WO2021179711A1 - 一种从金钗石斛中提取化合物的工艺及应用 - Google Patents

一种从金钗石斛中提取化合物的工艺及应用 Download PDF

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WO2021179711A1
WO2021179711A1 PCT/CN2020/134917 CN2020134917W WO2021179711A1 WO 2021179711 A1 WO2021179711 A1 WO 2021179711A1 CN 2020134917 W CN2020134917 W CN 2020134917W WO 2021179711 A1 WO2021179711 A1 WO 2021179711A1
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extract
fractions
formula
ethyl acetate
volume ratio
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French (fr)
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曾军
陈惠琴
戴好富
曹雪
梅文莉
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中国热带农业科学院热带生物技术研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/22—Separation; Purification; Stabilisation; Use of additives
    • C07C231/24—Separation; Purification
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C235/34—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms

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  • the invention belongs to the field of biotechnology, and particularly relates to a process and application for extracting compounds from Dendrobium nobile.
  • Diabetes Diabetes mellitus, DM
  • DM Diabetes mellitus
  • Type II patients account for more than 90% of the total number of diabetes, but the precise molecular mechanism of its occurrence and development is not yet fully understood. In recent years, many clinical experimental evidences have accumulated that the pathogenesis of type 2 diabetes is related to insulin resistance caused by obesity and inflammation.
  • Inflammation is a series of immune responses of the body and cells to harmful external physical, chemical and biological stimuli.
  • the common manifestations of the reaction include fever, redness, pain, and dysfunction.
  • inflammation is the defensive response of tissues and organs to injury or infection, which is beneficial to the body, but a long-term inflammatory response can induce the body to produce excessive and abnormal reactions, and even induce many diseases, such as diabetes and atherosclerosis. Sclerosis, Alzheimer's disease, cancer, etc.
  • steroid hormones and non-steroids are often used clinically to treat inflammation, and the human body will produce a series of toxic side effects after taking it, such as liver and gastrointestinal damage and cardiovascular system damage.
  • plant-derived drugs have slow and mild effects, strong durability, low toxic and side effects, and are not easily tolerated by the body. Therefore, it is necessary to find substances with hypoglycemic and anti-inflammatory activities from plants to provide for the development of new diabetes and inflammatory drugs. Lead compound.
  • the purpose of the present invention is to provide a process and application for extracting compounds from Dendrobium nobile.
  • the process of the present invention extracts compounds of formula I and II from the stems of the medicinal plant Dendrobium nobile, which can effectively inhibit the key enzymes of blood sugar levels.
  • the anti-inflammatory activity was evaluated based on the model of LPS-induced NO production by RAW264.7.
  • the compounds shown in I and II can inhibit the production of NO and reflect the anti-inflammatory activity.
  • the present invention provides a process for extracting compounds from Dendrobium nobile, including the following steps:
  • Extract A Prepares the extract A and water into a suspension, sequentially extract with petroleum ether, ethyl acetate and n-butanol, screen the three extracts, and concentrate the ethyl acetate extract to make extract B;
  • the volume ratio of extract A to water in step B) is 1: (0.5-2);
  • the volume ratio of the extract A to petroleum ether is 1: (0.5-2); the volume ratio of the extract A to ethyl acetate is 1: (0.5-2); the extract A and n-butanol The volume ratio is 1: (0.5 ⁇ 2).
  • the gradient elution of the mixed lotion of petroleum ether and ethyl acetate in step C) is specifically: the volume ratio of petroleum ether and ethyl acetate uniformly decreases from 20:1 to 0:1 within 80-120 hours,
  • the total time of the gradient elution is preferably 90 to 110 hours, more preferably 100 hours.
  • the obtained fractions are concentrated under reduced pressure, and subjected to thin-layer chromatography spot plate detection, and similar fractions are combined according to the color development.
  • the gradient elution in step D) is specifically: sequentially eluting with a methanol aqueous solution with a mass concentration of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, each The elution time of each elution gradient is equal, and the total elution time is 24 to 48 hours, more preferably 32 to 36 hours.
  • the volume ratio of methanol to chloroform in the step E) is 1: (0.5-2).
  • the present invention provides an application of the compound represented by formula I and the compound represented by formula II extracted by the above-mentioned process in the preparation of medicines for preventing and treating diabetes and relieving inflammation.
  • the drug for preventing and treating diabetes is an ⁇ -glucosidase activity inhibitor; the drug for relieving inflammation is an inhibitor of NO production.
  • the present invention provides a pharmaceutical preparation, which comprises a compound represented by formula I or a compound represented by formula II obtained by the above-mentioned process extraction and pharmaceutically acceptable excipients.
  • the dosage form of the pharmaceutical preparation is tablet, capsule, pill, granule, decoction, ointment, lotion, oral liquid, drop pill or syrup.
  • the present invention provides a process for extracting compounds from Dendrobium nobile, including the following steps: A) Crushing Dendrobium nobile, leaching with 1 to 3 times the volume of an aqueous ethanol solution for 2 to 5 times, and the resulting extract is filtered Combine and concentrate to obtain extract A; B) Prepare extract A and water into a suspension, and then extract with petroleum ether, ethyl acetate and n-butanol, and concentrate the ethyl acetate extract to make extract B C) Take the extract B, pass it through a decompression column, eluted with a mixed washing liquid of petroleum ether and ethyl acetate, and finally wash the column with acetone, collect the fractions, concentrate and combine the obtained fractions, and finally obtain 16 streams Fr.1 ⁇ Fr.16; D) Pass the fraction Fr.13 through a reversed-phase column and perform gradient elution with a 30-100% methanol aqueous solution to obtain 14 fractions, denoted
  • the research of the present invention shows that the compounds represented by formula I and II can inhibit the activity of ⁇ -glucosidase, and the anti-inflammatory activity is evaluated based on the model of LPS-induced NO production by RAW264.7, and the compounds represented by I and II can inhibit the production of NO , Reflects anti-inflammatory activity. Therefore, it is shown that the compound can be used to prepare foods and/or medicines for the treatment and/or prevention of diabetes and inflammation.
