A method for extracting effective active ingredients from Buddleja buddlejae and its application

Abstract

The present invention discloses a method for extracting effective active ingredients from Buddleja buddhist flower and its application. The method comprises the following steps: first subjecting the crushed Buddleja buddhist flower to ethanol reflux extraction and concentration, and then extracting the obtained Buddleja buddhist flower alcohol extract with petroleum ether, ethyl acetate, and n-butanol respectively. The remaining aqueous phase is concentrated under reduced pressure, purified by a D101 macroporous resin column, and the target product obtained after drying is structurally identified as a verbascoside monomer compound. The method of the present invention can better control the quality standards of Buddleja buddhist flower and pave the way for a more in-depth study of its pharmacological effects by effectively separating the verbascoside compounds from the Buddleja buddhist flower, thereby laying the foundation for the clinical development of new drugs with anti-tumor, antioxidant, and immunomodulatory activities.

Description

Technical Field

[0001] The invention relates to the technical field of plant active ingredient extraction, in particular to a method for extracting effective active ingredients from Buddleja buddlejae and application thereof. Background Art

[0002] Buddlea officinalis Maxim, a member of the genus Buddlea in the family Masaceae, is primarily found in Henan, Guangxi, Gansu, Shanxi, Sichuan, Shaanxi, and Yunnan. It grows 1-4 meters tall, with leaves that are oblong-lanceolate or narrowly elliptical. It blooms from March to April, boasting vibrant, vibrant blooms and a unique fragrance. In southern China, Buddlea officinalis is often grown as an ornamental plant in gardens and yards. Its high pigment content makes it a popular dye for making multicolored sticky rice during the "March 3rd" festival in Guangxi Zhuang Autonomous Region. Buddlea officinalis is an important natural medicinal herb in my country, with a sweet and slightly cold nature and high medicinal value. The entire plant is used as a medicinal herb. Recent research has shown that the chemical components of Buddlea officinalis primarily include flavonoids, phenylalanols, phenols, and alkaloids, with flavonoids and phenylalanols being the most prominent.

[0003] Buddleja buddhistata, a natural product unique to my country, has the effects of removing heat and protecting the liver, improving eyesight and removing cataracts. It also exhibits multiple pharmacological effects, including lowering blood sugar, providing antioxidant and anti-inflammatory benefits, inhibiting endothelial cell proliferation, and regulating immune activity. As a natural medicine, it effectively kills tumor cells, has few side effects, is inexpensive, can reverse drug resistance, regulate human immune activity, and enhance immune function. Currently, the development and utilization of natural medicines is gaining increasing attention both domestically and internationally, and the market prospects for natural medicines cannot be underestimated.

[0004] Verbascoside (VB), also known as ergosterol, verbascoside, and acteoside, is a phenylethanol glycoside compound composed of 4,5-dihydroxyphenylethanol (hydroxytyrosol) linked via ester and glycosidic bonds to rhamnose (C1) via a glycosidic bond at the C3 position and β-D-pyranose. It exhibits significant antioxidant, anti-inflammatory, memory-enhancing, neuroprotective, and anti-tumor activities. Currently, research on the active ingredients of Buddleja buddhistia mainly focuses on the extraction of verbascoside, and no relevant literature has documented the extraction of verbascoside from Buddleja buddhistia. Summary of the Invention

[0005] The present invention aims to provide a method for extracting effective active ingredients of verbascoside from Buddleja buddhist flower and its application in anti-tumor immunomodulatory activity, antioxidant and other aspects. In this method, Buddleja buddhist flower is first extracted with ethanol, and then extracted with petroleum ether, ethyl acetate and n-butanol respectively. The remaining aqueous phase is concentrated under reduced pressure and then purified by a D101 macroporous resin column. The obtained fraction is dried to obtain a target product. The target product is determined to be a verbascoside compound through testing. By effectively separating the verbascoside compound from Buddleja buddhist flower, it is possible to better control the quality standards of Buddleja buddhist flower and pave the way for more in-depth research on its pharmacological effects, thereby laying a foundation for the clinical development of new drugs with anti-tumor, antioxidant and immunomodulatory activities.

