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No.	1 (CD₃OD)		1 (DMSO-d₆)
2′		159.2			157.1
3′		135.5		133.3
4′		179.4		177.5
5′		162.5		160.9
6′	6.43 (1H, s)	100.9	6.43 (1H, d, J = 2.1 Hz)	99.8
7′		164.6		162.8
8′	6.60 (1H, s)	95.8	6.66 (1H, d, J = 2.1 Hz)	94.4
9′		157.8		155.9
10′		107.4		105.5
1′		121.6		119.5
2′	7.57 (1H, s)	107.2	7.45 (1H, d, J = 2.1 Hz)	105.8
3′		148.8		147.6
4′		139.0		138.0
5′		146.3		145.5
6′	7.28 (1H, s)	111.2	7.16 (1H, d, J = 2.1 Hz)	109.8
3′-OCH₃	3.91 (3H, s)	57.1	3.83 (3H, s)	55.9
Glc-1′′	5.44 (1H, d, J = 7.1 Hz)	103.4	5.54 (1H, d, J = 7.1 Hz)	100.8
2′′	3.53 (1H, m)	77.7	3.26 (1H, m)	76.5
3′′	3.45 (1H, overlapped)	76.9	3.26 (1H, m)	75.7.
4′′	3.42 (1H, m)	78.2	3.38 (1H, overlapped)	77.1
5′′	3.71 (1H, m)	71.2	3.72 (1H, d, J = 11.0 Hz)	70.4
6′′	3.85 (1H, m)	62.3	3.72 (1H, d, J = 11.0 Hz)	60.5
	3.69 (1H, d, J = 11.2 Hz)		3.65 (1H, dd, J = 11.0, 2.0 Hz)
Glc-1′′′	5.02 (1H, d, J = 7.1 Hz)	101.4	5.04 (1H, d, J = 7.1 Hz)	99.3
2′′′	3.47 (1H, m)	74.6	3.25 (1H, m)	73.4
3′′′	3.87 (1H, m)	76.9	3.70 (1H, overlapped)	76.4
4′′′	3.71 (1H, m)	71.0	3.53 (1H, m)	70.3
5′′′	3.55 (1H, m)	75.9	3.53 (1H, m)	74.2
6′′′	3.83 (1H, dd, J = 10.8, 6.2 Hz)	67.7	3.72 (1H, d, J = 11.0 Hz)	65.9
	3.52 (1H, d, J = 10.8 Hz)		3.50 (1H, overlapped)
Rha-1′′′′	4.60 (1H, d, J = 1.7 Hz)	102.0	4.51 (1H, d, J = 1.7 Hz)	100.4
2′′′′	3.50 (1H, overlapped)	72.2	3.53 (1H, m)	68.2.
3′′′′	3.71 (1H, m)	70.3	3.66 (1H, overlapped)	69.6
4′′′′	4.88 (1H, overlapped)	75.1	4.76 (1H, t, J = 9.6 Hz)	74.2
5′′′′	3.58 (1H, m)	67.8	3.51 (1H, overlapped)	66.2
6′′′′	0.88 (3H, d, J = 6.2 Hz)	17.6	0.80 (3H, d, J = 6.2 Hz)	17.1
1′′′′′		127.2		125.1
2′′′′′	7.51 (1H, d, J = 8.0 Hz)	131.4	7.54 (1H, d, J = 8.5 Hz)	130.3
3′′′′′	6.84 (1H, d, J = 8.0 Hz)	116.9	6.81 (1H, d, J = 8.5 Hz)	115.8
4′′′′′		161.2		159.7
5′′′′′	6.84 (1H, d, J = 8.0 Hz)	116.9	6.81 (1H, d, J = 8.5 Hz)	115.8
6′′′′′	7.51 (1H, d, J = 8.0 Hz)	131.4	7.54 (1H, d, J = 8.5 Hz)	130.3
7′′′′′	7.55 (1H, d, J = 15.8 Hz)	147.0	7.50 (1H, d, J = 16.0 Hz)	144.7
8′′′′′	6.27 (1H, d, J = 15.8 Hz)	115.3	6.24 (1H, d, J = 16.0 Hz)	114.3
9′′′′′		169.2		166.3

No.	1 (CD₃OD)		1 (DMSO-d₆)
2′		159.2			157.1
3′		135.5		133.3
4′		179.4		177.5
5′		162.5		160.9
6′	6.43 (1H, s)	100.9	6.43 (1H, d, J = 2.1 Hz)	99.8
7′		164.6		162.8
8′	6.60 (1H, s)	95.8	6.66 (1H, d, J = 2.1 Hz)	94.4
9′		157.8		155.9
10′		107.4		105.5
1′		121.6		119.5
2′	7.57 (1H, s)	107.2	7.45 (1H, d, J = 2.1 Hz)	105.8
3′		148.8		147.6
4′		139.0		138.0
5′		146.3		145.5
6′	7.28 (1H, s)	111.2	7.16 (1H, d, J = 2.1 Hz)	109.8
3′-OCH₃	3.91 (3H, s)	57.1	3.83 (3H, s)	55.9
Glc-1′′	5.44 (1H, d, J = 7.1 Hz)	103.4	5.54 (1H, d, J = 7.1 Hz)	100.8
2′′	3.53 (1H, m)	77.7	3.26 (1H, m)	76.5
3′′	3.45 (1H, overlapped)	76.9	3.26 (1H, m)	75.7.
4′′	3.42 (1H, m)	78.2	3.38 (1H, overlapped)	77.1
5′′	3.71 (1H, m)	71.2	3.72 (1H, d, J = 11.0 Hz)	70.4
6′′	3.85 (1H, m)	62.3	3.72 (1H, d, J = 11.0 Hz)	60.5
	3.69 (1H, d, J = 11.2 Hz)		3.65 (1H, dd, J = 11.0, 2.0 Hz)
Glc-1′′′	5.02 (1H, d, J = 7.1 Hz)	101.4	5.04 (1H, d, J = 7.1 Hz)	99.3
2′′′	3.47 (1H, m)	74.6	3.25 (1H, m)	73.4
3′′′	3.87 (1H, m)	76.9	3.70 (1H, overlapped)	76.4
4′′′	3.71 (1H, m)	71.0	3.53 (1H, m)	70.3
5′′′	3.55 (1H, m)	75.9	3.53 (1H, m)	74.2
6′′′	3.83 (1H, dd, J = 10.8, 6.2 Hz)	67.7	3.72 (1H, d, J = 11.0 Hz)	65.9
	3.52 (1H, d, J = 10.8 Hz)		3.50 (1H, overlapped)
Rha-1′′′′	4.60 (1H, d, J = 1.7 Hz)	102.0	4.51 (1H, d, J = 1.7 Hz)	100.4
2′′′′	3.50 (1H, overlapped)	72.2	3.53 (1H, m)	68.2.
3′′′′	3.71 (1H, m)	70.3	3.66 (1H, overlapped)	69.6
4′′′′	4.88 (1H, overlapped)	75.1	4.76 (1H, t, J = 9.6 Hz)	74.2
5′′′′	3.58 (1H, m)	67.8	3.51 (1H, overlapped)	66.2
6′′′′	0.88 (3H, d, J = 6.2 Hz)	17.6	0.80 (3H, d, J = 6.2 Hz)	17.1
1′′′′′		127.2		125.1
2′′′′′	7.51 (1H, d, J = 8.0 Hz)	131.4	7.54 (1H, d, J = 8.5 Hz)	130.3
3′′′′′	6.84 (1H, d, J = 8.0 Hz)	116.9	6.81 (1H, d, J = 8.5 Hz)	115.8
4′′′′′		161.2		159.7
5′′′′′	6.84 (1H, d, J = 8.0 Hz)	116.9	6.81 (1H, d, J = 8.5 Hz)	115.8
6′′′′′	7.51 (1H, d, J = 8.0 Hz)	131.4	7.54 (1H, d, J = 8.5 Hz)	130.3
7′′′′′	7.55 (1H, d, J = 15.8 Hz)	147.0	7.50 (1H, d, J = 16.0 Hz)	144.7
8′′′′′	6.27 (1H, d, J = 15.8 Hz)	115.3	6.24 (1H, d, J = 16.0 Hz)	114.3
9′′′′′		169.2		166.3