  • Figure 1 is a 1 H NMR spectrum of the compound represented by formula I in Example 1 of the present invention.
  • Figure 2 is a 13 C NMR+DEPT135 spectrum of the compound represented by formula I in Example 1 of the present invention.
  • Figure 3 is the HSQC profile of the compound represented by formula I in Example 1 of the present invention.
  • Figure 4 is a 1 H NMR spectrum of the compound represented by formula II in Example 1 of the present invention.
  • Figure 5 is a 13 C NMR+DEPT135 spectrum of the compound represented by formula II in Example 1 of the present invention.
  • Figure 6 is the HSQC spectrum of the compound represented by formula II in Example 1 of the present invention.
  • the present invention provides a process for extracting compounds from Dendrobium nobile, including the following steps:
  • Extract A Prepares the extract A and water into a suspension, sequentially extract with petroleum ether, ethyl acetate and n-butanol, screen the three extracts, and concentrate the ethyl acetate extract to make extract B;
  • Dendrobium nobile Lindl. belongs to the Orchidaceae Dendrobium (Dendrobium Sw.) plant. It is also called nobile stone, nobile bud, and yellow grass. It is a traditional Chinese medicine of my country and is known as the "Dendrobium Sw.” It is often used as medicine with fresh or dried stems. Dendrobium nobile in my country is mainly distributed in the subtropical regions south of the Yangtze River, such as Guizhou, Yunnan, and Guangxi. In the present invention, it is preferable to use the dried stems of Dendrobium nobile as a raw material for extraction.
  • the dried stems of Dendrobium nobile are pulverized, and the ethanol aqueous solution is added for extraction, and the obtained extract is filtered and then combined and concentrated into extract A.
  • the crushed particle size of the dried stems of Dendrobium nobile is preferably 0.1-1 cm, more preferably 0.5-0.6 cm;
  • the volume ratio of ethanol to water in the ethanol aqueous solution is preferably (15-20):1 , More preferably (18-19):1;
  • the volume of the ethanol aqueous solution is 1 to 3 times the volume of the crushed Dendrobium nobile, preferably 2 times the volume;
  • the number of times of extraction is preferably 2 to 5 The number of times is preferably 3 to 4 times;
  • the concentration ratio of the leachate is preferably (3 to 5):1, more preferably 4:1.
  • the present invention prepares the extract A and water into a suspension, and then sequentially extracts with petroleum ether, ethyl acetate and n-butanol. Each extract layer is extracted until the extract is colorless and the extraction is stopped. The ethyl acetate extract was concentrated to make extract B.
  • Petroleum ether, ethyl acetate and n-butanol increase in polarity.
  • the n-butanol extract may contain some highly polar glycoside compounds.
  • select the fraction with medium polarity that is, the ethyl acetate extract.
  • the volume ratio of A to ethyl acetate is preferably 1: (0.5 to 2), more preferably 1: (1 to 1.5); specifically, in the embodiment of the present invention, extract A: water: ethyl acetate
  • the concentration ratio of the ethyl acetate extract is preferably (2-5):1, more preferably (3-4):1.
  • the present invention passes the extract B through a pressure-reduced column, eluted with a mixed washing liquid of petroleum ether and ethyl acetate, and then washes the column with acetone, collects the fractions, concentrates and combines the obtained fractions, and finally Obtain 16 flow points, denoted as Fr.1 ⁇ Fr.16.
  • the reduced pressure column is silica gel column chromatography
  • the silica gel column is silica gel H
  • the particle size of the silica gel H is 100-200 mesh.
  • the volume ratio of the mixed solution of petroleum ether and ethyl acetate is (20-0):1.
  • the specific process of the gradient elution is:
  • the volume ratio of the mixed solution of petroleum ether and ethyl acetate is initially 20:1. As the gradient elution progresses, the volume ratio uniformly decreases until it drops to 0:1.
  • the total time of the gradient elution is preferably 80 ⁇ 120 hours, more preferably 90 to 110 hours, most preferably 100 hours;
  • the temperature of the gradient elution is preferably room temperature, that is, 20 to 35°C, preferably 25 to 30°C, the volume of the eluent used for each gradient Preferably it is 3 to 4L.
  • fractions Preferably, collect once every 500 mL.
  • TLC thin layer chromatography
  • the obtained fractions are detected by thin layer chromatography (TLC) spot plate, observed by naked eyes, and merged together with the same or similar main points, and a total of 16 fractions are obtained.
  • TLC thin layer chromatography
  • Fr.13-1 ⁇ Fr.13-14 After analysis by high performance liquid chromatography, the characteristic peak of Fr.13 is more obvious. Pass it through a reversed-phase column (filler is C 18 ), and the order is 30%, 40%, 50%, 60%, 70%, 80%, 90%. %, 100% methanol aqueous solution gradient elution, 100mL collection once, each gradient with 1.5L mixed solution, each fraction obtained by thin layer chromatography (TLC) spot plate detection, visual observation, the same or similar main points are merged into Together, 14 fractions were obtained, denoted as Fr.13-1 ⁇ Fr.13-14.
  • TLC thin layer chromatography
  • the elution time of each gradient is equal, and the total time of the elution is preferably 24 to 48 hours, more preferably 32 to 36 hours.
