AU2021102578A4 - Biopolymer nanosphere containing nadh, method of preparing the same and use thereof - Google Patents

Abstract

A biopolymer nanosphere containing NADH comprises a biopolymer carrier and NADH dispersed on the biopolymer carrier. The biopolymer carrier has a three-dimensional network structure, so that the NADH is protected, which solves the problem of decomposing easily after contacting light or oxygen, extends the storage time of the effective ingredients of NADH and reduces the storage difficulty. A method of preparing the biopolymer nanosphere is disclosed. A physically modified biopolymer material has a moderate chain length and is easy to form spherical particles so as to form a honeycomb three-dimensional interpenetrating network structure, which facilitates loading of small NADH molecules on inner and outer surfaces of the network structure. The biopolymer nanosphere can be used to prepare a tablet, a granule, a capsule or a soft capsule and other pharmaceuticals to serve as a human body healthcare or pet therapeutic medicine or a functional food product. (Fig. 1) A raw material of biopolymer and an excipient are mixed and ground to physically modify the raw material S II of biopolymer to obtain a biopolymer carrier. A raw material of NADH is ground and sieved to obtain S12 the NADH. The NADH and the biopolymer carrier are mixed and stirred to obtain the biopolymer nanosphere containing S13 NADH. Fig. 1 1/6

Description

A raw material of biopolymer and an excipient are mixed and ground to physically modify the raw material S II of biopolymer to obtain a biopolymer carrier.

A raw material of NADH is ground and sieved to obtain S12 the NADH.

The NADH and the biopolymer carrier are mixed and stirred to obtain the biopolymer nanosphere containing S13 NADH.

Fig. 1

1/6

BIOPOLYMER NANOSPHERE CONTAINING NADH, METHOD OF PREPARING THE SAME AND USE THEREOF

Technical Field of the Invention The present invention relates to the technical field of health care products, in particular to a biopolymer nanosphere containing nicotinamide adenine dinucleotide (NADH), preparation methods and use thereof.

Background of the Invention The reduced state of nicotinamide adenine dinucleotide (NADH) is a proton-transmitting coenzyme, also known as reduced coenzyme I. In the process of hundreds to thousands of metabolic reactions in living cells, NADH as an electron donor has redox activity and plays an important role of electron transfer during glycolysis, citric acid cycle and photosynthesis. In the organism, NADH participates in the process of production of vitamin derivation and adenosine triphosphate (ATP), and NADH is the coenzyme of more than 250 kinds of dehydrogenases that have been identified. It has the function of maintaining cell growth, differentiation and energy metabolism.

NADH is considered to have beneficial effects on both the body and intelligence without side effects. It can be used as a medicine to improve health and quality of life. Clinical studies have shown that parenteral administration of NADH has a positive effect on the treatment of Parkinson's disease and major depression. NADH can promote the cognitive ability and exercise ability of patients with Parkinson's disease and Alzheimer's disease, while relieving fatigue, lethargy, and pigmented fatigue syndrome and enhancing the vitality of users.

In the middle of the last century, NADH has successfully treated various neurological diseases such as Parkinson's disease, Alzheimer's disease and delayed dementia through transfusion, but because NADH liquid is highly sensitive (very sensitive to light and oxygen), its chemical properties are not stable (this requires that NADH be used immediately after being prepared), which makes it difficult to be widely used. In addition, NADH is decomposed immediately once contacting with gastric acid, so that it cannot be orally administrated.

Summary of the Invention In view of the foregoing, the present invention provides a biopolymer nanosphere containing NADH, which has high stability, is easy to preserve, and can be orally administrated, and can be produced on a large scale.

In addition, it is necessary to provide a method of preparing the above-mentioned biopolymer nanosphere containing NADH and use of the above-mentioned biopolymer nanosphere containing NADH.

The present invention provides a biopolymer nanosphere containing NADH, comprising a biopolymer carrier and NADH dispersed on the biopolymer carrier.

The present invention further provides a method of preparing the biopolymer nanosphere containing NADH. The method includes the following steps: mixing and grinding a raw material of a biopolymer and an excipient to physically modify the raw material of the biopolymer, to obtain a biopolymer carrier, grinding and sieving a raw material of NADH to obtain the NADH, and mixing and stirring the NADH and the biopolymer carrier to obtain biopolymer nanosphere containing NADH, the NADH being dispersed on the biopolymer carrier.

Preferably, the excipient comprises, based on parts by weight, at least one of 0.05-30 parts of sodium alginate and 0.05-30 parts of xanthan gum.

The present invention provides use of the biopolymer nanosphere containing NADH in preparation of a medication and a functional food for prevention and treatment of sub-health and tumors.

The present invention further provides a pharmaceutical formulation. The pharmaceutical formulation comprises the biopolymer nanosphere containing NADH, and a pharmaceutically acceptable excipient.

