Preparation method of Chinese angelica medicinal composition nanosuspension and myocardial ischemia resisting and blood enriching effects of Chinese angelica medicinal composition nanosuspension

Abstract

The invention relates to the field of Chinese angelica medicinal composition nanosuspension traditional Chinese medicine preparations, in particular to preparation of a Chinese angelica medicinal composition nanosuspension taking vitamin E polyethylene glycol succinate as an auxiliary material and application research of the Chinese angelica medicinal composition nanosuspension in myocardial ischemia resistance, blood enriching, antipyretic and analgesic. The preparation method comprises the following steps: taking 1g of Chinese angelica medicinal slices and 5g of astragalus membranaceus medicinal slices, adding water for reflux extraction, concentrating to obtain a concentrated solution, uniformly mixing the concentrated solution with a vitamin E polyethylene glycol succinate aqueous solution, adding a rotor and zirconium oxide beads, magnetically stirring, uniformly grinding, and sucking out the suspension to obtain the Chinese angelica medicinal composition nano suspension. The Chinese angelica medicinal composition nanosuspension disclosed by the invention is simple in preparation process and good in slow-release effect, tests show that the Chinese angelica medicinal composition nanosuspension can improve the effects of resisting myocardial ischemia and anemia, relieving fever and easing pain and the like, no organic reagent is used in the whole process, the medicine safety is high, and the market application and popularization prospects are relatively good.

Description

Technical Field

[0001] The present application relates to a preparation method of a nano-suspension of an angelica pharmaceutical composition and its effects of anti-myocardial ischemia, blood nourishing, antipyretic and analgesic effects. Background Art

[0002] Qi and blood are essential substances for constructing the body and maintaining life activities. The relationship between qi and blood and the body's health, diseases and life is extremely close. Red blood cells in the blood have functions such as oxygen transportation and immunity. Oxygen is an important factor in life activities. Most of the energy for human life activities is provided by aerobic metabolism, and oxygen cannot be stored in the human body. Therefore, the oxygen transportation function of red blood cells is of great significance to human life activities. Hypoxia is also an important inducement for many diseases, and the improvement of diseases also requires regulating metabolism. At present, blood-nourishing drugs mainly include genetically engineered drugs, chemical drugs, traditional Chinese patent medicines, etc. Clinically, there are mainly compound polysaccharide iron, dextran iron, and blood-generating oral liquid, etc. Sucrose iron and sodium gluconate iron are used more abroad, and the main domestic chemical drugs are polysaccharide iron complex, ferrous succinate, etc. There are only two domestic companies that have been approved to market ferrous lactate capsules. Traditional Chinese patent medicines with Chinese medicinal materials such as donkey-hide gelatin and angelica as the monarch drug have always dominated the consumption. The nano-suspension of the angelica pharmaceutical composition has an obvious improvement effect on the fluidity and deformability of red blood cells in hypoxic mice. The nano-suspension of the angelica pharmaceutical composition has an obvious improvement effect on the functions of red blood cells in acute and chronic hypoxic states. In addition to increasing the material basis of blood, it can achieve the dual effects of supplementing qi and generating blood by improving the functions of red blood cells. The nano-suspension of the angelica pharmaceutical composition can significantly inhibit the ischemic changes in the electrocardiogram of rats induced by pituitary posterior lobe hormone. At the same time, it has good effects on acetic acid writhing and carrageenan-induced paw swelling in rats, and has an inhibitory effect on the pain response caused by peripheral inflammation, indicating that it also has antipyretic and analgesic effects while nourishing blood.

[0003] The nano-drug delivery system has become one of the research hotspots in modern pharmaceutics and is widely used in the delivery of hydrophobic drugs. As a good functional nano-carrier, the nano-drug delivery system is widely used in the pharmaceutical field, which can increase the efficacy of poorly soluble pharmaceutical compositions, improve the targeting and biocompatibility of pharmaceutical compositions, reduce the toxic and side effects of pharmaceutical compositions, prolong the circulation time of pharmaceutical compositions in the body, and resist multi-drug resistance.

[0004] The nano-suspension pharmaceutical composition is formed by adding a small amount of surfactant to the particles of the pharmaceutical composition and dispersing them in a dispersion medium to form a dispersion of the pharmaceutical composition, which is used to improve the clinical application of poorly soluble pharmaceutical compositions and has the advantages of high drug loading and less excipient dosage.

[0005] Vitamin E polyethylene glycol succinate is a safe pharmaceutical excipient recognized by the US FDA. It has amphiphilic properties and can be used as an emulsifier, solubilizer, etc. in pharmaceutical preparations. In this invention, an Angelica sinensis pharmaceutical composition is prepared into a nano - preparation to improve the bioavailability of the drug in vivo. Summary of the Invention

[0006] The present invention provides a nano - suspension of an Angelica sinensis pharmaceutical composition using vitamin E polyethylene glycol succinate as an excipient, with a simple process, less excipient dosage, and good sustained - release effect.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing a nano - suspension of an Angelica sinensis pharmaceutical composition using vitamin E polyethylene glycol succinate as an excipient, with a simple process, less excipient dosage, and good sustained - release effect.

[0009] (1) Take 1 g of Angelica sinensis cut - pieces and 5 g of Astragalus membranaceus cut - pieces, reflux - extract with water twice, each time for 1 hour, combine the extraction solutions, and concentrate to 1:1 (ml:g) to obtain a concentrated solution (i.e., aqueous phase I);

[0010] (2) Dissolve vitamin E polyethylene glycol succinate in water to obtain aqueous phase II.