[0006] In order to achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] S1: After crushing the Buddleja flos, ethanol was added to carry out heating and reflux extraction, and the extraction was repeated 3 times. The filtrate was combined, and the Buddleja flos suspension was filtered with a filter cloth to remove the residue. The obtained filtrate was heated and evaporated by a rotary evaporator. After the rotary evaporation of the filtrate was completed, it was further evaporated to dryness in a water bath until the filtrate was in a non-flowing paste to obtain the Buddleja flos alcohol extract;

[0008] S2: The alcohol extract of Buddleja chinensis was added to sterile water to prepare a suspension, petroleum ether and the suspension were thoroughly mixed, and the mixture was allowed to stand at room temperature until obvious stratification occurred. The upper petroleum ether suspension was rotary evaporated and heated in a water bath to evaporate to a paste without obvious organic solvent taste, thereby obtaining a petroleum ether extract, which was stored in a refrigerator at 4°C for later use; the remaining aqueous layer was extracted with ethyl acetate and n-butanol in accordance with the above extraction method to obtain an ethyl acetate extract and an n-butanol extract, respectively. The remaining aqueous phase was concentrated under reduced pressure to remove the organic solvent, and the obtained aqueous phase was the crude extract A of Buddleja chinensis;

[0009] S3: The crude extract A of Buddleja buddhistata is purified by D101 macroporous resin column, and the fractions are collected and finally concentrated, freeze-dried and dried under reduced pressure to obtain the target product of Buddleja buddhistata. The target product is then subjected to structural identification to finally determine the structure of the target product.

[0010] The target product was detected by infrared, hydrogen spectrum, carbon spectrum, mass spectrum and ultraviolet spectrum, and it was confirmed that the product was a monomer compound of verbascoside with a molecular formula of C 29 H 36 O 15 , molecular weight is 624.59, and the structural formula is as follows:

[0011]

[0012] The solution of the present invention is further optimized: the D101 macroporous resin column purification treatment adopts water and ethanol gradient eluent.

[0013] The scheme of the present invention is further optimized: the volume ratios of water and ethanol in the gradient elution are 100:0, 80:20, 60:40 and 40:60, respectively.

[0014] The scheme of the present invention is further optimized: the D101 macroporous resin is also pretreated, and the specific pretreatment steps are as follows: first, the D101 macroporous resin is soaked in triethanolamine for 24 hours, and then it is loaded into the chromatography column by the wet column packing method, and 95% ethanol solution is passed through the resin layer at a flow rate of 2BV / h until the effluent is no longer white and turbid when distilled water is added. The ethanol is washed with distilled water at the same flow rate, and then a mixed solution of oxalic acid and sodium persulfate is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and rinsed with distilled water at the same flow rate until the pH value is neutral, and then 2BV of 2% NaOH solution is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and rinsed with distilled water at the same flow rate until the pH value is neutral.

[0015] The solution of the present invention is further optimized: the mass ratio of oxalic acid to sodium persulfate is 4:1, and the mass concentration of the mixed solution is 3-5%.

[0016] The scheme of the present invention is further optimized: the mass ratio of the Buddleja bud alcohol extract and sterile water in step S2 is 1:1-3, wherein when the different solvent extracts are subjected to rotary evaporation and water bath heating, it is important to note that the temperature setting must not exceed the boiling point of the solvent, the temperature of petroleum ether is (60-90°C), the temperature of ethyl acetate is set to 70°C, and the temperature of n-butanol and water is set to 90°C.

[0017] Compared with the prior art, the advantages and beneficial effects of the present invention include:

[0018] 1. This method first extracts Buddleja buddhist flower with ethanol, and then extracts with petroleum ether, ethyl acetate, and n-butanol respectively. The remaining aqueous phase is concentrated under reduced pressure, purified by a D101 macroporous resin column, and separated by high performance liquid chromatography. After drying, the target monomer compound is obtained. The target monomer compound is determined to be a verbascoside compound after testing. The effective separation of the verbascoside compound of Buddleja buddhist flower can better control the quality standards of Buddleja buddhist flower, pave the way for more in-depth research on its pharmacological effects, and lay the foundation for the clinical development of new drugs with anti-tumor, antioxidant, and immunomodulatory activities.

