CN114516893B

CN114516893B - Fructus momordicae neoflavonoid compound, preparation method thereof and application thereof in bee attractant

Info

Publication number: CN114516893B
Application number: CN202210224878.8A
Authority: CN
Inventors: 胡兴华; 霍华珍; 叶家桐; 谢运昌; 朱晓珍; 邓涛; 蔡爱华; 李典鹏
Original assignee: Guangxi Institute of Botany of CAS
Current assignee: Guangxi Institute of Botany of CAS
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2022-07-26
Anticipated expiration: 2042-03-09
Also published as: NL2032687B1; CN114516893A

Abstract

The invention discloses a momordica grosvenori neoflavonoid compound, a preparation method thereof and application thereof in a bee attractant, belonging to the technical field of neoflavonoid compounds; the compound is a compound having the structure:

extracting male leaves of Momordica grosvenori, performing high-speed countercurrent chromatographic separation on the obtained extract, collecting fractions in stages, identifying and combining the fractions by UPLC-MS or TLC, recovering solvent to obtain 6 parts I-VI respectively, wherein yellow powdered countercurrent chromatographic purified product of part IV is neoflavonoid compound with structure shown in the formula; the novel flavonoid compound is derived from the momordica grosvenori, is a novel natural compound, has good attractant activity for bees, provides an ideal candidate compound for developing a novel bee attractant, opens up a novel bee attractant and a novel application field of the momordica grosvenori, and lays a foundation for further development and utilization of momordica grosvenori leaves.

Description

Fructus momordicae neoflavonoid compound, preparation method thereof and application thereof in bee attractant

Technical Field

The invention belongs to the technical field of neoflavonoid compounds, and particularly relates to a momordica grosvenori neoflavonoid compound, a preparation method thereof and application thereof in a bee attractant.

Background

The flavonoid compound is formed by connecting two benzene rings (ring A and ring B) through three central carbon atoms and has C ₆ -C ₃ -C ₆ A series of compounds of the skeleton are widely distributed, mainly exist in a glucoside form in plants, and play important roles in the growth, development and result of the plants and the defense of pathogens and animal invasion. Meanwhile, the flavonoid compound is an important bioactive component and has various physiological functions and pharmacological actions of regulating immunity, improving blood circulation, resisting viruses, inflammation and tumors, resisting aging, protecting liver and kidney, reducing blood sugar, reducing blood fat and the like. The new flavonoid compound not only enriches the variety and the source of natural compounds, but also has important significance for developing new health products, natural medicines and chemical products.

The natural pollination mode of plants comprises aeolian media, insect media, aquatic media, bird media and the like, wherein most flowering plants are pollinated by taking insects as media. However, some plants have low efficiency of natural pollination due to their own special physiological structures, such as Siraitia grosvenorii (Swingle) c.jeffrey), which not only has short life of male flowers and pollen grains firmly adhere to the lower hook of anthers, but also is difficult to pollinate through wind vectors; and because the female flowers lack components such as flowery odour, honey source and the like, the female flowers are difficult to attract insects to pollinate the female flowers, so that the serious natural pollination limitation is caused, and the fruit setting and maturing rate is extremely low. In order to improve the yield of plants with limited natural pollination in agricultural production, the problem is solved by adopting an artificial supplementary pollination mode, but the production link is extremely labor-consuming and time-consuming and has many concurrent problems, and particularly, the phenomena of large input and low yield of the planting industry are frequently caused by the rising labor cost in recent years. Therefore, there is an urgent need to find a method that can solve the pollination problem of such plants at a low cost.

The honeybees are excellent pollination insects and are bee species widely raised in the world, and the honeybee attractant is used for attracting the honeybees to pollinate plants with limited natural pollination, so that the yield and income are increased, the uniformity of fruit growth is facilitated, and deformed fruits caused by artificial pollination are reduced. However, in the prior art, most of the attractant formula is aromatic volatile components or plant-derived flower juice or syrup, the volatile components are easily diffused to cause unstable bee pollination inducing effect, and the attractant containing a large amount of sugar is sprayed on the plant to easily cause various diseases of the plant and attract other pests. It is therefore necessary to find other active ingredients from natural products which have an attractive effect on bees.

The grosvenor momordica fruit is a plant of the genus arhat fruit of the family cucurbitaceae, is a male and female heterostrain, has dual purposes of medicine and food, is widely applied in the world as a natural sweetener, and has greatly increased demand and daily and increased planting area year by year. Meanwhile, a large amount of byproduct resources such as stems, leaves and rattans of the momordica grosvenori are generated in the planting process but are not effectively utilized, and although the leaves are used as tea in folk, the leaves are generally discarded as wastes, so that the resources are wasted and the environment is polluted. Therefore, if more natural active ingredients can be searched from the byproduct resources of the momordica grosvenori, the waste is changed into valuable, and the method has important practical significance for environmental protection and promotion of the development of the local momordica grosvenori industry.

