Pyrrolidinyl diaminopyrimidine oxide supramolecular nano-vesicle as well as preparation method and application thereof

Abstract

The invention provides a pyrrolidinyl diaminopyrimidine oxide supramolecular nano-vesicle as well as a preparation method and application thereof, and relates to the technical field of cosmetics. Comprising the following components: pyrrolidinyl diaminopyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, a cosolvent and a solvent, the prepared pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles can significantly improve the solubility and hair follicle targeting property of pyrrolidinyl diaminopyrimidine oxide, have the advantages of high encapsulation efficiency, large drug loading capacity, good stability and high safety, can effectively promote the anti-hair loss active factor pyrrolidinyl diaminopyrimidine oxide to accurately act on hair follicle cells, and can effectively inhibit hair loss. And the efficient anti-falling effect is achieved. The pyrrolidinyl diaminopyrimidine oxide supramolecular nano-vesicle provided by the invention is simple in preparation process, accurate in dosage and suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a pyrrolidino diaminopyrimidine oxide supramolecular nanovesicle, a preparation method and an application thereof. Background Art

[0002] Alopecia is a common and frequently occurring skin disease characterized by abnormal, excessive hair loss and thinning hair. Androgenetic alopecia and alopecia areata are the most common causes. Modern medicine believes that a variety of pathogenic factors, including genetics, autoimmunity, psychological factors, endocrine factors, cytokines, and trace elements, can induce hair loss by dysregulating the hair growth cycle and miniaturizing hair follicles. Hair loss treatment and hair follicle regeneration remain pressing challenges in both the medical and scientific communities.

[0003] With the advancement of medical technology, in addition to traditional plant-derived ingredients, a growing number of high-tech chemical ingredients are being developed to more effectively address hair loss. Pyrrolidinodiaminopyrimidine oxide, as an advanced chemical ingredient, exhibits faster results than traditional plant-based ingredients, making it well-suited for use in anti-hair loss products, demonstrating significant market potential and application prospects.

[0004] Pyrrolidine diaminopyrimidine oxide (also known as "Kopyrrol") is a new hair growth promoter with a similar structure to minoxidil, a first-line anti-hair loss drug. Its molecular formula is C8H 13 N5O, with a molecular weight of 195.22. Pyrrolidinodiaminopyrimidine oxide (PDPO) has a similar anti-hair loss mechanism to minoxidil. As a potassium channel opener, it effectively promotes the proliferation of dermal papilla cells, stimulates hair follicle regeneration, and nourishes hair follicles by dilating blood vessels, increasing nutrients and oxygen to the hair root. It also accelerates new hair growth by optimizing the hair follicle growth cycle. Experimental studies have shown that PDPO also reduces serum 5α-reductase, androgen receptor, and dihydrotestosterone levels in mice, significantly improving pathological changes in hair follicles on the back of the mice, and exhibits a dose-response relationship, demonstrating its promising anti-hair loss efficacy. Substituted PDPOs, through salt formation, yield active compounds with improved solubility, enhanced transdermal absorption, and a longer duration of action. They are also less toxic than minoxidil, allowing for use at higher concentrations without undesirable side effects. These advantages, combined with their excellent water solubility and targeted targeting of hair follicles, are prerequisites for the wider application of PDPO in water-based cosmetics.

[0005] Glycyrrhizic acid, one of the main active ingredients in the traditional Chinese medicine licorice (Licorice), is primarily composed of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid. Due to its polymeric behavior and amphiphilic structure, which allows it to form micelles, glycyrrhizic acid is used as an absorption enhancer and delivery vehicle to improve transdermal absorption of active ingredients. Glycyrrhizic acid has shown promise in preventing hair loss. Research suggests that glycyrrhizic acid can inhibit 5α-reductase, reducing testosterone-induced excess oil secretion on the scalp, thereby helping to control hair loss. Furthermore, glycyrrhizic acid has anti-inflammatory properties, soothing the scalp, and alleviating irritation, thereby providing relief from itching and preventing hair loss. In practical applications, glycyrrhizic acid is often combined with other ingredients, such as baicalin, to form nanomicelles to improve its solubility, stability, and bioavailability, allowing it to more effectively penetrate the scalp and hair follicles. For example, research by Professor Liu Qiang's team at Southern Medical University has shown that, through nano-encapsulation technology, a combination of glycyrrhizic acid and baicalin can significantly promote hair growth and has a certain therapeutic effect on alopecia areata. Overall, glycyrrhizic acid, as an ingredient with multiple biological activities, shows positive prospects in preventing hair loss.

[0006] Traditional Chinese herbal extracts such as ginger, Platycladus orientalis, and Polygonum multiflorum are mild but slow to take effect. In contrast, modern chemical ingredients such as pyrrolidinodiaminopyrimidine oxide have shown great potential with their clear mechanism of action and rapid effect. This compound can not only work alone, but also significantly enhance the anti-hair loss effect when used in combination with other anti-hair loss raw materials such as diaminopyrimidine oxide, plant extracts and biological agents. For example: compounded with diaminopyrimidine oxide (Kopexil): used in combination with diaminopyrimidine oxide, which is also a minoxidil analogue. Patent application WO2007 / 101357A2 shows that when pyrrolidinodiaminopyrimidine oxide (Kopyrrol) is combined with diaminopyrimidine oxide (Kopexil), it effectively promotes hair growth, which is much stronger than the effect of using diaminopyrimidine oxide or minoxidil alone, and can play a synergistic role. Researchers studied the combination of Kopyrrol, Kopexil, and their mixture. A mouse model of androgenic alopecia was established by applying testosterone (0.05% by mass) to the back of the mouse. The mice were evaluated for hair loss and related indicators, including epidermal growth factor and serum 5α-reductase activity. Combination with plant extracts: Plant extracts are commonly added to current hair loss prevention and care products. Plant extracts are popular among consumers due to their natural origin, and experiments have shown that their inclusion in formulas can further enhance the effectiveness of pyrrolidine diaminopyrimidine oxide. A related company has developed a hair growth-promoting nanocomplex composed of pyrrolidine diaminopyrimidine oxide and baicalin in a 5:2 mass ratio. The two exhibit a synergistic effect, significantly promoting hair growth and providing a more comprehensive approach to preventing and treating hair loss. Combination with bioactive agents: Certain amino acid or small peptide bioactive agents also have anti-hair loss and care benefits. Therefore, these bioactive agents have the potential to be combined with pyrrolidine diaminopyrimidine oxide to enhance their effectiveness.

[0007] Acetyl tetrapeptide-3 accelerates the synthesis of extracellular matrix proteins, increases hair follicle volume and length, strengthens hair roots, repairs the epidermal-dermal junction, and anchors hair within follicles. Adenosine increases the expression of fibroblast growth factor in dermal papilla cells, dilates blood vessels, improves blood circulation, and reduces hair loss. Kopyrrol, adenosine, and acetyl tetrapeptide-3 were used as the main hair growth and development active ingredients. Pyrrolidine diaminopyrimidine oxide / adenosine / active peptide composite nanoliposomes (nanobricoside) were prepared by combining these three active ingredients with different mechanisms. Their hair growth and development efficacy was evaluated in vitro and in animal studies. Cell survival rates were observed after 48-hour treatment of dermal papilla cells with different concentrations of free active ingredients and nanocobicoside. Compared with the normal control group, both free active ingredients and nanocobicoside significantly promoted dermal papilla cell proliferation after 48 hours of culture (P < 0.05). Compared with the free active substance, the cell proliferation effect of nano-cobixin was more obvious, with significant differences.

