Biomimetic mineralized peptide-graphene bone joint health-care paste as well as preparation system and preparation method thereof

Abstract

The invention relates to the technical field of pharmacy, in particular to biomimetic mineralized peptide-graphene bone joint health-care paste as well as a preparation system and a preparation method thereof. The health-care cream comprises an olibanum serratum resin extract, thyme flower / leaf oil and other extracts, hexanediol, p-hydroxyacetophenone, triethanolamine, chlorphenesin and a biomimetic mineralized peptide-graphene composite material. The graphene oxide layer is introduced into the bone joint health-care ointment, so that the conductivity of a medicine carrying system is improved, directional migration of calcium ions and phosphorus ions is promoted, and the mineralization process is accelerated; in addition, the bone targeting peptide is modified on the surface of the carrier, so that the traditional Chinese medicine extract, phosphorus ions and calcium ions are effectively and efficiently permeated into the bone joint part, and the bone joint treatment effect is improved. The health-care ointment can quickly resist inflammation, relieve pain, promote cartilage / bone repair and promote microcirculation, so that microcrack new sites are increased, continuous anti-inflammation is realized, and pain, stiffness and structural damage caused by gonitis, scapulohumeral periarthritis and rheumatism are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a biomimetic mineralized peptide-graphene bone and joint health care ointment, its preparation system, and preparation method. Background Technology

[0002] Osteoarthritis, such as knee osteoarthritis (KOA) and hip osteoarthritis, has become an epidemic among people aged 40 and over. Globally, the prevalence in people over 60 is >37%, and in China, the prevalence of symptomatic knee osteoarthritis is as high as 8.1% in people over 50. Currently, treatment options mainly focus on two main directions: calcium and phosphorus supplementation, and external application of traditional Chinese medicine. However, both of these approaches have shortcomings: existing health patches may reduce calcium intake... 2 + / PO43- is directly mixed into the ointment, making it impossible to dynamically release according to the local bone metabolism. Calcium and phosphorus cannot penetrate the skin barrier, and the real-time effectiveness of the herbal active ingredients is poor, leading to problems such as applying too much, absorbing too little, and slow results. Furthermore, excessive free calcium ions can easily form hydroxyapatite microcrystals on the skin surface or soft tissue, inducing stinging, itching, and even calcified nodules. Moreover, although existing herbal formulas contain hundreds of active substances such as flavonoids, saponins, and organic acids, they lack a "bone-targeted" delivery system, preventing the active substances from reaching the subchondral bone effectively. Summary of the Invention

[0003] One of the objectives of this invention is to overcome the shortcomings of existing technologies by providing a biomimetic mineralized peptide-graphene bone and joint health care ointment. This ointment can efficiently deliver traditional Chinese medicine ingredients and phosphorus and calcium ions to the bone and joint locations, effectively improving drug utilization and treatment efficiency. It can quickly reduce inflammation and pain, alleviate heat and swelling pain, significantly improve blood supply to the joint capsule, provide sustained anti-inflammatory effects, and promote cartilage / bone repair and microcirculation. This increases the number of microcrack formation sites and significantly improves pain, stiffness, and structural damage in knee arthritis, frozen shoulder, and rheumatism.

[0004] The second objective of this invention is to provide a preparation system for a biomimetic mineralized peptide-graphene bone and joint health care cream.

[0005] The third objective of this invention is to provide a method for preparing a biomimetic mineralized peptide-graphene bone and joint health care cream.

[0006] To achieve one of the above objectives, the present invention provides the following technical solution:

[0007] We offer a biomimetic mineralized peptide-graphene joint health cream, comprising the following ingredients by weight percentage:

[0008] Ethanol 5%-20%, xanthan gum 0.1%-1%, carbomer 0.1%-0.5%, Boswellia carterii resin extract 0.1%-1%,

[0009] CO40 0.1%-1%, Thyme flower / leaf oil 0.01%-0.1%, Glycerin 5%-20%, Vanillyl butyl ether 0.01%-0.1%, Angelica sinensis extract 0.1%-1%, Centella asiatica extract 0.1%-1%, Opuntia ficus-indica stem extract 0.1%-1%, Houttuynia cordata extract 0.1%-1%, Chamomile extract 0.1%-1%, Portulaca oleracea extract 0.1%-1%, Scutellaria baicalensis extract 0.1%-1%, Paeonia suffruticosa root extract 0.1%-1%, Hexanediol 1%-5%, p-hydroxyacetophenone 0.01%-0.1%, Triethanolamine 0.1%-1%, Chlorphenesin 0.01%-0.1%, Biomimetic mineralized peptide-graphene composite material 0.01%-1%,

[0010] The biomimetic mineralized peptide-graphene composite material is prepared by the following method:

[0011] Step 1: Mix graphene oxide and biomimetic mineralized peptide at a mass ratio of 1 to 2:1, stir at room temperature for 1 to 2 hours, add 1-ethyl-3-(3-dimethylaminopropyl)carboxydiimide and N-hydroxysuccinimide, and continue stirring for 2 to 4 hours to complete amidation and obtain graphene oxide-peptide.

[0012] Step 2: Place graphene oxide-peptide in an aqueous phase, add 0.1-0.2 mol L-1 of NaBH, and reduce in a water bath at 50℃-60℃ for 1-3 hours to obtain black, uniformly dispersed reduced graphene-peptide hybrid nanosheets.

