药学学报

No.	δ_H (J in Hz)	δ_C
1	1.15 (1H, m), 1.82 (1H, m)	48.8 (t)
2	4.23 (1H, m)	67.1 (d)
3	5.37 (1H, br s)	126.2 (d)
4	-	138.3 (s)
5	1.89 (1H, m)	44.9 (d)
6	1.31 (1H, overlapped), 2.20 (1H, m)	33.9 (t)
7	-	75.6 (s)
8	1.77 (1H, m), 2.07 (1H, m)	32.5 (t)
9	1.34 (1H, m), 1.38 (1H, m)	38.9 (t)
10	-	36.5 (s)
11	-	147.7 (s)
12	5.06 (1H, br s), 5.08 (1H, br s)	114.1 (t)
13	1.81 (3H, s)	19.0 (q)
14	0.94 (3H, s)	16.8 (q)
15	1.68 (3H, s)	21.1 (q)

No.	δ_H (J in Hz)	δ_C
1	1.15 (1H, m), 1.82 (1H, m)	48.8 (t)
2	4.23 (1H, m)	67.1 (d)
3	5.37 (1H, br s)	126.2 (d)
4	-	138.3 (s)
5	1.89 (1H, m)	44.9 (d)
6	1.31 (1H, overlapped), 2.20 (1H, m)	33.9 (t)
7	-	75.6 (s)
8	1.77 (1H, m), 2.07 (1H, m)	32.5 (t)
9	1.34 (1H, m), 1.38 (1H, m)	38.9 (t)
10	-	36.5 (s)
11	-	147.7 (s)
12	5.06 (1H, br s), 5.08 (1H, br s)	114.1 (t)
13	1.81 (3H, s)	19.0 (q)
14	0.94 (3H, s)	16.8 (q)
15	1.68 (3H, s)	21.1 (q)

益智的化学成分及其神经保护作用

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邱成省 , 王吉 , 穆利萍 , 张荣平 , 陈兴龙 ^*

药学学报 | 研究论文 2023,58(9): 2746-2753

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药学学报 |研究论文 2023 , 58 (9) : 2746 -2753

益智的化学成分及其神经保护作用

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邱成省, 王吉, 穆利萍, 张荣平, 陈兴龙^*

作者信息

云南中医药大学中药学院暨云南省南药可持续利用研究重点实验室, 云南昆明 650500

通讯作者:

*陈兴龙, E-mail: chxlkib@163.com

Chemical constituents from the fruits of Alpinia oxyphylla and their neuroprotective effects

Cheng-xing QIU, Ji WANG, Li-ping MU, Rong-ping ZHANG, Xing-long CHEN^*

Affiliations

School of Chinese Materia Medica & Yunnan Key Laboratory of Southern Medicine Utilization, Yunnan University of Chinese Medicine, Kunming 650500, China

出版时间: 2023-09-12 doi: 10.16438/j.0513-4870.2023-0080

文章导航

摘要

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采用正相硅胶、MCI、RP-18、Sephadex LH-20、制备薄层色谱及半制备HPLC等分离手段, 并结合HR-ESI-MS、NMR、ECD、X-单晶衍射等结构鉴定方法, 从益智干燥果实90%乙醇提取物的乙酸乙酯部位中分离得到14个化合物, 分别鉴定为: (2R, 5R, 7R, 10S)-2, 7-dihydroxyl-eudesmane-3(4), 11(12)-diene (1)、α-rotunol (2)、diketone I (3)、(1S, 4S, 5R, 7S)-1-hydroxyl-eremophilane-9(10), 11(12)-diene-8-one (4)、cyperusol A₁ (5)、(6R, 9S, 10S)-10-hydroxyl-11, 12, 13-trinor-cadinane-4(5)-ene-3-one (6)、(2E, 4E)-6-hydroxy-2, 6-dimethylhepta-2, 4-dienal (7)、益智醇(8)、益智酮A (9)、(5R)-5-羟基-1, 7-二苯基-3-庚酮(10)、(5S)-5-羟基-7-(4″-羟基)-苯基-1-苯基-3-庚酮(11)、(5S)-5-羟基-7-(4″-羟基-3″-甲氧基)-苯基-1-苯基-3-庚酮(12)、7-(4″-羟基-3″-甲氧基)-苯基-1-苯基-3, 5-庚二酮(13)、bis-(2-ethylhexyl) terephthalate (14)。化合物1~6为倍半萜类化合物, 其中化合物1为新的桉烷型倍半萜类化合物, 化合物7为单萜类化合物, 化合物8~13为二苯基庚烷类化合物, 化合物2~6、14为首次从益智中分离得到。H₂O₂氧化损伤SH-SY5Y细胞实验表明, 化合物2、6、7、12和13在低、中浓度下具有神经保护作用, 尤其是化合物6在低、中、高浓度下细胞存活率均高于阳性药表没食子儿茶素没食子酸酯(EGCG), 显示较好的神经保护作用。