Disclosure of Invention

The invention provides a momordica grosvenori neoflavonoid compound, a preparation method thereof and application thereof in a bee attractant aiming at the defects in the prior art.

In order to realize the purpose, the invention provides the following technical scheme:

the invention provides a novel flavonoid compound which is characterized by being a compound with a structure shown in a formula (I) or a pharmaceutically acceptable salt thereof:

the chemical name of the compound with the structure shown in the formula (I) is as follows: rhamnocitrin-3-O- α -L-rhamnose-4' -O- β -D-glucoside;

the english name: Rhamnocitrin-3-O- α -L-rhamnosyl-4' - β -D-glucoside;

the molecular formula is: c ₂₈ H ₃₂ O ₁₅ ，[M+HCOO] ^- 653.1739, average molecular weight 608.55;

the physical and chemical properties are as follows: yellow amorphous powder, easily soluble in water and methanol, and insoluble in petroleum ether, chloroform, ethyl acetate, n-butanol, and ethanol. This is designated XY8 in the present invention.

The invention also provides a preparation method of the neoflavonoid compound, which comprises the following steps: extracting male leaves of momordica grosvenori to obtain an extract, then carrying out high-speed countercurrent chromatographic separation on the extract, collecting fractions in sections, identifying and merging the fractions by UPLC-MS or TLC, recovering a solvent, and respectively obtaining 6 parts I-VI, wherein the yellow powdery countercurrent chromatographic purified substance of the part IV is the neoflavonoid compound with the structure shown in the formula (I).

Further, the extraction takes water and/or lower alcohol as a solvent; the stationary phase and the mobile phase separated by the high-speed counter-current chromatography are a two-phase solvent system which consists of one or more than two of water and an ester solvent, an alcohol solvent, a ketone solvent, an ether solvent, a halogenated hydrocarbon solvent, an alkane solvent, a nitrile solvent or an acid solvent, and the distribution coefficient of the two-phase solvent system to the neoflavonoid compound is 0.2-5.0.

When the male leaves of the momordica grosvenori are extracted, the male leaves of the momordica grosvenori can be fresh or dry; the extraction procedure (e.g., extraction method, extraction time) is the same as that of the prior art, and preferably heating extraction (40-80 deg.C) or reflux extraction is used.

Further, the lower alcohol comprises 10-100% by volume of methanol or 10-100% by volume of ethanol, preferably 30-80% by volume of methanol or 30-80% by volume of ethanol; the ester solvent is selected from one or more of ethyl acetate, butyl acetate and amyl acetate; the alcohol solvent is selected from one or more of methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, n-pentanol and 2-pentanol; the ketone solvent is selected from one or more of acetone, butanone, cyclopentanone, methyl isobutyl ketone and cyclohexanone; the ether solvent is selected from one or more of diethyl ether, methyl tert-butyl ether, dioxane and tetrahydrofuran; the halogenated hydrocarbon solvent is selected from one or more of dichloromethane, dichloroethane, trichloromethane and carbon tetrachloride; the alkane solvent is selected from one or more of n-hexane, n-heptane, isooctane, petroleum ether and gasoline; the nitrile solvent is acetonitrile; the acid solvent is formic acid and/or acetic acid.

Still further, the petroleum ether includes petroleum ether having a boiling range of 30 to 60 ℃ and petroleum ether having a boiling range of 60 to 90 ℃.

Because the proportion of the high-speed countercurrent chromatography two-phase solvent system is related to the proportion of two phases, the total composition proportion of the two-phase solvent can be changed according to the proportion of the upper phase and the lower phase without influencing the composition of the upper phase or the composition of the lower phase and the distribution coefficient of the two-phase solvent.

Further, the stationary phase and the mobile phase when the high-speed countercurrent chromatography separation is performed are preferably:

a mixed solution was prepared with ethyl acetate, methanol, water and acetic acid in a volume ratio of 3.5:1:3.5: 0.05.

The solvent system for high-speed countercurrent chromatographic separation can adopt an upper phase as a stationary phase and a lower phase as a mobile phase; alternatively, the lower phase may be the stationary phase and the upper phase the mobile phase.

Further, the temperature of the high-speed countercurrent chromatographic separation is 10-35 ℃, the host machine rotates forwards or backwards, the rotating speed is 750-900 rpm, and the flow rate of the mobile phase is 0.5-15.0 mL/min.