[0019] 2. The crude extract of Buddleja buddleja is purified by a D101 macroporous resin column to achieve the effect of decolorization and impurity removal. At the same time, the D101 macroporous resin is soaked with triethanolamine. By introducing a large number of active functional groups such as hydroxyl groups and amino groups into the surface of the macroporous adsorption resin, the polarity of the adsorption macroporous resin is improved, thereby improving its higher adsorption capacity and selectivity for organic compounds; the macroporous adsorption resin is also oxidized by oxalic acid and sodium persulfate, which not only improves the adsorption performance of the macroporous adsorption resin, but also further decolorizes the jasmine monosaccharide compounds, thereby improving the purification effect of the crude target extract of Buddleja buddleja.

[0020] 3. The method of the present invention successively utilizes petroleum ether, ethyl acetate, and n-butanol for extraction, and the remaining aqueous phase is concentrated under reduced pressure and extracted with different organic solvents, respectively, to separate the other effective components of Buddleja buddhist flower. After the other effective components are separated, it is more conducive to the separation of verbascoside, and at the same time it is beneficial to improve the extraction rate and purity of verbascoside. After high performance liquid chromatography detection, the product purity of the verbascoside compound of Buddleja buddhist flower reaches 99.78%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the infrared detection spectrum of verbascoside from Buddleja buddleja.

[0022] Figure 2 This is the hydrogen spectrum of glycosides from the verbascoside of Buddleja buddleja;

[0023] Figure 3 This is the carbon spectrum of verbascoside from Buddleja buddleja;

[0024] Figure 4 This is the mass spectrum of verbascoside from Buddleja buddleja;

[0025] Figure 5 This is the UV detection spectrum of verbascoside from Buddleja buddleja.

[0026] Figure 6 This is the HPLC chromatogram of verbascoside from Buddleja buddleja.

[0027] Figure 7 The inhibitory effect of verbascoside on tumor cells;

[0028] Figure 8 The pro-apoptotic effect of verbascoside monomer on BEL-7404 tumor cells;

[0029] Figure 9 The pro-apoptotic effect of verbascoside monomer on SGC-7901 tumor cells;

[0030] Figure 10 The results show that the monomeric compound of verbascoside promotes apoptosis of MCF-7 tumor cells. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Example 1

[0033] A method for extracting effective active ingredients from Buddleja buddhistae, comprising the following steps:

[0034] S1: Weigh the thoroughly dried and crushed Buddleja buddleja, add 3 times the amount of Buddleja buddleja and perform heating reflux extraction, extract for 2 hours each time, repeat the extraction 3 times, combine the filtrate, filter the Buddleja buddleja suspension with filter cloth, remove the residue, and the obtained filtrate is heated and evaporated at 55 ° C using a rotary evaporator. After the rotary evaporation of the filtrate is completed, it is further evaporated to dryness in a water bath until the filtrate is in a non-flowing paste to obtain a Buddleja buddleja alcohol extract;

[0035] S2: Dilute the Buddleja buddleja alcohol extract 3 times with water, mix the petroleum ether and the suspension thoroughly, let it stand at room temperature, wait for obvious stratification, take the upper petroleum ether suspension for rotary evaporation, and evaporate it with a water bath until it is paste-like and has no obvious smell of organic solvent, to obtain the petroleum ether extract, which is stored in a refrigerator at 4°C for later use; then extract the remaining water layer with ethyl acetate and n-butanol according to the above extraction method, respectively, to obtain ethyl acetate extract and n-butanol extract, and concentrate the remaining aqueous phase under reduced pressure to remove the organic solvent, and the obtained aqueous phase is the crude extract A of Buddleja buddleja. The volume ratio of petroleum ether to suspension is 3:1, the volume ratio of ethyl acetate to suspension is 2:1, and the volume ratio of n-butanol to suspension is 1:1. When rotary evaporation and water bath heating, pay attention to the temperature setting not to exceed the boiling point of the solvent. The temperature of petroleum ether is 75°C, the temperature of ethyl acetate is set to 70°C, and the temperature of n-butanol and water is set to 90°C.