[0008] Chinese invention patent application publication number CN114732751A discloses an anti-hair loss essence containing pyrrolidino diamino pyrimidine oxide, which includes the following components in parts by weight: 0.01-20 parts by weight of pyrrolidino diamino pyrimidine oxide; 0.01-20 parts by weight of adenosine; 0.01-20 parts by weight of diamino pyrimidine oxide; 0.01-20 parts by weight of niacinamide; 0.01-20 parts by weight of urea; 100-200 parts by weight of a solvent; and 0.01-0.1 parts by weight of tripeptide-1 copper.

[0009] Chinese invention patent application publication number CN110917062A discloses a hair growth-promoting nanoemulsion and its preparation method. The nanoemulsion comprises a hair growth-promoting complex composed of pyrrolidinopyrimidine oxide and baicalin, and a nanocomposite with a particle size of 10 to 133 μm composed of polyoxyethylene castor oil, lecithin, ethyl oleate, glutathione, and ethanol. The mass ratio of the pyrrolidinopyrimidine oxide to the baicalin is 5:2. The hair growth-promoting nanocomposite provided by this solution, composed of pyrrolidinopyrimidine oxide and baicalin as effective hair growth promoting substances, can effectively promote hair growth. When prepared as a nanoemulsion, it exhibits low irritation, excellent stability, and enhanced efficacy.

[0010] Chinese invention patent application publication number CN118319801A discloses a composition containing ginseng root extract, its preparation method, and its use. The composition comprises ginseng root extract, adenosine, diaminopyrimidine oxide, and pyrrolidinopyrimidine oxide. The composition, comprising ginseng root extract as the primary active ingredient and adenosine, diaminopyrimidine oxide, and pyrrolidinopyrimidine oxide as antioxidant enhancers, exhibits a synergistic effect, significantly enhancing antioxidant efficacy and combating oxidative stress.

[0011] Chinese invention patent application publication number CN118512476A discloses a pharmaceutical composition with comprehensive scalp environment improvement efficacy, its preparation method, and its use. The composition contains 0.1-10% of a hair follicle repair protein composition, 0.001-0.1% of caffeine, 0.001-0.1% of a pyrrolidinodiaminopyrimidine oxide, 0.001-0.1% of a diaminopyrimidine oxide, 0.1-10% of a plant extract, 0.001-0.1% of an amino acid, and a pharmaceutically acceptable carrier. The plant extract is selected from any one or a combination of emblica fruit extract, arborvitae leaf extract, ginseng extract, burdock root extract, trifolium repens leaf extract, mugwort leaf extract, and ginger root extract, and the amino acid is selected from any one or a combination of arginine, cystine, ornithine, histidine, tyrosine, citrulline, serine, and niacinamide. This pharmaceutical composition has a significant repair effect and can comprehensively improve a patient's scalp environment.

[0012] Chinese invention patent application publication number CN118512478A discloses a pharmaceutical composition with anti-hair loss efficacy, its preparation method, and its application. The composition comprises 0.1-10% of a hair follicle repair protein composition, 0.1-10% of a pyrrolidinodiaminopyrimidine oxide, 0.1-10% of a diaminopyrimidine oxide, 0.1-10% of a traditional Chinese medicine extract, 0.1-10% of an amino acid, and a pharmaceutically acceptable carrier. The traditional Chinese medicine extract is selected from any one or a combination of emblica fruit extract, Platycladus orientalis leaf extract, ginger root extract, tea leaf extract, asparagus root extract, trifolium repens leaf extract, and mugwort leaf extract, and the amino acid is selected from any one or a combination of silk amino acids, arginine, cystine, niacinamide, tyrosine, histidine, ornithine, and citrulline. The composition effectively prevents hair loss and has the advantages of high absorption rate, no scalp irritation, no trauma, and rapid effect.

[0013] Although these innovative combinations provide more efficient and comprehensive solutions for hair loss treatment to a certain extent, they still cannot fully exert the anti-hair loss efficacy of pyrrolidinopyrimidine oxide as the main active ingredient.

[0014] Although pyrrolidinodiaminopyrimidine oxide was initially discovered to have similar effects to minoxidil, pharmaceutical research and development was not pursued. Furthermore, because diaminopyrimidine oxide and pyrrolidinodiaminopyrimidine oxide are crystalline powders with poor water solubility, hair growth solutions prepared from them must contain large amounts of alcohol, which can easily lead to discomfort such as dryness and itching of the scalp. Furthermore, due to the skin's inherent barrier, the active ingredient has difficulty penetrating into the hair follicles and directly acting on the target area, resulting in low bioavailability. Subsequently, after a series of studies, Zhang Yan et al., in Chinese invention patent application publication number CN108210937A, disclosed a pyrrolidinodiaminopyrimidine oxide inclusion complex with β-cyclodextrin or / and a β-cyclodextrin derivative, which improved the solubility of the poorly water-soluble pyrrolidinodiaminopyrimidine oxide anti-hair loss and hair growth agent in aqueous solution. While cyclodextrin encapsulation increases the water solubility of pyrrolidinodiaminopyrimidine oxide, its large particle size prevents deep penetration into the hair follicle structure, limiting its target site and resulting in suboptimal hair loss prevention.

[0015] Chinese invention patent publication number CN109528725B discloses a nanocomposition containing diaminopyrimidine oxide and pyrrolidinopyrimidine oxide, as well as its preparation method and application. The composition comprises the following components by weight: 5-20% diaminopyrimidine oxide, 2.5-10% pyrrolidinopyrimidine oxide, 0.1-10% 5α-reductase inhibitor, 0.1-10% androgen receptor antagonist, 0.1-5% vasodilator, 0.1-10% anti-inflammatory and antibacterial agent, 0.1-5% antioxidant, 0.5-10% phospholipid, 0.1-40% softener, 0.1-5% stabilizer, 0.01-1% preservative, and the balance water. The nanocomposition provided by the invention achieves transdermal co-delivery of multiple anti-hair loss active ingredients, penetrating deeply into hair follicles and achieving synergistic effects. It has both anti-hair loss and hair growth efficacy and can be widely used in anti-hair loss and hair growth cosmetics. However, the particle size of the composition of this solution is large, the skin permeability and absorption rate are poor, and the hair follicle targeting is poor.

[0016] The paper "Microneedle Delivery Platform Integrated with Codelivery Nanoliposomes for Effective and Safe Androgenetic Alopecia Treatment" published in "ACS Applied Materials & Interfaces" uses diaminopyrimidine oxide (kopexil) and pyrrolidinyl diaminopyrimidine oxide (kopyrrol) as the core anti-hair loss ingredients encapsulated in nanoliposomes (KK-NLPs). However, after the anti-hair loss active substances diaminopyrimidine oxide (kopexil) and pyrrolidinyl diaminopyrimidine oxide (kopyrrol) are simultaneously encapsulated in the nanoliposomes, the particle size is large and there are certain defects in transdermal absorption, which prevents the anti-hair loss efficacy of diaminopyrimidine oxide (kopexil) and pyrrolidinyl diaminopyrimidine oxide (kopyrrol) from being fully utilized.

[0017] In view of this, the present invention is proposed. Summary of the Invention

[0018] One of the objectives of the present invention is to provide a supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2. The supramolecular nanovesicle composed of glycyrrhizic acid and acetyl tetrapeptide-2 provided by the present invention is used to encapsulate pyrrolidinodiaminopyrimidine oxide. Acetyl tetrapeptide-2 and glycyrrhizic acid act as carriers to act as reservoirs, continuously releasing the anti-hair loss functional factor pyrrolidinodiaminopyrimidine oxide, maintaining it at an effective concentration for a long time, and synergistically enhancing its effect, thereby achieving a highly effective anti-hair loss effect.

[0019] A second object of the present invention is to provide a method for preparing supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2.

[0020] A third objective of the present invention is to provide supramolecular nanovesicles of pyrrolidinopyrimidine oxide. The supramolecular nanovesicles provided by the present invention can significantly improve the solubility and hair follicle targeting of pyrrolidinopyrimidine oxide. They have high encapsulation efficiency, large drug loading capacity, good stability, and high safety. They can effectively promote the precise action of the anti-hair loss active factor pyrrolidinopyrimidine oxide on hair follicle cells, achieving a highly effective anti-hair loss effect.