[0011] (3) Adopt the medium grinding method to combine the drug carriers, and after dilution, obtain the final nano - suspension of the Angelica sinensis pharmaceutical composition.

[0012] The nano - suspension of the pharmaceutical composition prepared by the present invention through this method can increase the release rate of poorly - soluble drugs in vivo, improve the distribution of drugs in vivo, and enhance the anti - myocardial ischemia, blood - enriching, antipyretic and analgesic effects of the drugs.

[0013] The above - mentioned preparation method also includes freeze - drying: adding 2% of a freeze - drying protectant to the nano - suspension of the Angelica sinensis pharmaceutical composition to extend the storage time of the drug; among them, the freeze - drying time is 12 hours, and the vacuum pressure is 0.15 mbr.

[0014] Preferably, the water used in step (1) is distilled water. The Angelica sinensis pharmaceutical composition and vitamin E polyethylene glycol succinate are preferably in a mass ratio of 1:1, 2:1, 4:1, 6:1, and the best ratio is 4:1. The concentration of the Angelica sinensis pharmaceutical composition in the nano - suspension is 4.5 - 5.5 mg / ml, the ultrasonic power is 100 w, and the ultrasonic temperature is 60 °C.

[0015] Preferably, in step (3), the grinding medium is zirconia beads with a diameter of 0.4 - 0.6 mm, the dosage is 10 g, the magnetic stirrer is 2 cm, the rotation speed is 300 - 400 r / min, and the water - bath temperature is 60 °C.

[0016] In addition, the residual liquid medicine on the zirconia beads should be diluted and fixed in volume after ultrasonic oscillation to reduce the loss of drug amount caused by drug adsorption due to grinding wear of the zirconia beads, and the drug content per unit volume is increased to the greatest extent.

[0017] The present invention prepares a nano-suspension of Angelica drug composition by using the medium grinding method, screens the optimal prescription, and performs freeze-drying to make a preparation, maintaining the storage stability of the nano-suspension of Angelica drug composition. The nano-suspension of Angelica drug composition has effects such as antipyretic, analgesic, anti-myocardial ischemia, and blood-enriching. The present invention conducts research on the effects of antipyretic, analgesic, anti-myocardial ischemia, and blood-enriching of the nano-suspension of Angelica drug composition.

[0018] The preparation method of the drug of the present invention is further illustrated by the following examples.

[0019] Example: Preparation of nano-suspension of Angelica drug composition

[0020] 1) The embodiment of the present invention discloses a method for preparing a nano-suspension of Angelica drug composition with vitamin E polyethylene glycol succinate as a carrier. The preparation process is simple, and the obtained nano-suspension of Angelica drug composition has a high drug loading amount and a good sustained-release effect.

[0021] The present invention is further specifically described by the following examples.

[0022] Example: Preparation of nano-suspension for enriching blood with Angelica

[0023] The nano-suspension of Angelica blood-enriching drug composition is prepared by using the medium grinding method. First, the drug-to-carrier ratio is screened. According to the mass ratio of the concentrated solution to vitamin E polyethylene glycol succinate of 1:1, 2:1, 4:1, and 6:1 respectively, they are dispersed in water with 10 ml of the concentrated solution and ground at room temperature for 1 hour. The particle sizes of different nano-suspensions are measured, and the best drug-to-carrier ratio is screened as 4:1.

[0024] The mixture with a mass ratio of Angelica drug composition to vitamin E polyethylene glycol succinate of 4:1 is ground at a rotation speed of 300 r / min at water bath temperatures of 0 °C, 30 °C, 60 °C, and 80 °C respectively for 1 hour. When the temperature is 60 °C, the particle size of the drug mixture is the smallest, so 60 °C is selected as the temperature for preparing the nano-suspension of Angelica blood-enriching drug composition.

[0025] For the selection of the grinding time, the time is set to 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h to prepare the nano-suspension with a mass concentration of 1 mg / mL. As the grinding time increases, the particle size gradually becomes smaller, and the PDI gradually increases after 1.5 h. Therefore, the grinding time is selected as 2 h to prepare the suspension.

[0026] Under the conditions that the drug mass ratio is 4:1, the grinding temperature is 60 °C, and the grinding time is 2 h, the rotation speeds of 200, 250, 300, and 350 r / min are respectively set to investigate the rotation speed of the magnetic stirrer. When the rotation speed is greater than or equal to 300 r / min, the particle size is better, so 300 r / min is selected as the rotation speed.

[0027] In summary, the optimal preparation process of the Angelica drug composition nano-suspension is as follows: using the medium grinding method, first add 8 mg of vitamin E polyethylene glycol succinate to 4 mL of water, heat in a water bath at 60 °C, and dissolve by ultrasonic wave. Then add 8 ml of the concentrated solution into a vial, add the vitamin E polyethylene glycol succinate solution into the vial, shake the vial, add a rotor and zirconia beads, heat in a water bath at 60 °C, rotate at 300 r / min, and stir for 2 h. Aspirate the suspension to obtain the Angelica drug composition nano-suspension. Detailed implementation mode

[0028] The following experimental examples are used to further elaborate the efficacy of the Angelica drug composition nano-suspension described in the invention. These experimental examples include the anti-myocardial ischemia, blood-enriching, and antipyretic and analgesic effect tests of the drug of the present invention (hereinafter referred to as the drug suspension).

[0029] 1. Research on the antipyretic and analgesic mechanism of the inventive drug suspension

[0030] Test drug: Drug suspension, provided by the Preparation Research Laboratory of Jilin Academy of Chinese Medicine. Preparation method: Prepare the drug suspension into the required concentration with distilled water before use, shake well at room temperature and administer by gavage.

