WO2016101335A1 - 一种从樟子松树皮中提取醇溶性物质的方法 - Google Patents

一种从樟子松树皮中提取醇溶性物质的方法 Download PDF

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WO2016101335A1
WO2016101335A1 PCT/CN2015/000921 CN2015000921W WO2016101335A1 WO 2016101335 A1 WO2016101335 A1 WO 2016101335A1 CN 2015000921 W CN2015000921 W CN 2015000921W WO 2016101335 A1 WO2016101335 A1 WO 2016101335A1
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alcohol
chloroform
hexane
filtrate
soluble substance
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孙昊东
孙大庆
王桂华
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孙昊东
孙大庆
王桂华
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/13—Coniferophyta (gymnosperms)
    • A61K36/15—Pinaceae (Pine family), e.g. pine or cedar
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/40—Separation, e.g. from natural material; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/01—Preparation of ethers
    • C07C41/34—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/23—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/40—Unsaturated compounds
    • C07C59/58—Unsaturated compounds containing ether groups, groups, groups, or groups
    • C07C59/64—Unsaturated compounds containing ether groups, groups, groups, or groups containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
    • C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
    • C07D493/20—Spiro-condensed systems

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  • the invention belongs to the process of extracting active ingredients of plants, and in particular relates to a method for extracting natural products in the bark of Pinus sylvestris var.
  • Pinus sylvestris var. mongolica is one of the main afforestation tree species in the Three North Region of China. It is widely distributed and rich in resources. The bark of Pinus sylvestris var. mongolica is an important forestry by-product, and its resource utilization rate is low.
  • the current method for extracting alcohol-soluble substances from the bark of Pinus sylvestris var. mongolica is mainly to extract directly with ethanol. Using this method, the flavonoids in the alcohol-soluble natural product obtained are obtained. The content is low.
  • the method of directly extracting the active ingredient in the bark of Pinus sylvestris L. with an alcohol substance has a defect of low extraction rate.
  • the invention provides a method for extracting natural products in the bark of Pinus sylvestris var.
  • the method for extracting an alcohol-soluble substance from the bark of Pinus sylvestris var. chinensis comprises the following steps:
  • the bark of Pinus sylvestris var. chinensis is pulverized into particles of 20 to 80 mesh size, and is placed in n-hexane/chloroform for sonication.
  • the conditions of the ultrasonic treatment are: temperature 30 to 60 ° C, time 15 to 60 min, frequency 15 ⁇ 40KHz;, the mixture A is obtained;
  • the temperature in the step (1) is 5 to 15 ° C higher than the temperature in the step (3), and the frequency in the step (1) is 5 to 10 KHz higher than the ultrasonic frequency in the step (3).
  • the invention promotes the volume ratio of n-hexane and chloroform in the n-hexane/chloroform to be 1:5 to 5:1. Since the oil content in the bark of Pinus sylvestris is low, the extraction effect by using a conventional organic solvent such as petroleum ether or diethyl ether is not Well, the extraction effect of the composite extractant n-hexane/chloroform was significantly improved.
  • the mass to volume ratio of the particles to n-hexane/chloroform is from 1:10 to 1:30.
  • the concentration of the ethanol solution in the present invention is 40% to 80%.
  • the weight-to-volume ratio of the particles to the ethanol solution is 1:10 to 1:50.
  • the extraction method of the present invention includes the following preferred schemes:
  • the filter residue is charged into an ethanol solution, the concentration of the ethanol solution is 50%-80%, and the mass-to-volume ratio of the particles to the ethanol solution is 1:20-1:25, and the solution is sonicated.
  • the conditions of the ultrasonic treatment are: temperature 30 ⁇ 35 ° C, ultrasonic time 15 ⁇ 60min, frequency 15 ⁇ 25KHz, to obtain a mixture B;
  • the temperature in the step (1) is 5 to 15 ° C higher than the temperature in the step (3), and the frequency in the step (1) is 5 to 10 KHz higher than the ultrasonic frequency in the step (3).
  • the invention provides a method for extracting natural products in the bark of Pinus sylvestris var. mongolica by ultrasonic assisted solvent, which has the following advantages:
  • the method of segmentation extraction adopted by the invention first extracts lipids and then extracts alcohol-soluble substances, thereby effectively reducing the interference of oils and fats in the alcohol extract, increasing the extraction rate of active substances such as flavonoids in the alcohol extract, and reducing the subsequent The difficulty of the purification process.
