solid, [ꢀ] +29ꢁ (c 0.20 in CH OH). It gave a violet coloration
D
3
–
with FeCl for a phenol. The negative mode electron spray ionization mass spectrum (ESI-MS) gave a quasi-molecular [M – H]
3
–
peak at m/z 461, while the HR-FAB-MS showed the [M – H] peak at m/z 461.1014, corresponding to the molecular formula
C H O The negative mode FAB-MS further shows a peak at m/z 299 resulting from the loss of a hexose moiety. The
22 22 11.
1
H NMR spectrum (Table 1) exhibited two singlets at ꢂ 8.26 (1H, s, H-5) and 7.29 (1H, s, H-8), which were consistent with a
6,7-dioxygenated A ring of isoflavone. The protons of the 1,3,4-trisubstituted ring B were observed at 7.92 (1H, d, J = 1.8 Hz,
1
H-2ꢃ), 7.23 (1H, d, J = 8.2 Hz, H-5ꢃ), and 7.51 (1H, dd, J = 8.2, 1.8 Hz, H-6ꢃ). The H NMR spectrum gave the downfield singlet
of an olefinic proton at ꢂ 8.21. The signal of the methoxyl group appeared at 3.79 (3H, s). It also gave the signal for an anomeric
proton at 4.89 (1H, d, J = 7.6 Hz, H-1ꢃꢃ), suggesting the ꢄ-configuration of the hexose unit. Further signals of the hexose moiety
were observed between 3.47 and 3.97. Acid hydrolysis of 1 provided the sugar, which could be identified as D-glucose
through co-TLC and the sign of its optical rotation. On the basis of these resonances, 1 was deduced to be an isoflavone glucoside.
1) Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, P. O. Box 2457, Riyadh
11451, Saudi Arabia, e-mail: shagufta792000@yahoo.com, shakhan@ksu.edu.sa; 2) Department of Chemistry, COMSATS
Institute of Information Technology, Abbottabad-22060, Pakistan; 3) Department of Pharmacognosy, Faculty of Pharmacy,
Cairo University, 11562, Cairo, Egypt; 4) International Center for Chemical and Biological Sciences, H.E.J. Research Institute
of Chemistry, University of Karachi, 75270, Karachi, Pakistan. Published in Khimiya Prirodnykh Soedinenii, No. 3, May–June,
2014, pp. 380–382. Original article submitted January 1, 2013.
440
0009-3130/14/5003-0440 02014 Springer Science+Business Media New York

1
13
TABLE 1. H NMR and C NMR Data of Caragiside D (1) (DMSO-d , ꢂ, ppm, J/Hz)
6
C atom
C atom
ꢂH
ꢂC
ꢂH
ꢂC
2
3
4
5
6
7
8
9
10
8.21 (s)
152.6
124.0
175.3
109.8
146.3
154.4
104.0
152.4
118.2
126.3
119.1
149.7
–
148.0
113.6
121.8
102.4
75.6
77.4
70.8
4
5
6
ꢃ
ꢃ
ꢃ
–
–
7.23 (d, J = 8.2)
7.51 (dd, J = 8.2, 1.8)
4.89 (d, J = 7.6)
3.53 (m)
8.26 (s)
1
2
3
4
5
6
ꢃꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
–
–
3.49 (m)
3.47 (m)
4.56 (m)
7.29 (s)
–
–
–
76.9
62.1
3.97 (dd, J = 2.5, 11.2)
3.73 (dd, J = 4.9, 11.2)
3.79 (s)
1
2
3
ꢃ
ꢃ
ꢃ
7.92 (d, J = 1.8)
–
56.6
3 -OMe
ꢃ
H
H
1
O
7
HO
H
9
H
3
OCH
3'
3
10
HO
5
1'
O
6''
OH
O
H
O
5'
1''
5''
3''
OH
OH
H
HO
H
1
Fig. 1. Important HMBC correlations of caragiside D (1).
13
The C NMR spectrum of 1 (Table 1) showed 22 signals comprising one methyl, one methylene, eleven methines, and nine
quaternary carbons. The signals at ꢂ 152.6, 124.0 175.3, 152.4, and 118.2 were typical of C-2, C-3, C-4, C-9, and C-10 of an
isoflavone moiety. Apart from further peaks of the aromatic carbons, the spectrum showed the signal of one anomeric
carbon at ꢂ 102.4, oxymethine carbons ranging between ꢂ 70.8–77.4, and an oxymethylene carbon at ꢂ 62.1. The point
of attachment of the sugar moiety was shown to be C-4ꢃ through HMBC correlations, in which the anomeric proton (ꢂ 4.89)
3
showed a J correlation to C-4ꢃ (ꢂ 148.0) (Fig. 1). The position of the methoxy group on ring A was confirmed by HMBC
3
experiment, in which the OCH proton (ꢂ 3.79) showed a J correlation to C-3ꢃ (ꢂ 149.7). All these data were in complete
3
agreement with the assigned structure of caragiside D (1) as 6,7-dihydroxy-3ꢃ-methoxy-4ꢃ-O-(ꢄ-D-glucopyranosyl) isoflavone.
EXPERIMENTAL
General Experimental Procedures. Optical activity was measured on a JASCO P-2000 Series polarimeter (JASCO
1
13
Corporation, 2967-5, Tokyo, Japan). The IR spectra were recorded on a Shimadzu 460 spectrometer. The H, C NMR, and
2D NMR spectra were recorded on a Bruker AMX-500 spectrometer with tetramethylsilane (TMS) as an internal standard.
