WO2023115742A1 - 一种芸苔素甾醇类似物、新晶型、制备方法及应用 - Google Patents

一种芸苔素甾醇类似物、新晶型、制备方法及应用 Download PDF

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WO2023115742A1
WO2023115742A1 PCT/CN2022/083660 CN2022083660W WO2023115742A1 WO 2023115742 A1 WO2023115742 A1 WO 2023115742A1 CN 2022083660 W CN2022083660 W CN 2022083660W WO 2023115742 A1 WO2023115742 A1 WO 2023115742A1
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crystal form
new crystal
brassinolide
sterol
present
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PCT/CN2022/083660
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English (en)
French (fr)
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何其明
任丹
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成都新朝阳作物科学股份有限公司
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION 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
    • A01N45/00Biocides, 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
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01PBIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00Plant growth regulators
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07JSTEROIDS
    • C07J9/00Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane

Definitions

  • the invention relates to the technical field of agricultural chemistry, in particular to a brassinoid sterol analogue, a new crystal form, a preparation method and an application.
  • Natural brassinosterols are a general term for a class of lactone compounds and their sterol analogues. In addition to the identified BL, more than 60 different natural structures have been discovered, collectively called brassinosterols or brassinosterols ( Brassinosteroids, BRs). Our company has more than 20 years of research history on natural brassinolide sterols, and has developed a series of preparation processes for extracting natural brassinolide sterols from natural raw materials. Natural brassinoid sterol analogues (BR1-BR6) with different structures were isolated. On this basis, the applicant discovered a new structure of natural brassinosteroid sterol analogues through continuous in-depth development and continuous research.
  • Compound polymorphism means that the active ingredient of a drug can form two or more molecular assembly modes during the crystallization process, and the appearance of polymorphism is the result of kinetic competition in the process of crystallization thermodynamics and molecular recognition.
  • crystallization conditions such as: solvent composition, temperature, concentration, supersaturation, pH value, stirring speed, impurities, etc.
  • the change of combination mode and force can make the crystal have different unit cell parameters and space groups, thus forming polymorphism.
  • the present invention provides a new brassinolide sterol analogue and its new crystal form.
  • a brassinolide with significantly improved biological activity can be obtained New crystal forms of sterol analogues.
  • the present invention realizes through following technical scheme:
  • a brassinolide sterol analog the chemical formula of the brassinolide sterol analog is C 27 H 46 O 7 , and the chemical name is (20R,22R)-2 ⁇ ,3 ⁇ ,14 ⁇ -14,20,22,25- Hexahydroxy-5 ⁇ ,8 ⁇ ,9 ⁇ -cholestan-6-one, the structural formula is as follows:
  • a new crystal form of brassinolide sterol analogues said new crystal form has an X-ray powder diffraction spectrum at 30.988°, 29.076°, 19.135°, 133.38° represented by Cu-K ⁇ radiation and 2 ⁇ 0.2° diffraction angle °, 210.159°, 37.182°, 35.958°, 158.388°, 12.865°, 13.476°, 9.729°, 20.971°, 21.105°, 14.088°, 34.735°, 22.882°, 7.894°, 9.11815.388°, 12 Displayed at .865° There are diffraction characteristic peaks.
  • the DSC spectrum of the new crystal form has characteristic exothermic peaks at 110.2°C and 175.2°C.
  • the infrared spectrum of the new crystal has characteristics at wavenumbers of 3432.18cm -1 , 2963.72cm -1 , 2361.83cm -1 , 1703.15cm -1 , 1629.34cm -1 , 1390.85cm -1 , 1067.19cm -1 , 666.88cm -1 peak.
  • the present invention provides a method for preparing a new crystal form of brassinolide sterol analogues.
  • the preparation method of the crude extract is the same as the "Application of Natural Brassinolide Lactone Analogues" No. 201210026285.7, including the following steps: 1) using 80 to 100 % (V/V) ethanol aqueous solution to extract the broken rapeseed pollen, retain the filtrate after solid-liquid separation (optionally wherein the filtrate is further concentrated), and obtain the alcohol-soluble extract; 2) mix the alcohol-soluble extract with 0-60% (V/V V) ethanol aqueous solution is mixed, then add ethyl acetate to extract, keep ethyl acetate layer and add esterase to carry out incomplete reaction, then dry, obtain ester-soluble extract; 3) ester-soluble extract is loaded on silica gel chromatographic column, use The mixed solution of methanol and ethyl acetate is eluted, and the eluate containing natural brassin
  • the present invention provides an application of a brassinolide sterol analogue or a new crystal form thereof in the field of agriculture, preferably in promoting plant growth.
