CN114558059B - Radix Sophorae Flavescentis-Coptidis rhizoma extract with antitumor activity, and quality detection method and application thereof - Google Patents

Abstract

The application discloses a kuh-seng-coptis extract with anti-tumor activity, and a quality detection method and application thereof. According to the structure and the property characteristics of different components in the kuh-seng-coptis chinensis drug pair, the optimal mobile phase composition, elution program, flow rate, chromatographic column, mass spectrum conditions and other analysis conditions are screened out through a large number of experiments. Multiple experiments prove that 10 alkaloid compounds with different structures and types can be simultaneously obtained by adopting a UPLC-TQ-MS method, the method has high detection sensitivity and good stability, and can objectively, comprehensively and accurately evaluate the quality of the kuh-seng-coptis medicine on medicinal materials, extracts and preparations thereof, thereby having important significance in controlling the quality and ensuring the clinical curative effect. Experiments show that the kuh-seng-coptis chinensis with the weight ratio of 1:4 has better activity of resisting colon cancer.

Description

Radix Sophorae Flavescentis-Coptidis rhizoma extract with antitumor activity, and quality detection method and application thereof

Technical Field

The application relates to the technical field of traditional Chinese medicines, in particular to a kuh-seng-coptis extract with anti-tumor activity, and a quality detection method and application thereof.

Background

The liquid chromatography-mass spectrometry technology uses liquid chromatography as a separation system, the mass spectrum as a detection system, a chromatographic separation technology is used for separating a mixed sample into single substances, the substances are ionized after entering the mass spectrum system, ion fragments with different mass numbers are separated through a mass spectrum mass analyzer, the substance structure is analyzed through a mass spectrum detector, the structure of the ion fragments is judged through a mass-charge ratio in a comparison chart library by judging the breaking mode of chemical bonds, and therefore the quality of an unknown sample is determined. The liquid chromatography-mass spectrometry combines the high separation capacity of high performance liquid chromatography for complex samples with the advantages of high selectivity, high sensitivity of mass spectrometry and capability of providing relative molecular mass and structural information. The chemical components of the traditional Chinese medicine are complex and various, and how to efficiently separate and identify the chemical components of the traditional Chinese medicine is a key problem in the basic research of the drug effect substances of the traditional Chinese medicine, so that the problem is solved by using a liquid chromatography-mass spectrometry technology. After simple pretreatment, a small amount of medicines are detected on a machine, and the quick and accurate analysis of the traditional Chinese medicine components is carried out simply, efficiently and easily in operation, so that the liquid chromatography-mass spectrometry technology is an indispensable experimental method in the basic research of the traditional Chinese medicine efficacy substances.

The radix Sophorae Flavescentis is dry root of Sophora flavescens ait. It mainly contains alkaloid components such as matrine, oxymatrine, sophoridine, methyl cytisine, sophocarpine, tetrandrine and flavonoid components such as kurarinone, and has antiinflammatory, antipyretic, antibacterial, antipruritic, antitumor, antiarrhythmic and myocardial ischemia effects. The Coptidis rhizoma is dried rhizome of Coptis chinensis Franch (Coptis chinensis Franch.) of Ranunculaceae, coptis deltoidea C.Y. Cheng et Hsiao or Coptis teeta wall, and has antibacterial, antiinflammatory, antiviral, and antitumor effects. The two medicinal materials of coptis and kuh-seng respectively have the effects of clearing heat and drying dampness, purging fire and removing toxin, clearing heat and drying dampness and scrubbing humidity, and the two medicinal materials are combined with donkey-hide gelatin in ancient book general Ji Fang to form coptis and kuh-seng decoction for mainly treating: the patient suffers from poor and ill condition, and the patient can not heal the disease due to the symptoms of intestinal slip, diarrhea, purulent blood, tens of days, angina in the abdomen, fever with fire, headache with broken pulse with astringency. Is used for colorectal cancer treatment in clinic and has good curative effect.

