Lactoferrin-modified liposomes, preparation methods and applications in health

Abstract

The present application discloses a lactoferrin-modified liposome, a preparation method thereof and an application thereof. The liposome is loaded with Gastrodia elata active substances, and the Gastrodia elata active substances include barisonoside H, barisonoside K and barisonoside W. Through in vitro experiments and characterization, it is found that the liposome has a smoother drug release performance and drug loading performance for Gastrodia elata active substances after being modified with lactoferrin. The liposome has application prospects in related health fields such as hyperlipidemia and improvement of intestinal flora.

Description

Technical Field

[0001] The present application relates to the technical field of lactoferrin, and in particular to lactoferrin-modified liposomes, preparation methods, and applications in health. Background Art

[0002] Lactoferrin is an iron-sugar binding protein that mainly exists in the colostrum of animals and belongs to the transferrin family in terms of classification. Lactoferrin not only plays an important role in natural immunity, but also has functions such as promoting bone growth, anti-microbial activity, anti-inflammation, and anti-cancer. Lactoferrin can stabilize reduced iron ions, improve the utilization rate of iron by intestinal cells, and reduce the irritation of iron ions to the intestine. Lactoferrin can regulate the absorption of iron by intestinal mesangial cells and balance the stable state of iron in the human body.

[0003] At present, many researchers have reported on the amino acid sequence, spatial structure and molecular mechanism of lactoferrin's antibacterial and antiviral effects, which provides a basis for the expression of lactoferrin by genetic recombination system. In addition, the transformation or modification of other small molecule drugs or ingredients by lactoferrin is also an important research direction. Summary of the invention

[0004] In view of this, the purpose of the present application is to provide at least a lactoferrin-modified liposome to improve at least one of the technical problems of the prior art such as poor bioavailability and poor pharmacokinetics of Gastrodia elata active ingredients to a certain extent.

[0005] In a first aspect, the embodiments of the present application disclose a lactoferrin-modified liposome, wherein the liposome is loaded with Gastrodia elata active substances, and the Gastrodia elata active substances include balisonoside H, balisonoside K and balisonoside W.

[0006] In the examples of the present application, the average particle size of the liposome is 164.22 to 170.57 nm, the Zeta potential is -10.88 to -10 mV, and the modification amount of lactoferrin is 175.9 to 276.7 mg / g.

[0007] In the examples of the present application, the drug loading capacity of the liposome for barisenoside H is 14.05-19.09 mg / g; the drug loading capacity of the liposome for barisenoside K is 17.41-22.33 mg / g; and the drug loading capacity of the liposome for barisenoside W is 18.06-24.7 mg / g.

[0008] In the embodiments of the present application, the Gastrodia elata active ingredients further include gastrodin, citric acid, p-hydroxybenzyl alcohol and balisonoside E.

[0009] In the embodiments of the present application, the drug loading capacity of the liposomes for gastrodin is not higher than 0.16 mg / g; the drug loading capacity of the liposomes for citric acid is not higher than 0.36 mg / g; the drug loading capacity of the liposomes for p-hydroxybenzyl alcohol is not higher than 0.4 mg / g; the drug loading capacity of the liposomes for parisonoside E is not higher than 0.66 mg / g.

[0010] In a second aspect, the present application discloses a method for preparing the liposomes described in the first aspect, comprising the following steps:

[0011] Obtaining the primary extract of Gastrodia elata;

[0012] obtaining a first purified product of Gastrodia elata, wherein the first purified product of Gastrodia elata is obtained by purifying the primary extract of Gastrodia elata through a chromatography column filled with HP20 macroporous resin;

[0013] obtaining a second day Gastrodia elata purified product, wherein the second day Gastrodia elata purified product is obtained by purifying the first Gastrodia elata purified product through a chromatography column filled with HPD826 macroporous resin;

[0014] obtaining liposomes loaded with the purified second anesthetic product; and

[0015] Lactoferrin-modified liposomes loaded with the second day anesthetic purified product were obtained.

[0016] In the examples of the present application, the second day hemp purified product contains 88.3-203.8 mg / g of balisonoside H, 196.4-282 mg / g of balisonoside K and 259.9-370.8 mg / g of balisonoside W.

[0017] In the embodiment of the present application, the second hemp purified product further comprises 10.6-12.6 mg / g gastrodin, 4.2-5.6 mg / g citric acid, 2.7-3.8 mg / g p-hydroxybenzyl alcohol and 13.2-14.2 mg / g barisin E;

[0018] The second day hemp purified material does not contain balisonoside A, balisonoside B and balisonoside C.

[0019] In a third aspect, the present application discloses a lyophilized agent, comprising the liposome described in the first aspect or the liposome prepared by the preparation method described in the second aspect, and a lyophilization protective agent for stabilizing and protecting the liposome. The lyophilization protective agent comprises at least one of sucrose, lactose and mannitol, and the amount used is 2 to 10 wt% of the mass of the liposome.

[0020] In a fourth aspect, the embodiments of the present application disclose the use of the liposome described in the first aspect or the liposome prepared by the preparation method described in the second aspect in the preparation of health products related to lowering blood lipids and improving intestinal flora.

[0021] Compared with the prior art, this application has at least the following beneficial effects:

[0022] In the present application, Gastrodia elata active substances including PH, PK and PW were obtained by extracting Gastrodia elata balisong glycoside compounds.

