Nano-cellulose, preparation method thereof and application of nano-cellulose in reduction of bile acid level

Abstract

The invention belongs to the technical field of disease medicine preparation, and particularly relates to nanocellulose, a preparation method thereof and application of the nanocellulose to reduction of bile acid level. The nano-crystalline cellulose is nano-crystalline cellulose fiber or nano-crystalline cellulose crystal; wherein the nano-crystalline cellulose fibers are in a slender whisker shape, the average length of the nano-crystalline cellulose fibers is 250-400 nm, and the nano-crystalline cellulose fibers are partially gathered and wound; the nanocellulose crystal is in a short rod crystal shape, the surface of the nanocellulose crystal is connected with a sulfuric acid half ester group, and the length of the nanocellulose crystal is 50-200 nm. The nanocellulose provided by the invention can achieve the purpose of treating intrahepatic cholestasis in the gestation period by improving the TBA level and improving the filial generation live birth rate; the traditional Chinese medicine composition has no obvious influence on liver and kidney functions, blood sugar, blood fat and the like, and is high in safety; in addition, the nanocellulose does not have potential target organ poison or accumulative effect, the biological safety performance is improved, and the nanocellulose can be used for a long time and is suitable for special crowds during pregnancy.

Description

Technical Field

[0001] The invention belongs to the technical field of disease medicine preparation, and specifically relates to nanocellulose and a preparation method thereof and application in reducing bile acid levels. Background Art

[0002] ICP (intrahepatic cholestasis of pregnancy) is a liver disease that occurs in the second and third trimesters of pregnancy. It is mainly manifested by skin itching and jaundice. The cause is not yet fully understood and may be related to female hormones, genetics, environment, and drug factors. The disease has a high incidence rate, and the purpose of treatment is to relieve symptoms, restore liver function, and ensure the safety of the fetus.

[0003] Currently, ICP (intrahepatic cholestasis of pregnancy) can be treated with drugs, such as ursodeoxycholic acid (UDCA), which can achieve the therapeutic purpose by reducing the toxicity of bile acid and improving liver function. However, UDCA can only relieve the itching symptoms of some ICP patients, but has no significant effect on reducing bile acid in pregnant women, reducing or avoiding adverse perinatal outcomes such as meconium-stained amniotic fluid, fetal distress, spontaneous premature birth, intrauterine growth restriction, and even intrauterine death.

[0004] In recent years, some synthetic nanomaterials (such as nanoparticles and nanosheets) have been reported to significantly improve related hepatobiliary diseases by directly adsorbing bile acids or regulating intestinal flora, but these materials may have biocompatibility and long-term safety issues. Plant-derived nanocellulose (such as nanocellulose crystals and nanocellulose fibers) is an insoluble nanoscale dietary fiber. It is derived from plant fibers and has the characteristics of high biocompatibility, good safety and high specific surface area. It can adsorb bile acid molecules and regulate intestinal flora, etc., and has attracted the attention of researchers in recent years. Summary of the invention

[0005] Based on this, the present invention has developed a plant-derived nanocellulose, nanocellulose crystals (CNC) and nanocellulose fibers (CNF), both of which can effectively treat intrahepatic cholestasis of pregnancy.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On one hand, the present invention provides a nanocellulose, which is nanocellulose fiber or nanocellulose crystal; wherein the nanocellulose fiber has an elongated whisker-like morphology, an average length of the nanocellulose fiber is 250nm-400nm, an average diameter is 40nm-60nm, and some of the nanocellulose fibers are aggregated and entangled; the nanocellulose crystal has a short rod crystal-like morphology, and has a sulfate half-ester group connected to the surface, an average length of the nanocellulose crystal is 50nm-200nm, and an average diameter is 10nm-40nm.

[0008] Preferably, the nanocellulose satisfies one or more of the following conditions: (1) the surface of the nanocellulose is also modified with carboxyl groups and / or sulfate groups; (2) the nanocellulose is in the form of a suspension or a powder.

[0009] On the other hand, the present invention also provides a method for preparing the nanocellulose of the present invention, the preparation method comprising: when the nanocellulose is nanocellulose fiber, the preparation method of the nanocellulose fiber comprises: mixing microcrystalline cellulose and alkali solution, ball milling, and removing the alkali solution to obtain a nanocellulose fiber suspension; when the nanocellulose is nanocellulose crystal, the preparation method of the nanocellulose crystal comprises: hydrolyzing the microcrystalline cellulose with sulfuric acid to obtain a hydrolyzate; centrifuging the hydrolyzate until the supernatant of the centrifugation becomes a turbid suspension, which is the nanocellulose crystal suspension.