[0036] S3: The crude extract A of Buddleja buddleja was purified by passing it through a D101 macroporous resin column (100 cm×6.0 cm), which contained 600 g of D101 macroporous resin and was eluted with water and ethanol in different ratios (100:0, 80:20, 60:40 and 40:60 v / v; 3000 mL for each ratio).

[0037] The D101 macroporous resin is also pretreated, and the specific pretreatment steps are as follows: first, the D101 macroporous resin is soaked in triethanolamine for 24 hours, and then it is loaded into the chromatography column by the wet column packing method, and 95% ethanol solution is passed through the resin layer at a flow rate of 2BV / h until the effluent is no longer white and turbid when added with distilled water, and the ethanol is washed with distilled water at the same flow rate, and then a mixed solution of oxalic acid and sodium persulfate with a mass ratio of 4:1 with a mass concentration of 3% is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and rinsed with distilled water at the same flow rate until the pH value is neutral, and then 2BV of 2% NaOH solution is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and rinsed with distilled water at the same flow rate until the pH value is neutral.

[0038] S4: The fraction obtained after purification by macroporous resin is finally concentrated at 50°C, freeze-dried, and dried under reduced pressure at 40°C to obtain the target product.

[0039] The target product was tested by infrared, hydrogen spectrum, carbon spectrum, mass spectrum and ultraviolet spectrum respectively. The test results are shown in the following table and Figure 1-Figure 5 The compound was finally determined to be verbascoside, with the molecular formula C 29 H 36 O 15 , molecular weight is 624.59, and the structural formula is as follows:

[0040]

[0041] 1 H-NMR

[0042]

[0043]

[0044] 13 C-NMR

[0045]

[0046]

[0047]

[0048] The purity of the isolated verbascoside compound obtained in this example was tested by high performance liquid chromatography. The results of the high performance liquid chromatography test are shown in the following table and Figure 6 As shown, the purity of the product reached 99.78%.

[0049]

[0050]

[0051] Example 2

[0052] A method for extracting effective active ingredients from Buddleja buddhistae, comprising the following steps:

[0053] S1: Weigh thoroughly dried and crushed Buddleja buddleja flower, add 2 times the amount of Buddleja buddleja flower for heating and reflux extraction, extract for 1 hour each time, repeat the extraction 3 times, combine the filtrates, filter the Buddleja buddleja flower suspension with filter cloth, remove the residue, and heat the filtrate to evaporate and concentrate it at 60°C using a rotary evaporator. After the rotary evaporation of the filtrate is completed, further evaporate it to dryness using a water bath until the filtrate becomes a non-flowing paste to obtain the Buddleja buddleja flower alcohol extract;

[0054] S2: Dilute the alcohol extract of Buddleja buddleja 3 times with water, mix the petroleum ether and the suspension thoroughly, let it stand at room temperature, and wait until obvious stratification occurs. Take the upper petroleum ether suspension and perform rotary evaporation. Heat it in a water bath and evaporate it until it becomes a paste with no obvious smell of organic solvent. The petroleum ether extract is obtained and stored in a refrigerator at 4°C for later use. The remaining aqueous layer is then extracted with ethyl acetate and n-butanol according to the above extraction method to obtain ethyl acetate extract and n-butanol extract respectively. The remaining aqueous phase is concentrated under reduced pressure to remove the organic solvent. The obtained aqueous phase is the crude extract A of Buddleja bud ...

[0055] S3: The crude extract A of Buddleja buddleja was purified by passing it through a D101 macroporous resin column (100 cm×6.0 cm), which contained 600 g of D101 macroporous resin and was eluted with water and ethanol in different ratios (100:0, 80:20, 60:40 and 40:60 v / v; 3000 mL for each ratio).