No.	2
2′	5.29 (1H, dd, J = 13.0, 3.0 Hz)	80.0
3′	2.66 (1H, dd, J = 17.4, 3.0 Hz)	46.3
′	2.95 (1H, dd, J = 17.4, 13.0 Hz)
4′		192.6
5′		162.5
6′	6.31 (1H, d, J = 2.2 Hz)	102.0
7′		166.7
8′	6.04 (1H, d, J = 2.2 Hz)	100.4
9′		162.9
10′		105.7
1′		131.3
2′	7.30 (1H, d, J = 8.4 Hz)	128.9
3′	6.80 (1H, d, J = 8.4 Hz)	116.3
4′		158.9
5′	6.80 (1H, d, J = 8.4 Hz)	116.3
6′	7.30 (1H, d, J = 8.4 Hz)	128.9
Glc-1′′	4.74 (1H, d, J = 7.5 Hz)	104.8
2′′	3.51 (1H, m)	74.1
3′′	3.57 (1H, m)	77.6
4′′	3.46 (1H, m)	77.2
5′′	3.38 (1H, m)	72.1
6′′	4.03(1H, dd, J = 11.5, 2.0 Hz)	69.9
	3.70 (1H, d, J = 11.5 Hz)
Rha-1′′′	4.79 (1H, d, J = 1.7 Hz)	102.5
2′′′	3.91 (1H, m)	72.3
3′′′	3.36 (1H, d, J = 6.0 Hz)	71.4
4′′′	3.71 (1H, d, J = 3.2 Hz)	74.7
5′′′	3.68 (1H, m)	67.8
6′′′	1.23 (3H, d, J = 6.2 Hz)	18.0

No.	2
2′	5.29 (1H, dd, J = 13.0, 3.0 Hz)	80.0
3′	2.66 (1H, dd, J = 17.4, 3.0 Hz)	46.3
′	2.95 (1H, dd, J = 17.4, 13.0 Hz)
4′		192.6
5′		162.5
6′	6.31 (1H, d, J = 2.2 Hz)	102.0
7′		166.7
8′	6.04 (1H, d, J = 2.2 Hz)	100.4
9′		162.9
10′		105.7
1′		131.3
2′	7.30 (1H, d, J = 8.4 Hz)	128.9
3′	6.80 (1H, d, J = 8.4 Hz)	116.3
4′		158.9
5′	6.80 (1H, d, J = 8.4 Hz)	116.3
6′	7.30 (1H, d, J = 8.4 Hz)	128.9
Glc-1′′	4.74 (1H, d, J = 7.5 Hz)	104.8
2′′	3.51 (1H, m)	74.1
3′′	3.57 (1H, m)	77.6
4′′	3.46 (1H, m)	77.2
5′′	3.38 (1H, m)	72.1
6′′	4.03(1H, dd, J = 11.5, 2.0 Hz)	69.9
	3.70 (1H, d, J = 11.5 Hz)
Rha-1′′′	4.79 (1H, d, J = 1.7 Hz)	102.5
2′′′	3.91 (1H, m)	72.3
3′′′	3.36 (1H, d, J = 6.0 Hz)	71.4
4′′′	3.71 (1H, d, J = 3.2 Hz)	74.7
5′′′	3.68 (1H, m)	67.8
6′′′	1.23 (3H, d, J = 6.2 Hz)	18.0

黑果枸杞中两个新的黄酮苷类化合物

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代向阳 ¹ , 朱研洁 ¹ , 张晓祎 ¹ , 赵珍珠 ¹^,² , 孙彦君 ¹^,² , 冯卫生 ¹^,²^,^* , 陈辉 ¹^,²^,^*

药学学报 | 研究论文 2025,60(7): 2263-2270

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药学学报 |研究论文 2025 , 60 (7) : 2263 -2270

黑果枸杞中两个新的黄酮苷类化合物

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代向阳¹, 朱研洁¹, 张晓祎¹, 赵珍珠¹^,², 孙彦君¹^,², 冯卫生¹^,²^,^*, 陈辉¹^,²^,^*

作者信息

¹河南中医药大学药学院, 河南郑州 450046

²呼吸疾病中医药防治省部共建协同创新中心, 河南郑州 450046

通讯作者:

冯卫生，Tel: 86-371-60190296, E-mail: fwsh@hactcm.edu.cn

陈辉，Tel: 86-371-65962746, E-mail: chenhuiyxy@hactcm.edu.cn

作者简介:

作者贡献:

陈辉负责实验设计; 代向阳负责实验实施、数据处理和论文撰写; 朱研洁和张晓祎参与实验实施; 陈辉、冯卫生、赵珍珠和孙彦君负责文章的审阅和提出论文写作的建议。

Two new flavonoid glycosides from the fruits of Lycium ruthenicum Murr.

Xiang-yang DAI¹, Yan-jie ZHU¹, Xiao-yi ZHANG¹, Zhen-zhu ZHAO¹^,², Yan-Jun SUN¹^,², Wei-sheng FENG¹^,²^,^*, Hui CHEN¹^,²^,^*

Affiliations

¹School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China

²Co-construction of Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases by Henan & Education Ministry of China, Zhengzhou 450046, China

出版时间: 2025-07-12 doi: 10.16438/j.0513-4870.2025-0161

文章导航

摘要

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采用D101大孔吸附树脂、硅胶、ODS、Sephadex LH-20等柱色谱以及半制备液相等现代色谱分离技术对黑果枸杞果实水提物进行系统的分离纯化, 得到9个黄酮苷类化合物。运用现代波谱学方法(1D NMR、2D NMR、MS等) 鉴定结构分别为laricetrin-3-O-β-D-葡萄糖基-7-O-[(4′′′′-反式对羟基肉桂酰基)-α-L-鼠李糖(1′′′′→6′′′)]-β-D-葡萄糖苷(1)、(2S)-柚皮素-5-O-α-L-鼠李糖(1′′′→6′′)-β-D-葡萄糖苷(2)、山柰酚-3-O-洋槐糖苷(3)、槲皮素-3-O-芸香糖苷(4)、异鼠李素-3-O-芸香糖苷(5)、山柰酚-3-O-β-D-葡萄糖-(1→4)-α-L-鼠李糖苷(6)、柚皮素-7-O-β-D-葡萄糖苷(7)、(2S)-helichrysin A (8)、2′-O-β-D-glucopyranoside (9)。化合物1和2为两个新的黄酮苷, 分别命名为枸杞黄酮苷A和枸杞黄酮苷B, 化合物6和9为首次从黑果枸杞中分离得到。对部分化合物进行α-葡萄糖苷酶抑制活性筛选, 结果显示, 化合物9对α-葡萄糖苷酶具有一定的抑制活性, IC₅₀为(76.14 ± 4.64) μmol·L^-1。

关键词

黑果枸杞 / 化学成分 / 黄酮苷 / 枸杞黄酮苷A / α-葡萄糖苷酶抑制活性

Abstract

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Nine flavonoid glycosides were isolated from water extract of the fruits of Lycium ruthenicum Murr. by using various chromatographic methods, including D-101 macroporous adsorption resin, silica gel, ODS, Sephadex LH-20 and semi-preparative HPLC. The structures of the isolated compounds were identified as laricetrin-3-O-β-D-glucopyranoside-7-O-[4′′′′-O-(E)-p-coumaroyl-α-L-rhamnopyranosyl-(1′′′′→6′′′)]-β-D-glucopyranoside (1), (2S)-naringenin-5-O-α-L-rhamnopyranosyl(1′′′→6′′)-β-D-glucopyranoside (2), kaempferol-3-O-robinobioside (3), quercetin-3-O-rutinose (4), isorhamnetin-3-rutinoside (5), kaempferol-3-β-D-glucopyranosyl(1→4)-α-L-rhamnopyranoside (6), prunin (7), (2S)-helichrysin A (8), 2′-O-β-D-glucopyranoside (9) by spectroscopy methods (1D NMR, 2D NMR, MS, etc.). Compounds 1 and 2 were new flavonoid glycosides, named lyciuflavonoid A and lyciuflavonoid B. Compounds 6 and 9 were isolated from L. ruthenicum for the first time. Compound 9 has an inhibitory effect on α-glucosidase with an IC₅₀ of (76.14 ± 4.64) μmol·L^-1.

