Two New Flavonol Glycosides from Polygala sibirica L. var megalopha Fr.

Abstract

Two new flavonol glycosides, named polygalin H (1) and polygalin I (2), as well as the known compound polygalin D (3), were isolated from the whole plant of Polygala sibirica L. var megalopha Fr. Their structures were elucidated on the basis of spectroscopic data analysis. These flavonol glycosides exhibited strong inhibitory activities against xanthine oxidase in vitro. Their half-maximal inhibitory concentrations (IC₅₀) were calculated, which were 9.48, 8.31, 16.00 μM, respectively.

1. Introduction

Polygala sibirica L. var megalopha Fr. (Polygalaceae), a native medicinal plant of Yunnan Province (China), has long been used in folk medicine for the treatment of fever, inflammation, arthralgia and viper bites [1,2,3,4]. So far, it was reported that polysaccharides and some novel flavonol and xanthone derivatives had been isolated from this plant [5,6], and some reports on high inhibitory activities against xanthine oxidase by these flavonoids and xanthone derivatives were revealed recently [7,8,9]. All of this attracted us to further investigate its chemical constituents and seek more compounds as potential xanthine oxidase inhibitors. In the present work, two new compounds, named as polygalin H (1) and polygalin I (2), and one known compound polygalin D (3) [10,11], were isolated. In this paper, we describe the isolation, structural elucidation of the two new compounds, as well as the inhibition of xanthine oxidase by the three compounds.

2. Results and Discussion

Compound 1 was obtained as a yellow amorphous powder. Its molecular formula was determined to be C₃₀H₃₆O₁₇ by MALDI-TOF/TOF-MS (m/z 691.1077, calcd. for 691.1640, [M + Na]⁺). The compound exhibited IR absorption bands at 3440 and 1613 cm⁻¹ and UV maximum absorptions (209, 258, 273 and 343 nm), characteristic of a flavonoid. Besides the eleven sugar signals in the ¹³C-NMR spectrum, the remaining peaks corresponded to signals of three methoxyl [δ_C 57.3 (q), 56.5 (q), 55.6 (q)], one oxygenated methylene (δ_C 60.8, t), four methines and eleven quaternary carbons. In contrast to polygalin C [11], compound 1 has one additional methoxy signal and another oxygenated methylene signal. The oxygenated methylene moiety was deduced to be linked at C-6 by the HMBC correlations between the oxygenated methylene protons H-11 (δ_H 4.39, s, 2H) and C-6 (δ_C 108.0), C-5 (δ_C 159.3), C-7 (δ_C 163.9), which was further confirmed by the evidence that the chemical shift of C-6 (δ_C 108.0) in compound 1 moved 10.2 ppm downfield compared with polygalin C. Simultaneously, in the HMBC spectrum, the long-range correlations between the three methoxyl (δ_H 3.21, 3.91, 3.87) and their corresponding carbons C-5 (δ_C 159.3), C-7 (δ_C 163.9), C-4′ (δ_C 150.2), indicated the positions of the methoxy moieties. Therefore, the additional methoxy moiety compared with polygalin C was determined to be linked at C-5. These assignments were further confirmed by the ROSEY spectrum (see Figure 1).

Figure 1. Key COSY, HMBC and ROESY correlations of polygalin H (1) and polygalin I (2).

Figure 1. Key COSY, HMBC and ROESY correlations of polygalin H (1) and polygalin I (2).

Acid hydrolysis of 1 with 1 M HCl afforded two monosaccharides, which were determined to be d-galactopyranose and d-apiofuranose by TLC and GC analysis. In the HMBC spectrum, the long-range correlations indicated that H-1″ (δ_H 5.62, d, J = 7.7 Hz) of the galactose was attached to C-3 (δ_C 133.9) of the aglycone and the H-1‴ (δ_H 5.31, s) of the apiose was linked at C-2″ (δ_C 74.9) of the galactose. In addition, the coupling constant (J = 7.7 Hz) of the anomeric proton in galactose revealed its β-orientation, while the apiose was also deduced to be in an β-orientation by comparing its ¹³C-NMR spectral data (δ_C 108.8, 76.1, 79.2, 73.9, 64.2) with literature data [12,13]. Thus, compound 1 was elucidated as 3,3′-dihydroxy-6-hydroxymethyl-5,7,4′-trimethoxyflavone-3-O-β-d-apiofuranosyl-(1→2)-β-d-galactopyranoside (see Figure 1), and was named polygalin H.