  • the present invention has the following advantages and beneficial effects:
  • a brassinoid sterol analog and its crystal form provided in the embodiment of the present invention have significant biological activity
  • Fig. 1 provides the powder diffraction pattern of new crystal form for the embodiment of the present invention
  • Fig. 2 provides the H NMR spectrum of the new crystal form for the embodiment of the present invention
  • Fig. 3 provides the infrared spectrogram of the new crystal form for the embodiment of the present invention
  • Fig. 4 provides the TG figure of the new crystal form for the embodiment of the present invention.
  • Fig. 5 provides the DSC figure of the new crystal form for the embodiment of the present invention.
  • Fig. 6 is a solubility curve diagram of the new crystal form in methanol provided by an embodiment of the present invention.
  • ester-soluble extract is loaded on a silica gel chromatographic column, eluted with a mixture of methanol and ethyl acetate, and the eluate containing natural brassinolide analogs is collected, then dried to obtain natural brassinolide Analog crude extract;
  • Example 1 The difference from Example 1 is that the recrystallization method is different, specifically as follows: Weigh 15g of brassinolide sterol analogue, dissolve it in 0.5L of methanol solution, add 1L of acetonitrile solvent and stir evenly, and seal the hole with a parafilm and let it stand Volatile, about 20 days, although small crystal particles appear on the test tube wall, but it is not suitable for X-ray test.
  • Example 1 The difference from Example 1 is that the recrystallization method is different, as follows: Weigh 5g of brassinolide sterol analogues, dissolve them in 0.5L ethanol solution, filter and let stand for volatilization for a week, the solution turns into a yellow viscous liquid without crystals Precipitate.
  • Example 1 The difference from Example 1 is that the recrystallization method is different, specifically as follows: Weigh 10g of brassinolide sterol analogs, dissolve them in 0.3L of methanol solution, add 0.8L of acetonitrile solvent and stir evenly, filter and place in a long test tube Slowly volatilize, no crystals are precipitated, and after the solvent volatilizes completely, a yellow thick liquid substance remains.
  • thermogravimetric curve shows that the compound of the present invention has good stability from room temperature to 300°C.
  • the DSC result of the new crystal form of the present invention contains two exothermic peaks with physical interference, and the peak positions are respectively at 110.2°C and 175.2°C.
  • the new crystal form of the present invention has good solubility in methanol.
  • the biological activity assay of the new crystal form provided by Example 1 of the present invention comprises the following steps:
  • the new crystal form of the present invention (prepared as a 10mg/ml mother liquor with absolute ethanol, ready for use during experiments).
  • each petri dish After the pollen medium is melted, add different concentrations of agents according to the experimental design, pour it into a 90 ⁇ 90cm round petri dish, pour 25ml of the medium into each petri dish quantitatively, and use it on the culture medium after the medium is solidified Sprinkle the pollen evenly and put it in the refrigerator at 4°C overnight (15 hours), so that the pollen can fully absorb the drug, and then transfer it to a biochemical incubator at 28°C to induce germination for 1.5 hours, and then irradiate with ultraviolet light for 1 hour to fix the germination state of the pollen. During observation, each petri dish was divided into four areas, and 5 visual fields were observed in each area, that is, 20 visual fields were observed in each treatment group, and germination was considered when the pollen tube length reached more than 1 times the pollen particle diameter.
  • the pollen germination effects of different treatment groups are shown in Table 3. It can be seen from Table 3 that the chemical solvents at various concentrations have no obvious effect on pollen germination.
  • the pollen germination rate of the new crystal form was better than that of the clean water control CK at the concentration of 0.003 and 0.3ppm, and the effect was the best at the concentration of 0.03ppm.
  • the new crystal form of the present invention has the biological activity of promoting pollen germination.
  • the embodiment of the present invention provides the application of the new crystal form in promoting plant growth, specifically taking the experiment of promoting Arabidopsis root growth as an example, the steps are as follows:
  • the new crystal form of the present invention (prepared as a 10mg/ml mother liquor with absolute ethanol, ready for use during experiments).
  • the root lengths of different treatment groups 3 days after drug treatment and 7 days after drug treatment are shown in Table 5. From the measurement results, it can be seen that the solvent will not affect the growth of Arabidopsis thaliana. From the experimental results, it can be seen that when the new crystal form of the present invention is at a concentration of 10nmol/L, the length of 3 days and 7 roots after the drug is significantly better than that of the clear water control CK. In summary, the new crystal form of the present invention has the effect of promoting the root growth of Arabidopsis thaliana.