[0023] The present application example further encapsulates the Gastrodia elata active substance in liposomes and modifies the liposomes with lactoferrin. In vitro experiments and characterizations show that the lactoferrin-modified liposomes have better and smoother drug release performance and drug loading performance for the Gastrodia elata active substance.

[0024] The examples of the present application further conducted an in vivo experiment to confirm that the pharmacokinetic process in rats was poor compared to the Gastrodia elata active ingredient liposomes that were not modified with lactoferrin, and the liposomes of other Gastrodia elata active ingredients that were modified with lactoferrin (such as Gastrodia elata active ingredients mainly composed of PA, PB and PC), which were not conducive to biological utilization. In addition, the lactoferrin-modified Gastrodia elata active ingredient liposomes with PH, PK and PW provided in the examples of the present application could reduce the blood lipid level of rats with hyperlipidemia, improve liver fat accumulation and arteriosclerosis symptoms, and promote the development of the damaged intestinal flora environment of rats towards a probiotic trend, indicating that the lactoferrin-modified Gastrodia elata active ingredient liposomes prepared in the examples of the present application have application prospects in the fields of related medicines and health products such as hyperlipidemia and improvement of intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a liquid chromatogram of the second day hemp purification product provided in Example 1 of the present application.

[0026] Figure 2 This is the liquid chromatogram of the first day of hemp purification provided in Comparative Example 1 of the present application.

[0027] Figure 3 This is a liquid chromatogram of the purified hemp product on the second day provided in Comparative Example 2 of the present application.

[0028] Figure 4 This is a typical electron micrograph of liposomes loaded with Gastrodia elata active ingredients provided in this application.

[0029] Figure 5 This is a typical electron micrograph of lactoferrin-modified liposomes loaded with Gastrodia elata active ingredients provided in this application.

[0030] Figure 6 This is the in vitro dissolution curve provided in Example 1 of the present application.

[0031] Figure 7 This is the in vitro dissolution curve provided in Example 2 of the present application.

[0032] Figure 8 This is the in vitro dissolution curve provided in Example 3 of the present application.

[0033] Fig. 9 This is the in vitro dissolution curve provided for Comparative Example 1 of the present application.

[0034] Fig.10 This is the in vitro dissolution curve provided for Comparative Example 2 of the present application.

[0035] Fig.11 This is the in vitro dissolution curve provided for Comparative Example 3 of the present application.

[0036] Fig.12 This is the in vitro dissolution curve provided for Comparative Example 4 of the present application.

[0037] Fig.13 This is the in vitro dissolution curve provided for Comparative Example 5 of the present application.

[0038] Fig.14 This is the in vitro dissolution curve provided for Comparative Example 6 of the present application.

[0039] Fig.15 This is the in vitro dissolution curve provided for Comparative Example 7 of the present application. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained commercially; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0041] Extraction of Balisong Glycosides from Gastrodia Elata

[0042] 1. Materials and Methods

[0043] Ingredients: pure Gastrodia elata powder, Bohui Herbal Hall.

[0044] 1. Extraction

[0045] Accurately weigh 300g of the above-mentioned Gastrodia elata powder, fully disperse it, add 3L 95% methanol, vortex mix for 1min, ultrasonic extract at 40°C for 40min, filter to obtain the filtrate, and ultrasonically extract with 95% ethanol again, ultrasonically extract 3 times in total, filter respectively, combine the filtrate, recover ethanol with a rotary evaporator, add hot water to the concentrate and transfer, add ethyl acetate according to the ratio of concentrate: ethyl acetate of 1:3, extract 2 to 4 times, combine the lower aqueous solution, heat and concentrate, then transfer the concentrate to a constant temperature water bath and evaporate it to an extract, put it into an electric constant temperature blast drying oven and continue to dry to constant weight, and obtain 173g of Gastrodia elata primary extract.

[0046] 2. Purification

[0047] The purification process of a specific embodiment 1 is:

[0048] (1) 173 g of the primary extract of Gastrodia elata was fully dissolved in 9% ethanol solution, filtered, and loaded onto a pretreated chromatography column (diameter 2.5 cm, column height 25 m) filled with HP20 macroporous resin (Solarbio). After loading, the column was allowed to stand for 10 min to allow the active ingredients in the primary extract of Gastrodia elata to be fully adsorbed by the resin;

[0049] The elution procedure is distilled water (10 BV), 20% to 65% ethyl acetate aqueous solution eluting 20 BV, and finally 75% ethyl acetate aqueous solution eluting 10 BV, collecting the ethyl acetate eluate, concentrating under reduced pressure, and freeze-drying to obtain 43 g of the first day hemp purified product;

[0050] (2) The first hemp purified product was dissolved in 95% ethanol solution, filtered, and loaded onto a fully swollen chromatography column (diameter 2.5 cm, column height 25 m) filled with HPD826 (Tianjin Yunkai Resin Technology Co., Ltd.). After loading, the column was allowed to stand for 10 min to allow the active ingredients in the first hemp purified product to be fully adsorbed by the resin;

[0051] The elution procedure was distilled water (10 BV), 20% to 65% ethyl acetate aqueous solution eluted 20 BV, and finally 75% ethyl acetate aqueous solution eluted 10 BV. The ethyl acetate eluate was collected, concentrated under reduced pressure, and freeze-dried to obtain 27 g of the purified second day hemp.