关键词

益智 / 化学成分 / 倍半萜 / 二苯基庚烷 / 神经保护作用

Abstract

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Fourteen compounds were isolated from the ethyl acetate fraction of 90% EtOH extracts of the dried fruits of Alpinia oxyphylla by silica gel, MCI, RP-18, Sephadex LH-20, TLC and semi-preparative HPLC column chromatography. Their structures were identified by HR-ESI-MS, UV, IR, NMR, ECD and X ray single crystal diffraction spectroscopic data as: (2R, 5R, 7R, 10S)-2, 7-dihydroxyl-eudesmane-3(4), 11(12)-diene (1), α-rotunol (2), diketone I (3), (1S, 4S, 5R, 7S)-1-hydroxyl-eremophilane-9(10), 11(12)-diene-8-one (4), cyperusol A₁ (5), (6R, 9S, 10S)-10-hydroxyl-11, 12, 13-trinor-cadinane-4(5)-ene-3-one (6), (2E, 4E)-6-hydroxy-2, 6-dimethylhepta-2, 4-dienal (7), oxyphyllacinol (8), yakuchinone A (9), (5R)-5-hydroxy-1, 7-diphenylhept-3-heptanone (10), (5S)-5-hydroxy-7-(4″-hydroxyphenyl)-1-phenylhept-3-heptanone (11), (5S)-5-hydroxy-7-(4″-hydroxyl-3″-methoxyphenyl)-1-phenyl-3-heptanone (12), 7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-3, 5-heptadione (13), bis-(2-ethylhexyl) terephthalate (14). Compounds 1-6 were sesquiterpenoids in which compound 1 is a new eudesmane sesquiterpenoid and compound 7 was a monoterpenoid. Compounds 8-13 were diarylheptanoids, and compounds 2-6 and 14 were isolated from A.oxyphylla for the first time. The experiments on H₂O₂ induced SH-SY5Y cells showed that compounds 2, 6, 7, 12 and 13 had neuroprotective effects at low and medium concentrations. In particular, compound 6 showed obvious neuroprotective effect at low, medium and high concentrations whose cell viability was higher than that of the positive control.

Key words

Alpinia oxyphylla / chemical constituent / sesquiterpenoids / diarylheptanoids / neuroprotective effect

引用本文

邱成省, 王吉, 穆利萍, 张荣平, 陈兴龙. 益智的化学成分及其神经保护作用. 药学学报, 2023 , 58 (9) : 2746 -2753 . DOI: 10.16438/j.0513-4870.2023-0080

Cheng-xing QIU, Ji WANG, Li-ping MU, Rong-ping ZHANG, Xing-long CHEN. Chemical constituents from the fruits of Alpinia oxyphylla and their neuroprotective effects[J]. Acta Pharmaceutica Sinica, 2023 , 58 (9) : 2746 -2753 . DOI: 10.16438/j.0513-4870.2023-0080

正文

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益智为姜科山姜属植物益智(Alpinia oxyphylla Miq.) 的干燥成熟果实, 又名益智仁, 主产于海南、云南、广东、广西等地^[1]。益智的化学成分主要包括倍半萜、二苯基庚烷及挥发油等^[2]。现代药理学研究认为益智提取物具有神经保护^[3]、抗氧化^[4]、抗炎^[5]和改善记忆障碍^[6]的功效, 其中神经保护作用是重点研究对象。Wong等^[7]研究发现益智的乙醇提取物对神经细胞tau蛋白的磷酸化有抑制作用, Ma等^[8]研究发现益智挥发油能够改善小鼠学习记忆障碍。Guan等^[9]研究发现益智中原儿茶酸对H₂O₂诱导的PC12细胞氧化损伤具有保护作用。但是这些研究大多集中在益智提取物或其挥发性成分, 对益智特征性成分倍半萜的神经保护作用研究仅涉及nootkatone^[10]等少数倍半萜, 其他倍半萜类成分的神经保护作用研究较少。

本课题组长期致力于研究天然来源的神经保护剂, 已发现多个神经保护作用较强的小分子^{[11, 12]}。在前期研究中发现益智乙醇提取物对H₂O₂氧化损伤的SH-SY5Y细胞具有较好的神经保护作用, 为了揭示其神经保护作用, 本研究从中分离鉴定了14个化合物(图 1) 并评价其神经保护活性, 其中化合物1为新化合物, 化合物2~6、14为首次从益智中分离得到, 药理活性筛选表明化合物6具有明显的神经保护作用。

结果与讨论

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1 结构鉴定

化合物1 白色针状结晶, 熔点186 ℃; [α]$ {}_{\mathrm{D}}^{26.6} $ = -45.93 (c 0.30, MeOH); UV (MeOH) λ_max (log ε) 230 (3.24) nm; IR (KBr) ν_max 3 272, 2 974, 2 938, 2 853, 1 642, 1 434, 1 376, 1 188, 1 058, 1 026, 994, 900, 831, 619 cm^-1; 由高分辨质谱HR-ESI-MS m/z 275.140 1 [M+K]⁺ (计算值为275.140 8) 确定分子式为C₁₅H₂₄O₂, 不饱和度为4, 推测化合物1为倍半萜类化合物; ¹H NMR谱(表 1) 在低场区提示有一个连羟基质子信号δ_H 4.23 (1H, m, H-2)、3个烯烃质子信号δ_H 5.37 (1H, br s, H-3), 5.06 (1H, br s, H-12a) 和5.08 (1H, br s, H-12b); 高场区显示有3个甲基质子信号δ_H 1.81 (3H, s, H-13)、0.94 (3H, s, H-14) 和1.68 (3H, s, H-15)。¹³C NMR和DEPT谱显示有15个碳信号, 分别为位于δ_C 16.8、19.0和21.1处的3个甲基, 位于δ_C 32.5、33.9、38.9、48.8和114.1处的5个亚甲基, 位于δ_C 44.9、67.1和126.2处的3个次甲基和位于δ_C 36.5、75.6、138.3和147.7处的4个季碳。其中在δ_C 114.1处为1个烯烃亚甲基, 在δ_C 67.1和126.2处分别为连一个羟基的次甲基和1个烯烃次甲基, 在δ_C 138.3和147.7处为两个烯烃季碳和在δ_C 75.6处为1个连羟基季碳(表 1)。在HMBC谱中, H-13和C-7相关, H-14和C-1/C-5/C-9相关, H-15和C-3/C-5, 可以确定化合物1为桉烷型倍半萜。然后依据HMBC谱中H-3和C-1/C-5相关, H-2和C-4相关以及¹H-¹H COSY谱中H-1/H-2/H-3相关, 可知该化合物在C-3和C-4之间形成双键并且C-3为羟基取代(图 2); 接着依据H-5和C-7相关, H-12和C-7相关, 以及H-13和C-7相关确定另外一个羟基连接在C-7上(图 2)。化合物1的相对构型依据ROESY谱中H-2和H-14以及H-5和H-12相关, 推测具有2R, 5R, 7R, 10S和2S, 5S, 7S, 10R两种可能的构型, 最后依据计算ECD和X-单晶衍射(图 3) 将化合物1的绝对构型确定为2R, 5R, 7R, 10S, 并将其命名为(2R, 5R, 7R, 10S)-2, 7-dihydroxyl-eudesmane-3(4), 11(12)-diene。

