CN116898906A - Application of haw extract in preparing medicine for treating cancer pain - Google Patents

Abstract

The application of the haw extract in preparing the medicine for treating cancer pain, wherein the haw extract comprises 0.21-0.25% of total flavone, 11-14% of total triterpene and 2.0-2.5% of total phenol, and the content of tannic acid in the haw extract is not higher than 2.5%. The research shows that the haw extract exists in nano crystal form and has higher bioavailability than the single active ingredient; the 10 hawthorn extracts taken each time are equivalent to eating 3120g hawthorn fruits, and can produce the effects of relieving and even treating cancer pain, especially refractory cancer pain.

Description

Application of haw extract in preparing medicine for treating cancer pain

Technical Field

The invention relates to the field of application of hawthorn extracts, in particular to application of hawthorn extracts in preparation of medicines for treating cancer pain.

Background

In recent years, the incidence of cancer in China has increased significantly, and pain is one of the most common and intolerable symptoms for cancer patients. Pain incidence rate of initial cancer patients is about 25%, and pain incidence rate of advanced cancer patients can reach 60% -80%, wherein 1/3 of patients are severe pain, and life quality of cancer patients can be seriously affected, so that pain relieving effect is important in cancer treatment process.

With the continuous progress and development of the palliative treatment discipline, the WHO three-step analgesic treatment principle and NCCN adult cancer pain guideline are mastered by doctors at all levels gradually, and 80% -90% of pain symptoms of tumor patients can be relieved through standard and effective treatment. However, 10% -20% of the patients still have pain which belongs to refractory cancer pain, and the conventional drug treatment is almost ineffective and is easy to cause intolerable adverse reactions, so that the pain is a troublesome problem faced by doctors and patients. Finding more effective analgesic means to enrich refractory cancer pain becomes an important direction of clinical research.

Modern pharmacological in vitro and in vivo experiments show that the hawthorn has various pharmacological effects of reducing blood fat, resisting cardiac muscle, resisting ischemia, resisting thrombus, resisting atherosclerosis, resisting inflammation, aiding digestion, resisting hypertension, resisting tumor, resisting depression, protecting nerves, reducing blood sugar and the like. Studies such as Can OD (optical density) and the like show that the hawthorn fruit extract Can increase the response time of mice to tail clamp (mechanical stimulation) and hot plate (thermal stimulation) stimulation in a dose-dependent manner, reduce the number of twists and stretching behaviors in a twister test (chemical stimulation), and prove that the hawthorn fruit extract has a certain analgesic effect on mechanical, thermal and chemical nociceptive nerve pathways.

Because the hawthorn is rich in tannic acid, the tannic acid can cause the combination and aggregation of protein, calcium and magnesium to form precipitate and easily form 'gastrolith', and the heavy metal content in the hawthorn is higher, so that the potential safety hazard exists when the hawthorn is eaten excessively; therefore, the intake of hawthorn is generally not more than 50 g/person/day, and the dosage of hawthorn in the traditional Chinese medicine formula is generally not more than 10g.

Disclosure of Invention

The invention provides an application of a haw extract in preparing a medicine for treating cancer pain.

Preferably, the cancer pain is refractory cancer pain. Preferably, the refractory cancer pain is caused by one of breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, esophageal cancer, gastric cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, and ovarian cancer.

Preferably, the active ingredient of the medicine is a haw extract, the haw extract comprises 0.21-0.25% of total flavone, 11-14% of total triterpene and 2.0-2.5% of total phenol, the content of tannic acid in the haw extract is not higher than 2.5%, and the content of heavy metal is not higher than 1.6mg/kg.

Preferably, the haw extract comprises 0.22-0.24% of total flavone, 12.5-13.5% of total triterpene and 2.1-2.4% of total phenol, and the content of tannic acid in the haw extract is not higher than 2.3%. Preferably, the haw extract comprises 0.22-0.23% of total flavone, 12.8-13.2% of total triterpene and 2.2-2.3% of total phenol, and the content of tannic acid in the haw extract is not higher than 2.2%.

Preferably, the ratio of total flavone to tannic acid in the haw extract is not less than 1:10. Preferably, the average particle size of the haw extract dissolved in the ultrapure water system is 103.18nm.

Preferably, the content of arsenic in the haw extract is not higher than 0.06mg/kg, the content of lead is not higher than 0.16mg/kg, and the content of mercury is not higher than 0.05mg/kg. Preferably, the content of arsenic in the haw extract is not higher than 0.04mg/kg, the content of lead is not higher than 0.07mg/kg, and the content of mercury is not higher than 0.01mg/kg.