  • Fr.13-7 Take the fraction Fr.13-7 and pass it on a Sephadex LH-20 gel column with a mixture of methanol and chloroform for elution. At room temperature, one drop is taken in 2 to 3 seconds, and a tube per 5 mL is collected. The fractions were concentrated under reduced pressure, and subjected to thin-layer chromatography (TLC) spot plate detection, and the similar fractions were combined to obtain 18 fractions, denoted as Fr.13-7-1 ⁇ Fr.13-7-18.
  • TLC thin-layer chromatography
  • the volume ratio of methanol and chloroform is preferably 1:(0.5-2), more preferably 1:(1 ⁇ 1.5).
  • the present invention also provides the use of a compound represented by formula I or a compound represented by formula II in the preparation of a medicine for preventing and treating diabetes and relieving inflammation.
  • the medicine for preventing and treating diabetes is an ⁇ -glucosidase activity inhibitor
  • the diabetes is type II diabetes.
  • the compounds represented by formula I and formula II in the present invention inhibit the activity of ⁇ -glucosidase, reduce the decomposition of oligosaccharides in the digestive tract, delay the absorption of glucose by the intestinal tract, and thereby reduce meals. Post-hyperglycemia risk, to achieve the effect of lowering blood sugar levels;
  • the drug for alleviating inflammation in the present invention is an inhibitor of NO production.
  • the compound represented by formula I or the compound represented by formula II in the present invention can inhibit the production of NO, exhibit anti-inflammatory activity, and relieve body redness, swelling, and fever. , Pain and other symptoms.
  • the present invention also provides a pharmaceutical preparation, which comprises the compound represented by formula I or the compound represented by formula II extracted by the above process and pharmaceutically acceptable excipients.
  • the dosage form of the pharmaceutical preparation is preferably an oral preparation, more preferably a tablet, capsule, pill, granule, decoction, ointment, lotion, oral liquid, drop pill or syrup.
  • the capsule is a hard capsule or a soft capsule; more preferably, the tablet is an oral tablet or an oral tablet; more preferably, an oral tablet refers to a tablet for oral administration, and most of such tablets
  • the drug in the drug is absorbed through the gastrointestinal tract to exert its effect, and the drug in some tablets is used to exert its effect locally in the gastrointestinal tract.
  • oral tablets are ordinary compressed tablets, dispersible tablets, effervescent tablets, chewable tablets, coated tablets or sustained and controlled release tablets.
  • the pharmaceutically acceptable excipients include fruit powder, edible flavors, sweeteners, sour agents, fillers, lubricants, preservatives, suspending agents, food colorings, diluents, emulsifiers, disintegrants or plasticizers One or a mixture of two or more of the agents.
  • the present invention provides a process for extracting compounds from Dendrobium nobile, including the following steps: A) Crushing Dendrobium nobile, leaching with 1 to 3 times the volume of an aqueous ethanol solution for 2 to 5 times, and the resulting extract is filtered Combine and concentrate to obtain extract A; B) Prepare extract A and water into a suspension, and then extract with petroleum ether, ethyl acetate and n-butanol, and concentrate the ethyl acetate extract to make extract B C) Take the extract B, pass it through a decompression column, eluted with a mixed washing liquid of petroleum ether and ethyl acetate, and finally wash the column with acetone, collect the fractions, concentrate and combine the obtained fractions, and finally obtain 16 streams Fr.1 ⁇ Fr.16; D) Pass the fraction Fr.13 through a reversed-phase column and perform gradient elution with a 30-100% methanol aqueous solution to obtain 14 fractions, denoted
  • the research of the present invention shows that the compounds represented by formula I and II can inhibit the activity of ⁇ -glucosidase, and the anti-inflammatory activity is evaluated based on the model of LPS-induced NO production by RAW264.7, and the compounds represented by I and II can inhibit the production of NO , Reflects anti-inflammatory activity. Therefore, it is shown that the compound can be used to prepare foods and/or medicines for the treatment and/or prevention of diabetes and inflammation.
  • Step 1 After pulverizing the dried stems of Dendrobium nobile (13kg), extract 3 times with 2 times the volume of ethanol aqueous solution, and the obtained extracts are filtered and concentrated into extract A;
  • the ethyl acetate extract is concentrated to make extract B;
  • Step 3 Take the extract B, pass it through a decompression column, eluted with a mixture of petroleum ether and ethyl acetate (20:1 ⁇ 0:1, V/V), and finally wash the column with acetone, and collect one for every 500 mL Bottle, each fraction obtained is tested by thin-layer chromatography (TLC) spot plate, and similar fractions are combined to obtain 16 fractions, denoted as Fr.1 ⁇ Fr.16;
  • TLC thin-layer chromatography
  • Step 4 After analysis by high performance liquid chromatography, the characteristic peak of Fr.13 is more obvious. Pass it through a reversed-phase column to determine 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% Gradient elution with methanol aqueous solution, 100mL to collect a bottle, each gradient with 1.5L of mixed solution, the obtained fractions were detected by thin layer chromatography (TLC) spot plate, and similar fractions were combined to obtain 14 fractions, denoted as Fr .13-1 ⁇ Fr.13-14;
  • TLC thin layer chromatography
  • each group was added with 40 ⁇ L (2.5mmol/L 4-nitrophenyl- ⁇ -D-glucopyranoside) PNPG solution; (blank: add 40 ⁇ L (0.1mol) /L)PBS solution)
  • Inhibition rate (OD DMSO -OD sample )/(OD DMSO -OD PBS ) ⁇ 100%
  • the results are shown in Table 2.
  • the compound has a certain degree of inhibitory activity on ⁇ -glucosidase and is better than the positive control acarbose.
  • the control group was indomethacin, and the negative control group was DMSO.