[0021] A fourth object of the present invention is to provide a method for preparing pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles.

[0022] The fifth object of the present invention is to provide a pyrrolidino diamino pyrimidine oxide supramolecular nanovesicle or the use of the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicle prepared by the preparation method in the preparation of anti-hair loss care products.

[0023] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted: In a first aspect, the present invention provides a supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2, comprising the following components by mass percentage: 10.0%-20.0% glycyrrhizic acid, 1.0%-2.0% acetyl tetrapeptide-2 and the remainder a solvent.

[0024] Furthermore, the supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 include the following components by mass percentage: 15.0% glycyrrhizic acid, 1.5% acetyl tetrapeptide-2 and the balance solvent.

[0025] Furthermore, the particle size of the supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 is 2-20 nm, preferably 2-10 nm.

[0026] In a second aspect, the present invention provides a method for preparing supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2, comprising the following steps: The glycyrrhizic acid, acetyl tetrapeptide-2 and solvent in the formulated amounts are mixed to obtain supramolecular nanovesicles formed by self-assembly of the glycyrrhizic acid and acetyl tetrapeptide-2.

[0027] In a third aspect, the present invention provides a pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicle, comprising pyrrolidinodiaminopyrimidine oxide, and the supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 encapsulated outside the pyrrolidinodiaminopyrimidine oxide, or the supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 prepared by the preparation method.

[0028] Furthermore, the pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles include the following components: Pyrrolidinodiaminopyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, cosolvent and solvent; Preferably, the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles comprise the following components by mass percentage: 2.0%-2.8% pyrrolidino diamino pyrimidine oxide, 2.0%-5.0% glycyrrhizic acid, 0.2%-0.5% acetyl tetrapeptide-2, 10.1%-25.3% propylene glycol, 5.2%-9.3% Tween 20 and the balance solvent; Preferably, the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles comprise the following components by mass percentage: 2.36% pyrrolidino diamino pyrimidine oxide, 4.87% glycyrrhizic acid, 0.49% acetyl tetrapeptide-2, 19.68% propylene glycol, 9.13% Tween 20 and the balance solvent.

[0029] Furthermore, the cosolvent includes at least one of propylene glycol and Tween 20.

[0030] In a fourth aspect, the present invention provides a method for preparing the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles, comprising mixing a formulated amount of pyrrolidino diamino pyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, a cosolvent and a solvent to obtain the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles.

[0031] Further, the following steps are included: (a) dissolving a formulated amount of pyrrolidinodiaminopyrimidine oxide and a cosolvent in a solvent to obtain a solution a; (b) dissolving the formulated amounts of glycyrrhizic acid and acetyl tetrapeptide-2 in a solvent to obtain a glycyrrhizic acid solution and an acetyl tetrapeptide-2 solution, respectively, and then mixing the glycyrrhizic acid solution and the acetyl tetrapeptide-2 solution to obtain a solution b; (c) Mixing solution a and solution b in step 1 and stirring at 60-85°C for 10-60 minutes to form a transparent liquid; (d) After a transparent liquid is formed, the temperature is lowered and stirring is continued for 6-8 h; (e) Filtration to obtain glycyrrhizic acid and acetyl tetrapeptide-2-encapsulated pyrrolidinopyrimidine oxide supramolecular nanovesicles.

[0032] In a fifth aspect, the present invention provides a use of the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles or the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles prepared by the preparation method in the preparation of anti-hair loss care products.

[0033] Compared with the prior art, the present invention has the following beneficial effects: The supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 provided by the present invention use acetyl tetrapeptide-2 and glycyrrhizic acid as carriers, play the role of a reservoir, can encapsulate components with anti-hair loss function, and continuously release the components with anti-hair loss function, so that the components are maintained at an effective concentration for a long time, synergistically enhancing the effect, and thus playing a highly efficient anti-hair loss effect.

[0034] The pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles provided by the present invention are encapsulated in supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2. Glycyrrhizic acid and acetyl tetrapeptide-2 are used as carriers to increase the solubility and bioavailability of the pyrrolidino diaminopyrimidine oxide, and the skin retention amount is greatly improved. The pyrrolidino diaminopyrimidine oxide can inhibit the activity of 5α-reductase, and can also exert its own effect of inhibiting 5α-reductase and reducing excessive scalp oil secretion caused by testosterone. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 This is a contour map showing the effects of glycyrrhizic acid dosage and reaction temperature on the encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinodiaminopyrimidine oxide nanovesicles in Example 5; Figure 2 This is a contour map showing the effects of glycyrrhizic acid dosage and reaction time on the encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinodiaminopyrimidine oxide nanovesicles in Example 5; Figure 3 This is a contour map showing the effects of reaction temperature and reaction time on the encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinodiaminopyrimidine oxide nanovesicles in Example 5; Figure 4 This is a response surface diagram showing the effects of glycyrrhizic acid dosage and reaction temperature on the encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinodiaminopyrimidine oxide nanovesicles in Example 5; Figure 5 This is a response surface diagram showing the effects of glycyrrhizic acid dosage and reaction time on the encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinodiaminopyrimidine oxide nanovesicles in Example 5; Figure 6 The response surface diagram of the effects of reaction temperature and reaction time on the encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles provided in Example 5; Figure 7 This is a sample of supramolecular nanovesicles self-assembled from glycyrrhizic acid and acetyl tetrapeptide-2 prepared in Example 1; Figure 8This is a sample of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles prepared in Example 5; Figure 9 This is the particle size distribution diagram of the pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles prepared in Example 5; Figure 10 It is the 5α-reductase inhibition rate of Example 5 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0037] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0038] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] The first aspect of the present invention provides a supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2, the components of which include glycyrrhizic acid, acetyl tetrapeptide-2 and a solvent. The supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 is used to encapsulate components with anti-shedding function, such as pyrrolidinodiaminopyrimidine oxide.

[0040] In some preferred embodiments, the supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 include the following components by mass percentage: 10.0%-20.0% glycyrrhizic acid, 1.0%-2.0% acetyl tetrapeptide-2 and the balance solvent.

[0041] Taking the total mass of the supramolecular nanovesicles self-assembled by the glycyrrhizic acid and acetyl tetrapeptide-2 as 100%, the amount of glycyrrhizic acid added is 10.0%-20.0%, for example, 10.0%, 15.0%, 20.0%, etc.; Taking the total mass of the supramolecular nanovesicles self-assembled by glycyrrhizic acid and acetyl tetrapeptide-2 as 100%, the added amount of acetyl tetrapeptide-2 is 1.0%-2.0%, for example, 1.0%, 1.5%, 2.0%, etc.

[0042] In some preferred embodiments, the supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 include the following components by mass percentage: 15.0% glycyrrhizic acid, 1.5% acetyl tetrapeptide-2 and the balance solvent.

[0043] In some preferred embodiments, the particle size of the supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 is 2-20 nm, preferably 2-10 nm.

[0044] In the present invention, the particle size of the product after subsequent coating with pyrrolidinodiaminopyrimidine oxide is also 2-20 nm.

[0045] The second aspect of the present invention provides a method for preparing supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2, comprising the following steps: The glycyrrhizic acid, acetyl tetrapeptide-2 and solvent in the formulated amounts are mixed to obtain supramolecular nanovesicles formed by self-assembly of the glycyrrhizic acid and acetyl tetrapeptide-2.

