CN115040548A

CN115040548A - Halocynthia Roretzi antitumor extract and preparation method and application thereof

Info

Publication number: CN115040548A
Application number: CN202210236605.5A
Authority: CN
Inventors: 董波; 祝玉婷; 高宏伟; 李建辉; 韩善浩; 于海燕
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2022-09-13
Anticipated expiration: 2042-03-11
Also published as: CN115040548B

Abstract

The invention provides an echinocandin antitumor extract and a preparation method and application thereof, the echinocandin activity extract is prepared by boiling homogenate of the echinocandin, then filtering and collecting upper suspension, dialyzing the upper suspension, and collecting the external water solution of a dialysis bag; separating the dialyzate by normal phase silica gel column chromatography, eluting with a mixed solution of ethyl acetate, methanol and formic acid as a flowing phase, and collecting ethyl acetate: methanol: concentrating and drying the eluent with formic acid at a ratio of 5:1:0.02 to obtain the active extract of the inner capsule of the Halocynthia Roretzi. The invention prepares an extract with high-efficiency anti-tumor activity and low toxicity to normal cells from Halocynthia Roretzi. The experimental result shows that the echinococcus eupatorium extract has proliferation inhibition activity on various tumor cells, induces the tumor cells to generate apoptosis, has synergistic antitumor effect with doxorubicin, and only shows limited influence on normal cells under the action of single or combined action with doxorubicin.

Description

Halocynthia Roretzi antitumor extract and preparation method and application thereof

Technical Field

The invention belongs to the technical field of natural active substance preparation, and particularly relates to an ascidian antitumor extract, and a preparation method and application thereof.

Background

Halocynthia roretzi belongs to the phylum chordata (Chordates), the subdivision Urochordata (Urochordata), the class Ascidiacea (Ascidiacea). Halocynthia Roretzi is rich in various nutrients essential to human body, and is popular with people of Japan and Korea.

Halocynthia Roretzi not only contains abundant mineral elements and various rare amino acids, but also contains a large amount of polyunsaturated fatty acids, especially EPA and DHA, and has effects of regulating blood lipid, inhibiting platelet aggregation, lowering blood pressure, improving biological membrane liquid state, resisting tumor, resisting inflammation, regulating immunity, promoting smooth muscle contraction, preventing arteriosclerosis, and preventing senile dementia. As a marine product rich in nutrients, research on the efficacy of marine products has been increasing in recent years.

With the continuous improvement and deepening of people on the understanding of marine resources, a large number of active substances with natural structures in the sea are gradually discovered, the compounds with the natural structures often have good medicinal values, have very important effects of resisting tumors, microorganisms, viruses, immunoregulation and the like, and are still mostly not utilized by human beings, and the natural active products of marine organisms are expected to become effective and specific good medicines for treating human diseases. At present, over twenty or more marine derived anti-tumor compounds have entered clinical research phase worldwide, where three drugs, cytarabine from the sponge Cryptotethia cyprta, the analog eribulin of Halichondrin B in the sponge Halichondrial okadai, and trabectedin from ascidian Ecteinascidia turbinates, have completed clinical phase trials approved for clinical treatment of different cancer diseases.

Disclosure of Invention

The invention provides an ascidian antitumor extract, a preparation method and an application thereof, and the provided ascidian endocyst active extract has the effects of inhibiting tumor cell proliferation and causing tumor cell apoptosis. The active extract provided by the invention has a synergistic effect with broad-spectrum anti-tumor drug doxorubicin, and can be used for preparing an auxiliary drug and treating cancer together with doxorubicin.

The invention firstly provides a sea squirt extract with anti-tumor efficacy, which is prepared by boiling homogenate of an inner sac of the sea squirt, then filtering and collecting upper suspension, dialyzing the upper suspension, and collecting dialysate; separating the dialysate by normal phase silica gel column chromatography, eluting with mixed solution of ethyl acetate, methanol and formic acid as mobile phase, collecting ethyl acetate: methanol: concentrating and drying the eluent with formic acid at a ratio of 5:1:0.02 to obtain the active extract of the inner capsule of the Halocynthia Roretzi.