[0031] Experimental animals: 48 Balb / c mice, 8 weeks old, half male and half female, provided by the Experimental Animal Center of Shandong University, license: SCXK(Lu)2022-0001. Raised in a clean environment, fed with feed and water, and the temperature of the animal room is 25±2 °C.

[0032] Reagents: Glacial acetic acid, Chengdu Kelong Chemical Reagent Factory, batch number: 20210721; Sustained-release morphine hydrochloride tablets, Southwest Pharmaceutical Co., Ltd. of Taichi Group, batch number: 21210047; Aspirin enteric-coated tablets, Bayer Healthcare Co., Ltd., batch number: BJ20648; Prednisone acetate, Zhejiang Xianju Pharmaceutical Co., Ltd., batch number: 220621; LPS (Lipopolysaccharides from Escherichia coil055:B5), Lot#046M4021V, SIGMA Company, USA; 2,4-dinitrophenol (DNP), Chengdu Kelong Chemical Reagent Factory, batch number: 2022110101; Xylene, Chengdu Kelong Chemical Reagent Factory, batch number: 2021082301.

[0033] Experimental instruments: PV-200 Rat Toe Swelling Instrument, Chengdu Taimeng Technology Co., Ltd.; RB-200 Hot Plate Instrument, Chengdu Taimeng Technology Co., Ltd.; MC-347 Electronic Thermometer, Omron (Dalian) Co., Ltd.; BT125D Electronic Analytical Balance, Sartorius AG, Germany.

[0034] (1) Effects of drug suspension on LPS-induced fever in rats

[0035] After rats were intraperitoneally injected with 50 μg / kg of LPS, the body temperature of rats in the model group reached the maximum at the 4th hour after modeling, with an average temperature increase of 1.23 °C. Aspirin could significantly reduce the fever body temperature of rats induced by LPS, and there were statistically significant differences in temperature reduction compared with the model group at 0.5, 2, 4, and 6 h after modeling; the drug suspension also had a similar effect. Among them, the high-dose group had obvious effects at 2, 4, and 6 h after modeling, and the low-dose group had obvious effects at 4 and 6 h after modeling. The temperature reduction values were statistically different from those of the model group. At other time points, each dose of the drug suspension also showed a certain temperature reduction effect.

[0036] Table 1 Effects of drug suspension on LPS-induced fever in rats

[0037]

[0038]

[0039] Note: Compared with the control group * P < 0.05, ** P < 0.01

[0040] (2) Effects of drug suspension on 2,4-dinitrophenol-induced fever in rats

[0041] After 40 min of drug administration, except for the control group, after the remaining groups of rats were subcutaneously injected with 15 mg / kg of 2,4-dinitrophenol, the body temperature of rats in the model group was significantly increased compared with the control group at each body temperature measurement time point. Each dose of the drug suspension could significantly slow down the rising speed of the body temperature of rats induced by 2,4-dinitrophenol, and the difference was statistically significant compared with the model group. Aspirin had no obvious antipyretic effect on this model.

[0042] Table 2 Effects of drug suspension on 2,4-dinitrophenol-induced fever in rats (x-±s)

[0043]

[0044] Note: Compared with the control group * P < 0.05, ** P < 0.01

[0045] (3) Effects of the drug suspension on writhing response in mice induced by glacial acetic acid

[0046] SPF-grade KM mice, male, were adaptively fed for 2 days and randomly divided into 5 groups, namely the control group, the aspirin group at 50 mg / kg, and the drug suspension groups at 150 mg / kg, 100 mg / kg, and 25 mg / kg. They were given drugs by gavage, once in the morning and once in the afternoon every day for a total of 3 times. The administration volume was 0.1 ml / 10 g. The control group was given the corresponding volume of vegetable oil. One hour after the last administration, each group of mice was intraperitoneally injected with 0.7% (0.1 ml / 10 g) glacial acetic acid. The number of writhing responses of the mice within 5 - 20 min after intraperitoneal injection of glacial acetic acid was observed. The writhing response was counted once when abdominal depression, body distortion, and hind limb extension occurred simultaneously, and the analgesic inhibition rate was calculated.

[0047] Analgesic inhibition rate (%) = (average number of writhing responses in the control group - average number of writhing responses in the drug group) / average number of writhing responses in the control group × 100%

[0048] The test results showed that compared with the control group, the number of writhing responses of each group of mice decreased; and there were statistical differences between the aspirin group and the drug suspension group at 300 mg / kg and the control group. Moreover, for the three dose groups of the drug suspension at 50, 200, and 300 mg / kg, there was a certain dose - effect relationship between the analgesic inhibition rate and the administration dose.

[0049] Table 3 Effects of the drug suspension on writhing response in acetic acid-induced mice

[0050]

[0051] Note: Compared with the control group * P < 0.05, ** P < 0.01

[0052] (4) Effects of the drug suspension on pain response to heat stimulation in mice

[0053] SPF-grade KM mice, 18 - 22 g, female. After 2 days of adaptive feeding, they were screened. Before the first administration, the pain threshold of the mice before drug administration was measured. Fifty qualified mice were selected and randomly divided into 5 groups according to the average pain threshold before drug administration, namely the control group, the morphine hydrochloride group at 10 mg / kg, and the drug suspension groups at 300 mg / kg, 200 mg / kg, and 50 mg / kg. They were given drugs by gavage, 3 times in the morning and 3 times in the afternoon every day. The administration volume was 0.1 mml / 10 g. The control group was given the corresponding volume of vegetable oil by gavage. At 0.5, 1, and 2 h after the last administration, the mice were successively placed in the bell jar of the hot plate instrument to measure the pain threshold of heat stimulation at each time point after the last drug administration (the extreme value of the pain threshold of the mice after drug administration was 60.00 s).

