WO2021109836A1 - 一种茶皂苷类化合物、制备方法及应用 - Google Patents

一种茶皂苷类化合物、制备方法及应用 Download PDF

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WO2021109836A1
WO2021109836A1 PCT/CN2020/128618 CN2020128618W WO2021109836A1 WO 2021109836 A1 WO2021109836 A1 WO 2021109836A1 CN 2020128618 W CN2020128618 W CN 2020128618W WO 2021109836 A1 WO2021109836 A1 WO 2021109836A1
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tea
tea saponin
substituted
compound
preparation
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PCT/CN2020/128618
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French (fr)
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侯如燕
崔传坚
周春松
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江苏一片叶高新科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N45/00—Biocides, pest repellants or attractants, or plant growth regulators, containing compounds having three or more carbocyclic rings condensed among themselves, at least one ring not being a six-membered ring
    • A01N45/02—Biocides, pest repellants or attractants, or plant growth regulators, containing compounds having three or more carbocyclic rings condensed among themselves, at least one ring not being a six-membered ring having three carbocyclic rings

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  • the invention relates to a biochemical technology, in particular to a tea saponin compound, a preparation method and an application.
  • the use of natural plant metabolites to control harmful insects is a hot topic in pesticide research.
  • the active ingredients are mainly terpenes, alkaloids, flavonoids, steroids, and phenols.
  • plant-derived insecticides that have been industrialized, mainly including azadirachtin, celangulin, matrine, wolf toxin and osthole.
  • saponins synthesized by several plants are reported to have defensive effects.
  • Saponins or sapogenins can form a large number of hybrids in plants, and are characterized by their surface activity. Due to the stretching activity of saponins, they dissolve in water by forming a foaming solution.
  • saponins are mainly used in industry and medicine.
  • the pharmaceutical industry uses several saponins to obtain drugs, while the cosmetics industry uses several saponins to obtain detergent properties.
  • studies have shown that there are relatively few saponins with insecticidal activity, and the saponins extracted from different plants have large differences in their activities against harmful insects.
  • Camellia belongs to the Camellia family, including 280 species of plants. Worldwide, most naturally occurring species and hybrids are distributed in Southeast Asia, from the Himalayas to Japan, from southern China to Java and Sumatra. Several of the camellia plants have important economic value, including tea made from the leaves of tea and Assam tea. As the world's second-most consumption beverage, it has many benefits to human health; tea plum and camellia It is a famous ornamental plant; Camellia seed oil, as a protective agent for keeping skin and hair healthy, is a traditional cosmetic with a long history of use in Japan; Camellia oleifera is a unique oil crop in China. Camellia seeds have high economic value.
  • the oil contains a lot of unsaturated fatty acids and is a high-quality natural edible oil.
  • Camellia plants contain a large number of secondary metabolites.
  • tea saponin has a unique triterpene structure and a wide range of pharmacological functions.
  • Tea saponins are a kind of promising natural insecticides with good emulsification, foaming, penetration and uniform dispersibility, and have been used in papermaking, textiles, detergents, skin care, hair care and other fields.
  • tea saponin has a content of more than 10% in the seeds of Camellia oleifera. Chen et al. proved that a solution containing 25% of the active ingredients of Camellia oleifera extract can significantly increase the mortality of cabbage butterfly larvae (84%). How to find that feeding crude tea saponin extracts from Camellia oleifera can inhibit the growth and development of Plutella xylostella.
  • Camellia plants are rich in active substances, which have different degrees of biological activity to a variety of organisms.
  • it is mainly focused on the preliminary activity screening, and the research on its active ingredients has not been carried out yet.
  • the technical problem to be solved by the present invention is: how to ascertain the insecticidal properties of the tea saponin compound, and provide a tea saponin compound, a preparation method and an application.
  • a tea saponin compound of the present invention is characterized in that its general structural formula is as follows:
  • R1 is a substituted or unsubstituted five-carbon sugar or six-carbon sugar
  • R2 is a substituted or unsubstituted hydroxyl or carboxyl group
  • R3 is a substituted or unsubstituted hydroxyl or organic acid
  • R4 is a substituted or unsubstituted hydroxyl or organic acid
  • R5 is a substituted or unsubstituted hydroxyl or organic acid
  • R6 is a substituted or unsubstituted hydroxyl or organic acid
  • R7 is a substituted or unsubstituted hydroxyl or carboxyl group.
  • a method for preparing tea saponin compounds including the following steps:
  • the oil tea seed cake is crushed into powder; extracted with ethanol water for many times to obtain the crude extract extract, which is separately extracted with petroleum ether, ethyl acetate, n-butanol and concentrated under reduced pressure to obtain petroleum ether and ethyl acetate.