2 化合物1~13神经保护作用

采用MTT法测试化合物1~13对SH-SY5Y细胞的毒性及H₂O₂损伤SH-SY5Y细胞的神经保护活性。化合物14因量较少, 故未进行细胞毒性及神经保护作用研究。多数倍半萜类化合物和二苯基庚烷类化合物在低、中、高三个剂量浓度下细胞存活率与对照组接近(图 4), 未对SH-SY5Y神经细胞造成损伤, 化合物5和6在中、高浓度下能显著提高细胞存活率, 说明益智化学成分在5、10和20 μmol·L^-1浓度下对神经细胞无毒性, SH-SY5Y细胞模型可用于评价这些化合物的神经保护作用。接着, H₂O₂组与空白组相比细胞存活率显著下降(图 5), 说明H₂O₂损伤的SH-SY5Y细胞模型造模成功。化合物1、2、4、5、6、7、12、13和阳性药表没食子儿茶素没食子酸酯(EGCG) 与H₂O₂组相比细胞存活率显著提高(图 5), 说明大多数的化合物对H₂O₂损伤的SH-SY5Y细胞具有神经保护作用, 其中化合物2、6、7、12和13在多个浓度下与EGCG相比细胞存活率升高, 具有显著性差异, 说明益智中倍半萜类成分、二苯基庚烷类成分乃至一些其他类型的小分子都具有神经保护作用; 尤其是化合物6在低、中、高三个浓度下细胞存活率均超过80%, 强于阳性药EGCG对神经细胞的保护作用, 具有显著的神经保护活性(图 5)。

讨论

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益智是我国四大南药之一, 具有益气安神、镇静催眠、提高学习记忆能力、抗氧化等功效, 通常用来治疗神经系统和消化系统疾病。前期研究发现其提取物具有潜在神经保护作用, 但是神经保护活性成分尚不明确。氧化应激和活性氧的积累是导致神经退行性疾病的主要机制, 人体产生过多的活性氧(ROS) 结合脑内黑质区铁离子产生的活性氧自由基阻断线粒体电子转移, 造成氧化应激和神经元的过度损伤和缺失, 最终导致神经退行性疾病的发生^{[26, 27]}。从天然产物中寻求能够对抗氧化应激或者具有抗氧化功能的神经保护剂, 有望开发为对神经退行性疾病具有潜在治疗作用的先导化合物。本研究从益智中分离并鉴定了14个化合物, 包括6个倍半萜类化合物、6个二苯基庚烷类化合物、1个单萜类化合物和1个酯类化合物, 其中化合物1为桉烷型倍半萜类新化合物, 化合物2~6和14为首次从该植物中分离得到, 丰富了益智的化学成分。药理活性研究表明化合物6在低、中、高浓度下具有较好的神经保活作用且无细胞毒活性。因此, 将进一步研究这些化合物的神经保护作用机制, 同时, 以天然产物为对象, 发掘更多的结构相似的小分子化合物, 开发为对神经退行性疾病具有治疗作用的神经保护剂。

实验部分

收起

益智于2020年09月购自昆明市螺蛳湾药材市场, 经云南中医药大学张荣平教授鉴定为姜科植物益智(Alpinia oxyphylla Miq) 的干燥果实, 样品编号为No.2020-0915, 储藏于云南中医药大学中药学院暨云南省南药可持续利用研究重点实验室。

Shimadzu LC-MS-IT-TOF、UV-2401型紫外光谱仪(日本Shimadzu公司); Bruker AV 600型核磁共振波谱仪[布鲁克(北京) 科技有限公司]; NICOLET iS10型红外光谱仪[赛默飞世尔科技(中国) 有限公司]; Jascomodel 1020旋光仪(日本Horiba公司); NU3000 serials制备型高压液相色谱仪(江苏汉邦科技有限公司); LC-52半制备型高效液相色谱仪、YMC-ODS-A填料(北京赛普锐斯科技有限公司); MCI CHP-20P GEL (日本三菱化学公司); Sephadex LH-20 (瑞典Pharmacia公司); Basic C18色谱柱(10 mm × 250 mm, 5 μm, 日本YMC公司); 薄层色谱硅胶板和柱色谱硅胶(上海皓鸿生物医药科技有限公司); 氘代试剂(北京伊诺凯有限公司); 色谱纯乙腈(德国默克公司); 常用有机试剂为国产AR级试剂(云南利妍科技有限公司)。