  • the embodiment of the present invention provides the application of the new crystal form in promoting plant growth, specifically taking the experiment of promoting the growth of tobacco as an example, the steps are as follows:
  • the new crystal form of the present invention (prepared as a 10mg/ml mother liquor with absolute ethanol, ready for use during experiments).
  • the 7-day-old tobacco seedlings were transplanted and planted in the cultivation room, and the experiment was carried out after 2 days of slowing down the seedlings.
  • the treatment agent was prepared according to the experimental design, the leaves of the tobacco seedlings were evenly sprayed, and each treatment was repeated 3 times, with 2 plants in each repetition, that is, 6 seedlings in each treatment. Chemicals entering the soil affect the experimental results.
  • the RGB AREA_MM parameter value (leaf area/mm 2 ) of tobacco was recorded with a plant phenotype instrument before, 3 days and 7 days after the drug, and the leaf area growth rate was calculated to evaluate the growth-promoting effect of each agent.
  • the leaf area growth rate was calculated The formula is as follows:
  • the new crystal of the present invention has the effect of promoting the growth of tobacco.
  • the embodiment of the present invention provides the application of the new crystal form in promoting plant growth, specifically taking the experiment of promoting the growth of wheat in hydroponics as an example, the steps are as follows:
  • the new crystal form of the present invention (prepared as a 10mg/ml mother liquor with absolute ethanol, ready for use during experiments).
  • the selected ones with consistent germination were sown in hydroponic bottles respectively, and 8 seeds were placed in each hydroponic bottle, and different concentrations of medicaments were prepared according to the experimental design based on Hoagland's nutrient solution. , add 200ml of the prepared medicine to the bottle, repeat each treatment 3 times, place it in a culture room at 25°C for cultivation, and set the light intensity to 3200lux. When there were obvious differences among the treatments, the roots and shoots were taken out to measure the length.
  • the new crystal form of the present invention has a growth-promoting effect , where the concentration of 0.03ppm has the best growth-promoting effect, which is obviously better than that of the control CK. To sum up, the new crystal form of the present invention has the effect of promoting roots of wheat in the way of wheat hydroponics.
  • the embodiment of the present invention provides the application of the new crystal form in promoting plant growth, specifically taking the experiment of soaking seeds to promote the germination and growth of corn as an example, the steps are as follows:
  • the new crystal form of the present invention (prepared as a 10mg/ml mother liquor with absolute ethanol, ready for use during experiments).
  • test agent prepares the test agent according to the experimental design, select 20 normal plump corn seeds, put them in the agent, soak the seeds at 26°C for 24 hours, then wash the seeds with clean water, put them in the culture box and put them in a constant temperature incubator for germination
  • the germination temperature was set to 2 periods: 14h light, 28°C, 10h dark, 25°C, and cultured for 7 days. Each treatment was repeated 3 times.
  • 5 ml of clear water was regularly added with a pipette at 9 o'clock every day to keep the culture box moist.
  • Germination potential was measured after 3 days, and bud length, root length, root vigor and bud vigor were measured after 7 days.
  • Germination potential germinated number of treated seeds on the 3rd day/total number of treated seeds*100%
  • Germination rate germinated number of treated seeds/treated total number of seeds*100% on the 7th day
  • Bud vigor index germination rate on the 7th day*the seedling bud length on the 7th day;
  • Root vitality index germination rate on the 7th day * root length of the seedlings on the 7th day.
  • each treatment group is shown in table 11 on the impact measurement result of corn seed germination and growth, as can be seen from the experimental results, the new crystal form of the present invention has no significant impact on the germination potential of corn seeds, and the new crystal form of the present invention at 0.03ppm has no significant effect on the germination potential of corn seeds.
  • the rate has a certain hair promoting effect, but the effect is not significant.
  • the new crystal form of the present invention has the effect of improving the root vitality index and bud vitality index, and the new crystal form of the present invention at a concentration of 0.03ppm has the best effect, which is obviously better than the control CK.
  • the new crystal form of the present invention has obvious growth-promoting effect on corn by soaking seeds.