[0010] Preferably, the preparation method of the above-mentioned nanocellulose fibers meets one or more of the following conditions: (a) the diameter of the grinding balls used in ball milling is 5mm-15mm; (b) the alkali solution is sodium hydroxide solution; (c) the ball milling includes: ball milling for 18min-22min, resting for 8min-12min; and total ball milling for 1.5h-2.5h; (d) the ball milling rate is 350r / min-450r / min; The preparation method of nanocellulose crystals meets one or more of the following conditions: (i) the mass fraction of sulfuric acid is 55%-65%; (ii) the hydrolysis temperature is 45℃-55℃; (ii) the hydrolysis time is 1h-2h.

[0011] Preferably, the preparation method further comprises freeze-drying the nanocellulose fiber suspension or the nanocellulose crystal suspension to obtain nanocellulose fiber freeze-dried powder or nanocellulose crystal freeze-dried powder.

[0012] In another aspect, the present invention provides a medicine or functional food, which comprises the nanocellulose of the present invention.

[0013] Preferably, the above-mentioned medicine further comprises dietary fiber and / or probiotics.

[0014] In another aspect, the present invention further provides a use of the nanocellulose of the present invention in the preparation of a medicament for treating intrahepatic cholestasis of pregnancy.

[0015] Preferably, the above-mentioned application includes one or more combinations of the following applications: (i) application of the nanocellulose of the present invention in the preparation of a drug for reducing the bile acid level of the body; (ii) application of the nanocellulose of the present invention in the preparation of a drug for improving the survival rate of offspring during pregnancy; (iii) application of the nanocellulose of the present invention in the preparation of a drug for increasing the abundance of beneficial bacteria in the intestinal flora of the body; (iv) application of the nanocellulose of the present invention in the preparation of a drug for reducing the abundance of bacteria related to bile acid metabolism in the intestinal flora of the body.

[0016] In another aspect, the present invention also provides a use of the nanocellulose of the present invention in maintaining a healthy level of bile acid in the body.

[0017] Preferably, in the above application, the effective amount of nanocellulose is 30 mg / Kg-180 mg / Kg.

[0018] The beneficial effects of the present invention include: the nanocellulose provided by the present invention can achieve the purpose of treating intrahepatic cholestasis of pregnancy by improving the levels of TBA, AST and ALT, as well as improving the live fetal rate of offspring and the weight level at the end of pregnancy; and has no obvious effect on liver and kidney function, blood sugar and blood lipids, and has high safety; in addition, the nanocellulose in the present invention does not have potential target organ toxicity or cumulative effect, increases biosafety performance, can be used for a long time and is suitable for special populations during pregnancy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 FTIR spectra of CNC and CNF;

[0020] Figure 2 XRD patterns of CNC and CNF;

[0021] Figure 3 AFM images of CNC and CNF;

[0022] Figure 4 It is a schematic diagram of the verification process of the nanocellulose of the present invention;

[0023] Figure 5 The contents of TBA, AST and ALT in different groups of mice;

[0024] Figure 6 The survival rates of offspring of mice in different groups are shown in Figure 2.

[0025] Figure 7 The weight changes of mice in different groups;

[0026] Figure 8 The weight of mice in different groups at the end of pregnancy;

[0027] Fig. 9The safety test results of CNC and CNF mice are as follows;

[0028] Fig.10 The differential bacterial flora between the CNF intervention group and the ICP group;

[0029] Fig.11 These are the different bacterial flora between the CNC intervention group and the ICP group. DETAILED DESCRIPTION

[0030] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0031] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0032] An embodiment of the present invention provides a nanocellulose, wherein the nanocellulose is nanocellulose fiber or nanocellulose crystal; wherein the nanocellulose fiber has an elongated whisker-like morphology, an average length of the nanocellulose fiber is 250nm-400nm (such as 300nm, 350nm or 370nm, etc.), an average diameter of 40nm-60nm (such as 45nm, 50nm, 52nm, 57nm or 59nm, etc.), and some of the nanocellulose crystals are aggregated and entangled; the nanocellulose crystals have a short rod crystal-like morphology, and a sulfate half-ester group is connected to the surface, the average length of the nanocellulose crystals is 50nm-200nm (such as 75nm, 90nm, 120nm, 150nm or 180nm, etc.), and an average diameter of 10nm-40nm (such as 15nm, 17nm, 19nm, 23nm, 25nm, 28nm, 31nm, 35nm or 38nm, etc.).