Key words

Lycium ruthenicum Murr. / chemical constituent / flavonoid glycoside / lyciuflavonoid A / α-glucosidase inhibitory activity

引用本文

代向阳, 朱研洁, 张晓祎, 赵珍珠, 孙彦君, 冯卫生, 陈辉. 黑果枸杞中两个新的黄酮苷类化合物. 药学学报, 2025 , 60 (7) : 2263 -2270 . DOI: 10.16438/j.0513-4870.2025-0161

Xiang-yang DAI, Yan-jie ZHU, Xiao-yi ZHANG, Zhen-zhu ZHAO, Yan-Jun SUN, Wei-sheng FENG, Hui CHEN. Two new flavonoid glycosides from the fruits of Lycium ruthenicum Murr.[J]. Acta Pharmaceutica Sinica, 2025 , 60 (7) : 2263 -2270 . DOI: 10.16438/j.0513-4870.2025-0161

正文

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黑果枸杞(Lycium ruthenicum Murr.) 为茄科(Solanaceae) 枸杞属(Lycium) 多年生多刺灌木, 广泛分布于中国西北干旱沙漠和盐碱地区^[1]。据《维吾尔药志》及藏医学名著《晶珠本草》与《四部医典》记载: “黑果枸杞味甘、性平、清心热, 用于治疗心热病、心脏病、月经不调、停经等病症”^[2]。现代药理活性研究表明黑果枸杞在抗疲劳^[3]、抗辐射^[4]、抗炎^[5]、抗氧化、抗衰老^[6]、降血糖^[7]等方面具有显著的治疗作用。化学成分研究表明黑果枸杞中主要含有多糖、花青素、苯丙素类衍生物、常见黄酮类化合物、亚精胺、苯乙胺衍生物^{[8, 9]}类等成分, 其中以多糖和花青素类为其主要的药效成分, 目前已被广泛研究。已有文献^[10]研究表明, 黑果枸杞提取物对α-葡萄糖苷酶具有明显的抑制作用, 并发现其中的苯丙素和生物碱类成分具有一定的α-葡萄糖苷酶抑制活性, 但有关其余化学成分的α-葡萄糖苷酶抑制活性研究尚未见文献报道。本课题组前期曾对宁夏枸杞和中华枸杞果实的化学成分进行了系统研究, 部分化合物显示出较强的α-葡萄糖苷酶抑制活性^[11]。为促进枸杞属药用植物资源的进一步开发利用, 本实验对黑果枸杞果实水提物进行化学成分研究, 共分离鉴定了9个黄酮苷类化合物(图1), 包括2个新的黄酮苷laricetrin-3-O-β-D-葡萄糖基-7-O-[(4′′′′-反式对羟基肉桂酰基)-α-L-鼠李糖(1′′′′→6′′′)]-β-D-葡萄糖苷(1)、(2S)-柚皮素-5-O-α-L-鼠李糖(1′′′→6′′)-β-D-葡萄糖苷(2) 和2个首次从黑果枸杞中分离得到的黄酮苷山柰酚-3-O-β-D-葡萄糖-(1→4)-α-L-鼠李糖苷(6)、2′-O-β-D-glucopyranoside (9)。活性实验结果表明, 化合物9对α-葡萄糖苷酶具有一定的抑制活性, IC₅₀为(76.14 ± 4.64) μmol·L^-1。

结果与讨论

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1　结构鉴定

化合物1为黄色固体, [α]

$ {}_{\mathrm{D}}^{20} $

-12.5 (c 0.01, MeOH)。紫外光谱(UV) 显示该化合物在211、268和314 nm处有最大吸收, 红外光谱(IR) 显示有羟基(3 414 cm^-1), 羰基(1 694 cm^-1) 和苯环(1 598 cm^-1) 的特征吸收。HR-ESI-MS给出准分子离子峰m/z 971.242 9 [M+Na]⁺, 计算值为971.242 8 [M+Na]⁺, 推测分子式为C₄₃H₄₈O₂₄, 不饱和度为20。化合物1的¹H NMR (500 MHz, CD₃OD) 谱中(表1) 低场区共显示一组反式双键氢信号[δ_H 7.55 (1H, d, J = 15.8 Hz, H-7′′′′′), 6.27 (1H, d, J = 15.8 Hz, H-8′′′′′)], 一个对位取代苯环氢信号[δ_H 7.51 (2H, d, J = 8.0 Hz, H-2′′′′′, 6′′′′′), 6.84 (2H, d, J = 8.0 Hz, H-3′′′′′, 5′′′′′)] 以及两个1, 3, 4, 5-四取代苯环氢信号[7.57 (1H, s, H-2′), 7.28 (1H, s, H-6′)] 和[6.60 (1H, s, H-8), 6.43 (1H, s, H-6)]。此外, ¹H NMR谱中高场区还显示三个糖端基氢信号[δ_H 5.44 (1H, d, J = 7.1 Hz, H-1′′), 5.02 (1H, d, J = 7.1 Hz, H-1′′′)] 和[4.60 (1H, d, J = 1.7 Hz, H-1′′′′)] 以及一个甲氧基氢信号[3.91 (3H, s, 3′-OCH₃)]。¹³C NMR (125 MHz, CD₃OD) 谱共显示43个碳信号, 结合HSQC, 归属为两组吡喃葡萄糖基碳信号(δ_C 103.4, 77.7, 76.9, 78.2, 71.2, 62.3) 和(δ_C 101.4, 74.6, 76.9, 71.0, 75.9, 67.7), 一组鼠李糖基碳信号(δ_C 102.0, 72.2, 70.3, 75.1, 67.8, 17.6) 以及一组反式对羟基肉桂酸片段碳信号(δ_C 169.2, 161.2, 147.0, 131.4×2, 127.2, 116.9×2, 115.3)。除去以上碳信号, 还剩余16个碳信号, 包括一个羰基碳信号(δ_C 179.4), 12个芳香碳信号(δ_C 164.6, 162.5, 157.8, 148.8, 146.3, 139.0, 121.6, 111.2, 107.4, 107.2, 100.9, 95.8), 两个连氧烯碳信号(δ_C 159.2, 135.5) 和一个甲氧基碳信号(δ_C 57.1)。以上NMR信息推测化合物1为一个黄酮醇苷类化合物, 进一步与文献^[12]报道的数据进行对比分析, 确定化合物1苷元为laricetrin。

综合化合物1的2D NMR (¹H-¹H COSY、HSQC、HMBC和NOESY) 谱信息, 对化合物1的碳氢信号进行了归属(表1)。在化合物1的HMBC谱中(图2), H-7′′′′′与C-2′′′′′/C-9′′′′′, H-8′′′′′与C-1′′′′′/C-9′′′′′以及H-2′′′′′与C-4′′′′′/C-6′′′′′/C-7′′′′′存在远程相关信号, 结合¹H-¹H COSY谱(图2) 中H-2′′′′′/H-3′′′′′、H-7′′′′′/H-8′′′′′存在的相关, 证明化合物1中存在反式对羟基肉桂酸片段。此外, H-6与C-8/C-10, H-8与C-6/C-10, H-2′与C-2/C-4′/C-6′, H-6′与C-2/C-2′/C-4′以及3′-OCH₃与C-3′存在关键远程HMBC相关信号, 结合NOESY谱中3′-OCH₃与H-2′间的NOE相关信号, 进一步证明了化合物1的苷元为laricetrin。此外, 鼠李糖上的H-4′′′′ (δ_H 4.88) 与C-6′′′′ (δ_C 17.6)/C-9′′′′′ (δ_C 169.2) 存在明显远程相关, 说明鼠李糖的C-4′′′′位与反式对羟基肉桂酸的C-9′′′′′位通过酯键相连。鼠李糖端基H-1′′′′ (δ_H 4.60) 与葡萄糖Glc Ⅱ的C-6′′′ (δ_C 67.7) 存在明显远程相关, 说明鼠李糖和葡萄糖Glc Ⅱ的连接方式是C-1′′′′→C-6′′′连接。葡萄糖Glc Ⅰ端基H-1′′ (δ_H 5.44) 与苷元的C-3 (δ_C 135.5) 存在明显远程相关信号, 说明葡萄糖Glc Ⅰ连接在苷元3位碳上。化合物1以CD₃OD为溶剂测试的HMBC谱中, 葡萄糖Glc Ⅱ的端基氢与苷元之间未出现明显的相关信号, 但已有文献^[13]研究报道黄酮醇类化合物形成糖苷时苷元碳原子化学位移向高场区移动, 结合化合物1苷元C-7的化学位移向高场区移动了δ 5.0, 可判断化合物1的C-7位形成糖苷。为进一步证明上述结论的正确性, 换用DMSO-d₆为溶剂重新对化合物1进行了图谱测试。在化合物1以DMSO-d₆为溶剂测试的HMBC谱中, 观察到葡萄糖Glc Ⅱ的端基氢H-1′′′ (δ_H 5.04) 与苷元C-7 (δ_C 162.8) 存在明显的相关信号, 证明葡萄糖Glc Ⅱ连接在苷元7位碳上。根据葡萄糖端基氢的偶合常数(J_H-1′′ = 7.1 Hz和J_H-1′′′ = 7.1 Hz) 可确定其相对构型为β构型, 根据鼠李糖端基氢的偶合常数(J_{H-1′′′′} = 1.7 Hz) 可确定其相对构型为α构型^[14]。将化合物1进行酸水解及衍生化实验, 之后与D-葡萄糖、D-鼠李糖和L-鼠李糖的标准品衍生物进行LC-MS分析, 确定葡萄糖绝对构型为D型, 鼠李糖绝对构型为L型^[14]。综合以上信息, 确定化合物1为laricetrin-3-O-β-D-葡萄糖基-7-O-[(4′′′′-反式对羟基肉桂酰基)-α-L-鼠李糖(1′′′′→6′′′)]-β-D-葡萄糖苷, {laricetrin-3-O-β-D-glucopyranoside-7-O-[4′′′′-O-(E)-p-coumaroyl-α-L-rhamnopyranosyl-(1′′′′→6′′′)]-β-D-glucopyranoside)}, 命名为枸杞黄酮苷A (lyciuflavonoid A)。

