CN113476492A

CN113476492A - Dandelion seed extract and application thereof

Info

Publication number: CN113476492A
Application number: CN202110969607.0A
Authority: CN
Inventors: 盛春华; 张林波; 肖盛元; 刘帅
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2021-10-08

Abstract

The invention relates to a dandelion seed extract and application thereof, wherein the dandelion seed extract comprises phenylacetic acid lipid derivatives, cinnamic acid lipid derivatives and flavonoids compounds. The dandelion seed extract is extracted by ethanol, so that the active ingredients of the medicinal materials are not damaged, the extraction rate is high, the preparation method is simple, the cost is low, and the dandelion seed extract prepared by the method has an obvious tumor inhibition effect.

Description

Dandelion seed extract and application thereof

Technical Field

The invention belongs to the technical field of medicines, health products and food additives, and particularly relates to a dandelion seed extract and application thereof.

Background

The dandelion has been studied as a medicinal plant for years, and has been proved to have the effects of resisting bacteria, viruses, inflammation, tumors, swelling and pain, oxidation, allergy, liver protection, cholagogue and diuresis, reducing blood sugar and the like. The dandelion seed is mature and dry seed of Taraxacum officinale L.of Compositae family. The dandelion seeds are reported in documents to have strong antioxidant activity, can effectively remove various free radicals and have good capability of inhibiting linoleic acid peroxidation. However, the extraction method, the components and the anticancer effect of dandelion seeds are not reported at home and abroad. The dandelion is used as a plant with homology of medicine and food, and has wide application prospect. The dandelion is simple in planting process and low in cost, and dandelion seeds are expected to become anticancer drugs, so that the dandelion seed extract has higher value, but no effective ingredients in the dandelion seeds and an extraction method thereof are disclosed at present.

The present invention has been made in view of the above circumstances.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides the dandelion seed extract and the application thereof.

The first purpose of the invention is to provide a dandelion seed extract, which comprises phenylacetic acid lipid derivatives, cinnamic acid lipid derivatives and flavonoids compounds.

Furthermore, the dandelion extract also comprises seed oil lipid components.

Furthermore, the components of the seed oil lipid are higher fatty acid and phospholipid.

Further, the dandelion seed extract is prepared by the following method, which specifically comprises the following steps: (1) removing impurities from herba Taraxaci seed, drying, and pulverizing;

(2) loading pulverized semen Taraxaci into a packed column, soaking and extracting with ethanol, and collecting filtrate;

(3) evaporating the collected filtrate, and collecting extract to obtain the dandelion seed extract.

Further, the volume fraction of the ethanol in the step (2) is 50-100%.

Further, the volume fraction of ethanol was 70%.

Further, the soaking time in the step (2) is 3-20 h.

Further, the soaking time is 15 h.

Further, soaking and extracting for 9-15 times in the step (2).

Further, soaking and extracting for 9 times.

Further, the temperature for soaking in the step (2) is normal temperature.

Further, recovering ethanol from the filtrate collected in the step (3) by using a rotary evaporator until no alcohol smell exists, and collecting an extract to obtain the dandelion seed extract.

The second purpose of the invention is to provide the application of the dandelion seed extract in preparing antitumor drugs.

Furthermore, the tumor comprises lung cancer, melanoma or liver cancer.

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

the dandelion seed extract prepared by the method has thick color and luster, good fluidity and fragrant smell, and volatile oil is extracted; the dandelion seed extract has better application in preparing antitumor drugs and has obvious inhibition effect on tumors.

Drawings

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

FIG. 1 is a comparison graph of the inhibition effect of dandelion seed extract with different concentrations on lung cancer A549 cells;

FIG. 2 is a curve of tumor inhibition of different concentrations of dandelion seed extract on melanoma mice;

FIG. 3 is a graph showing the change of alpha-fetoprotein values in a liver cancer patient;

FIG. 4 is a chromatogram of the dandelion seed extract prepared in example 4;

FIG. 5 is an extracted ion current chromatogram for Compound 22;

fig. 6 is an extracted ion flow chromatogram of

compounds

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 26;

FIG. 7 is an extracted ion current chromatogram for

compounds

23, 25, 26;

FIG. 8 is a representative UV spectrum of

compounds

1, 2, and 3;

FIG. 9 is a representative UV spectrum of the remaining compounds in Taraxacum seed extract;

FIG. 10 is a mass spectrum of Compound 1;

FIG. 11 is a mass spectrum of Compound 2;

FIG. 12 is a mass spectrum of Compound 3;

FIG. 13 is a mass spectrum of

compounds

4, 5, 6, 7;