Chemical shifts are in ppm (ꢂ) relative to tetramethylsilane, and coupling constants (J) are reported in Hertz. ESI-MS were
recorded on an Agilent Triple Quadrupole 6410 QQQ LC/MS mass spectrometer with ESI ion source (gas temperature 350ꢁC,
nebulizer pressure 60 psi and gas flow rate 12 L/min), operating in the negative and positive scan modes of ionization through
the direct infusion method using CH OH–H O (1:1 v/v) at a flow rate of 0.4 mL/min. FAB and HR-FAB-MS (negative ion
3
2
mode, matrix: glycerol) were performed on a JEOL JMS-HX110 mass spectrometer. Column chromatography was carried out
on Sephadex LH-20 (Merck, Darmstadt, Germany). Thin layer chromatography (TLC) was performed on precoated TLC
plates (aluminum sheets, RP-18 F , Merck, Germany); detection was done at 254 nm and by spraying with ceric sulfate
254
reagent. UFLC analysis was performed on a Prominence Shimadzu LC Solution (Kyoto, Japan); the system was equipped with
a CBM-20A communications bus module, two LC-10AD pumps, a DGU-20A3 vacuum degasser, a CTO-10A(C) column
oven, and an SPD 10A(V) diode array detector. A Shim-pack VP-ODS (150 mm ꢅ 4.6 mm, 5.0 m, Shimadzu) analytical
column and a preparative Shim-pack VP-ODS (20 ꢅ 250 mm, 5 m, Shimadzu) column were used and kept at 40ꢁC. The
441

mobile phase consisted of water containing 0.1% triflouroacetic acid (A) and CH OH (B). The flow rate was set at 0.4 mL/min,
3
and the injection volume was 50 L for the analytical column at 4.0 mL/min flow rate; the injection volume was 200 L for the
preparative column. The DAD detection was achieved in the range of 254 nm.
Plant Material. The whole plant of Caragana conferta Benth. ex Bake (7.0 kg) was collected from Gilgit (Pakistan)
and identified by the senior scientist of the National Agriculture Research Center, Islamabad, Pakistan. A voucher specimen
has been deposited in the Herbarium of the Department of Botany, University of Karachi (voucher No. 319).
Extraction and Isolation. The shade-dried plant material (7.0 kg) was extracted with methanol (25 ꢅ 3 L) at room
temperature. The combined methanolic extract was evaporated under reduced pressure to obtain a thick gummy mass (350 g).
It was suspended in water and successively extracted with n-hexane (120 g), chloroform (70 g), ethyl acetate (50 g), and
n-butanol (60 g).Apart of the n-BuOH soluble fraction (50 g) was dissolved in water and loaded on a Sephadex LH-20 column
(flow rate 100 mL/h, 12 ꢅ 70 cm), and elution was successively carried out with water and mixtures of H O and MeOH in
2
decreasing order of polarity, leading to two major subfractions I–II. Subfraction I (5.0 g) obtained from H O–MeOH (6:4) was
2
a binary mixture, which was rechromatographed over Sephadex LH-20 (flow rate 40 mL/h, 8 ꢅ 70 cm) using H O–MeOH
2
(1:1) as eluent to afford syringaresinol 4-O-glucoside (4, 10 mg) and pinoresinol 4-O-glucoside (3, 12 mg). Subfraction II
(4.0 g) eluted with H O–MeOH (1:1) showed two major spots on TLC and was subjected to HPLC with flow rate 1.0 mL/min
2
using H O–MeOH (4:6) to afford caragiside A (2, 15 mg) and caragiside D (1, 13 mg).
2
25
–1
Caragiside D (1). Yellow gummy solid, [ꢀ] +29ꢁ (c 0.20, MeOH). IR (KBr, ꢆ, cm ): 3445, 2950, 1660, 1610,
D
1
13
1590, 462, 1120. H and C NMR data, see Table 1. ESI-MS (–ve ion mode) m/z 461; FAB-MS (–ve ion mode) m/z: 461, 299;
–
HR-FAB-MS (–ve ion mode) m/z 461.1014 ꢇM – Hꢈ (calcd for C H O ).
22 21 11
Acid Hydrolysis of Caragiside D (1). Caragiside D (1 mg) was dissolved in 0.5 mL of a solution of 1 N
HCl–methanol (1:1). The mixture was refluxed at 70ꢁC for 85 min. The reaction mixture was concentrated in vacuo, water was
added and the whole was extracted with ethyl acetate. The aqueous portion was filtered, the filtrate was evaporated, and
25
D-glucose was identified from the sign of its optical rotation ([ꢀ] +52.0ꢁ) and co-TLC (ethyl acetate–methanol–water,
D
10:4:1, R 0.12) with an authentic sample of D-glucose (Merck).
f
HPLC Analysis for the Purity of Compound 1. Caragiside D (1.0 mg) was dissolved in 3 mL Millipore water
(HPLC grade), and the solution was filtered through 0.45 m Millipore filter. Compound 1 was detected at Rt 13.195 min
using methanol–water (1:1) at 0.4 mL/min flow rate.
ACKNOWLEDGMENT
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud
University for its funding of this research through the Research Group Project No. RGP-VPP-221".
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