1 提取和分离

干燥的益智果实20.0 kg粉碎后以60 L 90%乙醇室温浸泡过夜, 然后回流提取2 h, 共3次, 合并3次提取液浓缩至无乙醇味, 以等体积水分散, 用2倍量乙酸乙酯萃取, 共3次, 合并乙酸乙酯层, 得到乙酸乙酯部位。乙酸乙酯部位580.0 g, 经硅胶柱色谱(5.0 kg, 23 cm × 75 cm, 石油醚-乙酸乙酯95:2、95:5、90:10、80:20) 洗脱获得6个流分Fr.1~Fr.6。

Fr.4 (11 g) 经中压MCI gel CHP 20P柱色谱(200 g, 5 cm × 50 cm, 甲醇-水50:50、70:30、90:10) 洗脱, 得到3个组分Fr.4-1~Fr.4-3。Fr.4-1 (8 g) 经硅胶柱色谱, 多次重结晶, 得到化合物10 (800 mg)。Fr.4-2 (0.8 g) 先通过硅胶柱色谱(8 g, 1.5 cm × 35 cm, 石油醚-乙酸乙酯, 95:5→80:20) 和Sephadex LH-20凝胶柱色谱(90 g, 1.5 cm × 190 cm, 甲醇) 分离, 再用制备TLC (石油醚-丙酮, 70:30) 纯化得到化合物13 (45 mg)。类似方法从Fr.4-3 (0.6 g) 中得到11 (60 mg)。

Fr.5 (150 g) 经中压MCI gel CHP 20P柱色谱(1 500 g, 15 cm × 40 cm, 甲醇-水30:70、50:50、70:30、90:10) 洗脱得到4个组分Fr.5-1~Fr.5-4。Fr.5-1 (35 g)经硅胶柱色谱(350 g, 6.0 cm × 30 cm, 石油醚-乙酸乙酯, 90:10→80:20) 得到4个流分Fr.5-1-1~Fr.5-1-4。Fr.5-1-2 (4.5 g) 经硅胶柱色谱(45 g, 1.5 cm × 35 cm, 石油醚-丙酮, 95:5→80:20) 分离, 再通过半制备型HPLC (乙腈-水, 20:80→70:30, 30 min) 纯化, 得到化合物1 (5 mg, t_R = 19.0 min) 和5 (5 mg, t_R = 22.0 min)。将Fr.5-1-3 (7.5 g) 先通过反复硅胶柱色谱和Sephadex LH-20凝胶柱色谱(220 g, 2.5 cm × 190 cm, 甲醇) 分离, 再用制备TLC (石油醚-乙酸乙酯, 50:50) 纯化得到化合物7 (5 mg) 和14 (3 mg)。Fr.5-2 (42 g) 采用硅胶柱色谱(420 g, 6.0 cm × 30 cm, 石油醚-乙酸乙酯, 85:15→40:60) 洗脱得到3个流分Fr.5-2-1~Fr.5-2-3。Fr.5-2-2 (15 g) 经硅胶柱色谱(150 g, 4.5 cm × 35 cm, 二氯甲烷-乙酸乙酯, 98:2→80:20) 得到化合物3 (4 mg) 和6 (15 mg)。Fr.5-2-3 (1.5 g) 经半制备型HPLC (乙腈-水, 40:60) 纯化得到化合物9 (35 mg, t_R = 49.0 min)。Fr.5-3 (45 g) 采用硅胶柱色谱(450 g, 6 cm × 35 cm, 石油醚-丙酮, 90:10→60:40) 分离得到3个流分Fr.5-3-1~Fr.5-3-3。Fr.5-3-1 (0.9 g) 通过半制备型HPLC (乙腈-水, 50:50) 纯化得到化合物2 (11 mg, t_R = 21.0 min)。Fr.5-3-2 (6.5 g) 先通过硅胶柱色谱(65 g, 2.5 cm × 35 cm, 二氯甲烷-乙酸乙酯, 95:5→80:20) 和Sephadex LH-20凝胶柱色谱分离, 再用制备TLC (二氯甲烷-丙酮, 90:10) 纯化得到化合物4 (5 mg)、8 (30 mg) 和12 (25 mg)。

2 结构鉴定

化合物1 白色针状结晶, 熔点186 ℃; [α]$ {}_{\mathrm{D}}^{26.6} $ -45.93 (c 0.30, MeOH); UV (MeOH) λ_max (log ε) 230 (3.24) nm; IR (KBr) ν_max 3 272, 2 974, 2 938, 2 853, 1 642, 1 434, 1 376, 1 188, 1 058, 1 026, 994, 900, 831, 619 cm^-1; HR-ESI-MS m/z 275.140 1 [M+K]⁺ (计算值为275.140 8); ¹H NMR (600 MHz)、¹³C NMR (150 MHz) 数据见表 1。

化合物2 黄色油状物, HR-ESI-MS m/z 235.170 4 [M+H]⁺ (计算值C₁₅H₂₂O₂, 235.169 3); ¹H NMR (CDCl₃, 600 MHz) δ_H: 5.91 (1H, s, H-3), 4.75 (2H, s, H-12), 2.70 (1H, s, H-1a), 2.10 (1H, s, H-1b), 2.00 (3H, s, H-15), 1.98 (1H, m, H-9a), 1.77 (1H, m, H-7), 1.75 (3H, s, H-13), 1.72 (1H, m, H-9b), 1.58 (1H, m, H-8a), 1.51 (3H, m, H-6/H-8b), 1.16 (3H, s, H-14); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 48.1 (t, C-1), 198.9 (s, C-2), 128.6 (d, C-3), 159.8 (s, C-4), 74.4 (s, C-5), 35.6 (t, C-6), 42.4 (d, C-7), 26.4 (t, C-8), 36.1 (t, C-9), 39.9 (s, C-10), 148.5 (s, C-11), 109.5 (t, C-12), 21.0 (q, C-13), 21.4 (q, C-14), 19.5 (q, C-15)。以上数据与文献^[13]报道基本一致, 故鉴定其为α-rotunol。