化合物2为黄色固体, [α]

$ {}_{\mathrm{D}}^{20} $

-6.43 (c 0.01, MeOH)。紫外光谱(UV) 显示该化合物在204和367 nm处有最大吸收。红外光谱(IR) 显示有羟基(3 367cm^-1) 和苯环(1 606, 1 519 cm^-1) 的特征吸收。HR-ESI-MS给出准分子离子峰m/z 603.168 1 [M+Na]⁺, 计算值为603.168 4 [M+Na]⁺, 推测分子式为C₂₇H₃₂O₁₄, 不饱和度为12。化合物2的¹H NMR (500 MHz, CD₃OD) 谱中(表2) 低场区共显示一个对位取代苯环氢信号[δ_H 7.30 (2H, d, J = 8.4 Hz, H-2′, 6′), 6.80 (2H, d, J = 8.4 Hz, H-3′, 5′)] 和一个1, 3, 4, 5-四取代苯环氢信号[6.31 (1H, d, J = 2.2 Hz, H-6), 6.04 (1H, d, J = 2.2 Hz, H-8)]。此外, ¹H NMR谱中高场区还显示两个糖端基氢信号[4.79 (1H, d, J = 1.7 Hz, H-1′′′)] 和[δ_H 4.74 (1H, d, J = 7.5 Hz, H-1′′)] 以及一个次甲基氢信号[5.29 (1H, dd, J = 13.0, 3.0 Hz, H-3)], 一组亚甲基氢信号[2.95 (1H, dd, J = 17.4, 13.0 Hz, H-3b), 2.66 (1H, dd, J = 17.4, 3.0 Hz, H-3a)]。¹³C NMR (125 MHz, CD₃OD) 谱共显示27个碳信号, 结合HSQC, 归属为一组葡萄糖基碳信号(δ_C 104.8, 74.1, 77.6, 77.2, 72.1, 69.9) 和一组鼠李糖基碳信号(δ_C 102.5, 72.3, 71.4, 74.7, 67.8, 18.0)。除去以上碳信号, 还剩余15个碳信号, 包括一个羰基碳信号(δ_C 192.6), 12个芳香碳信号(δ_C 166.7, 162.9, 162.5, 158.9, 131.3, 128.9×2, 116.3×2, 105.7, 102.0, 100.4), 一个次甲基碳信号(δ_C 80.0) 和一个亚甲基碳信号(δ_C 46.3)。以上NMR信息推测化合物2为一个二氢黄酮苷类化合物, 进一步与文献^[15]报道的数据进行对比分析, 确定化合物2苷元为柚皮素。

综合化合物2的2D NMR (¹H-¹H COSY、HSQC和HMBC) 谱信息, 对化合物2的碳氢信号进行了归属(表2)。在化合物2的HMBC谱中(图2), H-6与C-8/C-10, H-8与C-6/C-10, H-2′与C-2′/C-4′/C-6′以及H-2与C-3/C-4/C-2′, H-3与C-2/C-4存在远程相关信号, 结合 ¹H-¹H COSY谱中H-2/H-3、H-2′/H-3′存在的相关, 进一步证明了化合物2的苷元为柚皮素。此外, 在HMBC谱中鼠李糖上的端基H-1′′′ (δ_H 4.79) 与葡萄糖的C-6′′ (δ_C 69.9) 存在明显远程相关, 说明鼠李糖和葡萄糖的连接方式是C-1′′′→C-6′′连接。葡萄糖端基H-1′′ (δ_H 4.74) 与苷元的C-5 (δ_C 162.5) 存在明显远程相关, 说明葡萄糖连接在苷元的5位碳上。同时结合苷元C-6和C-8的化学位移分别向低场移动了δ 4.8和4.1, 进一步证明化合物2的C-5位形成糖苷^{[15, 16]}。根据糖端基氢的偶合常数(葡萄糖J_H-1′′ = 7.5 Hz, 鼠李糖J_H-1′′′ = 1.7 Hz), 判断葡萄糖相对构型为β构型, 鼠李糖相对构型为α构型^[16]。化合物2经酸水解及衍生化实验, 之后与D-葡萄糖、D-鼠李糖和L-鼠李糖的标准品衍生物进行LC-MS分析, 确定葡萄糖绝对构型为D型, 鼠李糖绝对构型为L型。化合物2的CD谱在302 nm处显示负的Cotton效应(苯环π-π*跃迁产生), 在333 nm处显示正的Cotton效应(羰基n-π*跃迁产生), 与文献^[17]报道已知化合物(2S)-naringenin 5-O-β-D-glucopyranosyl(1′′′→6′′)-β-D-glucopyranoside一致, 说明C-2的绝对构型为S型。此外, CD激子手性法显示出负的激子手性, 这与化合物2在302 nm处的Cotton效应一致, 进一步证明以上结论的正确性。综合以上信息确定化合物2为(2S)-柚皮素-5-O-α-L-鼠李糖(1′′′→6′′)-β-D-葡萄糖苷, [(2S)-naringenin-5-O-α-L-rhamnopyranosyl(1′′′→6′′)-β-D-glucopyranoside], 命名为枸杞黄酮苷B (lyciuflavonoid B)。

2　 α-葡萄糖苷酶抑制作用

实验结果表明在初始浓度为50.0 μmol·L^-1时, 化合物3、4和9具有弱的α-葡萄糖苷酶抑制活性, 其抑制率分别为(10.75 ± 1.72)%、(16.62 ± 1.69)%和(45.53 ± 2.29)%。化合物1、2、5、6和8抑制率均小于5.00%。对抑制率最高的化合物9进一步测试, 其IC₅₀为(76.14 ± 4.64) μmol·L^-1。阳性对照药槲皮素在浓度为10.0 μmol·L^-1时, 抑制率为(69.88 ± 2.07)%, IC₅₀为(5.61 ± 0.27) μmol·L^-1。化合物7的α-葡萄糖苷酶抑制活性已有文献^[18]报道, 其IC₅₀为(317 ± 2.12) μmol·L^-1。