[0013] Step 3: Disperse the reduced graphene-peptide hybrid sheets into simulated body fluid, add 0.01-0.05M NaCO solution dropwise, and slowly introduce CO gas to keep the ratio of calcium ions to phosphate ions at 1.5-1.7 without stirring to obtain a mixture. Let the mixture stand at 37℃-42℃ for 12-48 hours to allow hydroxyapatite nanocrystals to grow from bottom to top on the surface of the graphene sheets, thus obtaining a layered biomimetic mineralized peptide-graphene composite material.

[0014] In some embodiments, the biomimetic mineralized peptide is a casein phosphopeptide or a bone-targeting hydroxyapatite-binding peptide, and the concentration of the biomimetic mineralized peptide is 0.2–0.5 mg / mL. -1 Adjust the pH to 6-7 using a buffer solution.

[0015] In some embodiments, the simulated body fluid is 10 times the volume of the simulated body fluid.

[0016] In some embodiments, the aqueous phase comprises 8%–10% 0.01M PBS buffer, 5%–7% 1,2-hexanediol, 6%–8% p-hydroxyacetophenone, 1%–2% xanthan gum, and 1%–2% carbomer, with the remainder being water;

[0017] The PBS buffer salt contains 0.24 g L⁻¹ NaH₂PO₄ and 8.0 g L⁻¹ NaCl.

[0018] In some embodiments, the ratio of calcium ions to phosphate ions is determined by EDTA complexometric titration to determine the molar content of calcium ions, and by molar content of phosphate ions is determined by molybdenum blue colorimetry.

[0019] The beneficial effects of this biomimetic mineralized peptide-graphene bone and joint health care cream are as follows:

[0020] (1) In this invention, a nano-bionic mineralized peptide-graphene composite material is used. The graphene-peptide hybrid sheets in this material serve as encapsulation carriers, loading calcium and phosphorus minerals, such as hydroxyapatite precursors and active ingredients from traditional Chinese medicine, so that the particle size of the calcium and phosphorus loading is <100nm, ensuring the permeability of the stratum corneum. It penetrates the stratum corneum through intercellular pathways, and the flexible deformation ability of the carrier is used to improve the transdermal efficiency. In the microenvironment of osteoarthritis, ALP activity increases, and ALP triggers Ca2+. 2 + / PO43- release enables lesion-specific mineralization; simultaneously, the introduction of a graphene oxide layer enhances the conductivity of the drug delivery system, promotes directional ion migration, and accelerates the mineralization process; furthermore, the carrier surface is modified with bone-targeting peptides, which, through HA-CD44 binding, accumulate in the subchondral bone region with a targeting precision of 200μm, effectively avoiding the problem of instability in targeting that leads to poor efficacy. This results in rapid anti-inflammatory and analgesic effects, reducing the sensation of heat and swelling, significantly improving blood supply to the joint capsule, providing sustained anti-inflammatory and rapid anti-inflammatory and analgesic effects, promoting cartilage / bone repair, and improving microcirculation, thereby increasing the number of microcrack formation sites and significantly improving pain, stiffness, and structural damage in knee arthritis, frozen shoulder, and rheumatism.

[0021] (2) In this invention, ethanol is used to quickly open the lipid bilayer of the stratum corneum, improving the permeability of small molecules of traditional Chinese medicine; glycerin absorbs moisture and locks in water, reducing the risk of dry patch falling off, and as a polar solvent, it helps dissolve various plant extracts. Hexanediol strengthens the preservative system and reduces irritation; carbomer forms a three-dimensional network, making it easy to apply; xanthan gum, in conjunction with carbomer, provides yield value, preventing graphene and traditional Chinese medicine particles from settling; CO40 keeps essential oils such as thyme oil transparent in the ethanol-water system, and thyme flower / leaf oil has natural antibacterial and cooling properties, reducing the need for preservatives; vanillyl butyl ether is an instant warming agent, promoting local blood flow and enhancing the rapid onset of action. Boswellia carterii resin extract is used to inhibit 5-LOX, block the leukotriene pathway, and provide rapid analgesia. Angelica dahurica extract is used to dilate capillaries and improve joint microcirculation. Centella asiatica extract promotes type II collagen synthesis and accelerates cartilage repair. Opuntia ficus-indica stem extract is rich in flavonoids and is used for anti-oxidation and soothing irritation. Houttuynia cordata extract is used to combat Staphylococcus aureus and Staphylococcus epidermidis, reducing secondary infections; Chamomile extract, containing bisabolol and apigenin, is used for antihistamines and anti-inflammatory effects; Portulaca oleracea extract is used to reduce IL-6 and TNF-α, relieving joint inflammation and swelling. Baicalin from Scutellaria baicalensis extract inhibits NF-κB signaling, providing potent anti-inflammatory effects. Paeonol from Paeonia suffruticosa root extract scavenge free radicals and protect chondrocyte mitochondria; p-hydroxyacetophenone, a natural ketone antioxidant, synergizes with hexanediol to create a "paraben-free" preservative. Chlorphenesin is a broad-spectrum antibacterial agent, also effective against fungi, supplementing the coverage blind spots of ethanol / hexanediol. Triethanolamine neutralizes carbomer carboxyl groups, forming a transparent gel, maintaining pH 6.3–6.8, ensuring the activity of peptide-graphene and skin compatibility. The biomimetic mineralized peptide-graphene composite material has its own hydroxyapatite calcium library, releasing Ca on demand through the skin. 2 + / PO43-. Graphene sheets provide thermal conductivity and microcurrent channels, synergistically enhance penetration with thermosensitive agents, and peptide chains target ALP-highly expressed regions to achieve in-situ mineralization and repair of the cartilage-bone interface.