Preferably, the preparation method of the haw extract comprises the following steps: s1, preprocessing hawthorn fruits to obtain primary hawthorn liquid; s2, performing microbial fermentation on the primary hawthorn liquid to obtain fermentation supernatant; s3, purifying by using a weak base anion exchange resin column, a weak polarity macroporous adsorption resin column, cation exchange resin and a chromatographic column; s4, concentrating and drying.

Preferably, in the step S3, the weak base anion exchange resin column is one of LX6702, LX6702 and LX67, the weak polar macroporous adsorption resin column is AB-8, the cation exchange resin is at least one of LSC-200, LSC-AS and LCS-400, and the chromatographic column is LXB2000 or LXB-20SS. Preferably, the capacity ratio of the weak base anion exchange resin column to the weak polarity macroporous adsorption resin column is 1:3-5.

Preferably, step S3 includes:

s31, filtering the fermentation supernatant by a ceramic membrane with the aperture of 0.5 mu m, and performing supersaturation adsorption on the filtrate by a weak base anion exchange resin column at the flow rate of 1.5-3.0 BV/h; the effluent is put on a macroporous adsorption resin column with weak polarity at the flow rate of 1.5-2.5BV/h, washed to be colorless and the pH value is 7-8, eluted with 70 percent ethanol at the flow rate of 0.5-0.7BV/h and the eluent is collected;

the tannic acid and part of the active ingredients are adsorbed to a weak base anion exchange resin column in the early stage of the upper column, wherein the adsorption capacity of the tannic acid to the tannic acid is stronger than that of the active ingredients, so that the tannic acid can replace the active ingredients to be adsorbed when the tannic acid is in a supersaturated adsorption state, and the active ingredients flow out along with effluent liquid, so that the active ingredients can be reserved as much as possible, and the amount of the tannic acid can be reduced; and then the effluent is put on a macroporous adsorption resin column, at the moment, most heavy metal ions flow out along with the effluent in the process of putting on the column, and the effluent is washed by deionized water until the pH value of the effluent is 7-8, so that most heavy metals in the residual resin gaps can be washed away.

S32, the eluting mixed solution is subjected to serial connection of cation exchange resin columns at the flow rate of 1.2-2.0BV/h, then water washing is carried out until no alcohol smell exists, and effluent liquid and water washing liquid are collected.

After the alcohol aqueous solution is eluted, a small amount of heavy metal still remains in the eluent, the alcohol phase is directly loaded with cation exchange resin, heavy metal ions are exchanged and adsorbed by the cation exchange resin, and active ingredients in the alcohol phase are basically not exchanged and adsorbed by the cation exchange resin, so that a good heavy metal removal effect is achieved.

S33, concentrating the collected effluent and the water washing liquid in vacuum under reduced pressure until no alcohol smell exists, and adding phosphoric acid to adjust the pH to 3.4-3.6; and (3) loading the mixture on a chromatographic column according to the volume of 8-12%, eluting and collecting other effluent liquid except tannic acid.

The treated effluent is concentrated under reduced pressure to almost no alcoholic smell, at which time the components are relatively single relative to the initial fermentation supernatant, and residual tannins can be separated and removed by chromatography, and other effluents are combined for subsequent treatment. The method can effectively remove tannic acid and heavy metals, and maximally retain active ingredients in the hawthorn, so that large-dose oral administration is possible.

Preferably, step S1 includes: selecting high-quality haw fruits, washing, putting the haw fruits into a crusher for crushing, respectively adding deionized water into haw fragments according to 100g:200-400ml, heating to 60-80 ℃ and preserving heat for 10-30min; cooling to 35-45 ℃ and then mixing according to 100g: adding pectase 0.01-0.05g, heating for 2.0-3.5 hr, separating solid from liquid, and collecting primary fructus crataegi liquid.

Preferably, step S2 is: the primary haw juice is pasteurized and inoculated with 4-6% Pichia pastoris, and the fermentation supernatant is collected after solid-liquid separation. Can greatly remove saccharide, pectin and other components in the hawthorn through fermentation

Pichia pastoris has good acid resistance and has low requirement on growth culture conditions; as the strain is fermented by using the primary haw juice, the ingredients such as monosaccharide, oligosaccharide, polysaccharide and the like in the haw juice can be consumed to the greatest extent, and the total flavone and total phenolic acid ingredients in the haw juice are hardly reduced, so that the active ingredient content in the haw extract is greatly improved, and the haw extract is equivalent to 390 g haw.