[0046] Specifically, the preparation process of the supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 includes: (1) Accurately weigh the formulated amount of acetyl tetrapeptide-2 and dissolve it in a certain amount of deionized water (the amount of deionized water is not strictly limited, as long as it can be dissolved); (2) Weigh the formulated amount of glycyrrhizic acid and add a certain amount of deionized water to fully dissolve it (the amount of deionized water used is not strictly limited, as long as it can be dissolved); It should be noted that there is no strict requirement for the amount of water used when preparing the glycyrrhizic acid solution and the acetyl tetrapeptide-2 solution respectively. The final glycyrrhizic acid solution and the acetyl tetrapeptide-2 solution can meet the requirements of 10.0%-20.0% glycyrrhizic acid, 1.0%-2.0% acetyl tetrapeptide-2 and the remaining solvent for self-assembly.

[0047] (3) slowly adding the solution (glycyrrhizic acid solution) in step (2) dropwise to the solution (acetyl tetrapeptide-2 solution) in step (1) while stirring; (4) Continue stirring for 6 to 8 hours to obtain supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2.

[0048] The third aspect of the present invention provides a pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicle, comprising pyrrolidinodiaminopyrimidine oxide, and the supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 encapsulated outside the pyrrolidinodiaminopyrimidine oxide, or the supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 prepared by the above preparation method.

[0049] The pyrrolidino diamino pyrimidine oxide supramolecular nanovesicle provided by the invention is a pyrrolidino diamino pyrimidine oxide supramolecular nanovesicle wrapped based on glycyrrhizic acid and acetyl tetrapeptide-2.

[0050] The pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles include the following components: Pyrrolidinodiaminopyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, cosolvent and solvent.

[0051] In some preferred embodiments, the co-solvent includes at least one of propylene glycol and Tween 20.

[0052] In some preferred embodiments, the following components are included by mass percentage: 2.0%-2.8% pyrrolidinodiaminopyrimidine oxide, 2.0%-5.0% glycyrrhizic acid, 0.2%-0.5% acetyl tetrapeptide-2, 10.1%-25.3% propylene glycol, 5.2%-9.3% Tween 20 and the balance solvent.

[0053] Based on the total mass of the pyrrolidinopyrimidine oxide supramolecular nanovesicles as 100%, the added amount of the pyrrolidinopyrimidine oxide is 2.0%-2.8%, for example, 2.0%, 2.4%, 2.8%, etc.; Based on the total mass of the pyrrolidinopyrimidine oxide supramolecular nanovesicles as 100%, the added amount of the glycyrrhizic acid is 2.0%-5.0%, for example, 2.0%, 3.5%, 5.0%, etc.; Based on the total mass of the pyrrolidinopyrimidine oxide supramolecular nanovesicles as 100%, the added amount of the acetyl tetrapeptide-2 is 0.2%-0.5%, for example, 0.2%, 0.3%, 0.4%, 0.5%, etc.; Based on the total mass of the pyrrolidinopyrimidine oxide supramolecular nanovesicles as 100%, the added amount of propylene glycol is 10.1%-25.3%, for example, 10.1%, 15%, 20%, 25%, 25.3%, etc.; Based on the total mass of the pyrrolidinopyrimidine oxide supramolecular nanovesicles as 100%, the added amount of Tween 20 is 5.2%-9.3%, for example, 5.2%, 6%, 7%, 8%, 9.3%, etc.

[0054] Preferably, the pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles include the following components by mass percentage: 2.36% pyrrolidinodiaminopyrimidine oxide, 4.87% glycyrrhizic acid, 0.49% acetyl tetrapeptide-2, 19.68% propylene glycol, 9.13% Tween 20 and the balance solvent.

[0055] In the present invention, glycyrrhizic acid is used as one of the raw materials. Glycyrrhizic acid is one of the main active ingredients of the Chinese herbal medicine Licorice, mainly composed of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid. Due to its polymerization behavior and the ability to form micelles due to its amphiphilic structure, glycyrrhizic acid is used as an absorption enhancer and delivery carrier to improve the transdermal absorption of the active ingredient. Glycyrrhizic acid can inhibit 5α-reductase and reduce the excessive oil secretion of the scalp caused by testosterone, thereby helping to control hair loss. Acetyl Tetrapeptide-2 is an anti-hair loss follicle-targeted penetrating peptide that can penetrate the skin barrier and directly act on the hair follicles. It has the effects of promoting hair growth and preventing hair loss. Acetyl Tetrapeptide-2 can prevent hair loss. Its main mechanism of action is mainly in the following four aspects: (1) Stimulating hair follicle growth: Acetyl Tetrapeptide-2 can promote the normal growth cycle of hair follicles and reduce the chance of hair follicles entering the resting phase, thereby contributing to the healthy growth of hair; (2) Inhibiting 5α-reductase activity: This peptide can reduce the activity of 5α-reductase and reduce the production of dihydrotestosterone (DHT). DHT is a major factor in androgenic alopecia, so inhibiting its production can effectively slow the process of hair loss. (3) Antioxidant protection: Acetyl tetrapeptide-2 also has antioxidant properties, which can neutralize free radicals and protect hair follicles from oxidative stress damage. (4) Regulating immune response: In some cases, abnormal reactions of the immune system can also cause hair loss. Acetyl tetrapeptide-2 helps regulate these abnormal immune activities and reduce the adverse effects on hair follicles.

[0056] This invention utilizes supramolecular technology, which can significantly improve hair follicle targeting. This technology can recombine different molecules into new supramolecular structures that not only retain the functions of the original molecules but also enable the molecules within the new structure to synergize with each other, thereby enhancing efficacy and properties. This technology can improve the product's penetration efficiency and absorption rate, reduce the loss of active ingredients, and enhance the stability and mildness of the ingredients.

[0057] In the present invention, glycyrrhizic acid and acetyl tetrapeptide-2 form a supramolecule through intermolecular interactions such as hydrogen bonds and van der Waals forces. The resulting supramolecule may have new anti-hair loss biological activity or physicochemical properties, providing new materials or candidate molecules for the field of cosmetic anti-hair loss.

[0058] The fourth aspect of the present invention provides a method for preparing the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles, comprising mixing a formulated amount of pyrrolidino diamino pyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, a cosolvent and a solvent to obtain the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles.

[0059] In some preferred embodiments, the method comprises the following steps: (a) dissolving a formulated amount of pyrrolidinodiaminopyrimidine oxide and a cosolvent in a solvent to obtain a solution a; (b) dissolving the formulated amounts of glycyrrhizic acid and acetyl tetrapeptide-2 in a solvent to obtain a glycyrrhizic acid solution and an acetyl tetrapeptide-2 solution, respectively, and then adding the glycyrrhizic acid solution to the acetyl tetrapeptide-2 solution and mixing them to obtain a solution b; (c) Mixing solution a and solution b in step 1 and stirring at 60-85°C for 10-60 minutes to form a transparent liquid; (d) After a transparent liquid is formed, the temperature is lowered and stirring is continued for 6-8 h; (e) Filtration to obtain glycyrrhizic acid and acetyl tetrapeptide-2-encapsulated pyrrolidinopyrimidine oxide supramolecular nanovesicles.

[0060] Specifically, the preparation method of the pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles comprises the following steps: (1) Accurately weigh the formulated amount of pyrrolidinopyrimidine oxide, propylene glycol, Tween 20, and deionized water, place them in a beaker, and stir evenly; (2) Weigh the formulated amount of glycyrrhizic acid and dissolve it in a certain amount of deionized water to obtain a glycyrrhizic acid solution; weigh the formulated amount of acetyl tetrapeptide-2 and add a certain amount of deionized water to fully dissolve it to obtain an acetyl tetrapeptide-2 solution; slowly add the glycyrrhizic acid solution dropwise to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6 to 8 hours, and then slowly add it to the mixed solution in step (1) while stirring; (3) Mix the solutions in step (1) and step (2), place at 80°C, and continue stirring for 30 minutes to form a transparent liquid; (4) After a transparent liquid is formed, cool the mixture to 25°C and continue stirring for 6–8 h. (5) The pyrrolidinopyrimidine oxide supramolecular nanovesicles coated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration using a 0.22 µm microporous filter membrane.

