Method for preparing micro-nano particles in traditional Chinese medicine decoction piece decoction liquid

Abstract

The invention relates to a micro-nano material, in particular to a method for preparing micro-nano particles in traditional Chinese medicine decoction piece decoction, which comprises the following steps: providing the traditional Chinese medicine decoction to obtain supernate, and freezing at-80 DEG C to-20 DEG C for 12-24 hours; and then melting and unfreezing, and collecting the micro-nano particles. The invention newly finds that the freeze-thaw effect has a remarkable effect in the preparation of the traditional Chinese medicine micro-nano particles. The supernate obtained after decoction of the traditional Chinese medicine decoction pieces can generate micro-nano particles in various shapes through freeze thawing, and shape control can be achieved through process parameters. The uniform particles with various shapes can be widely applied to medicine, biological drug loading, tracing and the like, and also have important significance on research on the action mechanism of traditional Chinese medicine.

Description

Technical Field

[0001] The invention relates to micro-nano materials, and in particular to a method for preparing micro-nano particles in a decoction of traditional Chinese medicine pieces. Background Art

[0002] Traditional Chinese medicine is a treasure trove of drug research. The complex molecular system in Chinese herbal decoctions provides a huge exploration space for drug and material research. Traditional micro-nano particles are mostly synthesized in organic phases, and it is even more difficult to synthesize larger-scale particles with diverse morphologies.

[0003] Currently, there is no method for preparing micro-nano particles in decoctions of traditional Chinese medicines. Summary of the invention

[0004] The present invention newly discovered the significant effect of freeze-thaw effect in the preparation of micro-nano particles of traditional Chinese medicine. The supernatant after decoction of traditional Chinese medicine pieces can produce micro-nano particles with various morphologies through freeze-thaw effect, and the morphology can be controlled by process parameters. These uniform particles with various morphologies will have a wide range of applications in medicine, biological drug delivery, tracing, etc., and are also of great significance to the study of traditional Chinese medicine components.

[0005] The present invention provides a green, cheap, biocompatible micro-nano particle synthesized and extracted from the supernatant of a Chinese medicine decoction slice decoction in view of the deficiencies of the prior art. The prepared micro-nano particles are uniform and can produce a variety of morphologies. They have broad application prospects in the field of drug therapy. In contrast, the micro-nano particles synthesized by a Chinese medicine decoction slice decoction are simple to operate, low in cost, high in yield, and good in biocompatibility. The present invention fills the gap in the synthesis technology of spontaneously forming micro-nano particles using Chinese medicine ingredients to a certain extent.

[0006] A method for preparing micro-nano particles in a decoction of Chinese herbal medicines comprises: providing a decoction of Chinese herbal medicines, obtaining a supernatant, freezing the supernatant at -80°C to -20°C for 12-24 hours; then thawing the supernatant and collecting the micro-nano particles.

[0007] In the present invention, a conventional method can be used to prepare a Chinese medicine decoction. Specifically, for example, the Chinese medicine pieces are soaked in water, boiled, and a decoction is obtained. The amount of water added can usually cover the Chinese medicine pieces. The soaking time can usually be 1-5 hours. The boiling time can usually be 30-60 minutes. In some embodiments, double distilled water is used for soaking.

[0008] In some embodiments, the Chinese herbal medicine slices are Achyranthes bidentata slices, Angelica sinensis slices, Licorice slices, Rehmannia root slices, Aster root slices, Angelica dahurica slices, Notopterygium wilfordii slices, Cassia twig slices, White Peony root slices, Platycodon grandiflorum slices, Perilla leaf slices, Platycladus orientalis leaf slices, and Forsythia suspensa slices.

[0009] In some embodiments, the Chinese herbal medicine pieces are a combination of Achyranthes bidentata-Glycyrrhiza uralensis (weight ratio 1:1), a combination of Achyranthes bidentata-Angelica sinensis (weight ratio 1:1), and a combination of Achyranthes bidentata-Astragalus membranaceus (weight ratio 1:1).