[0054] Test results: In the hot plate experiment of mice, the positive drug morphine hydrochloride had a significant analgesic effect. The drug suspension at a dose of 300 mg / kg had a certain effect of increasing the pain threshold at each measurement time point, and there were statistical differences at 0.5 h and 2 h after drug administration.

[0055] Table 4 Effects of drug suspension on pain threshold of hot plate mice

[0056]

[0057]

[0058] Note: Compared with the control group * P < 0.05, ** P < 0.01.

[0059] (5) Effects of drug suspension on ear swelling in mice induced by xylene

[0060] Fifty SPF-grade KM mice, male, weighing 18 - 22 g, were randomly divided into 5 groups, namely the model group, the group treated with prednisone acetate at 10 mg / kg, and the groups treated with drug suspension at 300 mg / kg, 200 mg / kg, and 50 mg / kg. Each drug group was intragastrically administered the corresponding drug, and the model group was intragastrically administered the drug suspension with the corresponding volume. The drugs were administered once in the morning and once in the afternoon every day for a total of 3 times. Thirty minutes after the last drug administration, 30 μl of xylene was evenly applied to both the front and back sides of the left ear of each mouse to cause inflammation, and the right ear was not treated as a control. Thirty minutes after inflammation induction, the mice were sacrificed by cervical dislocation. The left and right ear pieces were taken with a puncher with a diameter of 8 mm and weighed. The swelling degree was represented by the difference in the weights of the left and right ear pieces, and the swelling inhibition rate was calculated.

[0061] Ear swelling degree = weight of left ear piece - weight of right ear piece

[0062] Ear swelling inhibition rate (%) = (average ear swelling degree of the model group - average ear swelling degree of the drug group) / average ear swelling degree of the model group × 100%

[0063] Test results: The average ear swelling degree of the model group was 25.27 mg, and the model was successfully established. The prednisone acetate drug group had a good anti-inflammatory effect, and there was a statistical difference compared with the model group. Each dose group of the drug suspension also had a good anti-inflammatory effect, and the difference was statistically significant compared with the model group.

[0064] Table 5 Effects of drug suspension on ear swelling in mice induced by xylene

[0065]

[0066] Note: Compared with the control group * P < 0.05, ** P < 0.01.

[0067] (6) Effect of the drug suspension on carrageenan-induced paw swelling in rats

[0068] SPF-grade SD rats, male, weighing 180 - 220 g, were randomly divided into 5 groups with 10 rats in each group, namely the model group, the prednisone acetate group at 7 mg / kg, and the drug suspension groups at three doses of 300 mg / kg, 200 mg / kg, and 50 mg / kg. Rats in each drug group were intragastrically administered the corresponding drug, and rats in the model group were intragastrically administered the Angelica drug suspension of the corresponding volume. Each rat was administered once in the morning and once in the afternoon every day for a total of 3 times. Before the last administration, the volume of the right hind paw of each group of rats before inflammation was measured. 30 minutes after the last administration, each group of rats was subcutaneously injected with 1% carrageenan at a volume of 0.1 ml / rat in the right hind paw for inflammation induction. At 1, 2, 3, and 4 hours after inflammation induction, the volume of the right hind paw after inflammation was measured with a foot volume measuring device, and the difference between the volume measured before and after inflammation was used as the degree of paw swelling.

[0069] The test results showed that at each measurement point after inflammation induction, the paws of rats in the model group were continuously swollen. Prednisone acetate showed obvious anti-inflammatory effects and a relatively long duration. Each dose group of the drug suspension had significant anti-swelling effects at each measurement time point. This indicates that the drug suspension has good curative effects on carrageenan-induced paw swelling in rats.

[0070] Table 6 Effect of the drug suspension on carrageenan-induced paw swelling in rats

[0071]

[0072] Note: Compared with control group 5 * P < 0.05, ** P < 0.01.

[0073] Results and discussion: In this study, it was found that the drug suspension had good antipyretic effects on the fever induced by LPS and 2,4-dinitrophenol, and aspirin's inhibition of COX had no effect on this model. The drug suspension had good effects on acetic acid-induced writhing and carrageenan-induced paw swelling in rats, inhibited the pain response caused by peripheral inflammation (acetic acid-induced writhing response in mice), and also had good preventive effects on the central pain model (mouse hot plate experiment).

[0074] 2. Study on the anti-myocardial ischemia effect of the invented drug suspension in rats

[0075] Drugs and reagents: Drug suspension, provided by Jilin Academy of Traditional Chinese Medicine, batch number 20230124. Prepared with 0.5% CMC to the required concentration for animal gavage and stored in a refrigerator at 4°C. Xiaoxintong Tablets, Isosorbide Dinitrate Tablets, produced by Tianjin Pacific Pharmaceutical Co., Ltd., batch number 221206. Prepared with 0.5% CMC to the required concentration for animal gavage and stored in a refrigerator at 4°C. Diao Xinxuekang Capsules, produced by Chengdu Diao Pharmaceutical Group Co., Ltd., batch number 220908. Prepared with 0.5% CMC to the required concentration for animal gavage. Stored in a refrigerator at 4°C. Posterior pituitary hormone injection, produced by Shanghai Hefeng Pharmaceutical Co., Ltd., batch number 220302. The content is 6 units / 1ml. Prepared with normal saline to the required concentration for intravenous administration to rats. Stored in a refrigerator at 4°C.