[0052] In the purification process of a specific embodiment 2, step (1) and step (2) are the same as those of embodiment 1, wherein an aqueous solution of tetrahydrofuran is used as an eluent in both step (1) and step (2).

[0053] In the purification process of a specific embodiment 3, its step (1) and step (2) are the same as those of embodiment 1, wherein an aqueous solution of 4-methyl-2-pentanone is used as an eluent in both step (1) and step (2).

[0054] In the purification process of a specific comparative example 1, only step (1) was performed, and the process of step (1) was the same as that of example 1.

[0055] In the purification process of a specific comparative example 2, only step (2) was performed, and the process of step (2) was the same as that of example 1.

[0056] 3. Active ingredient detection

[0057] Chromatographic conditions Chromatographic column: Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm);

[0058] Mobile phase: acetonitrile (A)-0.05% phosphoric acid aqueous solution (B), gradient elution: 0-10 min, 2% A; 10-30 min, 2%-30% A, 30-35 min, 30%-2% A;

[0059] Detection wavelength: 220nm; Volume flow rate: 1.0mL / min;

[0060] Column temperature: 25°C; injection volume: 10 μL.

[0061] Standards: Gastrodin (GA, CAS No.: 62499-27-8, Yuanye Biotechnology Co., Ltd.), citric acid (CA, CAS: 77-92-9, Shanghai Aladdin Biochemical Technology Co., Ltd.), p-hydroxybenzyl alcohol (HA, CAS: 623-05-2, Tianjin Fengyuan Biotechnology Co., Ltd.), barisonoside A (PA, CAS: 62499-28-9, Shanghai Yuanye Biotechnology Co., Ltd.), barisonoside B (PB, PHL83886, Reference Substance), Balisin C (PC, PHL83887, Reference Substance), Balisin E (PE, PHL83888, Reference Substance); Balison glycosides H, K, and W were prepared according to the method disclosed in “LI Z, WANC Y, OUYANG H, et al. A novel dereplication strategy for the identification of two new trace compounds in the extract of Gastrodiaelata using UHPLC / Q TOF-MS / MS[J]. J. Chromatogr B, 2015, 988: 45-52.”

[0062] Preparation of test solution: Take the first day hemp purified product or the second day hemp purified product, accurately weigh 2 g, accurately weigh, place in a stoppered conical flask, accurately add 50 mL of 95% ethanol, weigh, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 min, cool, weigh again, make up the lost mass with dilute ethanol, filter, accurately measure 10 mL of the filtrate, concentrate to nearly dryness without alcohol taste, add acetonitrile-water (3:97) mixed solution to dissolve the residue, transfer to a 25 mL second volumetric flask, dilute to the scale with acetonitrile-water (3:97) mixed solution, shake well, filter, and take the filtrate to obtain.

[0063] Standard curves were prepared according to the peak areas and concentrations of the standard products in the HPLC spectra, and the contents of the active ingredients in the test solution were calculated according to the standard curves, and then the contents of the active ingredients in the first day hemp purification product or the second day hemp purification product prepared in Example 1 and Comparative Examples 1 to 2 were calculated (e.g. Figures 1 to 3 All test data were expressed as mean and standard deviation, and SPSS13.0 software was used to process the data, and multiple comparisons and significant differences were performed on the data.

[0064] 2. Results

[0065] Table 1 Content of each active ingredient (mg / g)

[0066] Implementation Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 GA 12.2±0.4d 11.5±0.9d 11.8±0.4d 14.6±0.8c 118.2±24.7a CA 4.8±0.6d 5.2±0.4d 4.9±0.5d 5.2±0.4c 30.6±2.5c HA 3.5±0.3d 2.9±0.2d 3.3±0.4d 2.4±0.6c 102.5±18.5a PA － － － 96.7±3.2a 69.2±4.7b PB － － － 82.0±5.6b 108.3±17.4a PC － － － 67.6±4.7b 82.4±5.3ab PE 13.7±0.5d 14.5±0.3d 15.1±0.7d 11.5±1.3c 56.8±3.4b PH 146.2±42.7c 139.5±51.2c 172.6±31.2c 67.8±3.8b 51.6±2.7b PK 232.5±36.1b 246.8±41.5b 254.1±27.9b 103.9±8.3a 73.5±4.2b PW 309.3±61.5a 316.5±37.2a 302.5±42.6a 87.5±3.9b 81.0±8.6ab

[0067] Since only step (1) was performed in comparative example 1, a purified first Gastrodia elata product was obtained; and only step (2) was performed in comparative example 2, a purified second Gastrodia elata product was obtained. Table 1 lists the contents of each active ingredient in the purified second Gastrodia elata product obtained in Examples 1 to 3, the purified first Gastrodia elata product obtained in comparative example 1, and the purified second Gastrodia elata product obtained in comparative example 2. In Table 1, multiple comparisons and significant differences were made between the contents of the same active ingredient in different examples and comparative examples; in Table 1, "-" indicates that it was not detected. It can be seen from Table 1 that the purified Gastrodia elata products obtained in Examples 1 to 3 do not contain PA, PB, and PC; the contents of PA, PB, and PC contained in the purified Gastrodia elata products obtained in comparative examples 1 and 2 are both over 50 mg / g. In addition, the main components of the purified second Gastrodia elata product obtained in Examples 1 to 3 are PH, PK, and PW.