Preferably, the pharmaceutical formulation is one of tablets, capsules, granules, injections, tinctures, suppositories, patches, pills, syrups, mixtures, powders, lotions, film agents and dropping pills.

Preferably, the pharmaceutical formulation is capsules, comprising hard capsules and soft capsules.

The present invention further provides a method of preparing a pharmaceutical formulation. The method includes the following steps: providing the biopolymer nanosphere containing NADH, and adding a granulating excipient to the biopolymer nanosphere, followed by stirring, preparing, by sieving, wet granules with a uniform particle diameter, and drying the wet granules to obtain granules of the biopolymer nanosphere containing NADH.

Preferably, the method further includes pressing the granules of the biopolymer nanosphere containing NADH into tablets.

Preferably, the method further includes putting the granules of the biopolymer nanosphere containing NADH into capsule shells to form capsules.

Preferably, the method further includes mixing the granules of the biopolymer nanosphere containing NADH with edible oil and putting a resulting mixture into soft capsule shells to form soft capsules.

Preferably, the granulating excipient is at least one of microcrystalline cellulose, polyvinylpyrrolidone, sodium bicarbonate, magnesium stearate, calcium polyphosphate, a wetting agent, and an adhesive.

The present invention further provides a functional food which comprises the biopolymer nanosphere containing NADH and food additives.

The present invention has the following advantages. The biopolymer nanosphere containing NADH provided by the present invention extends the preservation time of the effective ingredients of NADH, reduces the preservation difficulty, improves the stability, and maintains the biological activity.

Brief Description of the Drawings Fig. 1 is a flow chart for the preparation of a biopolymer nanosphere containing NADH in a preferred embodiment of the present invention.

Fig. 2 is a flow chart for the preparation of a pharmaceutical formulation in a preferred embodiment of the present invention.

Fig. 3 is an XRD test diagram of the biopolymer nanosphere containing NADH in a preferred embodiment of the present invention at different NADH contents.

Fig. 4A and Fig. 4B are scanning electron microscope images of the biopolymer nanosphere containing NADH in Example 1 of the present invention.

Fig. 5 is a chart showing a trend in release rates of effective ingredients of tablets prepared from biopolymer nanosphere containing NADH, prepared in Example 2 of the present invention, when the tablets were being immersed in a simulated gastric fluid and a simulated colon fluid.

Fig. 6 is a chart showing a trend in release rates of effective ingredients of tablets prepared from biopolymer nanosphere containing NADH, prepared in Example 2 of the present invention when the tablets were being immersed in a simulated colon fluid.

Detailed Description of the Invention The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments, rather than all the embodiments, of the present invention. All other embodiments obtained, based on the embodiments of the present invention, by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the technical field of the present invention. The terms used herein in the specification of the present invention herein are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

A preferred embodiment of the present invention provides a biopolymer nanosphere containing NADH. The biopolymer nanosphere comprises a biopolymer carrier and NADH dispersed on the biopolymer carrier.

The biopolymer carrier presents a three-dimensional network structure, and has an average particle diameter of 200-1000 nm. The biopolymer carrier comprises at least one of chitosan (CS) and konjac glucomannan (KGM). KGM is a kind of natural polymer soluble dietary fiber. The fiber has a honeycomb three-dimensional interpenetrating network structure with pores in sizes ranging from a few tens of nanometers to thousands of nanometers. NADH enters through the pores and is loaded on the surface of the KGM fiber network. Preferably, the konjac glucomannan is at least one of konjac glucomannan, quaternary ammonium konjac glucomannan, carboxymethyl konjac glucomannan, and deacetylated konjac glucomannan. The biopolymer nanosphere comprises, based on parts by weight, 1-20 parts of the NADH and 20-80 parts of the biopolymer carrier.

Preferably, the biopolymer nanosphere further comprises an excipient. The excipient comprises, based on parts by weight, at least one of 0.05-30 parts of sodium alginate and 0.05-30 parts of xanthan gum. Sodium alginate is a kind of natural polysaccharide capable of concentrating solutions, forming gel and forming films. Sodium alginate can form a film structure, on an outer surface of the biopolymer carrier, to thereby produce waterproof and anti-oxidation effects, and avoid the problem of decomposition of NADH once the NADH is exposed to light or oxygen. The average particle diameter of the biopolymer nanosphere is 500-1000 nm.

Referring to Fig. 1, a preferred embodiment of the present invention further provides a method of preparing the biopolymer nanosphere containing NADH, which includes the following steps.

S11. A raw material of biopolymer and an excipient are mixed and ground to physically modify the raw material of biopolymer to obtain a biopolymer carrier.

Mixing and grinding the raw material of biopolymer and the excipient decreases the molecular chain length and reduces the molecular weight of the raw material of the biopolymer, forming a honeycomb three-dimensional interpenetrating network structure and which agglomerates to form nanospheres with an average particle diameter of 200-1000 nm.