[0076] Animals: Wistar rats, Beijing Weitonglihua Experimental Animal Technology Co., Ltd., Animal Certificate No.: SCXK (Beijing) 2022-0001. The animals were kept in an observation room controlled by a central air-conditioning system, with a room temperature of 25±2℃ and artificial lighting for 12h / day. They were kept in separate cages, 43×27×18cm, 5 rats per cage, free access to water, fed with rat chow, and fresh tap water once a day.

[0077] Instrument: MP-150 data acquisition system, a product of BIOPAC, USA. ECG100 electrocardiogram amplification module, a product of BIOPAC, USA.

[0078] Animal screening: Healthy Wistar rats (220-250g), male, were anesthetized with 12% chloral hydrate (360mg / kg, ip), fixed in supine position, needle electrodes were inserted subcutaneously in the limbs, the signal was amplified by ECG100, and the II lead electrocardiogram (ECGII) was recorded. The signal was converted into a digital signal by the MP-150 data acquisition system and stored in the computer, and the J point and T wave amplitude were measured by the software AcqKnowledgev.3.8.2; after the normal ECGII recording was completed, posterior pituitary hormone (0.75U / kg) was injected into the sublingual vein, and 1min ECGII was recorded. Rats with J point changes (elevation or decrease) exceeding 0.08mv were selected, and formal experiments were carried out 24h later.

[0079] 2.1 Study on anti-myocardial ischemia effect

[0080] Experimental method: Qualified rats were selected and randomly divided into 7 groups, with 10 rats in each group. The normal control group and the model control group were given 0.5% CMC by gavage. The 3 test substance groups were given drug suspensions by gavage at doses of 3, 10, and 30 mg / kg, respectively. The 2 positive drug groups were given isosorbide dinitrate and Dioscoreae bulbiferae Rhizoma by gavage at doses of 3.2 and 63 mg / kg, respectively, both equivalent to the clinical dosage, and the administration volume was 10 ml / kg for all. 1 hour after administration, the animals were anesthetized with 12% chloral hydrate (360 mg / kg, ip), fixed in the supine position, and the ECGII was recorded using the same method as above. Then, pituitrin (0.75 U / kg) was injected into the sublingual vein to induce myocardial ischemia, and the ECGII was recorded at 10, 20, 30, 40, 50, 60 s, 2, 5, 10, and 20, 30 min after modeling.

[0081] Statistical analysis: The experimental data were expressed as mean ± standard deviation (x-±s), and the t-test was used to compare the significance of the difference in mean values between groups.

[0082] Experimental results:

[0083] (1) Effect on the change of J point of electrocardiogram

[0084] After pituitrin was given to rats, the J point of the electrocardiogram changed significantly (elevated or depressed), indicating obvious myocardial ischemia. The drug suspensions (10 - 30 mg / kg) could also produce obvious anti-myocardial ischemia effects. The maximum changes in J point at 3, 10, and 30 mg / kg inhibited -16.9%, 6.8%, and 44.1% respectively, and the AUC 0-30min decreased by -0.2%, 52.5%, and 71.4% respectively. The positive drugs isosorbide dinitrate (3.2 mg / kg) and Dioscoreae bulbiferae Rhizoma (63 mg / kg) both produced obvious anti-myocardial ischemia effects. The maximum changes in J point inhibited 55.1% and 53.4% respectively, and the AUC 0-30min decreased by 83.4% and 74.9% respectively.

[0085] Table 7 Effects of drug suspensions on the maximum change of J point of electrocardiogram in rats with myocardial ischemia induced by pituitrin ( n = 10)

[0086]

[0087] Note: 1. Compared with the normal control: △ P < 0.05, △△ P < 0.01, △△△ P < 0.001;

[0088] 2. Compared with the model control: * P < 0.05, ** P < 0.01, ***P < 0.001.

[0089] Table 8 Effects of drug suspension on the AUC of J-point changes in electrocardiogram of rats with myocardial ischemia induced by pituitrin 0-30min ( n = 10)

[0090]

[0091] Note: 1. Compared with the normal control: △ P < 0.05, △△ P < 0.01, △△△ P < 0.001; 2. Compared with the model control: * P < 0.05, ** P < 0.01, *** P < 0.001.

[0092] (2) Effects on the changes in T-wave height of electrocardiogram

[0093] After the rats were given pituitrin, the height of the T-wave in the electrocardiogram changed significantly (increased or inverted), indicating obvious myocardial ischemia. The drug suspension (10 - 100 mg / kg) could produce obvious anti-myocardial ischemia effects. The maximum inhibitions of T-wave changes at 10, 30, and 100 mg / kg were 32.5%, 45.0%, and 63.9% respectively, and the areas under the T-wave change curves (AUC) 0-30min decreased by 34.0%, 73.3%, and 60.1% respectively. The drug suspension (3 - 30 mg / kg) could also produce obvious anti-myocardial ischemia effects. The maximum inhibitions of T-wave changes at 3, 10, and 30 mg / kg were 13.6%, 30.2%, and 60.9% respectively, and the AUC 0-30min decreased by 22.7%, 62.4%, and 83.2% respectively. The positive drugs isosorbide dinitrate (3.2 mg / kg) and dioscoreae bulbiferae total saponins (63 mg / kg) both produced obvious anti-myocardial ischemia effects. The maximum inhibitions of T-wave changes were 53.8% and 63.3% respectively, and the AUC 0-30min decreased by 72.5% and 96.0% respectively.

