CN112851621A - Total iridoid extract of caulis et folium piperis, extraction and purification method and application thereof - Google Patents
Total iridoid extract of caulis et folium piperis, extraction and purification method and application thereof Download PDFInfo
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- CN112851621A CN112851621A CN202110116605.7A CN202110116605A CN112851621A CN 112851621 A CN112851621 A CN 112851621A CN 202110116605 A CN202110116605 A CN 202110116605A CN 112851621 A CN112851621 A CN 112851621A
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- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
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- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- PCMORTLOPMLEFB-UHFFFAOYSA-N sinapinic acid Natural products COC1=CC(C=CC(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-UHFFFAOYSA-N 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- ABRULANJVVJLFI-DGHBBABESA-N swertisin Chemical compound COC1=CC=2OC(C=3C=CC(O)=CC=3)=CC(=O)C=2C(O)=C1[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O ABRULANJVVJLFI-DGHBBABESA-N 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- SGEWCQFRYRRZDC-VPRICQMDSA-N vitexin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(O)C2=C1OC(C=1C=CC(O)=CC=1)=CC2=O SGEWCQFRYRRZDC-VPRICQMDSA-N 0.000 description 1
- PZKISQRTNNHUGF-UHFFFAOYSA-N vitexine Natural products OC1C(O)C(O)C(CO)OC1OC1=C(O)C=C(O)C2=C1OC(C=1C=CC(O)=CC=1)=CC2=O PZKISQRTNNHUGF-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明提供了一种牛白藤总环烯醚萜提取物、提取纯化方法及其应用，所述提取纯化方法依次包括如下步骤：将牛白藤加入乙醇，然后加入壳聚糖，调节pH值为5.8‑6.5，提取0.5‑8小时，过滤，得到提取液和滤渣，再将滤渣加热回流提取，反复2‑4次，合并提取液，浓缩，得干膏提取物；然后将干膏提取物净化、提纯，得到牛白藤总环烯醚萜提取物。该提取方法提取率高，纯化效果好，并且得到的牛白藤总环烯醚萜提取物具有抗食管癌的作用。
The present invention provides a total iridoid extract of Boswellia officinalis, an extraction and purification method and an application thereof. The extraction and purification method sequentially includes the following steps: adding ethanol and then adding chitosan to adjust the pH value to 5.8 ‑6.5, extract for 0.5‑8 hours, filter to obtain the extract and filter residue, then heat and reflux the filter residue for extraction, repeat 2‑4 times, combine the extracts, and concentrate to obtain a dry paste extract; then purify the dry paste extract, Purified to obtain the total iridoid extract of Boswellia chinensis. The extraction method has the advantages of high extraction rate and good purification effect, and the obtained total iridoid extract of the bovine white vine has anti-esophageal cancer effect.
Description
Technical Field
The invention relates to an active component extracted from traditional Chinese medicine, a preparation method and application thereof, in particular to a total iridoid extract with an anti-tumor effect and taking a caulis sinomenii as a raw material, an extraction and purification method and application thereof.
Background
The iridoid compound is a large class of natural products and comprises nearly thousands of compounds, wherein more than 90 percent of the compounds are iridoid glycosides. The structural parent nucleus can be divided into cyclopentane iridoid and secoiridoid according to whether the structural parent nucleus has complete cyclopentane structural units, wherein the cyclopentane iridoid comprises jasminoidin, loganin, cantaxanthin, aucubin, and spicate vitexin, and the secoiridoid comprises swertiamarin, gentiopicroside, swertisin, picroside, and morroniside.
The traditional Chinese medicine composition can be widely stored in various traditional Chinese medicinal materials, such as gardenia, picrorhiza rhizome, eucommia bark, carpet bugle, honeysuckle and the like, and is one of the effective components of a plurality of common Chinese patent medicines, such as gardenia golden flower pills, honeysuckle antipyretic capsules, lung-heat clearing and fire suppressing tablets, nine-ingredient swertia herb pills, nine-ingredient dispersion and the like.
The common Chinese medicine of the bovine white vine also contains iridoid compounds, the bovine white vine refers to dried stem leaves of the bovine white vine of the genus Erysia of the family Rubiaceae, also called hairy fevervine herb, sweet tea, sparerib and the like, and is mainly produced in areas of Guangdong, Guangxi, Yunnan, Guizhou, Fujian, Taiwan and the like.