  • Ester part and n-butanol part of the extract take 120g of the n-butanol extraction part and dissolve it with methanol, mix with 100-200 mesh silica gel, and pack the column with 100-200 mesh silica gel dry method, dry load the sample; use ethyl acetate Ester: methanol eluent system for gradient elution, the concentration gradient is EtOAc-MeOH volume ratio 100:0 to 0:100 as a gradient elution, each eluent gradient elution 6 times the column volume, the obtained wash
  • the deliquation was separated and purified with an ODS reversed-phase silica gel column, eluted with a methanol-water eluent gradient, and the volume ratio of the gradient concentration of MeOH:H 2 O was 0:100 ⁇ 30:70 ⁇ 50:50 ⁇ 70:30 ⁇ 100: 0, to obtain tea saponin compounds.
  • a Camelliasaponin B1, Theasaponin H1, Oleiferasaponin C3 monomer compound isolated from the tea saponin compounds, and its general structural formulas are as follows:
  • tea saponin compounds then dissolve tea saponin compounds in 35% acetonitrile water, separate and purify by preparative liquid phase (Prep-HPLC, mobile phase: acetonitrile-water) gradient elution, and perform mobile phase based on UV absorption chromatogram Collected, combined with HPLC liquid phase analysis, combined the same components, concentrated under reduced pressure and repeatedly prepared, and separated Camelliasaponin B1, Theasaponin H1, and Oleiferasaponin C3 three tea saponin monomer compounds.
  • Prep-HPLC mobile phase: acetonitrile-water
  • the present invention proves that tea saponin compounds and multiple monomers have insecticidal properties, and develops new applications of Camellia oleifera. Tea saponin compounds are effective against tea looper larvae. It has a strong insecticidal effect, not only can destroy the outer epidermis and even the inner skeleton of the tea looper larva through contact action, but also the intestinal cells of the tea looper will be greatly destroyed after eating tea saponin. Through two different mechanisms of action, tea saponin compounds can effectively prevent pests. As a natural plant-derived insecticidal compound, tea saponin compounds have great potential to become green pesticides.
  • Figure 1 is a scanning electron microscope photograph of the surface of the larva of the tea looper
  • A blank treatment, a1: surface observed at 100 times magnification, a2: surface observed at 5000 times magnification, a3: surface near the villi observed at 5000 times magnification;
  • B Camelliasaponin B1 treatment, b1: magnification 100 The surface observed under magnification, b2: the surface observed under 5000 times magnification, b3: the surface near the villi observed under 5000 times magnification;
  • Figure 2 is a picture of chitin staining of the midgut section of the larvae of the tea looper larva
  • a blank processing
  • b Camelliasaponin B1 processing
  • Fig. 3 is a diagram showing the staining and structural changes of histocytes in the larvae of the tea looper after different treatments
  • a blank processing
  • b Camelliasaponin B1 processing.
  • Camellia seed cake of this embodiment produced and provided by Anhui Huangshan Yuzigui Camellia Processing Plant, and Camellia seed was purchased in the mountainous area of southern Anhui; Column chromatography silica gel powder (100-200 mesh, 200-300 mesh): China National Pharmaceutical Group; Phase silica gel: Japanese YMC ODS-A C18 material, diameter 50 ⁇ m; Sephadex LH-20 gel: imported from the United States Pharmaeia; Chromatographic pure methanol, acetic acid, acetonitrile: United States TEDIA company; Water: laboratory pure water mechanism ultra-pure water; Bifenthrin original drug (98% purity): Provided by China Yuanye Biological Co., Ltd.; other reagents and drugs are analytically pure produced by Sinopharm Group unless otherwise specified.
  • the camellia seed cake was crushed into powder, first extracted with 70% ethanol water three times (10L/10L/10L) to obtain the crude extract extract, and then separately extracted with petroleum ether, ethyl acetate, n-butanol and Concentrate under reduced pressure to obtain extracts of petroleum ether, ethyl acetate and n-butanol. Take about 120g of the n-butanol extraction part and dissolve it with methanol, mix the sample with 100-200 mesh silica gel, and pack the column with 100-200 mesh silica gel dry method, and dry load the sample.
  • the obtained eluate was separated and purified by ODS reversed-phase silica gel column, and eluted with a methanol-water eluent gradient, and the volume ratio of the gradient concentration of MeOH-H2O was 0:(100 ⁇ 30):(70 ⁇ 50):(50 ⁇ 70): (30 ⁇ 100):0, to obtain the total saponins of camellia oleifera, namely tea saponins compounds.
  • the total saponins of Camellia oleifera were dissolved in 35% acetonitrile water, and separated and purified by preparative liquid phase (Prep-HPLC, mobile phase: acetonitrile-water) gradient elution.
  • the mobile phase was collected according to the UV absorption chromatogram, combined with HPLC liquid analysis and combined the same
  • the components were repeatedly prepared by concentration under reduced pressure, and three tea saponin monomer compounds, Camelliasaponin B1, Theasaponin H1, and Oleiferasaponin C3, were separated.