[0068] Preparation and characterization of lactoferrin-modified liposomes loaded with purified Gastrodia elata

[0069] 1. Materials and Methods

[0070] 1. Preparation process

[0071] (1) Soybean lecithin (CAS: 8002-43-5-Calbiochem, Sigma-Aldrich, referred to as PC), cholesterol (CAS: 57-88-5, Aibixin (Shanghai) Biotechnology Co., Ltd., referred to as CHOL) and DSPE-PEG2000-NHS (distearoyl phosphatidylethanolamine-polyethylene glycol 2000 activated lipid, Xi'an Ruixi Biotechnology Co., Ltd., referred to as DPN) were fully dispersed in 200 mL of chloroform solution, and the purified Gastrodia elata (referred to as TM) prepared in Examples 1 to 3 and Comparative Examples 1 to 2 were added thereto, wherein the amount of PC added was 50 g, the amount of CHOL added was 10 g, the amount of DPN added was 6 g, and the amount of TM added was 10 g. The mixture was reacted in a water bath at 37° C. using a rotary evaporator until the solvent was completely evaporated, and a uniform thin film was formed in the container, which was the product. 150 mL of PBS was added to hydrate the thin film to obtain a liposome suspension. The liposome suspension was ultrasonically treated in an ice bath for 5 minutes, and then the liposomes were extruded back and forth ten times using a membrane extruder, so that the liposomes passed through polycarbonate membranes with pore sizes of 400 nm and 200 nm in turn. The obtained liposomes were passed through an HP20 chromatography column to remove free purified Gastrodia elata, thereby obtaining liposomes loaded with purified Gastrodia elata.

[0072] (2) Preparation of lactoferrin-modified liposomes

[0073] The liposome suspension loaded with purified Gastrodia elata prepared above was reacted with 35 mg / mL lactoferrin (human lactoferrin L4040, Sigma) solution at 4°C and slowly shaken overnight on a shaker at 50 rpm, wherein the volume ratio of the liposome suspension to the lactoferrin solution was 10:2. After sufficient reaction, unreacted lactoferrin was removed with a G-100 dextran gel column to obtain lactoferrin-modified liposomes loaded with purified Gastrodia elata (LF-Lipo).

[0074] The corresponding LF-Lipo was prepared using the purified Gastrodia elata provided in Examples 1 to 3 and Comparative Examples 1 and 2, respectively; in addition, liposomes (Lipo) loaded with purified Gastrodia elata were prepared using the purified Gastrodia elata provided in Example 1, Comparative Examples 1 and 2, respectively, as Comparative Examples 3 to 5.

[0075] 2. Characterization of liposomes

[0076] (1) Particle size and potential of LF-Lipo and Lipo

[0077] The morphology of liposomes was observed and photographed using a transmission electron microscope (Hitachi, Japan) at a voltage of 200 KV. The particle size and potential of LF-Lipo and Lipo were measured using a multi-angle particle size and high-sensitivity Zeta potential analyzer (Brookhaven Instruments, USA).

[0078] (2) Detection of the amount of lactoferrin modified in LF-Lipo and the drug loading capacity of purified Gastrodia elata

[0079] In the above-mentioned step of modifying liposomes with lactoferrin, the amount of lactoferrin adsorbed by the G-100 dextran gel column and the concentration of the initially added lactoferrin solution are used to calculate the amount of lactoferrin modified in LF-Lipo. The amount of lactoferrin adsorbed by the G-100 dextran gel column can be eluted by the gel column, and the eluate can be collected. The protein concentration in the eluate can be used to calculate the amount of gel column chromatography adsorption. Then the amount of lactoferrin modified in LF-Lipo = (LF initial amount - gel column chromatography adsorption amount) / liposome mass.

[0080] Similarly, the liposomes were passed through an HP20 chromatography column to remove free purified Gastrodia elata, and the drug loading capacity (%) of the purified Gastrodia elata in the liposomes prepared in each embodiment and comparative example = (initial amount of purified Gastrodia elata - amount adsorbed on the HP20 chromatography column) / mass of liposomes.

[0081] 3. In vitro drug release performance

[0082] The LF-Lipo and Lip prepared in Examples 1 to 3 and Comparative Examples 1 to 2, respectively, and the purified Gastrodia elata provided in Examples 1 to 3 and Comparative Examples 1 to 2, respectively, were put into a dissolution apparatus (DT12 automatic dissolution sampling system, Haineng Future Technology Group Co., Ltd.), and the dissolution and release rate determination method in General Rule 0931 of the 2015 edition of the Chinese Pharmacopoeia (Part 4) was determined by the paddle method, and 900 mL of ultrasonically degassed water, 900 mL of artificial gastric juice (16.4 mL of dilute hydrochloric acid was accurately measured, 800 mL of water was added to dissolve, and 1 g of pepsin was added to the mixture. 0g was dissolved and shaken well, and water was added to make up to 1L) as the dissolution medium, the rotation speed was 50rpm, the temperature was 37±0.5℃, 5mL of samples were taken at 30, 60, 120 and 180min respectively, and the dissolution medium of the same temperature and volume was added to the dissolution cup at the same time, the sample solution was taken out and filtered with a 0.22μm microporous filter membrane, and then the content of relevant Gastrodia elata active ingredients in the samples at each time point was determined according to the above-mentioned HPLC method, and the cumulative dissolution at each time point was calculated, wherein, cumulative dissolution=∑content of Gastrodia elata active ingredients in samples at each time point / initial amount of Gastrodia elata active ingredients.