S12. A raw material of NADH is ground and sieved to obtain the NADH.

The average particle diameter of the obtained NADH is 10-100 nm.

S13. The NADH and the biopolymer carrier are mixed and stirred to obtain the biopolymer nanosphere containing NADH. The NADH is dispersed on the biopolymer carrier.

It can be understood that in the present invention, the NADH and the biopolymer carrier are not fed sequentially and may be added in an arbitrary feeding sequence.

An XRD diagram as shown in Fig. 3 is obtained by adjusting an amount of the NADH added in step S13 and detecting the biopolymer nanosphere containing NADH at different NADH contents (0%, 8%, and 10%) using X-ray diffraction (XRD). As can be seen from Fig. 3, the biopolymer carrier (blank microsphere) without being loaded with NADH shows broad diffraction peaks of polysaccharide cellulose (the diffraction peaks at 20 angles of 18° and 22.50). After the biopolymer carrier is loaded with NADH, a number of new diffraction peaks appears, which are characteristic diffraction peaks of NADH, and an intensity of XRD diffraction peaks of a nanosphere containing 10% of NADH is higher than that of a nanosphere containing 8% of NADH, which further proves that NADH has been successfully loaded on the biopolymer carrier.

Further, the raw material of NADH in step S12 is prepared by the following process.

S121. Crude extract of nicotinamide riboside adenylyltransferase or pure enzyme thereof is extracted.

S122. The crude extract of nicotinamide riboside adenylyltransferase or pure enzyme thereof is immobilized and recombined.

S123. The immobilized and recombined nicotinamide riboside adenylyltransferase is used for catalyzing, and nicotinamide nucleotide and adenosine triphosphate disodium (ATP) are used as a substrate to prepare the raw material of NADH.

Specific steps of extracting the crude extract of nicotinamide riboside adenylyltransferase or pure enzyme thereof in S121 are as follows.

The plasmid pRSET bmj containing the gene of nicotinamide riboside adenylyltransferase is transformed into competent bacterial cells E. coliHB101, which is cultured on Luriabroth (LB) plates (containing 100 mg/L of kanamycin) at 37 °C for 24 h. Inoculating a single clone is performed in 5 ml of LB liquid medium (containing 100 mg/L of kanamycin), and culturing is performed at 30 °C for 20-24 h. The cells are collected by centrifugation and suspended in 1 mL of 100 mM Tris hydrochloric acid buffer solution (pH=7.5). Then the bacterial cells are lysed with ultrasonic, centrifuged (10 °C, 17800 g, 10 min), and a supernatant is collected, which is the crude protein (or crude extract). The recombined crude protein of nicotinamide riboside adenylyltransferase is heat-treated at 70 °C for 10 min, centrifuged ( 10 °C, 17800 g, 10 min), and a supernatant is collected, which is the partially purified protein.

Specific steps of immobilizing nicotinamide riboside adenylyltransferase in S122 are as follows. The crude protein or partially purified protein of nicotinamide riboside adenylyltransferase is taken and diluted using an enzyme-washing buffer solution (0.02 M of Tris HCl solution or 0.001 M of EDTA solution, with pH=7.0) so that a protein content is 5-10 mg/mL. The enzyme diluent and a PB solution (2.0 mol/L of potassium dihydrogen phosphate, with pH=7.5) in equal volume are mixed, with addition of an epoxy-type immobilized enzyme carrier LX 3000 (a carrier with 10 mg of enzyme per gram), and placed on a shaker (with a rotating speed of 100 rpm) to react at 25 °C for 20 h. After the reaction is completed, filtering is performed with a filter bag, and washing is performed 5-6 times with the enzyme-washing buffer solution to obtain immobilized nicotinamide riboside adenylyltransferase.

Specific steps of preparing the raw material of NADH with immobilized nicotinamide riboside adenylyltransferase in S123 are as follows. A substrate solution is prepared with 5 mM of nicotinamide nucleotide, 10 mM of adenosine triphosphate disodium (ATP), 100 mM of Tris hydrochloric acid buffer solution and 10 mM of

MgCl 2 with a final concentration, and the pH is adjusted to 7.5. 1 mL of the substrate solution is taken, with addition of 0.05 g of immobilized nicotinamide riboside adenylyltransferase, to react at 37 °C for 2-20 h. Centrifuging (10 °C, 17800 g, 15 min) is performed, and a supernatant is collected. A content of nicotinamide adenine dinucleotide in the obtained supernatant is measured by high pressure liquid chromatography (HPLC). Results show that a conversion rate of nicotinamide nucleotide into nicotinamide adenine dinucleotide exceeds 80%.

The present invention further provides use of the biopolymer nanosphere containing NADH in preparation of a medication and functional food for prevention and treatment of sub-health and tumors.