[0056] The D101 macroporous resin is also pretreated, and the specific pretreatment steps are as follows: first, the D101 macroporous resin is soaked in triethanolamine for 24 hours, and then it is loaded into the chromatography column by the wet column packing method, and 95% ethanol solution is passed through the resin layer at a flow rate of 2BV / h until the effluent is no longer white and turbid when added with distilled water, and then the ethanol is washed with distilled water at the same flow rate, and then a mixed solution of oxalic acid and sodium persulfate with a mass ratio of 4:1 with a mass concentration of 5% is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and then rinsed with distilled water at the same flow rate until the pH value is neutral, and then 2BV of 2% NaOH solution is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and then rinsed with distilled water at the same flow rate until the pH value is neutral.

[0057] S4: After the macroporous resin purification treatment, the collected fractions are concentrated at 50°C, freeze-dried, and dried under reduced pressure at 40°C to obtain the target product of Buddleja buddhistata.

[0058] The target monomer compound separated from Buddleja buddleja in this example is verbascoside compound, and after high performance liquid chromatography test, the product purity reaches 99.34%.

[0059] Example 3

[0060] A method for extracting effective active ingredients from Buddleja buddhistae, comprising the following steps:

[0061] S1: Weigh the thoroughly dried and crushed Buddleja buddleja, add 3 times the amount of Buddleja buddleja and perform heating reflux extraction, extract for 2 hours each time, repeat the extraction 3 times, combine the filtrate, filter the Buddleja buddleja suspension with filter cloth, remove the residue, and the obtained filtrate is heated and evaporated at 55 ° C using a rotary evaporator. After the rotary evaporation of the filtrate is completed, it is further evaporated to dryness in a water bath until the filtrate is in a non-flowing paste to obtain a Buddleja buddleja alcohol extract;

[0062] S2: Dilute the Buddleja buddleja alcohol extract 3 times with water, mix the petroleum ether with the suspension thoroughly, let it stand at room temperature, wait for obvious stratification, take the upper petroleum ether suspension for rotary evaporation, and evaporate it with a water bath until it is paste-like and has no obvious smell of organic solvent, to obtain the petroleum ether extract, which is stored in a refrigerator at 4°C for later use; then extract the remaining water layer with ethyl acetate and n-butanol according to the above extraction method, respectively, to obtain ethyl acetate extract and n-butanol extract, and concentrate the remaining aqueous phase under reduced pressure to remove the organic solvent, and the obtained aqueous phase is the crude extract A of Buddleja buddleja. The volume ratio of petroleum ether to suspension is 2:1, the volume ratio of ethyl acetate to suspension is 2:1, and the volume ratio of n-butanol to suspension is 1:1. When rotary evaporation and water bath heating, pay attention to the temperature setting not to exceed the boiling point of the solvent. The temperature of petroleum ether is 75°C, the temperature of ethyl acetate is set to 70°C, and the temperature of n-butanol and water is set to 90°C.

[0063] S3: The crude extract A of Buddleja buddleja was purified by passing it through a D101 macroporous resin column (100 cm×6.0 cm), which contained 600 g of D101 macroporous resin and was eluted with water and ethanol in different ratios (100:0, 80:20, 60:40 and 40:60 v / v; 3000 mL for each ratio).

[0064] The D101 macroporous resin is also pretreated, and the specific pretreatment steps are as follows: first, the D101 macroporous resin is soaked in triethanolamine for 24 hours, and then it is loaded into the chromatography column by the wet column packing method, and 95% ethanol solution is passed through the resin layer at a flow rate of 2BV / h until the effluent is no longer white and turbid when added with distilled water, and the ethanol is washed with distilled water at the same flow rate, and then a mixed solution of oxalic acid and sodium persulfate with a mass ratio of 4:1 with a mass concentration of 4% is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and rinsed with distilled water at the same flow rate until the pH value is neutral, and then 2BV of 2% NaOH solution is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and rinsed with distilled water at the same flow rate until the pH value is neutral.