  • R1 is a substituted or unsubstituted five-carbon sugar or six-carbon sugar, wherein the five-carbon sugar is one of arabinose and xylose; the six-carbon sugar is glucose, galactose, rhamnose, glucuronic acid and One of the methyl glucuronic acid esters;
  • R2 is one of substituted or unsubstituted hydroxy or carboxy
  • R3 is a substituted or unsubstituted hydroxyl or organic acid, wherein the substitution is one of angelic acid, tigeous acid, hexenoic acid, 2-methylbutyric acid and cinnamic acid;
  • R4 is a substituted or unsubstituted hydroxyl or organic acid, wherein the substitution is a methanol group, an aldehyde group, an ester acyl group, an acetoxy group, a cinnamoyl group, a hydrogenated cinnamoyl group, a vinyl group, a vinyl group, an angelyloxy group, and One of methylbutyryl;
  • R5 is a substituted or unsubstituted hydroxy or organic acid, wherein the substitution is a methanol group, an aldehyde group, an ester acyl group, an acetoxy group, a cinnamoyl group, a hydrogenated cinnamoyl group, a vinyl group, a vinyl group, an angelyloxy group, and One of methylbutyryl;
  • R6 is a substituted or unsubstituted hydroxyl or organic acid, wherein the substitution is a methanol group, an aldehyde group, an ester acyl group, an acetoxy group, a cinnamoyl group, a hydrogenated cinnamoyl group, a vinyl group, a vinyl group, an angelyloxy group, and One of methylbutyryl;
  • R7 is one of a substituted or unsubstituted hydroxyl group or a carboxyl group.
  • Tea looper belongs to the Lepidoptera, Geometridae, and is the main insect that harms tea gardens.
  • the food was fed from the agricultural garden of Anhui Agricultural University Tea tree leaves (Shu Chazao, tea tree species), in order to ensure the stability of the variety, it is passed down to the 30th generation. Screen the same batch of tea loopers that grow to the second instar.
  • the experimental group consists of 4 groups: Camelliasaponin B1, Theasaponin H1, Oleiferasaponin C3, each group diluted with water to 8 concentrations (5, 10, 15, 20, 25, 30, 40, 50 mg/L), each The concentration is 3 parallels, each with 20 tea loopers, and a Burkard automatic microtiter is used to accurately titrate 1 ⁇ L drop on the back of the tea loopers. Observe and record the number of insect deaths 24 hours after the titration, consider the failure to crawl normally as death, and calculate the virulence regression curve and LC 50 value according to the probability analysis method.
  • the tea looper was fed with leaves impregnated with 50mg/L Camelliasaponin B1. After 24 hours, the larvae were anesthetized in ether, the intestines were removed, and fixed in Bouns solution for 12 hours. After that, they were dried and transparentized in ethanol/xylene (1:1). After putting in paraffin, the sample was cut into 7 ⁇ m sections and stained with hematoxylin-eosin. The morphological changes of the midgut of the tested larvae were dried and compared with the tissues of a control group taken from an untreated diet and prepared in the same manner.
  • Toxicity regression equation shows the relationship between dose and lethal value
  • LC 50 means 50% lethal concentration, that is, the dose required to kill half of the tested species
  • Toxicity regression equation shows the relationship between dose and lethal value
  • LC 50 means 50% lethal concentration, that is, the dose required to kill half of the tested species
  • the waxy layer is the main barrier to protect insects from external compounds.
  • the main function of the wax layer is to prevent dryness, feel for the environment, mechanical support and movement.
  • Some natural products have been found to have a strong destructive effect on the waxy layer of insects, but there has not been any report on the effect of tea saponin on the waxy layer of insects.
  • the effects of these two treatments on the epidermis of the tea looper larvae were observed by scanning electron microscopy ( Figure 1). Compared with the blank treatment ( Figure 1A a1), the epidermal layer of the tea looper larvae treated with the Camelliasaponin B1 aqueous solution became rough and severely wrinkled ( Figure 1B b1), and the trunk was dehydrated and shrunk (magnification 100 times).
  • tea saponin can produce high toxicity in a short period of time in contact with tea looper. Tea saponin affects the water balance of the tea looper by destroying the waxy layer on the surface of the tea looper, causing the tea looper to lose water and penetrate into the body, causing the tea looper to die due to water loss.
  • tea saponin can not only effectively destroy the waxy layer of tea looper, but also has a strong damaging effect on its chitin layer.
  • the chitin layer in the outer circle of the intestine was also damaged. It proves the extremely strong penetrating ability of tea saponin and the destructive ability from the outside to the inside.
  • the main functions of the midgut of the tea looper larvae include the production of digestive enzymes and the nutrient absorption of digested products.
  • the trophic matrix (PM) of the insect midgut is mainly composed of chitin and protein, which is believed to support digestion and protect against food particles and intestinal pathogens.
  • the midgut epithelium was composed of a single layer of digested cells, showing a brush border and cytoplasm with eosinophilic regions (the black arrow in Figure 3).
  • tea saponin compounds have a strong insecticidal effect on the tea looper larvae, not only can destroy the outer skin and even the internal skeleton structure of the tea looper larva through contact action, but also the tea looper intestine after eating tea saponin The tract cells will also be greatly damaged. Through two different mechanisms of action, tea saponin compounds can effectively prevent pests. As a natural plant-derived insecticidal compound, tea saponin compounds have great potential to become green pesticides.