[0022] To achieve the second objective mentioned above, the present invention provides the following technical solution:

[0023] A preparation system for the above-mentioned biomimetic mineralized peptide-graphene bone and joint health care cream is provided, including a settling tank, wherein the bottom surface of the settling tank is inclined and the bottom surface is an elastic bottom surface;

[0024] A heater is provided in the settling tank, and the heater is used to heat the liquid in the settling tank.

[0025] An ultrasonic module is disposed outside the elastic bottom surface.

[0026] In some embodiments, the inclination of the settling tank is 15°.

[0027] The beneficial effects of the preparation system for the biomimetic mineralized peptide-graphene bone and joint health care cream of the present invention are as follows:

[0028] In this invention, the preparation system uses a stabilizing tank with an inclined bottom to avoid convection in the solution system, which would affect crystallization. A heater is installed in the stabilizing tank to achieve staged heating, allowing nucleation in the first stage and induction of crystal length in the later stage. Furthermore, because the bottom of the stabilizing tank is designed with an elastic bottom, microcracks can be generated on the surface of the sheets by stretching the elastic bottom, thereby increasing nucleation sites and improving the crystallization sites of the biomimetic mineralized peptide-graphene. In addition, ultrasonic assistance is added; the cavitation microjets generated by this ultrasonic assistance can remove the stabilizing crystal nuclei, allowing the nuclei to continue growing and increasing the crystal content.

[0029] The third objective of this invention is to provide a method for preparing a biomimetic mineralized peptide-graphene bone and joint health care cream.

[0030] A method for preparing a biomimetic mineralized peptide-graphene bone and joint health care cream is provided, characterized in that...

[0031] The biomimetic mineralized peptide-graphene composite material was prepared using the above-described preparation system, including:

[0032] In step three, the reduced graphene-peptide hybrid sheets are transferred to the settling tank, simulated body fluid is added, and 0.01M-0.05M NaCO solution and 0.01M-0.05M NaOH are added alternately, with each addition being 40μL-50μL. CO gas is slowly introduced to maintain the ratio of calcium ions to phosphate ions at 1.5-1.7 without stirring to obtain a mixture. The heater is then started, and the mixture is heated at 42℃ for 6-7 hours. Subsequently, the heater is adjusted to 37℃ and heated for 8-45 hours. The elastic bottom surface is stretched every 5 hours, and the ultrasonic module is activated to vibrate the elastic bottom surface every 6 hours.

[0033] In some embodiments, the heater is a microwave heater.

[0034] In some implementations, the following steps are included:

[0035] S1. Add ethanol to the first container, heat the ethanol in a water bath to 40℃~41℃, add CO40, thyme oil and vanillyl butyl ether in sequence, and continue stirring until a clear mixture without oil droplets is obtained, thus obtaining the first mixture;

[0036] Deionized water was added to the second container, followed by carbomer. After stirring and swelling, xanthan gum was added to obtain the second mixture.

[0037] S2. Gradually add the second mixture to the first mixture and stir to form a uniform milky white dispersion;

[0038] S3. Add the extracts of Boswellia serrata resin, Angelica sinensis, Centella asiatica, Opuntia ficus-indica stem extract, Houttuynia cordata extract, Chamomile extract, Portulaca oleracea extract, Scutellaria baicalensis extract, and Paeonia suffruticosa root extract to the dispersion and continue stirring to ensure there are no lumps.

[0039] S4. First, moisten the biomimetic mineralized peptide-graphene composite material with glycerin, use ultrasonic vibration, add triethanolamine dropwise to make the pH 6.4-6.5, make the system 25℃, then add hexanediol, p-hydroxyacetophenone, and chlorphenesin, and continue stirring to obtain the biomimetic mineralized peptide-graphene bone and joint health care cream.

[0040] The beneficial effects of the preparation method of the biomimetic mineralized peptide-graphene bone and joint health care cream of the present invention are as follows:

[0041] In this invention, heating to 40–41°C using an ethanol water bath helps protect the stability of the active ingredients and avoids degradation that may be caused by high temperatures. The gradual addition of ingredients with continuous stirring ensures uniform dispersion of each component. The second mixture is gradually added to the first mixture, and stirring forms a uniform milky dispersion, which helps to form a stable emulsion system. Adding various plant extracts to the dispersion and continuously stirring to ensure no lumps form contributes to the uniform distribution of the active ingredients in the final product and improves its bioavailability. By using glycerin to moisten the biomimetic mineralized peptide-graphene composite material and precisely controlling the pH value at 6.4–6.5 and the temperature at 25°C, the stability and bioactivity of the biomimetic mineralized peptide-graphene composite material are maintained. Combined with various plant extracts, this may enhance the product's biocompatibility and therapeutic efficacy, particularly in bone and joint health. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the preparation system for biomimetic mineralized peptide-graphene bone and joint health care cream.