  • Figure 1 LC/MS/MS spectrum of the alcohol-soluble substance obtained by the fractional extraction method
  • the Pinus sylvestris bark used in the following examples was collected from Daxinganling, Heilongjiang City.
  • Example 1 A method for extracting alcohol-soluble substances from the bark of Pinus sylvestris
  • the filter residue in the step 2) was placed in a 400 mL, 50% ethanol solution, and ultrasonically applied at 30 ° C for 30 min, and the ultrasonic frequency was 19 KHz.
  • the lipid substance in the bark of Pinus sylvestris var. chinensis obtained by this method was 18.60 mg/g, and the total amount of alcohol-soluble substance was 88.63 mg/g.
  • the flavonoids in the obtained alcohol-soluble substance were examined by aluminum nitrite-aluminum nitrate-sodium hydroxide coloring method (in terms of rutin), wherein the flavonoid content was 23.09 mg/g.
  • Example 2 A method for extracting alcohol-soluble substances from the bark of Pinus sylvestris
  • the filter residue in the step 2) is put into a 500 mL, 80% ethanol solution, and the ultrasonic wave is applied for 60 min at 35 ° C, and the ultrasonic frequency is 25 KHz.
  • the lipid substance in the bark of Pinus sylvestris var. mongolica extracted by this method was 24.72 mg/g, and the total amount of alcohol-soluble substance was 95.24 mg/g.
  • the flavonoids in the obtained alcohol-soluble substance were measured by aluminum nitrite-aluminum nitrate-sodium hydroxide colorimetric method (in terms of rutin), wherein the flavonoid content was 26.28 mg/g.
  • Example 3 A method for extracting alcohol-soluble substances from the bark of Pinus sylvestris
  • the filter residue in the step 2) was placed in a 500 mL, 70% ethanol solution, and ultrasonically applied for 55 min at 30 ° C, and the ultrasonic frequency was 25 KHz.
  • the lipid substance in the bark of Pinus sylvestris L. extracted according to this method was 24.35 mg/g, and the total amount of the alcohol-soluble substance was 93.52 mg/g.
  • the flavonoids in the obtained alcohol-soluble substance were measured by aluminum nitrite-aluminum nitrate-sodium hydroxide colorimetric method (in terms of rutin), wherein the flavonoid content was 25.86 mg/g.
  • Example 2 The only difference compared to Example 2 was that the n-hexane/chloroform in the step (1) was replaced with diethyl ether.
  • the lipid substance in the bark of Pinus sylvestris L. extracted according to this method was 10.72 mg/g, and the total amount of the alcohol-soluble substance was 100.24 mg/g.
  • the flavonoids in the obtained alcohol-soluble substance were measured by an aluminum nitrite-aluminum nitrate-sodium hydroxide coloring method (in terms of rutin), wherein the flavonoid content was 19.28 mg/g.
  • the difference is only that the ultrasonic conditions in the step (1) and the step (2) are: 60 min at 35 ° C, and the ultrasonic frequency is 25 KHz.
  • the lipid substance in the bark of Pinus sylvestris var. mongolica extracted by this method was 15.72 mg/g, and the total amount of alcohol-soluble substance was 99.52 mg/g.
  • the flavonoids in the obtained alcohol-soluble substance were measured by aluminum nitrite-aluminum nitrate-sodium hydroxide colorimetric method (in terms of rutin), wherein the flavonoid content was 21.28 mg/g.
  • the total amount of alcohol-soluble substances in the bark of Pinus sylvestris var. mongolica extracted in this way was 103.68 mg/g.
  • the flavonoids in the obtained alcohol-soluble substance were measured by an aluminum nitrite-aluminum nitrate-sodium hydroxide coloring method (in terms of rutin), wherein the flavonoid content was 20.15 mg/g.
  • Example 2 The LC/MS/MS spectrum of Example 2 is as shown in FIG. 1, and the composition of the alcohol-soluble substance of Example 2 is obtained by analyzing and comparing the composition of FIG.