The operations of preparation, sample loading, sectional collection and the like of taking a sample to be separated by the high-speed countercurrent chromatography are the same as those of the prior art. The following are preferred:

preparation of a sample to be separated by high-speed countercurrent chromatography: weighing the dry extract, taking the upper phase solution or the lower phase solution or both the upper phase solution and the lower phase solution, dissolving the dry extract in a volume which is 100-1000 times of the mass of the dry extract, and centrifuging to obtain the supernatant.

Taking a sample to be separated, wherein the volume of the sample to be separated is not more than the volume of a sample injection ring of the high-speed countercurrent chromatograph, injecting the sample into the high-speed countercurrent chromatograph, and collecting 1 fraction every 5-15 mL; UPLC-MS detection or TLC detection, merging fractions, recovering solvent to obtain parts I-VI 6, wherein the yellow powder purified by countercurrent chromatography of part IV is compound XY 8.

In order to reduce the burden on the high-speed counter-current chromatography, it is preferable to preliminarily purify the extract and then separate it by the high-speed counter-current chromatography. The primary purification operation may specifically be macroporous resin column chromatography and/or extraction operation of the extract, specifically:

when macroporous resin column chromatography is adopted for purification, the macroporous resin is medicinal macroporous adsorption resin capable of adsorbing and separating compound XY8, and the preferable model is Amberlite XAD16, X-5, AB-8 or D101. In eluting the macroporous resin column, an alcohol/water ratio of 0: 100-95: 5, and collecting the part containing compound XY8, preferably alcohol/water, in a ratio of 30: 70 volume ratio of the component mixed solvent. Wherein the alcohol is methanol or ethanol.

When the purification is carried out by extraction, impurity removal extraction and/or compound XY8 extraction are included. For impurity extraction, it is preferable to remove impurities such as chlorophyll and sterol by extraction with petroleum ether or n-hexane. For the extraction of compound XY8, compound XY8 is preferably transferred to an organic solvent using n-butanol or a mixed solvent containing n-butanol.

The purity of the obtained new flavonoid compound reaches more than 95.0 percent (HPLC detection), if the purity needs to be further improved, the new flavonoid compound can be subjected to reversed phase chromatographic column chromatography on the new flavonoid compound, and a mixed solvent consisting of acetonitrile and water is used as a mobile phase for separation, so that the purified compound with the structure shown in the formula (I) can be obtained. Wherein the volume ratio of acetonitrile to water in the mobile phase composition is preferably 20: 80-40: 60. The specific operation is as follows: taking acetonitrile-water with the volume ratio of 20: 80-40: 60 as a mobile phase, feeding a sample, separating and refining, detecting with 313 wavelengths, carrying out total operation time for 8-18 min, and collecting unimodal eluent. During sample injection, a sample is dissolved by methanol to prepare a solution with the mass concentration of 0.1-20 mg/mL.

The preferable conditions are ZORBAX SB-C18 chromatographic column (9.4mm multiplied by 250mm,5 mu m), acetonitrile (A) -0.1% formic acid (B) as a mobile phase system, and gradient elution program (0-15 min, 23-40% A, 15-15.01 min, 40-70% A, 15.01-23 min, 70% A, 23-23.01 min, 70-23% A, 23.01-30 min, 23% A); the column temperature is 30 ℃; flow rate 3.0 mL/min ^-1 (ii) a The sample amount was 80. mu.L, and the detection wavelength was 313 nm.

The invention also provides application of the neoflavonoid compound in preparation of the bee attractant.

The invention also provides a bee attractant which comprises the novel flavonoid compound.

The invention discovers that the following components are prepared in the research of the grosvenor momordica fruit: further experiments prove that although the leaves of the male plant of the momordica grosvenori contain higher content of flavonoid compounds, the specific composition and content of the flavonoid compounds of the male plant of the momordica grosvenori and the female plant of the momordica grosvenori are obviously different, which indicates that the leaves of the male plant of the momordica grosvenori possibly contain relatively higher content of compounds having attraction effect on the bees, and the novel flavonoid compounds having good attraction effect on the bees are obtained through further extraction and separation.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts high-speed counter-current chromatography to separate a novel flavonoid compound from male plant leaves of Siraitia grosvenorii (Swingle) C.Jeffrey) in Siraitia grosvenorii in Cucurbitaceae in Siraitia for the first time, and the flavonoid compound has attractant activity for bees and can be used for preparing bee attractants.