实验部分

收起

BrukerAM-500 MHz核磁共振谱用超导核磁共振仪(TMS为内标, 德国Bruker公司); TripleTOF 6600型高效液相色谱-高分辨质谱联用仪(美国ABSCIEX); 赛谱锐思LC-52型半制备液相色谱仪(中国赛谱锐思北京科技有限公司); RE-5210A型旋转蒸发仪(上海亚荣生化仪器厂); LC52型高压制备液相色谱仪(赛谱锐思北京科技有限公司, SP-5030型半制备型高压输液泵, UV200型紫外检测器, Easychrom型色谱工作站); 色谱柱为(Herbsil C18 Plus, 10 mm × 250 mm, 5 μm) 和C₁₈ ODS柱色谱材料(50 μm, 日本YMC有限公司); 薄层色谱硅胶GF254、柱色谱硅胶(200~300目, 青岛海洋化工厂); Sephadex LH-20 (Pharmacia Biotech公司); RP-C18 (40~60 μm, YMC); 大孔吸附树脂D101; 分析甲醇、色谱甲醇(天津市四友精细化学品有限公司); 其他试剂均为分析级。α-葡萄糖苷酶、底物4-nitrophenyl α-D-glucopyranoside和阳性对照quercetin均购买自Sigma公司。

黑果枸杞果实于2022年9月采自青海柴达木盆地, 经河南中医药大学药学院陈随清教授鉴定为茄科枸杞属植物黑果枸杞Lycium ruthenicum Murr.的干燥成熟果实。凭证样本(20220906A) 存放于河南中医药大学中药化学研究室。

1　提取分离

黑果枸杞干燥果实30.00 kg, 使用水溶液闪式提取3次, 每次5 s, 过滤得到滤液, 药渣使用水溶液超声提取3次, 每次15 min。合并滤液, 减压浓缩, 得到总浸膏26.14 kg。浸膏加水混悬, 经大孔吸附树脂D101柱色谱分离, 依次用水、50%乙醇、95%乙醇进行洗脱, 得到水洗脱部位20.00 kg、50%乙醇洗脱部位2.33 kg、95%乙醇洗脱部位152.50 g。50%乙醇洗脱部位加水混悬, 再次经大孔吸附树脂D101柱色谱分离, 依次用水、10%乙醇、30%乙醇、50%乙醇、70%乙醇、95%乙醇进行洗脱, 得到第二次水洗脱部位1.00 kg、10%乙醇洗脱部位100.96 g、30%乙醇洗脱部位200.48 g、50%乙醇洗脱部位500.25 g、70%乙醇洗脱部位20.26 g、95%乙醇洗脱部位10.50 g。第二次过大孔吸附树脂50%乙醇洗脱部位浸膏(500.25 g) 经中压ODS柱色谱分离, 依次用甲醇-水(10%→100%) 梯度洗脱, 得到6个流份(Fr. A~Fr. F)。Fr. B (40.2 g) 经硅胶柱色谱分离依次用二氯甲烷-甲醇(40∶1→0∶1) 梯度洗脱, 并经薄层检识, 得4个流份(Fr. B-1~Fr. B-4): Fr. B-1 (800.5 mg)、Fr. B-2 (1.5 g)、Fr. B-3 (2.2 g)、Fr. B-4 (6.5 g)。Fr. B-1 (800.5 mg) 经甲醇溶解后进一步经Sephadex LH-20凝胶柱色谱分离得3个流份(Fr. B-1-1~Fr. B-1-3)。Fr. B-1-1 (200.8 mg) 经制备型HPLC制备(甲醇∶水= 30∶70) 得化合物2 (t_R = 19.20 min; 4.30 mg)、8 (t_R = 33.10 min; 23.40 mg)、9 (t_R = 35.20 min; 2.56 mg)。Fr. B-1-2 (150.6 mg) 经制备型HPLC制备(甲醇∶水= 28∶72) 得化合物5 (t_R = 22.24 min; 7.82 mg)、6 (t_R = 28.23 min; 20.82 mg)、7 (t_R = 40.60 min; 19.30 mg)。Fr. B-1-3 (200.4 mg) 经制备型HPLC制备(甲醇∶水= 28∶72) 得化合物3 (t_R = 22.24 min; 3.52 mg)、4 (t_R = 28.23 min; 4.82 mg)、1 (t_R = 40.60 min; 60.30 mg)。

2　结构鉴定

化合物1 黄色固体, 易溶于甲醇; UV (MeOH) λ_max (log ε) 211 (3.59), 268 (3.28) 和314 (3.43) nm; IR ν_max 3 414, 2 910, 2 830, 1 694, 1 598, 1 515, 1 494, 1 339, 1 202, 1 167, 1 017, 833 cm^-1; ¹H NMR (500 MHz, CD₃OD) 和¹³C NMR (125 MHz, CD₃OD) 数据见表1, HR-ESI-MS m/z 971.242 9 [M+Na]⁺ (计算值为C₄₃H₄₈O₂₄Na⁺, 971.242 8)。

化合物2 黄色固体, 易溶于甲醇; UV (MeOH) λ_max (log ε) 204 (3.87), 367 (3.18) nm; IR ν_max 3 367, 2 930, 1 606, 1 519, 1 448, 1 362, 1 217, 1 066, 977, 899 cm^-1; ¹H NMR (500 MHz, CD₃OD) 和¹³C NMR (125 MHz, CD₃OD) 数据见表2, HR-ESI-MS m/z 603.168 1 [M+Na]⁺ (计算值为C₂₇H₃₂O₁₄Na⁺, 603.168 4)。

化合物3 黄色粉末, 易溶于甲醇, ESI-MS m/z 617 [M+Na]⁺, 分子式为C₂₇H₃₀O₁₅。¹H NMR (500 MHz, CD₃OD) δ_H 8.05 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.87 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.31 (1H, d, J = 2.0 Hz, H-8), 6.13 (1H, d, J = 2.0 Hz, H-6), 5.06 (1H, d, J = 7.2 Hz, H-1′′), 4.51 (1H, d, J = 1.7 Hz, H-1′′′), 3.80-3.23 (10H, m, H-2′′-6′′, H-2′′′-5′′′), 1.13 (3H, d, J = 6.2 Hz, H-6′′′)。¹³C NMR (125 MHz, CD₃OD) δ_C 159.2 (C-2), 135.5 (C-3), 179.1 (C-4), 163.1 (C-5), 100.7 (C-6), 166.8 (C-7), 95.5 (C-8), 158.6 (C-9), 105.3 (C-10), 122.7 (C-1′), 132.3 (C-2′, 6′), 116.1 (C-3′, 5′), 161.5 (C-4′), 104.8 (C-1′′), 73.1 (C-2′′), 78.1 (C-3′′), 71.4 (C-4′′), 77.1 (C-5′′), 68.6 (C-6′′), 102.4 (C-1′′′), 72.2 (C-2′′′), 72.5 (C-3′′′), 74.0 (C-4′′′), 69.7 (C-5′′′), 18.0 (C-6′′′)。以上数据与文献^[19]报道基本一致, 确定化合物为山柰酚-3-O-洋槐糖苷。

化合物4 黄色粉末, 易溶于甲醇, ESI-MS m/z 633 [M+Na]⁺, 分子式为C₂₇H₃₀O₁₆。¹H NMR (500 MHz, CD₃OD) δ_H 7.66 (1H, d, J = 2.2 Hz, H-2′), 7.62 (1H, dd, J = 8.5, 2.2 Hz, H-6′), 6.86 (1H, d, J = 8.5 Hz, H-5′), 6.37 (1H, d, J = 2.0 Hz, H-8), 6.18 (1H, d, J = 2.0 Hz, H-6), 5.08 (1H, d, J = 7.7 Hz, H-1′′), 4.51 (1H, d, J = 1.7 Hz, H-1′′′), 1.12 (3H, d, J = 6.2 Hz, H-6′′′), 3.20~3.80 (10H, m, H-2′′-6′′, H-2′′′-5′′′)。¹³C NMR (125 MHz, CD₃OD) δ_C 159.2 (C-2), 135.6 (C-3), 179.3 (C-4), 162.9 (C-5), 98.6 (C-6), 166.2 (C-7), 94.0 (C-8), 158.6 (C-9), 105.3 (C-10), 123.5 (C-1′), 116.8 (C-2′), 145.9 (C-3′), 149.9 (C-4′), 117.6 (C-5′), 123.1 (C-6′), 104.8 (C-1′′), 75.7 (C-2′′), 78.2 (C-3′′), 72.2 (C-4′′), 77.2 (C-5′′), 69.7 (C-6′′), 102.4 (C-1′′′), 71.4 (C-2′′′), 72.1 (C-3′′′), 73.9 (C-4′′′), 68.6 (C-5′′′), 17.9 (C-6′′′)。以上数据与文献^[20]报道基本一致, 确定化合物为槲皮素-3-O-芸香糖苷。