Disclosure of Invention

The application aims to: the application aims to solve the defects of the prior art, and provides a kuh-seng-coptis extract with anti-tumor activity, and a quality detection method and application thereof through a large number of experimental screening.

The technical scheme is as follows: in order to achieve the above purpose, the technical scheme adopted by the application is as follows:

an extract of kuh-seng-coptis with anti-tumor activity is prepared by the following method: soaking radix Sophorae Flavescentis and Coptidis rhizoma in proper weight ratio in water, extracting with water, mixing filtrates, and concentrating.

As a preferred scheme, the kuh-seng-coptis extract with anti-tumor activity is prepared by the following method: soaking radix Sophorae Flavescentis and Coptidis rhizoma in proper weight ratio in water for 0.5 hr, extracting with 10 times of water for 1-3 times, each time for 1-2 hr, mixing filtrates, and concentrating.

As a preferred scheme, the kuh-seng-coptis extract with anti-tumor activity is prepared by the following method: soaking radix Sophorae Flavescentis and Coptidis rhizoma in a certain weight ratio for 0.5 hr, extracting with 10 times of water for 2 times each for 1 hr, mixing filtrates, and concentrating to crude drug concentration of about 1g/ml.

As a preferable scheme, the kuh-seng-coptis extract with anti-tumor activity is 1:1-1:4 of kuh-seng and coptis medicinal materials.

A quality detection method of kuh-seng-coptis extract with anti-tumor activity is characterized by comprising the following steps:

preparation of the reference substance solution in step (1)

Accurately weighing sophocarpine, matrine, sophoridine, sophocarpine oxide, matrine oxide, coptisine, methylcoptisine, berberine, jateorhizine, palmatine hydrochloride, adding methanol to prepare mixed standard stock solution, and preserving at 4 ℃;

preparation of the sample solution in step (2)

Soaking radix Sophorae Flavescentis and Coptidis rhizoma in proper weight ratio in water for 0.5 hr, extracting with 10 times of water for 1-3 times for 1-2 hr, mixing filtrates, concentrating to crude drug concentration of about 1g/ml, and filtering with microporous membrane before sampling;

step (3) establishment of linear regression equation

Taking the mixed standard stock solution in the step (1), sequentially diluting 5 times, filtering through a 0.22 mu m cellulose membrane to obtain a series of mixed reference substance solutions, sequentially injecting UPLC-TQ-MS, taking the series of reference substance concentrations as an abscissa X and the corresponding peak areas as an ordinate Y, performing linear regression analysis on each chemical component, and calculating a linear regression equation;

step (4) content measurement

Taking the kuh-seng-coptis root medicine in the step (2) to test the solution, injecting the solution into UPLC-TQ-MS for analysis, substituting the peak area into the linear regression equation in the step (3), and calculating the content of each component in the solution.

As a preferred scheme, the quality detection method of the kuh-seng-coptis root drug pair comprises the following steps of:

accurately weighing sophocarpine, matrine, sophoridine, sophocarpine oxide, matrine oxide, coptisine, methyl coptisine, berberine, jateorhizine, palmatine hydrochloride, adding methanol to prepare mixed standard stock solutions with the concentration of 300, 600, 300 and 200 mug/mL respectively, preserving at 4 ℃, and passing through a 0.22 mu m microporous filter membrane before sample injection.

As a preferred scheme, the quality detection method of the kuh-seng-coptis root drug pair in the step (3) and the step (4) has the following chromatographic conditions of UPLC-TQ-MS:

chromatographic conditions: watersAcquity HSS T3 chromatography column; 0.1% formic acid aqueous solution is phase A, acetonitrile is phase B, gradient elution: 0-1 min,15% B, 1-5 min,15% B-50% B, 5-7 min,50% B, 7-10 min,50% B-15% B, 10-13 min,15% B; flow rate: 0.3mL/min; column temperature: 35 ℃; sample injection amount: 2. Mu.L.