Compound 2 was also obtained as a yellow amorphous powder. The MALDI-TOF/TOF-MS provided a molecular formula of C₃₀H₃₆O₁₆ at m/z 675.1942 [M + Na]⁺ (calcd. for 675.1901). The compound showed IR absorption bands at 3441 and 1609 cm⁻¹ and UV maximum absorptions (209, 258, 273 and 343 nm). Comparison of the ¹³C-NMR and ¹H-NMR spectral data of compound 2 with those of compound 1, showed that the two compounds had similar structures except for one additional proton signal (δ_H 7.03, H-3′) in compound 1. Moreover, the protons of H-2′, and H-6′ (δ_H 8.27, d, J = 8.5 Hz), H-3′ and H-5′ (δ_H 7.03, d, J = 8.5 Hz) in compound 2 exhibited a set of AA′BB′-system signals, which indicated that the hydroxyl moiety at C-3′ in compound 1 was replaced by a hydrogen (δ_H 7.03, d, J = 8.5 Hz) in compound 2. Thus, the compound 2 was elucidated to be 3-hydroxy-6-hydroxymethyl-5,7,4′-trimethoxyflavone-3-O-β-d-apiofuranosyl-(1→2)-β-d-galactopyranoside (see Figure 1), and named polygalin I.

It is noteworthy that the hydroxymethyl function in the skeletons of these flavonoids is reported for the first time. Based on the furocoumarin biosynthetic pathway [14], we put forward a hypothesis that polygalin H (1) is derived from polygalin C, which we also had obtained from P. Sibirica L. Var megalopha Fr. previously. In our hypothesis (see Figure 2), we speculate that compounds K and M were unstable, as they were never found among flavones [15,16,17], and the ultimate product, polygalin H, would be obtained through methylation after L was transformed into N. In our following work, the biosynthetic pathway, the chemical properties and bioactivities of flavonoids with this substitution pattern will be further investigated.

Figure 2. Hypothetical biosynthesis of polygalin H (1).

Figure 2. Hypothetical biosynthesis of polygalin H (1).

Four concentrations (2.5, 5.0, 10.0, 20.0 μM) of the flavonol glycosides were analyzed for inhibition of xanthine oxidase, and then the corresponding half-maximal inhibitory concentration (IC₅₀) value was calculated (see Table 1). The results showed that their IC₅₀s were 9.48, 8.31, 16.00 μM, respectively, which were a little higher than the IC₅₀ value (4.34 μM) of the positive control, allopurinol. Consequently, this indicated that those three flavonol glycosides had strong inhibitory activities against xanthine oxidase.

Table 1. IC₅₀ values of three flavonol glycosides for inhibition of xantine oxidase.

Compound	Final Concentration (μM)	Inhibition (%)	IC50 (μM)
1	20.0	76.87 ± 2.0	9.48
	10.0	43.61 ± 2.7
	5.0	30.71 ± 0.8
	2.5	15.53 ± 3.8
2	20.0	80.87 ± 4.6	8.31
	10.0	46.35 ± 6.6
	5.0	33.73 ± 6.1
	2.5	18.45 ± 5.4
3	20.0	58.79 ± 1.6	16.00
	10.0	33.81 ± 2.7
	5.0	23.74 ± 1.2
	2.5	14.12 ± 0.6
Allopurinol (positive control)	20.0	96.87 ± 1.2	4.34
	10.0	70.61 ± 4.0
	5.0	45.71 ± 0.8
	2.5	37.53 ± 3.8

Table 1. IC₅₀ values of three flavonol glycosides for inhibition of xantine oxidase.

Compound	Final Concentration (μM)	Inhibition (%)	IC50 (μM)
1	20.0	76.87 ± 2.0	9.48
	10.0	43.61 ± 2.7
	5.0	30.71 ± 0.8
	2.5	15.53 ± 3.8
2	20.0	80.87 ± 4.6	8.31
	10.0	46.35 ± 6.6
	5.0	33.73 ± 6.1
	2.5	18.45 ± 5.4
3	20.0	58.79 ± 1.6	16.00
	10.0	33.81 ± 2.7
	5.0	23.74 ± 1.2
	2.5	14.12 ± 0.6
Allopurinol (positive control)	20.0	96.87 ± 1.2	4.34
	10.0	70.61 ± 4.0
	5.0	45.71 ± 0.8
	2.5	37.53 ± 3.8