[0094] Table 9 Effects of drug suspension on the maximum changes in T-wave height of electrocardiogram in rats with myocardial ischemia induced by pituitrin n = 10)

[0095]

[0096] Note: 1. Compared with the normal control: △ P < 0.05, △△ P < 0.01, △△△ P < 0.001; 2. Compared with the model control: *P < 0.05, ** P < 0.01, *** P < 0.001.

[0097] Table 10 Effects of drug suspensions on the change value of T-wave height AUC of electrocardiogram in rats with myocardial ischemia induced by pituitrin 0-30min ( n = 10)

[0098]

[0099]

[0100] Note: 1. Compared with the normal control: △ P < 0.05, △△ P < 0.01, △△△ P < 0.001; 2. Compared with the model control: * P < 0.05, ** P < 0.01, *** P < 0.001.

[0101] Test conclusion:

[0102] (1) Drug suspensions at 3, 10, and 30 mg / kg can also inhibit the change of J point. The inhibition rates of the maximum change are -16.9%, 6.8%, and 44.1% respectively, and the inhibition rates of the change value AUC 0-30min are -0.2%, 52.5%, and 71.4% respectively. The positive drugs isosorbide dinitrate at 3.2 mg / kg and dioscoreae bulbiferae saponins at 63 mg / kg can also significantly inhibit these two phenomena. The inhibition rates of the maximum change are 55.1% and 53.4% respectively, and the inhibition rates of the change value AUC are 83.4% and 74.9% respectively.

[0103] (2) Drug suspensions at 3, 10, and 30 mg / kg can also inhibit the change of T-wave height. The inhibition rates of the maximum change are 13.6%, 30.2%, and 60.9% respectively, and the inhibition rates of the change value AUC 0-30min are 22.7%, 62.4%, and 83.2% respectively. The positive drugs isosorbide dinitrate at 3.2 mg / kg and dioscoreae bulbiferae saponins at 63 mg / kg can also significantly inhibit these two phenomena. The inhibition rates of the maximum change are 53.8% and 63.3% respectively, and the inhibition rates of the change value AUC 0-30min are 72.5% and 96.0% respectively.

[0104] (3) Compared with the positive drugs, the effect of the drug suspension at 50 mg / kg is equivalent to that of isosorbide dinitrate at 3.2 mg / kg and dioscoreae bulbiferae saponins at 63 mg / kg.

[0105] All the drug suspensions could significantly inhibit the ischemic changes in the electrocardiogram of rats induced by pituitary posterior lobe hormone. They not only reduced the maximum change values of the J point and T wave, but also had an inhibitory effect on the AUC of the change values of the J point and T wave within 30 min. 0-30min This indicates that both drugs have anti-myocardial ischemia activity.

[0106] 3. Mechanism study of drug suspensions in the treatment of anemia

[0107] Experimental animals: Wistar rats (SPF grade), male, 54 rats, weighing 200 - 220 g; KM mice (SPF grade), half male and half female, 60 mice, weighing 22 - 25 g. All animals were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., with the production license number SCXK (Jing) 2022 - 1 - 0001, and were housed in the IVC animal room of the Department of Pharmacology, School of Basic Medicine, Hebei Medical University. The experimental animal use license number was SYXK (Ji) 2022 - 0023.

[0108] Instruments and drugs: Sysmex XT - 2000i automatic hematology analyzer (SYSMEX Corporation, Japan); Carboplatin injection (batch number: WB2J2203008, Qilu Pharmaceutical Co., Ltd.); Ejiao Blood - nourishing Oral Liquid (batch number: 220316, Shandong Fujiao Group Co., Ltd.); Shengxue Tiaoyuan Granules (batch number: 221106, China National Pharmaceutical Corporation Guangdong Huanqiu Pharmaceutical Co., Ltd.); Kang'ai Injection (batch number: 210407, Changbai Mountain Pharmaceutical Co., Ltd.); Diyu Shengbai Tablets (batch number: 221205, Chengdu Di'ao Group Tianfu Pharmaceutical Co., Ltd.); Xuesusheng Granules (batch number: 220901, Hebei Yongfeng Pharmaceutical Co., Ltd.).

[0109] Test methods:

[0110] Mouse acute hemorrhagic model: 60 Kunming mice were randomly divided into 5 groups, with 12 mice in each group, half male and half female. They were respectively set as blank control group A, blank control group B, model control group, drug suspension group (experimental group), and Ejiao Buxue Koufuye group (positive control group). The dosing doses were calculated according to the body surface area based on the maximum clinically recommended doses of each drug. The drug suspension was suspended with 0.5% sodium carboxymethylcellulose, and the dosing dose was 6 g / kg body weight; the dosing dose of Ejiao Buxue Koufuye was 10 mL / kg body weight; the blank control group and the model control group were both intragastrically administered normal saline at 0.1 mL / 10 g body weight. Each group was administered once a day for 15 consecutive days, and the modeling started on the 7th day after administration. For the mice in the model control group and each dosing group, starting from the day of modeling, blood was drawn from the fundus venous plexus at 0.15 mL / 10 g, once every other day for a total of 3 times, and the modeling was completed. For the first blood draw, the blood indexes of the model control group, drug suspension group, and Ejiao Buxue Koufuye group were detected; for the 3rd blood draw, the blood indexes of blank control group A, model control group, drug suspension group, and Ejiao Buxue Koufuye group were detected, and the animals in blank control group A were sacrificed; on the 4th day after the completion of modeling (the 8th day from the start of modeling, the 15th day of administration), 0.20 mL of blood was drawn from the fundus venous plexus to detect the blood indexes of blank control group B, model control group, drug suspension group, and Ejiao Buxue Koufuye group, and the animals were sacrificed.