Mass spectrometry conditions: electrospray ionization, multi-reaction monitoring ion scanning mode measurement, and main mass spectrum parameters are as follows: the capillary voltage is 5500V, and the desolvation temperature is 550 ℃; the positive ion detection mode and the negative ion detection mode are adopted, and the mass-to-charge ratios of the selected detected ions are respectively as follows: sophocarpine m/z 247.2/996.1, matrine m/z 249.2/148.3, sophoridine m/z 249.3/152.1, sophocarpine m/z 263.3/245.1, matrine oxide m/z 265.3/205.2, coptisine m/z 319.7/292.0, methylcoptisine m/z 334.2/261.1, berberine m/z335.9/320.2, jateorhizine m/z 337.9/322.4, palmatine hydrochloride m/z 352.0/336.1;

as a preferred scheme, the quality detection method of the kuh-seng-coptis root drug pair comprises the following linear regression equation of 10 reference substances in the step (3):

compounds of formula (I)	Standard curve	R 2	Linear range/ng/mL
				Sophocarpine	Y＝26219*X+64815	1.00	0.5～300
Matrine	Y＝34340*X-86061	0.999	0.5～600
				Sophoridine	Y＝17207*X-68695	0.999	0.5～600
Sophora fruit alkali oxide	Y＝4979*X+9805	1.00	0.5～600
				Oxymatrine	Y＝257*X+41101	1.00	1～600
Coptisine	Y＝9711*X+14305	0.999	0.5～600
				Methyl coptisine	Y＝11.4*X+214	0.999	0.5～600
Berberine	Y＝97034*X+2117352	0.994	1～600
				Jatrorrhizine	Y＝3570*X-7033	0.999	0.5～300
Palmatine hydrochloride	Y＝206624*X-1321	0.999	0.5～200

The application relates to an application of kuh-seng-coptis extract with anti-tumor activity in preparing anti-tumor drugs.

As a preferred scheme, the kuh-seng-coptis extract with anti-tumor activity is applied to preparing the anti-colon cancer drugs.

The beneficial effects are that: compared with the prior art, the extract of the kuh-seng-coptis medicine pair provided by the application has the following advantages:

according to the structure and the property characteristics of 10 alkaloid components in the kuh-seng-coptis chinensis drug pair, the optimal mobile phase composition, elution program, flow rate, chromatographic column, mass spectrum conditions and other analysis conditions are screened out through a large number of experiments. Multiple experiments prove that the method can detect 10 alkaloid compounds with different structures simultaneously, has high detection sensitivity and good stability, can objectively, comprehensively and accurately evaluate the quality of kuh-seng-coptis chinensis medicine on medicinal materials, extracts and preparations thereof, and has important significance in controlling the quality and ensuring clinical curative effects.

Drawings

FIG. 1 is a mass spectrum of 10 alkaloid compounds.

FIG. 2 is a bar graph of kuh-seng-coptis medicine against colorectal cancer.

FIG. 3 is a chart showing the morphology of the tissue HE staining of kuh-seng-coptis chinensis against colorectal cancer.

Detailed Description

The present application is further illustrated below with reference to specific examples, which are to be construed as merely illustrative of the application and not limiting of its scope, as various equivalent modifications to the application will fall within the scope of the application as defined in the appended claims after reading the application.

The instrument and the material used in the embodiment of the application are as follows: AB Sciex Instruments Q-Trap 5500 LC-MS, analysis TF1.6 analysis software, BALB/c male mice, CT26.WT cell lines, experimental data expressed as mean+ -SD, were statistically analyzed using one-way analysis ofvariance (ANOVA) and Dunnett's multiple comparisons test using Graphpad prism 7 software.

Example 1

1. The kuh-seng-coptis extract with anti-tumor activity is prepared by the following method: respectively soaking radix Sophorae Flavescentis and Coptidis rhizoma at weight ratio of 1:2 and 1:4 for 0.5 hr, respectively extracting with 10 times of water for 2 times each for 1 hr, mixing filtrates, and concentrating to obtain radix Sophorae Flavescentis and Coptidis rhizoma extracts at weight ratio of 1:2 and 1:4.