[0065] S4: After the macroporous resin purification treatment, the fractions were concentrated at 50°C, freeze-dried, and dried under reduced pressure at 40°C to obtain the target monomer compound of Buddleja buddhatu. The specific method for obtaining verbascoside by HPLC separation is as follows: mobile phase: 50% methanol-0.1% phosphoric acid aqueous solution (volume ratio of 17:83); mobile phase rate: 1.0 mL / min; instrument model: Waters 2695-996; analytical column: Inertsil ODS-SP, 5 μm, 4.6 x 250 mm; column temperature: 35°C; detection wavelength: 332 nm.

[0066] The target product separated from Buddleja buddleja in this example is a verbascoside compound. After high performance liquid chromatography testing, the product purity reaches 99.15%.

[0067] Comparative Example 1

[0068] The present invention is different from Example 1 in that: step S2 is to dilute the Buddleja buddleja alcohol extract 3 times with water, and mix the petroleum ether and the suspension in a volume ratio of 3: 1. The mixture is allowed to stand at room temperature until obvious stratification occurs, and the upper petroleum ether suspension is rotary evaporated at 75 ° C. and heated in a water bath at 75 ° C. to a paste with no obvious taste of organic solvent, to obtain a petroleum ether extract, which is stored in a refrigerator at 4 ° C for later use; the remaining aqueous phase is concentrated under reduced pressure to remove the organic solvent, and the obtained aqueous phase is the crude extract A of Buddleja buddleja.

[0069] The target product separated from Buddleja buddleja in this example is a verbascoside compound. After high performance liquid chromatography testing, the product purity reaches 95.43%.

[0070] Comparative Example 2

[0071] The present invention is different from Example 1 in that: step S2 is to dilute the Buddleja buddleja alcohol extract 3 times with water, and thoroughly mix the ethyl acetate and the suspension in a volume ratio of 2: 1, and let it stand at room temperature until obvious stratification occurs. The upper ethyl acetate suspension is rotary evaporated at 70 ° C, and heated in a water bath at 70 ° C to evaporate to a paste with no obvious taste of organic solvent to obtain an ethyl acetate extract, which is stored in a refrigerator at 4 ° C for standby use; the remaining aqueous phase is concentrated under reduced pressure to remove the organic solvent, and the obtained aqueous phase is the Buddleja buddleja crude extract A.

[0072] The target product separated from Buddleja buddleja in this example is a verbascoside compound. After high performance liquid chromatography testing, the product purity reached 96.34%.

[0073] Comparative Example 3

[0074] The present invention is different from Example 1 in that: step S2 is to dilute the alcohol extract of the Buddleja flower with water 3 times, and mix the n-butanol and the suspension thoroughly in a volume ratio of 1: 1, and let it stand at room temperature until obvious stratification occurs. The upper n-butanol suspension is rotary evaporated at 90 ° C, and heated in a water bath at 90 ° C to evaporate to a paste with no obvious taste of organic solvent to obtain a n-butanol extract, which is stored in a refrigerator at 4 ° C for standby use; the remaining aqueous phase is concentrated under reduced pressure to remove the organic solvent, and the obtained aqueous phase is the crude extract A of the Buddleja flower.

[0075] The target product separated from Buddleja buddleja in this example is a verbascoside compound. After high performance liquid chromatography testing, the product purity reached 95.16%.

[0076] Comparative Example 4

[0077] The present invention is different from Example 1 in that the D101 macroporous resin column is not pretreated.

[0078] The target monomer compound separated from Buddleja buddleja in this example is verbascoside compound. After high performance liquid chromatography test, the product purity reaches 98.07%.

[0079] The glycoside compound isolated from Buddleja officinalis in Example 1 was subjected to anti-tumor test

[0080] 1. Experimental Methods

[0081] 1. Study on the inhibitory effect of verbascoside monomer compounds on tumor cell proliferation

[0082] (1) Preparation of drugs

[0083] Verbascoside was dissolved in DMSO and uniformly prepared into a stock solution with an initial concentration of 25 mM, which could be prepared into corresponding concentrations according to experimental needs later.