[0061] The present invention provides a method for preparing supramolecular nanovesicles of pyrrolidinodiaminopyrimidine oxide, which features a simple preparation process, accurate dosage, and suitability for large-scale production. This preparation process meets the requirements of pyrrolidinodiaminopyrimidine oxide as a novel anti-shedding raw material, offering not only mildness and safety, but also excellent water solubility and no side effects. Furthermore, it lays a foundation for the research and development of nanoformulations of traditional Chinese medicine.

[0062] The fifth aspect of the present invention provides a use of the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles or the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles prepared by the preparation method in the preparation of anti-hair loss care products.

[0063] The present invention provides the use of supramolecular nanovesicles of pyrrolidinopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2 in anti-hair loss shampoo and conditioner products. The supramolecular nanovesicles of pyrrolidinopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2 can be directly applied to various anti-hair loss shampoo and conditioner products, such as anti-hair loss shampoo, anti-hair loss shampoo, anti-hair loss cream, and anti-hair loss essence. These products, through further scientific formulation, can maximize the anti-hair loss effect of pyrrolidinopyrimidine oxide.

[0064] The present invention has the following beneficial effects: 1. The glycyrrhizic acid and acetyl tetrapeptide-2-encapsulated pyrrolidinopyrimidine oxide supramolecular nanovesicles of the present invention are light yellow transparent liquids with good physiological compatibility and safety. The particle size is 2 to 20 nm. They can carry the active molecule pyrrolidinopyrimidine oxide and efficiently penetrate into hair follicle cells. They can also ensure that pyrrolidinopyrimidine oxide has a long retention time in hair follicle cells, achieving a highly effective anti-hair loss effect.

[0065] 2. Using traditional Chinese medicine nano-assembly technology (using traditional Chinese medicine nano-supramolecular self-assembly technology) to give full play to the characteristics and advantages of "drug-excipient combination", acetyl tetrapeptide-2 and glycyrrhizic acid can act as reservoirs as carriers, continuously releasing the anti-hair loss functional factor pyrrolidinopyrimidine oxide, so that it can maintain an effective concentration for a long time, synergistically enhance its effect, and play a highly effective anti-hair loss role.

[0066] 3. The pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles of the present invention can inhibit the activity of 5α-reductase to varying degrees at low, medium and high concentrations. After being encapsulated by acetyl tetrapeptide-2 and glycyrrhizic acid, even a low concentration (0.02%) of pyrrolidinodiaminopyrimidine oxide can achieve an inhibition rate of 40.12% on 5α-reductase.

[0067] 4. Experiments have confirmed that the pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles of the present invention have a significantly better inhibitory effect on 5α-reductase activity than comparative examples 1-2.

[0068] 5. The experiments confirmed that the pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles of the present invention meet the requirements in terms of appearance, high temperature and high humidity environment, strong direct light environment, and freezing environment.

[0069] 6. The pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles of the present invention are highly safe. No toxic organic solvents are used in the preparation method, no organic solvent residues are left, and the irritation to the skin is small, so the safety is high.

[0070] 7. The preparation process of the present invention is simple and easy to control, and is suitable for industrial and large-scale production.

[0071] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0072] In the embodiments of the present invention, the solvent is deionized water.

[0073] Example 1 Example 1 provides a supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2. Several formulas are provided in Example 1, the purpose of which is to explore the ratio of acetyl tetrapeptide-2 and glycyrrhizic acid in the supramolecular nanovesicle system.

[0074] The formula is shown in Table 1: Table 1. Ratio of acetyl tetrapeptide-2 and glycyrrhizic acid in supramolecular nanovesicle system

[0075] Preparation method: The corresponding raw materials are respectively taken according to the component ratios in the above-mentioned formulas 1-6 to prepare the supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2. The specific preparation process includes: (1) Accurately weigh the formulated amount of acetyl tetrapeptide-2 and dissolve it in a certain amount of deionized water; (2) Weigh the formulated amount of glycyrrhizic acid and add a certain amount of deionized water to fully dissolve it; (3) slowly adding the solution (glycyrrhizic acid solution) in step (2) dropwise to the solution (acetyl tetrapeptide-2 solution) in step (1) while stirring; (4) Continue stirring for 6-8 h to obtain supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 ( Figure 7 The particle sizes of the vesicles prepared in each formulation in Example 1 were all within the range of 2-20 nm.

[0076] The particle size, Zeta potential and polydispersity index (PDI) were measured using a nanoparticle size analyzer. The results are shown in Table 2.

[0077] When the dosage of glycyrrhizic acid is 15 parts and acetyl tetrapeptide-2 is 1.5 parts, that is, the ratio of glycyrrhizic acid to acetyl tetrapeptide-2 is 10:1 (the corresponding product picture is Figure 7 ), at this time, the supramolecular nanovesicles had a small particle size of 5.46 nm, a small Zeta potential and a small PDI, with a Zeta potential of -4.12 mV and a PDI of 0.239. Therefore, the ratio of glycyrrhizic acid to acetyl tetrapeptide-2 was finally selected as 10:1 for subsequent experiments.

[0078] Table 2. Effect of the ratio of acetyl tetrapeptide-2 to glycyrrhizic acid on the particle size, zeta potential, polydispersity index (PDI), and encapsulation efficiency of supramolecular nanovesicles

[0079] Example 2 Example 2 provides a pyrrolidino diaminopyrimidine oxide supramolecular nanovesicle. Example 2 provides several formulas for the purpose of exploring the dosage of acetyl tetrapeptide-2 and glycyrrhizic acid in the supramolecular nanovesicle system.

[0080] The formula is shown in Table 3: Table 3. Table of the proportions of acetyl tetrapeptide-2 and glycyrrhizic acid in the supramolecular nanovesicle system

[0081] Preparation method: The corresponding raw materials are respectively taken according to the component ratios in the above formulas 7-12 to prepare the pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles. The specific preparation process includes: (1) Accurately weigh the formulated amount of pyrrolidinopyrimidine oxide, propylene glycol, Tween 20, and deionized water, place them in a beaker, and stir evenly; (2) Weigh the formulated amount of acetyl tetrapeptide-2 and dissolve it in a certain amount of deionized water to obtain an acetyl tetrapeptide-2 solution; weigh the formulated amount of glycyrrhizic acid and add a certain amount of deionized water to fully dissolve it to obtain a glycyrrhizic acid solution; slowly add the glycyrrhizic acid solution dropwise to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6 to 8 hours, and then slowly add it to the mixed solution in step (1) while stirring; (3) Mix the solutions in step (1) and step (2), place at 80°C, and continue stirring for 30 minutes to form a transparent liquid; (4) After a transparent liquid is formed, cool the mixture to 25°C and continue stirring for 6–8 h. (5) The pyrrolidinopyrimidine oxide supramolecular nanovesicles coated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration using a 0.22 µm microporous filter membrane.

[0082] The encapsulation efficiency was calculated using high-performance liquid chromatography, and the particle size, zeta potential, and polydispersity index (PDI) were determined using a nanoparticle size analyzer. The results are shown in Table 4.

[0083] When the dosage of acetyl tetrapeptide-2 was 0.2 parts and the dosage of glycyrrhizic acid was 2 parts, the encapsulation efficiency was the highest at 86.39%, the particle size was small at 8.16 nm, the Zeta potential and PDI were small, the Zeta potential was -3.78 mV, and the PDI was 0.264. Therefore, 0.2 parts of acetyl tetrapeptide-2 and 2 parts of glycyrrhizic acid were finally selected for subsequent experiments.