[0033] It should be noted that the nanocellulose in the present invention is based on plant-derived nanocellulose (such as nanocellulose crystals CNC and nanocellulose fibers CNF), which has natural origin, low toxicity and good biocompatibility; the special surface structure (such as hydroxyl, carboxyl) and nanoscale size enable it to regulate intestinal flora. The key mechanisms include: improving intestinal flora and improving intestinal ecology: by increasing the abundance of specific beneficial bacteria (such as Alloprevotella and Prevotellaceae_UCG-001) and reducing the abundance of bile acid metabolism-related flora (such as Lactobacillus, Clostridiumsensu stricto1 and Acetatifactor), the intestinal ecological environment and bile acid metabolism are indirectly regulated. In addition, the nanocellulose in the present invention activates the bile acid metabolic pathway in the intestine to reduce bile acid reabsorption by regulating the structure and abundance of intestinal flora, inhibits liver bile acid synthesis, and increases liver bile acid excretion; while improving intestinal tryptophan, short-chain fatty acids and other metabolism to maintain intestinal health and promote the normal metabolic process of the intestine.

[0034] In some specific examples, the nanocellulose satisfies one or more of the following conditions:

[0035] (1) The surface of the nanocellulose is also modified with carboxyl groups and / or sulfate groups; specifically, the nanocellulose in the present invention can also be modified with carboxyl groups and / or sulfate groups to enhance the adsorption capacity of the nanocellulose for bile acid; in addition, the method of modifying the nanocellulose with carboxyl groups and / or sulfate groups is a method known in the art;

[0036] (2) The form of nanocellulose includes suspension or powder. Specifically, the form of nanocellulose in the present invention can be suspension or powder. The preparation methods of suspension and powder are well known in the art.

[0037] The present invention also provides a method for preparing the nanocellulose of the present invention, the method comprising:

[0038] When the nanocellulose is nanocellulose fiber, the method for preparing the nanocellulose fiber comprises: mixing microcrystalline cellulose and alkali solution, ball milling, and removing the alkali solution to obtain a nanocellulose fiber suspension;

[0039] When the nanocellulose is nanocellulose crystals, the preparation method of the nanocellulose crystals includes: hydrolyzing microcrystalline cellulose with sulfuric acid to obtain a hydrolyzate; centrifuging the hydrolyzate until the centrifugal supernatant becomes a turbid suspension, which is the nanocellulose crystal suspension.

[0040] In some specific examples, the method for preparing the nanocellulose fibers satisfies one or more of the following conditions:

[0041] (a) The diameter of the grinding balls used in the ball mill is 5 mm to 15 mm, such as 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm or 14 mm. Specifically, in the present invention, the grinding balls can all be grinding balls of the above single diameter, or can also be a mixture of grinding balls of the above multiple diameters.

[0042] (b) the alkali solution is a sodium hydroxide solution; specifically, the alkali solution is well known in the art, such as a sodium hydroxide solution;

[0043] (c) ball milling includes: ball milling for 18 min to 22 min, such as 19 min, 20 min or 21 min, etc., resting for 8 min to 12 min, such as 9 min, 10 min or 11 min, etc.; total ball milling for 1.5 h to 2.5 h, such as 1.7 h, 2 h or 2.2 h, etc.;

[0044] (d) The ball milling speed is 350 r / min-450 r / min, such as 370 r / min, 400 r / min or 430 r / min;

[0045] The method for preparing nanocellulose crystals satisfies one or more of the following conditions:

[0046] (i) the mass fraction of sulfuric acid is 55% to 65%, such as 58%, 60% or 62%;

[0047] (ii) the hydrolysis temperature is 45°C to 55°C, such as 47°C, 50°C or 53°C;

[0048] (ii) The hydrolysis time is 1 h to 2 h, such as 1.2 h, 1.5 h or 1.7 h.

[0049] In some specific examples, the preparation method further comprises freeze-drying the nanocellulose fiber suspension or the nanocellulose crystal suspension to obtain nanocellulose fiber freeze-dried powder or nanocellulose crystal freeze-dried powder.

[0050] It should be noted that the nanocellulose in the present invention can be prepared into freeze-dried powder by freeze-drying, which is more convenient for storage, transportation and use.

[0051] The embodiment of the present invention also provides a drug or functional food, which includes the nanocellulose of the present invention.