Iridoid compounds have various biological activities such as anti-tumor and anti-inflammatory activities, have great research value, and are receiving more and more attention in recent years.
The iridoid compound is extracted and separated by a solvent method, and the solvent is usually water, methanol, ethanol, dilute acetone, ethyl acetate and the like.
In the prior art, the extraction and separation method of the iridoid compound has low extraction rate and poor purification effect.
Disclosure of Invention
The invention mainly aims to overcome the defects of the prior art and provides a caulis fici Pumilae total iridoid extract, an extraction and purification method and application thereof. The method has high extraction rate and good purification effect of the caulis et folium fici Tikouae total iridoid extract.
In order to achieve the above objects, in a first aspect, the present invention provides a method for extracting and purifying a total iridoid extract of fevervine, which sequentially comprises the following steps:
(1) adding ethanol into caulis et folium Hedyotis Hedyotideae, adding chitosan, adjusting pH to 5.8-6.5, extracting for 0.5-8 hr, filtering to obtain extractive solution and residue, heating and reflux-extracting the residue for 2-4 times, mixing extractive solutions, and concentrating to obtain dry extract.
Preferably, the caulis sinomenii is caulis sinomenii powder.
Preferably, the concentration of the ethanol in percentage by weight is 55-65%, and the weight ratio of the caulis sinomenii powder to the ethanol is 1 (8-10).
Preferably, the weight ratio of the caulis Sinomenii to the chitosan is (10-20):1, and the chitosan can release iridoid compounds in the caulis Sinomenii better in the extraction process, thereby improving the yield.
During the extraction process, the chitosan can promote the release of the iridoid compounds and can adsorb part of the iridoid compounds, if the dosage of the chitosan is too high, the adsorption effect of the chitosan exceeds the release promotion effect in the extraction process, and the yield of the total iridoid extract in the extract after filtration is reduced; if the consumption of the fructan is too small, the corresponding effect cannot be achieved.
Preferably, ultrasonic extraction is used, and the extraction time is 0.5-0.6 hours.
Preferably, hydrochloric acid is used for adjusting the pH value.
In the extraction method of the total iridoid extract of the solanum lyratum thunb, if the pH value is lower than 5.8 or higher than 6.5, the yield is reduced and is equivalent to the yield of the traditional extraction method.
Preferably, the method for extracting and purifying the total iridoid extract of the caulis sinomenii further comprises the following steps:
(2) dissolving the dry extract prepared in the step (1) with hot water, filtering to prepare a crude extract of the iridoid of the bauhinia championii, purifying by adopting an extraction method, and adopting a solid-phase extraction column during extraction.
Wherein the temperature of the hot water is 50-60 ℃, the temperature is too low to be completely dissolved, the temperature is too high, chemical reaction is easy to occur, and the quality of the product is influenced.
Wherein the weight ratio of the dry extract to hot water is 0.1-0.4: 5.
The solid phase extraction column comprises A, B, C components from top to bottom, and isolation plates are arranged between each layer.
Preferably, A, B, C is 1 (0.8-1.2) to (0.8-1.2) by volume ratio.
Preferably, the component A of the filler is neutral Al2O3200-300 mesh, B component graphitized carbon black, 120-400 mesh, C component C18 alkyl filler bonded by silica gel as a matrix, the particle size is 40-63 μm, the average pore diameter is 60A, the carbon content is 16-18%, and C component end sealing.
Experiments prove that the C8 alkyl filler bonded by taking silica gel as a matrix has overlarge polarity, is not suitable for purifying the caulis sinomenii total iridoid extract, and has overlow yield; the phenyl filler bonded by taking silica gel as a matrix has too low polarity, is not suitable for purifying the caulis sinomenii total iridoid extract, and has too low yield.
The filling diameter and height ratio of the above fillers is 1: (2-3) column, prepared as a solid phase extraction column, preferably a PVC column; the method has the function of removing pigments, acidic interference impurities, sugar and fat-soluble impurities in the caulis spatholobi without adsorbing iridoid components.
The extraction method comprises the following steps: activating a solid phase extraction column by using methanol with 2-6 times of column volume and water with 2-6 times of column volume respectively, taking the crude extract of the caulis sinomenii iridoid with 2-3 times of column volume, passing the crude extract through the solid phase extraction column, drying the column by blowing, and collecting filtrate to obtain the purified crude extract of the total iridoid of the caulis sinomenii.
