WO2021103170A1 - 一种抑制肝糖异生的非苦味苦瓜三萜组合物及其应用 - Google Patents

一种抑制肝糖异生的非苦味苦瓜三萜组合物及其应用 Download PDF

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WO2021103170A1
WO2021103170A1 PCT/CN2019/125045 CN2019125045W WO2021103170A1 WO 2021103170 A1 WO2021103170 A1 WO 2021103170A1 CN 2019125045 W CN2019125045 W CN 2019125045W WO 2021103170 A1 WO2021103170 A1 WO 2021103170A1
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bitter
triterpene
charantoside
momordicoside
composition
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PCT/CN2019/125045
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English (en)
French (fr)
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邓媛元
贾栩超
张名位
魏振承
张雁
刘光
唐小俊
李萍
王佳佳
廖娜
王智明
钟立煌
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广东省农业科学院蚕业与农产品加工研究所
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Priority to JP2020569762A priority Critical patent/JP7050967B2/ja
Publication of WO2021103170A1 publication Critical patent/WO2021103170A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of food intensive and deep processing, and specifically relates to a non-bitter bitter gourd triterpene composition for inhibiting liver gluconeogenesis and its application.
  • Momordica charantia is a tropical-subtropical special medicinal and edible vegetable with a bitter taste and cold nature. It can be used to prevent and improve diabetes, obesity and other diseases related to energy metabolism imbalance. Triterpenoids are an important material basis for bitter gourd to regulate glucose and lipid metabolism, as well as its most important bitter substance. Although a large number of in vivo and in vitro experiments confirmed that bitter melon triterpene extract has significant blood glucose-lowering biological activity, the strong bitter taste limits its application in the food field.
  • Triterpenoids are generally considered to have a bitter taste. At present, there are nearly 150 triterpenoids isolated from Momordica charantia, but there is no research report on the bitterness intensity of a single compound. In addition, there are currently about 14 momordica charantia triterpenoids with hypoglycemic activity. Their hypoglycemic activity mainly includes activating AMPK signaling pathway, stimulating GLUT4 to transport glucose in the cell membrane, inhibiting the activity of ⁇ -glucosidase, and controlling diet. After blood glucose levels, etc., but the effect of triterpenoids on liver gluconeogenesis is not clear.
  • the pathological feature of type 2 diabetes is that the glucose content in the blood is too high, or/and the sugar utilization rate of the peripheral tissues is reduced. The excessive glucose content in the blood is derived from the liver glucose output. The incidence of hepatic gluconeogenesis in diabetic patients is as high as 90%. Inhibiting liver gluconeogenesis is an important target for the treatment of diabetes.
  • bitterness intensity of bitter melon triterpenes is currently unknown, and its effect on liver gluconeogenesis is also unclear.
  • the purpose of the present invention is to provide a bitter gourd triterpene composition, which has good anti-hepatic glycolytic activity and no obvious bitter taste.
  • the purpose of the present invention is also to provide the application of the above-mentioned bitter melon triterpene composition in the preparation of healthy foods with the effect of inhibiting liver gluconeogenesis.
  • a non-bitter bitter melon triterpene composition for inhibiting liver gluconeogenesis the active ingredient of which is mainly composed of Charantoside C and Momordicoside F1, said Charantoside C and Momordicoside
  • the mass ratio of F1 is 1:0.6 ⁇ 1.5.
  • a series of bitter melon triterpene compounds separated and obtained from bitter melon are quantitatively analyzed on the bitterness intensity of the series of bitter melon triterpene compounds separated and obtained by using an electronic tongue with coffee as standard products.
  • the rat primary hepatocytes were used as a model to analyze the hepatic gluconeogenesis inhibitory activity of the series of triterpenoids.
  • Two non-bitter triterpenoids Charantoside C and Momordicoside F1 with strong activity were obtained from the screening. On this basis, combined with the analysis and evaluation of activity and bitterness, a combination ratio with enhanced activity was invented.
  • the mass ratio of Charantoside C and Momordicoside F1 is 1:0.6 to 1.5.
  • the composition has significant inhibition of hepatic glucose anomalies and has no obvious bitter taste. It can be used as a functional ingredient of health food for diabetics.
  • the Charantoside C, Momordicoside F1 and the bitterness of the bitter melon triterpene composition are all lower than the electronic tongue ( ⁇ -ASTREE Liquid Taste Analyser, Alpha M.O.S., mecanic, France) detection threshold and are non-bitter substances.