FIG. 14 is a mass spectrum of

compounds

8, 9, 10;

FIG. 15 is a mass spectrum of

compounds

11, 12, 13;

FIG. 16 is a mass spectrum of

compounds

14, 15, 16, 17;

FIG. 17 is a mass spectrum of

compounds

18, 21;

FIG. 18 is a mass spectrum of

compounds

19, 20;

figure 19 is a mass spectrum of compound 22;

figure 20 is a mass spectrum of compound 23;

figure 21 is a mass spectrum of compound 24;

figure 22 is a mass spectrum of compound 25;

fig. 23 is a mass spectrum of compound 26.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Example 1

The preparation method of the dandelion seed extract comprises the following steps:

(1) sorting dandelion seeds, removing stones and impurities, drying by using a dryer, and crushing by using a crusher;

(2) loading pulverized semen Taraxaci into a packed column with height of 5cm, adding ethanol with the same volume, soaking and extracting with ethanol at room temperature for 3 hr for 9 times with ethanol volume fraction of 50%, collecting filtrate of each soaking and extracting, and mixing filtrates;

(3) and (3) recovering ethanol from the collected filtrate by a rotary evaporator until no alcohol smell exists, and collecting an extract to obtain the dandelion seed extract.

Example 2

(2) loading pulverized semen Taraxaci into a packed column with height of 5cm, adding ethanol with the same volume, soaking and extracting with ethanol at room temperature for 12 times with ethanol volume fraction of 70% for 15 hr, collecting filtrate of each soaking and extracting, and mixing filtrates;

(3) and (3) recovering ethanol from the collected filtrate by a rotary evaporator without alcohol smell, and collecting an extract to obtain the dandelion seed extract.

Example 3

(2) loading pulverized semen Taraxaci into a packed column with height of 5cm, adding ethanol with the same volume, soaking and extracting with ethanol at room temperature for 20 hr for 15 times with ethanol volume fraction of 100%, collecting filtrate of each soaking and extracting, and mixing filtrates;

Example 4

(2) loading pulverized semen Taraxaci into a packed column with height of 5cm, adding ethanol with the same volume, soaking and extracting with ethanol at room temperature for 15 hr at volume fraction of 90% for 9 times, collecting filtrate from each soaking and extracting, and mixing filtrates;

The extraction ratio in the following test examples (mass of the obtained extract/mass of dandelion seed raw material) × 100%

Test example 1

Investigating the influence of different ethanol concentrations on the extraction rate

The extraction rate at different ethanol concentrations is shown in Table 1, except that the ethanol concentration is changed and the other conditions are the same as in example 1.

TABLE 1

Ethanol volume fraction (%)	50	60	70	80	90
						Extraction ratio (%)	8.9	9.21	11.48	11.84	11.90

Test example 2

Investigating the influence of different soaking times on the extraction yield

The conditions were the same as in example 1 except that the soaking time was changed, and the extraction rates at different soaking times are shown in Table 2.

TABLE 2

Extraction time (h)	5	10	15	20	25
						Extraction ratio (%)	4.6	11.33	11.93	11.91	11.89

Test example 3

Investigating the influence of different soaking and extracting times on the extraction rate

The conditions were the same as in example 1 except that the number of soaking and extracting times was changed, and the extraction rates at the different soaking and extracting times are shown in Table 3.

TABLE 3

Number of times of extraction	3	6	9	12	15
						The extraction rate%	4	7.2	11.96	11.85	11.94

Through comprehensive analysis in tables 1-3, the optimal extraction conditions of the dandelion seed extract are determined to be that the volume fraction of ethanol is 90%, the soaking time is 15h, and soaking and extraction are carried out for 9 times.

Test example 4

Inhibition effect of dandelion seed extract on A549 (human lung adenocarcinoma cell) cells

A549 cell culture: the cell inoculation amount is 1 × 10⁴Each/ml was inoculated in a culture flask, cultured with DMEM medium and 10% fetal bovine serum, and cultured for 16 hours into the logarithmic growth phase. When in use, the cell suspension is prepared by 0.25% digestion.

cck8 cytotoxicity assay: a549 cells were inoculated onto a culture plate at a density of 5000 cells/well, and dandelion seed extract (prepared according to the method of example 4) was designed to be administered at concentrations of 0.1562, 0.3125, 0.625, 1.250 mg/ml. The cck8 test was used to examine the inhibitory effect of drugs on cell growth, and the cell growth state was used to examine the effect of drugs on cell growth. The results show that the taraxacum seed alcohol extract has good inhibition effect on the growth of A549 cells, and the inhibition rate is the maximum at the concentration of 1.25mg/ml and can reach 80 percent, which is shown in figure 1.