[0052] It should be noted that the nanocellulose in the present invention can achieve the purpose of treating intrahepatic cholestasis of pregnancy by improving TBA levels and improving the live fetal rate of offspring; therefore, it can be used as or prepared into a drug for treating intrahepatic cholestasis of pregnancy. It should also be noted that the nanocellulose in the present invention can maintain the body's bile acid level in a healthy state, so it can also be prepared into a functional food for maintaining the body's healthy bile acid level. It should also be noted that the nanocellulose in the present invention has no obvious effect on liver and kidney function, blood sugar, and blood lipids, and is highly safe.

[0053] In some specific examples, the above-mentioned medicine or functional food also includes dietary fiber and / or probiotics.

[0054] It should be noted that the nanocellulose in the present invention can be used in combination with dietary fiber and / or probiotics to further enhance the effect of regulating intestinal flora and bile acid metabolism; in addition, in addition to dietary fiber and / or probiotics, other carriers such as buffer solutions can also be included.

[0055] An embodiment of the present invention also provides a use of the nanocellulose of the present invention in the preparation of a drug for treating intrahepatic cholestasis of pregnancy.

[0056] In some specific examples, the above-mentioned applications include one or more combinations of the following applications: (i) application of the nanocellulose of the present invention in the preparation of a drug for reducing the bile acid level of the body; (ii) application of the nanocellulose of the present invention in the preparation of a drug for improving the survival rate of offspring during pregnancy; (iii) application of the nanocellulose of the present invention in the preparation of a drug for increasing the abundance of beneficial bacteria in the intestinal flora of the body; (iv) application of the nanocellulose of the present invention in the preparation of a drug for reducing the abundance of bacteria related to bile acid metabolism in the intestinal flora of the body.

[0057] It should be noted that the nanocellulose in the present invention can achieve the purpose of treating intrahepatic cholestasis of pregnancy by effectively reducing bile acid levels, improving the survival rate of offspring during pregnancy, increasing the abundance of beneficial bacteria in the body's intestinal flora, and reducing the abundance of bacteria related to bile acid metabolism in the body's intestinal flora.

[0058] In some specific examples, in the above applications, the effective amount of nanocellulose is 30 mg / Kg-180 mg / Kg, such as 40 mg / Kg, 80 mg / Kg, 100 mg / Kg, 130 mg / Kg, 150 mg / Kg or 170 mg / Kg, etc.

[0059] An embodiment of the present invention also provides an application of the nanocellulose in the present invention in maintaining a healthy level of bile acid in a body.

[0060] In some specific examples, in the above applications, the effective amount of nanocellulose is 30 mg / Kg-180 mg / Kg, such as 40 mg / Kg, 80 mg / Kg, 100 mg / Kg, 130 mg / Kg, 150 mg / Kg or 170 mg / Kg, etc.

[0061] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0062] 1. Preparation of plant-derived nanocellulose

[0063] Example 1 Extraction of CNC

[0064] (1) Weigh 5 g of microcrystalline cellulose (MCC) and dissolve it in 100 mL of sulfuric acid (64%, w / v), stir it with a magnetic stirrer at 45°C for 1.5 h until the cellulose is completely hydrolyzed, and then add 5 times the volume of distilled water to terminate the reaction to obtain a hydrolyzate;

[0065] (2) washing the hydrolyzate by high-speed centrifugation at 5000 r / min (washing with distilled water) until the supernatant becomes milky turbid, thereby obtaining a milky suspension, i.e., a CNC suspension;

[0066] (3) The CNC suspension was dialyzed in distilled water for 48 h, with the water changed every 4 h. The suspension was stored at 4 °C and a portion of the sample was vacuum dried for characterization.

[0067] Example 2 Extraction of CNF

[0068] (1) 1 g of MCC, 20 mL of 2 wt% NaOH solution and 90 g of zirconia balls (with diameters of 15 mm, 12 mm, 10 mm, 8 mm and 5 mm, respectively, and a number ratio of 1:1:7:11:63) were mixed in a ball mill, and the ball mill was processed at room temperature at a milling speed of 400 r / min for 2 h (forward operation for 10 min, stop for 10 min, and then reverse operation for 10 min, and the total working time was 2 h);

[0069] (2) The product obtained in step (1) was dialyzed at 4° C. for 48 h to remove the alkali solution to obtain a suspension, and the obtained suspension was stored at 4° C. for further use and partially freeze-dried for characterization.

[0070] 2. Characterization of plant-derived nanocellulose

[0071] (a) Fourier transform infrared spectroscopy (FTIR)

[0072] The freeze-dried dried CNC and CNF samples were placed in an infrared spectrometer (Nicoleti S10, Thermo Corporation, USA) for testing; the number of scans was 32 times, and the resolution was 4 cm -1 , the scanning wave number range is 4000~600cm -1 , the test temperature is 25℃.

