CN106220643A - 灵芝内酯d及其药物组合物与其在制药和食品中的应用 - Google Patents
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Abstract
提供了灵芝内酯D(1)及其药物组合物与其在制药和食品中的应用和其制备方法。从赤芝中分离得到一个新化合物灵芝内酯D，研究表明其可抑制TGF‑β1介导的肾小管上皮细胞α‑SMA，纤连蛋白和I型胶原表达，提示灵芝内酯D可明显抑制肾脏组织纤维化；也发现其可选择性抑制TGF‑β1诱导的肾小管上皮细胞Smad3磷酸化而对Smad2，PI3K，ERK，p‑38的磷酸化及Smad4，Smad7的蛋白表达无明显影响，鉴于Smad3磷酸化是介导多种器官纤维化的共同信号通路，提示其对于包括肾脏纤维化在内的器官纤维化的治疗作用。
Description
技术领域：
本发明属于药物技术和食品领域，具体地涉及灵芝内酯D及其药物组合物，以及其在制备治疗肾脏纤维化或慢性肾病或器官纤维化的药物或食品中的应用。
背景技术：
久病(慢性病)易导致器官纤维化，肾脏纤维化与肺、肝纤维化类似，是导致器官衰竭的必经过程。遗憾的是由于自身衰老，药物误用，环境污染等多种因素，器官纤维化明显增多。以肾病为例，我国慢性肾病的发病率已超过10％，慢性肾病经肾脏纤维化进展至终末期肾衰竭，目前我国的尿毒症患者也明显增多，其医疗开支惊人。防止纤维化可延缓器官衰竭，已有研究表明TGF‐β1介导的Smad3磷酸化是多种器官纤维化的主要病理机制之一，因此抑制Smad3磷酸化是抗纤维化和防止器官衰竭的关键，但相关药物缺乏。
灵芝作为著名中药在我国已有2000多年的应用历史，在中国传统文化中其被称为仙草，中医也将其归为安神药，世界称其为可治疗百病的药材，足见其药理作用广泛。本发明人在中医“久病必虚”，“久病及肾”，“虚则补之”的理论指导下，近些年提出灵芝之补不仅仅体现在其可调节免疫，而且还可能通过抗氧化应激和炎症实现微观的“以通为补”作用，这可能是灵芝发挥作用的另一重要途径。为此，本发明人在国家自然科学基金(U1202222,81525026,21472199)等多个项目的资助下，多年来系统地开展了灵芝属中药的研究。对赤芝(Ganoderma lucidum)的子实体研究中，发现了灵芝内酯D及其抗慢性肾病或抗器官纤维化作用，而现有技术中未见有本发明涉及的化合物及其功能的相关报道。
发明内容：
本发明的目的在于提供具有抗慢性肾病或抗肾脏纤维化或抗器官纤维化的化合物灵芝内酯D(又名赤芝内酯D，灵芝莱克酮D；Lingzhilactone D)及其在制备抗慢性肾病和/或抗肾脏纤维化和/或抗器官纤维化的药物中的应用，以及以该化合物为有效成分的药物组合物。本发明的另一目的也在于提供灵芝内酯的天然制备方法。
本发明的上述目的是由下述的技术方案得以实现的：
下述结构式所示的灵芝内酯D(1)，
制备灵芝内酯D(1)的方法，取赤芝，粉碎，用95％乙醇回流提取，合并提取液并减压回收溶剂得粗提取物，将粗提取物混悬于水中，然后用等体积乙酸乙酯萃取三次，合并萃取液，减压浓缩得乙酸乙酯萃取物，该萃取物经硅胶200‐300目柱层析，99:1，98:2，97:3，96:4，95:5，94:6，93:7，92:8，90:10，85:15，80:20，50:50的氯仿‐甲醇系统梯度洗脱，每种溶剂梯度为1.5倍柱体积，按照每份500mL收集得7个合并组份，其中组分3经MCI gel CHP20P柱层析，甲醇‐水45％‐100％，梯度变化程序为5％，洗脱得组分3.1‐3.11，其中组分3.2经Sephadex LH‐20甲醇洗脱，TLC跟踪，合并相同斑点得到组分3.2.1‐3.2.4，组分3.2.4经RP‐18柱层析，甲醇‐水,40％‐70％和半制备HPLC，甲醇‐水，40％纯化得灵芝内酯D(1)，该化合物是一个消旋体，手性HPLC 250mm×4.6mm,i.d.,5μm,Daicel Chiralpak IC拆分，n‐hexane/ethanol,70:30得(+)‐灵芝内酯D和(-)‐灵芝内酯D。