  • the above two compounds Charantoside C and Momordicoside F1 were quantitatively analyzed by electronic tongue, and their bitterness intensity was lower than the electronic tongue detection threshold (1 ⁇ 10 -4 g/mL caffeine), and the predicted values of the caffeine standard curve were 1.50 ⁇ 10 -6 and 3.39 ⁇ 10 -10 g/mL caffeine (that is, the bitterness of the ethanol solution of Charantoside C and Momordicoside F1 at 1 g/mL is equivalent to 1.50 ⁇ 10 -6 and 3.39 ⁇ 10 -10 g/mL caffeine, respectively Bitterness of ethanol solution), which is a non-bitter compound.
  • bitter melon triterpene composition composed of Charantoside C and Momordicoside F1 according to the mass ratio of 1:0.6 ⁇ 1.5, the bitterness intensity is also lower than the electronic tongue detection threshold, and the bitterness intensity of the 1g/mL composition is predicted by the standard curve of caffeine Caffeine is 3.40 ⁇ 10 -8 to 5.67 ⁇ 10 -6 g/mL, which is also a non-bitter substance.
  • bitter melon triterpene compounds Charantoside C, Momordicoside F1 and the bitter melon triterpene composition prepared from these two compositions in the present invention are non-bitter.
  • the above-mentioned second object of the present invention can be achieved by the following technical solution: the application of the above-mentioned bitter melon triterpene composition in the preparation of a healthy food with the property of inhibiting hepatic gluconeogenesis.
  • the experimental results in the examples of the present invention show that the hepatic gluconeogenesis inhibitory activity of Charantoside C and Momordicoside F1 at a concentration of 20 ⁇ M is 22.94% and 38.34%, respectively, which is higher than that of berberine (the main component of the traditional Chinese herbal medicine for the treatment of diabetes) at 20 ⁇ M.
  • the inhibition rate was 15.61%, and the inhibitory activity of hepatic gluconeogenesis was significant.
  • a bitter melon triterpene composition composed of Charantoside C and Momordicoside F1 in a ratio of 1:0.6 to 1.5 by mass can inhibit liver gluconeogenesis
  • the activity is significantly higher than that of a single compound at the same dose.
  • the added mass percentage of the bitter melon triterpene composition in the health food is 0.1-2%.
  • the preparation methods of Charantoside C and Momordicoside F1 are as follows:
  • n-butanol phase is roughly purified with a macroporous resin, it is eluted with water, 20% by volume ethanol-water and 80% by volume ethanol-water, and the volume percentage is The 80% ethanol-water elution part is the triterpene-rich part;
  • the pretreatment described in step (1) includes washing, deseeding, slicing, drying, crushing and sieving treatments.
  • the extraction volume ratio of bitter melon triterpene extract to petroleum ether, ethyl acetate, and n-butanol are all 1:3, and petroleum ether is used to extract once, ethyl acetate is used to extract twice, and n-butyl Alcohol extraction 3 times.
  • D101 macroporous resin is used for crude purification in step (4).
  • the present invention has the following beneficial effects:
  • the bitter melon triterpene extract in the prior art has significant hypoglycemic activity, but its effect on liver gluconeogenesis is unknown, and its bitter taste is strong, which limits its application in food.
  • the present invention is obtained from the previous separation Among the 19 bitter melon triterpene compounds, two non-bitter triterpene compounds with hepatic gluconeogenesis inhibitory activity were screened, and a combination ratio with enhanced activity was obtained, breaking through the long-term bitter melon triterpene extract due to its strong bitter taste and consumption. The application of low acceptance in the field of food intensive processing is limited.
  • the triterpene composition of the present invention can inhibit hepatic gluconeogenesis, and its effect is better than berberine. It is the main active ingredient of bitter melon triterpene extract to inhibit hepatic gluconeogenesis, and the composition has no obvious bitter taste and can As a functional ingredient added to food, it has been developed as a health food for diabetics with good flavor and high acceptance.
  • the non-bitter bitter gourd triterpene composition for inhibiting liver gluconeogenesis provided by the present embodiment has an active ingredient mainly composed of Charantoside C and Momordicoside F1, and the mass ratio of Charantoside C and Momordicoside F1 is 1:1.5.
  • composition is added to a functional oral liquid at a mass percentage of 2.0%, which can effectively inhibit liver gluconeogenesis and control postprandial blood glucose increase.
  • Charantoside C and Momordicoside F1 in this example can be obtained by chemical synthesis, or can be directly prepared by the method in Example 4 below.
  • the non-bitter bitter gourd triterpene composition for inhibiting hepatic gluconeogenesis provided in this embodiment has an active ingredient mainly composed of Charantoside C and Momordicoside F1, and the mass ratio of Charantoside C and Momordicoside F1 is 1:1.
  • the composition is added to brewed beverages at a mass percentage of 1.0%, which can effectively inhibit liver gluconeogenesis and control postprandial blood glucose rise.
  • the non-bitter bitter gourd triterpene composition for inhibiting liver gluconeogenesis provided in this embodiment has an active ingredient mainly composed of Charantoside C and Momordicoside F1, and the mass ratio of Charantoside C and Momordicoside F1 is 1:0.6.