[0010] In some embodiments, the method for obtaining the supernatant can be to filter or centrifuge the decoction. Among them, the preferred method is centrifugation (e.g., 10000g). In some embodiments, after obtaining the Chinese medicine decoction, it is immediately centrifuged (10000g) to remove the residue and collect the supernatant.

[0011] The present invention finds that in the decoction of Chinese herbal medicine slices, the components in the supernatant of the decoction are aggregated through the freeze-thaw effect to form micro-nano particles with a variety of specific morphologies.

[0012] In some embodiments, the freezing temperature is -80°C, -70°C, -60°C, -50°C, -40°C, -30°C or -20°C.

[0013] In some embodiments, the freezing time is 12h, 14h, 16h, 18h, 20h, 22h or 24h.

[0014] In some embodiments, the supernatant is frozen at -20°C overnight.

[0015] Further studies have found that modifying the supernatant before freezing can further regulate or change the morphology of the micro-nanoparticles.

[0016] In some embodiments, the method further comprises adding a modifying agent to the supernatant before freezing. The modifying agent may be selected from one or more of PEG 400, PEG 3000 or gelatin.

[0017] In some embodiments, based on the total weight of the modifier and the supernatant, the content of the modifier is 5-25 wt %, such as 10-20 wt %.

[0018] In some embodiments, after modification with PEG 400, spindle-shaped micro-nanoparticles can be obtained.

[0019] In some embodiments, after modification with PEG 3000, thick spindle-shaped micro-nanoparticles can be obtained.

[0020] In some embodiments, after modification with gelatin, thin tetrahedral micro-nanoparticles can be obtained.

[0021] In some embodiments, the micro-nano particles can be collected by centrifugation after thawing, specifically by centrifugation at 10000 g, and usually the centrifugation time can be 10-30 min.

[0022] In some embodiments, after collecting the micro-nano particles, the steps of resuspending by blowing with double distilled water, centrifuging and washing are further performed, so that relatively pure micro-nano particles can be obtained.

[0023] In some embodiments, the method for preparing micro-nano particles in a decoction of Chinese herbal medicine pieces comprises:

[0024] 1) taking Chinese herbal medicine slices, soaking them in water, and boiling them to obtain a decoction;

[0025] 2) centrifuging the decoction (immediately) (10000 g), removing the residue, and collecting the supernatant; or further adding a modifier to the supernatant;

[0026] 3) Freeze the supernatant of step 2) at -80°C to -20°C for 12-24h; thaw; centrifuge again (10000g), collect particles, and then resuspend them by blowing with double distilled water; centrifuge again (10000g), wash, and obtain micro-nanoparticles.

[0027] The present invention also provides another method for preparing micro-nano particles, comprising: providing a supernatant of achyranthes bidentata decoction and a supernatant of gypsum decoction; mixing the two to precipitate the micro-nano particles.

[0028] In some embodiments, the gypsum is raw gypsum.

[0029] In some embodiments, the mass ratio of Achyranthes bidentata and gypsum (raw gypsum) is controlled to be (1-5):(1-5), for example, 1:1.

[0030] In some embodiments, the supernatant of Achyranthes bidentata decoction and the supernatant of Gypsum Fibrosum decoction are mixed to directly precipitate particles.

[0031] The Achyranthes bidentata decoction supernatant and the Gypsum Fibrosum decoction supernatant can be prepared according to conventional methods.

[0032] In some embodiments, the method for preparing the supernatant of the Achyranthes bidentata decoction comprises: taking Achyranthes bidentata slices, soaking and decocting them in water to obtain a decoction; and filtering or centrifuging the decoction to obtain the supernatant of the Achyranthes bidentata decoction.

[0033] In some embodiments, the preparation method of the gypsum decoction supernatant includes: taking gypsum (raw gypsum) slices, soaking and decocting in water to obtain a decoction; filtering or centrifuging the decoction to obtain the gypsum decoction supernatant. Specifically, the decoction can be filtered with a 0.22 μm pore size filter membrane.

[0034] The present invention also includes micro-nano particles prepared by the above method.

[0035] The micro-nano particles prepared by the invention are micron- and nanometer-level particles with a size of 500nm-3μm.

