CN113577123A

CN113577123A - Preparation method and application of dandelion extract

Info

Publication number: CN113577123A
Application number: CN202110966558.5A
Authority: CN
Inventors: 董自波; 王倩楠; 石锦峰; 王晗毓; 郑家勤; 李媛媛; 许锦涛
Original assignee: Jiangsu Ocean University
Current assignee: Jiangsu Ocean University
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2021-11-02

Abstract

The invention provides a preparation method of dandelion extract and its application in treating or preventing esophageal cancer. The preparation method comprises: (1) activating the dandelion powder by adding water and a cellulase solution, filtering out the decoction after decoction, and concentrating it into an extract; (2) diluting the extract obtained in step (1) with D101 macroporous The resin was purified by column concentration. The invention improves the extraction rate and purification efficiency of organic acids, the dandelion extract has obvious preventive and therapeutic effects on inflammatory mice, and has good ability to inhibit proliferation and cell migration for esophageal cancer cells.

Description

Preparation method and application of dandelion extract

Technical Field

The invention belongs to the field of traditional Chinese medicines, and particularly relates to application of dandelion and an extract thereof in preparation of an anti-esophagitis and esophageal cancer prevention and treatment medicine.

Background

Esophageal cancer is a malignant tumor with high mortality rate, has multiple and complex pathogenic factors, and is often related to dietary habits, obesity, gastroesophageal reflux disease and barrett's esophagus. Researches find that the esophagus is often accompanied with the transformation of inflammatory cells and cytokines and the activation of inflammatory pathways in chronic inflammation, so that the microenvironment suitable for tumor proliferation and metastasis is modified, and the esophagus plays an important role in the processes of inflammation-metaplasia-canceration. Therefore, the relationship between inflammation and the occurrence and development of esophageal cancer is clarified, and a new idea can be provided for the prevention and treatment of esophageal cancer.

The dandelion is used as a traditional Chinese medicine in China, is cheap and easy to obtain, mainly comprises flavonoids, sterols, triterpenes, phenolic acids, pigments, sesquiterpene lactones, coumarins and the like, has the effects of resisting inflammation, viruses and tumors, can inhibit cell proliferation and improve the microenvironment of inflammation, and most importantly can relieve the drug resistance of organisms to hormones and antibiotic drugs, improve the immunity of the organisms and improve the life quality of patients.

Chinese patent 202110279290.8 discloses a Chinese medicinal preparation for treating esophageal cancer inflammation, which comprises 5-15 g of selfheal, 5-15 g of radix scrophulariae, 3-10 g of cortex phellodendri, 5-15 g of honeysuckle, 5-15 g of fructus forsythiae, 5-15 g of dandelion, 0.5-1.5 g of Chinese honeylocust fruit, 0.5-2 g of borneol, 2-10 g of mint, 2-10 g of burdock, 2-10 g of subprostrate sophora, 2-10 g of stiff silkworm and 2-10 g of liquorice. The components are complex, and the treatment effect is not ideal.

Chinese patent 202110493457.0 discloses a Chinese medicinal preparation for treating chronic gastritis, which is prepared from the following raw materials in parts by weight: 320 portions of galangal 280-containing materials, 320 portions of trichosanthes 280-containing materials, 320 portions of atractylodes macrocephala 280-containing materials, 220 portions of curcuma zedoary 180-containing materials, 220 portions of salvia miltiorrhiza 180-containing materials, 80-120 portions of cuttlebone, 80-120 portions of rhubarb, 40-80 portions of nardostachyos root and rhizome, 80-120 portions of bitter orange, 40-80 portions of white paeony root, 40-80 portions of chicken's gizzard-membrane, 60-100 portions of dandelion, 40-80 portions of scutellaria baicalensis and 40-80 portions of platycodon grandiflorum; the Chinese medicinal preparation has effects of warming spleen for dispelling cold, nourishing yin, regulating stomach function, activating qi-flowing, and removing blood stasis. But their actual pharmacology is unknown.