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Abstract

本发明公开了一种茶皂苷类化合物、制备方法及应用，所述茶皂苷类化合物结构通式如式（I）。本发明探明了茶皂苷类化合物以及其多个单体具有杀虫的性能，开发了山茶属植物油茶的新应用，茶皂苷类化合物对茶尺蠖幼虫具有很强的杀虫作用，不仅可以通过触杀作用破坏茶尺蠖幼虫的外表皮甚至内骨骼的结构，而且取食茶皂苷后的茶尺蠖肠道细胞也将受到极大破坏。通过两种不同的作用机理，茶皂苷类化合物可以有效预防害虫，作为一种天然植物来源的杀虫化合物，茶皂苷类化合物具有成为绿色农药的巨大潜力

Description

一种茶皂苷类化合物、制备方法及应用 技术领域
本发明涉及一种生物化学技术，尤其涉及的是一种茶皂苷类化合物、制备方法及应用。
背景技术
利用天然的植物代谢产物进行有害昆虫防治是农药研究的热点方向。目前，已证实具有杀虫活性成分的植物有一千多种，其活性成分主要为萜烯类、生物碱、类黄酮、甾体、酚类等。其中，已进行产业化生产的植物源杀虫剂约20余种，主要包括印楝素、苦皮藤素、苦参碱、狼毒素及蛇床子素等。
在植物防御中涉及的物质中，据报道，由几种植物合成的皂苷是具有防御作用的物质。皂苷或皂苷元可在植物中形成大量的杂化物，以其表面活性为特征性质，由于皂苷的拉伸活性，它们通过形成泡沫溶液而溶于水。目前皂苷主要用于工业和药业中，制药工业使用几种皂苷来获得药物，而化妆品工业则使用几种皂苷来获得洗涤剂特性。而在皂苷类化合物用于杀虫活性研究方面，研究表明具有杀虫活性的皂苷类物质相对较少，且不同植物提取的皂苷对有害昆虫的活性有较大的差异。
山茶属为山茶科,包括280种植物。在全世界范围内，所有自然产生的物种和杂交种大部分分布在亚洲的东南部地区，从喜马拉雅山到日本，从中国南部到爪哇和苏门答腊。其中几种山茶属植物具有重要的经济价值，包括以茶和阿萨姆茶的叶子制成的茶，作为世界上消费量仅次于水的饮料，对人类健康具有许多益处；茶梅和山茶是著名的观赏植物；山茶种子油作为保持皮肤和毛发健康的保护剂，是日本具有悠久使用历史的传统化妆品；油茶是我国特有的油料作物，油茶籽具有很高的经济价值，榨出来的茶油中含有大量不饱和脂肪酸，是一种优质天然食用油。山茶属植物中蕴含着大量的次级代谢产物，茶皂苷作为其中一类丰富的次级代谢产物，具有着独特的三萜结构和广泛的药理功能。
茶皂苷是一类极具前景的天然杀虫剂，具有很好的乳化、起泡、渗透与均匀的分散性，并已在造纸、纺织、洗涤剂、护肤、护发等领域有所应用。同时茶皂苷在山茶属植物油茶的种子中，具有超过10％的含量。陈等证明，含有25％的油茶提取物有效成分的溶液可显着提高白菜蝶的幼虫死亡率(84％)。何等发现喂养来自油茶的含茶皂苷的粗提物能抑制小菜叶蛾的生长发育。综上所述，山茶属植物体内含有丰富的活性物质，对多种生物具有不同程度的生物活性。但总体而言，主要集中于初步的活性筛选方面，尚未开展其活性成分研究。在现有技术中，仅有涉及油茶粗提物用于杀虫的零星报道。
发明内容
本发明所要解决的技术问题在于：如何探明茶皂苷类化合物的杀虫特性，提供了一种茶皂苷类化合物、制备方法及应用。
本发明是通过以下技术方案解决上述技术问题的，本发明的一种茶皂苷类化合物，其特征在于，其结构通式如下：
Figure PCTCN2020128618-appb-000001
其中，R1为取代或未取代的五碳糖或六碳糖；
R2为取代或未取代的羟基或羧基；
R3为取代或未取代的羟基或有机酸；
R4为取代或未取代的羟基或有机酸；
R5为取代或未取代的羟基或有机酸；
R6为取代或未取代的羟基或有机酸；
R7为取代或未取代的羟基或羧基。
一种茶皂苷类化合物的制备方法，包括以下步骤：
将油茶籽饼粕粉碎成粉；用乙醇水浸提多次，得到粗提物浸膏，分别用石油醚、乙酸乙酯、正丁醇分级萃取并减压浓缩，得到石油醚部分、乙酸乙酯部分和正丁醇部分浸膏；取正丁醇萃取部分120g用甲醇溶解，与100～200目的硅胶等质量拌样，用100～200目的硅胶干法装柱，干法上样；以乙酸乙酯：甲醇的洗脱剂系统进行梯度洗脱，浓度梯度以EtOAc-MeOH体积比100:0到0:100作为梯度洗脱，每个洗脱剂梯度洗脱6倍柱体积，将得到的洗脱液用ODS反相硅胶柱分离纯化，以甲醇-水洗脱剂梯度洗脱，梯度浓度MeOH：H 2O体积比为0:100～30:70～50:50～70:30～100:0，得到茶皂苷类化合物。
一种茶皂苷类化合物在制备杀虫剂中的应用。
一种从所述的茶皂苷类化合物中分离出来的Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3单体化合物，其结构通式分别如下:
Figure PCTCN2020128618-appb-000002
所述的单体化合物的制备方法，具体过程如下:
首先制备茶皂苷类化合物，然后将茶皂苷类化合物用35％乙腈水溶解，经制备液相(Prep-HPLC，流动相：乙腈-水)梯度洗脱分离纯化，根据紫外吸收色谱图进行流动相收集， 结合HPLC液相分析合并相同组分，减压浓缩反复制备，分离得到Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3三种茶皂苷单体化合物。
所述的单体化合物在制备杀虫剂中的应用。
本发明相比现有技术具有以下优点：本发明探明了茶皂苷类化合物以及其多个单体具有杀虫的性能，开发了山茶属植物油茶的新应用，茶皂苷类化合物对茶尺蠖幼虫具有很强的杀虫作用，不仅可以通过触杀作用破坏茶尺蠖幼虫的外表皮甚至内骨骼的结构，而且取食茶皂苷后的茶尺蠖肠道细胞也将受到极大破坏。通过两种不同的作用机理，茶皂苷类化合物可以有效预防害虫，作为一种天然植物来源的杀虫化合物，茶皂苷类化合物具有成为绿色农药的巨大潜力。
附图说明
图1是茶尺蠖幼虫表面的扫描电子显微镜照片；
其中，A：空白处理，a1：放大100倍观察到的表面，a2：放大5000倍观察到的表面，a3：放大5000倍观察到的绒毛附近的表面；B：Camelliasaponin B1处理，b1：放大100倍下观察到的表面，b2：放大5000倍下观察到的表面，b3：放大5000倍下观察到的绒毛附近的表面；
图2是茶尺蠖幼虫中肠切片的几丁质染色图片；
其中，a：空白处理；b：Camelliasaponin B1处理；
图3是不同处理后的茶尺蠖幼虫的组织细胞染色和结构变化图；
其中，a：空白处理；b：Camelliasaponin B1处理。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
一、试验药剂：
本实施例的油茶籽饼粕：由安徽黄山裕子贵油茶加工厂生产提供，油茶籽收购于安徽皖南山区；柱层析硅胶粉(100-200目，200-300目)：中国国药集团；反相硅胶：日本YMC ODS-A C18材料，直径50μm；Sephadex LH-20凝胶：美国Pharmaeia原装进口；色谱纯甲醇、乙酸、乙腈：美国TEDIA公司；水：实验室纯水机制的超纯水；联苯菊酯原药(98％纯度)：由中国源叶生物有限公司提供；其他试剂、药品如非特别说明均为国药集团生产分析纯。
二、茶皂苷类化合物及单体化合物的提取过程如下：
将油茶籽饼粕粉碎成粉，首先用70％的乙醇水浸提三次(10L/10L/10L)，得到粗提物 浸膏，然后分别用石油醚、乙酸乙酯、正丁醇分级萃取并减压浓缩，得到石油醚部分、乙酸乙酯部分和正丁醇部分浸膏。取正丁醇萃取部分约120g用甲醇溶解，与100～200目的硅胶等质量拌样，用100～200目的硅胶干法装柱，干法上样。以乙酸乙酯：甲醇的洗脱剂系统进行梯度洗脱，浓度梯度以EtOAc-MeOH体积比100:0到0:100作为梯度洗脱，每个洗脱剂梯度大约洗脱6倍柱体积，将得到的洗脱液用ODS反相硅胶柱分离纯化，以甲醇-水洗脱剂梯度洗脱，梯度浓度MeOH-H2O体积比为0:(100～30):(70～50):(50～70):(30～100):0，得到油茶总皂苷，即茶皂苷类化合物。将油茶总皂苷用35％乙腈水溶解，经制备液相(Prep-HPLC，流动相：乙腈-水)梯度洗脱分离纯化，根据紫外吸收色谱图进行流动相收集，结合HPLC液相分析合并相同组分，减压浓缩反复制备，分离得到Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3三种茶皂苷单体化合物。
茶皂苷类化合物、Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3的结构通式分别如下：
Figure PCTCN2020128618-appb-000003
R1为取代或未取代的五碳糖或六碳糖，其中，五碳糖为：阿拉伯糖、木糖的其中之一；六碳糖为：葡萄糖、半乳糖、鼠李糖、葡萄糖醛酸和葡糖糖醛酸甲基酯的其中之一；
R2为取代或未取代的羟基或羧基其中之一；
R3为取代或未取代的羟基或有机酸，其中，所述取代为当归酸、惕各酸、己烯酸、2-甲基丁酸和肉桂酸其中之一；
R4为取代或未取代的羟基或有机酸，其中，所述取代为甲醇基团、醛基、酯酰基、乙酰氧基、肉桂酰基、氢化肉桂酰基、乙烯酰基、乙烯基、当归酰氧基和甲基丁酰基的其中之一；