[0036] The present invention also includes the use of the micro-nano particles in preparing drugs for immunomodulation and / or tumor treatment.

[0037] The present invention also includes the use of the micro-nano particles as drug carriers. The drug includes curcumin.

[0038] The micro-nano particles prepared by the present invention have excellent water dispersibility and biocompatibility, can adsorb drugs, fluorescent molecules, etc., and have broad application prospects in the medical fields such as drug loading and tracing.

[0039] Compared with the prior art, the present invention has at least one of the following significant advantages:

[0040] 1) For the first time, the freeze-thaw method was used to synthesize and extract uniform micro-nano particles of various morphologies from Chinese herbal medicine decoctions; the morphologies were diverse under different compatibility and modification conditions;

[0041] 2) Low production cost, Chinese herbal medicine pieces are cheap and easy to obtain, with low toxicity and good biocompatibility;

[0042] 3) The synthesis method is simple and can be synthesized using traditional Chinese medicine decoction;

[0043] 4) The micro-nano particles themselves are aggregated from the ingredients in the traditional Chinese medicine decoction and can be used directly as medicine;

[0044] 5) The obtained product has small particles, excellent water dispersibility and biocompatibility, can adsorb drugs, fluorescent molecules, etc., and will be used in drug delivery, tracing, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a SEM image of particles synthesized from the decoction of Achyranthes bidentata slices in Example 1 of the present invention.

[0046] Figure 2 This is a SEM image of particles synthesized from the decoction of Angelica sinensis slices in Example 2 of the present invention.

[0047] Figure 3 This is a SEM image of particles synthesized from liquorice slice decoction in Example 3 of the present invention.

[0048] Figure 4 This is a SEM image of particles synthesized from the decoction of Rehmannia root slices according to Example 4 of the present invention.

[0049] Figure 5 This is a SEM image of particles synthesized from the decoction of Aster tataricus slices in Example 5 of the present invention.

[0050] Figure 6 This is a SEM image of particles synthesized from the decoction of Angelica dahurica slices in Example 6 of the present invention.

[0051] Figure 7This is a SEM image of particles synthesized from the decoction of Notopterygium wilfordii slices in Example 7 of the present invention.

[0052] Figure 8 This is a SEM image of particles synthesized from cinnamon twig decoction liquid in Example 8 of the present invention.

[0053] Fig. 9 This is a SEM image of particles synthesized from the decoction of white peony root slices in Example 9 of the present invention.

[0054] Fig.10 This is a SEM image of particles synthesized from the decoction of Platycodon grandiflorum slices in Example 10 of the present invention.

[0055] Fig.11 This is a SEM image of particles synthesized from the decoction of Perilla leaves in Example 11 of the present invention.

[0056] Fig.12 This is a SEM image of particles synthesized from the decoction of Platycladus orientalis leaves in Example 12 of the present invention.

[0057] Fig.13 This is a SEM image of particles synthesized from the decoction of Forsythia suspensa slices in Example 13 of the present invention.

[0058] Fig.14 This is a SEM image of particles synthesized by mixing the supernatant of Achyranthes bidentata slices and Gypsum slices decoction in Example 14 of the present invention.

[0059] Fig.15 This is a SEM image of particles synthesized by co-decocting Achyranthes bidentata slices and Angelica sinensis slices in Example 15 of the present invention.

[0060] Fig.16 This is a SEM image of particles synthesized by co-decocting Achyranthes bidentata slices and Licorice slices in Example 16 of the present invention.

[0061] Fig.17 This is a SEM image of particles synthesized by co-decocting Achyranthes bidentata slices and Astragalus membranaceus slices in Example 17 of the present invention.

[0062] Fig.18 This is a SEM image of particles synthesized from Achyranthes bidentata slices modified with PEG 400 in Example 18 of the present invention.

[0063] Fig.19 This is a SEM image of particles synthesized from Achyranthes bidentata slices modified with PEG 3000 in Example 19 of the present invention.

[0064] Fig. 20 This is a SEM image of particles synthesized from Achyranthes bidentata slices modified with gelatin in Example 20 of the present invention.