Example 2

The kuh-seng-coptis extract with anti-tumor activity is prepared by the following method: soaking radix Sophorae Flavescentis or Coptidis rhizoma in appropriate amount of water for 0.5 hr, extracting with 10 times of water for 2 times each for 1 hr, mixing filtrates, and concentrating to obtain radix Sophorae Flavescentis or Coptidis rhizoma extract.

Example 3

A quality detection method of radix Sophorae Flavescentis-Coptidis rhizoma extract with antitumor activity comprises the following steps:

preparation of the reference substance solution in step (1)

Accurately weighing sophocarpine, matrine, sophoridine, sophocarpine oxide, matrine oxide, coptisine, methyl coptisine, berberine, jateorhizine, palmatine hydrochloride, adding methanol to prepare mixed standard stock solutions with the concentration of 300, 600, 300 and 200 mug/mL respectively, preserving at 4 ℃, and passing through a 0.22 mu m microporous filter membrane before sample injection.

Preparation of the sample solution in step (2)

The extracts of the kuh-seng and the coptis chinensis in the weight ratio of 1:2 and 1:4 in the embodiment 1 and the kuh-seng or the coptis chinensis in the embodiment 2 are concentrated to the crude drug concentration of about 1g/ml, and the crude drug is filtered through a microporous filter membrane before sample injection;

step (3) establishment of linear regression equation

Taking the mixed standard stock solution in the step (1), sequentially diluting 5 times, filtering through a 0.22 mu m cellulose membrane to obtain a series of mixed reference substance solutions, sequentially injecting UPLC-TQ-MS, taking the series of reference substance concentrations as an abscissa X and the corresponding peak areas as an ordinate Y, performing linear regression analysis on each chemical component, and calculating a linear regression equation;

step (4) content measurement

Taking the kuh-seng-coptis root medicine in the step (2) to test the solution, injecting the solution into UPLC-TQ-MS for analysis, substituting the peak area into the linear regression equation in the step (3), and calculating the content of each component in the solution.

The chromatographic conditions of UPLC-TQ-MS in the step (3) and the step (4) are as follows:

chromatographic conditions: watersAcquity HSS T3 chromatography column; 0.1% formic acid aqueous solution is phase A, acetonitrile is phase B, gradient elution: 0-1 min,15% B, 1-5 min,15% B-50% B, 5-7 min,50% B, 7-10 min,50% B-15% B, 10-13 min,15% B; flow rate: 0.3mL/min; column temperature: 35 ℃; sample injection amount: 2. Mu.L.

Mass spectrometry conditions: electrospray ionization, multi-reaction monitoring ion scanning mode measurement, and main mass spectrum parameters are as follows: the capillary voltage is 5500V, and the desolvation temperature is 550 ℃; the positive ion detection mode and the negative ion detection mode are adopted, and the mass-to-charge ratios of the selected detected ions are respectively as follows: sophocarpine m/z 247.2/996.1, matrine m/z 249.2/148.3, sophoridine m/z 249.3/152.1, sophocarpine m/z 263.3/245.1, matrine oxide m/z 265.3/205.2, coptisine m/z 319.7/292.0, methylcoptisine m/z 334.2/261.1, berberine m/z335.9/320.2, jateorhizine m/z 337.9/322.4, palmatine hydrochloride m/z 352.0/336.1;

object to be measured	De-clustering voltage/V	Collision voltage/V
			Sophocarpine	40	40
Matrine	50	40
			Sophoridine	50	40
Sophora fruit alkali oxide	40	20
			Oxymatrine	76	40
Coptisine	48	30
			Methyl coptisine	40	47
Berberine	48	30
			Jatrorrhizine	22	22
Palmatine hydrochloride	40	47

The linear regression equation for the 10 controls in step (3) is as follows:

TABLE 3 content of kuh-seng-coptis root Compounds

Example 4 efficacy experiment of Sophora flavescens anti-colorectal cancer in situ transplantation tumor

1. Animal modeling and grouping

Seed mouse model

3 days after the BALB/c mice were fed adaptively, 5 mice were subcutaneously injected with a CT26.WT cell suspension (number of inoculated cells 1X 10) ⁶ And a right forelimb in the armpits. Colon cancer mice were operated with reference to literature: when the volume of the subcutaneous tumor increases to about 100mm ³ At the time, mice were sacrificed and tumors were peeled off, and the fish-like tissues with a strong growth ability were cut into 1mm ³ Is placed in 100 U.mL each containing penicillin and streptomycin ^-1 Pre-chilled RPMI1640 medium.