[0043] Figure Labels

[0044] 1. Settling tank; 2. Heater; 3. Ultrasonic module; 4. Bottom surface. Detailed Implementation

[0045] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention have been shown, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0046] The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a” and “the” as used in this invention and the appended claims are intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0047] It should be understood that although the terms "first," "second," "third," etc., may be used in this invention to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this invention, first information can also be referred to as second information, and similarly, second information can also be referred to as first information. Thus, features defined as "first" or "second" can explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0048] Osteoarthritis, such as knee osteoarthritis (KOA) and hip osteoarthritis, has become an epidemic among people aged 40 and over. Globally, the prevalence in people over 60 is >37%, and in China, the prevalence of symptomatic knee osteoarthritis is as high as 8.1% in people over 50. Currently, treatment options mainly focus on two main directions: calcium and phosphorus supplementation, and external application of traditional Chinese medicine. However, both of these approaches have shortcomings: existing health patches may reduce calcium intake... 2 + / PO43- is directly mixed into the ointment, making it impossible to dynamically release according to the local bone metabolism state. Calcium and phosphorus cannot penetrate the skin barrier, and the real-time effectiveness of the traditional Chinese medicine ingredients is poor, leading to problems such as applying too much, absorbing too little, and slow onset of effect. Furthermore, excessive free calcium ions easily form hydroxyapatite microcrystals on the skin surface or soft tissue, inducing stinging, itching, and even calcified nodules. Moreover, although existing traditional Chinese medicine formulas contain hundreds of active substances such as flavonoids, saponins, and organic acids, they lack a "bone-targeted" delivery system, preventing the active substances from reaching the subchondral bone effectively. Based on this, the following specific implementation method is disclosed:

[0049] This embodiment discloses a biomimetic mineralized peptide-graphene bone and joint health care cream, characterized by comprising the following raw materials by weight percentage:

[0050] Ethanol 5%-20%, xanthan gum 0.1%-1%, carbomer 0.1%-0.5%, Boswellia carterii resin extract 0.1%-1%,

[0051] CO40 0.1%-1%, Thyme flower / leaf oil 0.01%-0.1%, Glycerin 5%-20%, Vanillyl butyl ether 0.01%-0.1%, Angelica sinensis extract 0.1%-1%, Centella asiatica extract 0.1%-1%, Opuntia ficus-indica stem extract 0.1%-1%, Houttuynia cordata extract 0.1%-1%, Chamomile extract 0.1%-1%, Portulaca oleracea extract 0.1%-1%, Scutellaria baicalensis extract 0.1%-1%, Paeonia suffruticosa root extract 0.1%-1%, Hexanediol 1%-5%, p-hydroxyacetophenone 0.01%-0.1%, Triethanolamine 0.1%-1%, Chlorphenesin 0.01%-0.1%, Biomimetic mineralized peptide-graphene composite material 0.01%-1%,

[0052] The percentages of the above components can be adjusted according to actual conditions.

[0053] The biomimetic mineralized peptide-graphene composite material is prepared by the following method:

[0054] Step 1: Mix graphene oxide and biomimetic mineralized peptide at a mass ratio of 1 to 2:1, stir at room temperature for 1 to 2 hours, add 1-ethyl-3-(3-dimethylaminopropyl)carboxydiimide and N-hydroxysuccinimide, and continue stirring for 2 to 4 hours to complete amidation, to obtain graphene oxide-peptide.

[0055] Graphene oxide and biomimetic mineralized peptides were mixed at a specific mass ratio, and the mixture was stirred to ensure full contact between the two, forming a graphene oxide-peptide complex.

[0056] 1-Ethyl-3-(3-dimethylaminopropyl)carboxydiimide and N-hydroxysuccinimide are commonly used catalysts for amidation reactions. 1-Ethyl-3-(3-dimethylaminopropyl)carboxydiimide first reacts with the carboxyl group to generate an active intermediate, and then N-hydroxysuccinimide stabilizes this intermediate, ultimately promoting the formation of an amide bond between the amine and carboxyl groups.

[0057] Through amidation, the connection between the biomimetic mineralized peptide and graphene oxide becomes more stable, which helps to maintain the bioactivity of the biomimetic mineralized peptide.

[0058] Step 2: Place graphene oxide-peptide in an aqueous phase, add 0.1-0.2 mol L-1 of NaBH, and reduce in a water bath at 50℃-60℃ for 1-3 hours to obtain black, uniformly dispersed reduced graphene-peptide hybrid nanosheets.

[0059] Graphene oxide contains numerous oxygen functional groups, such as hydroxyl, carboxyl, and epoxy groups. Adding NaBH as a reducing agent reduces these oxygen functional groups, yielding reduced graphene. The reduced graphene-peptide hybrid nanosheets exhibit better stability and dispersibility in aqueous solutions. This is because the reduction process removes the oxygen functional groups from the graphene oxide, reducing their interaction with water molecules and thus improving their dispersibility in aqueous solutions.

[0060] Step 3: Disperse the reduced graphene-peptide hybrid sheets into simulated body fluid, add 0.01-0.05M NaCO solution dropwise, and slowly introduce CO gas into the system to keep the ratio of calcium ions to phosphate ions at 1.5-1.7 without stirring to obtain a mixture. Let the mixture stand at 37℃-42℃ for 12-48 hours to allow hydroxyapatite nanocrystals to grow from bottom to top on the surface of the graphene sheets, finally obtaining a layered biomimetic mineralized peptide-graphene composite material.