Preferably, step S4 is: concentrating the collected effluent liquid at 50-65deg.C under vacuum pressure of-0.07 to-0.095 MPa until the solid content is 18-25%, and drying to obtain fructus crataegi extract. The haw extract obtained by the above process exists in nano crystal form, and the average particle diameter of the haw extract dissolved in ultrapure water system is 103.18nm, which has higher bioavailability than the single active ingredient.

Preferably, the haw extract is prepared into tablets suitable for clinical use, the administration dosage is 10-60 tablets/day, and the tablet specification is 0.8 g/tablet.

The invention also provides a pharmaceutical preparation, which takes the haw extract as an active ingredient.

Compared with the prior art, the content of active ingredients such as flavonoid, total phenol, total triterpene and the like in the haw extract prepared by the invention is obviously higher than that of haw, and the content of tannic acid and heavy metal in the haw extract is low to an ideal level, so that the haw extract can be taken for a large amount for a long time without toxic and side effects, the effective action of the haw on human body is greatly enhanced, and obvious health care and medical effects are shown; the research shows that the haw extract exists in nano crystal form and has higher bioavailability than the single active ingredient; the 10 hawthorn extracts taken each time are equivalent to eating 3120g hawthorn fruits, and can produce the effects of relieving and even treating cancer pain, especially refractory cancer pain.

Drawings

FIG. 1 is a flow chart of a preparation process of the haw extract in the embodiment 1-3;

FIG. 2 is a particle size distribution of a sample prepared in example 4 of the present invention;

FIG. 3 shows the results of bioavailability of samples from the in vitro gastrointestinal digestion experiments of example 5 of the present invention, wherein orange, green, red and blue represent the flavone standard, polyphenol standard, hawthorn extract A (representing polyphenols in the extract), and Hawthorn extract B (representing flavones in the extract), respectively.

Detailed Description

In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. It should be noted that, on the premise of no conflict, the technical features in the embodiments of the present invention may be combined with each other.

As medicinal and edible traditional Chinese medicines, the traditional Chinese medicine theory records that hawthorn has the effects of promoting digestion, invigorating stomach, promoting qi circulation, removing blood stasis, resolving turbidity, reducing blood fat and the like, and modern clinical pharmacology also proves that the hawthorn has the effects of reducing blood fat, reducing blood pressure, resisting bacteria and the like and is widely applied to the fields of foods, health care products, cosmetics and the like; because heavy metals are gradually enriched and are not easy to metabolize in the growing process of hawthorns, when hawthorns polluted by the heavy metals are ingested, the heavy metals are absorbed by a human body and are gradually enriched, and when heavy metal poisoning occurs seriously, the damage to tissues and organs such as liver, kidney, spleen and stomach, nerves and the like is caused; in addition, the haw contains great amount of organic acid, fruit acid, haw acid, citric acid, etc. and has irritation to gastric mucosa to expand gastric pantothenic acid. In particular, tannic acid in hawthorn can be combined with gastric acid to form gastrolith, and stomach Dan Hennan is digested and accumulated more to cause gastric ulcer, gastrorrhagia and even gastric perforation, so that the hawthorn is eaten or has limited dosage, and the application of the hawthorn is limited.

Although only 10% -20% of cancer patients' pain belongs to refractory cancer pain, the daily activities, self-care ability, interaction ability and overall quality of life of the patients are seriously affected; the adverse reaction that the conventional drug treatment effect is almost ineffective and is easy to be intolerable is a 'soft rib' for cancer pain treatment. Although researches show that various compounds such as flavonoid, procyanidine and the like are separated and identified from hawthorn and have anxiolytic, sedative and analgesic effects, no evidence exists that the hawthorn and the procyanidine compound has an effect on cancer pain, especially refractory cancer pain.