化合物3 无色油状物, HR-ESI-MS m/z 233.153 3 [M+H]⁺ (计算值C₁₅H₂₀O₂, 233.153 6); ¹H NMR (CDCl₃, 600 MHz) δ_H: 6.27 (1H, s, H-1), 4.85 (1H, s, H-12a), 4.80 (1H, s, H-12b), 2.76 (1H, m, H-6a), 2.38 (2H, m, H-3), 2.25 (1H, m, H-4), 2.11 (2H, m, H-8), 1.78 (3H, s, H-13), 1.51 (1H, m, H-6b), 1.11 (3H, s, H-14), 1.05 (3H, d, J = 7.2 Hz, H-15); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 126.5 (d, C-1), 199.7 (s, C-2), 42.3 (t, C-3), 40.7 (d, C-4), 40.2 (s, C-5), 46.4 (t, C-6), 38.2 (d, C-7), 41.5 (t, C-8), 202.3 (s, C-9), 160.6 (s, C-10), 146.7 (s, C-11), 110.9 (t, C-12), 20.5 (q, C-13), 17.9 (q, C-14), 15.4 (q, C-15)。以上数据与文献^[14]报道基本一致, 故鉴定其为diketone I。

化合物4 粉红色油状物, HR-ESI-MS m/z 235.168 7 [M+H]⁺ (计算值C₁₅H₂₂O₂, 235.169 3); ¹H NMR (CDCl₃, 600 MHz) δ_H: 5.84 (1H, s, H-9), 4.98 (1H, s, H-12a), 4.83 (1H, s, H-12b), 4.34 (1H, m, H-1), 3.24 (1H, m, H-7), 1.99 (1H, m, H-2a), 1.96 (1H, m, H-6a), 1.90 (1H, m, H-3a), 1.88 (1H, m, H-6b), 1.85 (1H, m, H-3b), 1.73 (3H, s, H-13), 1.66 (1H, m, H-2b), 1.47 (1H, m, H-4), 1.35 (3H, s, H-14), 0.95 (3H, d, J = 7.0 Hz, H-15); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 73.2 (d, C-1), 33.0 (t, C-2), 24.9 (t, C-3), 43.5 (d, C-4), 39.1 (s, C-5), 43.1 (t, C-6), 51.6 (d, C-7), 200.1 (s, C-8), 126.7 (d, C-9), 167.5 (s, C-10), 143.6 (s, C-11), 114.5 (t, C-12), 20.1 (q, C-13), 18.4 (q, C-14), 15.3 (q, C-15)。以上数据与文献^[15]报道基本一致, 故鉴定其为(1S, 4S, 5R, 7S)-1-hydroxyl-eremophilane-9(10), 11(12)-diene-8-one。

化合物5 无色油状物, [α]$ {}_{\mathrm{D}}^{19.8} $ +15.79 (c 0.04, MeOH), HR-ESI-MS m/z 251.162 5 [M+H]⁺ (计算值C₁₅H₂₂O₃, 251.169 2); ¹H NMR (CDCl₃, 600 MHz) δ_H: 4.75 (2H, d, J = 2.0 Hz, H-12), 2.65 (1H, m, H-6a), 2.61 (2H, s, H-3), 2.50 (1H, m, H-6b), 2.39 (1H, m, H-7), 2.08 (1H, m, H-9a), 1.86 (2H, m, H-8), 1.75 (3H, s, H-13), 1.70 (1H, m, H-9b), 1.49 (3H, s, H-14), 1.46 (3H, s, H-15); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 144.0 (s, C-1), 207.1 (s, C-2), 51.8 (t, C-3), 76.1 (s, C-4), 174.7 (s, C-5), 28.1 (t, C-6), 43.4 (d, C-7), 27.4 (t, C-8), 36.2 (t, C-9), 71.8 (s, C-10), 149.0 (s, C-11), 110.2 (t, C-12), 20.2 (q, C-13), 27.2 (q, C-14), 26.6 (q, C-15)。以上数据与文献^[16]报道基本一致, 故鉴定其为cyperusol A₁。

化合物6 黄色油状物, HR-ESI-MS m/z 213.086 3 [M+Na]⁺ (计算值C₁₂H₁₄O₂, 213.088 6); ¹H NMR (CDCl₃, 600 MHz) δ_H: 7.78 (1H, d, J = 2.0 Hz, H-4), 7.56 (1H, d, J = 8.0 Hz, H-1), 7.41 (1H, dd, J = 8.0, 2.0 Hz, H-2), 2.83 (1H, m, H-7a), 2.66 (1H, m, H-7b), 2.35 (3H, s, H-14), 2.24 (2H, m, H-8), 1.61 (3H, s, H-15); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 127.3 (d, C-1), 135.3 (d, C-2), 137.9 (s, C-3), 125.3 (d, C-4), 130.5 (s, C-5), 197.7 (s, C-6), 36.0 (t, C-7), 38.6 (t, C-8), 70.2 (s, C-9), 146.8 (s, C-10), 29.2 (q, C-14), 21.1 (q, C-15)。以上数据与文献^[17]报道基本一致, 故鉴定其为(6R, 9S, 10S)-10-hydroxyl-11, 12, 13-trinor-cadinane-4(5)-ene-3-one。