  • the test compound was diluted in half by 5 concentration gradients. Represents the concentration of test compound in abscissa and the ordinate represents the rate of inhibition, IC 50 values determined plotting test compound.
  • Inhibition rate (%) (C 2 -C 1 )/(C 2 -C 0 ) ⁇ 100%;
  • C 0 , C 1 , C 2 are the absorbance values of the blank control group (without LPS), the experimental group, and the negative control group (with LPS) measured at 540 nm, respectively. Calculate the inhibition rate at each concentration and draw the compound concentration-inhibition rate curve, and calculate the half inhibitory concentration (IC 50 value) of the compound on LPS-induced NO production by RAW264.7. The results are shown in Table 3, the compound can effectively inhibit the production of NO in RAW264.7 cells, show a certain anti-inflammatory activity and the effect is significantly better than the positive control indomethacin.

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Abstract

一种从金钗石斛中提取化合物的工艺，A)将金钗石斛粉碎，用乙醇水溶液浸提，浸提液合并浓缩，得到浸膏A；B)将所述浸膏A与水混合，乙酸乙酯萃取，将乙酸乙酯萃取液浓缩制成浸膏B；C)取浸膏B，过减压柱，经梯度洗脱，获得16个流分；D)将流分13过反相柱，进行梯度洗脱，得到14个流分；E)第7流分过葡聚糖凝胶柱，以甲醇和氯仿的混合液进行洗脱，得到18个流分，合并第15～17流分，经半制备HPLC纯化，得到式I和式II所示化合物。研究表明式I和Ⅱ所示的化合物能够抑制α-葡萄糖苷酶的活性和抑制NO的产生，体现抗炎活性。表明该化合物能够用于制备治疗和/或预防糖尿病和炎症的食品以及药品。

Description

一种从金钗石斛中提取化合物的工艺及应用
本申请要求于2020年03月11日提交中国专利局、申请号为202010159498.1、发明名称为“一种从金钗石斛中提取化合物的工艺及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，尤其涉及一种从金钗石斛中提取化合物的工艺及应用。
背景技术
近年来，随着人们生活水平的提高，糖尿病的患病率也在逐年增加，据2017年国际糖尿病联盟(IDF)发布的数据显示，全世界约有4.25亿人(20-79岁)患有糖尿病，其中中国有1.14亿，位居世界第一，根据这一趋势，到2045年，全球将有6.29亿人是糖尿病患者。此外，糖尿病的发病率高达12％，并且在逐渐趋于年轻化。糖尿病(Diabetes mellitus,DM)是一种多种因素和病因造成的代谢紊乱疾病，它的特征在于慢性的高血糖症。其发病与饮食、遗传、环境因素和免疫系统功能紊乱等有密切关系。糖尿病主要分为三类，Ⅰ型、Ⅱ型和妊娠糖尿病，其中Ⅱ型患者占总糖尿人数的90％以上，但目前对其发生发展的精确分子机制尚不完全清楚。近几年，积累的诸多临床实验证据揭示Ⅱ型糖尿病的发病机制与肥胖及炎症所引起的胰岛素抵抗有关。
炎症是机体、细胞对外界物理、化学和生物等有害刺激做出的一系列免疫应答反应，反应的常见表现有发热、红肿、疼痛以及功能障碍等。一般来说，炎症是组织器官对损伤或者感染做出的防御反应，对机体是有益的，但是长期的炎症反应会诱导机体产生过激的异常反应，甚至会诱发许多疾病，如糖尿病、动脉粥样硬化、老年痴呆症、癌症等。目前临床上常使用甾体激素和非甾体类(阿司匹林、双氯芬酸和布洛芬等)治疗炎症，人体服用后会产生一系列的毒副作用，如肝脏和胃肠道损伤以及心血管系统损伤等。此外，植物源药物药效缓慢温和、持久性强，毒副作用小且不易产生机体耐受，因此有必要从植物中寻找具有降血糖和抗炎活性的物质，为新型糖尿病和炎症药物的研发提供先导化合物。
发明内容
本发明的目的在于提供一种从金钗石斛中提取化合物的工艺及应用，本发明中的工艺从药用植物金钗石斛的茎中提取得到式I和Ⅱ化合物，能有效抑制血糖水平关键酶的活性，以LPS诱导RAW264.7产生NO为模型进行抗炎活性评价，I和Ⅱ所示的化合物能够抑制NO的产生，体现抗炎活性。