R5为取代或未取代的羟基或有机酸，其中，所述取代为甲醇基团、醛基、酯酰基、乙酰氧基、肉桂酰基、氢化肉桂酰基、乙烯酰基、乙烯基、当归酰氧基和甲基丁酰基的其中之一；
R6为取代或未取代的羟基或有机酸，其中，所述取代为甲醇基团、醛基、酯酰基、乙酰氧基、肉桂酰基、氢化肉桂酰基、乙烯酰基、乙烯基、当归酰氧基和甲基丁酰基的其中之一；
R7为取代或未取代的羟基或羧基其中之一。
Figure PCTCN2020128618-appb-000004
Figure PCTCN2020128618-appb-000005
三、茶尺蠖的饲养
茶尺蠖：属于鳞翅目，尺蛾科，是危害茶园的主要昆虫。实验所用的茶尺蠖于安徽舒城茶园捕获，在人工气候室(温度23±2℃，光照：黑暗＝16h：8h，相对湿度75％)环境中饲养，饲喂食物为安徽农业大学农业园的茶树叶片(舒茶早，茶树种)，为了确保品种的稳定性，将其传代到第30代。筛选同一批出生长至二龄的茶尺蠖备用。
具有农药联苯菊酯抗性的茶尺蠖培养：实验所用的茶尺蠖于安徽舒城茶园捕获，在人工气候室(温度23±2℃，光照：黑暗＝16h：8h，相对湿度75％)环境中饲养，饲喂食物为安徽农业大学农业园的茶树叶片(舒茶早，茶树种)，在茶叶叶片中添加15mg/L的联苯菊酯原药，选取存活下来的昆虫进行传代，每两代之后联苯菊酯的添加量增加一倍，使用相同的饲养方法将其传代到第30代，每两代。筛选同一批出生长至二龄的茶尺蠖备用。
四、茶皂苷对茶尺蠖的触杀试验
将2000只二龄茶尺蠖随机分组，选择80个茶尺蠖用水处理作为空白组。实验组共4组：分别为油茶总皂苷、Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3，每组用水稀释为8个浓度(5、10、15、20、25、30、40、50mg/L)，各浓度为3个平行，每个平行用20只茶尺蠖,用Burkard自动微量滴定仪精确滴定1μL液滴于茶尺蠖背部。在滴定后24小时后观察并记录昆虫死亡的数量，以不能正常爬行为死亡，并按机率分析法求出毒力回归曲线与LC 50值。
将2000只具有农药联苯菊酯抗性的二龄茶尺蠖随机分组，选择80个茶尺蠖用水处理作为空白组。实验组共4组：分别为油茶总皂苷、Camelliasaponin B1、Theasaponin H1、 Oleiferasaponin C3，每组用水稀释为8个浓度(5、10、15、20、25、30、40、50mg/L)，各浓度为3个平行，每个平行用20只茶尺蠖,用Burkard自动微量滴定仪精确滴定1μL液滴于茶尺蠖背部。在滴定后24小时后观察并记录昆虫死亡的数量，以不能正常爬行为死亡，并按机率分析法求出毒力回归曲线与LC 50值。
五、扫描电子显微镜下观察茶尺蠖受皂苷影响后的形态
使用Burkard自动微量滴定仪将1μL的水和1μL Camelliasaponin B1水溶液(10mg/L)分别滴到两只茶尺蠖的背面。24小时后，将样品在2.5％戊二醛中固定过夜，并用磷酸盐缓冲液脱水洗涤4次，每次30分钟。逐步进行30％，50％，70％，80％，90％，100％酒精脱水，每次30分钟。将乙酸异戊酯更换两次，每次30分钟。将样品置于真空冷冻干燥设备(真空度为0.1mbar，温度为-42℃)中进行干燥，并且用导电胶带将样品粘附至样品台。在扫描电子显微镜下观察。使用扫描电子显微镜(aHitachi SU-8100)评估样品的形态，形状和大小特征，并在100～5000的放大倍数下进行。
六、受茶皂苷影响后茶尺蠖的几丁质染色
使用Burkard自动微量滴定仪将1μL的水和1μL Camelliasaponin B1水溶液(10mg/L)分别滴到两只茶尺蠖的背面。茶尺蠖在2.5％戊二醛中固定过夜，用150mM NaCl，10mM Na 2HPO 4、10mM NaH 2PO 4(pH 7.2)洗涤3×5分钟，然后在30％蔗糖戊二醛中固定8h。茶尺蠖被埋在O.C.T.复合材料(最佳切削温度复合材料，Sakura，美国)。使用Leica CM1950切片机(Leica Microsystems，Wetzlar，Germany)切割切片(8μm)，并在PBS(137mM NaCl，2.7mM KCl，10mM Na 2HPO 4、2mM KH 2PO 4，pH 7.4)中洗涤3×10min。将切片与DAPI二盐酸盐溶液(Beyotime)孵育，用PBST(含0.1％Tween的PBS)洗涤3×10分钟，与小麦胚芽凝集素-FITC标记的(WAG)(Sigma)孵育，然后与PBS洗涤3×10分钟PBST。用Olympus BX51显微镜拍摄载玻片。