As a concrete description of the embodiment, the interception specification of the dialysis bag used in the dialysis is 100-500D;

the normal phase silica gel column is 300-400 meshes;

the invention also provides an application of the prepared active extract of the inner capsule of the Halocynthia Roretzi, which is an application in preparing anti-tumor products;

the invention also provides the application of the prepared active extract of the inner capsule of the Halocynthia Roretzi and the broad-spectrum antitumor drug in preparing antitumor products;

the broad-spectrum anti-tumor drug is doxorubicin.

The invention also provides a product for inhibiting tumor cell proliferation and inducing tumor cell apoptosis, wherein the product contains the active extract of the echinocandis scaber inner capsule with pharmacological efficacy concentration;

furthermore, the preparation also comprises a broad-spectrum anti-tumor drug;

the broad-spectrum anti-tumor drug is doxorubicin.

The provided articles may further comprise any one or more of 7-hydroxycoumarin, erucamide, stearamide, cetyl and dimethyl phthalate.

The invention prepares an extract with high-efficiency anti-tumor activity and low toxicity to normal cells from Halocynthia Roretzi. The experimental result shows that the echinococcus eupatorium extract has proliferation inhibition activity on various tumor cells, induces the tumor cells to generate apoptosis, has synergistic antitumor effect with doxorubicin, and only shows limited influence on normal cells under the action of single or combined action with doxorubicin. The active component of the anti-tumor extract provided by the invention is a mixture of 7-hydroxycoumarin and several fatty amides. The method for extracting the antitumor active substances has the advantages of mild extraction conditions and simple and convenient process.

Drawings

FIG. 1: halocynthia Cylindrica anatomical diagram, wherein bar is 1.5 cm.

FIG. 2: antitumor activity profile of Halocynthia Roretzi inner capsule activity extract (WB-H-S1), wherein FIG. 2-1 shows that WB-H-S1 has proliferation inhibition effect on various tumor cells and shows dose dependence; FIG. 2-2 shows the toxicity test of WB-H-S1 on non-tumor cells (mouse fibroblast L929). P < 0.05.

FIG. 3: the Echinocactus giganteus inner sac active extract (WB-H-S1) causes apoptosis of tumor cells, wherein FIG. 3-1 is a flow analysis chart; fig. 3-2 is a data statistics map. P < 0.01

FIG. 4: the proliferation inhibition of the hepatoma carcinoma cells (HepG 2) is realized by combining the ascidian tun active extract (WB-H-S1) with the antitumor drug Doxorubicin (DOX), wherein the proliferation inhibition of the HepG2 cells is realized by combining WB-H-S1 with different concentrations and Doxorubicin (DOX) with the concentration of 5 mu g/mL in a figure 4-1; FIG. 4-2 shows the proliferation inhibition of HepG2 cells by different concentrations of WB-H-S1 in combination with 10. mu.g/mL Doxorubicin (DOX); FIGS. 4-3 show the cytotoxicity of 100. mu.g/mL Echinacea purpurea inner capsule active extract (WB-H-S1) in combination with 10. mu.g/mL Doxorubicin (DOX) in mouse fibroblasts. P < 0.05, P < 0.01.

FIG. 5: high performance liquid chromatography analysis chart of Echinacea purpurea inner sac active extract (WB-H-S1).

FIG. 6: isolation and characterization of active compounds from the Echinocactus giganteus active extract (WB-H-S1), wherein FIG. 6-1 is the inhibition of proliferation of HepG2 cells by each of the liquid phase fractions (a-l) and the remaining fractions (r), FIG. 6-2 is a chemical structural diagram of active compounds,. P < 0.01; fig. 6-3 is the chemical structural formula of erucamide, stearamide, hexadecamamide, and a kind of carboxylic ester, dimethyl phthalate contained in the extract.