[0061] Controlling Ca in simulated body fluids 2 The ratio of + to PO43-, the addition of sodium carbonate solution, and the introduction of CO gas can adjust the pH of the solution and promote the growth of Ca2+. 2 The combination of + and PO43- promotes the deposition of hydroxyapatite (HAP). HAP is a major component of human bone and has excellent biocompatibility and bioactivity.

[0062] Hydroxyapatite (HAP) possesses excellent bioactivity, promoting cell adhesion and proliferation. Depositing HAP nanocrystals on the surface of graphene sheets enhances the bioactivity of the composite material, thereby replenishing phosphorus and calcium ions to areas of bone and joint pain and providing more active sites. By controlling the calcium content... 2 The optimal ratio of HAP (+) to PO43- enables a bottom-up mineralization process, where HAP nanocrystals grow from the bottom of the graphene sheet and gradually cover the entire surface. This bottom-up mineralization process allows for better control over the distribution and morphology of the HAP nanocrystals, thus facilitating absorption.

[0063] In this embodiment, the biomimetic mineralized peptide is a casein phosphopeptide or a bone-targeting hydroxyapatite-binding peptide, and the concentration of the biomimetic mineralized peptide is 0.2–0.5 mg / mL. -1 Adjust the pH to 6-7 using a buffer solution.

[0064] Casein phosphopeptides or bone-targeting hydroxyapatite-binding peptides are derived from organisms and have good biocompatibility. They can reduce the immune response and inflammation caused by the materials in the body. The pH is adjusted to 6-7 using a buffer solution. This pH range is close to the pH value of human blood, which helps to simulate the in vivo environment and promote the mineralization process.

[0065] In this embodiment, the simulated body fluid is 10 times the volume of simulated body fluid.

[0066] In this embodiment, the aqueous phase comprises 8%–10% 0.01M PBS buffer, 5%–7% 1,2-hexanediol, 6%–8% p-hydroxyacetophenone, 1%–2% xanthan gum, and 1%–2% carbomer, with the remainder being water;

[0067] The PBS buffer salt contains 0.24 g L⁻¹ NaH₂PO₄ and 8.0 g L⁻¹ NaCl.

[0068] The composition of the aqueous phase and the function of each component are as follows:

[0069] 0.01M PBS buffer (8%–10%) provides a stable pH environment, maintains the acid-base balance of the solution, and prevents pH changes from affecting bioactive substances. This buffer contains NaHPO (0.24 g / L) and NaCl (8.0 g / L), which mimics the ionic environment of extracellular fluid and helps maintain the activity and stability of biomolecules.

[0070] 1,2-Hexanediol (5%–7%) acts as a humectant and solvent, increasing the viscosity of solutions and improving product stability and moisturizing properties. 1,2-Hexanediol is a polyol with good moisturizing and solubility properties, helping to maintain the activity of biomolecules.

[0071] p-Hydroxyacetophenone (6%–8%) acts as an antioxidant, capable of capturing free radicals and protecting biomolecules from oxidative damage.

[0072] Xanthan gum acts as a thickener and stabilizer, increasing the viscosity and stability of solutions and improving the rheological properties of products.

[0073] Carbomer (1%–2%) acts as a thickener and stabilizer, improving the viscosity and stability of solutions and enhancing the rheological properties of products.

[0074] In this embodiment, the ratio of calcium ions to phosphate ions was determined by EDTA complexometric titration to obtain the molar content of calcium ions, and the molar content of phosphate ions was determined by molybdenum blue colorimetry.

[0075] Both EDTA complexometric titration and molybdenum blue colorimetric method are relatively rapid methods. EDTA complexometric titration accurately determines the concentration of calcium ions in solution by forming a stable complex with EDTA (ethylenediaminetetraacetic acid).

[0076] The molybdenum blue colorimetric method involves the reaction of phosphate ions with molybdate to form a blue molybdenum phosphate complex, the intensity of which is directly proportional to the concentration of phosphate ions. Both methods can accurately determine the molar content of calcium ions and phosphate ions, thus ensuring that their ratio meets specific requirements.

[0077] In this embodiment, as Figure 1 As shown, a specific preparation system is used to prepare biomimetic mineralized peptide-graphene. The preparation system includes a settling tank 1, the bottom surface 4 of which is inclined and is an elastic bottom surface.

[0078] Heater 3 is provided in the settling tank 1, and the heater is used to heat the liquid in the settling tank.

[0079] Ultrasonic module 3, wherein the ultrasonic module 3 is disposed outside the elastic bottom surface.

[0080] In the aforementioned preparation system, by setting an inclined elastic bottom surface, the biomimetic mineralized peptide-graphene crystals naturally accumulate towards the lower side under the competition of gravity and buoyancy, forming a continuous concentration gradient. The elastic rebound can periodically stretch the graphene sheets, generating microcracks and microfolds, increasing the density of nucleation sites. The addition of a heater allows for rapid nucleation at high temperatures followed by slow growth at low temperatures, increasing the crystal aspect ratio from 2.5 to 5.0, achieving temperature gradient induction, and ultimately obtaining a sheet-and-rod hybrid HA nanoarray.

[0081] The ultrasound module uses 40kHz, 20W, 5s pulses / 10s pauses to periodically peel away non-growing crystals with cavitation microjets, re-transporting solutes to active sites, causing HA crystals to grow in parallel orientation along the graphene sheets, ultimately obtaining a layered-vertical dual-orientation composite crystal layer, increasing the crystal load per unit area by 2.8 times.