However, the dandelion extract has complex and various components, and the relationship between the biological activity, dose-effect and structure-effect of each component is not clear yet, thus bringing obstruction to the research of the dandelion in the aspect of medicine.

Disclosure of Invention

In order to solve the problems, the invention provides a preparation method of a dandelion extract and application thereof in treating or preventing esophageal cancer.

In one aspect, the invention provides a method for preparing a dandelion extract.

The method comprises the following steps:

(1) activating herba Taraxaci powder with water and cellulase solution, decocting, filtering decoction, and concentrating into extract;

(2) diluting the extract obtained in the step (1), and then concentrating and purifying the extract by using a D101 macroporous resin column.

The dandelion powder in the step (1) can be any one or more dry powder of dandelion whole grass, dandelion root, dandelion leaves and dandelion flowers.

The ratio of the addition amount of water to the amount of dandelion powder in the step (1) is 1: 2-5 (volume/mass).

The dilution ratio of the extract in the step (2) is 1: diluting with 10 distilled water.

The ratio of the volume of the sample injection liquid (mL) to the mass of the macroporous resin (g) in the step (2) is 10-14, and preferably 12.

The pH value of the injection liquid in the step (2) is preferably 2.

In the step (2), the adsorption flow rate is 1-2BV/h, the concentration of the desorption liquid ethanol is 40-60%, the desorption flow rate is 1.5-3BV/h, and the consumption of the desorption liquid is 1-4BV when the D101 macroporous resin is used for column-passing concentration and purification.

Preferably, the adsorption flow rate is 1.5BV/h, the ethanol concentration of the desorption solution is 50%, the desorption flow rate is 2BV/h, and the consumption of the desorption solution is 3BV when the D101 macroporous resin is used for column chromatography concentration and purification in the step (2).

In another aspect, the present invention provides a dandelion extract.

The dandelion extract is prepared by the preparation method.

In another aspect, the invention provides an application of dandelion in preparing a medicament related to esophagitis or esophageal cancer.

The medicine includes but is not limited to the prevention and/or treatment and/or prognosis repair of esophagitis or esophageal cancer.

The dandelion is prepared into an extract by the method and then is used for preparing the medicine.

In another aspect, the invention provides an application of the dandelion extract in preparing a medicine related to esophagitis or esophageal cancer.

In yet another aspect, the present invention provides a medicament for the prevention and/or treatment and/or prognostic repair of oesophagitis or oesophageal cancer.

The medicine comprises the dandelion extract.

Preferably, the medicament is a liquid.

The concentration of the dandelion extract in the medicine is 2500-. The crude drug refers to a purified product.

The crude drug refers to unprocessed Chinese medicinal material, and 2500 μ g crude drug/mL indicates that each 1mL purified product is equivalent to 2500 μ g of the original medicinal material.

Dosage forms of the drug include, but are not limited to: decoction, pill, powder, unguent, pill, medicated liquor, syrup, extract, medicated tea, suppository, paste, cake, liniment, oil, granule, oral liquid, capsule, dripping pill, aerosol, enema, and injection.

The medicine also comprises other pharmaceutically acceptable carriers or excipients.

The invention has the beneficial effects that:

1. the method optimizes the extraction process of the dandelion organic acid, improves the extraction rate of the organic acid, and has convenient operation and high repeatability.

2. The macroporous adsorption resin can directly separate the extracting solution, so that the purification efficiency is improved, the resin can be repeatedly recycled, the utilization rate is increased, the cost is reduced, and the benefit maximization is realized.

3. The dandelion extract has obvious functions of preventing and treating inflammation mice, reducing the in-vivo inflammation of the mice, relieving the disease symptoms of the mice, well relieving the drug resistance of the mice to antibiotic drugs and improving the immunity of the mice. Has good capability of inhibiting proliferation and cell migration of esophageal cancer cells.

Drawings

FIG. 1 shows the comparison of the efficiency of water and ethanol extraction.

FIG. 2 is a comparison of the efficiency of extraction rate of different water extraction methods.

FIG. 3 is an HPLC chromatogram in which A is a mixed control and B is a D101 purified sample.