Test example 5

Inhibition effect of dandelion seed extract on mouse melanoma

B16 cell (mouse melanoma cell) culture: the cells are adherent cells, and the culture condition is high-glucose DMEM culture medium + 10% FBS + 1% double antibody. Passage according to 1: 3.

Animal experiments: the dandelion seed alcohol extract prepared in the embodiment 4 is prepared into a solution with the concentration of 1.6, 3.2 and 4.8mg/ml by using normal saline, and the mouse is administrated by gavage at the fourth day, wherein the concentration of the solution is 0.5 ml/mouse. The control group was given 0.5ml of physiological saline. 5 mice were sacrificed each day, tumor tissues were separated, weighed, tumor inhibition rate was calculated, and inhibition rate curves were plotted (fig. 2). The result shows that the dandelion seed extract has good inhibition effect on melanoma, and when the concentration of the extract is 4.8mg/ml, the inhibition rate on the tumor is the highest.

Test example 6

Inhibition effect of dandelion seed extract on primary liver cancer

A patient Liu is diagnosed with primary liver cancer in 7 months in 2018 after a certain number of people in Liu, men, and 64 years old, and the significant increase of alpha-fetoprotein is detected. The patient began to take the dandelion seed extract prepared in example 4 twice a day 2g each time from 3 months in 2019. It can be observed that the value of alpha fetoprotein is reduced obviously after the patient takes the extract, the value is reduced to the normal level after one year (figure 3), the liver cancer does not progress obviously, and the disease is controlled.

Alpha fetoprotein is a short for alpha fetoprotein, and is a carbohydrate protein. At present, the diagnostic significance of alpha-fetoprotein on primary liver cancer is well recognized by the medical field, and the level change of alpha-fetoprotein has high correlation with primary liver cancer. After the dandelion alcohol extract is taken, the illness state of a patient is controlled, the alpha fetoprotein level is obviously reduced, and the dandelion seed alcohol extract is proved to have a certain inhibition effect on primary liver cancer.

Test example 7

Inhibition effect of dandelion seed extract on lung cancer

The patient is diagnosed with lung cancer in xu a certain time, woman, age 76, 12 months in 2020. Because of the age and poor physical quality of the patients, the patients cannot tolerate the radiotherapy. 2g of dandelion seed extract prepared in example 4 is taken twice a day at the beginning of 1 month in 2021, and the tumor marker value is obviously reduced after about 3 months of administration, which is shown in table 4. The carbohydrate antigen CA72-4 was reduced by about 72%, and the carbohydrate antigen cyfra21-1, neuron-specific enolase, were restored to within the normal range. After the medicine is used, the mental state of a patient is good, the life quality is improved, the tumor is not obviously progressed, and the dandelion seed alcohol extract is proved to have a certain inhibiting effect on the lung cancer.

TABLE 4

Tumor marker	1 month in 2021	2021 year 4 month
			Carbohydrate antigen CA72-4(U/ml, less than or equal to 6.9U/ml)	211.5	59.2
Carbohydrate antigen cyfra21-1(ng/ml, 3.3ng/ml or less)	10.23	3.19
			Neuron specific enolase (ng/ml, 16.3ng/ml or less)	17.46	11.36

The inventor also carried out the above tests on the dandelion seed extract prepared in other examples, and the results are basically consistent and are not listed one by one due to limited space.

Test example 8

The dandelion seed extract prepared in example 4 was subjected to chromatographic analysis under the following specific conditions:

chromatographic conditions are as follows: aqueous phase a was 20% methanol + 0.05% ammonium formate + 0.05% formic acid;

phase B is pure acetonitrile.

Gradient as shown in Table 1, equilibration time 3min, run time 60min, injection volume 5uL, DAD200-400 nm.

Mass spectrum conditions: the scanning range of positive and negative ions is 30-1500Da, and the range of parent ions is 200-1200Da in an online automatic secondary ion mode.

TABLE 1

Wherein, after analysis, the dandelion seed extract typically comprises compounds 1-26.