The novel flavonoid compound is derived from the momordica grosvenori, is a novel natural compound, has a structure and activity which are not reported yet, has good attractant activity for bees, provides an ideal candidate compound for developing a novel bee attractant, opens up a novel bee attractant and a novel application field of the momordica grosvenori, and lays a foundation for further development and utilization of momordica grosvenori leaves.

The preparation method of the neoflavonoid compound is simple, the product purity is high, the operation is easy, the market prospect is wide, and the preparation method is suitable for large-scale popularization and application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a high-speed countercurrent chromatography separation spectrum of the extract from leaves of male plant of Momordica grosvenori Swingle in example 1.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.

The "parts" in the present invention mean parts by weight unless otherwise specified.

Example 1

Preparation of neoflavonoid XY 8:

1) obtaining extract of male leaves of Momordica grosvenori

Weighing 1Kg of dry leaves of male leaves of Siraitia grosvenorii (Swingle) Grosvenorii C.Jeffrey), pulverizing, adding 8 times of 70% (volume) methanol, extracting under reflux at 60 deg.C for 60min, filtering, and collecting filtrate. Adding 70% (volume) methanol 8 times of the weight of the filter residue, heating and refluxing at 60 deg.C for 60min for extraction, and filtering. The filtrates are combined, and the filtrate is evaporated and concentrated to 2L by a rotary evaporator under reduced pressure at 60 ℃. Adding methanol 2L into the concentrated solution, extracting with petroleum ether 2 times with dosage of 2L each time, collecting lower phase, recovering solvent from petroleum ether phase, discarding, and rotary-steaming the lower phase at 60 deg.C to 2L to obtain extractive solution LHGXY 01.

2) Preliminary purification of the extract

Taking the extract LHGXY01, loading on D101 macroporous adsorbent resin column

After the above, water, 10% (volume) methanol-water, 50% (volume) methanol-water and 95% (volume) methanol are sequentially used for gradient elution. LC-MS analysis showed that 50% by volume of the methanol eluted fraction contained the novel compound XY 8. Mixing the 50% (volume) methanol eluents, concentrating, and vacuum evaporating to obtain extract LHGXY02。

3) High speed countercurrent chromatography separation

3.1) measuring 700mL of ethyl acetate, 200mL of methanol, 700mL of pure water and 0.1mL of acetic acid, preparing a mixed solution with a volume ratio of 3.5:1:3.5:0.05, fully shaking the mixed solution to XY8 to obtain a distribution coefficient of 1.0, standing the mixed solution for 0.5h, completely layering and clarifying an upper layer and a lower layer, separating the upper layer and the lower layer to obtain an upper phase solution and a lower phase solution, and respectively ultrasonically degassing for 10min for later use;

3.2) taking LHGXY021.0g of extract, taking 15mL of lower phase solution in the step 3.1), dissolving LHGXY02, centrifuging the solution, and taking supernatant for later use;

3.3) taking the upper phase solution obtained in the step 3.1) as a stationary phase, pumping into a separation tube of a high-speed counter-current chromatograph, adjusting a main machine to rotate forward until the maximum rotation speed (900r/min) is reached after the stationary phase is filled in the whole separation tube, simultaneously pumping the lower phase solution obtained in the step 3.1) as a mobile phase, taking 15mL of supernatant obtained in the step 3.2) after the mobile phase flows out from a column port and the stationary phase does not flow out, and injecting the supernatant into a sample injection valve after a two-phase solvent reaches dynamic balance in the separation tube, separating a sample under the condition of the flow rate of 2mL/min, detecting under the wavelength of 313nm, and collecting 1 fraction per 7 mL. The obtained high-speed countercurrent chromatography separation spectrum is shown in figure 1, and all fractions are segmented into 6 parts I-VI. Fractions 19-24 of fraction IV were spin dried using a vacuum centrifuge concentrator, dissolved in chromatographic alcohol methanol and detected by UPLC-MS. Wherein fraction 20-23 is yellow powder (LHGXY03, 153.5mg) with molecular weight of [ M + HCOO] ^- 653.1739 (Compound XY8) was found to be 95.8% pure by HPLC.

Characterization of the yellow powder obtained above, LHGXY 03:

XY 8: a yellow amorphous powder; UV (MeOH) λ max 264, 313 nm; ¹ h (500MHz) and ¹³ c (125MHz) NMR data are shown in Table 1; electrospray high resolution mass spectrometry (negative ion mode) HRESIMS M/z 653.1739[ M + HCOO] ^- Molecular formula C ₂₈ H ₃₂ O ₁₅ Calculated 653.1723, molecular weight 608.55, unsaturation 13.