  • the fractional extraction method can be used to extract the alcohol-soluble substances in the bark of Pinus sylvestris var. mongolica, and the active ingredients of flavonoids can be obtained, and the ring-opening larchol and 9'-p-hydroxy (trans) cassia can also be obtained.
  • An active ingredient such as acid-7-hydroxy-cyclohexyl larchol ester.
  • Sulfonic acid is one of the active constituents of traditional Chinese medicine mustard, and has antifungal and anti-tumor effects.
  • 9'-p-hydroxy (trans) cinnamic acid-7-hydroxy-cyclohexyl lactate and ring-opening larch are a class of compounds similar in structure to estrogen, with antioxidant, anti-mitotic, antiviral , anti-cancer, weaker estrogen-like effects, lower total cholesterol and low-density lipoprotein cholesterol. Therefore, the alcohol-soluble substance has good functionality and can be further applied.

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Abstract

一种从樟子松树皮中提取醇溶性物质的方法，包括如下步骤：(1)将樟子松树皮粉碎成20～80目大小的颗粒，投入到正己烷/氯仿中，进行超声处理，得到混合物A；(2)对所述混合物A进行过滤，得到滤液a和滤渣；将滤液a进行蒸馏，蒸出正己烷/氯仿，得到脂类物质；(3)将所述滤渣投入到乙醇溶液中，对其进行超声处理；(4)对所述混合物B进行过滤，得到滤液b，对滤液b进行蒸馏，蒸出乙醇溶液，得到醇溶性物质的浓缩液，将所述浓缩液干燥得醇溶性物质。运用此方法提取的醇溶性物质中黄酮含量高，还含有芥子酸、开环异落叶松脂醇、9'-对羟基(反式)桂皮酸-7-羟基-开环异落叶松脂醇酯等有效成分，具有抗氧化、抗菌等功效。

Description

一种从樟子松树皮中提取醇溶性物质的方法 技术领域
本发明属于植物有效成分提取工艺，具体地说，涉及一种超声波辅助溶剂分段提取樟子松树皮中天然产物的方法。
背景技术
樟子松是我国三北地区主要优良造林树种之一，分布广泛，资源丰富，樟子松树皮作为重要的林业副产物，其资源利用率较低。
研究表明，樟子松树皮用乙醇提取的醇溶性物质中含有多糖、酚性物质、有机酸、鞣质、黄酮等有效成分。其中以黄酮为公认的具有多种保健功效的天然提取物。樟子松中的油脂含量较低，约为树皮干重的1％-3％。
由于樟子松树皮中油脂含量较低，因此没有引起人们的重视，当前提取樟子松树皮中醇溶性物质的方法主要为直接用乙醇提取，运用该方法，所得的醇溶性天然产物中黄酮的含量较低。
发明内容
针对当前的方法中，直接用醇类物质提取样樟子松树皮中的活性成分的方法存在的提取率低的缺陷。本发明提供一种分段提取樟子松树皮中天然产物的方法。
本发明所述从樟子松树皮中提取醇溶性物质的方法，包括如下步骤：