[0073] FTIR spectra of CNC and CNF are shown in Figure 1 As shown in the figure, the results show that due to the same extraction raw materials and relatively stable structure of cellulose, the FTIR spectra of CNC obtained by sulfuric acid hydrolysis and CNF obtained by mechanical ball milling are basically consistent, showing basically similar characteristic peaks; among which 3320cm -1 The broad absorption peak at 1641 cm -1 The absorption peak at 2897cm belongs to OH stretching vibration, which is caused by the large amount of hydroxyl groups in nanocellulose; -1 The peak at 1063 cm corresponds to the CH stretching vibration; -1 and 1052cm -1 The peaks observed at 892 cm-1 and 892 cm-2 belong to the pyranose cyclic ether band of nanocellulose. -1 The peak at 815 cm -1 A weak peak appears at , which corresponds to the symmetrical stretching of SOC. This is because during the hydrolysis of sulfuric acid, the sulfate ions (–OSO3 - ) will undergo esterification reaction with CNC molecules, especially introducing sulfate groups at the C6 position of cellulose. FTIR results show that the basic structures of CNC and CNF are the same, and both retain the original structure of cellulose.

[0074] (ii) X-ray diffraction (XRD)

[0075] The dried CNC and CNF samples after freeze-drying were pressed flat, placed on the cassette, placed in the diffraction slot of the X-ray diffractometer (X'Pert3 Powder 10300, Bruker, USA), and recorded under Cu-Kα radiation of 40 kV and 40 mA. The scattering angle (2θ) ranged from 10° to 50°, the scanning rate was 2° / min, and the step size was 0.02°. The spectrum was analyzed using MDIJADE 6 software, and the crystallinity index (CrI) of CNF and CNC was calculated using the Segal formula:

[0076]

[0077] Among them I 002 is the intensity of the diffraction peak at 2θ = 22.8°; Iam is the intensity of the scattering peak at 2θ = 18.5°.

[0078] The XRD patterns of CNC and CNF are shown in Figure 2 As shown, the results show that both CNC and CNF samples show typical diffraction peaks of cellulose I at 16.0°, 22.8° and 31.9°, corresponding to the (101), (002) and (040) crystal planes of cellulose, respectively. This further proves that sulfuric acid hydrolysis and mechanical ball milling have no effect on the crystalline structure of cellulose. In addition, the crystallinity calculation results show that the crystallinity of CNF and CNC is 70.92% and 71.05%, respectively. This may be because sulfuric acid hydrolysis destroys more amorphous regions of CNC and retains crystalline regions, resulting in slightly higher crystallinity of CNC than CNF. Previous studies have shown that mechanical ball milling treatment has a great damage to the crystallinity of cellulose, especially vibration ball milling, which can be used to produce amorphous cellulose. Compared with the conventional mechanical ball milling method using distilled water as the ball milling medium to prepare CNF, the crystallinity of CNF prepared using NaOH solution as the ball milling medium is relatively high. This is mainly because the heat and mechanical energy generated by ball milling make it easy for hydroxide ions to enter the internal glycosidic bonds in the amorphous region of cellulose, accelerating the degradation of the amorphous region of cellulose; at the same time, the ball milling treatment accelerates the formation of the CNF network structure, avoiding excessive damage to its crystalline region by the ball milling shear force, thereby significantly improving the crystallinity of CNF.

[0079] 3. Atomic force microscopy (AFM)

[0080] The microscopic morphology of CNC and CNF was observed by AFM (Nano Man VS, Bruker, USA); the CNC and CNF suspensions were diluted to 0.01% (w / v), then dropped onto freshly peeled mica sheets, dried at room temperature, and tested. The average size of the samples was measured using Nano Measure software for the collected AFM images.

[0081] The microstructure of CNF and CNC samples was characterized. Figure 3 As shown, CNF presents a relatively long nanofibril structure and is partially aggregated and entangled, while CNC presents a short rod or needle structure; the average lengths of CNF and CNC samples were calculated by Image J software, and the average lengths of CNF and CNC were 340nm and 114nm, respectively; CNF is longer than CNC, which may make it easy for CNF to entangle together to form a fiber network structure; sulfuric acid hydrolysis makes CNC carry more negatively charged sulfonate groups, and the strong electrostatic repulsion between CNCs can lead to good dispersibility, thereby obtaining CNCs with small size and more uniform distribution.