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Abstract

本发明公开了一种芸苔素甾醇类似物、新晶型、制备方法及应用,所述芸苔素甾醇类似物的化学式为C27H46O7,化学命名为(20R,22R)-2β,3β,14α-14,20,22,25-六羟基-5α,8α,9α-胆甾-6-酮,结构式如aa所示:本发明通过对花粉粗提物纯化方法进行改进,通过重结晶得到了一种高生物活性的新晶型,能够应用在农业领域促进植物的生长。

Description

一种芸苔素甾醇类似物、新晶型、制备方法及应用 技术领域
本发明涉及农用化学技术领域,具体而言,涉及一种芸苔素甾醇类似物、新晶型、制备方法及应用。
背景技术
上世纪30年代,美国农业部(USDA)研究人员发现植物花粉提取物能促进植物生长。美国科学家Mitchell于1970年首先提出油菜素(Brassins)的概念,这种来源于油菜花粉提取物的未知成分在极低浓度下就能有效促进植物茎伸长和细胞分裂。随后,1979年科学家Grove从油菜花粉中提取到具有油菜素活性的化合物并通过X-射线单晶衍射分析确定其化学结构式,命名为芸苔素内酯(Brassinolide,BL)。芸苔素甾醇具有多种独特的调节植物生长的生理活性,与已知的生长素、细胞分裂素、赤霉素、脱落酸、乙烯等植物生长调节剂相比,生物活性更强,使用量更低,安全性更高,在农业上增产增收的效果更好。
天然芸苔素甾醇是一类内酯类化合物及其甾醇类似物的统称,除已经鉴定的BL外,人们陆续发现了超过60种不同的天然结构,统称为油菜素甾醇或芸苔素甾醇(Brassinosteroids,BRs)。本公司对天然芸苔素甾醇有着20多年的研究历史,开发了一系列从天然原料中提取天然芸苔素甾醇的制备工艺,在201210026285.7号《天然芸苔素内酯类似物的应用》中我们分离得到了不同结构的天然芸苔素甾醇类似物(BR1~BR6),本申请在此基础上通过不断的深入开发和持续研究,发现了一个新的天然芸苔素甾醇类似物结构。
化合物多晶型现象是指药物活性成分在结晶的过程中可以形成两种或者两种以上的分子组装模式,多晶型的出现是结晶热力学和分子识别过程中动力学竞争的结果。结晶过程中,由于结晶条件的变化,例如:溶剂组成、温度、浓度、过饱和度、pH值、搅拌速度、杂质等都会导致晶体内部质点元的构型、构象发生变化,或者相互之间的结合方式和作用力改变,都可以使晶体出现不同的晶胞参数和空间群,从而形成多晶型现象。一般来讲,一种化合物都会存在多种晶型,然而并不是所有制备的晶型都具有生物活性,因此筛选优势晶型非常重要。本申请在201210026285.7号《天然芸苔素内酯类似物的应用》专利的基础上,对花粉粗提物采用重结晶的方式得到了一个新晶体,其具有显著的生物活性。
有鉴于此,特提出本申请。
发明内容
本发明提供一种新的芸苔素甾醇类似物及其新晶型,通过对提取的天然芸苔素甾醇类似 物进行重结晶条件改进和优化,能够得到一种生物活性显著提高的芸苔素甾醇类似物新晶型。
本发明通过下述技术方案实现:
一种芸苔素甾醇类似物,所述芸苔素甾醇类似物的化学式为C 27H 46O 7,化学命名为(20R,22R)-2β,3β,14α-14,20,22,25-六羟基-5α,8α,9α-胆甾-6-酮,结构式如下所示:
Figure PCTCN2022083660-appb-000001
一种芸苔素甾醇类似物新晶型,所述新晶型在以Cu-Kα辐射、2θ±0.2°衍射角表示的X-射线粉末衍射光谱图在30.988°、29.076°、19.135°、133.38°、210.159°、37.182°、35.958°、158.388°、12.865°、13.476°、9.729°、20.971°、21.105°、14.088°、34.735°、22.882°、7.894°、9.11815.388°、12.865°处显示有衍射特征峰。
新晶型的核磁氢谱数据如下: 1H NMR(400MHz,DMSO):δ4.37(s,1H),4.31(s,1H),4.15(s,1H),4.07(s,1H),3.72-3.74(m,2H),3.49-3.55(m,2H),3.10(d,J=10.20Hz,1H),2.65(t,J=14.52Hz,1H),2.41(s,1H),2.16-2.29(m,2H),1.99-2.12(m,2H),1.43-1.87(m,12H),1.21-1.29(m,5H),0.87-1.30(m,14H)。