化合物5 黄色粉末, 易溶于甲醇, ESI-MS m/z 647 [M+Na]⁺, 分子式为C₂₈H₃₂O₁₆。¹H NMR (500 MHz, CD₃OD) δ_H 7.93 (1H, d, J = 2.2 Hz, H-2′), 7.61 (1H, dd, J = 8.5, 2.2 Hz, H-6′), 6.90 (1H, d, J = 8.5 Hz, H-5′), 6.36 (1H, d, J = 2.0 Hz, H-8), 6.17 (1H, d, J = 2.0 Hz, H-6), 5.20 (1H, d, J = 7.2 Hz, H-1′′), 4.52 (1H, d, J = 1.6 Hz, H-1′′′), 3.94 (3H, s, 3′-OCH₃), 3.20~3.82 (10H, m, H-2′′-6′′, H-2′′′-H-5′′′), 1.09 (3H, d, J = 6.2 Hz, H-6′′′)。¹³C NMR (125 MHz, CD₃OD) δ_C 158.7 (C-2), 135.4 (C-3), 179.1 (C-4), 163.0 (C-5), 100.0 (C-6), 167.4 (C-7), 95.2 (C-8), 158.6 (C-9), 104.6 (C-10), 122.9 (C-1′), 114.5 (C-2′), 150.9 (C-3′), 148.3 (C-4′), 116.1 (C-5′), 123.9 (C-6′), 102.5 (C-1′′), 75.9 (C-2′′), 78.2 (C-3′′), 72.3 (C-4′′), 77.4 (C-5′′), 68.5 (C-6′′), 100.4 (C-1′′′), 71.6 (C-2′′′), 72.1 (C-3′′′), 73.8 (C-4′′′), 69.9 (C-5′′′), 17.9 (C-6′′′), 56.7 (3′-OCH₃)。以上数据与文献^[21]报道基本一致, 确定化合物为异鼠李素-3-O-芸香糖苷。

化合物6 黄色粉末, 易溶于甲醇, ESI-MS m/z 617 [M+Na]⁺, 分子式为C₂₇H₃₀O₁₅。¹H NMR (500 MHz, CD₃OD) δ_H 7.75 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.92 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.41 (1H, d, J = 2.0 Hz, H-8), 6.23 (1H, d, J = 2.0 Hz, H-6), 5.16 (1H, d, J = 1.4 Hz, H-1′′), 4.30 (1H, d, J = 7.8 Hz, H-1′′′), 4.01 (1H, m, H-2′′), 3.80 (1H, m, H-3′′), 3.70 (1H, m, H-6′′′a), 3.65 (1H, d, J = 11.2 Hz, H-6′′′b), 3.43 (1H, m, H-4′′), 3.42 (1H, m, H-3′′′), 3.40 (1H, m, H-4′′′), 3.30 (1H, m, H-5′′), 3.20 (1H, m, H-5′′′), 2.95 (1H, m, H-2′′′), 0.88 (3H, d, J = 6.2 Hz, H-6′′)。¹³C NMR (125 MHz, CD₃OD) δ_C 157.0 (C-2), 135.4 (C-3), 177.6 (C-4), 161.5 (C-5), 98.8 (C-6), 164.5 (C-7), 93.8 (C-8), 156.5 (C-9), 104.2 (C-10), 120.4 (C-1′), 130.5 (C-2′, 6′), 115.4 (C-3′, 5′), 160.0 (C-4′), 101.9 (C-1′′), 70.2 (C-2′′), 70.3 (C-3′′), 81.9 (C-4′′), 68.9 (C-5′′), 17.2 (C-6′′), 104.7 (C-1′′′), 74.5 (C-2′′′), 76.6 (C-3′′′), 69.9 (C-4′′′), 76.9(C-5′′′), 62.2 (C-6′′′)。以上数据与文献^[22]报道基本一致, 确定化合物为山柰酚-3-O-β-D-葡萄糖-(1→4)-α-L-鼠李糖苷。

化合物7 黄色粉末, 易溶于甲醇, ESI-MS m/z 457 [M+Na]⁺, 分子式为C₂₁H₂₂O₁₀。¹H NMR (500 MHz, CD₃OD) δ_H 7.33(2H, d, J = 8.4 Hz, H-2′, 6′), 6.84 (2H, d, J = 8.4 Hz, H-3′, 5′), 6.22 (1H, s, H-8), 6.20 (1H, s, H-6), 5.40 (1H, m, H-2), 4.99 (1H, d, J = 6.9 Hz, H-1′′), 3.89 (1H, brd, J = 11.9 Hz, H-6′′a), 3.70 (1H, dd, J = 11.9, 4.7 Hz, H-6′′b), 3.49~3.34 (4H, m, H-2′′-5′′), 3.18 (1H, dd, J = 17.2, 13.0 Hz, H-3a), 2.77 (1H, dd, J = 17.2, 3.3 Hz, H-3b)。¹³C NMR (125 MHz, CD₃OD) δ_C 80.6 (C-2), 44.2 (C-3), 198.6 (C-4), 165.0 (C-5), 98.0 (C-6), 167.1 (C-7), 96.9 (C-8), 164.6 (C-9), 105.0 (C-10), 130.8 (C-1′), 129.1 (C-2′, 6′), 116.3 (C-3′, 5′), 159.1 (C-4′), 101.2 (C-1′′), 74.6 (C-2′′), 77.8 (C-3′′), 71.1 (C-4′′), 78.3 (C-5′′), 62.3 (C-6′′)。以上数据与文献^[23]报道基本一致, 确定化合物为柚皮素-7-O-β-D-葡萄糖苷。

化合物8 黄色粉末, 易溶于甲醇, ESI-MS m/z 457 [M+Na]⁺, 分子式为C₂₁H₂₂O₁₀。¹H NMR (500 MHz, CD₃OD) δ_H 7.28 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.80 (2H, d, J = 8.5 Hz, H-3′, 5′), 6.46 (1H, d, J = 2.2 Hz, H-8), 6.11 (1H, d, J = 2.2 Hz, H-6), 5.29 (1H, dd, J = 13.2, 3.0 Hz, H-2), 4.75 (1H, d, J = 7.5 Hz, H-1′′), 3.94 (1H, dd, J = 12.0, 2.0 Hz, H-6′′a), 3.75 (1H, dd, J = 12.0, 5.2 Hz, H-6′′b), 3.54~3.42 (4H, m, H-2′′-5′′), 2.97 (1H, dd, J = 17.3, 13.2 Hz, H-3a), 2.68 (1H, dd, J = 17.3, 3.0 Hz, H-3b)。¹³C NMR (125 MHz, CD₃OD) δ_C 80.3 (C-2), 46.4 (C-3), 193.2 (C-4), 162.5 (C-5), 100.6 (C-6), 167.4 (C-7), 99.7 (C-8), 166.7 (C-9), 107.1 (C-10), 131.1 (C-1′), 129.2 (C-2′, 6′), 116.4 (C-3′, 5′), 159.1 (C-4′), 105.2 (C-1′′), 74.8 (C-2′′), 77.3 (C-3′′), 71.4 (C-4′′), 78.8 (C-5′′), 62.7 (C-6′′)。以上数据与文献^[24]报道基本一致, 确定化合物为(2S)-helichrysin A。

化合物9 黄色粉末, 易溶于甲醇, ESI-MS m/z 457 [M+Na]⁺, 分子式为C₂₁H₂₂O₁₀。¹H NMR (500 MHz, CD₃OD) δ_H 8.01 (1H, d, J = 15.5 Hz, H-8), 7.66 (1H, d, J = 15.5 Hz, H-7), 7.60 (2H, d, J = 8.4 Hz, H-2, 6), 6.83 (2H, d, J = 8.4 Hz, H-3, 5), 6.21 (1H, d, J = 2.2 Hz, H-5′), 5.99 (1H, d, J = 2.2 Hz, H-3′), 5.13 (1H, d, J = 7.4 Hz, H-1′′), 3.92 (1H, m, H-6′′a), 3.75 (1H, d, J = 5.5 Hz, H-6′′b), 3.55 (1H, s, H-5′′), 3.51 (1H, d, J = 8.4 Hz, H-3′′), 3.43~3.46 (2H, m, H-2′′, 4′′)。¹³C NMR (125 MHz, CD₃OD) δ_C 128.5 (C-1), 131.7 (C-2, 6), 116.9 (C-3, 5), 161.1 (C-4), 144.1 (C-7), 125.9 (C-8), 194.5 (C-9), 107.4 (C-1′), 161.8 (C-2′), 95.7 (C-3′), 166.6 (C-4′), 98.4 (C-5′), 167.8 (C-6′), 101.8 (C-1′′), 75.0 (C-2′′), 78.5 (C-3′′), 71.1 (C-4′′), 78.6 (C-5′′), 62.4 (C-6′′)。以上数据与文献^[25]报道基本一致, 确定化合物为2′-O-β-D-glucopyranoside。