FIG. 4 is a chromatogram of a purified sample of HPD 600.

FIG. 5 is a graph showing the effect of different concentrations of dandelion on the quality of granuloma in rats.

FIG. 6 shows the effect of dandelion extract on reducing cell proliferation ability of KYSE 70.

FIG. 7 is a graph showing the results of the scratch test of the dandelion extract on KYSE70 cells.

FIG. 8 is a graph of Dandelion extract slowing the healing rate of KYSE70 cell scratch.

FIG. 9 is an HPLC chart of the content determination of chlorogenic acid, caffeic acid and chicoric acid before purification of the dandelion extract.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Example 1 preparation of aqueous Dandelion extract

The method comprises the following steps:

(1) weighing 5g of herba Taraxaci whole plant powder (sieved with 40 mesh sieve), placing in a round bottom flask, adding 15 times of water and 12.5mL of cellulase solution (with final concentration of 50U/mL, obtained from Shanghai Michelin Biochemical technology Co., Ltd., product number: C805042), activating at 50 deg.C for 30min, decocting in slightly boiling state for 1h, filtering off the decoction while it is hot, and collecting with a beaker. Standing at 4 deg.C for 24 hr, filtering, concentrating the filtrate with rotary evaporator to obtain extract with relative density of 1.1-1.15(50 deg.C), storing in 4 deg.C refrigerator, and performing three experiments in parallel.

(2) Determination of chlorogenic acid content in dandelion water extract

Preparation of control solutions: precisely weighing 6.493mg of chlorogenic acid reference substance, placing in a 100mL volumetric flask, adding ultrapure water to constant volume to scale, and obtaining a reference substance solution.

Preparation of a test solution: precisely weighing 99.342mg of herba Taraxaci extract, placing in a 100mL volumetric flask, adding appropriate amount of ultrapure water, performing ultrasonic treatment for 30min, fixing volume to scale, precisely sucking 1mL from the flask, and fixing volume with ultrapure water to 50mL volumetric flask.

Drawing a standard curve: precisely measuring 0.2, 0.4, 0.6, 0.8 and 1.0mL of chlorogenic acid reference substance (0.0649mg/mL) in a 10mL volumetric flask, and adding water to a constant volume to obtain a scale. The reference substance and the test solution are scanned by an ultraviolet spectrophotometer at full wavelength, and have maximum absorption at 323nm, so the absorbance of the reference solution is measured at 323nm respectively, the regression equation is obtained, Y is 0.1035x +0.0151, and the correlation coefficient r is 0.9991.

Determination of chlorogenic acid content: taking 0.5mL of the dandelion extract test solution in a 10mL volumetric flask, adding water to a constant volume to a scale, measuring the absorbance at 323nm, calculating the content of chlorogenic acid in the test solution according to a regression equation, and calculating the average extraction rate of chlorogenic acid in the dandelion extract according to the content of chlorogenic acid in the test solution to be 1.084%.

Example 2 purification of Dandelion extract

1) D101 macroporous resin purification of dandelion extract

The dandelion extract was extracted under the conditions of example 1 in a ratio of 1: diluting with 10 distilled water, passing through a column in D101 macroporous resin, wherein the ratio of sample injection liquid volume (mL) to macroporous resin mass (g) is 12, pH is 2, adsorption flow rate is 1.5BV/h, desorption liquid ethanol concentration is 50%, desorption flow rate is 2BV/h, and desorption liquid dosage is 3 BV. Collecting the separated liquid with a conical flask, and concentrating under reduced pressure for use.

The contents of the three-component organic acids before and after purification were compared by HPLC. The three organic acids content before purification was 71.91% (fig. 9) and after purification was 84.57%.