(1)将樟子松树皮粉碎成20～80目大小的颗粒，投入到正己烷/氯仿中，进行超声处理，所述超声处理的条件为：温度30～60℃，时间15～60min，频率15～40KHz；，得到混合物A；
(2)对所述混合物A进行过滤，得到滤液a和滤渣；将所述滤液a进行蒸馏，蒸出正己烷/氯仿，得到脂类物质；
(3)将所述滤渣投入到乙醇溶液中，对其进行超声处理，所述超声处理的条件为：温度30～55℃，超声时间15～60min，频率10～35KHz得到混合物B；
(4)对所述混合物B进行过滤，得到滤液b，对滤液b进行蒸馏，蒸出乙醇溶液，得到醇溶性物质的浓缩液，再将所述浓缩液置于35℃烘箱干燥12-24h，即得醇溶性物质；
所述步骤(1)中的温度比步骤(3)中的温度高5～15℃，所述步骤(1)中的频率比步骤(3)中的超声频率高5-10KHz。
本发明促所述正己烷/氯仿中正己烷和氯仿的体积比为1∶5～5∶1，由于樟子松树皮中油脂含量低，用传统的有机溶剂如石油醚、乙醚等提取效果不好，采用复合提取剂正己烷/氯仿提取效果明显改善。
本发明中所述颗粒与正己烷/氯仿的质量体积比为1∶10～1∶30。
本发明中所述乙醇溶液的浓度为40％～80％。
本发明中所述颗粒与乙醇溶液的重量体积比为1∶10～1∶50
本发明所述的提取方法包括如下的优选方案：
1)将樟子松树皮粉碎成20～80目大小的颗粒，投入到正己烷/氯仿中，所述正己烷/氯仿，正己烷氯仿的体积比为1∶1～1∶2，所述颗粒与正己烷/氯仿的质量体积比为1∶15-1∶20；进行超声处理，所述超声处理的条件为：温度35～50℃，时间15～60min，频率25～35KHz；，得到混合物A；
(2)对所述混合物A进行过滤，得到滤液a和滤渣；将滤液a进行蒸馏，蒸出正己烷/氯仿，得到脂类物质；
(3)将所述滤渣投入到乙醇溶液中，所述乙醇溶液的浓度为50％-80％，所述颗粒与乙醇溶液的质量体积比为1∶20-1∶25，对其进行超声处理，所述超声处理的条件为：温度30～35℃，超声时间15～60min，频率15～25KHz，得到混合物B；
(4)对所述混合物B进行过滤，得到滤液b，对滤液b进行蒸馏，蒸出乙醇溶液，得到醇溶性物质的浓缩液，再将所述浓缩液置于35℃烘箱干燥12-24h，即得醇溶性物质；
所述步骤(1)中的温度比步骤(3)中的温度高5～15℃，所述步骤(1)中的频率比步骤(3)中的超声频率高5-10KHz。
本发明提供的一种超声波辅助溶剂分段提取樟子松树皮中天然产物的方法，具有如下优点：
(一)本发明采用的分段提取的方法，先提取脂类再提取醇溶性物质，有效减少了醇提物中油脂的干扰，提高醇提物中黄酮等活性物质的提取率，降低了后续纯化工艺的难度。
(二)采用本发明方法成功提取分离出脂类与醇溶性物质两类产品，提取效率高，分离效果好，成功实现了将现代提取分离技术应用于樟子松树皮的精细化利用，对樟子松树皮的多元化、高值化、精细化利用具有重要的意义。
附图说明
图1：分段提取法所得醇溶性物质的LC/MS/MS谱图；
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
以下实施例中使用的樟子松树皮采集自黑龙江省大兴安岭。
实施例1一种从樟子松树皮中提取醇溶性物质的方法
包括如下步骤：
(1)将20g樟子松树皮粉碎成20～80目大小的颗粒，并投入到300mL的正己烷/氯仿中，正己烷与氯仿的体积比为1∶5，在35℃条件下超声波作用30min，超声频率为25KHz。
(2)趁热对处理后的混合物进行真空抽滤，得到滤液a和滤渣。将滤液a置于旋转蒸发仪进行减压蒸馏，蒸出正己烷/氯仿溶剂，得到脂类物质。
(3)将步骤2)中的滤渣投入400mL、50％乙醇溶液中，在30℃条件下超声波作用30min，超声频率为19KHz。
(4)趁热对处理后的混合物进行真空抽滤，得到滤液b。将滤液a置于旋转蒸发 仪进行减压蒸馏，蒸出乙醇溶液，得到醇溶性物质的浓缩液，再将剩余物置于35℃烘箱干燥12h。
按此方法提取的樟子松树皮中的脂类物质为18.60mg/g，醇溶性物质的总量为88.63mg/g。