本发明提供一种从金钗石斛中提取化合物的工艺，包括以下步骤：
A)将金钗石斛粉碎，用1～3倍体积的乙醇水溶液浸提2～5次，所得浸提液过滤后合并浓缩，得到浸膏A；
B)将所述浸膏A与水制备成混悬液，依次用石油醚、乙酸乙酯和正丁醇进行萃取，筛选三种萃取液后，将乙酸乙酯萃取液浓缩制成浸膏B；
C)取浸膏B，过减压柱，经石油醚和乙酸乙酯混合洗液梯度洗脱，然后用丙酮冲柱，收集流分，将所得各个流分浓缩合并，最终获得16个流分，记为Fr.1～Fr.16；
D)将流分Fr.13过反相柱，以30～100％的甲醇水溶液进行梯度洗脱，得到14个流分，记为Fr.13-1～Fr.13-14；
E)取流分Fr.13-7过葡聚糖凝胶柱，以甲醇和氯仿的混合液进行洗脱，得到18个流分，记为Fr.13-7-1～Fr.13-7-18，合并Fr.13-7-15～Fr.13-7-17流分，经半制备HPLC纯化，在t R＝13.5min得到式I所示化合物，在t R＝16.4min得到式II所示化合物；
Figure PCTCN2020134917-appb-000001
优选的，所述步骤B)中浸膏A与水的体积比为1：(0.5～2)；
所述浸膏A与石油醚的体积比为1：(0.5～2)；所述浸膏A与乙酸乙酯的体积比为1：(0.5～2)；所述浸膏A与正丁醇的体积比为1：(0.5～2)。
优选的，所述步骤C)中石油醚和乙酸乙酯混合洗液的梯度洗脱具体为：石油醚和乙酸乙酯的体积比在80～120小时内由20：1均匀降至0:1，所述梯度洗 脱的总时间优选为90～110小时，更优选为100小时。
优选的，所述步骤C)将得到的各个流分减压浓缩，经过薄层层析点板检测，根据显色合并相似流分。
优选的，所述步骤D)中梯度洗脱具体为：依次以质量浓度30％、40％、50％、60％、70％、80％、90％和100％的甲醇水溶液进行洗脱，每个洗脱梯度的洗脱时间相等，洗脱总时间为24～48小时，更优选为32～36小时。
优选的，所述步骤E)中甲醇与氯仿的体积比为1：(0.5～2)。
本发明提供一种如上文所述的工艺提取得到的式I所示化合物和式II所示化合物在制备防治糖尿病和缓解炎症的药物中的应用。
优选的，所述防治糖尿病的药物为α-葡萄糖苷酶活性抑制剂；所述缓解炎症的药物为NO的产生抑制剂。
本发明提供一种药物制剂，包括上文所述的工艺提取得到的式I所示化合物或式II所示化合物以及药学上可接受的辅料。
优选的，所述药物制剂的剂型为片剂、胶囊剂、丸剂、颗粒剂、汤剂、膏剂、露剂、口服液剂、滴丸剂或糖浆剂。
本发明提供了一种从金钗石斛中提取化合物的工艺，包括以下步骤：A)将金钗石斛粉碎，用1～3倍体积的乙醇水溶液浸提2～5次，所得浸提液过滤后合并浓缩，得到浸膏A；B)将所述浸膏A与水制备成混悬液，依次用石油醚、乙酸乙酯和正丁醇进行萃取，将乙酸乙酯萃取液浓缩制成浸膏B；C)取浸膏B，过减压柱，经石油醚和乙酸乙酯混合洗液梯度洗脱，最后用丙酮冲柱，收集流分，将所得各个流分浓缩合并，最终获得16个流分，记为Fr.1～Fr.16；D)将流分Fr.13过反相柱，以30～100％的甲醇水溶液进行梯度洗脱，得到14个流分，记为Fr.13-1～Fr.13-14；E)取Fr.13-7过葡聚糖凝胶柱，以甲醇和氯仿的混合液进行洗脱，得到18个流分，记为Fr.13-7-1～Fr.13-7-18，合并Fr.13-7-15～Fr.13-7-17流分，经半制备HPLC纯化，在t R＝13.5min得到式I所示化合物，在t R＝16.4min得到式II所示化合物。本发明研究表明式I和Ⅱ所示的化合物能够抑制α-葡萄糖苷酶的活性，以LPS诱导RAW264.7产生NO为模型进行抗炎活性评价，I和Ⅱ所示的化合物能够抑制NO的产生，体现抗炎活性。因此表明该化合物能够用于制备治疗和/或预防糖尿病和炎症的食品和/或药品。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1是本发明实施例1中式I所示化合物的 1H NMR图谱；
图2是本发明实施例1中式I所示化合物的 13C NMR+DEPT135图谱；
图3是本发明实施例1中式I所示化合物的HSQC图谱；
图4是本发明实施例1中式Ⅱ所示化合物的 1H NMR图谱；
图5是本发明实施例1中式Ⅱ所示化合物的 13C NMR+DEPT135图谱；
图6是本发明实施例1中式Ⅱ所示化合物的HSQC图谱。
具体实施方式
本发明提供了一种从金钗石斛中提取化合物的工艺，包括以下步骤：
A)将金钗石斛粉碎，用1～3倍体积的乙醇水溶液浸提2～5次，所得浸提液过滤后合并浓缩，得到浸膏A；
B)将所述浸膏A与水制备成混悬液，依次用石油醚、乙酸乙酯和正丁醇进行萃取，筛选三种萃取液后，将乙酸乙酯萃取液浓缩制成浸膏B；
C)取浸膏B，过减压柱，经石油醚和乙酸乙酯混合洗液梯度洗脱，最后用丙酮冲柱，收集流分，将所得各个流分浓缩合并，最终获得16个流分，记为Fr.1～Fr.16；
D)将流分Fr.13过反相柱，以30～100％的甲醇水溶液进行梯度洗脱，得到14个流分，记为Fr.13-1～Fr.13-14；