2. Colon cancer mouse in situ transplantation tumor model

After the mice are anesthetized with isoflurane, the left lower abdomen incision is disinfected, the cecum is gently pulled out, and the serosa is gently scraped by a No. 4 needle at the junction of the blind colon, so that micro-blood seepage is realized. Subsequently, 1mm will be prepared ³ Is adhered to the scraping site with a histocryl adhesive, after the cecum has been dried in air for about 10 seconds, is carefully received back into the abdomen, the abdominal wall is sutured, and the mice are returned to their cage. Wherein 7 mice in the sham surgery group all underwent the same surgical procedure except for tumor attachment. All mice were routinely housed in SPF animal houses. The whole operation process follows the principle of aseptic operation, and antibiotics are not used after the operation.

3. Animal administration

Mice were randomly divided into 6 groups (n=10 per group): blank group, model group and each administration group, administration group mice are successful model making mice, and are treated by adopting a gastric lavage administration mode for 21 days, and the specific grouping is as follows:

(1) blank (normal saline);

(2) model group (physiological saline);

(3) kuh-seng single group (0.6 g/kg/d);

(4) radix Sophorae Flavescentis: coptis group (1:2) (0.6 g/kg/d);

(5) radix Sophorae Flavescentis: coptis group (1:4) (0.6 g/kg/d);

(5) cinobufagin tablet as positive medicine (0.624 g/kg/d)

4. Sampling and detecting index

The eyebox of the mouse is taken to obtain blood, and after the blood is taken, the blood is centrifuged for 10min at the temperature of 4 ℃ as soon as possible and at the speed of 10000rpm/min, and the supernatant is taken.

5. Weighing tumor seeds, calculating tumor inhibition rate and organ index

All mice were sacrificed at day 2 after the end of dosing with cervical dislocation, the in situ transplanted tumors were completely stripped, weighed, and the tumor inhibition rate calculated. Thymus and spleen weights were weighed. The results are shown in FIG. 2. 1:4 kuh-seng-coptis drug pair shows better activity against rectal cancer. 6. Tissue HE staining

Paraffin embedding and cutting into 4 μm tumor sections for later use. Paraffin sections dewaxed to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II 5min-75% ethanol 5min, and washing with tap water. Hematoxylin staining: the slices are stained with hematoxylin dye solution for 3-5min, washed with running water, differentiated with differentiation solution, washed with running water, returned to blue, and washed with running water. Eosin staining: the slices are dehydrated in gradient alcohol of 85% and 95% for 5min respectively, and then are dyed in eosin dye solution for 5min. And (3) removing the water sealing piece: sequentially slicing, adding absolute ethyl alcohol I5 min-absolute ethyl alcohol II5 min-absolute ethyl alcohol III 5 min-dimethyl I5 min-dimethyl II5min, and sealing with neutral resin. Microscopic examination, image acquisition and analysis.

Interpretation of the results: the nucleus is blue and the cytoplasm is red. The results are shown in FIG. 3. 1:4 kuh-seng-coptis drug pair shows better activity against rectal cancer.

The UPLC-TQ-MS detection method provided by the application can accurately detect 10 alkaloid components in the kuh-seng-coptis medicine pair test sample at the same time, and the quality control method of the kuh-seng-coptis medicine pair test sample and the extract thereof provided by the application has the advantages of high precision, high sensitivity, high stability and high accuracy, can objectively, comprehensively and accurately evaluate the quality of the kuh-seng-coptis medicine pair test sample medicinal material, the extract thereof and the preparation thereof, and has important significance for accurately controlling the quality of the kuh-seng-coptis medicine pair test sample medicinal material and the preparation thereof.