用亚硝酸铝-硝酸铝-氢氧化钠显色法(以芦丁计)对所得的醇溶性物质中的黄酮进行检测，其中黄酮含量为23.09mg/g。
实施例2一种从樟子松树皮中提取醇溶性物质的方法
(1)将20g樟子松树皮粉碎成30～60目大小的颗粒，并投入到400mL的正己烷/氯仿中，正己烷与氯仿的体积比为1∶1，在50℃条件下超声波作用60min，超声频率为30KHz。
(2)趁热对处理后的混合物进行真空抽滤，得到滤液a和滤渣。将滤液a置于旋转蒸发仪进行减压蒸馏，蒸出正己烷/氯仿溶剂，得到脂类物质。
(3)将步骤2)中的滤渣投入500mL、80％乙醇溶液中，在35℃条件下超声波作用60min，超声频率为25KHz。
(4)趁热对处理后的混合物进行真空抽滤，得到滤液b。滤液b用旋转蒸发仪进行减压浓缩，蒸出乙醇溶剂，得到脂溶性物质的浓缩液，将浓缩液置于35℃烘箱干燥16h，得到醇溶性物质。
按此方法提取的樟子松树皮中的脂类物质为24.72mg/g，醇溶性物质的总量为95.24mg/g。
用亚硝酸铝-硝酸铝-氢氧化钠显色法(以芦丁计)测量对所得的醇溶性物质中的黄酮进行检测，其中黄酮含量为26.28mg/g。
实施例3一种从樟子松树皮中提取醇溶性物质的方法
(1)将20g樟子松树皮粉碎成30-70目大小的颗粒，并投入到350mL的正己烷/氯仿中，正己烷与氯仿的体积比为1∶2，在40℃条件下超声波作用50min，超声频率为35KHz。
(2)趁热对处理后的混合物进行真空抽滤，得到滤液a和滤渣。将滤液a置于旋转蒸发仪进行减压蒸馏，蒸出正己烷/氯仿溶剂，得到脂类物质。
(3)将步骤2)中的滤渣投入500mL、70％乙醇溶液中，在30℃条件下超声波作用55min，超声波频率为25KHz。
(4)趁热对处理后的混合物进行真空抽滤，得到滤液b。滤液b用旋转蒸发仪进行减压浓缩，蒸出乙醇溶剂，得到醇溶性物质的浓缩液，将浓缩液置于35℃烘箱干燥14h，得到醇溶性物质。
按此方法提取的樟子松树皮中的脂类物质为24.35mg/g，醇溶性物质的总量为93.52mg/g。
用亚硝酸铝-硝酸铝-氢氧化钠显色法(以芦丁计)测量对所得的醇溶性物质中的黄酮进行检测，其中黄酮含量为25.86mg/g。
对比例1
同实施例2相比，区别仅在于，将步骤(1)中的正己烷/氯仿替换为乙醚。按此方法提取的樟子松树皮中的脂类物质为10.72mg/g，醇溶性物质的总量为100.24mg/g。
用亚硝酸铝-硝酸铝-氢氧化钠显色法(以芦丁计)测量对所得的醇溶性物质中的黄酮进行检测，其中黄酮含量为19.28mg/g。
对比例2
同实施例2相比，其区别仅在于，所述步骤(1)和步骤(2)中的超声波条件均为：在35℃条件下作用60min，超声频率为25KHz。
按此方法提取的樟子松树皮中的脂类物质为15.72mg/g，醇溶性物质的总量为99.52mg/g。
用亚硝酸铝-硝酸铝-氢氧化钠显色法(以芦丁计)测量对所得的醇溶性物质中的黄酮进行检测，其中黄酮含量为21.28mg/g。
对比例3
同实施例2相比，略去第步骤(1)和(2)提取油脂的操作，其他操作相同。
按此方法提取的樟子松树皮中醇溶性物质的总量为103.68mg/g。
用亚硝酸铝-硝酸铝-氢氧化钠显色法(以芦丁计)测量对所得的醇溶性物质中的黄酮进行检测，其中黄酮含量为20.15mg/g。
实验例1
实验对象：实施例2提取的醇溶性物质。
实验方法：利用高效液相色谱串联二级质谱分析检测醇溶性物质中的物质组成。仪器型号：Thermo Scientific-Q Exactive，数据处理软件：Xcalibur。紫外检测器型号：Ultimate 3000，检测波长：280nm。柱型号：Hypersil Gold Dim(mm)1002.1。流动相：A为水，B为甲醇，流速：200μL/min。洗脱梯度：0min，5％B→10min，10％B→30min，15％B→45min，20％B→60min，30％B→70min，50％B→100min，50％B。离子源：ESI-，m/z扫描范围：0-1500，喷雾电压(-)：2500.00。进样量5μL。
实验结果：