[0082] In this embodiment, the inclination of the settling tank is 15°, which can be adjusted according to the actual situation.

[0083] In this embodiment, the biomimetic mineralized peptide-graphene composite material is prepared using the above-described preparation system, including:

[0084] In step three, the reduced graphene-peptide hybrid sheets are transferred to the settling tank, and simulated body fluid is added. 0.01M–0.05M NaCO solution and 0.01M–0.05M NaOH are added alternately, with each addition being 40μL–50μL. CO gas is slowly introduced into the system to maintain the calcium ion to phosphate ratio at 1.5–1.7. No stirring is required to obtain a mixture. The heater is then started, and the mixture is heated at 42°C for 6–7 hours. Subsequently, the heater temperature is lowered to 37°C and heated for 8–45 hours. The elastic bottom surface is stretched every 5 hours, and the ultrasonic module is activated every 6 hours to vibrate the elastic bottom surface.

[0085] In this embodiment, the heater is a microwave heater. A microwave heater enables the molecules inside a substance to vibrate rapidly, generating heat and thus achieving rapid and uniform heating. This heating method is more efficient than traditional heat conduction methods because it acts directly on the molecules of the substance being heated.

[0086] This embodiment includes the following steps:

[0087] S1. Add ethanol to the first container, heat the ethanol to 40-41°C in a water bath, add CO40, thyme oil and vanillyl butyl ether in sequence, and continue stirring until a clear mixture without oil droplets is obtained, thus obtaining the first mixture;

[0088] Deionized water was added to the second container, followed by carbomer. After stirring and swelling, xanthan gum was added to obtain the second mixture.

[0089] This step prepares a base emulsion or paste with appropriate consistency and stability.

[0090] S2. Gradually add the second mixture to the first mixture and stir to form a uniform milky white dispersion;

[0091] This step, in which the second mixture is gradually added to the first mixture and stirred to form a uniform milky white dispersion, prepares an emulsion product and helps to form a stable emulsion.

[0092] S3. Add the extracts of Boswellia serrata resin, Angelica sinensis, Centella asiatica, Opuntia ficus-indica stem extract, Houttuynia cordata extract, Chamomile extract, Portulaca oleracea extract, Scutellaria baicalensis extract, and Paeonia suffruticosa root extract to the dispersion and continue stirring to ensure there are no lumps.

[0093] This step involves adding various plant extracts to the already formed milky white dispersion and continuing to stir to ensure the mixture is homogeneous and free of lumps.

[0094] S4. First, moisten the biomimetic mineralized peptide-graphene composite material with glycerin, then use ultrasonic vibration to add triethanolamine dropwise to make the pH 6.4-6.5 and the temperature system 25℃. Then add hexanediol, p-hydroxyacetophenone, and chlorphenesin, and continue stirring to obtain the biomimetic mineralized peptide-graphene bone and joint health care cream.

[0095] This step, by adjusting pH and temperature and adding various auxiliary ingredients, produces a biomimetic mineralized peptide-graphene bone and joint health cream with good biocompatibility, stability, and moisturizing properties.

[0096] Experimental effect test

[0097] To further illustrate the experimental effects of the present invention, the following experimental tests were conducted.

[0098] Experimental Example 1

[0099] Preparatory work

[0100] Instruments: IKA C-MAG HS7 heated magnetic stirrer (with 5cm magnetic stir bar), ultrasonic cleaner (40kHz, 150W), benchtop pH meter (±0.01), vacuum degassing machine (-0.08MPa).

[0101] Clean area: Class 10,000 clean bench, all glass containers sterilized by moist heat at 121℃ for 20 minutes.

[0102] Raw material pre-inspection: 95% ethanol, CO40, essential oil, extract, and graphene composite material were all vacuum dried at 40℃ for 2 hours.

[0103] Weigh 15.0g of ethanol and put it into a beaker. Heat the beaker in a water bath at 40.5°C. While stirring at 300 rpm, add 0.5g of CO40, 0.05g of thyme flower / leaf oil, and 0.05g of vanillyl butyl ether in sequence, with a 30s interval between each addition. Continue heating at 40°C and 300 rpm for 5 minutes until the mixture is clear and free of oil droplets, thus obtaining the first mixture.

[0104] Add 55.9g of deionized water to another beaker, and while stirring (600rpm), sprinkle in 0.30g of carbomer. After sprinkling, let it stand at room temperature for 30min to fully swell. Continue stirring at 600rpm, sprinkle in 0.20g of xanthan gum, and stir for another 5min until there are no particles, to obtain the second mixture.

[0105] Maintain the second mixture at 25°C and 600 rpm;

[0106] Using a 50 mL separatory funnel, slowly add the second mixture dropwise to the first mixture (1 mL min⁻¹, for a total of 10 min). After the addition is complete, increase the speed to 800 rpm and continue for 5 min to obtain a uniform milky white dispersion.

[0107] Mix 0.50g of each of the following 9 extracts beforehand:

[0108] Toothed Boswellia resin, round-leaved Angelica sinensis, Centella asiatica, Opuntia ficus-indica stem, Houttuynia cordata, golden chamomile, Portulaca oleracea, Scutellaria baicalensis, and Paeonia suffruticosa root.

[0109] Add the dispersion in three portions at 800 rpm, with a 2-minute interval between each addition, ensuring no clumps are formed; continue at 800 rpm for 5 minutes.