  • the composition is added to the nutritional meal powder at a mass percentage of 0.1%, which can effectively inhibit liver gluconeogenesis and control the increase in blood sugar after a meal.
  • the following uses the primary isolated SD rat liver parenchymal cells as a model, metformin (metformin), berberine (Berberine), bitter melon triterpenoid extracts (triterpenoid extracts, prepared below) as positive controls to evaluate the bitter melon triterpene compound Charantoside C And Momordicoside F1, and the effects of the composition in different proportions on hepatic gluconeogenesis, further elucidating the beneficial effects of the present invention.
  • metformin metformin
  • berberine berberine
  • bitter melon triterpenoid extracts prepared below
  • Fresh bitter gourd is washed, seeded, sliced, dried at 55°C for 12 hours, and crushed through an 80-mesh sieve to obtain dry bitter gourd powder.
  • the bitter gourd dry powder was extracted twice with 70% ethanol according to the ratio of material to liquid at room temperature of 1:5 to obtain the bitter gourd triterpene extract.
  • n-butanol phase is crudely purified by D101 macroporous resin, eluted with water, 20% ethanol-water and 80% ethanol-water in sequence.
  • the 80% ethanol-water elution part is the triterpene-rich part, which is further separated and purified by a normal phase silica gel column.
  • chloroform/methanol 50:1 and chloroform/methanol 20:1 were used in sequence.
  • the chloroform/methanol 20:1 part was collected and separated by reversed-phase column chromatography, eluted with a gradient of 50% and 60% methanol with water.
  • the 60% methanol water eluate was collected, and further purified by a preparative high performance liquid chromatograph, and eluted with 80% acetonitrile to obtain multiple elution peaks.
  • the bitter melon triterpene extract obtained by extraction is concentrated by vacuum rotary evaporation to obtain triterpenoid extracts, which are used for cell experiments.
  • In situ perfusion digestion method was used to digest SD rat liver with type IV collagenase, and the hepatocytes were dispersed and transferred to 49% Percoll separation solution for centrifugation. The supernatant was discarded and the cells were inserted into a 24-well plate coated with 0.2% gelatin. Stick to the wall for 6h. After starvation with serum-free low-sugar medium for 8 hours, the supernatant was discarded, and the reaction solution (sugar-free DMEM medium containing 2mM sodium pyruvate and 20mM sodium lactate) was added after washing with PBS buffer, and waiting for sample test.
  • the triterpene compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 ⁇ M mother solution, which was diluted with the above reaction solution, and finally added to the cell culture well to a final concentration of 20 ⁇ M, and the final concentration of DMSO was 0.4%.
  • DMSO dimethyl sulfoxide
  • the positive control triterpene extract was prepared with DMSO at 50 mg/mL, the final concentration of the test was 50 ⁇ g/mL, and the final concentration of DMSO in the solution was maintained at 0.4%.
  • the negative control is a reaction solution containing the same concentration of DMSO (0.4%).
  • the positive control metformin hydrochloride was prepared with ultrapure water to 250 mM, the final concentration of the test was 2 mM, and the final concentration of DMSO was maintained at 0.4%.
  • the positive control berberine (Berberine) was prepared with ultrapure water to 40mM, the final concentration of the test was 20 ⁇ M, and the final concentration of DMSO was maintained at 0.4%.
  • the test substance was added to the cell reaction solution, and the culture was continued for 6 hours. Then the glucose concentration in each well of the culture medium (ie reaction solution) was measured by the standard curve method, and then the culture medium was discarded, the cells were lysed with 250 mM NaOH after washing with PBS and the protein concentration was measured by the Coomassie brilliant blue method. By reading the absorbance value, the standard curve method is used to determine the concentration of glucose and the concentration of protein in the culture medium in all sample wells, and the ratio is used to measure the average gluconeogenesis level of the cells in the well.
  • the LSD method was used to analyze the differences. The results are expressed as mean ⁇ standard deviation, and p ⁇ 0.05 indicates that the difference is significant.
  • Fig. 1 shows the effects of charantoside C and Momordicoside F1, the triterpenoids of momordica charantia, and their compositions in different proportions on liver gluconeogenesis.
  • the results showed that the inhibitory rate of hepatic gluconeogenesis of metformin was 61.01% when the usual amount of 2mM was used.
  • the inhibition rate of berberine at 20 ⁇ M was 15.61%.
  • the inhibitory rate of triterpene extract at a concentration of 50 ⁇ g/mL was 54.22%.
  • the hepatic gluconeogenesis inhibitory activity of Charantoside C and Momordicoside F1 at 20 ⁇ M is 22.94% and 38.34%, respectively, which are higher than berberine and close to the triterpene extract (the bitter melon triterpene extract is a mixture containing triterpenes, proteins, polysaccharides, etc.)