Detailed Description

The applicant researches and discovers that the water extract of the inner sac of the Halocynthia Roretzi has the activity of obviously inhibiting the proliferation of tumor cells. Active ingredients in the extract are separated and purified through dialysis and silica gel column chromatography, a simple and feasible preparation method is explored, the finally obtained active ingredients have certain proliferation inhibition activity on tumor cells to different degrees, the tumor cells are induced to undergo apoptosis, and the extract and doxorubicin have a synergistic anti-tumor effect. Further analyzing the structure of the main active ingredients in the extract by ultra performance liquid chromatography tandem mass spectrometry.

The preparation method of the active extract of the inner sac of the Halocynthia Roretzi provided by the invention comprises the following steps:

1. preparing a material to be extracted from Halocynthia Roretzi:

squeezing out a plurality of tunicaria intestinalis inner sacs, shearing into pieces and homogenizing.

2. Water extraction method is used for preparing water extract of the inner sac of Halocynthia Roretzi.

Mixing the homogenate of the inner capsule of the ascidians cecostata with pure water, putting the mixture into a container, decocting until the water is boiled and lasts for a period of time, filtering residues, collecting upper suspension, and concentrating to obtain an aqueous extract of the ascidians cecostata;

wherein the homogenate of the inner sac of Halocynthia Roretzi can be mixed with other solvent commonly used in active substance extraction, and boiled.

3. Preparing small molecular solution of water extract of Halocynthia Roretzi inner capsule by dialysis.

Separating the water extract of the inner sac of the Halocynthia Roretzi by using a dialysis bag with the interception specification of 100-500D, collecting the water solution outside the dialysis bag, and concentrating to obtain the micromolecule components of the water extract of the inner sac of the Halocynthia Roretzi.

4. Separating the components with antitumor activity from the small molecular components of water extract of Halocynthia Roretzi inner capsule by silica gel column chromatography.

Separating the sample by using 300-400 mesh normal phase silica gel in the step 3), and according to the sample to be separated: silica gel powder is filled into the column according to the proportion of 1: 50. Using an ethyl acetate/methanol/formic acid ternary system as a mobile phase, and carrying out gradient isocratic elution (EtOAc-MeOH-CH2O3(5:1:0.02 → 4:1:0.02 → 3:1:0.02 → 2:1:0.02 → 1:1:0.02) → MeOH) and collecting the eluent of the system EtOAc-MeOH-CH2O3 ═ 5:1:0.02, concentrating and drying to obtain the active extract (WB-H-S1) of the tunica ectenes endocystis.

Through analysis, the extract of the invention is found to contain 7-hydroxycoumarin, erucamide, stearamide, hexadecane amide and several fatty amides and a carboxylic ester, dimethyl phthalate.

The prepared active extract of the inner sac of the Halocynthia Roretzi has obvious activity of inhibiting the proliferation of tumor cells, can be used for preparing products for inhibiting the proliferation of the tumor cells and causing the apoptosis of the tumor cells, and the products contain the active extract of the inner sac of the Halocynthia Roretzi with pharmacological efficacy concentration; the preparation can also contain broad-spectrum antitumor drugs, for example, the doxorubicin preparation can also contain 7-hydroxycoumarin, erucamide, stearamide, hexadecane dimethyl phthalate.

The method of the present invention is described in detail below with reference to examples and the accompanying drawings.

Example 1

The active extract (WB-H-S1) of the inner sac of the Halocynthia Roretzi is prepared by separating the water extract of the inner sac of the Halocynthia Roretzi, and experiments such as inhibition of proliferation of various tumor cells show that the extract has anti-tumor proliferation activity and low toxicity to normal cells.

1. Preparation of Echinocactus giganteus active extract (WB-H-S1)

1) Collecting Halocynthia Roretzi, culturing in clean seawater tank for 2-3 days, discharging silt in intestinal tract, cleaning outer sheath, dissecting to obtain Halocynthia Roretzi inner sac (figure 1), placing in clean container, and crushing and homogenizing.