2) Determination of chlorogenic acid, caffeic acid and chicoric acid content in herba Taraxaci extract

Preparation of the reference: precisely weighing 4.40, 3.92 and 4.70mg of chicoric acid, caffeic acid and chlorogenic acid reference substances in three 10mL measuring bottles respectively, ultrasonically dissolving with 70% methanol, cooling to room temperature, adding 70% methanol to a constant volume to scale, and mixing uniformly; and precisely sucking 1mL of the reference substance stock solution, mixing the reference substance stock solution in a 10mL measuring flask, dissolving the reference substance stock solution with 70% methanol, and metering to a scale to prepare mixed reference substance solutions containing 44 mu g/mL, 39.2 mu g/mL and 47 mu g/mL of each of the chicoric acid, the caffeic acid and the chlorogenic acid.

Preparation of a test solution: taking 0.0586g of dandelion purified product, placing into a 10mL measuring flask, adding a proper amount of 70% methanol, carrying out ultrasonic extraction for 10min, cooling to room temperature, then adding 70% methanol to a constant volume, filtering, and taking a subsequent filtrate as a test solution.

Chromatographic conditions are as follows: the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid water solution, gradient elution (0-5min, 10% -15% A, 5-15min, 15% -40% A, 15-16min, 40% -10% A, 16-21min, 10% A) is carried out, the flow rate is 1.0 mL/min^-1The detection wavelength is 328nm, the column temperature is 35 ℃, and the sample injection amount is 20 mu L. Precisely absorbing the test sample, the mixed reference sample and the negative sample solution respectively, injecting sample under the chromatographic conditions, recording chromatogram, and showing HPLC chromatogram in FIG. 3, wherein A is the mixed reference sample and B is the purified test sample of D101. Chlorogenic acid, caffeic acid and chicoric acid were 32.06%, 3.22% and 49.29%, respectively.

Example 3 Taraxacum extract Effect on rat granuloma test

The rats of this example were SD rats weighing 200- & 250g and were provided by the university of Chinese medicine laboratory animal center of Nanjing.

The construction method of the rat model comprises the following steps: after anesthetizing the rat with chloral hydrate, the underarm portion was unhaired and sterilized with 75% ethanol, and a 50mg cotton ball soaked with 5mL of 75% ethanol was implanted under the rat forelimb armpit.

The rats are randomly divided into four groups of low, medium and high drug dose groups and a model group, the three groups of low and medium drug dose are respectively 1.56g crude drug/kg, 3.12g crude drug/kg and 6.24g crude drug/kg according to the conversion of dandelion, and the model group is administered with physiological saline. The rat model was continuously dosed for 7 days, 1mL of drug per 100g of rat weight was gavaged, the rat was decapitated on day 8, the cotton ball was taken out and oven-dried at 60 ℃ for 1 day, and the granuloma mass was calculated, as can be seen from fig. 5, the dry weight of the cotton ball granuloma could be reduced by dosing each dose of dandelion. The effect of the high dose is strongest in each dose of dandelion, which indicates that dandelion can inhibit granulation tissue hyperplasia.

Example 4 Experimental Effect of Taraxacum extract on proliferation of esophageal cancer cells KYSE70

KYSE70 cancer cells were purchased from Shanghai Meixuan Biotech Co., Ltd.

100 μ L of cell suspension was prepared in a 96-well plate at 5X 10 per well³The cells were incubated in an incubator for 24h (37 ℃, 5% CO)₂). Adding 10 μ L of herba Taraxaci extract into the well plate to obtain final concentration of 50, 250, 500, 1000, 2500, 5000, 7500, and 10000 μ g crude drug/mL. After 24h incubation, 10. mu.L of CCK8 solution was added, and after 2h incubation, the absorbance at 450nm was measured using a microplate reader. As can be seen from FIG. 6, dandelion significantly reduced the proliferation of esophageal cancer cells. Among them, 2500 + 10000. mu.g crude drug/mL had the best effect.