[0082] 3. Verification of plant-derived nanocellulose

[0083] In the following verification, the Beckman Coulter AU5821 analyzer (Shizuoka, Japan) was used to measure the levels of serum total bile acid (TBA), liver function (alanine aminotransferase ALT, aspartate aminotransferase AST), renal function (blood urea nitrogen BUN, blood uric acid sUA and blood creatinine sCR), blood sugar and blood lipids (glucose GLU, total cholesterol TCHO, high-density lipoprotein cholesterol HDL and low-density lipoprotein cholesterol LDL).

[0084] 1. Effects of nanocellulose on ICP rats

[0085] (1) Experimental Materials

[0086] CNC suspensions with measured concentrations of 2.1% (2.1 g CNC per 100 ml of suspension) and 2.4% CNF suspensions (2.1 g CNF per 100 ml of suspension) were prepared for dietary intervention tests; UDCA (ursodeoxycholic acid) was purchased from Sigma-Aldrich Company and used as the treatment control group; Sprague–Dawley (SD) rats (specific pathogen-free, 8 weeks, 190 g-220 g) were purchased from the Experimental Animal Center of Chongqing Medical University, with a temperature of 18°C-21°C, a constant humidity of 65%-70%, a light intensity of 12 hours (8:00-20:00), sound insulation, free food and water intake, and experiments were conducted after one week of adaptation to the environment.

[0087] (2) Experimental methods

[0088] 17α-ethinyl estradiol is a drug widely used at home and abroad to induce animal intrahepatic cholestasis models in vivo. 5 mg / kg of 17α-ethinyl estradiol was subcutaneously injected daily in pregnant rats from the 13th to the 16th day of pregnancy to induce the pregnancy cholestasis model, and then subcutaneous injections were continued from the 17th to the 20th day of pregnancy to maintain the ICP model; propylene glycol was used as a solvent to dissolve 17α-ethinyl estradiol in the experiment, and the final solution concentration was 1.0 mg / ml.

[0089] The female mice used for the formal experiment of nanocellulose intervention and ICP modeling were randomly divided into 5 groups (6 mice in each group): (1) Normal pregnancy group (NP group): Each mouse was gavaged with 1.0 ml of deionized water every day, and 1.5 ml of propylene glycol was injected subcutaneously behind the neck every day from the 13th to the 20th day of pregnancy; (2) ICP model group (ICP group): Each mouse was gavaged with 1.0 ml of deionized water every day, and 5 mg / (kg·d) of 17α-ethynyl estradiol was injected subcutaneously behind the neck every day from the 13th to the 20th day of pregnancy; (3) ICP+UDCA intervention group (ICP-UDCA group): Each mouse was gavaged with 1.0 ml of deionized water every day, and 5 mg / (kg·d) of 17α-ethynyl estradiol was injected subcutaneously behind the neck every day from the 13th to the 20th day of pregnancy. g / (kg·d) 17α-ethinyl estradiol was injected subcutaneously behind the neck. Then, from the 17th to the 20th day of pregnancy, each mouse was gavaged with UDCA at a dose of 25 mg / (kg·day) per day; (4) ICP+CNC intervention group (ICP-CNC group): CNC suspension was gavaged at a dose of 90 mg / (kg·d) per day. From the 13th to the 20th day of pregnancy, each mouse was subcutaneously injected with 17α-ethinyl estradiol at a dose of 5 mg / (kg·d) per day behind the neck; (5) ICP+CNF intervention group (ICP-CNF group): CNF suspension was gavaged at a dose of 90 mg / (kg·d) per day. From the 13th to the 20th day of pregnancy, each mouse was subcutaneously injected with 17α-ethinyl estradiol at a dose of 5 mg / (kg·d) per day behind the neck. In addition, the above rats were all based on a standard rat maintenance feed diet (the feed was derived from SDF-grade rat maintenance feed from Universal Biological). The mating process was to put male and female rats in estrus together in cages, and the first day of pregnancy was when a vaginal plug was observed.

[0090] The animals were weighed every two days during the experiment. On the 21st day of pregnancy, the rats were anesthetized with pentobarbital at 30 mg / kg after overnight fasting, and blood was collected from the post-annular venous plexus. The survival of the offspring was counted by cesarean section. The cecal contents were collected with sterile forceps and quickly frozen in liquid nitrogen and then stored in a -80°C refrigerator for subsequent 16S rRNA gene sequencing analysis. The blood samples were centrifuged at 4°C and 3000 rpm for 10 minutes to separate the serum, which was then stored at -80°C for subsequent analysis.