2) Putting the homogenate liquid of the inner sac of the Halocynthia Roretzi obtained in the step 1) and pure water in a ratio of 1:3 into a container, decocting until the water is boiled, continuing for 30min, filtering for multiple times through 100-mesh silk yarn to remove residues, collecting filtrate, and concentrating the collected filtrate at 95 ℃ under reduced pressure until the volume of the filtrate is 1/20 times of the original volume to obtain the water extract of the inner sac of the Halocynthia Roretzi.

3) Ultrafiltering the water extract of the inner sac of the Halocynthia Roretzi obtained in the step 2) by using a dialysis membrane with the molecular weight cutoff of 100Da, fixing one section of a dialysis bag by using a dialysis clamp, adding a proper amount of water extract of the inner sac of the Halocynthia Roretzi from the other end, fixing by using the dialysis clamp, and then putting into pure water, wherein the ratio of the water extract to the pure water is 1: 20, dialyze overnight with stirring purified water. The large molecules are trapped inside the dialysis bag and the small molecule compounds are shuttled to the portion of the aqueous solution outside the dialysis bag. After dialysis, collecting the external liquid of the dialysis bag, concentrating under reduced pressure at 95 deg.C, evaporating to dryness, weighing, and dissolving with pure water to obtain active components of water extract of Halocynthia Roretzi inner capsule.

4) And (3) fully and uniformly mixing the active component obtained in the step 3) with 200-mesh silica gel powder of 300 meshes, and drying the mixture (adding a small amount of sample into the silica gel powder for multiple times), wherein the mass ratio of the active component to the silica gel is 1: 3. According to the sample to be separated: silica gel powder is filled into the column according to the proportion of 1: 100. Using an ethyl acetate/methanol/formic acid ternary system as the mobile phase, gradient isocratic elution (EtOAc-MeOH-CH2O3(5:1:0.02 → 4:1:0.02 → 3:1:0.02 → 2:1:0.02 → 1:1:0.02) → MeOH), each gradient eluting at 2.5 column volumes, and the collection system was EtOAc-MeOH-CH2O3 ═ 5:1:0.02 eluent, i.e. the first elution gradient. Concentrating the collected eluate at 55 deg.C under reduced pressure, weighing, and dissolving with methanol to obtain Halocynthia Roretzi inner capsule active extract (WB-H-S1).

2. Detecting the antitumor activity of Halocynthia Roretzi inner capsule active extract (WB-H-S1).

MTT method for detecting proliferation inhibition of tumor cells by extract

1) Digesting logarithmic phase cells with pancreatin, centrifuging and collecting after terminating, making into cell suspension, counting cells, adjusting concentration to 5-10 × 10 ⁴ and/mL. After the cell suspension is prepared, the cell suspension is gently mixed, and the mixture is added into a 96-well plate, 100uL of the cell suspension is added into each well, and the density of the cells to be detected is 5000-10000/well.

2) And (3) placing the inoculated cell culture plate into an incubator for culture until a cell monolayer is paved on the bottom of a 96-well plate, and adding a medicine with a concentration gradient. Each drug was provided with 5 multiple wells. When adding drugs, drugs with different concentrations and culture media are mixed well in an EP tube, then culture supernatants in a 96-well plate are removed, and 100 mu L of culture media containing drugs with different concentrations are added into each well.

3) 5% CO2, incubating at 37 deg.C for 48 hr, and observing the effect of the medicine under an inverted microscope.

4) A solution of DMED (containing 0.5mg/mL MTT) was added to each well and incubation in the incubator was continued for 4 h.

5) The culture was terminated and the crystals were prepared for dissolution. Add 150. mu.L of dimethyl sulfoxide into each well, and shake for 10min at low speed on a shaking bed to dissolve the crystals sufficiently. The absorbance of each well was measured at OD 490nm in an ELISA detector. The magnitude of the absorbance represents the density of the number of viable cells remaining in each well.