3　化合物的酸水解及糖的鉴定

称取化合物1和2各1 mg, 分别加3.0 mol·L^-1盐酸溶液3.0 mL, 在90 ℃水浴加热2.5 h。将混合物真空浓缩干燥后加水(1 mL), 再加等体积的乙酸乙酯(1 mL × 3) 萃取三次。水层浓缩干燥得到糖部分。糖部分加入吡啶(1 mL)、L-半胱氨酸甲酯盐酸盐(1 mg), 在60 ℃水浴下加热1.5 h, 然后加入邻甲苯异硫氰酸酯(20 μL), 在60 ℃水浴再加热1.5 h, 得到糖部分的衍生物。另取D-葡萄糖、L-鼠李糖和D-鼠李糖加L-半胱氨酸甲酯盐酸盐(1 mg) 和吡啶(1 mL) 在60 ℃水浴下加热1.5 h, 再加入邻甲苯异硫氰酸酯(20 μL) 在60 ℃水浴下加热1.5 h。将得到的葡萄糖供试品衍生物和D-葡萄糖标准品、L-鼠李糖和D-鼠李糖标准品衍生物进行液质分析。液质分析条件: TripleTOF 6600 UPLC/MS(AB SCIEX); Kinetex C18 100Å (2.6 μm, 2.1 mm × 50 mm); 梯度洗脱: 经0.1%甲酸水(A)-甲醇(B) 等度洗脱(0~30 min, 28%B); 柱温40 ℃, 体积流量为0.3 mL·min^-1, 进样量为10 μL; 质谱条件: 正离子模式。通过对比供试品衍生物与各标准品衍生物的保留时间和质谱数据, 化合物1酸水解糖部分衍生物质谱数据(m/z 447.125 5 [M+H]⁺, t_R = 8.728 min; m/z 431.129 7 [M+H]⁺, t_R = 13.551 min), 化合物2酸水解糖部分衍生物质谱数据(m/z 447.125 0 [M+H]⁺, t_R = 8.722 min; m/z 431.130 9 [M+H]⁺, t_R = 13.546 min), D-葡萄糖标准品衍生物质谱数据(m/z 447.125 8 [M+H]⁺, t_R = 8.725 min), D-鼠李糖标准品衍生物质谱数据(m/z 431.130 3 [M+H]⁺, t_R = 6.855 min), L-鼠李糖标准品衍生物质谱数据(m/z 431.130 0 [M+H]⁺, t_R = 13.544 min)。从而确定了化合物1和2中的单糖结构片段为D-葡萄糖和L-鼠李糖。

4　 α-葡萄糖苷酶抑制活性筛选

采用4-硝基苯基-β-D-吡喃葡萄糖苷(PNPG) 法^[10]筛选化合物1~6、8和9对α-葡萄糖苷酶的抑制作用, 槲皮素为阳性对照药。将样品(终浓度50 μmol·L^-1) 与酶溶液(终浓度0.025 U·mL^-1)、缓冲液、底物(终浓度1 mmol·L^-1) 顺序加入96孔酶标板, 充分混匀, 设置三孔重复。同时设置不含药物的空白对照和槲皮素(终浓度10 μmol·L^-1) 阳性对照。37 ℃孵育50 min, 酶标仪测定405 nm处的OD值, 计算得出α-葡萄糖苷酶活性的抑制率。选择抑制率大于40%的样品, 从50 μmol·L^-1开始梯度稀释, 按照上述方法操作并计算其IC₅₀值。抑制率(%) = (1 - 实验孔OD_{405 nm} / 空白孔OD_{405 nm}) × 100%。IC₅₀按Reed & Muench法计算。

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doi: 10.16438/j.0513-4870.2025-0161

接收时间：2025-02-19
首发时间：2026-09-20
出版时间：2025-07-12

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收稿日期：2025-02-19
修回日期：2025-03-24

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¹河南中医药大学药学院, 河南郑州 450046

²呼吸疾病中医药防治省部共建协同创新中心, 河南郑州 450046

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冯卫生，Tel: 86-371-60190296, E-mail: fwsh@hactcm.edu.cn

陈辉，Tel: 86-371-65962746, E-mail: chenhuiyxy@hactcm.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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No.	1 (CD₃OD)		1 (DMSO-d₆)
2′		159.2			157.1
3′		135.5		133.3
4′		179.4		177.5
5′		162.5		160.9
6′	6.43 (1H, s)	100.9	6.43 (1H, d, J = 2.1 Hz)	99.8
7′		164.6		162.8
8′	6.60 (1H, s)	95.8	6.66 (1H, d, J = 2.1 Hz)	94.4
9′		157.8		155.9
10′		107.4		105.5
1′		121.6		119.5
2′	7.57 (1H, s)	107.2	7.45 (1H, d, J = 2.1 Hz)	105.8
3′		148.8		147.6
4′		139.0		138.0
5′		146.3		145.5
6′	7.28 (1H, s)	111.2	7.16 (1H, d, J = 2.1 Hz)	109.8
3′-OCH₃	3.91 (3H, s)	57.1	3.83 (3H, s)	55.9
Glc-1′′	5.44 (1H, d, J = 7.1 Hz)	103.4	5.54 (1H, d, J = 7.1 Hz)	100.8
2′′	3.53 (1H, m)	77.7	3.26 (1H, m)	76.5
3′′	3.45 (1H, overlapped)	76.9	3.26 (1H, m)	75.7.
4′′	3.42 (1H, m)	78.2	3.38 (1H, overlapped)	77.1
5′′	3.71 (1H, m)	71.2	3.72 (1H, d, J = 11.0 Hz)	70.4
6′′	3.85 (1H, m)	62.3	3.72 (1H, d, J = 11.0 Hz)	60.5
	3.69 (1H, d, J = 11.2 Hz)		3.65 (1H, dd, J = 11.0, 2.0 Hz)
Glc-1′′′	5.02 (1H, d, J = 7.1 Hz)	101.4	5.04 (1H, d, J = 7.1 Hz)	99.3
2′′′	3.47 (1H, m)	74.6	3.25 (1H, m)	73.4
3′′′	3.87 (1H, m)	76.9	3.70 (1H, overlapped)	76.4
4′′′	3.71 (1H, m)	71.0	3.53 (1H, m)	70.3
5′′′	3.55 (1H, m)	75.9	3.53 (1H, m)	74.2
6′′′	3.83 (1H, dd, J = 10.8, 6.2 Hz)	67.7	3.72 (1H, d, J = 11.0 Hz)	65.9
	3.52 (1H, d, J = 10.8 Hz)		3.50 (1H, overlapped)
Rha-1′′′′	4.60 (1H, d, J = 1.7 Hz)	102.0	4.51 (1H, d, J = 1.7 Hz)	100.4
2′′′′	3.50 (1H, overlapped)	72.2	3.53 (1H, m)	68.2.
3′′′′	3.71 (1H, m)	70.3	3.66 (1H, overlapped)	69.6
4′′′′	4.88 (1H, overlapped)	75.1	4.76 (1H, t, J = 9.6 Hz)	74.2
5′′′′	3.58 (1H, m)	67.8	3.51 (1H, overlapped)	66.2
6′′′′	0.88 (3H, d, J = 6.2 Hz)	17.6	0.80 (3H, d, J = 6.2 Hz)	17.1
1′′′′′		127.2		125.1
2′′′′′	7.51 (1H, d, J = 8.0 Hz)	131.4	7.54 (1H, d, J = 8.5 Hz)	130.3
3′′′′′	6.84 (1H, d, J = 8.0 Hz)	116.9	6.81 (1H, d, J = 8.5 Hz)	115.8
4′′′′′		161.2		159.7
5′′′′′	6.84 (1H, d, J = 8.0 Hz)	116.9	6.81 (1H, d, J = 8.5 Hz)	115.8
6′′′′′	7.51 (1H, d, J = 8.0 Hz)	131.4	7.54 (1H, d, J = 8.5 Hz)	130.3
7′′′′′	7.55 (1H, d, J = 15.8 Hz)	147.0	7.50 (1H, d, J = 16.0 Hz)	144.7
8′′′′′	6.27 (1H, d, J = 15.8 Hz)	115.3	6.24 (1H, d, J = 16.0 Hz)	114.3
9′′′′′		169.2		166.3

No.