  • the bitter melon triterpene extract is a mixture containing triterpenes, proteins, polysaccharides, etc.
  • Many substances because of the synergistic effect between the substances, and the large amount of the extract, usually the activity of the extract is better than the monomer, and the two compounds in this application are isolated monomeric compounds, and their activity is close to the triterpene extraction The results show that these two compounds are the main active monomers in the bitter melon triterpene extract).
  • bitter melon triterpene compounds Charantoside C and Momordicoside F1 have significant liver gluconeogenesis inhibitory activity, and are bitter melon triterpene extracts. It is the main component that exerts hepatic gluconeogenesis inhibitory activity.
  • the electronic tongue ( ⁇ -ASTREE Liquid Taste Analyser, Alpha M.O.S., Toulouse, France) is used to detect the bitterness of the bitterness of the bitter melon triterpene compounds Charantoside C and Momordicoside F1, and the composition in different proportions, to further illustrate the beneficial effects of the present invention as follows:
  • the processed samples are directly placed in the electronic tongue special beaker for analysis. Each sample is repeated 7 times during the experiment.
  • the analysis conditions of the experiment are as follows: sample volume: 25mL, sample collection time: 120s, and each analysis time: 180s.
  • bitterness intensity is defined as: the bitterness intensity of a 1g/mL sample ethanol solution is equivalent to the bitterness intensity of a caffeine ethanol solution (g/mL).
  • the compounds Charantoside C and Momordicoside F1 were quantitatively analyzed by the electronic tongue, and their bitterness intensity was lower than the electronic tongue detection threshold.
  • the predicted values of the caffeine standard curve were 1.50 ⁇ 10 -6 and 3.39 ⁇ 10 -10 g/mL caffeine (i.e. 1g/
  • the bitterness intensity of mL of Charantoside C and Momordicoside F1 ethanol solution is equivalent to 1.50 ⁇ 10 -6 and 3.39 ⁇ 10 -10 g/mL caffeine ethanol solution bitterness intensity), which are non-bitter compounds.