[0084] Table 4. Effects of acetyl tetrapeptide-2 and glycyrrhizic acid dosage on the particle size, zeta potential, polydispersity index (PDI), and encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles

[0085] Example 3 Example 3 provides a pyrrolidino diaminopyrimidine oxide supramolecular nanovesicle. Example 3 provides several formulas for the purpose of exploring the temperature when the supramolecular nanovesicle system is stirred and mixed.

[0086] The formula is shown in Table 5: Table 5. Temperature parameters for stirring and mixing of supramolecular nanovesicle system

[0087] Preparation method: The corresponding raw materials are respectively taken according to the component ratios in the above formulas 13-18 to prepare the pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles. The specific preparation process includes: (1) Accurately weigh the formulated amount of pyrrolidinopyrimidine oxide, propylene glycol, Tween 20, and deionized water, place them in a beaker, and stir evenly; (2) Weigh the formulated amount of acetyl tetrapeptide-2 and dissolve it in a certain amount of deionized water to obtain an acetyl tetrapeptide-2 solution; weigh the formulated amount of glycyrrhizic acid and add a certain amount of deionized water to fully dissolve it to obtain a glycyrrhizic acid solution; slowly add the glycyrrhizic acid solution dropwise to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6 to 8 hours, and then slowly add it to the mixed solution in step (1) while stirring; (3) Mix the solutions in step (1) and step (2), place them under different reaction temperature conditions after thorough mixing, and continue stirring for 30 minutes to form a transparent liquid; (4) After a transparent liquid is formed, cool the mixture to 25°C and continue stirring for 6–8 h. (5) The pyrrolidinopyrimidine oxide supramolecular nanovesicles coated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration using a 0.22 µm microporous filter membrane.

[0088] The encapsulation efficiency was calculated using high-performance liquid chromatography, and the particle size, zeta potential, and polydispersity index (PDI) were measured using a nanoparticle size analyzer. The results are shown in Table 6.

[0089] When the reaction temperature is 80℃, the Zeta potential is as low as -3.59 mV, and the encapsulation efficiency is the highest at 89.23%. The particle size is small and the PDI is also the smallest, with a particle size of 6.36 nm and a PDI of 0.257. Therefore, when the reaction temperature is 80℃, the glycyrrhizic acid-pyrrolidinopyrimidine oxide nanovesicles are relatively stable.

[0090] Table 6. Effect of reaction temperature on the particle size, zeta potential, polydispersity index (PDI), and encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles

[0091] Example 4 Example 4 provides a pyrrolidino diaminopyrimidine oxide supramolecular nanovesicle. Example 4 provides several formulas for the purpose of exploring the reaction time in the supramolecular nanovesicle system.

[0092] The formula is shown in Table 7.

[0093] Table 7. Reaction time parameters under supramolecular nanovesicle system

[0094] Preparation method: The corresponding raw materials are respectively taken according to the component ratios in the above formulas 19-24 to prepare the pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles. The specific preparation process includes: (1) Accurately weigh the formulated amount of pyrrolidinopyrimidine oxide, propylene glycol, Tween 20, and deionized water, place them in a beaker, and stir evenly; (2) Weigh the formulated amount of acetyl tetrapeptide-2 and dissolve it in a certain amount of deionized water to obtain an acetyl tetrapeptide-2 solution; weigh the formulated amount of glycyrrhizic acid and add a certain amount of deionized water to fully dissolve it to obtain a glycyrrhizic acid solution; slowly add the glycyrrhizic acid solution dropwise to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6 to 8 hours, and then slowly add it to the mixed solution in step (1) while stirring; (3) The solutions in step (1) and step (2) were mixed, placed at 80°C after thorough mixing, and continued to stir for different reaction times to form a transparent liquid; (4) After a transparent liquid is formed, cool the mixture to 25°C and continue stirring for 6–8 h. (5) The pyrrolidinopyrimidine oxide supramolecular nanovesicles coated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration using a 0.22 µm microporous filter membrane.

[0095] The encapsulation efficiency was calculated using high-performance liquid chromatography, and the particle size, zeta potential, and polydispersity index (PDI) were measured using a nanoparticle size analyzer. The results are shown in Table 8.

[0096] When the reaction time was 30 min, the encapsulation efficiency of the nanovesicles was the highest, reaching 84.32%, the PDI was the lowest, reaching 0.261, and the particle size and Zeta potential were also smaller, with a particle size of 6.16 nm and a Zeta potential of -4.38 mV.

[0097] Table 8. Effect of reaction time on the particle size, zeta potential, polydispersity index (PDI), and encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles

[0098] Example 5 According to the results of Examples 2 to 4, the selected factors for investigation were glycyrrhizic acid dosage (X1), reaction temperature (X2) and reaction time (X3), the encapsulation efficiency (Y) was used as the investigation index, the dosage of pyrrolidinopyrimidine oxide was fixed at 1 part, the range of each investigation factor was determined, and the Box-Benhken design-response surface methodology was used to optimize the preparation process of pyrrolidinopyrimidine oxide supramolecular nanovesicles (Table 9).

[0099] Table 9. Box-Behnken response surface factor and level design table

[0100] Preparation method: (1) Accurately weigh the formulated amount of pyrrolidinopyrimidine oxide, propylene glycol, Tween 20, and deionized water, place them in a beaker, and stir evenly; (2) Weigh the formulated amount of acetyl tetrapeptide-2 and dissolve it in a certain amount of deionized water to obtain an acetyl tetrapeptide-2 solution; weigh the formulated amount of glycyrrhizic acid and add a certain amount of deionized water to fully dissolve it to obtain a glycyrrhizic acid solution; slowly add the glycyrrhizic acid solution dropwise to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6 to 8 hours, and then slowly add it to the mixed solution in step (1) while stirring; (3) mixing the solutions in step (1) and step (2), placing them under different reaction temperature conditions after thorough mixing, and continuing to stir for different reaction times to form a transparent liquid; (4) After a transparent liquid is formed, cool the mixture to 25°C and continue stirring for 6–8 h. (5) The pyrrolidinopyrimidine oxide supramolecular nanovesicles coated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration using a 0.22 µm microporous filter membrane.

[0101] The encapsulation efficiency was calculated using high-performance liquid chromatography, and the particle size, zeta potential, and polydispersity index (PDI) were determined using a nanoparticle size analyzer. The factors investigated, experimental design, and results are shown in Table 10, and the analysis of variance is shown in Table 11.

[0102] Table 10. Box-Benhken response surface method experimental design and results

[0103] Table 11. Results of variance analysis

[0104] The encapsulation efficiency data in Table 10 were input into Design-Expert 8.0.6.1 software to obtain the fitting equation Y =86.00-0.28 X 1+2.94 X 2+0.10 X 3+0.88 X 1 X 2-0.98 X 1 X 3+4.51 X 2 X 3-4.06 X 1 2 -8.71 X 2 2 -7.00 X 3 2 ( r =0.9892, P < 0.05). Through the significance test, we can see that X2, X2X3, X 1 2 、 X 2 2 、 X 3 2 right Y The value has a significant impact ( P < 0.05), the lack-of-fit term was not significant ( P ≥ 0.05), indicating that the regression model has good predictive power and the regression equation has a good fit in the entire regression region. The order of influence of each factor on the encapsulation efficiency is X 2> X3>X1, that is, reaction temperature>reaction time>glycyrrhizic acid dosage. Design-Expert 8.0.6.1 software was used to draw the effect surface diagram and contour map between each factor. The results are shown in Figures 1-6 According to the analysis results, the predicted optimal process conditions for the preparation of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles were obtained by Design-Expert 8.6.0.1 as follows: glycyrrhizic acid dosage was 1.99 parts, reaction temperature was 80.93℃, reaction time was 31.36 min, and encapsulation efficiency was 86.28%.