化合物7 无色油状物, C₉H₁₄O₂; ¹H NMR (CDCl₃, 600 MHz) δ_H: 9.45 (1H, s, H-1), 6.84 (1H, d, J = 11.2 Hz, H-3), 6.74 (1H, dd, J = 15.0, 11.2 Hz, H-4), 6.31 (1H, d, J = 15.0 Hz, H-5), 1.87 (3H, s, H-9), 1.41 (6H, s, H-7/H-8); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 195.2 (s, C-1), 138.0 (s, C-2), 150.9 (d, C-3), 122.0 (d, C-4), 148.4 (d, C-5), 71.3 (s, C-6), 29.9 (q, C-7), 29.9 (q, C-8), 9.7 (q, C-9)。以上数据与文献^[18]报道基本一致, 故鉴定其为(2E, 4E)-6-hydroxy-2, 6-dimethylhepta-2, 4-dienal。

化合物8 黄色油状物, [α]$ {}_{\mathrm{D}}^{19.9} $ -6.54 (c 0.11, MeOH), HR-ESI-MS m/z 315.193 5 [M+H]⁺ (计算值C₂₀H₂₆O₃, 315.195 5); ¹H NMR (CDCl₃, 600 MHz) δ_H: 7.22 (2H, m, H-3″/H-5″), 7.12 (3H, m, H-2″/H-4″/H-6″), 6.78 (1H, d, J = 8.0 Hz, H-5′), 6.66 (1H, d, J = 1.8 Hz, H-2′), 6.62 (1H, dd, J = 8.0, 1.8 Hz, H-6′), 3.79 (3H, s, 3′-OCH₃), 3.57 (1H, m, H-3), 2.66 (1H, m, H-1a), 2.55 (3H, m, H-1b/H-7), 1.64 (4H, m, H-2/H-4), 1.44 (3H, m, H-5/H-6a), 1.31 (1H, m, H-6b); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 31.5 (t, C-1), 39.4 (t, C-2), 71.4 (d, C-3), 37.5 (t, C-4), 25.3 (t, C-5), 31.8 (t, C-6), 35.9 (t, C-7), 134.1 (s, C-1′), 111.2 (d, C-2′), 146.6 (s, C-3′), 143.8 (s, C-4′), 114.4 (d, C-5′), 120.9 (d, C-6′), 142.6 (s, C-1″), 128.3 (d, C-2″), 128.4 (d, C-3″), 125.7 (d, C-4″), 128.4 (d, C-5″), 128.3 (d, C-6″)。以上数据与文献^[19]报道基本一致, 故鉴定其为益智醇。

化合物9 无色油状物, HR-ESI-MS m/z 313.177 5 [M+H]⁺ (计算值C₂₀H₂₄O₃, 313.179 8); ¹H NMR (CDCl₃, 600 MHz) δ_H: 7.26 (2H, m, H-3″/H-5″), 7.16 (3H, m, H-2″/H-4″/H-6″), 6.81 (1H, d, J = 7.8 Hz, H-5′), 6.67 (1H, d, J = 1.6 Hz, H-2′), 6.65 (1H, dd, J = 7.8, 1.6 Hz, H-6′), 3.84 (3H, s, 3′-OCH₃), 2.81 (2H, t, J = 7.5 Hz, H-1), 2.67 (2H, t, J = 7.5 Hz, H-2), 2.59 (2H, t, J = 7.9 Hz, H-7), 2.39 (1H, t, J = 7.7 Hz, H-4), 1.59 (4H, m, H-5/H-6); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 29.7 (t, C-1), 44.7 (t, C-2), 210.4 (s, C-3), 43.0 (t, C-4), 23.5 (t, C-5), 31.1 (t, C-6), 35.8 (t, C-7), 133.2 (s, C-1′), 111.2 (d, C-2′), 146.5 (s, C-3′), 56.0 (q, 3′-OCH₃), 144.0 (s, C-4′), 114.4 (d, C-5′), 120.9 (d, C-6′), 142.3 (s, C-1″), 128.4 (d, C-2″), 128.5 (d, C-3″), 125.9 (d, C-4″), 128.5 (d, C-5″), 128.4 (d, C-6″)。以上数据与文献^[20]报道基本一致, 故鉴定其为益智酮A。

化合物10 无色油状物, [α]$ {}_{\mathrm{D}}^{20.1} $ +1.04 (c 0.27, MeOH), HR-ESI-MS m/z 283.168 6 [M+H]⁺ (计算值C₁₉H₂₂O₂, 283.169 3); ¹H NMR (CDCl₃, 600 MHz) δ_H: 7.25 (4H, m, H-3′/H-5′/H-3″/H-5″), 7.15 (6H, m, H-2′/H-4′/H-6′/H-2″/H-4″/H-6″), 4.02 (1H, m, H-5), 2.86 (2H, t, J = 7.6 Hz, H-1), 2.76 (1, H, m, H-7a), 2.70 (2H, t, J = 7.6 Hz, H-2), 2.64 (1H, m, H-7b), 2.50 (2H, d, J = 5.7 Hz, H-4), 1.77 (1H, m, H-6a), 1.65 (1H, m, H-6b); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 29.5 (t, C-1), 45.0 (t, C-2), 211.0 (s, C-3), 49.4 (t, C-4), 66.9 (d, C-5), 38.1 (t, C-6), 31.8 (t, C-7), 140.7 (s, C-1′), 128.3 (d, C-2′), 128.6 (d, C-3′), 126.2 (d, C-4′), 128.6 (d, C-5′), 128.3 (d, C-6′), 141.8 (s, C-1″), 128.4 (d, C-2″), 128.5 (d, C-3″), 125.9 (d, C-4″), 128.5 (d, C-5″), 128.4 (d, C-6″)。以上数据与文献^[21]报道基本一致, 故鉴定其为(5R)-5-羟基-1, 7-二苯基-3-庚酮。