  • bitterness intensity of the bitter melon triterpene composition in Example 1 is lower than the electronic tongue detection threshold, and the bitterness intensity prediction value of the 1g/mL composition 4 (predicted value of the caffeine standard curve) is 1.22 ⁇ 10 -8 g/mL caffeine , Is a non-bitter compound.
  • bitterness intensity of the bitter melon triterpene composition in Example 2 is lower than the electronic tongue detection threshold, and the bitterness intensity prediction value of 1g/mL composition 3 (predicted value of the caffeine standard curve) is 7.51 ⁇ 10 -7 g/mL caffeine , Is a non-bitter compound.
  • the bitterness intensity of the bitter melon triterpene composition in Example 3 is lower than the electronic tongue detection threshold, and the bitterness intensity prediction value of 1g/mL composition 2 (predicted value of the caffeine standard curve) is 5.24 ⁇ 10 -6 g/mL caffeine , Is a non-bitter compound.

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Abstract

本发明提供了一种抑制肝糖异生的非苦味苦瓜三萜组合物，其活性成分主要由Charantoside C和Momordicoside F1组成，所述Charantoside C和Momordicoside F1的质量份配比为1:0.6～1.5。还公开了上述苦瓜三萜组合物在制备具有抑制肝糖异生效果的健康食品中的应用。

Description

一种抑制肝糖异生的非苦味苦瓜三萜组合物及其应用 技术领域
本发明属于食品精深加工技术领域，具体涉及一种抑制肝糖异生的非苦味苦瓜三萜组合物及其应用。
背景技术
苦瓜(Momordica charantia L.)是热带-亚热带特色药食两用蔬菜，性味苦寒，可用于预防和改善糖尿病、肥胖等多种能量代谢失衡相关疾病。三萜类化合物是苦瓜调节糖脂代谢的重要物质基础，同时也是其最主要的苦味物质。虽然大量体内外实验确证了苦瓜三萜提取物具有显著的降血糖生物活性，但强烈的苦味限制了其在食品领域的应用。
三萜类化合物被普遍认为具有苦味。目前从苦瓜中分离得到的三萜类化合物近150个，但单一化合物的苦味强度并无研究报道。此外，目前已知的具有降糖活性的苦瓜三萜化合物约14个，其降糖活性主要包括激活AMPK信号通路、刺激GLUT4在细胞膜对葡萄糖的转运，抑制α-葡萄糖苷酶的活性，控制餐后血糖水平等，但关于三萜化合物对肝糖异生的影响并不清楚。2型糖尿病的病理特征在于血液中葡萄糖含量过高，或/且外周组织的糖利用率降低。而血液中过高的葡萄糖含量正来源于肝脏葡萄糖输出。肝糖异生在糖尿病患者中的发生率高达90％。抑制肝糖异生是治疗糖尿病的重要靶点。
综上所述，目前关于苦瓜三萜的苦味强度尚未可知，且其对肝糖异生的影响亦不清楚。
发明内容
本发明的目的在于提供一种苦瓜三萜组合物，该组合物具有良好的抑制肝糖异生活性，且无明显苦味。
本发明的目的还在于提供上述苦瓜三萜组合物在制备具有抑制肝糖异生效果健康食品中的应用。
本发明的上述第一个目的可以通过以下技术方案来实现：一种抑制肝糖异生的非苦味苦瓜三萜组合物，其活性成分主要由Charantoside C和Momordicoside F1组成，所述Charantoside C和Momordicoside F1的质量份配比为1:0.6～1.5。
本发明以咖啡因为标准品，采用电子舌对苦瓜中分离获得的系列苦瓜三萜化合物进行了苦味强度定量分析。同时以大鼠原代肝细胞为模型，分析了该系列三萜化合物的肝糖异生抑制活性。从中筛选获得2个具有强活性的非苦味三萜化合物Charantoside C和Momordicoside F1。在此基础之上，结合活性与苦味分析评价，发明了具有增强其活性的组合比例，Charantoside C和Momordicoside F1的质量份配比为1:0.6～1.5。该组合物具有显著的抑制肝糖异生活性，且无明显苦味。可以作为糖尿病人专用健康食品的功能性配料。
其中Charantoside C和Momordicoside F1的化学结构式分别如下：
Figure PCTCN2019125045-appb-000001
其中所述Charantoside C、Momordicoside F1以及所述苦瓜三萜组合物的苦味强度均低于电子舌(α-ASTREELiquidTaste Analyser，Alpha M.O.S.,Toulouse,France)检测阈值，属于非苦味物质。