治疗肾脏纤维化或慢性肾病或器官纤维化的药物组合物，含有治疗有效 量的灵芝内酯D(1)和药学上可接受的载体。
所述的灵芝内酯D(1)在制备治疗肾脏纤维化或慢性肾病或器官纤维化药物中的应用。
所述的灵芝内酯D(1)在制备治疗肾脏纤维化或慢性肾病或器官纤维化的保健食品中的应用。
本发明化合物可以单独直接应用或组合应用，也可以与其它药物包括植物提取物组成复方的形式使用，可以使用不同的药用辅料，制成多种固体制剂和液体制剂。将本发明的药物组合物以单位体重服用量的形式使用。本发明的药物可经不同给药途径给药。使用量可根据给药途径、患者的年龄、体重、所治疗疾病的类型和严重程度等变化进行一次或多次使用。
附图说明：
图1表示化合物(+)‐灵芝内酯D抑制TGF‐β1诱导的肾小管上皮细胞纤维化作用。A：(+)‐灵芝内酯D的mRNA水平抗肾脏纤维化作用，分组从左至右分别为：对照组(不加TGF‐β1和受试药物)，(+)‐1(10μM)组(不加TGF‐β1，只加(+)‐1的药物组，TGF‐β1+(+)‐1(0μM)组，TGF‐β1+(+)‐1(1μM)组，TGF‐β1+(+)‐1(3μM)组，TGF‐β1+(+)‐1(10μM)组；B：(+)灵芝内酯D的蛋白水平抗肾脏纤维化作用，分组从左至右分别为：对照组(不加TGF‐β1和受试药物)，(+)‐1(10μM)组(不加TGF‐β1，只加(+)‐1的药物组，TGF‐β1+(+)‐1(0μM)组，TGF‐β1+(+)‐1(1μM)组，TGF‐β1+(+)‐1(3μM)组，TGF‐β1+(+)‐1(10μM)组。
图2化合物(+)‐灵芝内酯D选择性抑制TGF‐β1诱导的肾小管上皮细胞Smad3磷酸化作用。图2a：灵芝呋喃A抑制Smad3而非Smad2的磷酸化；图2b：A：灵芝呋喃A不抑制Smad4，Smad7的表达；B：灵芝呋喃A对ERK，PI3K，p38蛋白的磷酸化水平无抑制作用。
具体实施方式：
下面结合附图，用本发明的实施例来进一步说明本发明的实质性内容， 但并不以此来限定本发明。根据本发明的实质对本发明进行的简单改进都属于本发明的范围。
实施例1：
化合物1的分离纯化：
取赤芝(80kg)，粉碎，用95％乙醇回流提取(2x 360L x 2h)，合并提取液并减压回收溶剂得粗提取物，将粗提取物混悬于水中，然后用等体积乙酸乙酯萃取三次，合并萃取液，减压浓缩得乙酸乙酯萃取物，该萃取物经硅胶(200‐300目)柱层析，氯仿‐甲醇系统梯度(99:1，98:2，97:3，96:4，95:5，94:6，93:7，92:8，90:10，85:15，80:20，50:50)洗脱，每种溶剂梯度为1.5倍柱体积，按照每份500mL收集得7个合并组份。其中组分3经MCI gel CHP 20P柱层析，甲醇‐水(45％‐100％，梯度变化程序为5％)洗脱得组分3.1‐3.11。其中组分3.2(8.4g)经Sephadex LH‐20(甲醇)洗脱，TLC跟踪，合并相同斑点得到组分3.2.1‐3.2.4。组分3.2.4(350mg)经RP‐18柱层析(甲醇‐水,40％‐70％)和半制备HPLC(甲醇‐水，40％)纯化得灵芝内酯D(1)(15mg)。该化合物是一个消旋体，手性HPLC(250mm×4.6mm,i.d.,5μm,DaicelChiralpak IC)拆分(n‐hexane/ethanol,70:30)得(+)‐灵芝内酯D(5.7mg)和(-)‐灵芝内酯D(5.9mg)。
化合物1的结构确证：
化合物1的结构式如下所示：
化合物1的结构鉴定数据：
Table 1.1H(600 MHz)and 13C(150 MHz)NMR data of 1 in acetone‐d6(δinppm,J in Hz).