Example 5 effects of Taraxacum extract on KYSE70 cell scratch test

1mL of cell suspension was prepared in 24-well plates at 2X 10/well⁵Each cell is provided with 4 parallel holes, the sample adding needs to be slow, the sample adding needs to be rotary, the cell culture plate needs to be slightly rotated to ensure that the cells are evenly spread and saturatedHumidity, 37 ℃ and 5% CO₂Culturing in an incubator for 24 h. When the cell aggregation degree is 90%, scribing three parallel scratches on the cell surface with 200 μ L gun head, sucking out old culture medium, washing with 500 μ L sterile PBS buffer solution for 2 times, adding 1% serum culture medium, and culturing at saturated humidity, 37 deg.C and 5% CO₂The cultivation was continued in the incubator and the scratch area was measured at 0, 6, 12, 24 and 48h, respectively, at intervals. Wound closure rate formula: (S)₀-St)/S₀X 100, wherein S₀Is the area at which the initial scratch of the wound is formed and St is the area of the scratch at the time point of measurement. As can be seen from FIGS. 7 and 8, the dandelion organic acid can significantly slow down the healing rate of KYSE70 cell scratch and significantly slow down the migration of esophageal cancer cells.

Example 6 anticancer effect of extracts of different parts of Dandelion

Referring to the experimental method of example 1-2, the preparation of water extracts of different parts of dandelion is specifically as follows: dandelion root, dandelion leaf, dandelion flower.

The obtained product was tested for anti-cancer effect and compared with the water extract prepared from the whole herb, the results were as follows:

(1) granuloma experiment in rat

Group of	Dosage of drug	Granuloma/g
			Blank space	--	0.1081±0.0364
All grass of herbaceous plants	6.24g/kg	0.0682±0.0307
			Root of Taraxacum Mongolicum	6.24g/kg	0.0801±0.0395
Dandelion leaf	6.24g/kg	0.0657±0.0316
			Dandelion flower	6.24g/kg	0.0925±0.0349

(2) Experimental effect on proliferation of esophageal cancer cells KYSE70

Group of	Dosage of drug	Survival rate/%)
			Blank space	--	100％
All grass of herbaceous plants	5000μg/mL	88.45％
			Root of Taraxacum Mongolicum	5000μg/mL	90.55％
Dandelion leaf	5000μg/mL	87.49％
			Dandelion flower	5000μg/mL	94.31％

(3) Effect on KYSE70 cell scratching experiment

Group of

Dosage of

drug

6h

12h

24h

48h

Blank space

--

8.4317％

21.3853％

21.3906％

23.8004％

All grass of herbaceous plants

5000μg/mL

-0.7194％

-1.7425％

-3.9739％

-18.5266％

Root of Taraxacum Mongolicum

5000μg/mL

-0.0254％

-1.4906％

-2.7822％

-8.2393％

Dandelion leaf

5000μg/mL

-1.0488％

-2.0647％

-5.6265％

-19.7053％

Dandelion flower

5000μg/mL

1.2317％

-0.8643％

-2.4722％

-5.3481％

Comparative example 1 preparation of ethanol extract of Taraxacum officinale

Weighing 5g of dandelion whole plant powder (sieved by a 40-mesh sieve), placing the dandelion whole plant powder into a round-bottom flask, adding 15 times of 80% ethanol and 12.5mL of cellulase solution, activating at 50 ℃ for 30min, decocting for 1h in a slightly boiling state, filtering decoction while hot, and collecting the decoction by using a beaker. Standing at 4 deg.C for 24 hr, filtering, concentrating the filtrate with rotary evaporator until no alcohol smell is present, storing in 4 deg.C refrigerator, and performing three experiments in parallel.

The chlorogenic acid is used as a reference substance, the extract is subjected to ultraviolet spectrophotometry, the content of the chlorogenic acid in the extract is detected, and the extraction rate is calculated, namely the average extraction rate of extraction is 1.013%.

As can be seen from fig. 1, the extraction efficiency of water is superior to that of ethanol.

Comparative example 2 preparation of an extract of Dandelion by ultrasonic mode

Weighing 0.5g of powder sample, placing in a 100mL conical flask, adding 15 times of water, placing in an ultrasonic cleaner for 20min, transferring the supernatant to a clean conical flask, cleaning the residue with distilled water for 2 times, and combining the supernatants. And (4) placing the supernatant into a centrifuge with the speed of 3000r/min for centrifugation for 5min, transferring the supernatant into a 50mL volumetric flask for constant volume, refrigerating for standby, and performing three experiments in parallel.