The present invention further provides a pharmaceutical formulation comprises the biopolymer nanosphere containing NADH and a pharmaceutically acceptable excipient. Preferably, the pharmaceutical formulation is one of tablets, capsules, granules, injections, tinctures, suppositories, patches, pills, syrups, mixtures, powders, lotions, film agents and dropping pills. Preferably, the pharmaceutical formulation is capsules which include hard capsules and soft capsules. The pharmaceutically acceptable excipient comprises a granulating excipient. Preferably, the granulating excipient is at least one of microcrystalline cellulose, polyvinylpyrrolidone, sodium bicarbonate, magnesium stearate, calcium polyphosphate, a wetting agent and an adhesive.

Preferably, an appropriate daily dose of oral NADH formulation is 5-500 mg.

Preferably, an appropriate daily dose of the oral NADH formulation is 25-100 mg.

Referring to Fig. 2, a preferred embodiment of the present invention further provides a method of preparing the pharmaceutical formulation, which includes the following steps.

S21. The biopolymer nanosphere containing NADH is provided, and a granulating excipient is added to the biopolymer nanosphere, followed by stirring.

S22. Sieving is performed to obtain wet granules with a uniform particle diameter.

S23. The wet granules are dried to obtain granules of the biopolymer nanosphere containing NADH.

Preferably, the method further includes pressing the granules of the biopolymer nanosphere containing NADH into tablets.

Preferably, the method further includes putting the granules of the biopolymer nanosphere containing NADH into capsule shells to form capsules.

Preferably, the method further includes mixing the granules of the biopolymer nanosphere containing NADH with edible oil to obtain a mixture, and putting a resulting mixture of the granules of the biopolymer nanosphere containing NADH and the edible oil into a soft capsule shell to form soft capsules.

Preferably, the granulating excipient is at least one of microcrystalline cellulose, polyvinylpyrrolidone, sodium bicarbonate, magnesium stearate, calcium polyphosphate, a wetting agent and an adhesive.

The wetting agent comprises one or both of ethanol with a volume concentration of 70% and water, and the adhesive comprises at least one of a pre-gelled starch with a mass concentration of 5-20%, a starch pulp with a mass concentration of 10-15% and a hydroxypropyl methylcellulose solution with a mass concentration of 10%. Condition for the drying is vacuum drying at 10-50 °C for 0.5-24 h.

It can be understood that, in the present invention, the biomolecular nanosphere containing NADH and the granulating excipient are not fed sequentially, and can be added in an arbitrary feeding sequence. In the present invention, the preparation of the biopolymer nanosphere containing NADH may be carried out simultaneously with the preparation of the pharmaceutical formulation. That is, when the NADH is mixed with the biopolymer carrier, the granulating excipient may be added, and stirred together.

The present invention further provides a functional food which comprises the biopolymer nanosphere containing NADH and food additives. The food additives include one or more of pectin, fumaric acid, polydextrose, maltose, phospholipid, citric acid, hydroxypropyl starch, lactic acid, sorbitol, milk powder, maltodextrin, honey, corn starch, corn oil, sesame oil, sucrose, vitamin C, vitamin E, xylitol and gelatin.

The present invention is described in detail below by way of examples.

Example 1

Preparation of biopolymer nanosphere containing NADH:

Si1. Konjac glucomannan (KGM) and sodium alginate were mixed and ground with a colloid grinder to obtain a biopolymer carrier.

S12. A raw material of NADH was ground and sieved to obtain the NADH, haveing an average particle diameter of 10-100 nm.

S13. The NADH obtained in step S12 and the biopolymer carrier were mixed and followed by stirring evenly to obtain the biopolymer nanosphere containing NADH. The biopolymer nanosphere comprises, based on parts by weight, 20 parts of the biopolymer carrier and 1 part of the NADH, and the material of the biopolymer carrier was konjac glucomannan (KGM). The biopolymer nanosphere containing NADH further comprised an excipient, which comprised, based on parts by weight, 0.05 parts of sodium alginate.

The above-mentioned biopolymer nanosphere was used to prepare a pharmaceutical formulation. The pharmaceutical formulation was granules.

Preparation of granules of the pharmaceutical formulation is as follows:

S21. A granulating excipient was added to the biopolymer nanosphere containing NADH followed by stirring. The granulating excipient comprises, based on parts by weight, 10 parts of a wetting agent and 5 parts of an adhesive. The wetting agent was ethanol with a volume concentration of 70%, and the adhesive was a starch pulp with a mass concentration of 10%.

S22. A wet granulation process was performed using a medium steel sieve. Specifically, a weighed material was placed on a set of sieves with pore sizes thereof decreasing from inside to outside, so that the particles for the material were left on the sieves respective layers depending on the particle diameters thereof, to obtain the wet granules.

S23. The wet granules were dried at a drying temperature of 40 °C for 3 h to obtain granules of the biopolymer nanosphere containing NADH.