3. Experimental Section

3.1. General Information

Optical rotations were recorded using a P-1020 digital polarimeter (Jasco, Tokyo, Japan). The UV spectra were measured on a UV-2401PC spectrophotometer (Shimadzu, Suzhou, China). The IR spectra were recorded as KBr pellets on a Tensor-27 spectrometer (Bruker, Bremen, Germany). The NMR spectra were recorded on an AM-400 spectrometer (Bruker) with TMS as an internal standard. ESI-MS and HR-EI-M-TOF-MS were measured on a Bruker HTC/Esquire spectrometer and a Bruker Daltonics Flex spectrometer, respectively. GC analysis was carried out on an HP-5890 II system (Gentech Scientific, Alto, CA, USA) equipped with a FID detector and a HP-20M capillary column (25 m × 0.32 mm × 0.3 μm). Column chromatography was performed with silica gel (Qingdao Marine Chemical Industry Factory, Qingdao, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Fairfield, CT, USA) and reversed-phase C18 silica gel (40–60 μm, Merck, Darmstadt, Germany). TLC was performed with silica gel GF254 (Qingdao Marine Chemical Industry Factory). Fractions were monitored by TLC and spots were visualized by heating after spraying with 5% H₂SO₄ in ethanol.

3.2. Plant Material

The whole plant of P. Sibirica L. Var megalopha Fr. were collected in Yongshan, Yunnan Province, People’s Republic of China. A voucher specimen (HUA002) was identified by Prof. Fan Du (Southwest Forestry University) and was deposited at the College of Forestry, Southwest Forestry University.

3.3. Extraction, Isolation and Characterization

The 75% ethanol extract of the whole plant of Polygala sibirica L. var megalopha Fr. (5.0 kg), was chromatographed over a D₁₀₁ macroporous resin, eluted successively with a gradient of H₂O, 35% EtOH, 65% EtOH, 95% EtOH,. The 65% EtOH eluate was subjected to silica gel column chromatography and eluted with CHCl₃–MeOH (50:1→0:1) to afford thirteen fractions (Fr.1→Fr.13). Fr.8 (56 g) was chromatographed on a silica gel column and RP-C18 to yield eleven fractions (Fr.8-1→Fr.8-11). Fr.8-7 (35 mg) was purified by Sephadex LH-20 with CHCl₃–MeOH (1:1) and semi-preparative HPLC with a mixture of acetonitrile and water (34:66, v/v) to give compound 1 (20 mg). Fr.8-5 (4.654 g) was repeatedly subjected to silica gel column and Sephadex LH-20 chromatography to yield five fractions (Fr.8-5A→Fr.8-5E). Fr.8-5C (40.8 mg) was purified by semi-preparative HPLC with a mixture of acetonitrile and water (29:71, v/v) to afford 2 (4.6 mg) and 3 (4.3 mg).

Polygalin H (1). Yellow amorphous power; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ −89(c 0.0011, MeOH); UV (CH₃OH) λ_max (log ε): 343 (0.17), 273 (0.19), 258 (0.19), 209 (0.45); IR (KBr) ν_max: 3440, 2926, 1613, 1512, 1484, 1354, 1263, 1213, 1142, 1078, 1024 cm⁻¹; for ¹H- and ¹³C-NMR, see Table 2; MALDI-TOF/TOF-MS: m/z 691.1077 [M + Na]⁺ (calcd. for 691.1640, C₃₀H₃₆O₁₇Na⁺).

Polygalin I (2). Yellow amorphous power; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ −89 (c 0.0011, MeOH); UV (CH₃OH) λ_max (log ε): 343 (0.17), 273 (0.19), 258 (0.19), 209 (0.45); IR (KBr) ν_max: 3441, 2925, 1609, 1510, 1484, 1351, 1258, 1212, 1180, 1077, 1024 cm⁻¹; for ¹H- and ¹³C-NMR, see Table 2; MALDI-TOF/TOF-MS: m/z 675.1942 [M + Na]⁺ (calcd. for 675.1901, C₃₀H₃₆O₁₆Na⁺).