新晶型的DSC图谱在110.2℃、175.2℃有放热特征峰。
新晶型的红外光谱在波数为3432.18cm -1、2963.72cm -1、2361.83cm -1、1703.15cm -1、1629.34cm -1、1390.85cm -1、1067.19cm -1、666.88cm -1有特征峰。
本发明提供一种芸苔素甾醇类似物新晶型的制备方法,粗提物的制备方法同201210026285.7号《天然芸苔素内酯类似物的应用》,包括如下步骤:1)用80~100%(V/V)乙醇水溶液提取破碎的油菜花粉,固液分离后保留滤液(任选其中滤液进一步浓缩),获得醇溶性提取液;2)将醇溶性提取液与0~60%(V/V)乙醇水溶液混合,然后加入乙酸乙酯萃取,保留乙酸乙酯层并加入酯酶进行不完全反应,然后干燥,获得酯溶性提取物;3)酯溶性提取物上样于硅胶色谱柱,用甲醇和乙酸乙酯的混和液洗脱,收集含有天然芸苔素内酯类似物的洗脱液,然后干燥,获得天然芸苔素内酯类似物粗提物;4)称量10~20份天然芸苔素内酯类似物粗提物溶解在20~30倍体积的甲醇和丙酮的混合溶剂中,甲醇和丙酮体积比为1~4:1,再加入乙酸乙酯直至溶液刚好开始转向浑浊,再补加甲醇使其完全溶解,并用封口膜 密封扎孔静置挥发,析出的颗粒状白色晶体即为芸苔素甾醇类似物新晶型。
本发明提供一种芸苔素甾醇类似物或其新晶型在农业领域的应用,优选为在促进植物生长方面的应用。
本发明与现有技术相比,具有如下的优点和有益效果:
1、本发明实施例提供的一种芸苔素甾醇类似物及其晶型,具有显著的生物活性;
2、本发明实施例提供的一种芸苔素甾醇类似物新晶型的制备方法,通过对花粉粗提物纯化方法进行改进,通过重结晶得到了一种高生物活性的新晶型,能够应用在农业领域促进植物的生长。
附图说明
为了更清楚地说明本发明示例性实施方式的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,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,还可以根据这些附图获得其他相关的附图。
图1为本发明实施例提供新晶型的粉末衍射图;
图2为本发明实施例提供新晶型的核磁氢谱;
图3为本发明实施例提供新晶型的红外光谱图;
图4为本发明实施例提供新晶型的TG图;
图5为本发明实施例提供新晶型的DSC图;
图6为本发明实施例提供新晶型在甲醇中的溶解度曲线图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例和附图,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
在以下描述中,为了提供对本发明的透彻理解阐述了大量特定细节。然而,对于本领域普通技术人员显而易见的是:不必采用这些特定细节来实行本本发明。在其他实施例中,为了避免混淆本本发明,未具体描述公知的方法。
在整个说明书中,对“一个实施例”、“实施例”、“一个示例”或“示例”的提及意味着:结合该实施例或示例描述的特定特征、结构或特性被包含在本本发明至少一个实施例中。因此,在整个说明书的各个地方出现的短语“一个实施例”、“实施例”、“一个示例”或“示例”不一定都 指同一实施例或示例。此外,可以以任何适当的组合和、或子组合将特定的特征、结构或特性组合在一个或多个实施例或示例中。此外,本领域普通技术人员应当理解,在此提供的示图都是为了说明的目的,并且示图不一定是按比例绘制的。这里使用的术语“和/或”包括一个或多个相关列出的项目的任何和所有组合。
实施例1
本发明实施例提供的一种芸苔素甾醇类似物新晶型的制备方法,包括如下步骤:
1、天然芸苔素内酯类似物粗提物的提取方法
1)用80~100%(V/V)乙醇水溶液提取破碎的油菜花粉,固液分离后保留滤液(任选其中滤液进一步浓缩),获得醇溶性提取液;
2)将醇溶性提取液与0~60%(V/V)乙醇水溶液混合,然后加入乙酸乙酯萃取,保留乙酸乙酯层并加入酯酶进行不完全反应,然后干燥,获得酯溶性提取物;
3)酯溶性提取物上样于硅胶色谱柱,用甲醇和乙酸乙酯的混和液洗脱,收集含有天然芸苔素内酯类似物的洗脱液,然后干燥,获得天然芸苔素内酯类似物粗提物;