Example 2

Example 2 differed from Example 1 in that the biopolymer nanosphere comprised 80 parts of the biopolymer carrier and 20 parts of the NADH. A material of the biopolymer carrier was a mixture of quaternary ammonium konjac glucomannan (QKGM) and carboxymethyl konjac glucomannan (CKGM) in a mass ratio 1:1. The excipient comprised 30 parts of sodium alginate.

Quaternary ammonium konjac glucomannan (QKGM) and carboxymethyl konjac glucomannan (CKGM) were prepared by quaternizing and carboxymethylating konjac glucomannan (KGM), respectively. It was also possible to mix quaternary ammonium konjac glucomannan (QKGM) and carboxymethyl konjac glucomannan (CKGM) together with 0.1-1 mol/L HCl or 0.1-1 mol/L NaOH followed by stirring them for 2-10 h to reduce a molecular weight of konjac glucomannan, to facilitate subsequent physical modification.

The pharmaceutical formulation was tablets. The tablets were prepared by the following method.

The granulation method described in Example 1 was used to prepare granules, and then the obtained granules were compressed with a tablet press. By adjusting parameters and a pressure of a tableting die, tablets with a target diameter, thickness and hardness could be obtained. The difference between the granulation method of Example 2 and that of Example 1 was that the granulated excipient was different. The granulating excipient comprised 40 parts of an adhesive, 30 parts of magnesium stearate and 30 parts of microcrystalline cellulose. The adhesive was a mixture of pre-gelled starch with a mass concentration of 5% and a hydroxypropyl methylcellulose solution with a mass concentration of 10%.

Example 3

Example 3 differed from Example 1 in that the biopolymer nanosphere comprised 45 parts of the biopolymer carrier and 5 parts of the NADH, and the material of the biopolymer carrier was deacetylated konjac glucomannan (da-KGM). The excipient comprised 15 parts of sodium alginate.

Deacetylated konjac glucomannan (da-KGM) was prepared by deacetylating KGM. Before step SI1, the method further included mixing the deacetylated konjac glucomannan (da-KGM) with 0.1-1 mol/L HCl or 0.1-1 mol/L NaOH and stirring them for 2-10 hours to reduce the molecular weight of the konjac glucomannan to facilitate subsequent physical modification.

The pharmaceutical formulation was capsules, and the capsules were prepared by the following method.

The granulation method described in Example 1 was used to prepare granules. After preparation of the granules, the prepared granules were put into commercially available edible hard capsule shells by a quantitative filling method to obtain a capsule formulation. The granulation method used in Example 3 differs from that used in Example 1 in that the granulating excipient was different. The granulating excipient comprised 20 parts of an adhesive, 0.5 parts of magnesium stearate, 0.5 parts of microcrystalline cellulose, 1 part of polyvinylpyrrolidone, 2 parts of sodium bicarbonate and 20 parts of a wetting agent. The wetting agent was ethanol with a volume concentration of 70%, and the adhesive was a starch pulp with a mass concentration of 10%.

Example 4

Example 4 differed from Example 1 in that the biopolymer nanosphere comprised 60 parts of the biopolymer carrier and 5.2 parts of the NADH. The excipient comprised 30 parts of xanthan gum.

Before step Sll, the method further included mixing KGM with 0.1-1 mol/L HCl or 0.1-1 mol/L NaOH and stirring them for 2-10 hours to reduce the molecular weight of the konjac glucomannan, to facilitate subsequent physical modification.

The pharmaceutical formulation was soft capsules. The soft capsules were prepared by the following method.

The granulation method described in Example 1 was used to prepare granules. After preparation of the granules, obtained prepared granules were mixed with an appropriate amount of edible oil and a biosurfactant, and a resulting mixture was then encapsulated in commercially available soft capsule shells to obtain a soft capsule formulation. The edible oil could be olive oil, sesame oil, soybean oil, corn oil, camellia oil, grape seed oil, etc. The difference between the granulation method of Example 4 and that of Example 1 was that the granulating excipient was different. In Example 4, the granulating excipient comprised 16 parts of an adhesive, 2 parts of magnesium stearate, 1 part of calcium polyphosphate and 15 parts of a wetting agent. The wetting agent was ethanol with a volume concentration of 70%, and the adhesive was a hydroxypropyl methylcellulose solution with a mass concentration of 10%.

Example 5

Example 5 differed from Example 1 in that the biopolymer nanosphere comprised 40 parts of the biopolymer carrier and 12 parts of the NADH. The excipient comprised 0.05 parts of xanthan gum and 13 parts of sodium alginate.

Before step Si1, the method further included mixing KGM with 0.1-1 mol/L HCl or 0.1-1 mol/L NaOH and stirring them for 2-10 hours to reduce the molecular weight of the konjac glucomannan to facilitate subsequent physical modification.