Polygalin D (3). Yellow needles (MeOH); m.p. 251–253 °C; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ −47 (c 0.51, MeOH); UV (CH₃OH) λ_max (log ε) nm: 268 (4.12), 355 (3.98); IR (KBr) cm⁻¹: 3410, 2943, 1657, 1601, 1497, 1353, 1218 and 1025. HRESIMS (positive) m/z: 647.1587 (calcd. for C₂₈H₃₂O₁₆, 647.1588]; ¹H-NMR (DMSO-d₆, 600 MHz) δ_H: 7.82 (1H, dd, J = 9.0, 2.0 Hz, H-6′), 7.59 (1H, d, J = 2.0 Hz, H-2′), 7.03 (1H, d, J = 9.0 Hz, H-5′), 6.72 (1H, d, J = 2.5 Hz, H-8), 6.38 (1H, d, J = 2.5 Hz, H-6), 3.88 (3H, s, 4′-OMe), 3.87 (3H, s, 7-OMe), 5.63 (1H, d, J = 8.0 Hz ,Glc-H-1); ¹³C-NMR (DMSO-d₆, 150 MHz) δ: 177.8 (s, C-4), 165.1 (s, C-7), 161.0 (s,C-5), 156.2 (s, C-9), 155.7 (s, C-2), 150.1 (s, C-4′), 145.0 (s, C-3′), 133.7 (s, C-3), 122.5 (s, C-6′), 121.8 (s, C-1′), 115.5 (s, C-2′), 111.1 (s, C-5′), 108.6 (d, Api-C-1), 105.0 (s, C-10), 98.5 (d, Glc-C-1), 97.8 (s, C-6), 92.1 (s, C-8), 79.2 (s, Api-C-3), 77.5 (d, Glc-C-2), 77.0 (d, Glc-C-3, 5), 76.1 (d, Api-C-2), 73.9 (t, Api-C-4), 70.2 (d, Glc-C-4), 64.2 (t, Api-C-5), 60.8 (t, Glc-C-6), 56.1 (q, 7-OMe), 55.6 (q, 4′-OMe).

Table 2. The ¹H-NMR and ¹³C-NMR data of 1, 2 in DMSO-d₆ (600 MHz).

No.	1		2
	δH, J (Hz)	δC	δH, J (Hz)	δC
2		155.6 s		155.4 s
3		133.9 s		133.8 s
4		177.7 s		177.7 s
5		159.3 s		159.3 s
6		108.0 s		108.0 s
7		163.9 s		163.9 s
8	6.81 (s)	90.3 d	6.86 (s)	90.4 d
9		156.1 s		156.1 s
10		104.5 s		104.6 s
11	4.39 (s)	60.8 t	4.39 (s)	60.8 t
1′		122.5 s		122.4 s
2′	7.58 (s)	115.3 d	8.27 (d, 8.5)	130.9 d
3′		146.1 s	7.03 (d, 8.5)	113.7 d
4′		150.2 s		161.3 s
5′	6.97 (d, 8.7)	111.2 d	7.03 (d, 8.5)	113.7 d
6′	7.94 (d, 8.7)	122.2 d	8.27 (d, 8.5)	130.9 d
5-OMe	3.21 (s)	57.3 q	3.22 (s)	57.3 q
7-OMe	3.91 (s)	56.5 q	3.91 (s)	56.5 q
4′-OMe	3.87 (s)	55.6 q	3.85 (s)	55.4 q
Gal-1″	5.62 (d, 7.7)	98.9 d	5.61 (d, 7.6)	98.9 d
2″	3.76 (m)	74.9 d	3.76 (m)	74.9 d
3″	3.57 (m)	73.8 d	3.83 (m)	73.7 d
4″	3.63 (s)	68.3 d	3.63 (m)	68.2 d
5″	3.33 (m)	75.8 d	3.34 (m)	75.7 d
6″	3.42 (m)	60.0 t	3.41 (m)	60.0 t
	3.27 (m)		3.25 (m)
Api-1‴	5.31 (s)	108.8 d	5.31 (s)	108.8 d
2‴	3.80 (m)	76.1d	3.81 (m)	76.1 d
3‴		79.2 s		79.2 s
4‴	3.83 (d, 9.2)	73.9 t	3.84 (m)	73.8 t
	3.49 (d, 9.0)		3.57 (m)
5‴	3.42 (m)	64.2 t	3.41 (m)	64.2 t
	3.37 (m)		3.34 (m)

Table 2. The ¹H-NMR and ¹³C-NMR data of 1, 2 in DMSO-d₆ (600 MHz).