[0105] According to the prescription and process conditions, three batches of pyrrolidinodiaminopyrimidine oxide nanovesicles were prepared (the optimal component ratio of Example 5: the components, by mass percentage, include: 2.36% pyrrolidinodiaminopyrimidine oxide, 4.87% glycyrrhizic acid, 0.49% acetyl tetrapeptide-2, 19.68% propylene glycol, 9.13% Tween 20 and the remaining solvent, and the preparation method in Example 5 was used for preparation, wherein the reaction temperature was 80.93°C and the reaction time was 31.36 min). The encapsulation efficiencies of the three batches of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles were 86.71%, 86.47%, and 86.34%, respectively. The test results were compared with the predicted values ​​of the fitting equation, and the relative deviation was calculated according to the formula [relative deviation = (predicted value - measured value) / predicted value]. The results showed that the relative deviation between the measured value and the predicted value was less than 5%, indicating that the preferred preparation process is stable and reliable. In addition, the pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles are stable in an aqueous environment and are light yellow and transparent ( Figure 8 ), particle size distribution as Figure 9 As shown, the average diameter is (6.74 ± 0.18) nm, the zeta potential is (-3.26 ± 1.41) mV, and the PDI is (0.255 ± 0.007).

[0106] Comparative Example 1 This comparative example provides a pyrrolidino diaminopyrimidine oxide solution, the components and the mass percentages of the components are: 2.36% pyrrolidinopyrimidine oxide, 19.68% propylene glycol, 9.13% Tween 20 and the balance deionized water.

[0107] The preparation was carried out using the formula of the comparative example 1, and the preparation method included the following steps: (1) Accurately weigh the formulated amount of pyrrolidinopyrimidine oxide, propylene glycol, Tween 20, and deionized water, place them in a beaker, and stir evenly; (2) After thorough mixing, place at 80°C and continue stirring for 30 min to form a transparent liquid; (3) After a transparent liquid is formed, cool the mixture to 25°C and continue stirring for 6–8 h. (4) Filter through a 0.22 µm microporous filter membrane to obtain a pyrrolidinodiaminopyrimidine oxide solution.

[0108] Comparative Example 2 This comparative example provides a pyrrolidinodiaminopyrimidine oxide nanovesicle (non-targeted), the components and the mass percentages of the components are as follows: 2.36% pyrrolidinopyrimidine oxide, 4.87% glycyrrhizic acid, 19.68% propylene glycol, 9.13% Tween 20 and the balance deionized water.

[0109] The above-mentioned comparative example 2 formulation was used for preparation, and the preparation method included the following steps: (1) Accurately weigh the formulated amount of pyrrolidinopyrimidine oxide, propylene glycol, Tween 20, and deionized water, place them in a beaker, and stir evenly; (2) Weigh the formulated amount of glycyrrhizic acid and slowly add it to the mixed solution in step (1) while stirring; (3) Mix the solutions in step (1) and step (2), place at 80°C after thorough mixing, and continue stirring for 30 minutes to form a transparent liquid; (4) After a transparent liquid is formed, cool the mixture to 25°C and continue stirring for 6–8 h. (5) Filter through a 0.22 µm microporous filter membrane to obtain pyrrolidinodiaminopyrimidine oxide nanovesicles (non-targeted).

[0110] Test example Experimental Example 1 Stability Experiment of Pyrrolidine Diaminopyrimidine Oxide Supramolecular Nanovesicles The pyrrolidinopyrimidine oxide supramolecular nanovesicles prepared by the optimal preparation process in Example 5 (the optimal components of Example 5 include: 2.36% pyrrolidinopyrimidine oxide, 4.87% glycyrrhizic acid, 0.49% acetyl tetrapeptide-2, 19.68% propylene glycol, 9.13% Tween 20, and the remainder solvent) were stored under high temperature and humidity, direct exposure to strong light, and repeated freeze-thaw conditions in a frozen environment. The samples were taken out after 1, 2, 4, 8, 14, and 21 days, and their appearance was observed. The particle size, zeta potential, PDI, and encapsulation efficiency of the pyrrolidinopyrimidine oxide supramolecular nanovesicles were also measured.

[0111] The results of stability investigation showed that the average particle size, Zeta potential, PDI and encapsulation efficiency of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles did not change much after being placed under high temperature and humidity, strong direct light, and frozen environment with repeated freeze-thaw cycles for 21 days (Tables 12-14), indicating that the pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles had good stability after being placed under high temperature and humidity, strong direct light, and frozen environment with repeated freeze-thaw cycles for 21 days.

[0112] Table 12. Stability of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles under high temperature and high humidity conditions

[0113] Table 13. Stability of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles under strong light conditions

[0114] Table 14. Stability of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles under freezing conditions

[0115] Experimental Example 2: 5α-reductase inhibitory activity test of pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles 1. Test sample stimulates standard enzyme (1) Prepare standardized enzyme, dilute to a concentration of 80 U / L, and spread on a cell culture plate with a volume of 100 μL added to each well.

[0116] (2) Prepare samples (Example 5 and Comparative Examples 1-2). Based on the preliminary experiment, the test sample concentration is configured to stimulate the standard enzyme. The blank control is stimulated with sterile enzyme-free water at a stimulation concentration of 100 μL. The time is 6 hours. Then, the supernatant is transferred to an EP tube and centrifuged to obtain the supernatant for testing.

[0117] (3) Use a kit to detect the amount of enzyme.

[0118] 2. Kit detection (1) Sample addition: Accurately add 50 μL of the standard and sample to the ELISA coated plate. Add the sample to the bottom of the ELISA plate well, trying not to touch the well wall, and gently shake to mix.

[0119] (2) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min.

[0120] (3) Liquid preparation: Dilute the 30-fold concentrated washing solution with 30-fold distilled water and set aside.

[0121] (4) Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds and then discard it. Repeat this 5 times and pat dry.

[0122] (5) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0123] (6) Incubation: The operation is the same as (2).

[0124] (7) Washing: The operation is the same as (4).

[0125] (8) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and incubate at 37°C in the dark for 15 minutes.

[0126] (9) Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0127] (10) Measurement: Set the blank to zero and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0128] 3. 5α-reductase inhibition rate The calculation formula of the enzyme activity of the sample to be tested is as follows:

[0129] The 5α-reductase inhibition rates obtained in Example 5 (product prepared using the optimal components of Example 5) and Comparative Examples 1-2 are shown in Tables 15 and Figure 10 As shown, after being encapsulated by acetyl tetrapeptide-2 and glycyrrhizic acid, the activity of pyrrolidino diamino pyrimidine oxide in inhibiting 5α-reductase was greatly improved. At low concentrations, the inhibition rate of pyrrolidino diamino pyrimidine oxide solution (Comparative Example 1) was 11.57%, the inhibition rate of glycyrrhizic acid-pyrrolidino diamino pyrimidine oxide nanovesicles (Comparative Example 2) was 4.47%, and the inhibition rate of pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles encapsulated by acetyl tetrapeptide-2 and glycyrrhizic acid (Example 5) was 40.12%, and the anti-hair loss and oil control efficacy was significantly improved. The results also show that the inhibitory effect of pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles encapsulated by acetyl tetrapeptide-2 and glycyrrhizic acid on 5α-reductase is not a simple "1+1=2" effect after mixing, but the result of synergistic enhancement of "drug-adjuvant combination".

[0130] Table 15. 5α-reductase inhibition rate of Example 5 and Comparative Examples 1-2

[0131] Application Examples Application Example 1 This application example provides an anti-hair loss essence containing pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles. The components of the anti-hair loss essence are shown in Table 16 below: Table 16. Anti-hair loss essence containing pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles

[0132] The preparation method is prepared using the formula in Application Example 1, and the preparation method includes the following steps: (1) Add the water in phase A1, disperse the remaining raw materials in sequence and add them under stirring, heat to 80-85°C, stir and keep the temperature constant for 15 minutes, homogenize and disperse evenly, and start stirring to cool down; (2) Cool down to 70-75°C, add the pre-dissolved A2 phase raw materials, and continue stirring and cooling; (3) Cool down to 40-45°C, add the pre-dissolved phase B raw materials, stir and mix evenly, then add the phase C raw materials and stir until completely dissolved; (4) Mix the raw materials of phase D in advance and add them. After stirring evenly, add the raw materials of phase E in sequence and stir until transparent.