In Example 5, the pharmaceutical formulation was granules. The granules were prepared by the method described in Example 1. The difference was that the granulating excipient comprised 5 parts of an adhesive, 12 parts of microcrystalline cellulose, and 15 parts of a wetting agent. The wetting agent was a mixture of ethanol with a volume concentration of 70% and water, and the adhesive was a pre-gelled starch with a mass concentration of 15%.

Example 6

Example 6 differed from Example 1 in that the biopolymer nanosphere comprised 70 parts of the biopolymer carrier and 15 parts of the NADH, the material of the biopolymer carrier was chitosan (CS). The excipient comprised 15 parts of xanthan gum and 24 parts of sodium alginate.

The biopolymer nanosphere containing NADH described in Example 6 could be prepared by the method of Example 1. The difference was that, before step Sll, chitosan was quaternified for chemical modification to reduce a molecular weight of chitosan, so as to facilitate subsequent physical modification.

In Example 6, the pharmaceutical formulation was a tablet, and the tablet was prepared by the following method.

The granulation method described in Example 1 was used to prepare granules, and then the obtained granules were compressed with a tablet press. By adjusting parameters and a pressure of a tableting die, tablets with a target diameter, thickness and hardness could be obtained. The granulating excipient comprised 16 parts of an adhesive, 8 parts of microcrystalline cellulose, 10 parts of polyvinylpyrrolidone, 20 parts of sodium bicarbonate and 13 parts of a wetting agent. The wetting agent was a mixture of ethanol with a volume concentration of 70% and water, and the adhesive was a starch pulp with a mass concentration of 10%.

Example 7

Example 7 differed from Example 1 in that the biopolymer nanosphere comprised 55 parts of the biopolymer carrier and 8 parts of the NADH. The material of the biopolymer carrier was chitosan (CS). The excipient comprised 20 parts of xanthan gum and 5 parts of sodium alginate. In Example 7, the material of the biopolymer carrier was chitosan (CS), and the CS was a natural polymer material which has a honeycomb three-dimensional interpenetrating network structure with pores in sizes ranging tens of nanometers to thousands of nanometers. NADH entered through the pores and is loaded on a surface of a chitosan network.

In Example 7, the pharmaceutical formulation was granules. The granules were prepared by the method described in Example 1. The difference was that the granulating excipient was different. The granulating excipient comprised 8 parts of an adhesive and 6 parts of microcrystalline cellulose, 4 parts of polyvinylpyrrolidone, 5 parts of sodium bicarbonate, 10 parts of magnesium stearate, 10 parts of calcium polyphosphate and 15 parts of a wetting agent. The wetting agent was a mixture of ethanol with a volume concentration of 70% and water, and the adhesive was a mixture of a starch pulp with a mass concentration of 10% and a pre-gelled starch with a mass concentration of 15%.

Table 1 Biopolymer nanosphere containing NADH prepared in Example 1-7 of the present invention and specific treatment conditions thereof.

Biopolymer nanosphere and specific treatment conditions Pharmaceutical thereof formulations Material(s) Biopolymer NADH in and mass Components Examples carrier in ratio thereof of excipient Forms parts by of in parts by weeight weight biopolymer weight carrier 0.05 parts of Example 1 20 1 KGM sodium Granules alginate

QKGMand 30 parts of Example 2 80 20 sodium tablets CKGM(1:1) agnt alginate

15 parts of Example 3 45 5 da-KGM sodium capsules alginate 30 parts of Example 4 60 5.2 KGM xanthan soft capsules gum 0.05 parts of xanthan gum and 13 Example 5 40 12 KGM granules parts of sodium alginate 15 parts of xanthan

Example 6 70 15 CS gmand24 tablets parts of sodium alginate 20 parts of xanthan

Example 7 55 8 CS gumand5 granules parts of sodium alginate

Scanning electron microscopy tests were performed on the biopolymer nanosphere containing NADH prepared in Example 1, and the test results are shown in Fig. 4A and Fig. 4B. It can be seen from Fig. 4A and Fig. 4B that the biopolymer nanosphere has a honeycomb three-dimensional interpenetrating network structure.

The pre-gelled starch in the granulating excipient in Example 2 not only has good disintegration and adhesion, but also can significantly improve the hardness, disintegration and surface brightness of the tablet. More importantly, the pre-gelled starch improves the dissolution rate, reduces the difficulty of granulation, and improves the granulation and compressibility of the granules. The compressed tablet has high hardness, small brittleness and a smooth surface. Hydroxypropyl methylcellulose is a derivative of a mixed ether of hydroxypropyl and methoxy cellulose, and its intramolecular substitution group is ether. It is used in tablets and is mainly used as an adhesive and a disintegrant to improve disintegration and increase dissolution. Microcrystalline cellulose has good fluidity and compressibility, has the properties of adhesion, lubrication and disintegration aid, and has no interaction with a medication, so that the compressed tablet has a smooth and beautiful appearance and is easy to disintegrate.