Example 1

A preparation method of fructus crataegi extract comprises:

s1, preprocessing, selecting high-quality hawthorn fruits, washing, putting the hawthorn fruits into a crusher for crushing, respectively adding deionized water into 100 g/300 ml of hawthorn fragments, heating to 70 ℃, and preserving heat for 20min; cooling to 50 ℃ and then mixing according to 100g: adding pectase 0.02g, heating at constant temperature for 3.5 hr, adding perlite 2.0% for filtering, and collecting primary fructus crataegi liquid;

s2, fermenting, namely sterilizing the primary hawthorn liquid by Pasteur, inoculating 5% Pichia pastoris, fermenting and culturing for 25 hours, and collecting fermentation supernatant after solid-liquid separation;

s3, purifying the fermentation supernatant obtained in the step S3 by using a macroporous adsorption resin, namely, using a ceramic membrane with the aperture of 0.5 mu m, filtering and removing impurities at the temperature of 20 ℃ under the membrane pressure of 2MPa, and loading the filtrate on a weak base anion exchange resin column (model: LX6702, height-diameter ratio of 2:1 and resin consumption of 100 ml) at the flow rate of 4 BV/h; the effluent is put on a weak-polarity macroporous adsorption resin column (model: AB-8, height-diameter ratio 4:1, resin dosage 330 ml) at a flow rate of 1.2BV/h, washed with water at a flow rate of 2BV/h until the eluent is colorless and pH value is about 8, eluted with 70% ethanol aqueous solution at a flow rate of 0.5BV/h and collected, washed with water until no alcohol smell exists, collected water eluent, and mixed;

s4, purifying the eluting mixture obtained in the step S3 by cation exchange resin, namely, introducing the eluting mixture into a cation exchange resin column (model: LSC-200, diameter-to-height ratio of 1:4, resin dosage of 133 ml) at a flow rate of 1BV/h, then washing the eluting mixture with deionized water at a flow rate of 2BV/h until no alcohol smell exists, and collecting effluent.

S5, chromatographic purification, namely placing the effluent liquid obtained in the step S4 into a single-effect concentrator, concentrating under reduced pressure of-0.09 MPa at 60 ℃ until no alcohol smell exists, and adding phosphoric acid to adjust the pH to 3.5; eluting with 50% acetone at 0.1BV/h according to 10% bed volume of chromatographic column (model LXB2000, height-diameter ratio 5:1, resin dosage 1600 ml), and collecting other effluent except tannic acid;

s6, concentrating and drying the effluent liquid collected in the step S5 at 60 ℃ under the vacuum pressure of-0.09 MPa until the solid content is 20%, adding a proper amount of starch, uniformly mixing, granulating, drying, finishing, adding a proper amount of carboxymethyl starch sodium and magnesium stearate, uniformly mixing, tabletting, and coating with a film to obtain the finished product.

Through detection, the haw extract obtained in the embodiment contains 0.234% of total flavone, 11.3% of total triterpene and 2.1% of total phenol, the content of tannic acid in the haw extract is 1.17%, the removal rate is 99.4%, and the Pb content is as follows: 0.026mg/kg, arsenic content of 0.05mg/kg, mercury content of not more than 0.01mg/kg.

Example 2

A preparation method of fructus crataegi extract, as shown in figure 1, comprises:

s1, preprocessing, selecting high-quality hawthorn fruits, washing, putting the hawthorn fruits into a crusher for crushing, respectively adding deionized water into 100 g/400 ml of hawthorn fragments, heating to 75 ℃, and preserving heat for 15min; cooling to 40 ℃ and then mixing according to 100g: adding pectase 0.03g, heating for 2.5 hr, adding perlite 2.5%, filtering, and collecting primary fructus crataegi solution;

s2, fermenting, namely sterilizing the primary hawthorn liquid by Pasteur, inoculating 4.5% Pichia pastoris, fermenting and culturing for 27h, and collecting fermentation supernatant after solid-liquid separation;

s3, purifying the fermentation supernatant obtained in the step S3 by using a macroporous adsorption resin, filtering and removing impurities at the temperature of 25 ℃ under the pressure of 1.8MPa by using a ceramic membrane with the aperture of 0.5 mu m, and loading the filtrate on a weak base anion exchange resin column (model: LX6703, height-diameter ratio of 2.2:1 and resin consumption of 80 ml) at the flow rate of 3.5 BV/h; the effluent is put on a weak-polarity macroporous adsorption resin column (model: AB-8, height-diameter ratio 4:1, resin dosage 310 ml) at a flow rate of 1.2BV/h, washed with water at a flow rate of 1.8BV/h until the eluent is colorless and pH value is about 7.8, then eluted with 70% acetone aqueous solution at a flow rate of 0.6BV/h and the eluent is collected, then washed until no alcohol smell exists, the water eluent is collected, and the collected eluent is mixed;

s4, purifying the cation exchange resin, namely sequentially loading the elution mixed solution obtained in the step S3 on a cation exchange resin column (model: LSC-200, diameter-to-height ratio is 1:3 and resin dosage is 150 ml) at a flow rate of 1.2BV/h, then washing with deionized water at a flow rate of 2BV/h until no alcohol smell exists, and collecting effluent.