[0091] (3) Experimental results

[0092] 1) Serum indicators

[0093] Serum was taken for determination of total bile acid (TBA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). The results are as follows:

[0094] First, the TBA, AST and ALT indicators of each group of mice were as follows Figure 5As shown, the results showed that compared with the ICP model group, the CNF intervention group and the CNC intervention group could reduce the TBA, AST and ALT contents, and the reduction effect was better than that of the UDCA treatment control group; and the TBA and AST levels of the CNF intervention group were close to those of the normal control group.

[0095] Second, the survival of the offspring of each group of mice was as follows Figure 6 As shown, the results showed that compared with the ICP model group, the CNC intervention group and the CNF intervention group could significantly improve the offspring survival rate; and the effect was better than that of the UDCA treatment control group; especially in the CNF intervention group, the offspring survival rate could reach 90%.

[0096] Third, the body weight of mice in each group on the 1st to 21st day of pregnancy and the body weight at the end of pregnancy were as follows: Figure 7 and Figure 8 As shown, the results showed that the CNC intervention group and the CNF intervention group could significantly improve the weight level at the end of pregnancy, and it was close to the level of the normal pregnancy group.

[0097] 2) Intestinal flora

[0098] In the present invention, Novogene was commissioned to test the intestinal flora of the ICP-CNF group and the ICP-CNC group respectively, and the steps were as follows:

[0099] (a) Fecal bacterial DNA was extracted using the TIANamp Soil DNA Kit (Cat. No. DP336) produced by TIANGEN Biotechnology and following the manufacturer's instructions;

[0100] (b) DNA quality and quantity were tested by using a nucleic acid fragment analyzer system (Agilent 5400);

[0101] (c) The universal primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) were used to amplify the V3-V4 region of the 16S rRNA gene; 15 μL was used for all PCR reactions. High-fidelity PCR mix (Biolabs, USA), 0.2 μmol / L of forward and reverse primers, and about 10 ng of template DNA were used; the thermal cycling program included: an initial denaturation step at 98°C for 1 min, followed by 30 cycles of a denaturation step (98°C, 10 s), an annealing step (50°C, 30 s), an extension step (72°C, 30 s), and a final extension step at 72°C for 5 min;

[0102] (d) PCR products were purified using magnetic bead purification method;

[0103] (e) Utilization The sequencing library was prepared using the Ultra II DNA library preparation kit (Cat. No. E7645B, Biolabs, USA) and sequenced on the NovaSeq6000 PE250 platform by Novogene (Beijing, China).

[0104] The intestinal flora of the ICP-CNF group and ICP-CNC group were Fig.10 and Fig.11 The results showed that both CNF and CNC could indirectly regulate the intestinal ecological environment and bile acid metabolism by increasing the abundance of specific beneficial bacteria and reducing the abundance of bacteria related to bile acid metabolism; for example, CNF could increase the abundance of specific beneficial bacteria (such as Alloprevotella and

[0105] For example, CNC can increase the abundance of specific beneficial bacteria (such as Desulfovibrio and Bilophila) and reduce the abundance of bile acid metabolism-related bacteria (such as [Eubacterium]ruminantium_group, Acetatifactor and Lachnospiraceae_NK4B4_group).

[0106] (II) Biosafety Verification of Nanocellulose (Long-term Oral Safety Experiment)

[0107] (1) Experimental Materials

[0108] Thirty female SD rats aged 10-11 weeks, weighing 210 g-240 g, were purchased from the Experimental Animal Center of Chongqing Medical University.

[0109] (2) Experimental methods

[0110] Preparation of CNC and CNFC suspensions: CNC and CNF with measured concentrations of 2.1% and 2.4% were prepared for oral gavage.

[0111] Animal grouping and dosing: After one week of adaptive feeding, the rats were fasted at 8 pm on the day before the experiment and were allowed to drink water freely. During the experiment, the rats were randomly divided into a control group, a CNC observation group and a CNF observation group according to their body weight, with 10 rats in each group. The CNF observation group was gavaged with a CNF suspension at a dose of 90 mg / Kg every day, the CNC observation group was gavaged with a CNF suspension at a dose of 90 mg / Kg every day, and the control group was gavaged with an equal amount of normal saline every day. After continuing the above gavage method for 8 weeks, fasting blood was collected the next morning, and serum was collected for testing after centrifugation at 4°C, 3000 rpm for 10 minutes.