[0065] Fig.21This is the SEM and DLS data graph of Experiment 1 of the present invention, observing the effect of freezing time on the morphology and particle size of Achyranthes bidentata particles.

[0066] Fig. 22 This is the SEM and DLS data graph of Experiment 1 of the present invention, observing the effect of 4°C storage time on the stability of Achyranthes bidentata granules.

[0067] Fig.23 This is the SEM and DLS data graph of Experiment 2 of the present invention, which explores the effects of freezing rate and freezing temperature on the morphology and particle size of Achyranthes bidentata particles.

[0068] Fig.24 This is the SEM image of particles precipitated directly after adding anhydrous calcium chloride solution to the supernatant of Achyranthes bidentata in Experiment 3 of the present invention.

[0069] Fig.25 This is the relationship between the concentration of calcium chloride added to the Achyranthes bidentata supernatant and the particle size curve of the Achyranthes bidentata particles in Experiment 3 of the present invention.

[0070] Fig.26 This is the effect of the concentration of Achyranthes bidentata granules on the proliferation activity of RAW264.7 cells cultured in vitro in Experiment 4 of the present invention.

[0071] Fig. 27 This is the comparison of curcumin loaded on Achyranthes bidentata granules and their drug release rate with free curcumin in Experiment 5 of the present invention.

[0072] Fig.28 This is a comparison of tumor tissue volume of the anti-tumor activity of Achyranthes bidentata granules on C57BL / 6 mice in Experiment 6 of the present invention. DETAILED DESCRIPTION

[0073] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0074] Example 1

[0075] (1) Weigh 50g of Achyranthes bidentata slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0076] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0077] (3) Freeze the collected supernatant at -20°C overnight.

[0078] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double-distilled water and centrifuged and washed in a handheld centrifuge to obtain the Achyranthes bidentata micro-nanoparticles.

[0079] Example 2

[0080] (1) Weigh 50g of Angelica sinensis slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0081] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0082] (3) Freeze the collected supernatant at -20°C overnight.

[0083] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0084] Example 3

[0085] (1) Weigh 50g of licorice slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0086] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0087] (3) Freeze the collected supernatant at -20°C overnight.

[0088] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0089] Example 4

[0090] (1) Weigh 50g of Rehmannia root slices, place in a pot, add double distilled water to cover the slices, and soak for 1 hour. Start the pot and boil for 40 minutes. Collect the decoction after boiling.

[0091] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0092] (3) Freeze the collected supernatant at -20°C overnight.

[0093] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0094] Example 5

[0095] (1) Weigh 50g of Aster tataricus slices, place in a pot, add double distilled water to cover the slices, and soak for 1 hour. Start the pot and boil for 40 minutes. Collect the decoction after boiling.

[0096] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0097] (3) Freeze the collected supernatant at -20°C overnight.

[0098] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0099] Example 6

[0100] (1) Weigh 50g of Angelica dahurica slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0101] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0102] (3) Freeze the collected supernatant at -20°C overnight.

[0103] (4) The supernatant was thawed and centrifuged at 10,000 g. When a precipitate was visible, the precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nanoparticles.

[0104] Example 7

[0105] (1) Weigh 50g of Notopterygium wilfordii slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0106] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0107] (3) Freeze the collected supernatant at -20°C overnight.

[0108] (4) The supernatant was thawed and centrifuged at 10,000 g. When a precipitate was visible, the precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nanoparticles.

[0109] Example 8

[0110] (1) Weigh 50g of cinnamon twig slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0111] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0112] (3) Freeze the collected supernatant at -20°C overnight.

[0113] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0114] Example 9

[0115] (1) Weigh 50g of white peony root slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0116] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0117] (3) Freeze the collected supernatant at -20°C overnight.

[0118] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0119] Example 10

[0120] (1) Weigh 50g of Platycodon grandiflorum slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0121] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0122] (3) Freeze the collected supernatant at -20°C overnight.

[0123] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0124] Embodiment 11

[0125] (1) Weigh 50g of Perilla leaf slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0126] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0127] (3) Freeze the collected supernatant at -20°C overnight.