[0083] 4. Statistical analysis

[0084] All test data were expressed as mean and standard deviation. SPSS13.0 software was used to process the data and perform multiple comparisons and significant differences.

[0085] 2. Results

[0086] Typical LF-Lipo electron microscope images prepared in this application are as follows Figure 5 As shown, a typical Lipo electron microscope image is as follows Figure 4 As shown. The average particle size and Zata potential results of the liposomes prepared in each embodiment and comparative example are shown in Table 2. As can be seen from Table 2, the Zeta potential of the liposomes prepared in Comparative Examples 3 to 7 that are not modified by lactoferrin has increased, but is still below zero. The average particle size of the liposomes prepared in Examples 1 to 3 and Comparative Examples 1 to 7 is not much different, among which the average particle size of Comparative Examples 1 to 2 and Comparative Examples 6 to 7 is slightly larger. In addition, Table 2 also shows the LF modification amount of each embodiment and comparative example. As can be seen from Table 2, the lactoferrin modification amount of Examples 1 to 3 is significantly higher than that of the comparative example, and more lactoferrin is connected to its liposomes.

[0087] Table 2

[0088]

[0089]

[0090] Table 3 Drug loading of LF-Lipo for each Gastrodia elata active ingredient (mg / g)

[0091] Implementation Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 GA 0.11±0.05b － － 0.14±0.05c 8.27±0.93b CA 0.24±0.12b 0.08±0.01b － 0.08±0.02c 2.18±0.46c HA 0.32±0.08b － 0.18±0.02b 0.06±0.01c 16.82±2.02a PA － － － 12.15±0.82a 14.23±1.56a PB － － － 10.86±0.57a 17.43±1.39a PC － － － 13.58±1.09a 13.64±1.23a PE 0.52±0.14b 0.63±0.11b 0.58±0.17b 0.42±0.21c 0.63±0.18c PH 15.26±1.21a 17.82±1.02a 18.13±0.96a 7.24±0.62b 8.38±0.35b PK 18.23±0.82a 21.08±1.25a 20.82±1.07a 8.72±0.45b 7.54±0.29b PW 19.12±1.06a 23.19±1.51a 22.28±1.34a 9.10±0.71ab 7.86±0.46b

[0092] Table 4 Lipo drug loading for each Gastrodia elata active ingredient (mg / g)

[0093] Implementation Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Comparative Example 7 GA 0.14±0.03b － － 0.18±0.08c 9.12±0.82b CA 0.29±0.07b 0.06±0.01c 0.15±0.03b 0.10±0.03c 4.02±0.53c HA 0.26±0.02b 0.05±0.01c 0.13±0.01b 0.09±0.04c 17.32±2.18a PA － － － 11.85±1.22a 15.16±1.37a PB － － － 11.13±0.94a 18.24±1.22a PC － － － 14.02±1.22a 14.52±1.19a PE 0.48±0.23b 0.63±0.11c 0.58±0.17b 0.62±0.18c 0.63±0.18c PH 15.25±1.32a 15.52±1.39b 16.82±1.23a 5.19±0.37b 7.83±0.42b PK 18.33±2.95a 18.85±2.13ab 17.73±1.35a 8.03±0.52b 7.03±0.33b PW 18.72±1.26a 20.05±1.47a 19.58±2.12a 8.73±0.59b 7.12±0.58b

[0094] Table 3 lists the drug loading of each Gastrodia elata active ingredient by LF-Lipo liposomes prepared in Examples 1 to 3 and Comparative Examples 1 to 2; in Table 3, “-” indicates that no drug loading was detected; each row of data in Table 3 is multi-labeled and significantly different. It can be seen from Table 3 that the LF-Lipo liposomes prepared in Examples 1 to 3 have a drug loading of GA, CA, HA,

[0095] The drug loading of PA, PB, PC and PE was significantly lower than that of Comparative Examples 1-2, but the drug loading of PH, PK and PW was significantly higher than that of Comparative Examples 1-2.

[0096] Table 4 lists the drug loading of each Gastrodia elata active substance obtained in the process of preparing LF-Lipo liposomes in Examples 1 to 3 and Comparative Examples 1 to 2; in Table 4, "-" indicates that no drug loading was detected; each row of data in Table 4 is multi-labeled and significantly different. In Table 4, the drug loading of each Lipo for each Gastrodia elata active substance shows the corresponding trend of the drug loading of each Gastrodia elata active substance prepared by LF-Lipo liposomes in Examples 1 to 3 and Comparative Examples 1 to 2.

[0097] The dissolution and release curves of the LF-Lipo liposomes or Lipo liposomes of Examples 1 to 3 and Comparative Examples 1 to 7 for various Gastrodia elata active ingredients in artificial gastric juice are as follows: Figures 6 to 15 As shown. As a result, the dissolution effect of Comparative Examples 1 to 7 on the active ingredients of Gastrodia elata showed that the dissolution amount in 180 min was insufficient, or the dissolution rate in the first 30 min was too fast, and the dissolution rate in the next 150 min was too slow, resulting in uneven dissolution rate, etc., especially the dissolution effect on PH, PK and PW was not good. However, Examples 1 to 3 had better dissolution effect on the active ingredients of Gastrodia elata in the artificial gastric juice, especially for PH, PK and PW, showing more uniform dissolution rate and more complete cumulative dissolution amount.