S5, chromatographic purification, namely placing the effluent liquid obtained in the step S4 into a single-effect concentrator, concentrating under reduced pressure of-0.08 MPa at 65 ℃ until no alcohol smell exists, and adding phosphoric acid to adjust the pH to 3.6; loading onto a chromatographic column (model LXB-20SS, height-diameter ratio 3:1, resin consumption 1200 ml) according to 8% of column bed volume, eluting with 50% acetone at 0.1BV/h, and collecting other effluent except tannic acid;

s6, concentrating and drying the effluent liquid collected in the step S5 at 55 ℃ under the vacuum pressure of-0.07 MPa until the solid content is 25%, adding a proper amount of starch, uniformly mixing, granulating, drying, finishing, adding a proper amount of carboxymethyl starch sodium and magnesium stearate, uniformly mixing, tabletting, and coating with a film to obtain the finished product.

Through detection, the haw extract obtained in the embodiment contains 0.219% of total flavone, 13.1% of total triterpene and 2.4% of total phenol, the content of tannic acid in the haw extract is 1.55%, the tannic acid removal rate is 99.5%, and the Pb content is as follows: 0.16mg/kg, arsenic content of 0.04mg/kg, mercury content of not more than 0.01mg/kg.

Example 3

A preparation method of fructus crataegi extract comprises:

s1, preprocessing, selecting high-quality haw fruits, washing, putting the haw fruits into a crusher for crushing, respectively adding deionized water into haw fragments according to 100 g/200 ml, heating to 72 ℃, and preserving heat for 24min; cooling to 45 ℃ and then mixing according to 100g: adding pectase 0.01g, heating for 2.5 hr, adding perlite 3%, filtering, and collecting primary fructus crataegi solution;

s2, fermenting, namely sterilizing the primary haw juice by Pasteur, inoculating 5.5% Pichia pastoris, fermenting and culturing for 23 hours, and collecting fermentation supernatant after solid-liquid separation;

s3, purifying the fermentation supernatant obtained in the step S3 by using a macroporous adsorption resin, namely, using a ceramic membrane with the aperture of 0.5 mu m, filtering to remove impurities at the temperature of 30 ℃ under the pressure of 1.7MPa, and loading the filtrate on a weak base anion exchange resin column (model: LX67, height-diameter ratio of 2.5:1 and resin consumption of 90 ml) at the flow rate of 3.0 BV/h; the effluent is put on a weak-polarity macroporous adsorption resin column (model: AB-8, height-diameter ratio 3:1, resin dosage 350 ml) at a flow rate of 1.5BV/h, washed with water at a flow rate of 2.5BV/h until the eluent is colorless and pH value is about 8, then eluted with 70% ethanol aqueous solution at a flow rate of 0.7BV/h and collected, then washed with water until no alcohol smell exists and the water eluent is collected, and the collected eluents are mixed;

s4, purifying the cation exchange resin, namely, serially connecting cation exchange resin columns (model: LSC-AS+LCS-400, height-diameter ratio is 2:1 and resin consumption is 100 ml) with the elution mixture obtained in the step S3 at the flow rate of 1.2BV/h, then washing with deionized water at the flow rate of 2.5BV/h until no alcohol smell exists, and collecting effluent.

S5, chromatographic purification, namely placing the effluent liquid obtained in the step S4 into a single-effect concentrator, concentrating under reduced pressure of-0.07 MPa at 58 ℃ until no alcohol smell exists, and adding phosphoric acid to adjust the pH to 3.4; loading onto a chromatographic column (model LXB200, height-diameter ratio 4:1, resin consumption 1200 ml) according to 12% of column bed volume, eluting with 60% ethanol at 0.2BV/h, and collecting other effluent except tannic acid;

s6, concentrating and drying the effluent liquid collected in the step S5 at 58 ℃ under the vacuum pressure of-0.07 MPa until the solid content is 22%, adding a proper amount of starch, uniformly mixing, granulating, drying, finishing, adding a proper amount of carboxymethyl starch sodium and magnesium stearate, uniformly mixing, tabletting, and coating with a film to obtain the finished product.