[0128] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0129] Example 12

[0130] (1) Weigh 50 g of Platycladus orientalis leaf slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0131] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0132] (3) Freeze the collected supernatant at -20°C overnight.

[0133] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0134] Embodiment 13

[0135] (1) Weigh 50g of Forsythia suspensa slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0136] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0137] (3) Freeze the collected supernatant at -20°C overnight.

[0138] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0139] Embodiment 14

[0140] (1) Weigh 50g of Achyranthes bidentata slices and place them in a medicine pot. Add double distilled water to cover the slices and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0141] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0142] (3) At the same time, weigh 50g of raw gypsum slices and decoct the gypsum according to the methods (1) and (2). After obtaining the gypsum decoction, filter the decoction with a 0.22μm pore size filter membrane and add it drop by drop into the supernatant of Achyranthes bidentata. Stir while adding. White precipitate can be seen to gradually precipitate.

[0143] (4) The suspension is centrifuged at 10,000 g to obtain a white precipitate. The white precipitate is resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0144] Embodiment 15

[0145] (1) Weigh 50g of Achyranthes bidentata slices and 50g of Angelica sinensis slices, place them together in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction liquid after boiling.

[0146] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0147] (3) Freeze the collected supernatant at -20°C overnight.

[0148] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0149] Example 16

[0150] (1) Weigh 50g of Achyranthes bidentata slices and 50g of Licorice slices, place them in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0151] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0152] (3) Freeze the collected supernatant at -20°C overnight.

[0153] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0154] Embodiment 17

[0155] (1) Weigh 50g of Achyranthes bidentata slices and 50g of Astragalus membranaceus slices, place them in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction liquid after boiling.

[0156] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0157] (3) Freeze the collected supernatant at -20°C overnight.

[0158] (4) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0159] Embodiment 18

[0160] (1) Weigh 50g of Achyranthes bidentata slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0161] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0162] (3) Thoroughly mix the PEG 400 reagent with the supernatant to control the mass fraction of PEG 400 to 20%.

[0163] (4) Freeze the above solution at -20°C overnight.

[0164] (5) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0165] Embodiment 19

[0166] (1) Weigh 50g of Achyranthes bidentata slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0167] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0168] (3) Mix PEG 3000 with the supernatant, control the mass fraction of PEG 3000 to 20%, and heat in a water bath at 100°C for 1 h until PEG melts and is fully mixed with the supernatant.

[0169] (4) Freeze the above solution at -20°C overnight.

[0170] (5) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0171] Embodiment 20

[0172] (1) Weigh 50g of Achyranthes bidentata slices, place in a medicine pot, add double distilled water to cover the slices, and soak for 1 hour. Start the medicine pot and boil for 40 minutes. Collect the decoction after boiling.

[0173] (2) After collecting the decoction, centrifuge at 10,000 g to remove the residue and collect the supernatant.

[0174] (3) Mix gelatin with the supernatant to obtain a gelatin mass fraction of 10%, and heat in a 100°C water bath for 1 h to allow the gelatin and supernatant to be fully mixed.

[0175] (4) Freeze the above solution at -20°C overnight.

[0176] (5) The supernatant was thawed and centrifuged at 10,000 g to obtain a white precipitate. The white precipitate was resuspended in double distilled water and centrifuged and washed in a handheld centrifuge to obtain micro-nano particles.

[0177] Figure 1-20 They are SEM images of the micro-nano particles prepared in Examples 1-20 respectively.

[0178] Experiment 1 Effects of freezing time and storage time

[0179] Take 50g of Achyranthes bidentata slices, add 500mL of double distilled water, soak for 1 hour, and then boil for 40 minutes. Collect the decoction, centrifuge at 10000g, 4℃, for 30 minutes, discard the precipitate, and freeze the supernatant in a -20℃ refrigerator. After 1, 3, 5, 7, 28, and 50 days of freezing, thaw the supernatant, centrifuge at 10000g, 4℃, for 30 minutes, collect the particles, and observe their morphology and particle size by SEM (scanning electron microscope) and DLS (dynamic light scattering). Experimental results ( Fig.21 ) showed that the freezing time had little effect on the morphology and particle size of the Achyranthes bidentata particles. The collected particles were sterilized by water bath at 100°C for 30 minutes and then stored at 4°C. SEM and DLS observations were performed on them at 1, 2, 3, 4, 5, 6, 7, and 9 days. The experimental results are as follows Fig. 22 As shown, the particles have good stability and little change in morphology and particle size.