1 (CD₃OD)

1 (DMSO-d₆)

δ_H

δ_C

δ_H

δ_C

2′

159.2

157.1

3′

135.5

133.3

4′

179.4

177.5

5′

162.5

160.9

6′

6.43 (1H, s)

100.9

6.43 (1H, d, J = 2.1 Hz)

99.8

7′

164.6

162.8

8′

6.60 (1H, s)

95.8

6.66 (1H, d, J = 2.1 Hz)

94.4

9′

157.8

155.9

10′

107.4

105.5

1′

121.6

119.5

2′

7.57 (1H, s)

107.2

7.45 (1H, d, J = 2.1 Hz)

105.8

3′

148.8

147.6

4′

139.0

138.0

5′

146.3

145.5

6′

7.28 (1H, s)

111.2

7.16 (1H, d, J = 2.1 Hz)

109.8

3′-OCH₃

3.91 (3H, s)

57.1

3.83 (3H, s)

55.9

Glc-1′′

5.44 (1H, d, J = 7.1 Hz)

103.4

5.54 (1H, d, J = 7.1 Hz)

100.8

2′′

3.53 (1H, m)

77.7

3.26 (1H, m)

76.5

3′′

3.45 (1H, overlapped)

76.9

3.26 (1H, m)

75.7.

4′′

3.42 (1H, m)

78.2

3.38 (1H, overlapped)

77.1

5′′

3.71 (1H, m)

71.2

3.72 (1H, d, J = 11.0 Hz)

70.4

6′′

3.85 (1H, m)

62.3

3.72 (1H, d, J = 11.0 Hz)

60.5

3.69 (1H, d, J = 11.2 Hz)

3.65 (1H, dd, J = 11.0, 2.0 Hz)

Glc-1′′′

5.02 (1H, d, J = 7.1 Hz)

101.4

5.04 (1H, d, J = 7.1 Hz)

99.3

2′′′

3.47 (1H, m)

74.6

3.25 (1H, m)

73.4

3′′′

3.87 (1H, m)

76.9

3.70 (1H, overlapped)

76.4

4′′′

3.71 (1H, m)

71.0

3.53 (1H, m)

70.3

5′′′

3.55 (1H, m)

75.9

3.53 (1H, m)

74.2

6′′′

3.83 (1H, dd, J = 10.8, 6.2 Hz)

67.7

3.72 (1H, d, J = 11.0 Hz)

65.9

3.52 (1H, d, J = 10.8 Hz)

3.50 (1H, overlapped)

Rha-1′′′′

4.60 (1H, d, J = 1.7 Hz)

102.0

4.51 (1H, d, J = 1.7 Hz)

100.4

2′′′′

3.50 (1H, overlapped)

72.2

3.53 (1H, m)

68.2.

3′′′′

3.71 (1H, m)

70.3

3.66 (1H, overlapped)

69.6

4′′′′

4.88 (1H, overlapped)

75.1

4.76 (1H, t, J = 9.6 Hz)

74.2

5′′′′

3.58 (1H, m)

67.8

3.51 (1H, overlapped)

66.2

6′′′′

0.88 (3H, d, J = 6.2 Hz)

17.6

0.80 (3H, d, J = 6.2 Hz)

17.1

1′′′′′

127.2

125.1

2′′′′′

7.51 (1H, d, J = 8.0 Hz)

131.4

7.54 (1H, d, J = 8.5 Hz)

130.3

3′′′′′

6.84 (1H, d, J = 8.0 Hz)

116.9

6.81 (1H, d, J = 8.5 Hz)

115.8

4′′′′′

161.2

159.7

5′′′′′

6.84 (1H, d, J = 8.0 Hz)

116.9

6.81 (1H, d, J = 8.5 Hz)

115.8

6′′′′′

7.51 (1H, d, J = 8.0 Hz)

131.4

7.54 (1H, d, J = 8.5 Hz)

130.3

7′′′′′

7.55 (1H, d, J = 15.8 Hz)

147.0

7.50 (1H, d, J = 16.0 Hz)

144.7

8′′′′′

6.27 (1H, d, J = 15.8 Hz)

115.3

6.24 (1H, d, J = 16.0 Hz)

114.3

9′′′′′

169.2

166.3

No.	2
2′	5.29 (1H, dd, J = 13.0, 3.0 Hz)	80.0
3′	2.66 (1H, dd, J = 17.4, 3.0 Hz)	46.3
′	2.95 (1H, dd, J = 17.4, 13.0 Hz)
4′		192.6
5′		162.5
6′	6.31 (1H, d, J = 2.2 Hz)	102.0
7′		166.7
8′	6.04 (1H, d, J = 2.2 Hz)	100.4
9′		162.9
10′		105.7
1′		131.3
2′	7.30 (1H, d, J = 8.4 Hz)	128.9
3′	6.80 (1H, d, J = 8.4 Hz)	116.3
4′		158.9
5′	6.80 (1H, d, J = 8.4 Hz)	116.3
6′	7.30 (1H, d, J = 8.4 Hz)	128.9
Glc-1′′	4.74 (1H, d, J = 7.5 Hz)	104.8
2′′	3.51 (1H, m)	74.1
3′′	3.57 (1H, m)	77.6
4′′	3.46 (1H, m)	77.2
5′′	3.38 (1H, m)	72.1
6′′	4.03(1H, dd, J = 11.5, 2.0 Hz)	69.9
	3.70 (1H, d, J = 11.5 Hz)
Rha-1′′′	4.79 (1H, d, J = 1.7 Hz)	102.5
2′′′	3.91 (1H, m)	72.3
3′′′	3.36 (1H, d, J = 6.0 Hz)	71.4
4′′′	3.71 (1H, d, J = 3.2 Hz)	74.7
5′′′	3.68 (1H, m)	67.8
6′′′	1.23 (3H, d, J = 6.2 Hz)	18.0

No.

δ_H

δ_C

2′

5.29 (1H, dd, J = 13.0, 3.0 Hz)

80.0

3′

2.66 (1H, dd, J = 17.4, 3.0 Hz)

46.3

′

2.95 (1H, dd, J = 17.4, 13.0 Hz)

4′

192.6

5′

162.5

6′

6.31 (1H, d, J = 2.2 Hz)

102.0

7′

166.7

8′

6.04 (1H, d, J = 2.2 Hz)

100.4

9′

162.9

10′

105.7

1′

131.3

2′

7.30 (1H, d, J = 8.4 Hz)

128.9

3′

6.80 (1H, d, J = 8.4 Hz)

116.3

4′

158.9

5′

6.80 (1H, d, J = 8.4 Hz)

116.3

6′

7.30 (1H, d, J = 8.4 Hz)

128.9

Glc-1′′

4.74 (1H, d, J = 7.5 Hz)

104.8

2′′

3.51 (1H, m)

74.1

3′′

3.57 (1H, m)

77.6

4′′

3.46 (1H, m)

77.2

5′′

3.38 (1H, m)

72.1

6′′

4.03(1H, dd, J = 11.5, 2.0 Hz)

69.9

3.70 (1H, d, J = 11.5 Hz)

Rha-1′′′

4.79 (1H, d, J = 1.7 Hz)

102.5

2′′′

3.91 (1H, m)

72.3

3′′′

3.36 (1H, d, J = 6.0 Hz)

71.4

4′′′

3.71 (1H, d, J = 3.2 Hz)

74.7

5′′′

3.68 (1H, m)

67.8

6′′′

1.23 (3H, d, J = 6.2 Hz)

18.0