七、受茶皂苷影响后的茶尺蠖肠道切片
用浸有50mg/L Camelliasaponin B1的叶子喂食茶尺蠖。24小时后，将幼虫在乙醚中麻醉，取出肠，并在Bouns溶液中固定12小时。此后，将它们在乙醇/二甲苯(1：1)中干燥并透明化。放入石蜡后，将样品切成7μm切片，并用苏木精-伊红染色。干燥受试幼虫中肠的形态学变化，并将其与取自未经处理饮食并以相同方式制备的对照组的组织进行比较。
七、统计分析
所有实验至少进行三次，数据报告为平均值和标准偏差。使用Statistics 8.1软件(Tallahassee，USA)通过单因素方差分析(ANOVA)进行统计学分析。具有95％置信水平(p<0.05)的最小显着性差异(LSD)被认为有统计学上的显著差异。
八、试验结果
1、茶皂苷杀虫活性评价
以2龄鳞翅目幼虫茶尺蠖为生测对象，24h后4种样品油茶总皂苷、Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3的杀虫毒力结果见表1。
由表1可知，四种样品油茶总皂苷、Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3对供试的茶尺蠖均具有明显的毒杀作用。在处理24h后，油茶总皂苷、Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3对茶尺蠖的毒力分别为36.943mg/L、26.120mg/L，48.061mg/L，48.483mg/L。
以具有农药联苯菊酯抗性的2龄鳞翅目幼虫茶尺蠖为生测对象，24h后4种样品油茶总皂苷、Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3的杀虫毒力结果见表1。
由表2可知，四种样品油茶总皂苷、Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3对具有联苯菊酯抗性的茶尺蠖均具有明显的毒杀作用。在处理24h后，油茶总皂苷、Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3对茶尺蠖的毒力分别为23.758mg/L、9.793mg/L，15.065mg/L，13.441mg/L。
通过以上数据可以看出，油茶总皂苷、Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3对具有联苯菊酯抗性的茶尺蠖及正常茶尺蠖均表现极强的毒杀活性，其中油茶中含量最高的皂苷Camelliasaponin B1单体具有最好的活性，显著高于具有多种皂苷混合物的油茶总皂苷。
表1不同样品对茶尺蠖毒性试验结果
Figure PCTCN2020128618-appb-000006
a.毒性回归方程表示剂量与致死值之间的关系
b.LC 50表示50％的致死浓度，即在杀死一半数量的被测物种所需的剂量
c.LC 50的95％置信区间。
表2不同样品对含有联苯菊酯抗性的茶尺蠖毒性试验结果
Figure PCTCN2020128618-appb-000007
a.毒性回归方程表示剂量与致死值之间的关系
b.LC 50表示50％的致死浓度，即在杀死一半数量的被测物种所需的剂量
c.LC 50的95％置信区间。
2、电镜观察Camelliasaponin B1处理的茶尺蠖幼虫表皮结果
蜡质层是保护昆虫免受外部化合物侵害的主要屏障。蜡质层的主要功能是防止干燥，对环境的感觉，机械支撑和运动。已经发现一些天然产物具有很强的昆虫蜡质层破坏性，但还未有过关于茶皂苷对昆虫蜡质层影响的报道。通过扫描电子显微镜观察到这两种处理对茶尺蠖幼虫表皮的影响(图1)。与空白处理相比(图1A a1)，用Camelliasaponin B1水溶液处理的茶尺蠖幼虫的表皮层变得粗糙并出现严重的皱纹(图1B b1)，躯干部分脱水并缩小(放大100倍)。在更微观的观察中(放大5000倍)，发现茶尺蠖表皮的蜡质层被消融(图1B b2)，并且在表皮中出现了空隙(图1B b3)。结果表明，茶皂苷在与茶尺蠖接触的短时间内可产生很高的毒性。茶皂苷通过破坏茶尺蠖表面的蜡质层而影响茶尺蠖的水平衡，使茶尺蠖失去水分并渗透到体内，导致茶尺蠖因失水而死亡。
3、Camelliasaponin B1处理后茶尺蠖幼虫几丁质染色及形态变化