[0180] Experiment 2 Effect of freezing speed and freezing temperature

[0181] Take 50g of Achyranthes bidentata slices, add 500mL of double distilled water, soak for 1 hour, and decoct for 40 minutes. Collect the decoction, centrifuge at 10000g, 4℃, for 30 minutes, discard the precipitate, collect the supernatant in a centrifuge tube and mark it as a and b. Tube a is directly frozen at -80℃, while tube b is first frozen at -20℃ for 30 minutes and then frozen at -80℃. Thaw after 12 hours and observe the morphology and particle size by SEM and DLS. The results show ( Fig.23 ), Fig.23 a (directly placed in -80℃), Fig.23 b (freeze at -20℃ for 30min, then put into -80℃ overnight) and Figure 1 (-20℃ freezing) The morphology difference is not obvious, and DLS test shows that the particle size distribution of samples a and b is consistent, which indicates that the freezing temperature and cooling rate have little effect on the morphology and particle size of Achyranthes bidentata particles.

[0182] Fig.21 , Fig. 22and Fig.23 In the figure, the abscissa Size represents the particle size, and the ordinate Intensity (Percent) represents the intensity (relative proportion).

[0183] Experiment 3 Effect of calcium content in decoction on particle size

[0184] Take 50g of Achyranthes bidentata slices, add 500mL of double distilled water, soak for 1 hour, and boil for 40min. Collect the decoction, centrifuge at 10000g, 4℃, for 30min, discard the precipitate, collect the supernatant and weigh 287.73g. Take 9mL of this supernatant respectively, add 1mL of calcium chloride solution, and make the concentration of calcium chloride added to the solution 0, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 500, 1000, 2500μg / mL. Then place it in a -20℃ freezer, thaw after 12h, collect the particles and test. It was found that when the concentration of calcium chloride added to the supernatant was above 500μg / mL, spherical particles were directly precipitated, and the particle size was large ( Fig.24 ). After DLS analysis of the above-mentioned gradient calcium chloride concentration samples, it was drawn Fig.25 . The results showed that when the calcium content added to the supernatant was below 10 μg / mL, the amount of calcium added had little effect on the particle size of the Achyranthes bidentata granules, and the average hydrated particle size of the Achyranthes bidentata granules was 751.37 nm. When the calcium chloride content added to the supernatant was between 10-100 μg / mL, the hydrated particle size of the Achyranthes bidentata granules increased with the increase in the amount of calcium added, and the fitted curve relationship was y=4.7059x+783.86. When the calcium chloride content added to the supernatant was above 500 μg / mL, the Achyranthes bidentata granules precipitated directly and had a larger particle size, with an average hydrated particle size of 2983.11 nm.

[0185] Fig.25 In the figure, the abscissa represents the mass concentration of the added calcium chloride in the total solution system, and the ordinate represents the average particle size of the sample obtained by DLS detection.

[0186] Experiment 4: Promoting macrophage proliferation

[0187] 3000 RAW264.7 cells / well were inoculated on a 96-well plate. The collected Achyranthes bidentata micro-nanoparticles prepared in Example 1 were sterilized in a 100°C water bath for 30 min, and then suspended in culture medium at concentrations of 100, 50, 25, 10, 5, 1, 0.5, and 0 μg / mL. 100 μL of each well was incubated with the cells. After 24 hours, the solution was aspirated, and DMEM culture medium containing 10% CCK8 reagent was added. After 2-3 hours, the absorbance was observed at 450 nm. Fig. 22The results showed that as the concentration of Achyranthes bidentata granules increased, the proliferation activity of RAW264.7 cells increased, and reached the peak of RAW264.7 proliferation activity at a concentration of 10 μg / mL. Then, as the concentration of Achyranthes bidentata granules increased further, the proliferation activity of RAW264.7 cells began to decline. This in vitro experiment shows that a certain concentration of Achyranthes bidentata granules can promote the proliferation of RAW264.7 macrophages, thereby regulating the body's immune ability.