Table 1: of yellow powder LHGXY03 ¹ HNMR and ¹³ c NMR data

Note: the solvent is deuterated methanol, the hydrogen spectrum is 500MHz, and the carbon spectrum is 125 MHz.

As can be seen from table 1, it is, ¹ HNMR δ 7.905(2H, d, J ═ 8.9Hz), 7.273(2H, d, J ═ 8.9Hz) are characteristic of flavone C ring 4' substituted; δ 6.614(1H, d, J ═ 2.1Hz), 7.273(1H, d, J ═ 2.1Hz) are characteristic of the meta position of flavone a ring disubstituted; the absence of a hydrogen signal on the B ring indicates that the 3' position of the B ring of the flavone is substituted; δ 3.90(1H, S) is characteristic of methoxy; δ 5.431(1H, d, J ═ 1.8Hz) and δ 5.431(3H, S) are characteristic of α -L-rhamnose; δ 5.10(1H, D, J ═ 7.5Hz) is characteristic of β -D-glucose; ¹³ the chemical shift of the CNMR carbon spectrum is close to that of 4 ', 3, 5, 7-tetrahydroxyflavone, and the compound is confirmed to be a substitution product of the 4', 3, 5, 7-tetrahydroxyflavone according to the correlation signals of the literature and HSQC. According to the HMBC correlation signal, the methoxyl is correlated with the aryl hydrogen at the 6-position and the 8-position, and the methoxyl is positioned at the 7-position; the terminal hydrogen of rhamnose has an association signal with the 3-carbon, indicating that rhamnose is located in the 3-position; the terminal hydrogen of glucose is associated with the 4 '-carbon, indicating that glucose is located at the 4' -position.

Therefore, the yellow amorphous powder obtained in the experimental example 1 can be determined to be the compound XY8, rhamnocitrin-3-O-alpha-L-rhamnose-4' -O-beta-D-glucoside, the structure of which is shown in the following formula (I), and the structure is confirmed by the nuclear magnetic resonance spectra HSQC and HMBC.

Example 2

Preparation of neoflavonoid XY 8:

1) obtaining extract of male leaves of Momordica grosvenori

Weighing 1Kg of dry leaves of male leaves of Siraitia grosvenorii (Swingle) Grosvenorii C.Jeffrey), pulverizing, adding 8 times of water, extracting at 50 deg.C under reflux for 60min, filtering, and collecting filtrate. Adding 8 times of water into the filter residue, heating and refluxing at 50 deg.C for 60min, and filtering. The filtrates are combined, and the filtrate is evaporated and concentrated to 2L by a rotary evaporator under reduced pressure at 60 ℃. Adding methanol 2L into the concentrated solution, extracting with petroleum ether 2 times with dosage of 2L each time, collecting lower phase, recovering solvent from petroleum ether phase, discarding, and rotary-steaming the lower phase at 60 deg.C to 2L to obtain extractive solution LHGXY 01.

Steps 2) to 3) were the same as in example 1.

In this example, step 3) was separated by high-speed countercurrent chromatography, and the resulting fractions were also fractionated into 6 fractions I to VI identical to those in example 1, after detection by UPLC-MS and combination, except that the contents of the fractions were different from those in example 1. Fractions 19-24 from fraction IV were spin dried using a vacuum centrifuge concentrator, dissolved in chromatographic alcohol methanol, and tested by UPLC-MS. Wherein fraction 20-23 is yellow powder (LHGXY03, 115.6mg) with molecular weight of [ M + HCOO] ^- 653.1739 (compound XY8) was found to be 95.8% pure by HPLC.

Example 3

Preparation of neoflavonoid XY 8:

1) obtaining extract of male leaves of Momordica grosvenori

Weighing 1Kg of dry leaves of male leaves of Siraitia grosvenorii (Swingle) Grosvenorii C.Jeffrey), pulverizing, adding 8 times of 100% (volume) ethanol, extracting at 40 deg.C under reflux for 60min, filtering, and collecting filtrate. Adding 100% (volume) ethanol 8 times the weight of the residue, heating and refluxing at 40 deg.C for 60min, and filtering. The filtrates are combined, and the filtrate is evaporated and concentrated to 2L by a rotary evaporator under reduced pressure at 60 ℃. Adding methanol 2L into the concentrated solution, extracting with petroleum ether 2 times with dosage of 2L each time, collecting lower phase, recovering solvent from petroleum ether phase, discarding, and rotary steaming at 60 deg.C to 2L to obtain extractive solution LHGXY 01.