具体的，上述两个化合物Charantoside C、Momordicoside F1经电子舌定量分析，其苦味强度低于电子舌检测阈值(1×10 -4g/mL咖啡因)，咖啡因标准曲线预测值分别为1.50×10 -6和3.39×10 -10g/mL咖啡因(即1g/mL的Charantoside C和Momordicoside F1乙醇溶液的苦味强度分别相当于1.50×10 -6和3.39×10 -10g/mL的咖啡因乙醇溶液苦味强度)，属于非苦味化合物。
由Charantoside C和Momordicoside F1按照1:0.6～1.5的质量份配比组成的苦瓜三萜组合物，其苦味强度同样低于电子舌检测阈值，1g/mL组合物苦味强度的咖啡因标准曲线预测值为3.40×10 -8～5.67×10 -6g/mL咖啡因，也属于非苦味物质。
因此，本发明中苦瓜三萜类化合物Charantoside C、Momordicoside F1以及由这两个组合物制成的苦瓜三萜组合物是非苦味的。
本发明的上述第二个目的可以通过以下技术方案来实现：上述苦瓜三萜组合 物在制备具有抑制肝糖异生活性的健康食品中的应用。
本发明实施例中的实验结果表明，Charantoside C和Momordicoside F1在20μM浓度时的肝糖异生抑制活性分别为22.94％和38.34％，高于黄连素(糖尿病治疗传统中草药黄连的主要成分)在20μM时的抑制率15.61％，肝糖异生抑制活性显著。
根据Charantoside C和Momordicoside F1结构差异及由结构引起的活性差异分析与预测，由Charantoside C和Momordicoside F1按照1:0.6～1.5的质量份配比组成的苦瓜三萜组合物，其肝糖异生抑制活性显著性高于相同剂量下的单一化合物。
优选的，所述苦瓜三萜组合物在健康食品中添加的质量百分含量为0.1～2％。
作为本发明一种优选的实施方式，所述Charantoside C和Momordicoside F1的制备方法如下：
(1)选取苦瓜，预处理后得苦瓜干粉；
(2)在苦瓜干粉中加入体积百分含量为70％的乙醇提取后，得苦瓜三萜提取液；
(3)将苦瓜三萜提取液依次用石油醚、乙酸乙酯、正丁醇萃取，然后浓缩，依次得到石油醚相、乙酸乙酯相和正丁醇相；
(4)将正丁醇相用大孔树脂粗纯化后，依次用水、体积百分含量为20％的乙醇-水及体积百分含量为80％的乙醇-水洗脱，其中体积百分含量为80％的乙醇-水洗脱部分为三萜富集部分；
(5)将体积百分含量为80％的乙醇-水洗脱部分用正相硅胶柱分离纯化，以氯仿/甲醇为洗脱剂，依次采用体积比为50:1的氯仿/甲醇和体积比为20:1的氯仿/甲醇洗脱；
(6)收集体积比为20:1的氯仿/甲醇洗脱液，采用反相柱色谱分离，分别采用体积百分含量为50％和体积百分含量为60％的甲醇水梯度洗脱；
(7)收集体积百分含量为60％的甲醇水梯度洗脱液，采用高效液相色谱仪进一步纯化，并用体积百分含量为80％的乙腈洗脱，得到苦瓜三萜化合物Charantoside C和Momordicoside F1。
在上述Charantoside C和Momordicoside F1的制备方法中：
优选的，步骤(1)中所述的预处理包括清洗、去籽、切片、烘干、粉碎和 过筛处理。
优选的，步骤(3)中苦瓜三萜提取液与石油醚、乙酸乙酯、正丁醇的萃取体积比均为1:3，采用石油醚提取1次，乙酸乙酯萃取2次，正丁醇萃取3次。
优选的，步骤(4)中采用D101大孔树脂粗纯化。
与现有技术相比，本发明具有如下有益效果：
(1)现有技术中苦瓜三萜提取物具有显著的降血糖活性，但对于肝脏葡萄糖异生的影响未知，且其苦味强烈，限制了其在食品中的应用，本发明从前期分离获得的19个苦瓜三萜化合物中筛选得到2个具有肝糖异生抑制活性的非苦味三萜化合物，并获得了具有增强其活性的组合比例，突破了长期以来苦瓜三萜提取物因苦味强烈、消费接受度低在食品精深加工领域的应用限制。
(2)本发明中的三萜组合物可以抑制肝脏葡萄糖异生，其效果优于黄连素，是苦瓜三萜提取物抑制肝糖异生的主要活性成分，并且该组合物无明显苦味，可以作为功能性配料添加在食品中，开发成为风味较好、接受度高的糖尿病人专用健康食品。
附图说明
图1是实施例4中苦瓜三萜组合物对肝实质细胞葡萄糖生成的影响，其中DMSO，二甲基亚砜(0.4％)；Metfoumin，二甲双胍(2mM)；Berberine黄连素(20μM)；triterpenoid extracts，苦瓜三萜提取物(50μg/mL)；组合物1,Charantoside C:Momordicoside F1＝1:0.25(质量份配比，下同)；组合物2,Charantoside C:Momordicoside F1＝1:0.6；组合物3,Charantoside C:Momordicoside F1＝1:1；组合物4,Charantoside C:Momordicoside F1＝1:1.5；组合物5,Charantoside C:Momordicoside F1＝1:4；组合物终浓度均为20μM。
具体实施方式
下面将结合具体的实施方式进一步说明本发明，但本发明要求保护的范围并不局限与下列实施方式。
以下实施例中采用的各原料，如无特殊说明，均为市售产品。
实施例1