化合物11 白色粉末, [α]$ {}_{\mathrm{D}}^{20.0} $ -14.22 (c 0.04, MeOH), HR-ESI-MS m/z 299.163 7 [M+H]⁺ (计算值C₁₉H₂₂O₃, 299.164 2); ¹H NMR (CDCl₃, 600 MHz) δ_H: 7.28 (2H, d, J = 7.6 Hz, H-2′/H-6′), 7.19 (1H, m, H-4′), 7.17 (2H, m, H-3′/H-5′), 7.04 (2H, d, J = 7.7 Hz, H-2″/H-6″), 6.74 (2H, d, J = 7.7 Hz, H-3″/H-5″), 4.03 (1H, m, H-5), 2.89 (2H, t, J = 7.5 Hz, H-1), 2.72 (2H, t, J = 7.6 Hz, H-2), 2.69 (1, H, m, H-7a), 2.58 (1H, m, H-7b), 2.52 (2H, d, J = 5.7 Hz, H-4), 1.76 (1H, m, H-6a), 1.62 (1H, m, H-6b); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 29.6 (t, C-1), 45.2 (t, C-2), 211.4 (s, C-3), 49.4 (t, C-4), 67.0 (d, C-5), 38.3 (t, C-6), 30.9 (t, C-7), 140.8 (s, C-1′), 128.4 (d, C-2′), 128.7 (d, C-3′), 126.4 (d, C-4′), 128.7 (d, C-5′), 128.4 (d, C-6′), 134.0 (s, C-1″), 129.7 (d, C-2″), 115.4 (d, C-3″), 153.9 (s, C-4″), 115.4 (d, C-5″), 129.7 (d, C-6″)。以上数据与文献^[22]报道基本一致, 故鉴定其为(5S)-5-羟基-7-(4″-羟基)-苯基-1-苯基-3-庚酮。

化合物12 黄色粉末, [α]$ {}_{\mathrm{D}}^{19.9} $ -22.66 (c 0.03, MeOH), HR-ESI-MS m/z 329.174 0 [M+H]⁺ (计算值C₂₀H₂₄O₄, 329.174 7); ¹H NMR (CDCl₃, 600 MHz) δ_H: 7.28 (2H, m, H-2′/H-6′), 7.18 (3H, m, H-3′/H-4′/H-5′), 6.82 (1H, d, J = 8.0 Hz, H-5″), 6.70 (1H, d, J = 2.0 Hz, H-2″), 6.67 (1H, dd, J = 8.0, 2.0 Hz, H-6″), 4.03 (1H, m, H-5), 3.87 (3H, s, 3″-OCH₃), 2.90 (2H, t, J = 7.5 Hz, H-1), 2.75 (2H, t, J = 7.5 Hz, H-2), 2.72 (1H, m, H-7a), 2.60 (1H, m, H-7b), 2.55 (2H, m, H-4), 1.77 (1H, m, H-6a), 1.62 (1H, m, H-6b); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 29.6 (t, C-1), 45.2 (t, C-2), 211.3 (s, C-3), 49.4 (t, C-4), 67.0 (d, C-5), 38.5 (t, C-6), 31.6 (t, C-7), 140.8 (s, C-1′), 128.4 (d, C-2′), 128.7 (d, C-3′), 126.4 (d, C-4′), 128.7 (d, C-5′), 128.4 (d, C-6′), 133.9 (s, C-1″), 111.2 (d, C-2″), 146.5 (s, C-3″), 56.0 (q, 3′-OCH₃), 143.9 (s, C-4″), 114.4 (d, C-5″), 121.1 (d, C-6″)。以上数据与文献^[23]报道基本一致, 故鉴定其为(5S)-5-羟基-7-(4″-羟基-3″-甲氧基)-苯基-1-苯基-3-庚酮。

化合物13 无色油状物, HR-ESI-MS m/z 327.157 6 [M+H]⁺ (计算值C₂₀H₂₂O₄, 327.159 1); ¹H NMR (CDCl₃, 600 MHz) δ_H: 7.29 (2H, d, J = 7.8 Hz, H-2′/H-6′), 7.19 (3H, m, H-3′/H-4′/H-5′), 6.83 (1H, d, J = 8.0 Hz, H-5″), 6.68 (1H, d, J = 1.9 Hz, H-2″), 6.66 (1H, dd, J = 8.0, 2.0 Hz, H-6″), 5.43 (2H, s, H-4), 3.86 (3H, s, 3″-OCH₃), 2.92 (2H, t, J = 7.8 Hz, H-1), 2.85 (2H, t, J = 7.8 Hz, H-7), 2.59 (2H, t, J = 7.8 Hz, H-2), 2.55 (2H, t, J = 7.8 Hz, H-6); ¹³C NMR (CDCl₃, 150 MHz) δ_C: 31.7 (t, C-1), 40.5(t, C-2), 203.5 (s, C-3), 99.8 (t, C-4), 193.3 (s, C-5), 40.1 (t, C-6), 31.5 (t, C-7), 140.8 (s, C-1′), 128.4 (d, C-2′), 128.7 (d, C-3′), 126.4 (d, C-4′), 128.7 (d, C-5′), 128.4 (d, C-6′), 132.7 (s, C-1″), 111.1 (d, C-2″), 146.5 (s, C-3″), 56.0 (q, 3″-OCH₃), 144.5 (s, C-4″), 114.5 (d, C-5″), 121.0 (d, C-6″)。以上数据与文献^[24]报道基本一致, 故鉴定其为7-(4″-羟基-3″-甲氧基)-苯基-1-苯基-3, 5-庚二酮。