E)取Fr.13-7过葡聚糖凝胶柱，以甲醇和氯仿的混合液进行洗脱，得到18个流分，记为Fr.13-7-1～Fr.13-7-18，合并Fr.13-7-15～Fr.13-7-17流分，经半制备HPLC纯化，在t R＝13.5min得到式I所示化合物，在t R＝16.4min得到式II所示化合物；
Figure PCTCN2020134917-appb-000002
金钗石斛(Dendrobium nobileLindl.)为兰科(Orchidaceae)石斛属(Dendrobium Sw.)植物，又名金钗石、扁金钗、扁黄草等，为我国传统名贵中药，素有“千金草”之称，常以新鲜或干燥茎入药。金钗石斛在我国主要分布于贵州、云南、广西等长江以南的亚热带地区。在本发明中，优选使用金钗石斛的干燥茎为原料进行提取。
本发明将金钗石斛干燥茎粉碎，加乙醇水溶液浸提，所得浸提液过滤后合并浓缩成浸膏A。
在本发明中，所述金钗石斛干燥茎的粉碎粒径优选为0.1～1cm，更优选为0.5～0.6cm；所述乙醇水溶液中乙醇与水的体积比优选为(15～20)：1，更优选为(18～19)：1；所述乙醇水溶液的体积为粉碎后的金钗石斛的体积的1～3倍，优选为2倍体积；所述浸提的次数优选为2～5次，优选为3～4次；所述浸提液浓缩的浓缩比优选为(3～5)：1，更优选为4:1。
得到浸膏A后，本发明将所述浸膏A与水制备成混悬液，然后依次用石油醚、乙酸乙酯和正丁醇进行萃取，各萃取层萃取至萃取液无色停止萃取，将乙酸乙酯萃取液浓缩制成浸膏B。
石油醚、乙酸乙酯和正丁醇极性依次增大，正丁醇萃取液中可能是一些极性大的糖苷类化合物，具体实验时选择极性中等的流分，即乙酸乙酯萃取液。在本发明中，所述浸膏A与水的体积比优选为1：(0.5～2)，更优选为1：(1～1.5)；所述浸膏A与石油醚的体积比为1：(0.5～2)，更优选为1：(1～1.5)；具体的，在本发明的实施例中，浸膏A：水：石油醚的体积比＝1：1：1；所述浸膏A与乙酸乙酯的体积比优选为1：(0.5～2)，更优选为1：(1～1.5)；具体的，在本发明的实施例中，浸膏A：水：乙酸乙酯的体积比＝1：1：1；所述浸膏A与正丁醇的体积比为1：(0.5～2)，更优选为1：(1～1.5)。具体的，本发明的实施 例中，浸膏A：水：正丁醇的体积比＝1：1：1。
在本发明中，所述乙酸乙酯萃取液的浓缩比优选为(2～5)：1，更优选为(3～4):1。
得到浸膏B后，本发明将浸膏B过减压柱，经石油醚和乙酸乙酯混合洗液梯度洗脱，然后用丙酮冲柱，收集流分，将所得各个流分浓缩合并，最终获得16个流分，记为Fr.1～Fr.16。
在本发明中，所述减压柱为硅胶柱层析，所述硅胶柱为硅胶H，所述硅胶H的粒径为100～200目。所述石油醚与乙酸乙酯的混合溶液的体积比为(20～0)：1，具体的，在本发明的实施例中，所述梯度洗脱的具体过程为：
石油醚与乙酸乙酯的混合溶液的体积比初始为20:1，随着梯度洗脱的进行体积比均匀减小，直至降为0:1，所述梯度洗脱的总时间优选为80～120小时，更优选为90～110小时，最优选为100小时；所述梯度洗脱的温度优选为室温，即20～35℃，优选为25～30℃，每个梯度所用洗脱液的体积优选为3～4L。
洗脱完成后，使用丙酮冲柱，收集流分，优选的，每500mL收集一次，将所得各个流分经过薄层层析(TLC)点板检测，合并相似流分，获得16个流分，记为Fr.1～Fr.16。
在本发明中，得到的各个流分经过薄层层析(TLC)点板检测，肉眼观察，主点相同或者类似的合并到一起，共获得16个流分。
经高效液相色谱分析后，Fr.13特征峰比较明显，将其过反相柱(填料为C 18)，依次以30％、40％、50％、60％、70％、80％、90％、100％甲醇水溶液梯度洗脱，100mL收集一次，每个梯度用1.5L混合液，所得各个流分经过薄层层析(TLC)点板检测，肉眼观察，主点相同或者类似的合并到一起，得到14个流份，记为Fr.13-1～Fr.13-14。
在本发明中，所述梯度洗脱中，每个梯度的洗脱时间相等，所述洗脱的总时间优选为24～48小时，更优选为32～36小时。
取流分Fr.13-7过葡聚糖Sephadex LH-20凝胶柱，以甲醇和氯仿的混合液进行洗脱，室温条件下，2～3秒一滴，每5mL收集一管，所得各个流分减压浓缩，经过薄层层析(TLC)点板检测，合并相似流分，得到18个流分，记为F r.13-7-1～Fr.13-7-18。
在本发明中，所述甲醇和氯仿的体积比优选为1：(0.5～2)，更优选为1：(1～1.5)。
Fr.13-7-15～Fr.13-7-17呈点相似，将其合并后经半制备HPLC(C 18色谱柱，35％甲醇-水洗脱)纯化，得到式I所示化合物(t R＝13.5min)和式Ⅱ化合物(t R＝16.4min)。
Figure PCTCN2020134917-appb-000003
本发明还提供了一种式I所示化合物或式II所示化合物在制备防治糖尿病和缓解炎症的药物中的应用，优选的，所述防治糖尿病的药物为α-葡萄糖苷酶活性抑制剂，所述糖尿病为II型糖尿病，本发明中的式I和式II所示化合物通过抑制α-葡萄糖苷酶的活性，减少低聚糖在消化道内分解，延缓肠道对葡萄糖的吸收，从而降低餐后高血糖风险，实现降低血糖水平的效果；
本发明中所述缓解炎症的药物为NO的产生抑制剂，本发明中的式I所示化合物或式II所示化合物能够抑制NO的产生，体现抗炎活性，缓解机体发红、肿胀、发热、疼痛等症状。