[0112] (3) Evaluation indicators

[0113] During the experiment, the general condition of the animals was observed, such as changes in behavior, body appearance, feces and urine excretion, photosensitivity, smell, appetite, etc., and any abnormal phenomena such as sudden death and behavioral stereotypy were observed and recorded. At the same time, the collected blood samples were tested for serum biochemical markers, including liver function (ALT and AST), kidney function (BUN, sU and sCR), blood sugar and blood lipids (GLU, TCHO, HDL and LDL). On the last day of the experiment, the rats were autopsied to observe whether there were any abnormal manifestations of the organs.

[0114] (4) Experimental results

[0115] No abnormal behavior or sudden death was observed in rats during long-term oral administration of CNC and CNF. The rats were killed by dislocation of the neck at the end of 4 and 8 weeks after gavage, and obvious abnormalities were found in both cases. In addition, the serum indexes such as Fig. 9 As shown, the results showed that there was no significant statistical difference between the CNC observation group and the CNF observation group and the control group, indicating that CNC and CNF had no significant effect on liver function, kidney function, blood sugar and blood lipids.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A nanocellulose, characterized in that Nanocellulose is nanocellulose fiber or nanocellulose crystal; wherein, the nanocellulose fiber has a slender whisker-like morphology, the average length of the nanocellulose fiber is 250nm-400nm, the average diameter is 40nm-60nm, and some are aggregated and entangled; the nanocellulose crystal has a short rod crystal morphology, and has sulfate half-ester groups connected to the surface, the average length of the nanocellulose crystal is 50nm-200nm, and the average diameter is 10nm-40nm.

2. The nanocellulose according to claim 1, characterized in that Nanocellulose meets one or more of the following conditions: (1) The surface of nanocellulose is also modified with carboxyl and / or sulfate groups; (2) Nanocellulose comes in the form of suspension or powder.

3. The method for preparing nanocellulose according to claim 1 or 2, characterized in that: The preparation method comprises: When the nanocellulose is nanocellulose fiber, the method for preparing the nanocellulose fiber comprises: mixing microcrystalline cellulose and alkali solution, ball milling, and removing the alkali solution to obtain a nanocellulose fiber suspension; When the nanocellulose is nanocellulose crystals, the preparation method of the nanocellulose crystals includes: hydrolyzing microcrystalline cellulose with sulfuric acid to obtain a hydrolyzate; centrifuging the hydrolyzate until the centrifugal supernatant becomes a turbid suspension, which is the nanocellulose crystal suspension.

4. The preparation method according to claim 3, characterized in that: The method for preparing nanocellulose fibers satisfies one or more of the following conditions: (a) The diameter of the grinding balls used in ball milling is 5mm-15mm; (b) the alkali solution is sodium hydroxide solution; (c) ball milling includes: ball milling for 18 min-22 min, resting for 8 min-12 min; total ball milling for 1.5 h-2.5 h; (d) The ball milling speed is 350 r / min-450 r / min; The method for preparing nanocellulose crystals satisfies one or more of the following conditions: (i) the mass fraction of sulfuric acid is 55%-65%; (ii) the hydrolysis temperature is 45°C to 55°C; (ii) The hydrolysis time is 1h-2h.

5. The method for preparing nanocellulose according to claim 4, characterized in that: The nanocellulose fiber suspension or the nanocellulose crystal suspension is freeze-dried to obtain nanocellulose fiber freeze-dried powder or nanocellulose crystal freeze-dried powder.

6. A medicine or functional food, characterized in that: The nanocellulose comprising claim 1 or 2.

7. The medicine or functional food according to claim 6, characterized in that The medicine or functional food may also include dietary fiber and / or probiotics.

8. Use of the nanocellulose according to claim 1 or 2 in the preparation of a medicament for treating intrahepatic cholestasis of pregnancy.

9. The use according to claim 8, characterized in that: Applications include one or a combination of the following: (i) Use of the nanocellulose according to claim 1 or 2 in the preparation of a drug for reducing bile acid levels in the body; (ii) Use of the nanocellulose according to claim 1 or 2 in the preparation of a drug for improving the survival rate of offspring during pregnancy; (iii) Use of the nanocellulose according to claim 1 or 2 in the preparation of a drug for increasing the abundance of beneficial bacteria in the intestinal flora of an organism; (iv) Use of the nanocellulose according to claim 1 or 2 in the preparation of a drug for reducing the abundance of bacteria related to bile acid metabolism in the intestinal flora of an organism.

10. Use of the nanocellulose according to claim 1 or 2 in maintaining a healthy level of bile acid in the body.

11. The use according to any one of claims 8 to 10, characterized in that The effective dosage of nanocellulose is 30mg / Kg-180mg / Kg.

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