[0098] In vivo experiments

[0099] 1. Materials and Methods

[0100] 1. Experimental Animals

[0101] SPF grade SD rats, catalog number hnslkjd001, half male and half female, weighing about 200 g, from Hunan Slake Jingda Experimental Animal Co., Ltd.; they were kept in a barrier environment with a relative humidity of 40-70% and fed a normal diet.

[0102] 2. Pharmacokinetic study in rats

[0103] Healthy SD rats were randomly divided into a drug administration group and a normal group. The drug administration group was divided into several groups, and the lactoferrin-modified liposome solutions provided in Examples 1 to 3 and Comparative Examples 1 to 2, or the liposome solutions provided in Comparative Examples 3 to 7 were injected into the rat tail vein at a dose of 1 mg / kg, and blood was collected from the tail vein before administration and 1, 2, 6, 12, and 24 hours after administration, placed in a heparinized EP tube, centrifuged at 8000 r / min for 5 minutes, and the plasma was separated and placed in an EP tube, and frozen at -20°C for testing.

[0104] 100 μL of rat plasma obtained at different time periods was added with 300 μL of ethyl acetate, vortexed for 1 min, centrifuged at 12000 r / min for 5 min, the supernatant was taken in a blank centrifuge tube, evaporated, 100 μL of methanol was added for redissolution, vortexed for 1 min, centrifuged at 12000 r / min for 5 min, and the supernatant was taken as the sample to be tested, and the content of various Gastrodia elata active substances therein was detected by the same HPLC method as in the above embodiment. The measured data was processed with DAS2.0 software to calculate the pharmacokinetic parameters.

[0105] 3. Establishment of hyperlipidemia model rats and grouping experiments

[0106] SD male rats were selected and gavaged with a high-fat emulsion (cholesterol, egg yolk powder and Tween 80 mixed in a ratio of 30:50:1) and prepared with distilled water to make a 5 mg / mL solution. Each rat was gavaged with 20 mL daily for 2 consecutive weeks. Blood was collected from the eye sockets and centrifuged at 3000 rpm for 15 min. The serum was separated to measure the basic levels of total cholesterol (TCHO), high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC) and triglycerides (TG) in rat serum. The success of the model was determined based on the TCHO level in the rat serum.

[0107] The hyperlipidemic rats with successful modeling were divided into a model group, a drug administration group and a drug administration control group. A normal group was set up and fed with normal basic feed. The drug administration group was given 1.5g / kg body weight / day of the liposomes provided in Examples 1 to 3 and Comparative Examples 1 to 7 respectively for 60 consecutive days. The drug administration control group was given atorvastatin at a dose of 12.0mg / kg / day body weight for 60 consecutive days.

[0108] 4. Detection indicators

[0109] (1) Body weight: Weigh once a week during the experiment

[0110] (2) Determination of liver index and fat body ratio

[0111] After the experiment, the animals were anesthetized with chloral hydrate solution, and the liver, kidney and peritoneal fat were separated and weighed to calculate the liver-to-body ratio and fat-to-body ratio. Liver-to-body ratio (%) = Ml / M × 100%,

[0112] Fat body ratio (%) = M2 / M×100%; wherein: M represents body weight, M1 represents liver wet weight, and M2 represents kidney and testicular peri-fat wet weight.

[0113] (3) Testing of blood lipids and liver function related indicators

[0114] Blood was collected from the abdominal aorta of anesthetized rats, and serum was separated. The contents of TCHO, HDLC, LDLC and TG in serum were determined using an automatic biochemical analyzer. The activity of hepatic lipase (HL, product number QY-SX1773, Shanghai Qiaoyu Biotechnology Co., Ltd.) and lipoprotein lipase (LPLD, ycextract) in serum were determined using ELISA kits. The cholinesterase activity (TCHO, product number KL-T-CHO-Mu, Shanghai Kanglang Biotechnology Co., Ltd.) and triglyceride content (TG, product number BH8381, Boyao Biotechnology) in liver homogenate were determined using kits, and the arteriosclerosis index was calculated (arteriosclerosis index = (TCHO-HDLG) / HDLC).

[0115] (4) Collecting intestinal contents

[0116] The rats were killed, and the cecal contents of each group of rats were collected aseptically. The intestinal flora of each group of rats was detected using 16S rDNA technology.

[0117] 5. Statistical analysis

[0118] All test data were expressed as mean and standard deviation. SPSS13.0 software was used to process the data and perform multiple comparisons and significant differences.

[0119] 2. Results

[0120] Table 5 In vivo pharmacokinetic parameters (PH)

[0121] Implementation Cmax(μg / mL) T1 / 2(h) MRT(h) F(％) Example 1 13.65±1.82a 5.82±0.82a 6.82±1.02a 42.24±1.32a Example 2 13.93±1.55a 6.24±0.64a 6.95±0.89a 43.32±1.56a Example 3 14.16±1.27a 6.19±0.73a 7.16±0.74a 45.68±1.38a Comparative Example 1 7.32±1.72b 1.72±0.48b 2.31±0.82b 26.83±2.15b Comparative Example 2 7.19±1.34b 1.58±0.56b 2.48±0.71b 27.28±1.83b Comparative Example 3 8.32±1.21b 1.92±0.35b 2.58±0.53b 25.23±1.06b Comparative Example 4 8.26±1.13b 1.82±0.47b 2.49±0.52b 24.93±1.54b Comparative Example 5 8.33±1.42b 1.87±0.44b 2.63±0.49b 25.37±1.62b Comparative Example 6 6.38±1.16b 1.49±0.35b 2.12±0.78b 21.08±1.38b Comparative Example 7 6.45±1.22b 1.32±0.26b 2.09±0.58b 21.14±1.45b