灵芝内酯D(Lingzhilactone D)(1).Yellow solid； UV(MeOH)λmax(logε)370(3.59),260(3.87),225(4.15)nm；CD(MeOH)Δε218–7.89,Δε375+1.23；(+)‐1}；UV(MeOH)λmax(logε)370(3.57),260(3.88),225(4.11)nm；CD(MeOH)Δε218+9.80,Δε375–3.85；(-)‐1}；ESIMS m/z 347[M-H]-；HREIMS m/z 347.0765[M-H]-(calcd for C17H15O8,347.0767).1H and 13C NMR data,seeTable.
(‐)‐1的晶体衍射数据：
Crystal data for cu_qylz2b_0m:C17H16O8,M＝348.30,monoclinic, α＝90.00°,β＝102.694(2)°,γ＝90.00°,T＝100(2)K,space group P21,Z＝2,μ(CuKα)＝1.020 mm‐1,5682reflections measured,2079independent reflections(Rint＝0.0482).The finalR1values were 0.1075(I>2σ(I)).The final wR(F2)values were 0.2890(I>2σ(I)).Thefinal R1values were 0.1080(all data).The final wR(F2)values were 0.2892(alldata).The goodness of fit on F2was 1.212.Flack parameter＝‐0.2(8).Thedeposition number CCDC 1491784for(‐)‐1can be obtained free of charge from TheCambridge Crystallographic Data Centre via www.
ccdc.cam.ac.uk/data_request/cif.
显示(‐)‐灵芝内酯D[(‐)‐1]的绝对构型
实施例2：
实施例1中化合物(+)‐灵芝内酯D，按常规法加注射用溶媒，精滤，灌封灭菌后可制成注射液。
实施例3：
实施例1中化合物(+)‐灵芝内酯D，将其溶于无菌注射用水中，用无菌漏斗过滤，分装，低温冷冻干燥后无菌熔封即得粉针剂。
实施例4：
实施例1中化合物(+)‐灵芝内酯D，按常规法配以各种药用辅料可制成片剂或胶囊剂。
使用实施例1中化合物(+)‐灵芝内酯D作为药物活性成分，使用几种常规赋形剂作为制备组合药物片剂或胶囊剂的辅料成分，按照常规方法制成每片或每粒胶囊含有药物成分10‐300mg的样品。
实施例5：
本发明化合物(+)‐灵芝内酯D的体外抗肾脏纤维化作用。
实验方法：参照文献(Journal of the American Society of Nephrology 2015,26,1827–1838)报道的方法进行。采用的细胞类型为大鼠肾小管上皮细胞(NRK‐52E)，培养基为含10％胎牛血清的DMEM(Gibco,Life Technologies)，(+)‐灵芝内酯D用DMSO溶解。NRK‐52E细胞采用图1中标识的浓度，TGF‐β1刺激的浓度为10ng/mL，常规培养，分组。(+)‐灵芝内酯D与细胞共孵育1小时(预防性给药)，然后用TGF‐β1刺激36或48小时，刺激完毕后(48小时)，提取细胞总蛋白，采用免疫印迹法(Western Blot)检测纤维化指标α‐SMA，纤连蛋白(fibronectin)和I型胶原(collagen I)蛋白水平；或刺激完毕后(36小时)采用实时荧光定量PCR(real‐time PCR)检测纤维化指标α‐SMA，纤连蛋白(fibronectin)和I型胶原(collagen I)的mRNA表达水平变化。统计方法为ANOVA.