The method for extracting and purifying the total iridoid extract of the caulis sinomenii further comprises the following steps:
(3) enriching and purifying total cyclic vinyl ether terpenoid:
and (3) respectively passing the purified crude extract of the total iridoid extract of the solanum lyratum thunb obtained in the step (2) through a polyamide resin adsorption column and a macroporous resin adsorption column, washing with water, then washing with 30-40% ethanol, and collecting ethanol eluent.
Preferably, the filling method of the polyamide resin adsorption column is as follows: the purified polyamide (200-300 mesh, acid-washed, alkali-washed and then water-washed to neutrality) is taken to fill the column, and the length-diameter ratio (1-2) of the resin is 1.
Preferably, the filling method of the macroporous resin adsorption column comprises the following steps: and (3) filling the purified macroporous resin AMBERLITE XDA-4 (acid washing, alkali washing and water washing to neutrality) into a column to ensure that the length-diameter ratio of the resin is 4-5: 1.
The polyamide resin adsorption column and the macroporous resin adsorption column are connected in series to obtain a series column, and a liquid drainage and liquid adding device is arranged between the two columns.
Taking iridoid compound extract with the same volume as the serial column, passing through the serial column, washing with water, washing with 30-40% ethanol 4-8 times the volume of the serial column, and collecting ethanol eluate; and (3) concentrating the obtained ethanol eluent to dryness to obtain the total iridoid extract of the solanum lyratum thunb.
In a second aspect, the invention also provides a caulis spatholobi total iridoid extract obtained by the preparation method of the caulis spatholobi total iridoid extract, which comprises chloranthus glaucoside, asperulosidic acid, deacetyl asperulosidic acid, paederosidic acid, asperuloside and hydroxychloroglaucoside.
The specific structural formula is as follows:
in a third aspect, the invention also provides application of the total iridoid extract of the solanum lyratum thunb in the prevention of esophageal cancer.
The invention has the following beneficial effects:
the method for extracting the caulis sinomenii total iridoid extract has high extraction rate and good purification effect, and the prepared caulis sinomenii total iridoid extract can be used for resisting esophageal cancer.
Drawings
FIG. 1 shows that the total iridoid extract of caulis et folium piperis prepared in example 1 acts on EC109, KYSE/140, KYSE410 and KYSE510 esophageal cancer cells in logarithmic phase, and CCK8 reagent is used for detecting the proliferation activity of esophageal cancer cells before and after drug intervention.
Detailed Description
The technical solutions in the embodiments of the present invention are described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the examples, the chitosan was produced by the Winhasi marine bioproduct Co., Ltd, the A, B, C filler components were all produced by the Aladdin reagent (Shanghai) Co., Ltd, and the polyamide resin and AMBERLITE XDA-4 were produced by the Aladdin reagent (Shanghai) Co., Ltd and the Shanghai spectral vibration Biotechnology Co., Ltd, respectively.
Example 1
Extracting and purifying iridoid compounds in the bovine caulis Sinomenii:
(1) extraction of iridoid compound
Taking 100g of caulis sinomenii powder, adding 55% ethanol according to the material-liquid ratio of 1:8, adding 10g of chitosan and pH 6, heating, boiling, refluxing and extracting for 6 hours, filtering to obtain an extracting solution and filter residues, then heating, refluxing and extracting the filter residues for 3 times, combining the extracting solutions, and concentrating to be nearly dry to obtain a dry extract.
(2) Purification of crude extract of iridoid from rattan
Taking 1g of the dry extract prepared in the step (1), dissolving with 25ml of hot water at 55-60 ℃, filtering to prepare a crude extract of the iridoid of the bauhinia variegata, and purifying by adopting a solid phase extraction method; the extraction method and conditions are as follows:
a) 3g of filler of the solid-phase extraction column, wherein the filler is formed by sequentially and hierarchically filling A, B, C components from A to C from top to bottom according to the ratio of 1:1:1, and has the function of removing pigments, acidic interference impurities, sugar and fat-soluble impurities in the solanum lyratum thunb without adsorbing iridoid components;
the component A of the filler is neutral Al2O3200 meshes to 300 meshes, the component B is graphitized carbon black, 120 meshes to 400 meshes, the component C is C18 filler bonded by taking silica gel as a matrix, the particle size is 40-63 mu m, the average pore diameter is 60A, the carbon content is 17 percent, and the tail is sealed; filling the above fillers into a PVC column with diameter of 1.0cm and height of 2cm to prepare a solid phase extraction column;
b) and (3) an elution mode: activating a solid phase extraction column with 5ml of methanol and 5ml of water respectively, taking 25ml of crude extract of the iridoid of the bovine stephania, passing through the solid phase extraction column, eluting with 40% ethanol, drying the column, and collecting filtrate to obtain purified extract of the iridoid.