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.

Claims (1)

1. Application of radix Sophorae Flavescentis-Coptidis rhizoma extract in preparing medicine for resisting colon cancer; the kuh-seng-coptis root extract is prepared by the following method: soaking radix sophorae flavescentis and coptis chinensis medicinal materials in a weight ratio of 1:4 in a proper amount of water for 0.5 hour, then adding 10 times of water for extraction for 1-3 times, each time for 1-2 hours, combining filtrates, and concentrating to obtain the traditional Chinese medicine composition;

the quality detection method of the kuh-seng-coptis chinensis extract comprises the following steps:

preparation of the reference substance solution in step (1)

Accurately weighing sophocarpine, matrine, sophoridine, sophocarpine oxide, matrine oxide, coptisine, methyl coptisine, berberine, jateorhizine, palmatine hydrochloride, adding methanol to prepare mixed standard stock solutions with the concentration of 300, 600, 300 and 200 mug/mL respectively, preserving at 4 ℃, and passing through a 0.22 mu m microporous filter membrane before sample injection;

preparation of the sample solution in step (2)

Taking radix sophorae flavescentis and coptis chinensis medicinal materials in a weight ratio of 1:4, adding a proper amount of water to soak for 0.5 hour, then adding 10 times of water to extract for 1-3 times, each time for 1-2 hours, combining filtrates, concentrating until the crude drug concentration is about 1g/ml, and filtering with a microporous filter membrane before sample injection;

step (3) establishment of linear regression equation

Taking the mixed standard stock solution in the step (1), sequentially diluting 5 times, filtering through a 0.22 mm cellulose membrane to obtain a series of mixed reference substance solutions, sequentially injecting UPLC-TQ-MS, taking the series of reference substance concentrations as an abscissa X and the corresponding peak areas as an ordinate Y, performing linear regression analysis on each chemical component, and calculating a linear regression equation;

step (4) content measurement

Taking the kuh-seng-coptis root medicine in the step (2) to test the solution, injecting the solution into UPLC-TQ-MS for analysis, substituting the peak area into the linear regression equation in the step (3), and calculating the content of each component in the solution;

the chromatographic conditions of UPLC-TQ-MS in the step (3) and the step (4) are as follows:

chromatographic conditions: waters Acquity HSS T3 chromatography column; 0.1% formic acid aqueous solution is phase A, acetonitrile is phase B, gradient elution: 0-1 min,15% B, 1-5 min,15% B-50% B, 5-7 min,50% B, 7-10 min,50% B-15% B, 10-13 min,15% B; flow rate: 0.3mL/min; column temperature: 35. c°; sample injection amount: 2. mu L;

mass spectrometry conditions: electrospray ionization, multi-reaction monitoring ion scanning mode measurement, and main mass spectrum parameters are as follows: capillary voltage 5500V, desolventizing temperature 550 ℃; the positive ion detection mode and the negative ion detection mode are adopted, and the mass-to-charge ratios of the selected detected ions are respectively as follows: sophocarpine m/z 247.2/996.1, matrine m/z 249.2/148.3, sophoridine m/z 249.3/152.1, sophocarpine m/z 263.3/245.1, matrine oxide m/z 265.3/205.2, coptisine m/z 319.7/292.0, methylcoptisine m/z 334.2/261.1, berberine m/z335.9/320.2, jateorhizine m/z 337.9/322.4, palmatine hydrochloride m/z 352.0/336.1;

object to be measured De-clustering voltage/V Collision voltage/V Sophocarpine 40 40 Matrine 50 40 Sophoridine 50 40 Sophora fruit alkali oxide 40 20 Oxymatrine 76 40 Coptisine 48 30 Methyl coptisine 40 47 Berberine 48 30 Jatrorrhizine 22 22 Palmatine hydrochloride 40 47

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