No.	1		2
	δH, J (Hz)	δC	δH, J (Hz)	δC
2		155.6 s		155.4 s
3		133.9 s		133.8 s
4		177.7 s		177.7 s
5		159.3 s		159.3 s
6		108.0 s		108.0 s
7		163.9 s		163.9 s
8	6.81 (s)	90.3 d	6.86 (s)	90.4 d
9		156.1 s		156.1 s
10		104.5 s		104.6 s
11	4.39 (s)	60.8 t	4.39 (s)	60.8 t
1′		122.5 s		122.4 s
2′	7.58 (s)	115.3 d	8.27 (d, 8.5)	130.9 d
3′		146.1 s	7.03 (d, 8.5)	113.7 d
4′		150.2 s		161.3 s
5′	6.97 (d, 8.7)	111.2 d	7.03 (d, 8.5)	113.7 d
6′	7.94 (d, 8.7)	122.2 d	8.27 (d, 8.5)	130.9 d
5-OMe	3.21 (s)	57.3 q	3.22 (s)	57.3 q
7-OMe	3.91 (s)	56.5 q	3.91 (s)	56.5 q
4′-OMe	3.87 (s)	55.6 q	3.85 (s)	55.4 q
Gal-1″	5.62 (d, 7.7)	98.9 d	5.61 (d, 7.6)	98.9 d
2″	3.76 (m)	74.9 d	3.76 (m)	74.9 d
3″	3.57 (m)	73.8 d	3.83 (m)	73.7 d
4″	3.63 (s)	68.3 d	3.63 (m)	68.2 d
5″	3.33 (m)	75.8 d	3.34 (m)	75.7 d
6″	3.42 (m)	60.0 t	3.41 (m)	60.0 t
	3.27 (m)		3.25 (m)
Api-1‴	5.31 (s)	108.8 d	5.31 (s)	108.8 d
2‴	3.80 (m)	76.1d	3.81 (m)	76.1 d
3‴		79.2 s		79.2 s
4‴	3.83 (d, 9.2)	73.9 t	3.84 (m)	73.8 t
	3.49 (d, 9.0)		3.57 (m)
5‴	3.42 (m)	64.2 t	3.41 (m)	64.2 t
	3.37 (m)		3.34 (m)

3.4. Acid Hydrolysis of Compound 1

A solution of 1 (4.8 mg) in 1 M HCl (3 mL) was heated in a water bath at 70 °C for 6 h. After cooling, the reaction mixture was neutralized with NaHCO₃ and extracted with CHCl3. Through TLC comparison with an authentic sample using EtOAc–EtOH–H2O (5:2:1) as a developing system, galactose and apiose were detected in the water layer (R_f = 0.35 and 0.48, respectively). The aqueous solution was further concentrated to dryness and dissolved in 1 mL pyridine. Then hydroxylammonium hydrochloride (1 mg) was added. The solution were kept in a 80 °C water bath for 30 min, followed by treatment with 1 mL Ac₂O and kept in a 90 °C water bath for another 40 min. The authentic monosaccharide samples were treated similarly as the hydrolysis products. Finally, 1 μL of these acetylated sugars were injected into a HP-20M capillary column using N₂ as carrier (oven temp. 210 °C) to be analyzed by GC using a FID detector (detector temp. 280 °C). Ultimately, the galactose and apiose were confirmed to be both d-configuration by comparing the retention times with those of standard samples: t_R: d-galactose 12.086 min, d-apiose 4.450 min, respectively.

3.5. Bioassay of Xanthine Oxidase Inhibitory Activity

The enzyme xanthine oxidase catalyses the oxidation of xanthine to uric acid. Inhibition of xanthine oxidase results in a decreased production of uric acid. The uric acid production was measured according to the increasing absorbance at 290 nm. Test solutions were prepared by adding xanthine (final concentrations 50 μM), hydroxylamine (final concentration 0.2 mM), EDTA (final concentration 0.1 mM), and flavonol glycosides in four concentrations (2.5, 5.0, 10.0, 20.0 μM). The reaction was started by adding 0.2 mL of xanthine oxidase (6.25 mU/mL) in a phosphate buffer solution (pH = 7.50, 200 mM). The mixture (total 1 mL) was incubated for 30 min at 37 °C. Prior to the measurement of uric acid prodution, the reaction was stopped by adding 0.1 mL of HCl (0.58 M) [7]. Allopurinol (2.5, 5.0, 10.0, 20.0 μM), a known xanthine oxidase inhibitor, was used as a positive control. The IC₅₀ values were calculated by the mean data values from three determinations.

4. Conclusions

The chemical investigation of P. sibirica L. var megalopha Fr. lead to the isolation of two new polygalin H (1) and polygalin I (2), as well as one known compound, polygalin D (3). The bioassay in vitro indicated that polygalin D had a high inhibitory activity against xanthine oxidase with an IC₅₀ of 16.00 μM. Moreover, those two new flavonol glycosides with rare hydroxymethyl function at C-6 in the skeleton of flavonoids showed stronger inhibitory activities against xanthine oxidase, with an IC₅₀ of 9.48 μM for 1 and 8.31 μM for 2, which deserved to be further studied.