(3) Enriching and purifying total cyclic vinyl ether terpenoid:
column 1: the purified polyamide (200-300 mesh, acid-washed, alkali-washed, and then water-washed to neutrality) was taken and 40ml of the column was packed so that the aspect ratio of the resin was 1: 1.
Column 2: 40ml of purified macroporous resin AMBERLITE XDA-4 (acid-washed, alkali-washed, and then water-washed to neutrality) was packed in a column to give a resin having a length-to-diameter ratio of 4: 1.
The column 1 and the column 2 are connected in series to form a funnel shape, and a liquid drainage and liquid feeding device is arranged between the two columns. Thus, column 1 is of larger diameter, a thick short column relative to column 2; the post 2 is of a smaller diameter and is an elongated post relative to the post 1. The thick short column (column 1, thinner filler, 200-300 meshes) can reduce the resistance while realizing the adsorption separation; the slender column (column 2, the filler is thicker, 10-20 meshes) can realize effective adsorption and enrichment and is not easy to block resin pores, particularly after passing through the column 1. The clever combination effectively improves the purification effect and efficiency.
And (3) taking 30ml of the iridoid compound extract purified in the step (2), passing through a series column of a column 1 and a column 2, washing with water, washing with 250ml of 40% ethanol for 3 times, and collecting ethanol eluent to obtain the purified total iridoid compound extract.
(4) Preparation of extract of total iridoid of caulis et folium fici Tikouae
And (4) concentrating the purified total iridoid extract obtained in the step (3) to dryness to obtain the compound.
(5) Identification of iridoid extract components of caulis et folium fici Tikouae
The total iridoid extract is identified by HPLC-MS method, and comprises 6 iridoid components such as monotropenoid glycoside, asperulosidic acid, deacetylasperulosidic acid, paederosidic acid, asperuloside and hydroxymonotropenoid glycoside.
(6) Total iridoid content determination
Precisely weighing about 0.05g of total iridoid extract, placing in a 100mL conical flask, adding 10mL of 60% ethanol, weighing, ultrasonically treating for 60min, shaking, filtering to obtain test solution, taking 5mg of gentiopicroside, diluting to 10mL with 60% ethanol to constant volume, and making into reference solution. The ultraviolet absorption values of the reference substance and the test substance are respectively measured, the concentration of the total iridoid can be known through the ratio of the absorption values of the reference substance and the test substance, and the results of the two measurements are shown in Table 1.
TABLE 1 Total iridoid extract content determination
Comparative example 1
Compared with the traditional extraction method:
according to the embodiment 1, 100g of the caulis sinomenii powder is taken, ethanol with the concentration of 65% is added according to the material-liquid ratio of 1:8, the hot reflux extraction, the ultrasonic extraction method and the supercritical fluid extraction are respectively adopted, finally, equal volume of water is added, the mixture is kept stand and centrifuged to remove the sediment, and the mixture is concentrated to be dry to obtain dry extract, wherein the extraction rate (%) is the percentage of the dry extract to the powder. The results are shown in Table 2-1:
TABLE 2-1 comparison of the quality of extracts obtained by different extraction methods
Compared with the traditional extraction method such as hot reflux extraction, an ultrasonic extraction method, supercritical fluid extraction and the like, the method adopted by the invention is simple and convenient, has higher extraction efficiency, and particularly, the chitosan is added, so that the iridoid compounds in the caulis sinomenii are better released while impurities are adsorbed.
Adjusting pH to 5.8-6.5 to increase solubility of iridoid such as gentiopicroside in extractive solution and greatly improve extraction efficiency (see Table 2-2), adjusting pH to 7 and keeping the pH unchanged in example 1, or adjusting pH to 5 and keeping the pH unchanged in example 1, and comparing the extraction rate in step (1) with the mass of dry extract accounting for the mass of caulis et folium fici Tikouae powder as extraction rate (%).