2、芸苔素甾醇类似物新晶型的重结晶方法
称量10g步骤1提取的天然芸苔素内酯类似物粗提物溶解在3倍体积的甲醇和丙酮(4:1)的混合溶剂中,再加入1L乙酸乙酯直至溶液刚好开始转向浑浊,再补加0.1L甲醇使其完全溶解,并用封口膜密封扎孔静置挥发,15天左右析出颗粒状的白色晶体。
对比例1
与实施例1的区别在于:重结晶方法不同,具体如下:称取15g芸苔素甾醇类似物,溶解在0.5L甲醇溶液中,再加入1L乙腈溶剂搅拌均匀,并用封口膜密封扎孔静置挥发,20天左右,试管壁虽然出现较小晶体颗粒,但是却不适合做X-ray测试。
对比例2
与实施例1的区别在于:重结晶方法不同,具体如下:称取5g芸苔素甾醇类似物,溶解在0.5L乙醇溶液中,过滤静置挥发一周,溶液转变成黄色粘稠液体,无晶体析出。
对比例3
与实施例1的区别在于:重结晶方法不同,具体如下:称取10g芸苔素甾醇类似物,溶解在0.3L甲醇溶液中,再加入0.8L乙腈溶剂搅拌均匀,过滤后置于长试管中缓慢挥发,无晶体析出,待溶剂挥发完全,仍剩下黄色的稠状液体物质。
对比例1-对比例3采用的重结晶体系与方法与本发明实施例1提供的重结晶方法不同,从结果可以看出,对比例1-对比例3均无法析出本发明的晶体。
对实施例1获得的白色晶体进行XRD、红外光谱和碳谱检测确定其结构式,并通过TG和DSC对晶体的热稳定性进行测试,结果如下:
1、本发明新晶型的XRD衍射结果,如图1所示,在以Cu-Kα辐射、2θ±0.2°衍射角表示的X-射线粉末衍射光谱图在30.988°、29.076°、19.135°、133.38°、210.159°、37.182°、35.958°、158.388°、12.865°、13.476°、9.729°、20.971°、21.105°、14.088°、34.735°、22.882°、7.894°、9.11815.388°、12.865°处显示有衍射特征峰。
2、本发明新晶型的核磁氢谱结果,如图2所示,数据如下: 1H NMR(400MHz,DMSO):δ4.37(s,1H),4.31(s,1H),4.15(s,1H),4.07(s,1H),3.72-3.74(m,2H),3.49-3.55(m,2H),3.10(d,J=10.20Hz,1H),2.65(t,J=14.52Hz,1H),2.41(s,1H),2.16-2.29(m,2H),1.99-2.12(m,2H),1.43-1.87(m,12H),1.21-1.29(m,5H),0.87-1.30(m,14H)。
3、本发明新晶型的红外光谱图结果,如图3所示,本发明新晶型的红外光谱显示在3432.18cm -1、2963.72cm -1、2361.83cm -1、1703.15cm -1、1629.34cm -1、1390.85cm -1、1067.19cm -1、666.88cm -1有特征峰。
4、本发明新晶型的TG结果,如图4所示,热重曲线表明本发明的化合物在室温到300℃均具有很好的稳定性。
5、本发明新晶型的DSC结果,如图5所示,图谱中含有存在物理干扰的2个放热峰,峰位置分别在110.2℃、175.2℃。
实施例2
本发明实施例1提供的新晶型的稳定性和溶解性研究
1、稳定性研究
取适量本发明新晶型,放置在80±2℃高温环境中进行稳定性实验,实验结果入下表1所示,从结果可以看出,本发明在高温环境下很稳定,不会出现晶型变化。
表1本发明新晶体的稳定性结果
Figure PCTCN2022083660-appb-000002
Figure PCTCN2022083660-appb-000003
2、溶解度研究
(1)在1mL甲醇体系中研究其在不同温度下的溶解度,温度梯度为10℃,在每个温度节点取20μL样,利用HPLC检测,称重记录下总质量,并换算成体积,表示为mg/mL,实验结果如图6所示;
从结果可以看出本发明新晶型在甲醇中具有良好的溶解度。
实施例3
本发明实施例1提供的新晶型的生物活性测定,包括如下步骤:
1、实验样品:
本发明新晶型(用无水乙醇配制为10mg/ml的母液,实验时现配现用)。
2、实验设计:
表2药剂浓度设计
Figure PCTCN2022083660-appb-000004
3、实验方法:
3.1花粉培养基的配制
称取蔗糖1.5g、0.01g硼酸、0.7g琼脂、无水氯化钙2mg,量取100ml蒸馏水,将称好的蔗糖、无水氯化钙、琼脂和蒸馏水倒入烧杯中,放到水浴锅中边加热、边搅动至完全溶解,然后加入硼酸溶解,加水至100ml,调节pH 6.5-6.8,备用。