[0122] Table 6 In vivo pharmacokinetic parameters (PK)

[0123]

[0124]

[0125] Table 7 In vivo pharmacokinetic parameters (PW)

[0126] Implementation Cmax(μg / mL) T1 / 2(h) MRT(h) F(％) Example 1 13.72±1.41a 5.41±0.82a 6.36±0.58a 43.08±1.82a Example 2 13.67±1.39a 5.46±0.76a 6.43±0.75a 42.96±1.75a Example 3 13.78±1.53a 5.38±0.49a 6.45±0.48a 43.14±1.53a Comparative Example 1 6.73±0.85b 1.19±0.18b 1.62±0.34b 20.76±1.28b Comparative Example 2 6.86±0.76b 1.22±0.26b 1.78±0.41b 20.35±1.19b Comparative Example 3 7.32±0.73b 1.42±0.23b 2.26±0.68b 21.52±1.37b Comparative Example 4 7.45±0.49b 1.46±0.32b 2.32±0.75b 21.73±1.42b Comparative Example 5 7.53±0.54b 1.44±0.22b 2.43±0.64b 21.86±1.55b Comparative Example 6 6.85±0.47b 1.12±0.14b 1.86±0.59b 20.34±1.46b Comparative Example 7 6.96±0.35c 1.15±0.18b 1.92±0.67b 20.28±1.35b

[0127] Tables 5 to 7 calculate the pharmacokinetic parameters of the liposomes provided in each of Examples 1 to 3 and Comparative Examples 1 to 7 in rats, and perform multiple comparisons and significant difference markings. As can be seen from the results, after the liposomes provided in Examples 1 to 3 were administered to rats, the pharmacokinetic parameters of PH in rat plasma: Cmax (μg / mL), T1 / 2 (h), MRT (h) and F (%) were significantly higher than those in Comparative Examples 1 to 7, indicating that after the liposomes provided in Examples 1 to 3 were administered to rats, the PH release amount reached the peak drug concentration, terminal elimination half-life, average residence time and bioavailability were higher than those in the Comparative Examples, indicating that the release of PH in rat plasma can be better utilized by rats, has a better pharmacodynamic process, and can provide help for promoting the efficacy of PH of Gastrodia elata active substance. In addition, the results in Tables 6 and 7 also show that after the liposomes provided in Examples 1 to 3 were administered to rats, the PH release amount reached the peak drug concentration, the terminal elimination half-life, the average residence time and the bioavailability were all higher than those in the comparative example, and the PK and PW released by the liposomes provided in Examples 1 to 3 had a more pharmacokinetic process in rat plasma, which was beneficial to the bioavailability of rats.

[0128] Table 8 Blood lipid levels in hyperlipidemic animals

[0129]

[0130] Table 8 shows the blood lipid levels of rats in each group after the animal experiment. As can be seen from Table 8, compared with the normal group, the total cholesterol, high-density lipoprotein, low-density lipoprotein and triglyceride levels of the model group rats were significantly increased, indicating that the model was successfully established. The four blood lipid indexes of the rats in the drug control group were significantly lower than those in the model group, indicating that atorvastatin administered to the hyperlipidemia model rats in the drug control group can improve the high blood lipid levels of the rats. In the drug administration group, the liposomes provided by Examples 1 to 3 had the most obvious effect on reducing the high blood lipid levels of rats, while the improvement effect of Comparative Examples 1 to 7 was limited.

[0131] In addition, Table 9 shows the liver-to-body ratio, fat body ratio and arteriosclerosis index of each group of rats after the animal experiment. As shown in Table 9, compared with the normal group, the liver-to-body ratio, fat body ratio and arteriosclerosis index of the model group were significantly increased, indicating that the modeling was successful and the model rats showed symptoms of arteriosclerosis. Although the liver-to-body ratio, fat body ratio and arteriosclerosis index of the rats in the drug control group were significantly lower than those in the model group, they were all significantly different from the normal group, indicating that atorvastatin did not significantly improve the liver fat and arteriosclerosis of rats. In the drug administration group, the liposomes provided in Examples 1 to 3 were significantly better than the liposomes and atorvastatin provided in Comparative Examples 1 to 7 in improving the liver fat and arteriosclerosis of rats, indicating that the liposomes loaded with Gastrodia elata active substances modified by lactoferrin provided in the examples of the present application can significantly reduce the serum fat level of rats and improve the symptoms of liver fat accumulation and arteriosclerosis.

[0132] Table 9

[0133]

[0134] Furthermore, the animal experiment of the present application also analyzed the intestinal microbial community structure of each group of rats, and the analysis found that the intestinal flora of rats mainly included Firmicutes, Verrucomicrobia, Bacteroidetes and Proteobacteria. At the level of bacteria, the intestinal flora of rats mainly included Clostridium XIVa, Bacteroides, Barnesiella, Lactobacillus, Romboutsia, Akkermansia, Eubacterium xylanophilum group, Roseburia and Ruminococcus.