本实施例提供的抑制肝糖异生的非苦味苦瓜三萜组合物，其活性成分主要由Charantoside C和Momordicoside F1组成，Charantoside C和Momordicoside F1的质量份配比为1:1.5。
该组合物以2.0％的质量百分含量添加于功能性口服液中，可以有效抑制肝糖异生，控制餐后血糖升高。
本实施例中的Charantoside C和Momordicoside F1可以通过化学合成获得，也可以直接采用下文实施例4中的方法制备获得。
实施例2
本实施例提供的抑制肝糖异生的非苦味苦瓜三萜组合物，其活性成分主要由Charantoside C和Momordicoside F1组成，Charantoside C和Momordicoside F1的质量份配比为1:1。
该组合物以1.0％的质量百分含量添加于冲调类饮料中，可以有效抑制肝糖异生，控制餐后血糖升高。
实施例3
本实施例提供的抑制肝糖异生的非苦味苦瓜三萜组合物，其活性成分主要由Charantoside C和Momordicoside F1组成，Charantoside C和Momordicoside F1的质量份配比为1:0.6。
该组合物以0.1％的质量百分含量添加于营养餐粉中，可以有效抑制肝糖异生，控制餐后血糖升高。
实施例4
下面以原代分离SD大鼠肝实质细胞为模型，二甲双胍(metformin)、黄连素(Berberine)、苦瓜三萜提取物(triterpenoid extracts，下文中制备获得)为阳性对照，评价苦瓜三萜化合物Charantoside C和Momordicoside F1，及其不同比例组合物对肝糖异生的影响，进一步阐述本发明的有益效果。过程如下：
1、试验材料
1.1样品制备
新鲜苦瓜清洗、去籽、切片，55℃烘干12小时，粉碎过80目筛，得苦瓜干粉。
苦瓜干粉用70％乙醇按照料液比1:5常温提取2次，得到苦瓜三萜提取液。
粗提液浓缩后依次用石油醚、乙酸乙酯、正丁醇萃取(萃取体积比1:3，石油醚提取1次，乙酸乙酯萃取2次，正丁醇萃取3次)，之后采用旋转蒸发仪真空浓缩，分别得到石油醚相、乙酸乙酯相和正丁醇相。
正丁醇相通过D101大孔树脂粗纯化，依次用水洗脱，20％乙醇-水洗脱及80％乙醇-水洗脱。
其中80％乙醇-水洗脱部分为三萜富集部分，进一步采用正相硅胶柱分离纯化。
以氯仿/甲醇为洗脱剂，依次采用氯仿/甲醇50:1和氯仿/甲醇20:1洗脱。
收集氯仿/甲醇20:1部分，采用反相柱色谱分离，50％、60％甲醇水梯度洗脱。
收集60％甲醇水洗脱液，经制备型高效液相色谱仪进一步纯化，80％乙腈洗脱，得到多个洗脱峰。
通过核磁等现有的波谱分析手段解析其化学结构，同时对比现有文献，确认其中两个洗脱峰为苦瓜三萜化合物Charantoside C和Momordicoside F1。
上述过程中，提取得到的苦瓜三萜提取液，经真空旋转蒸发浓缩得到三萜提取物(triterpenoid extracts)，细胞实验备用。
1.2实验细胞
采用原位灌注消化法，利用typeⅣ胶原酶消化SD大鼠肝脏，分散肝细胞后转移至49％Percoll分离液离心，弃去上清后将细胞接入以0.2％明胶包被的24孔板中贴壁6h。再以无血清低糖培养基饥饿8h后，弃去上清，PBS缓冲液清洗后加入反应液(含2mM丙酮酸钠和20mM乳酸钠的无糖DMEM培养基)，等待样品测试。
2、试验方法
三萜化合物溶解于二甲基亚砜(DMSO)中配制成10μM母液，用上述反应液稀释后，最终加入到细胞培养孔中的终浓度为20μM，DMSO终浓度0.4％。阳性对照三萜提取物用DMSO配置成50mg/mL，测试终浓度为50μg/mL，并维持溶液中DMSO终浓度0.4％。阴性对照为含相同浓度DMSO(0.4％)的反应液。阳性对照盐酸二甲双胍，用超纯水配置成250mM，测试终浓度为2mM，并维持DMSO终浓度0.4％。阳性对照黄连素(Berberine)，用超纯水配置成40mM，测试终浓度为20μM，并维持DMSO终浓度0.4％。细胞反应液中加入受试物，继续培养6h。之后以标准曲线法测定每孔培养基(即反应液)中葡萄糖浓度，而后弃去培养基，PBS换洗后以250mM NaOH裂解细胞并以考马斯亮蓝法测定蛋白浓度。通过读取吸光值，以标准曲线法测定所有样品孔内培养基中葡萄糖 浓度与裂解后蛋白质浓度，并以其比值衡量该孔细胞的平均糖异生水平。
2.1数据分析
采用LSD法分析差异性。结果以均数±标准差表示，p<0.05为差异有显著性。
2.2实验结果
苦瓜三萜化合物Charantoside C和Momordicoside F1，及其不同比例组合物对肝脏糖异生活性的影响见图1。结果发现二甲双胍在常用量2mM时的肝糖异生抑制率为61.01％。20μM时黄连素的抑制率为15.61％。50μg/mL浓度时的三萜提取物抑制率为54.22％。20μM的Charantoside C和Momordicoside F1的肝糖异生抑制活性分别为22.94％和38.34％，高于黄连素，接近三萜提取物(苦瓜三萜提取物是一个混合物，里面有三萜、蛋白、多糖等很多物质，因为物质间有协同增效作用，而且提取物剂量大，通常提取物的活性会好过单体，而本申请中的两个化合物是分离出来的单体化合物，其活性接近三萜提取物表明这两个化合物是苦瓜三萜提取物里面的主要活性单体)，因此，可以判定，苦瓜三萜化合物Charantoside C和Momordicoside F1具有显著的肝糖异生抑制活性，且是苦瓜三萜提取物发挥肝糖异生抑制活性的主要成分。