本发明还提供了一种药物制剂，包括上述工艺提取得到的式I所示化合物或式II所示化合物以及药学上可接受的辅料。
优选的，所述药物制剂的剂型优选为口服制剂，更优选为片剂、胶囊剂、丸剂、颗粒剂、汤剂、膏剂、露剂、口服液剂、滴丸剂或糖浆剂。
更优选的，所述胶囊剂为硬胶囊剂或软胶囊剂；更优选的，片剂为口服片剂或口腔片剂；更优选的，口服片剂指供口服的片剂，多数此类片剂中的药物是经胃肠道吸收而发挥作用，也有的片剂中的药物是在胃肠道局部发挥作用。在本发明提供的一些实施例中，口服片剂为普通压制片、分散片、泡腾片、咀嚼片、包衣片或缓控释片。
所述药学上可接受的辅料包括水果粉、食用香精、甜味剂、酸味剂、填充 剂、润滑剂、防腐剂、助悬剂、食用色素、稀释剂、乳化剂、崩解剂或增塑剂中的一种或两者以上的混合物。
本发明提供了一种从金钗石斛中提取化合物的工艺，包括以下步骤：A)将金钗石斛粉碎，用1～3倍体积的乙醇水溶液浸提2～5次，所得浸提液过滤后合并浓缩，得到浸膏A；B)将所述浸膏A与水制备成混悬液，依次用石油醚、乙酸乙酯和正丁醇进行萃取，将乙酸乙酯萃取液浓缩制成浸膏B；C)取浸膏B，过减压柱，经石油醚和乙酸乙酯混合洗液梯度洗脱，最后用丙酮冲柱，收集流分，将所得各个流分浓缩合并，最终获得16个流分，记为Fr.1～Fr.16；D)将流分Fr.13过反相柱，以30～100％的甲醇水溶液进行梯度洗脱，得到14个流分，记为Fr.13-1～Fr.13-14；E)取Fr.13-7过葡聚糖凝胶柱，以甲醇和氯仿的混合液进行洗脱，得到18个流分，记为Fr.13-7-1～Fr.13-7-18，合并Fr.13-7-15～Fr.13-7-17流分，经半制备HPLC纯化，在t R＝13.5min得到式I所示化合物，在t R＝16.4min得到式II所示化合物。本发明研究表明式I和Ⅱ所示的化合物能够抑制α-葡萄糖苷酶的活性，以LPS诱导RAW264.7产生NO为模型进行抗炎活性评价，I和Ⅱ所示的化合物能够抑制NO的产生，体现抗炎活性。因此表明该化合物能够用于制备治疗和/或预防糖尿病和炎症的食品和/或药品。
为了进一步说明本发明，以下结合实施例对本发明提供的一种从金钗石斛中提取化合物的工艺及应用进行详细描述，但不能将其理解为对本发明保护范围的限定。
实施例1
步骤1：将金钗石斛干燥茎(13kg)粉碎后，用2倍体积的乙醇水溶液浸提3次，所得浸提液过滤后合并浓缩成浸膏A；
步骤2：将所述浸膏A与水体积比1：1制成混悬液，依次用石油醚(浸膏A：水：石油醚＝1：1：1)、乙酸乙酯(浸膏A：水：乙酸乙酯＝1：1：1)、正丁醇(浸膏A：水：正丁醇＝1：1：1)进行萃取，各萃取层萃取至萃取液无色停止萃取，将乙酸乙酯萃取液浓缩制成浸膏B；
步骤3：取浸膏B，过减压柱，经石油醚、乙酸乙酯(20:1→0:1,V/V)的混合液梯度洗脱，最后用丙酮冲柱，每500mL收集一瓶，所得各个流分经过薄 层层析(TLC)点板检测，合并相似流分，获得16个流分，记为Fr.1～Fr.16；
步骤4：经高效液相色谱分析后，Fr.13特征峰比较明显，将其过反相柱，以30％、40％、50％、60％、70％、80％、90％、100％甲醇水溶液梯度洗脱，100mL收集一瓶，每个梯度用1.5L混合液，所得各个流分经过薄层层析(TLC)点板检测，合并相似流分，得到14个流份，记为Fr.13-1～Fr.13-14；
步骤5：Fr.13-7呈点性较好，取其过葡聚糖凝胶柱，以500mL甲醇：氯仿＝1：1的混合液进行洗脱，6mL收集一管，点板合并相似流分，得到18个流份，记为Fr.13-7-1～Fr.13-7-18；
步骤6：Fr.13-7-15～Fr.13-7-17呈点相似，合并后经半制备HPLC(C 18色谱柱，35％甲醇-水洗脱)纯化，得到式I和Ⅱ化合物(t R＝13.5min和t R＝16.4min)。
式I和式II所示结构化合物的鉴定图谱如图1～6所示。
式I和II所示结构鉴定数据如下：低分辨质谱m/z:352.4354[M+Na] +，分子式为C 18H 19NO 5； 1H NMR(500MHz)和 13C NMR(125MHz)数据如表1所示：
表1式I和Ⅱ所示化合物的NMR数据(溶剂为氘代甲醇)
Figure PCTCN2020134917-appb-000004
Figure PCTCN2020134917-appb-000005
式I和Ⅱ化合物对α-葡萄糖苷酶的抑制作用
待测样品溶液的配制：取450μL配置好的2U/ml的α-葡萄糖苷酶溶液(用pH＝6.8的PBS溶液稀释)于EP管中，将待测化合物溶解于DMSO后(浓度为2mM)，取45μL的该溶液加至EP管中，摇匀，分别取4次110μL混合均匀的待测溶液于96孔板中；(阴性与空白：取450μL配置好的2U/ml的α-葡萄糖苷酶溶液于EP管中，再取45μL的DMSO溶液加至EP管中，摇匀，分别取4次110μL混合均匀的待测溶液于96孔板中)
将96孔板于37℃放置15分钟后，各组均加入40μL(2.5mmol/L的4-硝基苯基-β-D-吡喃葡萄糖苷)PNPG溶液；(空白：加入40μL(0.1mol/L)PBS溶液)
将96孔板于37℃放置15分钟后，设置酶标仪于405nm波长下测量每孔的OD值吸光度.