TABLE 2-2 comparison of extract quality at different pH values
Compared with the traditional purification method, the purification method of the crude extract of the iridoid of the rattan comprises the following steps:
1g of the dry extract prepared in the step (1) in the example 1 is taken, dissolved in 25ml of hot water with the temperature of 55-60 ℃, filtered to prepare a crude extract of the iridoid of the bauhinia vines, and purified by a solid phase extraction method. Then concentrating to dry weight, measuring the content of the total rattan iridoid in the purified dry extract, simultaneously carrying out a standard experiment, and simultaneously evaluating the purification efficiency by using the content and the recovery rate, wherein the higher the content after purification is, the higher the recovery rate is, which indicates that the purification efficiency is higher, the packing volume and the column of each filler in the table 3 are consistent with the example 1 when the filler is used singly, and the packing volume and the column of A, B, C are consistent with the example 1 when the three components are combined according to the ratio of 1:1: 1.
TABLE 3 comparison of purification efficiency of different purification methods
Wherein the component A of the filler is neutral Al2O3200-300 meshes, the component B is graphitized carbon black, 120-400 meshes, the component C is C18 filler bonded by taking silica gel as a matrix, the particle diameter is 40-63 mu m, the average pore diameter is 60A, and the carbon content is 17%. The extract of nigella sativa contains more sinapic acid, which can be bound to gentiopicroside compounds, etc. via hydrogen bonds, where a: al (Al)2O3Can specifically adsorb erucic acid in the extractive solution, free gentiopicroside, and improve purification effect. The graphitized carbon black can absorb pigment and fatty acid, and the acidic substances are reduced and the purification effect of the graphitized carbon black is improved through the eluent which is used for removing erucic acid and is obtained by the step A. And (3) passing the eluent subjected to the treatment A and the treatment B through a reaction system C: when the C18 is filled, dead adsorption of C18 can be reduced, and the enrichment and purification effects of C18 are further improved.
The comparison result shows that the ABC combined purification method has the highest content measurement result which is far higher than other methods, and the recovery rate is high. Therefore, the ABC combined purification method has the best purification effect.
The purification method comparison of the crude extract of the iridoid of the rattan:
30ml of iridoid compound extract purified in the step (2) of the example 1 is taken, and the purification efficiency is evaluated by using the content and the recovery rate of the total iridoid in the purified and purified dry extract as compared with a macroporous resin method, a polyamide method and a combined method disclosed by the invention reported in a comparative document. The higher the purified content, the higher the recovery rate, indicating the higher the purification efficiency, the column volume was the same as in example 1 for polyamide and macroporous resin alone, and the column volume was the same as in column 1 and column 2 of example 1 for polyamide and macroporous resin in combination. The results are shown in Table 4.
TABLE 4 comparison of purification efficiency of different purification methods
The caulis Sinomenii contains various iridoid chemical components, and the content and purity of the obtained product are different by different extraction and purification (including purification) methods. The same extraction method is used for different extracts, and the results are different. The experimental results of the extraction and purification (including purification) of iridoid contained in the caulis sinomenii show that the XDA-4 has better effect in the macroporous resin, and the combination of the polyamide and the macroporous resin obviously improves the purification efficiency compared with the single use of the polyamide or the macroporous resin.
The method adopts a tandem chromatography column method for purifying the terpene components of the total iridoid in the caulis sinomenii for the first time. Compared with the method of passing through a column in a fractional manner (firstly passing through polyamide and then passing through macroporous resin), the method of the series chromatography column method (polyamide and macroporous resin) has the advantages of greatly improving the recovery rate (taking gentiopicroside as a labeling experiment) and being more convenient to operate because of reducing the experimental steps and reducing the used solvent amount. Proved by a tandem chromatography method of polyamide and macroporous resin column XDA-4, the effect is best.
Example 2
The extraction and purification method of example 2 is different from example 1 in that ultrasonic waves are used for heating reflux in step (1) and the reflux extraction time is 0.5 hours, and the method is otherwise the same as example 1.
Example 3
The extraction and purification method of example 3 is different from example 1 in that the amount of the caulis sinomenii powder used in step (1) is 120g, and the other steps are the same as example 1.
Example 4
The extraction and purification method of example 4 is different from example 1 in that the amount of chitosan used in step (1) is 8g, and the method is otherwise the same as example 1.
Example 5
The extraction and purification method of example 5 is different from example 1 in that pH in step (1) is 6.2, and the method is otherwise the same as example 1.