[0111] Observation indicators: Using Sysmex XT-2000i, select the manual mode for classification detection; the blood sample is the whole blood of the animal, anticoagulated with EDTAK 2 (40:1, v / v), and the detection is completed within 2 hours after blood collection. Five items including white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), platelets (PLT), and reticulocytes (RET) were selected as evaluation indicators. At the same time, the mental state and body weight changes of the animals were recorded every day. The data were analyzed using SPSS 22.0 statistical software, and the repeated measures analysis of variance was used for the comparison of measurement data. P < 0.05 was considered statistically significant.

[0112] Test results:

[0113] (1) Mouse acute hemorrhagic model

[0114] Changes in WBC of mice in each group: The WBC levels in each group first increased and then decreased. There was no statistically significant difference in the interaction between groups and between groups·time points (P > 0.05), while there was a statistically significant difference in time points (P < 0.05), as shown in Table 11.

[0115] Table 11 Effects of drug suspension on WBC in mice with hemorrhagic anemia

[0116]

[0117] Note: F between groups = 2.259, P > 0.05; F at time points = 7.346, P < 0.05

[0118] Changes in RBC and Hb of mice in each group: The levels of RBC and Hb in each group first decreased and then increased. There were significant differences in the interactions between groups, time points, and between groups and time points (P < 0.05). See Tables 12 and 13.

[0119] Table 12 Effects of drug suspension on RBC in mice with hemorrhagic anemia

[0120]

[0121] Note: F between groups = 2.301, P < 0.05; F at time points = 97.869, P < 0.05

[0122] Table 13 Effects of drug suspension on Hb in mice with hemorrhagic anemia

[0123]

[0124] Note: F between groups = 27.166, P < 0.05; F at time points = 95.536, P < 0.05

[0125] Changes in PLT of mice in each group: The levels of PLT in each group first increased and then decreased. There were significant differences in the interactions between groups, time points, and between groups and time points (P < 0.05). See Table 14.

[0126] Table 14 Effects of drug suspension on PLT in mice with hemorrhagic anemia

[0127]

[0128] Note: F between groups = 3.393, P < 0.05; F at time points = 15.907, P < 0.05

[0129] Changes in RET of mice in each group: The levels of RET in each group gradually increased. There were significant differences in the interactions between groups, time points, and between groups and time points (P < 0.05). See Table 15.

[0130] Table 15 Effects of drug suspension on RET in mice with hemorrhagic anemia

[0131]

[0132] Note: F between groups = 33.397, P < 0.05; F at time points = 125.382, P < 0.05

[0133] (2) Chemotherapy-induced anemia model in rats

[0134] Experimental method: 54 male Wistar rats were randomly divided into 6 groups of 9 rats each, which were set as blank control group, model control group, drug suspension group, Shengxue Tiaoyuan Granule group, Kang'ai Injection group, and Diyu Shengbai Tablet group. The dosage of each drug was calculated according to the body surface area based on the maximum clinically recommended dose. Solid drugs were suspended in 0.5% sodium carboxymethylcellulose and administered by gavage at a dose of 1 mL / 100 g body weight. The dosage of the drug suspension was 3 g / kg body weight; the dosage of Shengxue Tiaoyuan Granule was 3 g / kg body weight; the dosage of Diyu Shengbai Tablet was 0.1 g / kg body weight; the Kang'ai Injection group was administered by intraperitoneal injection at a dose of 0.5 mL / 100 g, and the dosage was 5 mL / kg body weight; the blank control group and the model control group were both given sodium carboxymethylcellulose by gavage at a dose of 1 mL / 100 g body weight. Each group was administered once a day for 33 consecutive days. On the 4th day after the administration of Kang'ai Injection and on the 7th day after the administration of other drugs, modeling was started. Rats in the model control group and each drug administration group were injected with carboplatin injection via the tail vein once at a dose of 0.4 mL / 100 g body weight, equivalent to a dosage of 40 mg / kg body weight, and the modeling was completed. On the day of modeling (0 d), 0.2 mL of blood was collected from the fundus venous plexus on the 4th, 8th, 14th, 21st, and 26th days after modeling for blood index detection.

[0135] Observation indicators: Sysmex XT-2000i was used, and the manual mode was selected for classification detection; the blood sample was animal whole blood anticoagulated with EDTAK 2 (40:1, v / v), and the detection was completed within 2 hours after blood collection. Five items including white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), platelets (PLT), and reticulocytes (RET) were selected as evaluation indicators. At the same time, the mental state and body weight changes of the animals were recorded every day. SPSS 22.0 statistical software was used to analyze the data, and repeated measures analysis of variance was used for comparison of measurement data. P<0.05 was considered statistically significant.

[0136] General observation results: After modeling, as the damage of the modeling drug to the animal body deepened, the animals in the model control group all showed symptoms such as decreased activity ability, fatigue, low mood, and reduced food intake, and no significant increase in body weight was observed.

[0137] Changes in WBC of rats in each group: The WBC count of rats in each group decreased first and then increased, reaching the lowest on the 8th day and then gradually rising. There were significant differences in the interaction between groups, time points, and groups·time points (P<0.05), as shown in Table 16.