[0188] Fig.26 In the figure, the horizontal axis represents the administration concentration of Achyranthes bidentata granules, and the vertical axis represents the cell survival rate.

[0189] Experiment 5 Curcumin loading and drug release

[0190] The collected Achyranthes bidentata micro-nanoparticles prepared in Example 1 were suspended in anhydrous ethanol to prepare an Achyranthes bidentata granules anhydrous ethanol suspension with a concentration of 2 mg / mL. Curcumin was dissolved in anhydrous ethanol to prepare a curcumin anhydrous ethanol solution with a concentration of 2 mg / mL. 1 mL of the Achyranthes bidentata granules anhydrous ethanol suspension and 1 mL of the curcumin anhydrous ethanol solution were taken and added to a round-bottom flask to mix. An appropriate amount of anhydrous ethanol was added to fully mix the Achyranthes bidentata granules in the solution. Place it on a rotary evaporator and evaporate it under reduced pressure at 80 rpm and 40°C. After the ethanol reagent was fully evaporated, it was visible that the round-bottom flask was covered with a yellow film. 2 mL of PBS buffer was added to the round-bottom flask, and the yellow film on the wall of the flask was scraped into the liquid in the suspension to obtain an Achyranthes bidentata granules-curcumin PBS suspension. A PBS suspension containing only curcumin was prepared in the same way. It was found that the sedimentation rate of the particles in the curcumin suspension was much faster than that of the Achyranthes bidentata granules-curcumin suspension ( Fig. 27 d). After standing for 5 minutes, the curcumin suspension settles completely, the supernatant is relatively colorless and transparent, and the upper layer of the Achyranthes bidentata granules-curcumin suspension is full of particles. At this time, the upper layer of the Achyranthes bidentata granules-curcumin suspension is carefully sucked out, and the precipitate is discarded to obtain purer Achyranthes bidentata granules-curcumin composite particles. The upper and lower particles are centrifuged to remove the solvent, and after being dissolved with anhydrous ethanol, the content of curcumin in the upper layer and the precipitate is measured at 426nm using an ultraviolet spectrophotometer, and the encapsulation rate of curcumin is calculated to be 46.0%.

[0191] Under SEM observation, free curcumin ( Fig. 27 a) is easy to crystallize in water and has poor dispersibility. Fig. 27 b) After the auxiliary preparation, the Achyranthes bidentata particles inhibited the crystallization of curcumin, and curcumin was wrapped on the surface of the Achyranthes bidentata particles ( Fig. 27 c). In summary, in water, the Achyranthes bidentata granules-curcumin composite particles have better dispersibility than free curcumin.

[0192] Prepare a PBS solution containing 30% (v / v) ethanol as the dissolution medium. Centrifuge the Achyranthes bidentata granules-curcumin composite particles to remove the solvent, add the release medium to resuspend, transfer to the dialysis bag, and add the same dissolution medium outside the dialysis bag. Take curcumin and control its mass to be consistent with the mass of curcumin loaded in the Achyranthes bidentata granules-curcumin, and set up the dialysis system in the same way as above. Stir at a constant speed under 37°C water bath conditions. Take out 9mL of the dissolution medium outside the dialysis bag at 15min, 30min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 20h, and 45h, respectively, and then immediately replenish an equal amount of dissolution medium to keep the total amount of liquid in the system unchanged. Use an ultraviolet spectrophotometer to measure the curcumin content in the solution at 426nm, and calculate the cumulative dissolution rate ( Fig. 27 e). It was found that the Achyranthes bidentata granules-curcumin composite particles released curcumin more slowly than free curcumin.

[0193] In summary, Achyranthes bidentata granules can be used as carriers to load target drug ingredients.

[0194] Fig. 27 In figure e, the abscissa represents the sampling time, and the ordinate represents the mass concentration of curcumin dissolved out of the dialysis bag.