[0110] Add 0.50g of biomimetic mineralized peptide-graphene composite material to 10.0g of glycerin and initially moisten with a glass rod;

[0111] Transfer to an ultrasonic cleaner (25℃, 40kHz, 50% power) and sonicate for 30 seconds to form a uniform slurry.

[0112] The slurry was poured into the main system at once (25℃, 800rpm), and 0.20g of triethanolamine (TEA) was added dropwise while measuring the pH. The final pH was 6.4. Then, 3.0g of hexanediol, 0.05g of p-hydroxyacetophenone, and 0.05g of chlorphenesin were added sequentially, with a 1min interval between each addition, and the process was carried out at 800rpm for 3min.

[0113] The preparation method of the biomimetic mineralized peptide-graphene composite material is as follows: 10 mL of simulated body fluid is added to a settling tank, and NaCO solution and NaOH are added alternately. The concentration range of NaCO solution and NaOH is 0.01 M to 0.05 M, and the amount added each time is 40 μL to 50 μL. CO gas is slowly introduced into the system. The ratio of calcium ions to phosphate ions is maintained between 1.5 and 1.7. No stirring is performed during the reaction.

[0114] First stage: Heat at 42℃ for 6 to 7 hours.

[0115] Second stage: Adjust the temperature to 37℃ and heat for 8 to 45 hours.

[0116] The elastic bottom surface is stretched every 5 hours, and the ultrasonic module is activated to vibrate the elastic bottom surface every 6 hours.

[0117] Comparative Example 1

[0118] The difference between Comparative Example 1 and Experimental Example 1 is that graphene was omitted, and 0.50g of biomimetic mineralized peptide was added. The other steps and operations were the same as in Experimental Example 1.

[0119] Experimental subjects: 60 healthy volunteers (KL II-III grade knee osteoarthritis, VAS≥40mm), randomized, double-blind, parallel controlled, treatment course of 4 weeks, 2g twice daily each time.

[0120] Grouping:

[0121] Group A: Experiment 1 (containing graphene)

[0122] Group B: Comparative Example 1 (no graphene, peptides only)

[0123] Group C: Blank matrix (containing no peptides or graphene)

[0124] Evaluation metrics and testing methods

[0125] Pain: VAS (0-100mm)

[0126] Cartilage repair: cartilage thickness, BML volume)

[0127] Transdermal efficiency: 24-hour cumulative permeability

[0128] Biomarkers: serum ALP, CTX-II, IL-6

[0129] Skin safety: Draize rating

[0130] Compliance: Remaining paste weight method

[0131] The results shown in Table 1 were obtained.

[0132] Table 1

[0133]

[0134] As shown in Table 1, the graphene + peptide group (A) was significantly better than the peptide-only group (B) and the blank matrix group (C) in all primary and secondary endpoints, and the differences were statistically significant (P<0.001).

[0135] The addition of graphene increased transdermal efficiency by 66% (68.4% vs 41.2%); the cartilage thickness was additionally increased by 0.16 mm.

[0136] The bone marrow lesion volume decreased by an additional 0.73 cm. 3 No skin irritation or systemic adverse reactions were observed in any of the three groups, indicating that the formula is safe and can proceed to the next phase of clinical expansion trials.

[0137] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of this application. It should also be understood that techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0138] In the description of this application, it should be understood that the orientation or positional relationship indicated by directional terms such as "front, back, up, down, left, right", "horizontal, vertical, horizontal" and "top, bottom" is generally based on orientation or positional relationship and is only for the convenience of describing this application and simplifying the description. Unless otherwise stated, these directional terms do not indicate or imply that the device or element referred to must have a specific orientation or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the scope of protection of this application; the directional terms "inner" and "outer" refer to the inner and outer contours relative to the outline of each component itself.

[0139] For ease of description, spatial relative terms such as "above," "on top of," "on the upper surface of," and "above" may be used herein to describe the spatial positional relationship of a device or feature to other devices or features. It should be understood that spatial relative terms are intended to encompass different orientations of the device in use or operation, in addition to the orientation depicted in the figures. The exemplary term "above" can include both "above" and "below." The device may also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein will be interpreted accordingly.

[0140] Furthermore, it should be noted that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore cannot be construed as limiting the scope of protection of this application.