Through detection, the haw extract obtained in the embodiment contains 0.22% of total flavonoids, 12.6% of total triterpenes and 2.3% of total phenols, wherein tannic acid in the haw extract is 0.75%, the tannic acid removal rate is 99.7%, the Pb content is 0.054mg/kg, the As content is 0.03mg/kg, and the Hg content is not higher than 0.01mg/kg.

Example 4 characterization of Hawthorn extract

The haw extract prepared in example 2 was diluted 20 times with ultrapure water, stirred and dissolved at 200rpm for 30min, and treated in two ways: 1) Centrifuging the sample at 5000rpm and 25 ℃ for 3min, sucking the supernatant of the sample, and measuring the particle size data of the supernatant, and marking the data as A; centrifuging the supernatant at 14000rpm and 25deg.C for 3min, collecting supernatant, measuring particle diameter, denoted as B, redissolving the precipitate with the same solvent, and measuring particle diameter, denoted as C; 2) The particle sizes of the supernatant and the precipitate, respectively, were determined by centrifugation at 14000rpm at 25℃for 3min, and were designated D and E, respectively.

1ml of sample cores are respectively dripped into a standard sample cell (quartz cell), the sample cells are detected by a dynamic light scattering technology through Zetasizer NanoZS (Markov instruments, inc. in England, after the instrument is started, the laser is preheated for half an hour, so that the laser is stabilized), the measurement temperature is 25.0 ℃, the laser wavelength is 633nm, the measurement angle theta=173 DEG, the sample cells are leached by filtered solvent for more than three times before use, the measurement is repeated for more than 3 times, and the hydration particle size of a measured sample is obtained, and the results are shown in Table 1 and figure 2.

TABLE 1 particle size distribution Table of diluted samples

The average particle diameters of the sample A, B are 234.47nm and 103.18nm respectively, the light scattering intensity of the sample B is not obviously changed, and the sample belongs to colloidal particles with more stable dynamics, which indicates that the particle diameter of the nano crystal corresponding to the haw extract is obviously reduced by centrifugation at high rotating speed again. The light scattering intensity value is positively correlated with particle size and number, indicating that there may be larger particles disintegrated into more smaller particles during centrifugation. If the supernatant obtained by centrifugation at 14000rpm directly, i.e. sample D, had an average particle size of 92.77nm, which is similar to that of sample B, but the light scattering intensity value was much lower than that of sample B, the solution obtained by precipitation and reconstitution, i.e. sample E, had very low light scattering, indicating that most of the particles remained in the supernatant.

Example 5 determination of bioavailability of Hawthorn extract

The efficacy of food or herbal medicine is not only dependent on how much of the active (pharmaceutical) ingredient is dosed, but is also directly related to the form in which the active ingredient is present. The stability and final absorption degree of the active ingredient in the gastrointestinal tract in the free or combined form are significantly different, and the efficacy exertion of the active ingredient is directly affected. Self-assembled micro-nano particles (MNPs) in chicken soup, coffee, tea, food or traditional Chinese medicine decoction are composed of polar and nonpolar components and are formed by interaction of chemical reaction and physical factors in the processing process, and once discovered, the self-assembled micro-nano particles are widely paid attention to people.

5.1 Experimental details

Simulated digestion experiments were performed in a dynamic bionic-simulated gastrointestinal digestive system AGDS that simulates the real shape and structure of the human stomach by 3D digital technology, which uses two sets of symmetrical rollers and one set of oppositely directed rollers to simulate the peristalsis of the human stomach, using a pH-stat workstation (Titrando-Metrohm, switzerland) to control pH throughout the digestion.

Establishing a digestion environment according to an international standard simulation in vitro gastrointestinal digestion (INFOGEST standard protocol) published by Minekus et al; simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) were prepared from fresh stock solutions per day, kept at 37℃prior to use, with final concentration of porcine gastric mucosal pepsin at SGF of 2000U/mL, pancreatin from porcine pancreas and bile salts (porcine bile extract) dissolved in SIF at final concentrations of 100U/mL and 10mM, respectively.

The experimental temperature was kept constant at 37℃and 20mL of 3 samples (haw extract and standard polyphenol catechins, standard flavone quercetin) and 20mLSGF were mixed separately using a magnetic stirrer at a speed of 95rpm and the pH was controlled at 3.0 using a pH-stat station throughout the digestion to simulate the gastric digestion environment. After 2 hours, SIF (pH 7) was added to AGDS in a ratio of 1:1 (v/v), intestinal phase simulation was performed under the same conditions (37 ℃ C. And 95 rpm) for 2 hours, and the pH was controlled to 7.4 throughout. Aliquots of digests were taken every 0.5h for analysis purposes.