2) High speed countercurrent chromatography

2.1) measuring 600mL of butyl acetate, 200mL of acetone, 600mL of pure water and 0.1mL of tetrahydrofuran, preparing to obtain a mixed solution, wherein the distribution coefficient of the mixed solution to XY8 is 0.5, fully shaking and standing for 0.5h, completely layering and clarifying an upper layer and a lower layer, separating the upper layer and the lower layer to obtain an upper phase solution and a lower phase solution, and respectively carrying out ultrasonic degassing for 10min for later use;

2.2) taking LHGXY021.0g of extract, taking 15mL of lower phase solution in the step 2.1), dissolving LHGXY02, centrifuging the solution, and taking supernatant for later use;

2.3) taking the upper phase solution obtained in the step 2.1) as a stationary phase, pumping into a separation tube of a high-speed counter-current chromatograph, adjusting a main machine to rotate positively to reach the maximum rotation speed (900r/min) after the stationary phase is filled in the whole separation tube, simultaneously pumping the lower phase solution obtained in the step 2.1) as a mobile phase, taking 15mL of supernatant obtained in the step 2.2) after the mobile phase flows out from a column port and the stationary phase does not flow out, and injecting a two-phase solvent into a sample injection valve after the two-phase solvent reaches dynamic balance in the separation tube, separating a sample under the condition of the flow rate of 2mL/min, detecting at the wavelength of 313nm, and collecting 1 fraction per 15 mL. The resulting stream, after separation by UPLC-MS detection and combination, was also segmented into 6 fractions i to vi identical to example 1, except that the content of each fraction was different from that of example 1. Fractions 23-26 of fraction IV were spin dried using a vacuum centrifuge concentrator, dissolved in chromatographic alcohol methanol and tested by UPLC-MS. Wherein fraction 24-26 is yellow powder (LHGXY03, 146.5mg) with molecular weight of [ M + HCOO] ^- 653.1739 (compound XY8) was 96.2% pure by HPLC.

Example 4

Preparation of neoflavonoid XY 8:

1) obtaining extract of male leaves of Momordica grosvenori

Weighing 1Kg of dry leaves of male leaves of Siraitia grosvenorii (Swingle) Grosvenorii C.Jeffrey), pulverizing, adding 8 times of 10% (volume) methanol, reflux extracting at room temperature for 60min, filtering, and collecting filtrate. Adding 8 times of 10% (volume) methanol into the filter residue, refluxing at normal temperature for 60min, and filtering. The filtrates are combined, and the filtrate is evaporated and concentrated to 2L by a rotary evaporator under reduced pressure at 60 ℃. Adding methanol 2L into the concentrated solution, extracting with petroleum ether 2 times with dosage of 2L each time, collecting lower phase, recovering solvent from petroleum ether phase, discarding, and rotary steaming at 60 deg.C to 2L to obtain extractive solution LHGXY 01.

2) Preliminary purification of the extract

Taking the extract LHGXY01, addingD101 macroporous adsorption resin column

After the above, water, 10% (volume) methanol-water, 50% (volume) methanol-water and 95% (volume) methanol are sequentially used for gradient elution. LC-MS analysis showed that 50% by volume of the methanol eluted fraction contained the novel compound XY 8. Mixing the 50% (volume) methanol eluents, concentrating, and evaporating to dryness under reduced pressure to obtain extract LHGXY 02.

3) High speed countercurrent chromatography separation

3.1) measuring 600mL of isobutanol, 150mL of n-hexane, 600mL of pure water, 50mL of trichloromethane and 50mL of acetonitrile, preparing to obtain a mixed solution, wherein the distribution coefficient of the mixed solution to XY8 is 2.5, fully shaking, standing for 0.5h, completely layering and clarifying an upper layer and a lower layer, separating the upper layer and the lower layer to obtain an upper phase solution and a lower phase solution, and respectively carrying out ultrasonic degassing for 10min for later use;

3.3) taking the upper phase solution obtained in the step 3.1) as a stationary phase, pumping into a separation tube of a high-speed counter-current chromatograph, adjusting a main machine to rotate positively to reach the maximum rotation speed (900r/min) after the stationary phase is filled in the whole separation tube, simultaneously pumping the lower phase solution obtained in the step 3.1) as a mobile phase, taking 15mL of supernatant obtained in the step 3.2) after the mobile phase flows out from a column port and the stationary phase does not flow out, and injecting a two-phase solvent into a sample injection valve after the two-phase solvent reaches dynamic balance in the separation tube, separating a sample at the flow rate of 2mL/min, detecting at the wavelength of 313nm, and collecting 1 fraction per 7 mL. The resulting stream, after separation by UPLC-MS detection and combination, was also segmented into 6 fractions i to vi identical to example 1, except that the content of each fraction was different from that of example 1. Fractions 22-27 of fraction IV were spin dried using a vacuum centrifuge concentrator, dissolved in chromatographic alcohol methanol and tested by UPLC-MS. Wherein the fraction 23-27 is yellow powder (LHGXY03, 120.7mg) with molecular weight of [ M + HCOO] ^- 653.1739 (compound XY8) was found to be 95.2% pure by HPLC.