Comparative example 2
The extraction and purification method of comparative example 2 is different from that of example 1 in that chitosan is not added in step (1), and the other steps are the same as those of example 1.
Comparative example 3
The extraction and purification method of comparative example 3 is different from example 1 in that the amount of chitosan added in step (1) is 25g, and the other steps are the same as example 1.
Comparative example 4
The preparation method of comparative example 4 is different from example 1 in that the raw material in step (1) is clove leaf powder instead of bovine fevervine root powder, and the other steps are the same as example 1.
The results of the measurements of the examples and comparative examples after the final extraction and purification are shown in Table 5.
TABLE 5 Total iridoid extract content and extraction yield
As can be seen from comparative examples 2-3, the total iridoid extract obtained by the method is greatly reduced in purity and extraction rate without adding chitosan or with too large amount; as can be seen from comparative example 4, the extraction method of the invention is suitable for the extraction and purification of the total iridoid extract of the caulis sinomenii.
Example 1 application of total iridoid extracts of solanum lyratum thunb to esophageal cancer resistance:
the invention adopts a pharmacological molecular biology method and evaluates the anti-esophageal cancer pharmacological action of the caulis sinomenii total cycloolefine ether terpene extract on esophageal cancer through a cell in vitro experiment. 4 kinds of esophageal cancer cells are taken as research objects, the activity change of tumor cells before and after the intervention of the total iridoid extract of the caulis sinomenii is detected by using a CCK-8(Cell Counting Kit-8) cytotoxicity activity detection reagent, and the influence of the drug intervention on the migration of the tumor cells is examined by using a Cell scratching experiment. Evaluating the killing effect of the medicament on tumor cells, and researching the influence of the total iridoid extract of the bovine solanum lyratum on the activity and migration of the tumor cells.
1. CCK8 is used for detecting and researching the influence of the total iridoid extract of the caulis sinomenii on the survival rate of esophageal cancer cells.
The bovine caulis Sinomenii total cyclonene ether terpenoid extract diluted by the culture medium into different drug concentrations acts on EC109, KYSE/140, KYSE410 and KYSE510 esophageal cancer cells in logarithmic phase, and CCK8 reagent is adopted to detect the proliferation activity of the esophageal cancer cells before and after drug intervention (see figure 1 specifically). The relative cell survival rate and the relative cell inhibition rate before and after intervention of the bovine caulis Sinomenii on esophageal cancer cells are measured by taking the same background drug and no cell as a blank group, taking non-administered tumor cells as a control group and taking 100 mug/mL cis-platinum and 100 mug/mL 5-fluorouracil as positive control groups respectively, and the Half inhibition Concentration (IC 50) is calculated (see Table 6 specifically). The experimental result shows that in the esophageal cancer cells, the caulis sinomenii with the concentration of more than 50 mug/mL has obvious inhibition effect on the esophageal cancer cells. The half inhibition concentration IC50 of the bovine rattan iridoid extract on esophageal cancer cells is between 32.81 and 104.9 mu g/mL. Under the action of effective medicine concentration of IC50, the caulis Sinomenii iridoid extract can remarkably reduce the relative survival rate of esophageal cancer cells, and shows certain dose dependence.
TABLE 6 Semithragma niveum total iridoid extract half inhibitory concentration IC50 on esophageal cancer cells
2. The scratch cell is adopted to research the influence of the caulis sinomenii iridoid extract on the movement of esophageal cancer cells:
after the bovine rattan with IC50 acted on the serum-free cultured tumor cells, the healing conditions of the scratch at different time points were detected, the scratch area was calculated, and the healing rate of the scratch was measured. The experimental result is as follows: after the medicine of IC50 intervenes in esophageal cancer cells, as the action time is prolonged, the tumor cells of the blank control group gradually move to the scratch, the scratch is gradually healed, while the tumor cells of the administration group have slower moving speed, the cell morphology changes at the later stage, and part of the cells are suspended and dead. Compared with the control group, the healing capacity of the tumor cells is obviously inhibited and the cell migration capacity is weakened under the action of the effective drug concentration of IC50, and the drug can inhibit the activity and migration of the tumor cells. According to CCK8 cell activity experiments and scarification experiments, the total iridoid extract of the caulis sinomenii is supposed to realize the anti-tumor effect by inhibiting cell migration/promoting tumor cell death.