5.2 detection method

TPC was determined by the modified Folin-Ciocalteu method by ultraviolet-visible spectrophotometry, specifically by extracting with methanol under stirring (200 rpm) for 1 hour, then centrifuging the mixture for 10 minutes (10000 rpm), transferring 0.125mL of the supernatant to a cuvette, adding 0.5mL of distilled water and 0.125mL of LFOLInCiocalteu reactant, standing for 6 minutes, and adding 1.0mL of distilled water and 1.25mL of sodium carbonate. Incubation at room temperature for 90 minutes followed by measuring absorbance at 760nm wavelength and comparing absorbance with the standard curve of Gallic Acid (GA) gives the measured substance content.

TFC determination was performed by colorimetric method, i.e., 3.0g of the sample was mixed with 3.0mL of water, and the mixture was centrifuged at 10,000rpm for 10 minutes (MedifrigerBL-S, P-Selecta). Thereafter, 0.25mL of the supernatant was mixed with 1mL of distilled water and 0.075mL of a 5% sodium nitrite solution. After 6 minutes of standing, 0.15mL of an aluminum chloride solution was added, and after another 5 minutes, 0.5mL of sodium hydroxide (1M) and 2.0mL of distilled water were added, and absorbance was read at 510nm using a spectrophotometer (JASCOV-630), and the total content of flavonoids was expressed as catechin equivalents (mg) per gram of dry matter.

To assess the effect of food matrix on the changes in polyphenols, flavonoids and total antioxidant activity that occur during the in vitro digestion of the gastrointestinal tract, it was calculated as bioavailability (Bioaccessibility) =x/Y X100%, where X is (TPC (mgGA/g dry matter) or FC (mg catechin equivalents/g dry matter) in the sample after the dialysis phase and Y is TPC or TFC (mg) in the total digested sample after the dialysis phase and expressed in the same units.

5.3 experimental results

The bioavailability release kinetics of polyphenols, flavonoids and hawthorn extract A, B under simulated gastrointestinal conditions are shown in figure 3. Wherein the curve of the haw extract A is the biological accessibility judged by TPC, and the curve of the haw extract B is the biological accessibility judged by TFC. It can be seen that the bioavailability of the haw extract A, B is significantly higher than that of flavone and polyphenol. The bioavailability of haw extract A, B increased rapidly (18.0±1.7% and 21.0±0.6% respectively) during the first half of the SGF phase, gradually rising and slowing down over the following 1.5 hours. At the end of the SGF phase, the bioavailability reached 33.4±0.9% and 37.4±0.8%, respectively. In contrast, flavones and polyphenols were slowly released in the SGF phase, only 13.4±0.6% and 11.5±0.9% were released at the end of the SGF phase, respectively. The bioavailability increased abruptly in all samples when exposed to SIF at pH 7. At the end of SIF, the bioavailability of haw extract A, B is as high as 74.4±0.6% and 81.4±1.5%, respectively, while the bioavailability of flavone and polyphenol is only 30.4±0.2% and 25.8±1.6%. This shows that the nano crystal form of haw extract can raise the bioavailability of loaded biological activity effectively, and the absorption rate of polyphenol flavone in haw extract is over 2 times that of free purified polyphenol flavone.

EXAMPLE 6 therapeutic Effect of Hawthorn extract on refractory cancer pain

6.1 dosing regimen:

on the basis of normal regulation of analgesic scheme (1 st day of initial regulation) of clinicians, the dosage climbing study is carried out by combining haw extract on 2 nd day, wherein the haw extract dosage group comprises 10 tablets, 15 tablets, 20 tablets, oral administration twice a day, each tablet is 0.8g, which is equivalent to 312g haw fruit.

Dose escalation regimen "3+3": according to the 3+3 design, whether or not the latter dose group test is performed after completion of each metering group is judged according to the following rule:

(1) If no DLT appears in 3 subjects in a dose group, the dose group is increased to the following dose group; (2) If DLT appears in 2 or more subjects in 3 subjects in a certain dose group, the dose group is decremented to the previous dose group; (3) If 1 out of 3 subjects in a certain dose group show DLT, the dose group is supplemented with 3 subjects, if 1/6 subjects show DLT, the later dose group is increased, if not less than 2/6 subjects show DLT, the previous dose group is decreased; (4) When decreasing to the previous dose group, if this dose group had only 3 subjects, then 3 subjects were again supplemented. If there are 6 subjects in the dose group, the up-dosing trial is completed and the dose is the MTD.