The tablets prepared in Example 2 were immersed in a simulated gastric fluid (SGF) with pH=2 for 2 hours, and then was immersed in a simulated colon fluid (SCF) with pH=6.8 for 6 hours. Release rates of the NADH at 2h, 4h, 6h and 8h were tested respectively. The test results are shown in Table 2 and Fig. 5.

Table 2. Release rates of an effective ingredient of tablets prepared from the biopolymer nanosphere containing NADH, prepared in Example 2, when the tablets were being immersed in the simulated gastric fluid and the simulated colon fluid.

Time (h) Release rates (%) 0 0 2 1.12 4 20.72 6 50.24 8 90.33

Remaining amount 0.30 Total amount 90.63

The tablet prepared in Example 2 were immersed in a simulated colon fluid SCF with pH=6.8 for 8 hours. Release rates of the NADH were tested at 2 hour, 4 hour, 6 hour and 8 hour respectively. Test results are shown in Table 3 and Fig. 6.

Table 3. Release rates of an effective ingredient of tablets prepared from the biopolymer nanosphere containing NADH, prepared in in Example 2. When the tables were being immersed in the simulated colon fluid.

Time (h) Release rate ( %) 0 0 2 39.57 4 66.85 6 85.5 8 93.27 Remaining amount 2.10 Total amount 95.37

The prepared formulation can release the effective ingredients of NADH within 8-10 hours after oral administration. The formulation stays in the gastric juice and releases the effective ingredients in the first 2 hours, and stays in the small intestine and releases the effective ingredients in following 6-8 hours. The above test results show that during immersion in the simulated gastric fluid and the simulated colon fluid, the effective ingredients NADH in the prepared tablets can be slowly released into the solutions, and the prepared tablets are a long-acting medication.

In Example 3, the microcrystalline cellulose in the excipient acts as a filler and a disintegrating agent, and the polyvinylpyrrolidone acts to protect and disperse the pharmaceutical formulation, thereby facilitating the release of the pharmaceutical formulation.

Compared with the existing technologies, the above-mentioned technical solutions of the present invention have the following advantages:

(1) The biopolymer nanosphere of the present invention comprises a biopolymer carrier and NADH dispersed on the biopolymer carrier. The biopolymer carrier is a polymer fiber polymeride, and its fiber skeleton forms a three-dimensional interpenetrating network structure through random arrangement, cross arrangement, crimp arrangement, etc., so that the NADH is protected. This solves the problem that NADH decomposes easily when being exposed to light or oxygen, extends the storage time of the effective ingredient NADH and reduces the storage difficulty.

(2) In the biopolymer nanosphere of the present invention, most of NADH is dispersed inside the three-dimensional interpenetrating network of the biopolymer carrier, and a small amount is loaded on the outer surface of the biopolymer carrier. This kind of biopolymer carrier with a three-dimensional interpenetrating network structure has biological activity and also has the function of loading the effective ingredients of NADH, which protects the NADH loaded between the network structures and solves the problem of rapid decomposition of NADH in the presence of gastric acid and inability to fully exert its effectiveness. The biopolymer carrier is a polymer carrier with slow-release effects in the intestinal tract, and provides an ideal medication carrier for using the NADH in treating Parkinson's disease, Alzheimer's disease, depression, cancer and other diseases. By slowly releasing of the effective ingredients NADH, DNA repair enzymes are activated to repair DNA, which can also prevent the occurrence of cancer.

(3) The biopolymer carrier of the present invention is chitosan and/or konjac glucomannan. Both chitosan and konjac glucomannan can form a honeycomb three-dimensional interpenetrating fiber network, and facilitate protection of NADH by loading NADH in the network. In addition, chitosan is a natural polymer material, which has good biological functions and compatibility, safety and microbial degradation and also has antibacterial properties because of its positive charge. Konjac glucomannan is a natural polymer soluble dietary fiber, which has properties of high viscosity, high water absorption and fast expansion and also has immunogenicity due to its special glycosidic bond structure.

(4) In the method of preparing the biopolymer nanosphere of the present invention, a physically modified biopolymer carrier is first prepared. By mixing the raw material of a biopolymer with xanthan gum and/or sodium alginate and grinding the resulting mixture, long chains of the biopolymer are broken into molecular chains of moderate lengths. The physically modified biopolymer material has a moderate chain length and is easier to form spherical particles and form a honeycomb three-dimensional interpenetrating network structure, which facilitates loading of small NADH molecules on the inner and outer surfaces of the network structure. In addition, the physically modified biopolymer carrier has a film-like structure formed on its outer surface, which has waterproofing and anti-oxidation effects. This solves the problem of decomposition of NADH when being exposed to light and oxygen and extends the storage time of the NADH. By grinding raw materials of NADH, NADH is ground to a particle diameter of tens of nanometers, so that it is easier for NADH to enter the three-dimensional interpenetrating network structure of the biopolymer material and thus to be protected.