为了研究Camelliasaponin B1处理后表皮受损的原因，用凝集素对茶尺蠖幼虫的几丁质层进行了染色(图2)。与空白处理相比(图2a)，Camelliasaponin B1处理的茶尺蠖的表皮几丁质变薄并消融(图2b箭头部分)，肠道(被几丁质层包围)也出现了溃疡(图2b红色箭头部分)。由于其两亲性结构，茶皂苷易于与胆固醇物质相互作用，并且还可以干扰蜕皮激素的合成，从而对昆虫表皮造成损害。结果证明，茶皂苷不仅可以有效地破坏茶尺蠖的蜡质层，而且对其几丁质层也具有很强的破坏作用。在染色中监测在肠道外圈的几丁质层也产生了损伤。它证明了茶皂苷的极强的渗透能力和从外到内的破坏能力。
4、Camelliasaponin B1处理后茶尺蠖幼虫中肠和形态变化
茶尺蠖幼虫中肠的主要功能包括消化酶的产生和消化产物的营养吸收。昆虫中肠的营养层基质(PM)主要由几丁质和蛋白质组成，被认为可以支持消化，并保护免受食物性食物颗粒和肠道病原体的侵害。在空白处理组(图3a)中，中肠上皮由单层消化细胞组成，表现出发达的刷状边界和具有嗜酸区域的细胞质(图3黑色箭头部分)。相比之下(图3b)，茶尺蠖幼取食Camelliasaponin B1后会导致细胞顶点的刷缘受损(图3b红色箭头部分)，产生染色质浓缩和核仁突显(图3b蓝色箭头部分)，并在肠壁上出现多处穿孔(图3b绿色箭头部分)。结果表明，茶皂苷Camelliasaponin B1可引起中肠上皮细胞的生理和形态损伤。取食Camelliasaponin B1后，细胞表现出空泡作用和囊泡释放作用，以进行能量排毒，而茶皂苷的过度毒性会使茶尺蠖中肠的微绒毛变短并导致死亡。
以上结果可以看出：茶皂苷类化合物对茶尺蠖幼虫具有很强的杀虫作用，不仅可以通过 触杀作用破坏茶尺蠖幼虫的外表皮甚至内骨骼的结构，而且取食茶皂苷后的茶尺蠖肠道细胞也将受到极大破坏。通过两种不同的作用机理，茶皂苷类化合物可以有效预防害虫，作为一种天然植物来源的杀虫化合物，茶皂苷类化合物具有成为绿色农药的巨大潜力。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (6)

  1. 一种茶皂苷类化合物，其特征在于，其结构通式如下：
    Figure PCTCN2020128618-appb-100001
    其中，R1为取代或未取代的五碳糖或六碳糖；
    R2为取代或未取代的羟基或羧基；
    R3为取代或未取代的羟基或有机酸；
    R4为取代或未取代的羟基或有机酸；
    R5为取代或未取代的羟基或有机酸；
    R6为取代或未取代的羟基或有机酸；
    R7为取代或未取代的羟基或羧基。
  2. 如权利要求1所述的一种茶皂苷类化合物的制备方法，其特征在于，制备方法如下：
    将油茶籽饼粕粉碎成粉；用乙醇水浸提多次，得到粗提物浸膏，分别用石油醚、乙酸乙酯、正丁醇分级萃取并减压浓缩，得到石油醚部分、乙酸乙酯部分和正丁醇部分浸膏；取正丁醇萃取部分120g用甲醇溶解，与100～200目的硅胶等质量拌样，用100～200目的硅胶干法装柱，干法上样；以乙酸乙酯：甲醇EtOAc-MeOH的洗脱剂系统进行梯度洗脱，浓度梯度以EtOAc-MeOH体积比100:0到0:100作为梯度洗脱，每个洗脱剂梯度洗脱6倍柱体积，将得到的洗脱液用ODS反相硅胶柱分离纯化，以甲醇-水MeOH：H 2O洗脱剂梯度洗脱，梯度浓度MeOH：H 2O体积比为0:100～30:70～50:50～70:30～100:0，得到茶皂苷类化合物。
  3. 如权利要求1所述的一种茶皂苷类化合物在制备杀虫剂中的应用。
  4. 一种从权利要求1所述的茶皂苷类化合物中分离出来的Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3单体化合物，其结构通式分别如下:
    Figure PCTCN2020128618-appb-100002
  5. 如权利要求4所述的单体化合物的制备方法，其特征在于，具体过程如下:
    首先制备茶皂苷类化合物，然后将茶皂苷类化合物用35％乙腈水溶解，经制备液相(Prep-HPLC，流动相：乙腈-水)梯度洗脱分离纯化，根据紫外吸收色谱图进行流动相收集， 结合HPLC液相分析合并相同组分，减压浓缩反复制备，分离得到Camelliasaponin B1、Theasaponin H1、Oleiferasaponin C3三种茶皂苷单体化合物。
  6. 如权利要求4所述的单体化合物在制备杀虫剂中的应用。
PCT/CN2020/128618 2019-12-06 2020-11-13 一种茶皂苷类化合物、制备方法及应用 WO2021109836A1 (zh)

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