The chlorogenic acid is used as a reference substance, the extract is subjected to ultraviolet spectrophotometry, the content of the chlorogenic acid in the extract is detected, and the extraction rate is calculated, namely the ultrasonic average extraction rate is 0.706%.

Comparative example 3 preparation of Dandelion extract by extraction method

Taking 5g of dandelion powder sample, adding 12.5mL of water and cellulase solution according to the proportion of 1: 15, placing in a 50 ℃ water bath for heating for 35min, extracting for 2 times at 70 ℃, combining the extract liquor, concentrating to 5mL, placing in a 4 ℃ refrigerator for later use, and performing three experiments in parallel.

The chlorogenic acid is used as a reference substance, the extract is subjected to ultraviolet spectrophotometry, the content of the chlorogenic acid in the extract is detected, and the extraction rate is calculated, namely the average extraction rate of extraction is 0.905%.

As can be seen from FIG. 2, the extraction efficiency of the heating reflux is superior to the leaching.

Comparative example 4 purification of Taraxacum Mongolicum extract HPD600 by macroporous resin

Extracting the dandelion extract under the conditions of 1: diluting with 10 distilled water, passing through column with HPD600 macroporous resin, wherein the ratio of sample injection liquid volume (mL) to macroporous resin mass (g) is 12, pH is 2, adsorption flow rate is 1.5BV/h, desorption solution ethanol concentration is 50%, desorption flow rate is 2BV/h, and desorption solution dosage is 3 BV. Collecting the separated liquid with a conical flask, and concentrating under reduced pressure for use.

The test solutions were prepared under the conditions of example 4, and under the same chromatographic conditions, as can be seen from FIG. 4, chlorogenic acid, caffeic acid and chicoric acid were 34.66%, 5.66% and 44.98%, respectively.

As can be seen from the comparison of FIG. 3 and FIG. 4, D101 has a better effect in enriching chlorogenic acid, caffeic acid and chicoric acid.

Comparative examples 5-11 comparison of purification conditions

A comparative example was set up with reference to example 2, which differs from example 2 only in part by the operation in the adsorption conditions of step 1), and was specifically set up as follows:

Claims

1. a preparation method of a dandelion extract is characterized by comprising the following steps:

2. The method according to claim 1, wherein the dandelion powder is a dry powder of any one or more of dandelion whole herb, dandelion root, dandelion leaf, and dandelion flower.

3. The preparation method according to claim 1, wherein the mass ratio of the volume of the injected liquid to the macroporous resin in the step (2) is 10-14.

4. The method according to claim 3, wherein the ratio of the volume of the injected liquid to the mass of the macroporous resin in step (2) is 12.

5. The preparation method according to claim 1, wherein in the step (2), the adsorption flow rate is 1-2BV/h, the concentration of ethanol in the desorption solution is 40-60%, the desorption flow rate is 1.5-3BV/h, and the dosage of the desorption solution is 1-4BV when the D101 macroporous resin is used for concentration and purification through a column.

6. The method according to claim 1, wherein the ratio of the amount of water added to the amount of dandelion powder in step (1) is 1: 2-5, wherein the ratio is a volume-to-weight ratio.

7. A dandelion extract characterized by being produced by the production method according to any one of claims 1 to 6.

8. Use of dandelion and/or an extract thereof for the preparation of a medicament for the prevention and/or treatment and/or prognostic repair of esophagitis or esophageal cancer, characterized in that the dandelion is applied to the medicament after the extract is prepared by the preparation method according to any one of claims 1 to 6.

9. A medicament for prevention and/or treatment and/or prognostic repair of esophagitis or esophageal cancer, characterized in that the medicament comprises the dandelion extract according to claim 7.

10. The drug as claimed in claim 9, wherein the drug is a liquid drug, and the concentration of the dandelion extract in the drug is 2500-10000 μ g crude drug/mL.