(5) The biopolymer nanosphere of the present invention can be used to prepare pharmaceutical formulations such as tablets, granules, capsules or soft capsules, etc. First, by preparing a biopolymer nanosphere containing NADH, the NADH is protected by the biopolymer carrier in the network structure. Then, the biopolymer nanosphere containing NADH is prepared into a pharmaceutical formulation, which solves the problem that the NADH in the formulation is decomposed by gastric juice and thus cannot be absorbed by the human body. Among the above formulations, the soft capsule disperses the biopolymer nanosphere containing NADH in the oil phase, which isolates the NADH from contacting with air and water and further solves the problem of easy decomposition of the NADH, and thus has a longer shelf life.

The above embodiments are only used to illustrate, rather than limiting the technical solutions of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced, without departing from the spirit and essence of the technical solutions of the present invention.

Claims (11)

Claims

1. A biopolymer nanosphere containing NADH, comprising a biopolymer carrier and NADH dispersed on the biopolymer carrier.

2. The biopolymer nanosphere containing NADH according to claim 1, wherein the biopolymer carrier has a three-dimensional network structure, and has an average particle diameter of 200-1000 nm.

3. The biopolymer nanosphere containing NADH according to claim 1, wherein the biopolymer carrier comprises at least one of chitosan and konjac glucomannan, wherein, preferably, the konjac glucomannan is at least one of konjac glucomannan, quaternary ammonium konjac glucomannan, carboxymethyl konjac glucomannan, and deacetylated konjac glucomannan.

4. The biopolymer nanosphere containing NADH according to claim 1, wherein the biopolymer nanosphere comprises, based on parts by weight 1-20 parts of NADH and 20-80 parts of the biopolymer carrier.

5. The biopolymer nanosphere containing NADH according to claim 1, wherein the biopolymer nanosphere further comprises an excipient, wherein the excipient comprises, based on parts by weight, at least one of 0.05-30 parts of sodium alginate and 0.05-30 parts of xanthan gum.

6. The biopolymer nanosphere containing NADH according to claim 1, wherein the biopolymer nanosphere has an average particle diameter of 500-1000 nm.

7. A method of preparing the biopolymer nanosphere containing NADH according to any one of claims 1 to 6, comprising the following steps: mixing and grinding a raw material of a biopolymer with an excipient to physically modify the raw material of the biopolymer, to obtain a biopolymer carrier, grinding and sieving a raw material of NADH to obtain the NADH, and mixing and stirring the NADH and the biopolymer carrier to obtain the biopolymer nanosphere containing NADH, wherein the NADH is dispersed on the biopolymer carrier, wherein, preferably, the excipient comprises, based on parts by weight, at least one of 0.05-30 parts of sodium alginate and 0.05-30 parts of xanthan gum.

8. Use of the biopolymer nanosphere containing NADH according to any one of claims 1 to 6 in preparation of a medication and a functional food for prevention and treatment of sub-health, and tumors.

9. A pharmaceutical formulation, comprising the biopolymer nanosphere containing NADH according to any one of claims 1 to 6, and a pharmaceutically acceptable excipient, wherein preferably, the pharmaceutical formulation is one of tablets, capsules, granules, injections, tinctures, suppositories, patches, pills, syrups, mixtures, powders, lotions, films and dripping pills, and preferably, the pharmaceutical formulation is capsules, and the capsules comprising hard capsules and soft capsules.

10. A method of preparing the pharmaceutical formulation according to claim 9, comprising the following steps: providing the biopolymer nanosphere containing NADH, and adding a granulating excipient to the biopolymer nanosphere, followed by stirring, preparing, by sieving, wet granules with a uniform particle diameter, and drying the wet granules to obtain granules of the biopolymer nanosphere containing NADH, wherein, preferably, the method further comprises pressing the granules of the biopolymer nanosphere containing NADH into tablets, preferably, the method further comprises putting the granules of the biopolymer nanosphere containing NADH into capsule shells to form capsules, preferably, the method further comprises mixing the granules of the biopolymer nanosphere containing NADH with edible oil and putting a resulting mixture into a soft capsule shells to form soft capsules, preferably, the granulating excipient is at least one of microcrystalline cellulose, polyvinylpyrrolidone, sodium bicarbonate, magnesium stearate, calcium polyphosphate, a wetting agent, and an adhesive.

11. A functional food, comprising the biopolymer nanosphere containing NADH according to any one of claims 1 to 6, and a food additive.

A raw material of biopolymer and an excipient are mixed and ground to physically modify the raw material S11 of biopolymer to obtain a biopolymer carrier.

A raw material of NADH is ground and sieved to obtain S12 the NADH. 2021102578

The NADH and the biopolymer carrier are mixed and stirred to obtain the biopolymer nanosphere containing S13 NADH.

Fig. 1

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