[0138] Table 16 Effects of drug suspension on WBC in rats with chemotherapy-induced anemia

[0139]

[0140] Note: F between groups = 17.625, P < 0.05; F at time points = 83.152, P < 0.05

[0141] Changes in RBC and Hb of rats in each group: The changes in RBC and Hb in the chemotherapy-induced anemia model showed high consistency. The WBC and Hb of rats in each group decreased first and then increased, reaching the lowest level on the 14th day and then gradually rising. The differences in the interaction between groups, time points, and group-time points were all statistically significant (P < 0.05), as shown in Tables 17 and 18.

[0142] Table 17 Effects of drug suspension on RBC in rats with chemotherapy-induced anemia

[0143]

[0144] Note: F between groups = 15.445, P < 0.05; F at time points = 54.596, P < 0.05

[0145] Table 18 Effects of drug suspension on Hb content in rats with chemotherapy-induced anemia

[0146]

[0147] Note: F between groups = 21.567, P < 0.05; F at time points = 112.632, P < 0.05

[0148] Changes in PLT of rats in each group: The PLT count of rats in each group decreased first and then increased, reaching the lowest level on the 8th day and then gradually rising. The differences in the interaction between groups, time points, and group-time points were all statistically significant (P < 0.05), as shown in Table 19.

[0149] Table 19 Effects of drug suspension on PLT in rats with chemotherapy-induced anemia

[0150]

[0151]

[0152] Note: F between groups = 18.674, P < 0.05; F at time points = 154.632, P < 0.05

[0153] Changes in RET of rats in each group: Except for the blank control group, the RET count decreased first and then increased. The lowest level of Kang'ai injection was on the 4th day, and the lowest level of the other groups was on the 8th day, and then gradually rose. The differences in the interaction between groups, different time points, and group-time points were all statistically significant (P < 0.05), as shown in Table 20.

[0154] Table 20 Effects of Angelica drug suspension on RET in rats with chemotherapy-induced anemia

[0155]

[0156] Note: F between groups = 12.654, P < 0.05; F at time points = 59.873, P < 0.05

[0157] Test results:

[0158] In the mouse model of acute hemorrhagic anemia, compared with the model control group, the counts of red blood cells, reticulocytes and the content of hemoglobin increased in the drug suspension group, and the difference was statistically significant (P < 0.05), while the effects on white blood cells and platelets were not statistically significant (P > 0.05). In the rat model of chemotherapy-induced anemia, compared with the model control group, during the chemotherapy drug injury period and the recovery period, the white blood cells, reticulocytes, red blood cell counts and hemoglobin content increased in the drug suspension group, and the difference was statistically significant (P < 0.05), while the effect on platelets was not statistically significant (P > 0.05). The drug suspension can improve the reduction of red blood cells and hemoglobin caused by acute blood loss in mice, is effective in reducing white blood cells, red blood cells and hemoglobin caused by chemotherapy drugs, and can significantly improve the hematopoietic function of the bone marrow, and is used to improve the reduction of RBC caused by different reasons and relieve anemia symptoms.

Claims

1. A method for preparing a nanosuspension of an angelica medicine composition by mixing vitamin E polyethylene glycol succinate with angelica and astragalus extract concentrates, characterized in that: The Angelica medicine composition nanosuspension is prepared using vitamin E polyethylene glycol succinate as an auxiliary material, and specifically comprises the following steps: (1) Take slices of Chinese Angelica and Astragalus, add water and reflux to extract twice, each time for 1 hour, combine the extracts, and concentrate to 1:1 (ml:g) to obtain a concentrated solution (i.e., aqueous phase I); (2) Dissolve vitamin E polyethylene glycol succinate in water to obtain aqueous phase II. (3) mixing the aqueous phase I and the aqueous phase II under ultrasonic conditions, and grinding in a water bath to obtain an initial solution; (4) diluting the initial solution to obtain a nanosuspension of the angelica medicinal composition.

2. The method for preparing a nanosuspension of an angelica medicine composition by mixing vitamin E polyethylene glycol succinate with angelica and astragalus extract concentrate according to claim 1, characterized in that: The method also includes freeze drying: adding 2% freeze-drying protective agent into the suspension of the pharmaceutical composition to extend the storage time of the drug; wherein the freeze drying time is 12 hours and the vacuum pressure is 0.15 mbr.

3. A method for preparing a nanosuspension of an angelica medicine composition by mixing vitamin E polyethylene glycol succinate with angelica and astragalus extract concentrate according to claim 1, characterized in that: In the step (1), the ratio of angelica to astragalus is 5:

1.

4. A method for preparing a nanosuspension of an angelica medicine composition by mixing vitamin E polyethylene glycol succinate with angelica and astragalus extract concentrate according to claim 1, characterized in that: In the step (1), the added mass ratio of the concentrate to vitamin E polyethylene glycol succinate is (1-6):1, and the concentration of the concentrate in the nanosuspension is 4.5-5.5 mg / ml, the ultrasonic power is 100 W, the ultrasonic temperature is 60° C., the ultrasonic time is 1-3 min, the grinding rate is 300-400 r / min, the grinding medium is 0.4-0.6 mm zirconium oxide beads, the grinding time is 1-3 h, and the water bath grinding temperature is (0-60° C.).

5. The method for preparing the nanosuspension of angelica medicinal composition according to claims 1 to 4, characterized in that: The angelica medicine composition nanosuspension is prepared by using vitamin E polyethylene glycol succinate as an auxiliary material.

6. Use of the nanosuspension of the angelica medicinal composition prepared by the method of claims 1 to 4 or the nanosuspension of the angelica medicinal composition as claimed in claim 5 in a pharmaceutical preparation.

7. The use according to claim 6, characterized in that: Also includes: Application of angelica medicine composition nanosuspension in anti-myocardial ischemia, blood tonification and antipyretic and analgesic.

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