[0135] Table 10 Relative abundance of intestinal microorganisms in each group of rats % (I)

[0136]

[0137]

[0138] Table 11 Relative abundance of intestinal microorganisms in each group of rats (%)

[0139]

[0140] Among them, Lactobacillus is a typical probiotic that has a beneficial effect on intestinal health and lipid metabolism indicators, can effectively increase TC and TG levels, and promote body weight gain. Lactobacillus can enhance the body's immunity and resistance, regulate the balance of intestinal microorganisms, promote the colonization of beneficial intestinal bacteria, and change the composition of short-chain fatty acids in the intestine. It has been widely used to regulate the intestinal microecological environment and improve metabolic diseases. Akkermansia can enhance the effect of cancer immunotherapy and is necessary for the effect of immunotherapy. Barnesiell is an anaerobic bacterium that can inhibit the growth of harmful bacteria in the intestine and provide the body with necessary nutrients, but sometimes it can also cause infection and abscesses. Eubacteriumxylanophilum group and Clostridium XIVa can cause intestinal inflammation.

[0141] Tables 10 and 11 show the abundance levels of Lactobacillus, Akkermansia, Barnesiell, Eubacterium xylanophilum group and Clostridium XIVa in the intestines of rats in each group. As shown in Tables 10 and 11, compared with the normal group, the abundance levels of Lactobacillus and Akkermansia in the intestines of the model group rats were significantly reduced, while the abundance levels of Barnesiell, Eubacterium xylanophilum group and Clostridium XIVa were significantly increased, indicating that the intestinal flora environment of the model rats was destroyed and developed in a trend that was not conducive to intestinal probiotics. In the drug administration group, after the intervention treatment with the liposomes provided in Examples 1 to 3, the abundance of Lactobacillus and Akkermansia in the intestines of the rats increased significantly to the same level as the normal group, while the abundance levels of Barnesiell, Eubacterium xylanophilum group and Clostridium XIVa decreased significantly to the same level as the normal group, while the intestinal flora of the rats after the intervention treatment with the liposomes and atorvastatin provided in Comparative Examples 1 to 7 did not achieve the improvement results. This shows that the liposomes provided in the examples of the present application can significantly improve the damaged intestinal flora environment of rats to develop towards a probiotic trend, which is beneficial to the healthy development of rats.

[0142] In summary, the present invention provides an example of extracting Gastrodia elata balisong glycoside compounds to obtain Gastrodia elata active substances with pH, ​​PK and PW.

[0143] The present application example further encapsulates the Gastrodia elata active substance in liposomes and modifies the liposomes with lactoferrin. In vitro experiments and characterizations show that the lactoferrin-modified liposomes have better and smoother drug release performance and drug loading performance for the Gastrodia elata active substance.

[0144] The examples of the present application further conducted an in vivo experiment to confirm that the pharmacokinetic process in rats was poor compared to the Gastrodia elata active ingredient liposomes that were not modified with lactoferrin, and the liposomes of other Gastrodia elata active ingredients that were modified with lactoferrin (such as Gastrodia elata active ingredients mainly composed of PA, PB and PC), which were not conducive to biological utilization. In addition, the lactoferrin-modified Gastrodia elata active ingredient liposomes with PH, PK and PW provided in the examples of the present application could reduce the blood lipid level of rats with hyperlipidemia, improve liver fat accumulation and arteriosclerosis symptoms, and promote the development of the damaged intestinal flora environment of rats towards a probiotic trend, indicating that the lactoferrin-modified Gastrodia elata active ingredient liposomes prepared in the examples of the present application have application prospects in the fields of related medicines and health products such as hyperlipidemia and improvement of intestinal flora.

[0145] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing lactoferrin-modified liposomes, characterized in that: The following steps are involved: Obtaining the primary extract of Gastrodia elata; obtaining a first purified product of Gastrodia elata, wherein the first purified product of Gastrodia elata is obtained by purifying the primary extract of Gastrodia elata through a chromatography column filled with HP20 macroporous resin; obtaining a second day Gastrodia elata purified product, wherein the second day Gastrodia elata purified product is obtained by purifying the first Gastrodia elata purified product through a chromatography column filled with HPD826 macroporous resin; obtaining liposomes loaded with the purified second anesthetic product; and obtaining lactoferrin-modified liposomes loaded with the second day anesthesia purified product; The second day hemp purified product contains 88.3-203.8 mg / g of balisonoside H, 196.4-282 mg / g of balisonoside K and 259.9-370.8 mg / g of balisonoside W; The second hemp purified product further comprises 10.6-12.6 mg / g of gastrodin, 4.2-5.6 mg / g of citric acid, 2.7-3.8 mg / g of p-hydroxybenzyl alcohol and 13.2-14.2 mg / g of barisin E; The second day hemp purified material does not contain balisonoside A, balisonoside B and balisonoside C.

2. The lactoferrin-modified liposome prepared by the preparation method according to claim 1.

3. A lyophilized agent comprising the liposome prepared by the preparation method according to claim 1, and a lyoprotectant for stabilizing and protecting the liposome.

4. Use of the liposomes prepared by the preparation method of claim 1 in preparing health products for lowering blood lipids and improving intestinal flora.

5. A health product for lowering blood lipids, characterized in that: The invention relates to a liposome prepared by the preparation method according to claim 1.

6. A health product for improving intestinal flora, characterized in that: The invention relates to a liposome prepared by the preparation method according to claim 1.

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