化合物14 无色油状物, HR-ESI-MS m/z 391.281 8 [M+H]⁺ (计算值C₂₄H₃₈O₄, 391.284 3); ¹H NMR (CDCl₃, 600 MHz) δ_H: 8.06 (4H, d, J = 7.8 Hz, H-3/H-4/H-6/H-7), 4.24 (4H, m, H-1′/H-1″), 1.18~1.64 (16H, m, H-3′/H-4′/H-5′/H-7′/H-3″/H-4″/H-5″/H-7″), 1.71 (2H, m, H-2′/H-2″), 0.93 (6H, t, J = 8.0 Hz, H-8′/H-8″), 0.90 (6H, t, J = 8.0 Hz, H-6′/H-6″); ¹³C NMR (150 MHz, CDCl₃) δ_C: 166.0 (s, C-1/C-8), 134.3 (s, C-2/C-5), 129.5 (d, C-3/C-4/C-6/C-7), 67.8 (t, C-1′/C-1″), 39.0 (d, C-2′/C-2″), 31.0 (t, C-3′/C-3″), 29.0 (t, C-4′/C-4″), 23.0 (t, C-5′/C-5″), 14.0 (q, C-6′/C-6″), 24.0 (t, C-7′/C-7″), 11.1 (q, C-8′/C-8″)。以上数据与文献^[25]报道基本一致, 故鉴定其为bis-(2-ethylhexyl) terephthalate。

3 晶体结构测定

化合物1选择0.70 mm × 0.37 mm × 0.06 mm大小的斜方晶, 利用布鲁克D8 QUEST型晶体衍射仪(铜靶) 进行测定, 总衍射次数为35 227, 其中观测到2 711次(R_int = 0.060 1), I > 2σ(I), R₁ = 0.055 2, wR(F²) = 0.131 3, F² = 0.131 4, Flack parameter = 0.28(9)。晶体数据如下: C₁₅H₂₄O₂, M = 236.34, a = 7.807 4(3) Å, b = 9.862 6(4) Å, c = 17.792 9(7) Å, α = 90°, β = 90°, γ = 90°, V = 1 370.08(9) Å³, T = 100.(2) K, 晶格尺寸P212121, Z = 4, μ(Cu Kα) = 0.576 mm^-1。

4 神经保护作用活性筛选

使用CCK-8法测试化合物1~13对H₂O₂损伤SH-SY5Y细胞的神经保护活性。将细胞分为对照组、H₂O₂组、药物A组和药物B组, 每组设5个复孔。将处于对数生长期的细胞SH-SY5Y细胞, 以每毫升1×10⁴个的密度接种于96孔细胞培养板上。对照组分为溶剂对照组和空白对照组; H₂O₂组用SH-SY5Y培养24 h后, 弃去旧培养基, 用300 μmol·L^-1 H₂O₂处理24 h; 药物A组用含有不同浓度(5、10和20 μmol·L^-1) 药物处理24 h, A组用于评价化合物对神经细胞有无毒性; 药物B组: 使用EGCG (阳性对照) 和不同浓度(5、10和20 μmol·L^-1) 药物预处理2 h后, 用300 μmol·L^-1 H₂O₂处理24 h, B组用于评价化合物的对受损的神经细胞有无保护作用。将细胞培养基更换为孔中含有10% CCK-8的DMEM, 孵育2 h后, 使用酶标仪在450 nm处读取每个孔的吸光度(A) 值, 计算各组细胞存活率。实验重复3次。

细胞存活率(%) = [A(药物组)-A(空白对照组)] / [A(溶剂对照组)-A(空白对照组)] × 100%

数据采用单因素分析, P < 0.05认为有统计学意义。

作者贡献: 邱成省负责化合物的分离、结构鉴定及文章的撰写; 王吉负责化合物的神经保护作用活性筛选; 穆利萍负责药材提取及完成部分数据分析; 张荣平和陈兴龙负责设计实验、提供实验指导、审阅文章, 并最终定稿。

利益冲突: 作者声明不存在任何利益冲突。

基金

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国家自然科学基金资助项目(32160107)
云南省基础研究计划-青年项目(202201AU070166)
“云岭学者”支持计划
云南省基础研究计划中医联合专项－面上项目(202101AZ070001-222)
云南省南药可持续利用重点实验室开放课题(202105AG070012XS2201)

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2023年第58卷第9期

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doi: 10.16438/j.0513-4870.2023-0080

接收时间：2023-01-29
首发时间：2025-11-21
出版时间：2023-09-12

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收稿日期：2023-01-29
修回日期：2023-03-01

基金

国家自然科学基金资助项目(32160107)

云南省基础研究计划-青年项目(202201AU070166)

“云岭学者”支持计划

云南省基础研究计划中医联合专项－面上项目(202101AZ070001-222)

云南省南药可持续利用重点实验室开放课题(202105AG070012XS2201)

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云南中医药大学中药学院暨云南省南药可持续利用研究重点实验室, 云南昆明 650500

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*陈兴龙, E-mail: chxlkib@163.com

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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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