计算化合物对α-糖苷酶的抑制活性，计算公式如下：
抑制率＝(OD DMSO-OD 样)/(OD DMSO-OD PBS)×100％
将化合物倍半稀释几个梯度后，以相同的检测方法检测不同浓度样品对α-葡萄糖苷酶的抑制率，利用GraphPad Prism 7软件计算其IC 50值。
结果如表2所示，化合物对α-葡萄糖苷酶有一定程度的抑制活性且好于阳性对照阿卡波糖。
表2化合物对α-葡萄糖苷酶的抑制活性
测试样品 IC 50±SD(μM)
金雀异黄酮(阳性对照) 8.54±0.69
阿卡波糖(阳性对照) 701.45±0.52
化合物I 22.98±1.46
化合物Ⅱ 46.42±1.12
式I和Ⅱ化合物抗炎活性评价
选取RAW264.7(小鼠单核巨噬细胞白血病细胞)，在96孔平底细胞培养板上接种100μL浓度为5×10 4个/mL的细胞，培养于37℃，5％CO 2，90％以上湿度的条件下，24h后加入50μL配制好的待测化合物溶液，继续在该条件下培养，1h后加入50μL配制的LPS(终浓度500ng/mL)溶液，24h后每孔取上清100μL于新的96孔板中，之后向每孔加入100μL(40mg/mL)的Griess试剂，十字交叉法混匀。于酶标仪540nm波长下测定并记录每孔的吸光度，按下面公式计算NO抑制率。对照组为吲哚美辛，阴性对照组为DMSO，把待测化合物倍半稀释5个浓度梯度。用横坐标表示待测化合物浓度，纵坐标表示抑制率，作图求出待测化合物的IC 50值。
抑制率(％)＝(C 2-C 1)/(C 2-C 0)×100％；
式中：C 0、C 1、C 2分别为540nm下测得的空白对照组(不加LPS)、实验组、阴性(加LPS)对照组的吸光值。计算各浓度下的抑制率并绘制化合物浓度—抑制率曲线图，计算得到化合物对LPS诱导RAW264.7产生NO的半抑制浓度(IC 50值)。结果如表3所示，化合物能有效抑制RAW264.7细胞NO的产生量，表现出一定的抗炎活性且效果显著好于阳性对照吲哚美辛。
表3化合物对RAW264.7产生NO的抑制作用
测试样品 IC 50±SD(μM)
吲哚美辛(阳性对照) 52.30±2.11
化合物I 11.07±0.61
化合物Ⅱ 10.43±1.17
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种从金钗石斛中提取化合物的工艺，包括以下步骤：
    A)将金钗石斛粉碎，用1～3倍体积的乙醇水溶液浸提2～5次，所得浸提液过滤后合并浓缩，得到浸膏A；
    B)将所述浸膏A与水制备成混悬液，依次用石油醚、乙酸乙酯和正丁醇进行萃取，筛选三种萃取液后，将乙酸乙酯萃取液浓缩制成浸膏B；
    C)取浸膏B，过减压柱，经石油醚和乙酸乙酯混合洗液梯度洗脱，然后用丙酮冲柱，收集流分，将所得各个流分浓缩合并，最终获得16个流分，记为Fr.1～Fr.16；
    D)将流分Fr.13过反相柱，以30～100％的甲醇水溶液进行梯度洗脱，得到14个流分，记为Fr.13-1～Fr.13-14；
    E)取流分Fr.13-7过葡聚糖凝胶柱，以甲醇和氯仿的混合液进行洗脱，得到18个流分，记为Fr.13-7-1～Fr.13-7-18，合并Fr.13-7-15～Fr.13-7-17流分，经半制备HPLC纯化，在t R＝13.5min得到式I所示化合物，在t R＝16.4min得到式II所示化合物；
    Figure PCTCN2020134917-appb-100001
  2. 根据权利要求1所述的工艺，其特征在于，所述步骤B)中浸膏A与水的体积比为1：(0.5～2)；
    所述浸膏A与石油醚的体积比为1：(0.5～2)；所述浸膏A与乙酸乙酯的体积比为1：(0.5～2)；所述浸膏A与正丁醇的体积比为1：(0.5～2)。
  3. 根据权利要求1所述的工艺，其特征在于，所述步骤C)中石油醚和乙酸乙酯混合洗液的梯度洗脱具体为：石油醚和乙酸乙酯的体积比在80～120小时内由20：1均匀降至0:1。
  4. 根据权利要求1所述的工艺，其特征在于，所述步骤C)将得到的各个流分减压浓缩，经过薄层层析点板检测，根据显色合并相似流分。
  5. 根据权利要求1所述的工艺，其特征在于，所述步骤D)中梯度洗脱具体为：依次以质量浓度30％、40％、50％、60％、70％、80％、90％和100％的甲醇水溶液进行洗脱，每个洗脱梯度的洗脱时间相等，洗脱总时间为24～48小时。
  6. 根据权利要求1所述的工艺，其特征在于，所述步骤E)中甲醇与氯仿的体积比为1：(0.5～2)。
  7. 一种如权利要求1～6所述的工艺提取得到的式I所示化合物和式II所示化合物在制备防治糖尿病和缓解炎症的药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述防治糖尿病的药物为α-葡萄糖苷酶活性抑制剂；所述缓解炎症的药物为NO的产生抑制剂。
  9. 一种药物制剂，包括权利要求1～6所述的工艺提取得到的式I所示化合物或式II所示化合物以及药学上可接受的辅料。
  10. 根据权利要求9所述的药物制剂，其特征在于，所述药物制剂的剂型为片剂、胶囊剂、丸剂、颗粒剂、汤剂、膏剂、露剂、口服液剂、滴丸剂或糖浆剂。
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