实验结果：图1A显示，与阳性对照组相比，(+)‐灵芝内酯D可浓度依赖性地降低TGF‐β1介导的NRK‐52E细胞α‐SMA，纤连蛋白(fibronectin)和I型胶原(collagen I)的mRNA表达水平。图1B表明，与阳性对照组相比，(+)‐灵芝内酯D可浓度依赖性地降低TGF‐β1介导的NRK‐52E细胞α‐SMA，纤连蛋白(fibronectin)和I型胶原(collagen I)的蛋白表达水平。
实施例6：
本发明化合物对TGF‐β1介导的NRK‐52E细胞Smad3磷酸化水平的抑制作用。
实验方法：参照文献(Journal of the American Society of Nephrology 2015,26,1827–1838；CN 201310577167.X)报道的方法进行。采用的细胞类型为大鼠肾小管上皮细胞(NRK‐52E)，培养基为含10％胎牛血清的DMEM(Gibco,Life Technologies)，(+)‐灵芝内酯D用DMSO溶解。NRK‐52E细胞采用图2中标识 的浓度，TGF‐β1刺激的浓度为10ng/mL，常规培养，分组。(+)‐灵芝内酯D与细胞共孵育1小时(预防性给药)，然后用TGF‐β1刺激1小时，刺激完毕后，提取细胞总蛋白，采用免疫印迹法(Western Blot)检测Smad2，Smad3磷酸化水平，Smad4，Smad7蛋白表达水平及p38，PI3K，ERK的磷酸化水平。统计方法为ANOVA。
实验结果：图2a显示，与阳性对照组相比，(+)‐灵芝内酯D可浓度依赖性地降低TGF‐β1介导的NRK‐52E细胞Smad3磷酸化，而对Smad2磷酸化水平无影响。图2b的A图显示，(+)‐灵芝内酯D对TGF‐β1介导的NRK‐52E细胞Smad4，Smad7表达无影响，对p38，PI3K，ERK的磷酸化水平也无明显影响。
综上所述，(+)‐灵芝内酯D可明显抑制肾脏组织纤维化；NRK‐52E细胞上(+)‐灵芝内酯D还可选择性抑制Smad3磷酸化水平，提示其对于包括肾脏纤维化在内的器官纤维化的治疗作用。
Claims (5)
1.下述结构式所示的灵芝内酯D(1)，
2.制备权利要求1所述的灵芝内酯D(1)的方法，取赤芝，粉碎，用95％乙醇回流提取，合并提取液并减压回收溶剂得粗提取物，将粗提取物混悬于水中，然后用等体积乙酸乙酯萃取三次，合并萃取液，减压浓缩得乙酸乙酯萃取物，该萃取物经硅胶200‐300目柱层析，99:1，98:2，97:3，96:4，95:5，94:6，93:7，92:8，90:10，85:15，80:20，50:50的氯仿‐甲醇系统梯度洗脱，每种溶剂梯度为1.5倍柱体积，按照每份500mL收集得7个合并组份，其中组分3经MCI gel CHP 20P柱层析，甲醇‐水45％‐100％，梯度变化程序为5％，洗脱得组分3.1‐3.11，其中组分3.2经Sephadex LH‐20甲醇洗脱，TLC跟踪，合并相同斑点得到组分3.2.1‐3.2.4，组分3.2.4经RP‐18柱层析，甲醇‐水,40％‐70％和半制备HPLC，甲醇‐水，40％纯化得灵芝内酯D(1)，该化合物是一个消旋体，手性HPLC 250mm×4.6mm,i.d.,5μm,Daicel ChiralpakIC拆分，n‐hexane/ethanol,70:30得(+)‐灵芝内酯D和(-)‐灵芝内酯D。
3.治疗肾脏纤维化或慢性肾病或器官纤维化的药物组合物，含有治疗有效量的权利要求1所述的灵芝内酯D(1)和药学上可接受的载体。
4.权利要求1所述的灵芝内酯D(1)在制备治疗肾脏纤维化或慢性肾病或器官纤维化药物中的应用。
5.权利要求1所述的灵芝内酯D(1)在制备治疗肾脏纤维化或慢性肾病或器官纤维化的保健食品中的应用。
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| CN114835724A (zh) * | 2022-03-29 | 2022-08-02 | 中国科学院昆明植物研究所 | （+/-）-sproganoapplanain A及其药物组合物与应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018177301A1 (zh) | 2017-04-01 | 2018-10-04 | 郑州大学 | 15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物及其在制备抗纤维化药物中的应用 |
| CN114835724A (zh) * | 2022-03-29 | 2022-08-02 | 中国科学院昆明植物研究所 | （+/-）-sproganoapplanain A及其药物组合物与应用 |
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