[0195] Experiment 6 Antitumor Activity

[0196] Take 50g of Achyranthes bidentata slices, add 500mL of double distilled water, soak for 1 hour, and then boil for 40 minutes. Collect the decoction, centrifuge at 10000g, 4℃, for 30 minutes, discard the precipitate, collect the supernatant and freeze for 24 hours, then thaw and collect the Achyranthes bidentata micro-nano particles. C57BL / 6 mice were divided into groups A and B, with 2 mice in each group. 5×10 5 One week later, mice in group A were given PBS peritumorally every 2 days, and mice in group B were given Achyranthes bidentata granules every 2 days. The tumor size was observed after 6 administrations. Fig.28 As shown, the results showed that the peritumoral administration of Achyranthes bidentata granules reduced the tumor volume compared with the peritumoral administration of PBS group.

[0197] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing micro-nano particles in a decoction of Chinese herbal medicine, characterized in that: include: Provide Chinese medicine decoction, obtain supernatant, and freeze at -80℃ to -20℃ for 12-24h; Then thaw and collect the micro-nano particles.

2. The method for preparing micro-nano particles in a decoction of Chinese herbal medicine according to claim 1, characterized in that: The Chinese herbal medicine slices are Achyranthes bidentata slices, Angelica sinensis slices, Licorice slices, Rehmannia root slices, Aster root slices, Angelica dahurica slices, Notopterygium wilfordii slices, Cassia twig slices, White Peony root slices, Platycodon grandiflorum slices, Perilla leaf slices, Platycladus orientalis leaf slices, Forsythia suspensa slices, a combination of Achyranthes bidentata and Licorice, a combination of Achyranthes bidentata and Angelica sinensis, or a combination of Achyranthes bidentata and Astragalus membranaceus.

3. The method for preparing micro-nano particles in a decoction of Chinese herbal medicine according to claim 1 or 2, characterized in that: The method for obtaining the supernatant is to filter or centrifuge the decoction.

4. The method for preparing micro-nano particles in a decoction of a Chinese herbal medicine according to any one of claims 1 to 3, characterized in that: The freezing temperature is -80°C, -70°C, -60°C, -50°C, -40°C, -30°C or -20°C; and / or, The freezing time is 12h, 14h, 16h, 18h, 20h, 22h or 24h.

5. The method for preparing micro-nano particles in a decoction of a Chinese herbal medicine according to any one of claims 1 to 3, characterized in that: The supernatant was frozen at -20°C overnight.

6. The method for preparing micro-nano particles in a decoction of a Chinese herbal medicine according to any one of claims 1 to 5, characterized in that: The method further comprises, prior to freezing, adding a modifying agent to the supernatant; Optionally, the modification is selected from one or more of PEG 400, PEG 3000 or gelatin; Optionally, based on the total weight of the modifier and the supernatant, the content of the modifier is 5-25 wt %.

7. The method for preparing micro-nano particles in a decoction of a Chinese herbal medicine according to any one of claims 1 to 6, characterized in that: include: 1) taking Chinese herbal medicine slices, soaking them in water, and boiling them to obtain a decoction; 2) centrifuging the decoction to remove the residue and collecting the supernatant; or further adding a modifier to the supernatant; 3) Freeze the supernatant of step 2) at -80°C to -20°C for 12-24h; thaw; centrifuge again to collect particles; then resuspend with double distilled water; centrifuge again and wash to obtain micro-nanoparticles.

8. A method for preparing micro-nano particles, characterized in that: include: Providing the supernatant of Achyranthes bidentata decoction and the supernatant of Gypsum Fibrosum decoction; The two are mixed to precipitate micro-nano particles; Optionally, the mass ratio of Achyranthes bidentata and gypsum is controlled to be (1-5):(1-5), for example 1:

1.

9. Micro-nano particles prepared by the method according to any one of claims 1 to 8; optionally, the size of the micro-nano particles is 500nm-3μm.

10. Use of the micro-nano particles according to claim 9 in the preparation of drugs for immunomodulation and / or tumor treatment or as drug carriers.

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