当Charantoside C和Momordicoside F1按不同比例组合后，其肝糖异生活性有显著增加。其中组合物4(Charantoside C:Momordicoside F1＝1:1.5，抑制率为43.54％)抑制率显著高于单一化合物相同剂量下的抑制率。
实施例5
下面采用电子舌(α-ASTREELiquidTaste Analyser，Alpha M.O.S.,Toulouse,France)检测苦瓜三萜化合物Charantoside C和Momordicoside F1，及其不同比例组合物的苦味强度，进一步阐述本发明的有益效果过程如下：
1、试验材料
1.1样品制备
称取1mg Charantoside C、Momordicoside F1，及其不同比例组合物，加30mL无水乙醇，溶解后将样品放置于电子舌专用烧杯(25mL)中待测。无水乙醇样品为空白对照。同时以无水乙醇配制不同浓度的咖啡因标准品，通过浓度量化的咖啡因标准曲线对各样品进行苦味的预测。咖啡因浓度量化标准曲线的相关系数R 2为0.9823。
1.2电子舌测定过程
处理好的样品直接放置于电子舌专用烧杯中进行分析，每个样品在实验时重复7次，实验的分析条件如下：样品体积：25mL，样品采集时间：120s，每次分析时间：180s。
1.3数据处理
采用Alpha soft软件进行数据分析。苦味强度表述定义为：1g/mL样品乙醇溶液的苦味强度相当于咖啡因乙醇溶液的苦味强度(g/mL)。
1.4实验结果
化合物Charantoside C和Momordicoside F1经电子舌定量分析，其苦味强度低于电子舌检测阈值，咖啡因标准曲线预测值分别为1.50×10 -6和3.39×10 -10g/mL啡因(即1g/mL的Charantoside C和Momordicoside F1乙醇溶液的苦味强度分别相当于1.50×10 -6和3.39×10 -10g/mL的咖啡因乙醇溶液苦味强度)，属于非苦味化合物。
实施例1中的苦瓜三萜组合物的苦味强度低于电子舌检测阈值，1g/mL组合物4的苦味强度预测值(咖啡因标准曲线预测值)为1.22×10 -8g/mL咖啡因，属于非苦味化合物。
实施例2中的苦瓜三萜组合物的苦味强度低于电子舌检测阈值，1g/mL组合物3的苦味强度预测值(咖啡因标准曲线预测值)为7.51×10 -7g/mL咖啡因，属于非苦味化合物。
实施例3中的苦瓜三萜组合物的苦味强度低于电子舌检测阈值，1g/mL组合物2的苦味强度预测值(咖啡因标准曲线预测值)为5.24×10 -6g/mL咖啡因，属于非苦味化合物。
以上实施例仅用于阐述本发明，并不限制本发明的保护范围。本技术领域的普通技术人员依据以上公开的范围，均可实现本发明的目的。

Claims (8)

  1. 一种抑制肝糖异生的非苦味苦瓜三萜组合物，其特征是：其活性成分主要由Charantoside C和Momordicoside F1组成，所述Charantoside C和Momordicoside F1的质量份配比为1:0.6～1.5。
  2. 根据权利要求1所述的抑制肝糖异生的非苦味苦瓜三萜组合物，其特征是：所述Charantoside C、Momordicoside F1以及所述苦瓜三萜组合物均属于非苦味物质。
  3. 权利要求1或2所述苦瓜三萜组合物在制备具有抑制肝糖异生效果的健康食品中的应用。
  4. 根据权利要求3所述的应用，其特征是：所述苦瓜三萜组合物在健康食品中添加的质量百分含量为0.1～2％。
  5. 根据权利要求3所述的应用，其特征是：所述Charantoside C和Momordicoside F1的制备方法如下：
    (1)选取苦瓜，预处理后得苦瓜干粉；
    (2)在苦瓜干粉中加入体积百分含量为70％的乙醇提取后，得苦瓜三萜提取液；
    (3)将苦瓜三萜提取液依次用石油醚、乙酸乙酯、正丁醇萃取，然后浓缩，依次得到石油醚相、乙酸乙酯相和正丁醇相；
    (4)将正丁醇相用大孔树脂粗纯化后，依次用水、体积百分含量为20％的乙醇-水及体积百分含量为80％的乙醇-水洗脱，其中体积百分含量为80％的乙醇-水洗脱部分为三萜富集部分；
    (5)将体积百分含量为80％的乙醇-水洗脱部分用正相硅胶柱分离纯化，以氯仿/甲醇为洗脱剂，依次采用体积比为50:1的氯仿/甲醇和体积比为20:1的氯仿/甲醇洗脱；
    (6)收集体积比为20:1的氯仿/甲醇洗脱液，采用反相柱色谱分离，分别采用体积百分含量为50％和体积百分含量为60％的甲醇水梯度洗脱；
    (7)收集体积百分含量为60％的甲醇水梯度洗脱液，采用高效液相色谱仪进一步纯化，并用体积百分含量为80％的乙腈洗脱，得到苦瓜三萜化合物 Charantoside C和Momordicoside F1。
  6. 根据权利要求5所述的应用，其特征是：步骤(1)中所述的预处理包括清洗、去籽、切片、烘干、粉碎和过筛处理。
  7. 根据权利要求5所述的应用，其特征是：步骤(3)中苦瓜三萜提取液与石油醚、乙酸乙酯、正丁醇的萃取体积比均为1:3，采用石油醚提取1次，乙酸乙酯萃取2次，正丁醇萃取3次。
  8. 根据权利要求5所述的应用，其特征是：步骤(4)中采用D101大孔树脂粗纯化。
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