Annexin V/PI double staining is used for detecting apoptosis. In the early stage of apoptosis, membrane Phosphatidylserine (PS) turns from the inner side of lipid membrane to the outer side. Annexin V is a phospholipid binding protein and thus can bind to the cell membrane of early apoptotic cells via extracellular exposure of phosphatidylserine. During apoptosis, membrane phosphatidylserine eversion occurs earlier than nuclear changes, and Propidium Iodide (PI) cannot pass through the cell membrane at this stage. When the cell is in late apoptosis or necrosis, the phosphatidylserine is turned out, but the permeability of the cell membrane is obviously increased, and the nucleic acid dyes such as PI enter the cell to be combined with the nucleic acid in the cell nucleus to emit red fluorescence. Annexin V-FITC (green fluorescence) is therefore often used in combination with a nucleic acid dye (PI) to identify dead or viable cells, to distinguish early apoptotic cells from late apoptotic or dead cells, and a flow detector to quantify the staining of each cell.

3. Halocynthia Roretzi inner sac active extract (WB-H-S1) for inhibiting tumor cell proliferation

The live extract of the inner sac of Halocynthia Roretzi (WB-H-S1) was mixed in culture medium at 100, 200, 300, 400 μ g/mL concentration, and incubated with five different tumor cell lines (cervical cancer HeLa cell, liver cancer HepG2 cell, fibroma HT-1080 cell, melanoma B16F2 cell, thyroid cancer BHT101 cell) for 48 hours, and the results are shown in FIG. 2-1, in which the proliferation of five cells was inhibited to different degrees. And five tumor cells were almost cell-free to survive with 48 hours of co-incubation when the sample was at a greater concentration. In contrast, WB-H-S1 did not cause significant inhibition of the proliferative growth of normal cells, i.e., mouse fibroblasts (FIGS. 2-2), indicating that it is less toxic to normal cells.

4. The Echinacea purpurea inner sac active extract (WB-H-S1) causes tumor cell apoptosis

After the active extract (WB-H-S1) of the Halocynthia rudis cyst is treated with HepG2 cells for 24 hours at the concentration of 200 mug/mL, Annexin V/PI is used for staining the cells, and then the detection result of a flow cytometer shows that the proportion of early apoptosis cells, namely cells with single positive Annexin V staining, is increased by 21.19 percent through the treatment of WB-H-S1, the number of living cells is reduced by 21.4 percent (figure 3-1 and figure 3-2), and the statistics of three experimental results show that the significant difference shows that the active extract (WB-H-S1) of the Halocynthia rudis caused to apoptosis in the tumor cells.

5. The combination of the Halocynthia Roretzi inner capsule active extract (WB-H-S1) and doxorubicin enhances the tumor activity of doxorubicin.

The Echinocactus auricula active extract (WB-H-S1) was combined with doxorubicin at two different concentrations (5, 10. mu.g/mL) at 50, 70, 90, 100, 150. mu.g/mL, respectively. The results show (figure 4-1; figure 4-2) that the active extracts at different concentrations can significantly enhance the inhibitory activity of the anticancer drug doxorubicin on HepG2 cells, and the synergistic effect of the two is stronger with the higher concentration. In contrast, the combination of the two enhanced the inhibition of tumor cells, but did not alter the toxicity of doxorubicin to normal cells (FIGS. 4-3). These experiments show that the combination with the active extract can reduce the dosage of doxorubicin and the side effects of anticancer drugs while maintaining the original inhibitory activity.

Example 2: isolation of Echinacea purpurea inner sac active extract (WB-H-S1) and identification of active compound

Semi-preparative high performance liquid phase was used to isolate the echinococcus eupatorium extract (WB-H-S1) under the conditions described in Table 1.

Table 1: semi-preparative liquid phase gradient elution system table

Time min	A Pump (%)	B Pump (%)
			0	5	95
10	10	90
			25	20	80
30	70	30
			45	100	0

The structure of the active compound is identified by using ultra high performance liquid tandem secondary mass spectrometry, wherein mobile phases are an aqueous solution (A liquid) containing 0.1% formic acid and 100% methanol (B liquid) containing 0.1% formic acid, and a mobile phase in a negative ion mode is an aqueous solution (A liquid) containing 10mM ammonia formate and 95% methanol (B liquid) containing 10mM ammonia formate. The chromatographic gradients are shown in table 2 below.