TABLE 7 esophageal cancer cell scratch healing Rate before and after administration of Total iridoid extract of Mucuna birdwoodiana
The first discovery shows that the total iridoid extract of the bovine solanum lyratum can inhibit the survival rate of tumor cells and inhibit the migration of the tumor cells, thereby realizing the effect of resisting esophageal cancer.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the present specification and directly/indirectly applied to other related technical fields within the spirit of the present invention are included in the scope of the present invention.
Claims (10)
1. A method for extracting and purifying a caulis et folium piperis total iridoid extract is characterized by sequentially comprising the following steps (1): adding ethanol into caulis et folium Hedyotis Hedyotideae, adding chitosan, adjusting pH to 5.8-6.5, extracting for 0.5-8 hr, filtering to obtain extractive solution and residue, and concentrating the extractive solution to obtain dry extract.
2. The method for extracting and purifying the caulis et folium piperis nivei total iridoid extract according to claim 1, wherein the concentration of the ethanol in percentage by weight is 55-65%, the weight ratio of the caulis et folium piperis nivei to the ethanol is 1 (8-10), and the weight ratio of the caulis et folium piperis nivei to the chitosan is (10-20): 1.
3. The method for extracting and purifying the extract of caulis et folium piperis niveae total iridoid as claimed in claim 1, wherein the ultrasonic extraction is adopted for 0.5-0.6 hours.
4. The method for extracting and purifying the extract of caulis spatholobi total iridoid as claimed in claim 1, further comprising the following step (2): dissolving the dry extract prepared in the step (1) with hot water, filtering to prepare a crude extract of the iridoid of the bauhinia championii, purifying by adopting an extraction method, and adopting a solid-phase extraction column during extraction;
wherein the temperature of the hot water is 50-60 ℃; the weight ratio of the dry extract to the hot water is (0.1-0.4) to 5;
the solid phase extraction column comprises A, B, C filler components from top to bottom, wherein A, B, C filler components are filled according to the volume ratio of 1 (0.8-1.2) to (0.8-1.2); the component of the filler A is neutral Al2O3200-300 meshes, wherein the component of the filler B is graphitized carbon black, 120-400 meshes, the component of the filler C is C18 alkyl filler bonded by taking silica gel as a matrix, the particle size is 40-63 mu m, the average pore diameter is 60A, the carbon content is 16-18%, and the component C seals the tail;
the extraction method comprises the following steps: activating a solid phase extraction column by using methanol with 2-6 times of column volume and water with 2-6 times of column volume respectively, taking the crude extract of the caulis sinomenii iridoid with 2-3 times of column volume, passing the crude extract of the caulis sinomenii iridoid through the solid phase extraction column, drying the column by blowing, and collecting filtrate to obtain the purified crude extract of the total iridoid of the caulis sinomenii.
5. The method for extracting and purifying the caulis spatholobi total iridoid extract according to claim 4, wherein the diameter and height ratio of the solid phase extraction column is 1: (2-3).
6. The method for extracting and purifying the extract of caulis et folium piperis niveae total iridoid as claimed in claim 4, further comprising the following step (3): and (3) respectively passing the purified crude extract of the total iridoid extract of the solanum lyratum thunb obtained in the step (2) through a polyamide resin adsorption column and a macroporous resin adsorption column, washing with water, then washing with 30-40% ethanol, and collecting ethanol eluent.
7. The method for extracting and purifying the caulis sinomenii total iridoid extract according to claim 6, wherein the filling method of the polyamide resin adsorption column is as follows: filling the purified polyamide into a column, wherein the length-diameter ratio (1-2) of the resin is 1;
the filling method of the macroporous resin adsorption column comprises the following steps: the column was packed with the purified macroporous resin AMBERLITE XDA-4, the aspect ratio of the resin (4-5) was 1.
8. The method for extracting and purifying the caulis Sinomenii total iridoid extract of claim 6, wherein the polyamide resin adsorption column and the macroporous resin adsorption column are connected in series.
9. The extract of total iridoid of Mucuna nivea prepared by the extraction and purification method according to any one of claims 1-8, which comprises monotropein, asperulosic acid, deacetylasperulosic acid, paederosidic acid, asperuloside and hydroxymonotropein.
10. The use of the extract of caulis et folium piperis longi total iridoid as claimed in claim 9 for the treatment of esophageal cancer.
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