Fig. 4 is a chromatogram of dandelion seed extract prepared in example 4, ultraviolet light is 280nm, fig. 5 is an extraction ion chromatogram (molecular weight 286.0415) of compound 22, fig. 6 is an extraction ion chromatogram of

compounds

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 26, fig. 7 is an extraction ion chromatogram of

compounds

23, 25, 26, fig. 8 is a representative ultraviolet spectrum of

compounds

1, 2, 3, and fig. 9 is a representative ultraviolet spectrum of the remaining compounds in dandelion seed extract. Among them, compound 1 (molecular weight 318.1), compound 2 (molecular weight 342.1), compound 3 (molecular weight 284.1),

compound

4, 5, 6, 7 (molecular weight 474.1),

compound

8, 9, 10 (molecular weight 354.1),

compound

11, 12, 13 (molecular weight 516.1),

compound

14, 15, 16, 17 (molecular weight 448.1),

compound

18, 19, 20, 21 (molecular weight 570.1), compound 22 (molecular weight 286.1), compound 23 (molecular weight 554.1), compound 24 (molecular weight 270.1), compound 25 (molecular weight 538.1), compound 26 (molecular weight 254.1)

FIGS. 10-23 show negative ions [ M-H ] of main ingredient of herba Taraxaci seed extract]^-Representative Collision induced Cleavage (CAD) secondary mass spectrograms (all parent ions are [ M-H ]]^-) FIG. 10 is Compound 1 and FIG. 11 is Compound 2; FIG. 12 is Compound 3; FIG. 13 is

compound

4, 5, 6, 7; figure 14 is

compounds

8, 9, 10; figure 15 is

compounds

11, 12, 13; FIG. 16 is

compound

14, 15, 16, 17; fig. 17 is

compound

18, 21; figure 18 is

compounds

19, 20; figure 19 is compound 22; FIG. 20 is Compound 23; figure 21 is compound 24; FIG. 22 is Compound 25; figure 23 is compound 26.

The analysis shows that the dandelion seed extract prepared by the invention comprises the following compounds:

phenylacetic acid lipid derivatives: the characteristic absorption peak of the oxygen-substituted benzene ring at 250-290nm can be observed in the ultraviolet spectrogram of the compound, and the alcohol part mainly comprises saccharide, quinic acid and the like, such as the compounds 1-3.

Cinnamic acid lipid derivatives: the ultraviolet spectrum of the compound can observe a characteristic ultraviolet spectrogram of cinnamic acid, which comprises an absorption peak (250-290nm) of an oxygen-substituted benzene ring and an absorption peak (300-350nm) of phenylacrylic acid; alcohol moieties include quinic acid, tartaric acid, and the like; such as compounds 4-13.

And (3) flavonoid compounds: the ultraviolet spectrum of the compounds is similar to that of cinnamic acid esters, and the ultraviolet characteristic spectrum I band (300-350nm) and the ultraviolet characteristic spectrum II band (250-300nm) of flavone can be observed, and the compounds can be divided into two categories of monoflavone and glycosides thereof and biflavones thereof, wherein the former category is 14-17, 22, 24 and 26; the latter are, for example, compounds 18 to 21, 23 and 25.

The inventor also finds that the dandelion seed extract also contains other common seed oil lipid components such as higher fatty acid, phospholipid and the like through research, and the components are not listed due to limited space.

The inventor can know that the dandelion seed extract contains a plurality of compound components and further has an obvious tumor inhibition effect through the analysis.

The inventor also carried out the above analysis on the components of the dandelion seed extract prepared in other examples, and the results are basically consistent and are not listed one by one due to limited space.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. The dandelion seed extract is characterized by comprising phenylacetic acid lipid derivatives, cinnamic acid lipid derivatives and flavonoids compounds.

2. The dandelion seed extract according to claim 1, wherein the dandelion extract further comprises a seed oil and fat component, preferably the seed oil and fat component is a higher fatty acid and a phospholipid.

3. The dandelion seed extract according to claim 1 or 2, which is prepared by the following method, and specifically comprises the following steps:

(1) removing impurities from herba Taraxaci seed, drying, and pulverizing;

4. A dandelion seed extract according to claim 3, wherein the volume fraction of ethanol in step (2) is 50-100%, preferably the volume fraction of ethanol is 70%.

5. The dandelion seed extract according to claim 3, wherein the soaking time in the step (2) is 3-20h, preferably 15 h; soaking and extracting for 9-15 times, preferably, soaking and extracting for 9 times.

6. The dandelion seed extract according to claim 1, wherein the soaking temperature in the step (2) is normal temperature.

7. The dandelion seed extract according to claim 1, wherein the filtrate collected in step (3) is subjected to ethanol recovery without alcohol smell by a rotary evaporator, and the extract is collected to obtain the dandelion seed extract.

8. Use of the dandelion seed extract according to any one of claims 1-7 in the preparation of an antitumor medicament.

9. The use of claim 8, wherein the tumor comprises lung cancer, melanoma and liver cancer.