[0133] Application Example 2 This application example provides an anti-hair loss essence containing pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles. The components of the anti-hair loss essence are shown in Table 17 below.

[0134] Table 17. Anti-hair loss essence containing glycyrrhizic acid-pyrrolidinopyrimidine oxide nanovesicles

[0135] The preparation was carried out using the formula in Application Example 2, and the preparation method was the same as that in Application Example 1.

[0136] Application Example 3 This application example provides an anti-hair loss essence containing glycyrrhizic acid-pyrrolidinopyrimidine oxide nanovesicles. The components of the anti-hair loss essence are shown in Table 18 below.

[0137] Table 18. Anti-hair loss essence containing pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles

[0138] The preparation method is prepared using the formula in Application Example 3, and the preparation method includes the following steps: (1) Add the water in phase A1, disperse the remaining raw materials in sequence and add them under stirring, heat to 80-85°C, stir and keep the temperature constant for 15 minutes, homogenize and disperse evenly, and start stirring to cool down; (2) Cool down to 70-75°C, add the pre-dissolved A2 phase raw materials, and continue stirring and cooling; (3) Cool down to 40-45°C, add the pre-dissolved phase B raw materials, stir and mix evenly, then add the phase C raw materials and stir until completely dissolved; (4) Mix the raw materials of phase D in advance and add them. After stirring evenly, add the raw materials of phase E in sequence and stir until transparent.

[0139] Test Example 3 Test of 5α-reductase inhibitory activity of an anti-hair loss essence containing pyrrolidinopyrimidine oxide supramolecular nanovesicles.

[0140] The samples are Application Examples 1-3, and the remaining steps are the same as Test Example 2. The results are shown in Table 19 below.

[0141] Table 19. 5α-reductase inhibition rate of anti-hair loss essence in application examples 1-3

[0142] According to the results analysis in Table 19: From the results of application examples 1-3, the gel prepared by containing pyrrolidinopyrimidine oxide supramolecular nanovesicles has significant anti-hair loss and oil control effects. In application example 1, when the mass fraction of pyrrolidinopyrimidine oxide nanovesicles is 3 wt%, the anti-hair loss and oil control effects are already very obvious, and with the increase of the addition ratio of pyrrolidinopyrimidine oxide supramolecular nanovesicles, the 5α-reductase inhibition rate of the anti-hair loss essence also increases accordingly, indicating that pyrrolidinopyrimidine oxide supramolecular nanovesicles can effectively improve the anti-hair loss and oil control effects of the essence.

[0143] Test Example 4: Subjective Human Perception Experiment Subjects were not allowed to wash their hair within 48 h ± 5 h before each test, and were not allowed to comb their hair on the day of the test. After arriving at the test site and acclimatizing to the environment, the same trained technician used a comb of the same specifications to comb their hair evenly 60 times (30 times on each side) in the order from left front to left back and right front to right back. The fallen hair was then collected and counted.

[0144] Thirty healthy subjects aged 18 to 60 years with severe hair loss and mild thinning, with hair length between 5 and 40 cm, and who had lost more than 10 hair strands using the 60-comb technique, were selected. The subjects were divided into three groups of 10 each. Each group was assigned to use the hair care products described in Examples 1-3, once in the morning and once in the evening. Each application followed the instructions, followed by rinsing and scalp massage for 1-3 minutes. The subjects performed the 60-comb technique before and 4, 8, and 12 weeks after use. The number of hair strands lost was recorded. The weekly hair loss reduction percentage was calculated using the formula: (number of hair strands lost before use - number of hair strands lost after 12 weeks) / number of hair strands lost before use * 100% (Table 20).

[0145] Table 20. 5α-reductase inhibition rate of anti-hair loss essence in application examples 1-3

[0146] The results show that the essence containing pyrrolidino diaminopyrimidine oxide supramolecular nanovesicles prepared by the process defined in the present invention can effectively strengthen hair and prevent hair loss.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2, characterized in that: The invention comprises the following components by mass percentage: 10.0%-20.0% of glycyrrhizic acid, 1.0%-2.0% of acetyl tetrapeptide-2 and the balance of solvent.

2. The supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 according to claim 1, characterized in that The supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 include the following components by mass percentage: 15.0% glycyrrhizic acid, 1.5% acetyl tetrapeptide-2 and the balance solvent.

3. The supramolecular nanovesicles self-assembled from glycyrrhizic acid and acetyl tetrapeptide-2 according to claim 2, characterized in that: The particle size of the supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 is 2-20 nm.

4. The method for preparing supramolecular nanovesicles formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 according to any one of claims 1 to 3, characterized in that: The following steps are involved: The glycyrrhizic acid, acetyl tetrapeptide-2 and solvent in the formulated amounts are mixed to obtain supramolecular nanovesicles formed by self-assembly of the glycyrrhizic acid and acetyl tetrapeptide-2.

5. A pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicle, characterized in that: The invention comprises a pyrrolidinodiaminopyrimidine oxide, and a supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 as claimed in any one of claims 1 to 3, or a supramolecular nanovesicle formed by self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 prepared by the preparation method according to claim 4, which is wrapped outside the pyrrolidinodiaminopyrimidine oxide.

6. The pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicle according to claim 5, characterized in that The pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles include the following components: Pyrrolidinodiaminopyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, cosolvent and solvent; Preferably, the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles comprise the following components by mass percentage: 2.0%-2.8% pyrrolidino diamino pyrimidine oxide, 2.0%-5.0% glycyrrhizic acid, 0.2%-0.5% acetyl tetrapeptide-2, 10.1%-25.3% propylene glycol, 5.2%-9.3% Tween 20 and the balance solvent; Preferably, the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles comprise the following components by mass percentage: 2.36% pyrrolidino diamino pyrimidine oxide, 4.87% glycyrrhizic acid, 0.49% acetyl tetrapeptide-2, 19.68% propylene glycol, 9.13% Tween 20 and the balance solvent.

7. The pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicle according to claim 6, characterized in that The cosolvent includes at least one of propylene glycol and Tween 20.

8. The method for preparing pyrrolidinodiaminopyrimidine oxide supramolecular nanovesicles according to any one of claims 5 to 7, characterized in that: The pyrrolidinyl diaminopyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, a cosolvent and a solvent in a prescribed amount are mixed to obtain the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle.

9. The preparation method according to claim 8, characterized in that The following steps are involved: (a) dissolving a formulated amount of pyrrolidinodiaminopyrimidine oxide and a cosolvent in a solvent to obtain a solution a; (b) dissolving the formulated amounts of glycyrrhizic acid and acetyl tetrapeptide-2 in a solvent to obtain a glycyrrhizic acid solution and an acetyl tetrapeptide-2 solution, respectively, and then mixing the glycyrrhizic acid solution and the acetyl tetrapeptide-2 solution to obtain a solution b; (c) Mixing solution a and solution b in step 1 and stirring at 60-85°C for 10-60 minutes to form a transparent liquid; (d) After a transparent liquid is formed, the temperature is lowered and stirring is continued for 6-8 h; (e) Filtration to obtain glycyrrhizic acid and acetyl tetrapeptide-2-encapsulated pyrrolidinopyrimidine oxide supramolecular nanovesicles.

10. Use of the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles according to any one of claims 5 to 7 or the pyrrolidino diamino pyrimidine oxide supramolecular nanovesicles prepared by the preparation method according to claim 8 or 9 in the preparation of anti-hair loss care products.

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