3.2花粉培养
将花粉培养基融化后,根据实验设计加入不同浓度的药剂,倒入90×90cm的圆形培养皿中,每个培养皿中定量倒入25ml培养基,待培养基凝固后用在培养基上均匀撒上花粉放入4℃冰箱中过夜(15小时),使花粉充分吸收药剂,再转入28℃生化培养箱中诱导萌发1.5小时后,紫外线照射1小时固定花粉萌发状态。观察时每个培养皿划分四个区域,每个区域观 察5个视野,即每个处理组观察20个视野,以花粉管长度达到花粉粒径的1倍以上视为萌发。
4、实验结果:
不同处理组花粉萌发效果如表3所示,从表3中可以看到各浓度下的药剂溶剂对花粉的萌发无明显影响。新晶型在0.003和0.3ppm浓度下花粉萌发率均优于清水对照CK,其中0.03ppm浓度下效果最佳。综上,本发明新晶型具有促花粉萌发的生物活性。
表3不同处理组对花粉萌发效果的影响
Figure PCTCN2022083660-appb-000005
实施例4
本发明实施例提供新晶型在促进植物生长方面的应用,具体的以促拟南芥根生长实验为例,步骤如下:
1、实验样品:
本发明新晶型(用无水乙醇配制为10mg/ml的母液,实验时现配现用)。
2、实验设计:
表4药剂浓度设计
Figure PCTCN2022083660-appb-000006
3、实验方法:
根据实验设计,小心挑取生长正常、根长一致、苗龄三天左右的拟南芥无菌幼苗,移入含不同浓度的药剂1/2MS培养基中(9x9方形培养皿),每个培养皿中定量倾倒25ml培养基,每个培养皿中6棵幼苗,每个处理组设置3个重复。各处理组置于长日照,16h/8h(白天/ 黑夜),光强为2000lx,22℃的培养室进行培养。每天划线记录各处理组根系长度,药后7天停止实验并拍照,采用ImageJ软件测量第三天和第七天各个处理组根长。
4、实验结果:
药后3天和药后7天不同处理组根长如表5,从测定结果上看溶剂不会对拟南芥生长造成影响。从实验结果可以看出本发明新晶型在10nmol/L浓度时,药后3天和药后7根长均显著优于清水对照CK。综上,本发明新晶型具有促拟南芥根生长的效果。
表5不同处理组拟南芥根长测定结果
Figure PCTCN2022083660-appb-000007
实施例5
本发明实施例提供新晶型在促进植物生长方面的应用,具体的以促烟草生长实验为例,步骤如下:
1、实验样品:
本发明新晶型(用无水乙醇配制为10mg/ml的母液,实验时现配现用)。
2、实验设计:
表6药剂浓度设计
Figure PCTCN2022083660-appb-000008
3、实验方法:
将7天苗龄烟草幼苗在培养室内进行移栽定植,缓苗2天后进行试验。处理药剂按照实验设计配制后对烟草幼苗叶片进行均匀喷施,每个处理3次重复,每个重复2株,即每个处理6株幼苗,喷施时以药液不滴落为宜,防止药剂进入土壤影响实验结果。分别于药前、药后3天和7天用植物表型仪记录烟草的RGB AREA_MM参数值(叶面积/mm 2),计算叶面积增长率评价各药剂的促长效果,叶面积增长率计算公式如下:
增长率(%)=(叶面积终-叶面积初)×100%/叶面积初
4、实验结果:
药后3天和药后7天不同处理组叶面积增长率如表7,从实验结果可以看出,药后3天各处理对烟草促长效果不明显,但与CK相比本发明新晶型表现出促长的趋势。药后7天,本发明新晶型表现出明显的促生长效果,其中浓度为0.003ppm的本发明新晶型促长效果最好。综上,本发明新晶型具有促进烟草生长的效果。
表7不同处理组烟草促长测定结果
Figure PCTCN2022083660-appb-000009
实施例6
本发明实施例提供新晶型在促进植物生长方面的应用,具体的以水培促小麦生长实验为例,步骤如下:
1、实验样品:
本发明新晶型(用无水乙醇配制为10mg/ml的母液,实验时现配现用)。
2、实验设计:
表8药剂浓度设计
Figure PCTCN2022083660-appb-000010
3、实验方法:
将供试小麦种子用清水浸种催芽24h后,挑选发芽一致的分别播于水培瓶中,每个水培瓶中8颗种子,以霍格兰营养液为基底按照试验设计配制不同浓度的药剂,向瓶中加入准备好的药剂200ml,每处理重复3次,放置到25℃的培养间中培养,光强设置为3200lux。在各处理出现明显差异时取出测定根长和芽长。