[0084] (2) Plating of tumor cells

[0085] After the grown cells are digested, centrifuge to obtain a cell pellet, add 2 mL of DMEM / RPMI-1640 complete medium, pipette and mix thoroughly, accurately draw 10 μL of cell suspension with a pipette and count on a cell counter. According to the result, the cell concentration is adjusted to 1×10 5 Cell / well, inoculate the prepared cell suspension into 96-well plates, 100 μL per well. When plating cells, ensure that the number of cells in each well is uniform to avoid experimental errors. Observe after 24 hours of culture.

[14] .

[0086] (3) Grouping and drug administration

[0087] Blank control group: cell culture medium

[0088] Negative control group: DMSO + cell solution + cell culture medium

[0089] 0 drug-dosing group: cell solution + cell culture medium

[0090] Experimental group: drug (verbascoside) + cell solution + cell culture medium

[0091] (4) Administration

[0092] Verbascoside was administered to MCF-7, BEL-7404, and SGC-7901 tumor cells at concentrations of 100, 200, 400, 600, and 800 μM. The culture medium in a confluent 96-well plate was discarded, and 100 μL of the drug culture medium at different concentrations was added sequentially. Six replicates were set up for each concentration, and the plates were cultured in a cell incubator. Dosing was repeated for 24, 48, and 72 hours. For longer cell culture times, PBS was added around the periphery of the 96-well plate to maintain moisture.

[0093] (5) Detection

[0094] After the drug administration is completed, the 96-well plate is transferred to the clean bench, and the liquid in the wells is aspirated with a pipette. Pay attention to the replacement of the pipette tip to prevent cross contamination. 10 μL of CCK-8 liquid is added to each well. Avoid the generation of bubbles during the addition process. Continue to incubate in the cell culture incubator for 0.5 h, and then detect with an enzyme marker.

[0095] Cell inhibition rate = 1-[OD(0 drug addition)-OD(drug addition)] / [OD(0 drug addition)-OD blank)]×100%

[0096] 2. Study on the pro-apoptotic effect of verbascoside monomer compounds on tumor cells

[0097] The cultured tumor cells were digested and transferred to a six-well plate. 2 mL of the mixed cell suspension was added to each well of the plate, and the cells were then cultured in a cell culture incubator. The cells were observed under a microscope and administered after they had adhered and grown to fill the entire field of view. The concentration of verbascoside administered to MCF-7, BEL-7404, and SGC-7901 was 400 μM. After administration, the cells were placed in an incubator and cultured for an additional 48 hours. Cell apoptosis was assessed by flow cytometry. Flow Jo V10 software was used to analyze and calculate the cell apoptosis rate.

[0098] 2. Experimental results of the effect of Buddleja verbascoside on tumor cells

[0099] 1. Proliferation inhibition

[0100] From Table 1 and Figure 7 Verbascoside has a certain inhibitory effect on three tumor cells: MCF-7, BEL-7404, and SGC-7901. The survival rate of tumor cells decreases with the increase of drug concentration. Verbascoside has the most obvious inhibitory effect on SGC-7901 tumor cells.

[0101] Table 1 IC values ​​of verbascoside monomer compounds on tumor cells 50 Value (unit: μM)

[0102]

[0103] 2. Pro-apoptotic effect

[0104] from Figures 8-10 It can be seen that the apoptosis rate of BEL-7404 tumor cells promoted by the verbascoside monomer compound is 1.97%; the apoptosis rate of SGC-7901 tumor cells is 5.39%; and the apoptosis rate of MCF-7 tumor cells is 2.94%.

[0105] Note: Figure 8 The first row in the middle shows, from left to right: blank control group, 0.1% DMSO group, and 400 μM verbascoside. Figure 9 The first row in the middle, from left to right, includes: blank control group, 0.1% DMSO group, and 400 μM verbascoside. Figure 10 The first row in the middle, from left to right, includes: blank control group, 0.1% DMSO group, and 400 μM verbascoside.