Table 2: ultra-high performance liquid phase gradient elution system table

Time min	A Pump (%)	B Pump (%)
			0	90	10
1	90	10
			13	2	98
18	70	30
			18.5	90	10
20	90	10

The mass spectral parameters are shown in table 3.

Table 3: high resolution mass spectrum detection parameter table

Scanning mode	Positive ion scanning
		Detection mode	Full mass/dd-MS2
Resolution ratio	70000(Full mass)；17500(dd-MS2)
		Electrospray voltage	3.8kV(Positive)
Capillary temperature	300℃
		Flow rate of sheath gas	40Arb
Temperature of atomizer	350℃

In order to find the active ingredient of WB-H-S1, a semi-preparative high performance liquid extract was used for systematic separation and purification. The fingerprint spectrum presented by gradient elution shows 12 groups of peaks, each peak is correspondingly numbered as a-l (figure 5), fractions a-l corresponding to the peaks are respectively collected, and the solvent is removed. In addition, the remaining elution fraction (r) is collected in addition to the fraction eluted by the main peak, in order to avoid possible active ingredient leakages. Dissolving the collected components with corresponding solvents respectively, and detecting the tumor cell proliferation inhibition activity of the components obtained by liquid phase separation by using HepG2 cells and an MTT method. The results showed that the compounds with peak e and peak j have inhibitory activity against HepG2 cells (FIG. 6-1). The e and j peaks of the separately collected compounds were identified by UHPLC-MS/MS, and the e peak was identified as 7-hydroxycoumarin and the j peak was identified as a mixture containing erucamide, stearamide, hexadecylamide, several fatty amides and one carboxylate, dimethyl phthalate, by comparing retention time, secondary mass spectrum fragment ions to standard databases (fig. 6-2, 6-3).

The above are specific embodiments of the present invention, which are only exemplary cases, and the present invention is not limited to the above embodiments. Any equivalent modifications and substitutions to the practice are also within the scope of the invention. Accordingly, any equivalent alterations, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. An Halocynthia Roretzi extract with anti-tumor effect is prepared by boiling homogenate of inner bag of Halocynthia Roretzi, filtering, collecting upper layer suspension, dialyzing, and collecting water solution outside dialysis bag; separating the dialysate by normal phase silica gel column chromatography, eluting with mixed solution of ethyl acetate, methanol and formic acid as mobile phase, collecting ethyl acetate: methanol: concentrating and drying the eluent with formic acid at a ratio of 5:1:0.02 to obtain the active extract of the inner capsule of the Halocynthia Roretzi.

2. The Halocynthia Roretzi extract as claimed in claim 1, wherein the cut-off size of the dialysis bag used in dialysis is 100D-500D.

3. The Halocynthia Roretzi extract as claimed in claim 1, wherein the normal phase silica gel column is 300-400 mesh normal phase silica gel column.

4. Use of Halocynthia Roretzi extract as claimed in claim 1 in the preparation of anti-tumor preparation.

5. Use of Halocynthia Roretzi extract as claimed in claim 1 in combination with a broad-spectrum antitumor agent for preparing an antitumor preparation.

6. The use according to claim 5, wherein the broad spectrum anti-neoplastic drug is doxorubicin.

7. A preparation for inhibiting tumor cell proliferation and inducing tumor cell apoptosis, comprising the Halocynthia Roretzi extract of claim 1 in a pharmacologically effective concentration.

8. The article of claim 7, further comprising a broad spectrum anti-tumor drug.

9. The article of manufacture of claim 8, wherein said broad spectrum anti-neoplastic agent is doxorubicin.

10. The article of claim 7, further comprising 7-hydroxycoumarin, erucamide, stearamide, cetyl, and dimethyl phthalate.