4、实验结果:
药后7天不同处理组促小麦生长测定结果如表9所示,从实验结果可以看出,各处理组 在芽长上无明显差异,在根长上,本发明新晶型具有促生长效果,其中浓度为0.03ppm促生长效果最佳,明显优于对照CK。综上,在小麦水培的方式下本发明新晶型具有对小麦具有促根的效果。
表9不同处理组小麦促生长测定结果
Figure PCTCN2022083660-appb-000011
实施例7
本发明实施例提供新晶型在促进植物生长方面的应用,具体的以浸种促玉米萌发和生长实验为例,步骤如下:
1、实验样品:
本发明新晶型(用无水乙醇配制为10mg/ml的母液,实验时现配现用)。
2、实验设计:
表10药剂浓度设计
Figure PCTCN2022083660-appb-000012
3、实验方法:
首先将试验药剂按照实验设计配制备用,选取正常饱满玉米种子20粒,置于药剂中,26℃浸种处理24h,然后用清水将种子洗净,置于培养盒中放入恒温培养箱中进行催芽培养,发芽温度设置为2个时段∶14h光照、28℃,10h黑暗、25℃,培养7天。每个处理重复3次,在整个试验过程中,每天9点定时利用移液枪添加5ml清水,以保持培养盒湿润。3d后测量发芽势,7d后测量芽长、根长、根活力、芽活力。
发芽势=第3天处理种子萌发数/处理种子总数*100%;
发芽率=第7天处理种子萌发数/处理种子总数*100%;
芽活力指数=第7天发芽率*第7天的幼苗芽长;
根活力指数=第7天发芽率*第7天的幼苗根长。
4、实验结果:
各处理组对玉米种子萌发和生长的影响测定结果如表11所示,从实验结果可以看出,本发明新晶型对玉米种子发芽势无明显影响,0.03ppm的本发明新晶型对发芽率有一定的促明发效果,但效果不显著。从根、芽活力指数上看,本发明新晶型有提升根活力指数和芽活力指数的作用,其中0.03ppm浓度下的本发明新晶型作用效果最佳,明显优于对照CK。
综上,采用浸种的方式,本发明新晶型对玉米具有明显的促生长效果。
表11不同处理组对玉米萌发和生长影响测定结果
Figure PCTCN2022083660-appb-000013
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。

Claims (6)

  1. 一种芸苔素甾醇类似物,其特征在于,所述芸苔素甾醇类似物的化学式为C 27H 46O 7,化学命名为(20R,22R)-2β,3β,14α-14,20,22,25-六羟基-5α,8α,9α-胆甾-6-酮,结构式如下所示:
    Figure PCTCN2022083660-appb-100001
  2. 一种如权利要求1所述芸苔素甾醇类似物的新晶型,其特征在于,所述新晶型以Cu-Kα辐射、2θ±0.2°衍射角表示的X-射线粉末衍射光谱图在30.988°、29.076°、19.135°、133.38°、210.159°、37.182°、35.958°、158.388°、12.865°、13.476°、9.729°、20.971°、21.105°、14.088°、34.735°、22.882°、7.894°、9.11815.388°、12.865°处显示有衍射特征峰。
  3. 根据权利要求2所述的芸苔素甾醇类似物新晶型,其特征在于,新晶型的红外光谱在波数为3432.18cm -1、2963.72cm -1、2361.83cm -1、1703.15cm -1、1390.85cm -1、1067.19cm -1、有特征峰。
  4. 一种如权利要求2-3任一所述的芸苔素甾醇类似物新晶型的制备方法,包括如下步骤:1)从油菜花粉中提取天然芸苔素内酯类似物粗提物;2)称量10~20份天然芸苔素内酯类似物粗提物溶解在20~30倍体积的甲醇、丙酮或两者混合溶剂中,再加入乙酸乙酯直至溶液刚好开始转向浑浊,再补加甲醇使其完全溶解,并用封口膜密封扎孔静置挥发,析出的颗粒状白色晶体即为芸苔素甾醇类似物新晶型。
  5. 如权利要求1所述芸苔素甾醇类似物或权利要求2-3任一所述新晶型在农业领域的应用。
  6. 根据权利要求5所述的芸苔素甾醇类似物或其新晶型在农业领域的应用,其特征在于,在促进植物生长方面的应用。
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