[0106] The experimental results show that the monomer compounds of verbascoside can inhibit the proliferation of tumor cells and promote the apoptosis of tumor cells. Therefore, the monomer compounds of verbascoside have good anti-tumor effects and are of great value for the development of tumor treatment drugs.

[0107] The above description is a further detailed description of the present invention in conjunction with specific / preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. Those skilled in the art of the present invention may make various substitutions or modifications to the described embodiments without departing from the scope of the present invention, and such substitutions or modifications should be considered to fall within the scope of protection of the present invention.

Claims

1. A method for extracting effective active ingredients from Buddleja flos, characterized in that: The steps include: S1: After crushing the Buddleja flos, ethanol was added to carry out heating and reflux extraction, and the extraction was repeated 3 times. The filtrate was combined, and the Buddleja flos suspension was filtered with a filter cloth to remove the residue. The obtained filtrate was heated and evaporated by a rotary evaporator. After the rotary evaporation of the filtrate was completed, it was further evaporated to dryness in a water bath until the filtrate was in a non-flowing paste to obtain the Buddleja flos alcohol extract; S2: The alcohol extract of Buddleja chinensis was added to sterile water to prepare a suspension, petroleum ether and the suspension were thoroughly mixed, and the mixture was allowed to stand at room temperature until obvious stratification occurred. The upper petroleum ether suspension was rotary evaporated and heated in a water bath to evaporate to a paste without obvious organic solvent taste, thereby obtaining a petroleum ether extract, which was stored in a refrigerator at 4°C for later use; the remaining aqueous layer was extracted with ethyl acetate and n-butanol in accordance with the above extraction method to obtain an ethyl acetate extract and an n-butanol extract, respectively. The remaining aqueous phase was concentrated under reduced pressure to remove the organic solvent, and the obtained aqueous phase was the crude extract A of Buddleja chinensis; S3: The crude extract A of Buddleja buddleja is purified by D101 macroporous resin column, and the collected fractions are concentrated, freeze-dried, and dried under reduced pressure to obtain the target product of Buddleja buddleja. The target product is then subjected to structural identification to finally determine the structure of the target product; The target product was structurally identified as a verbascoside compound; The mass ratio of the Buddleja buddleja alcohol extract and sterile water in step S2 is 1:1-3. During rotary evaporation and water bath heating, the temperature of petroleum ether is 60-90°C, the temperature of ethyl acetate is set to 70°C, and the temperature of n-butanol and water is set to 90°C; The D101 macroporous resin column purification process uses a gradient eluent of water and ethanol; The structure identification of the target product is to use infrared, hydrogen spectrum, carbon spectrum, mass spectrum and ultraviolet combined detection to identify the structure of the target product.

2. The method for extracting the effective active ingredient of Buddleja flos according to claim 1, wherein: The volume ratios of water and ethanol in the gradient elution were 100:0, 80:20, 60:40 and 40:60, respectively.

3. The method for extracting the effective active ingredient of Buddleja flos according to claim 1, wherein: The D101 macroporous resin is also pretreated, and the specific pretreatment steps are as follows: first, the D101 macroporous resin is soaked in triethanolamine for 24 hours, then it is loaded into the chromatography column using the wet column packing method, and 95% ethanol solution is passed through the resin layer at a flow rate of 2BV / h until the effluent is no longer white and turbid when added with distilled water, and then the ethanol is washed away with distilled water at the same flow rate, and then a mixed solution of oxalic acid and sodium persulfate is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and then rinsed with distilled water at the same flow rate until the pH value is neutral, and then 2BV of 2% NaOH solution is passed through the resin layer at a flow rate of 5BV / h and soaked for 3 hours, and then rinsed with distilled water at the same flow rate until the pH value is neutral.

4. The method for extracting the effective active ingredients of Buddleja flos according to claim 3, wherein: The mass ratio of the oxalic acid to the sodium persulfate is 4:1, and the mass concentration of the mixed solution is 3-5%.

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