[0141] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biomimetic mineralized peptide-graphene bone and joint health care cream, characterized in that, The raw materials include the following weight percentages: Ethanol 5%-20%, Xanthan gum 0.1%-1%, Carbomer 0.1%-0.5%, Boswellia carterii resin extract 0.1%-1%, CO40 0.1%-1%, Thyme flower / leaf oil 0.01%-0.1%, Glycerin 5%-20%, Vanillyl butyl ether 0.01%-0.1%, Angelica sinensis extract 0.1%-1%, Centella asiatica extract 0.1%-1%, Opuntia ficus-indica stem extract 0.1%-1%, Houttuynia cordata extract 0.1%-1%, Chamomile extract 0.1%-1%, Portulaca oleracea extract 0.1%-1%, Scutellaria baicalensis extract 0.1%-1%, Paeonia suffruticosa root extract 0.1%-1%, Hexanediol 1%-5%, p-hydroxyacetophenone 0.01%-0.1%, Triethanolamine 0.1%-1%, Chlorphenesin 0.01%-0.1%, Biomimetic mineralized peptide-graphene composite material 0.01%-1%, The biomimetic mineralized peptide-graphene composite material is prepared by the following method: Step 1: Mix graphene oxide and biomimetic mineralized peptide at a mass ratio of 1 to 2:1, stir at room temperature for 1 to 2 hours, add 1-ethyl-3-(3-dimethylaminopropyl)carboxydiimide and N-hydroxysuccinimide, and continue stirring for 2 to 4 hours to complete amidation and obtain graphene oxide-peptide. Step 2: Place graphene oxide-peptide in an aqueous phase, add 0.1-0.2 mol L-1 of NaBH, and reduce in a water bath at 50℃-60℃ for 1-3 hours to obtain black, uniformly dispersed reduced graphene-peptide hybrid nanosheets. Step 3: Disperse the reduced graphene-peptide hybrid sheets into simulated body fluid, add 0.01-0.05M NaCO solution dropwise, and slowly introduce CO gas to keep the ratio of calcium ions to phosphate ions at 1.5-1.7 without stirring to obtain a mixture. Let the mixture stand at 37℃-42℃ for 12-48 hours to allow hydroxyapatite nanocrystals to grow from bottom to top on the surface of the graphene sheets, thus obtaining a layered biomimetic mineralized peptide-graphene composite material.

2. The biomimetic mineralized peptide-graphene bone and joint health care cream according to claim 1, characterized in that, The biomimetic mineralized peptide is a casein phosphopeptide or a bone-targeting hydroxyapatite-binding peptide, and the concentration of the biomimetic mineralized peptide is 0.2–0.5 mg / mL. -1 Adjust the pH to 6-7 using a buffer solution.

3. The biomimetic mineralized peptide-graphene bone and joint health care cream according to claim 1, characterized in that, The simulated body fluid is 10 times the volume of the simulated body fluid.

4. The biomimetic mineralized peptide-graphene bone and joint health care cream according to claim 1, characterized in that, The aqueous phase comprises 8%–10% 0.01M PBS buffer, 5%–7% 1,2-hexanediol, 6%–8% p-hydroxyacetophenone, 1%–2% xanthan gum, and 1%–2% carbomer, with the remainder being water. The PBS buffer salt contains 0.24 g L⁻¹ NaH₂PO₄ and 8.0 g L⁻¹ NaCl.

5. The biomimetic mineralized peptide-graphene bone and joint health care cream according to claim 1, characterized in that, The ratio of calcium ions to phosphate ions was determined by EDTA complexometric titration to obtain the molar content of calcium ions, and by molar content of phosphate ions was determined by molybdenum blue colorimetry.

6. The preparation system of the biomimetic mineralized peptide-graphene bone and joint health care cream according to any one of claims 1 to 5, characterized in that, It includes a settling tank, the bottom surface of which is inclined and is an elastic bottom surface; A heater is provided in the settling tank, and the heater is used to heat the liquid in the settling tank. An ultrasonic module is disposed outside the elastic bottom surface.

7. The preparation system for the biomimetic mineralized peptide-graphene bone and joint health care cream according to claim 6, characterized in that, The inclination of the settling tank is 15°.

8. A method for preparing a biomimetic mineralized peptide-graphene bone and joint health care ointment, characterized in that, The preparation of the biomimetic mineralized peptide-graphene composite material using the preparation system of claim 6 or 7 includes: In step three, the reduced graphene-peptide hybrid sheets are transferred to the settling tank, simulated body fluid is added, and 0.01M-0.05M NaCO solution and 0.01M-0.05M NaOH are added alternately, with each addition being 40μL-50μL. CO gas is slowly introduced to maintain the ratio of calcium ions to phosphate ions at 1.5-1.7 without stirring to obtain a mixture. The heater is then started, and the mixture is heated at 42℃ for 6-7 hours. Subsequently, the heater is adjusted to 37℃ and heated for 8-45 hours. The elastic bottom surface is stretched every 5 hours, and the ultrasonic module is activated to vibrate the elastic bottom surface every 6 hours.

9. The preparation method of the biomimetic mineralized peptide-graphene bone and joint health care ointment according to claim 8, characterized in that, The heater is a microwave heater.

10. The preparation method of the biomimetic mineralized peptide-graphene bone and joint health care ointment according to claim 8, characterized in that, Includes the following steps: S1. Add ethanol to the first container, heat the ethanol in a water bath to 40℃~41℃, add CO40, thyme oil and vanillyl butyl ether in sequence, and continue stirring until a clear mixture without oil droplets is obtained, thus obtaining the first mixture; Deionized water was added to the second container, followed by carbomer. After stirring and swelling, xanthan gum was added to obtain the second mixture. S2. Gradually add the second mixture to the first mixture and stir to form a uniform milky white dispersion; S3. Add the extracts of Boswellia serrata resin, Angelica sinensis, Centella asiatica, Opuntia ficus-indica stem extract, Houttuynia cordata extract, Chamomile extract, Portulaca oleracea extract, Scutellaria baicalensis extract, and Paeonia suffruticosa root extract to the dispersion and continue stirring to ensure there are no lumps. S4. First, moisten the biomimetic mineralized peptide-graphene composite material with glycerin, use ultrasonic vibration, add triethanolamine dropwise to make the pH 6.4-6.5, make the system 25℃, then add hexanediol, p-hydroxyacetophenone, and chlorphenesin, and continue stirring to obtain the biomimetic mineralized peptide-graphene bone and joint health care cream.

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