实施例2的LC/MS/MS谱图如图1，对图1进行分析比对得到实施例2的醇溶性物质中的成份组成如表1
表1
Figure PCTCN2015000921-appb-000001
由表1可以看出，实施例2提取的醇溶性物质分离得到13个色谱峰，通过HPLC-MS-MS分析，初步鉴定出其中8种主要成分，为黄杉素、橙皮素、异鼠李素、落叶松内酯iarixinol的黄酮成分，和芥子酸、开环异落叶松脂醇、9’-对羟基(反式)桂皮酸-7-羟基-开环异落叶松脂醇酯1、9’-对羟基(反式)桂皮酸-7-羟基-开环异落叶松脂醇酯2。
实验结论：
上述结果表明，采取分段提取法对樟子松树皮中的醇溶性物质进行提取，可以得到黄酮类的活性成分，还可得到开环异落叶松脂醇、9’-对羟基(反式)桂皮酸-7-羟基-开环异落叶松脂醇酯等活性成分。芥子酸为中药芥子的有效成分之一，具有抗真菌、抗肿毒功效。9’-对羟基(反式)桂皮酸-7-羟基-开环异落叶松脂醇酯和开环异落叶松酯为结构类似于雌激素的一类化合物，具有抗氧化、抗有丝分裂、抗病毒、抗癌、较弱的雌激素样作用、降低总胆固醇和低密度脂蛋白胆固醇。因此，该醇溶性物质具有良好的功能性，可进一步地应用。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (8)

  1. 一种从樟子松树皮中提取醇溶性物质的方法，包括如下步骤：
    (1)将樟子松树皮粉碎成20～80目大小的颗粒，投入到正己烷/氯仿中，进行超声处理，所述超声处理的条件为：温度30～60℃，时间15～60min，频率15～40KHz，得到混合物A；
    (2)对所述混合物A进行过滤，得到滤液a和滤渣；将所述滤液a进行蒸馏，蒸出正已烷/氯仿，得到脂类物质；
    (3)将所述滤渣投入到乙醇溶液中，对其进行超声处理，所述超声处理的条件为：温度30～55℃，时间15～60min，频率10～35KHz得到混合物B；
    (4)对所述混合物B进行过滤，得到滤液b，对滤液b进行蒸馏，蒸出乙醇溶液，得到醇溶性物质的浓缩液，再将所述浓缩液置于35℃烘箱干燥12-24h，即得醇溶性物质。
  2. 根据权利要求1中所述的方法，其特征在于，所述正已烷/氯仿中正已烷和氯仿的体积比为1∶5～5∶1。
  3. 根据权利要求1中所述的方法，其特征在于，所述颗粒与正已烷/氯仿的质量体积比为1∶10～1∶30。
  4. 根据权利要求1中所述的方法，其特征在于，所述乙醇溶液的浓度为40％～80％。
  5. 根据权利要求1中所述的的方法，其特征在于，所述颗粒与乙醇溶液的质量体积比为1∶10～1∶50。
  6. 根据权利要求1中所述的方法，其特征在于，所述步骤(1)中的温度比步骤(3)中的温度高5～15℃，频率比步骤(3)中的频率高5-10KHz。
  7. 根据权利要求1-6任一项所述的方法，其特征在于，包括如下步骤：1)将樟子松树皮粉碎成20～80目大小的颗粒，投入到正己烷/氯仿中，所述正已烷/氯仿，正已烷氯仿的体积比为1∶1～1∶2，所述颗粒与正已烷/氯仿的质量体积比为1∶15-1∶20；进行超声处理，所述超声处理的条件为：温度35～50℃，时间15～60min，频率25～35KHz；，得到混合物A；
    (2)对所述混合物A进行过滤，得到滤液a和滤渣；将滤液a进行蒸馏，蒸出正已烷/氯仿，得到脂类物质；
    (3)将所述滤渣投入到乙醇溶液中，所述乙醇溶液的浓度为50％-80％，所述颗粒与乙醇溶液的质量体积比为1∶20-1∶25，对其进行超声处理，所述超声处理的条件为：温度30～35℃，超声时间15～60min，频率15～25KHz，得到混合物B；
    (4)对所述混合物B进行过滤，得到滤液b，对滤液b进行蒸馏，蒸出乙醇溶液，得到醇溶性物质的浓缩液，再将所述浓缩液置于35℃烘箱干燥12-24h，即得醇溶性物质。
  8. 权利要求1-7任一项方法提取的醇类物质。
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