MTD is defined as the MTD when more than or equal to 33% of subjects present in the higher dose group following the current dose group present DLT (i.e., 2/3 or 2/6) and 0/6 or 1/6 of subjects present in the current dose group present DLT.

Dose Limiting Toxicity (DLT) definition: adverse events (such as nausea and emesis, etc. can be improved by optimal support treatment or increased index of alkaline phosphatase, etc.) of CTCAE 3 degrees or more occur in the first 14 days of administration of the hawkthorn red extract; if a 1-case DLT event occurs in any one group, that group will extend to 6 subjects. If any of the groups had 2 cases of DLT events, the study was stopped.

6.2 inclusion criteria

1) Patients over 18 years of age with pathological diagnosis as malignant tumor; 2) Refractory cancer pain, i.e. the patient is present at the same time: 1) The digital score of the persistent pain is more than or equal to 4 minutes and/or the number of times of the breakthrough pain is more than or equal to 3 times/day; 2) Following the guidelines for the treatment of relevant cancer pain, patients treated with opioids alone and/or in combination with co-analgesic drugs are still not satisfied with pain relief and/or have intolerable adverse reactions for 1-2 weeks; 3) Patients who need chemotherapy, targeting, immunotherapy or bisphosphonate treatment after one week of elution undergo stable treatment; 4) No mental illness; 5) ECOG-PS is less than or equal to 2 minutes; 6) No drug test (including the test drug) was enrolled for 1 month prior to the test; 7) The subject voluntarily and signed an informed consent.

6.3 exclusion criteria

1) The patient is diagnosed with non-cancerous pain or pain of unknown cause; 2) Postoperative pain in the patient; 3) Paralytic ileus patients; 4) Brain metastasis leads to painful patients; 5) Allergic/addicted patients to opioids; 6) Abnormal and clinically significant laboratory results, such as creatinine ∈2.5 times greater than or equal to the upper limit of normal, ALT or AST ∈2.5 times greater than or equal to the upper limit of normal (liver metastasis patient or primary liver cancer ∈5 times greater than or equal to the upper limit of normal), or liver function Child C grade; 7) Patients who cannot take drugs orally; 8) Uncontrollable nausea and vomiting; 9) Patients with medical history such as gastritis and gastric ulcer; 10 Patients in need of analgesia with non-steroidal drugs and steroidal hormonal drugs; 11 A diabetic who is unable to control blood glucose stably; 12 Pregnant and lactating women; a subject with a pregnancy program (also including male subjects) within 1 month after the test; 13 Alcoholism patient; 14 A patient with cognitive dysfunction; 15 Major depressive patients.

Performing effect evaluation by using an Edmonton symptom evaluation system (ESAS), and performing security analysis on adverse event AE according to NCI (common acute and subacute toxicity grading Standard) which is classified into 1-5 grades (NCI-CTCAE 5.0); the hawthorn extract is effective and has less side effect on at least one case of patients with refractory cancer pain in the group.

Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.

Claims (7)

1. Application of fructus crataegi extract in preparing medicine for treating cancer pain is provided.

2. The use according to claim 1, wherein the cancer pain is refractory cancer pain.

3. The use according to claim 2, wherein the refractory cancer pain is caused by one of breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, esophageal cancer, stomach cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer.

4. The use according to any one of claims 1 to 3, wherein the active ingredient of the medicament is a haw extract, the haw extract comprises 0.21-0.25% total flavonoids, 11-14% total triterpenes, 2.0-2.5% total phenols, the tannins content in the haw extract is not higher than 2.5%, and the heavy metal content is not higher than 1.6mg/kg.

5. The use according to claim 4, wherein the content of arsenic in the haw extract is not higher than 0.06mg/kg, the content of lead is not higher than 0.16mg/kg, and the content of mercury is not higher than 0.05mg/kg.

6. The use according to claim 5, wherein the preparation method of the haw extract comprises: s1, preprocessing hawthorn fruits to obtain primary hawthorn liquid; s2, performing microbial fermentation on the primary hawthorn liquid to obtain fermentation supernatant; s3, purifying by using a weak base anion exchange resin column, a weak polarity macroporous adsorption resin column, cation exchange resin and a chromatographic column; s4, concentrating and drying.

7. The use according to claim 1, wherein the haw extract is formulated into a tablet suitable for clinical use at a dosage of 10-60 tablets/day and a tablet specification of 0.8 g/tablet.