4) Further purifying the compound XY8 obtained in step 3.3) by reverse phase chromatography: dissolving XY8 compound in methanolPreparing a sample solution with the concentration of 10mg/mL by hydrolysis under the elution conditions of a ZORBAX SB-C18 chromatographic column (9.4mm multiplied by 250mm and 5 mu m), an acetonitrile (A) -0.1% formic acid (B) as a mobile phase system, and a gradient elution program (0-15 min, 23-40% A, 15-15.01 min, 40-70% A, 15.01-23 min, 70% A, 23-23.01 min, 70-23% A, 23.01-30 min and 23% A); the column temperature is 30 ℃; flow rate 3.0 mL/min ^-1 (ii) a The sample size was 80. mu.L, and the detection wavelength was 313 nm. And collecting the unimodal eluate, and detecting that the purity of the obtained purified XY8 compound is up to 98.5%.

Example 5

The new compound XY8 purified in the step 4) of the example 4 and other flavonoid compounds separated from the leaves of the grosvener siraitia are used for measuring the bee attracting activity.

1.1 Experimental materials

The test insects: the experimental Apis cerana cerana (Apis cerana cerana) is taken from Meixi meadow of Guangxi Guilin city, prefecture and is fasted for 0.5h before experiment. Worker bees are randomly selected during indoor bioassay.

Test monomeric compounds to be tested: compound 1, XY 8; compound 2, kaempferol-3, 7-O-alpha-L-dirhamnoside; compound 3, kaempferol-3-O- α -L-rhamnoside; compound 4, kaempferol-3-O-alpha-L-rhamnose-7-O- [ beta-D-glucosyl- (1-2) -alpha-L-rhamnoside](ii) a Compound 5, quercetin-3-O-beta-D-glucose-7-O-alpha-L-rhamnoside. The compounds 2-5 are other known flavonoid compounds separated from the leaves of the Momordica grosvenori Swingle, and the purity of all the compounds is more than or equal to 98.0%. Using ultrapure water as solvent, 10 are prepared for each compound ^-2 g/mL、10 ^-4 g/mL、10 ^-6 g/mL、10 ^-8 g/mL 4 doses. For each behavior assay, 1ml of a solution of each dose of test compound was aspirated as a taste source, and 1ml of ultrapure water was used as CK.

1.2Y-shaped olfactometer and measurement procedure

The Y-shaped tube is made of colorless transparent glass, the length of the two arms and the base part are both 15cm, the inner diameter is 1.5cm, and the included angle of the two arms is 120 degrees. The two arms are respectively connected with a flavor source bottle (or a control bottle), a humidifying bottle, an air filtering bottle (filled with activated carbon) and a flowmeter, and all the parts are connected by odorless Teflon pipes. Absorbing 1ml taste source ampoule before measurement, the ampoule being placed in the taste source ampoule; then absorbing 1ml of ultrapure water into the open ampoule, which is contained in the control bottle. Air is pumped from the tube opening of the base of the Y-shaped tube by an air pump, and the flow of each arm is adjusted to be 120ml/min during measurement. Before measurement, air is pumped for 10min to fill the Y-shaped tube odor source arm with the information substance to be measured. When in test, 1 Chinese bee is introduced from the tube opening at the base of the Y-shaped tube by using a test tube. After the bees enter, the bees advance against the wind, or crawl linearly, or advance spirally, a selection is made at the intersection of the Y-shaped pipes, the bees enter one arm of the Y-shaped pipes, crawling for 5cm is recorded as selecting the arm, and the counting is not performed if no reaction occurs after more than 5 min.

Measuring 20 heads of Chinese bees for each treatment dosage of 5 flavor sources, wherein each head is used for 1 time; and (3) scrubbing the inner wall and the outer wall of the Y-shaped tube by using absolute ethyl alcohol every 10 times of the test, drying, and replacing the connecting positions of two arms of the Y-shaped tube, the flavor source bottle and the comparison bottle so as to eliminate the influence caused by possible asymmetry of the Y-shaped tube. After the measurement of the taste source for each dose is finished, glass parts such as Y-tubes, taste source bottles and the like are washed by potassium dichromate washing liquor and dried in an oven at 100 ℃. Drying the activated carbon at 100 ℃ for 4h to remove the adsorbed peculiar smell molecules.

The bioassay was performed in a dark room with 1 15W incandescent lamp 1.5cm above the Y-tube to provide illumination. The biometric time per day ranged from 8:00 to 16:00, during which the bees were active. Room temperature 25 deg.C, relative humidity (70 + -3)%.

1.3 data analysis methods

Data analysis was done using SPSS 22.0 software. When the indoor Y-shaped tube olfaction behaviors of Chinese bees are measured, if the taste testing source has no influence on the behaviors of the Chinese bees, the probability of the taste testing source and the probability of CK of the Chinese bees are both 50%, and if test H is assumed ₀ 50: 50, making chi for the difference between the number of the bee trend flavor source and CK ² Test, when P<When the ratio is 0.05, the difference reaches a remarkable level, and when P is in<At 0.01, the difference was very significant.

The results of the experiment are shown in table 2.

TABLE 25 difference in number of Apis cerana lured by monomeric compounds and ultrapure water

Note: n.s. not significant; significant; significance of

As is clear from Table 2, compound 1(XY8) was found to be 10 among 5 monomer compounds ^-2 The difference between the attraction effect on the Chinese bees and CK (ultrapure water) under the concentration of g/mL reaches an extremely significant level (P)<0.01) at 10 ^-4 g/mL、10 ^-6 The difference between the attraction effect on Chinese bees and CK (ultrapure water) under the concentration of g/mL reaches a remarkable level (P)<0.05); while the compounds 2-5 have no significant difference between the attraction effect on apis cerana bees and CK (ultrapure water) under the concentration set in the test. The new compound XY8 has better attraction activity to Chinese bees.

The above description is only for the preferred embodiment of the present invention, and the protection scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention, the technical solution and the inventive concept of the present invention equivalent or change within the technical scope of the present invention.

Claims

1. The novel flavonoid compound is characterized by being a compound with a structure shown in a formula (I) or a pharmaceutically acceptable salt thereof:

2. the process for preparing a neoflavonoid compound according to claim 1, characterized by comprising the steps of: extracting male leaves of momordica grosvenori to obtain an extract, then carrying out high-speed countercurrent chromatographic separation on the extract, collecting fractions in sections, identifying and merging the fractions by UPLC-MS or TLC, recovering a solvent, and respectively obtaining 6 parts I-VI, wherein the yellow powdery countercurrent chromatographic purified substance of the part IV is the neoflavonoid compound with the structure shown in the formula (I).

3. The method according to claim 2, wherein the extraction is carried out using water and/or a lower alcohol as a solvent; the stationary phase and the mobile phase separated by the high-speed counter-current chromatography are a two-phase system consisting of one or more of water and an ester solvent, an alcohol solvent, a ketone solvent, an ether solvent, a halogenated hydrocarbon solvent, an alkane solvent, a nitrile solvent or an acid solvent, and the distribution coefficient of the stationary phase and the mobile phase to the neoflavonoid compound is 0.2-5.0.

4. The method according to claim 3, wherein the lower alcohol comprises methanol at a volume concentration of 10 to 100% or ethanol at a volume concentration of 10 to 100%; the ester solvent is selected from one or more of ethyl acetate, butyl acetate and amyl acetate; the alcohol solvent is selected from one or more of methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, n-pentanol and 2-pentanol; the ketone solvent is selected from one or more of acetone, butanone, cyclopentanone, methyl isobutyl ketone and cyclohexanone; the ether solvent is selected from one or more of diethyl ether, methyl tert-butyl ether, dioxane and tetrahydrofuran; the halogenated hydrocarbon solvent is selected from one or more of dichloromethane, dichloroethane, trichloromethane and carbon tetrachloride; the alkane solvent is selected from one or more of n-hexane, n-heptane, isooctane, petroleum ether and gasoline; the nitrile solvent is acetonitrile; the acid solvent is formic acid and/or acetic acid.

5. The preparation method according to claim 2, wherein the temperature of the high-speed countercurrent chromatography is 10-35 ℃, the rotation speed of a main machine is 750-900 rpm, and the flow rate of the mobile phase is 0.5-15.0 mL/min.

6. The method of claim 2, further comprising the step of purifying the extract prior to the high-speed counter-current chromatography.

7. The preparation method according to claim 6, wherein the purification is carried out by performing macroporous resin column chromatography and/or extraction on the extract.

8. The method according to claim 2, further comprising the step of purifying the neoflavonoid compound by reverse phase chromatography.

9. Use of neoflavonoids according to claim 1 for the preparation of a bee attractant.

10. An attractant for bees, comprising the neoflavonoid compound according to claim 1.