Pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles, preparation method and application thereof

Abstract

The application provides a pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle and a preparation method and application thereof, relates to the technical field of cosmetics, and comprises the following components: pyrrolidinyl diaminopyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, a cosolvent and a solvent. The prepared pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle can significantly improve the solubility and hair follicle targeting property of pyrrolidinyl diaminopyrimidine oxide, has high encapsulation efficiency, large drug loading, good stability and high safety, can effectively promote the precise action of the anti-hair loss active factor pyrrolidinyl diaminopyrimidine oxide on hair follicle cells, and realizes a high-efficiency anti-hair loss effect. The pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle provided by the application has a simple preparation process, accurate dosage and is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle, its preparation method, and its application. Background Technology

[0002] Hair loss is a common and frequently occurring skin disease characterized by abnormal and excessive hair loss and thinning of hair, with androgenetic alopecia and alopecia areata being the most common. Modern medicine believes that various pathogenic factors, including genetics, autoimmunity, psychological factors, endocrine disorders, cytokines, and trace elements, induce hair loss by abnormally regulating the hair growth cycle, leading to hair follicle miniaturization. Hair loss treatment and hair follicle regeneration remain challenges that the medical and scientific communities urgently need to overcome.

[0003] With the development of medical technology, in addition to traditional plant extracts, more and more high-tech chemical ingredients are being developed to more effectively address hair loss. Pyrrolidinyl diaminopyrimidine oxide, as an advanced chemical ingredient, shows results faster than traditional plant-based ingredients, making it ideal for use in anti-hair loss products and demonstrating enormous market potential and application prospects.

[0004] Pyrrolidinyl diaminopyrimidine oxide (also known as "Kopyrrol") is a novel hair growth promoter with a structure similar to minoxidil, a first-line anti-hair loss drug. Its molecular formula is C8H. 13 N5O, with a molecular weight of 195.22, is a pyrrolyl diaminopyrimidine oxide with 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 to increase nutrients and oxygen to the hair roots. It can also accelerate new hair growth by optimizing the hair follicle growth cycle. Experimental studies have found that pyrrolyl diaminopyrimidine oxide also reduces serum 5α-reductase, androgen receptor, and dihydrotestosterone levels in mice, significantly improving pathological changes in hair follicles on the back of mice, exhibiting a dose-response relationship and demonstrating good anti-hair loss effects. The substituted diaminopyrimidine oxide, through salt formation, yields an active compound with improved solubility, increased transdermal absorption, and a longer duration of action. It also exhibits lower toxicity than minoxidil, allowing for higher concentrations without undesirable side effects. Based on these advantages, its good water solubility and targeted nature to hair follicles are prerequisites for the wider application of pyrrolyl diaminopyrimidine oxide in water-based cosmetics.

[0005] Glycyrrhizic acid is one of the main active ingredients in the traditional Chinese medicine licorice. It is primarily composed of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid. Due to its polymerization behavior and its amphiphilic structure, which allows it to form micelles, glycyrrhizic acid is used as an absorption enhancer and delivery carrier to improve the transdermal absorption of the active ingredients. Glycyrrhizic acid has shown potential in preventing hair loss. According to relevant studies, glycyrrhizic acid can reduce excessive sebum secretion caused by testosterone by inhibiting 5α-reductase, thus helping to control hair loss. In addition, glycyrrhizic acid has anti-inflammatory and soothing effects on the scalp, reducing scalp irritation and thus relieving itching and preventing hair loss. In practical applications, glycyrrhizic acid is often combined with other components such as baicalin to form nanomicelles to improve its solubility, stability, and bioavailability, thereby allowing it to penetrate the scalp and hair follicles more effectively to exert its effects. For example, research by Professor Liu Qiang's team at Southern Medical University showed that the combination of glycyrrhizic acid and baicalin, through nano-encapsulation technology, can significantly promote hair growth and has a certain therapeutic effect on alopecia areata. In general, glycyrrhizic acid, as a component with multiple biological activities, shows positive promise in preventing hair loss.

[0006] Traditional Chinese herbal extracts such as ginger, arborvitae leaves, and Polygonum multiflorum, while gentle, have a slow onset of action. In contrast, modern chemical ingredients such as pyrrolyl diaminopyrimidine oxide (PKO) demonstrate great potential due to their well-defined mechanisms of action and rapid effects. This compound can not only function alone, but its combined use with other anti-hair loss ingredients such as PKO, plant extracts, and biological agents can significantly enhance its anti-hair loss effect. For example, in combination with PKO (Kopexil): PKO is used in conjunction with PKO, which is also an analogue of minoxidil. Patent application WO2007 / 101357A2 shows that when pyrrolyl diaminopyrimidine oxide (Kopyrrol) is combined with PKO (Kopexil), it effectively promotes hair growth, with a much stronger effect than using PKO or minoxidil alone, demonstrating a synergistic effect. Researchers conducted studies on the combination of these two substances, using Kopyrrol, Kopexil, and their mixtures as research subjects. A mouse androgenetic alopecia model was established by applying testosterone (0.05% by mass) to the back of the head, and epigenetic indicators and serum 5α-reductase levels were observed. Combination with plant extracts: Current anti-hair loss shampoos and conditioners often contain plant extracts. Plant extracts are generally favored by consumers due to their natural origin, and experiments have verified that plant extracts in the formula can further enhance the effects of pyrrolidinyl diaminopyrimidine oxide. Related companies have developed a nanocomposite that promotes hair growth, formed by pyrrolidinyl diaminopyrimidine oxide and baicalin in a 5:2 mass ratio. The two have a synergistic effect, significantly promoting hair growth and providing more comprehensive prevention and treatment of hair loss. Combination with bioactive agents: Certain amino acids or small molecule peptides also have anti-hair loss and hair care effects. Therefore, these bioactive agents also have the potential to be combined with pyrrolidinyl diaminopyrimidine oxide to make the product have a better anti-hair loss effect.

[0007] Acetyl tetrapeptide-3 accelerates the synthesis of extracellular matrix proteins, increases the volume and length of hair follicles, strengthens hair roots, repairs the epidermal-dermal junction, and anchors hair within the follicle. Adenosine increases the expression level of fibroblast growth factor in dermal papilla cells, dilates blood vessels, increases blood circulation, and reduces hair loss. Using kopyrrol, adenosine, and acetyl tetrapeptide-3 as the main active ingredients for hair growth, a pyrrolidinyl diaminopyrimidine oxide / adenosine / active peptide composite nanoliposome (nano-kopyridine peptide) was prepared. Its hair growth efficacy was evaluated using in vitro cell experiments and animal experiments. The changes in cell viability after treating dermal papilla cells with different concentrations of free active ingredient and nano-kopyridine peptide for 48 hours were observed. Compared with the normal group, both free active ingredient and nano-kopyridine peptide significantly promoted dermal papilla cell proliferation after 48 hours of culture (P<0.05). Compared with free active substances, nano-comparable glycoside peptides showed a more significant cell proliferation effect, exhibiting a marked difference.

[0008] Chinese invention patent application CN114732751A discloses an anti-hair loss essence containing pyrrolyl diaminopyrimidine oxide, comprising the following components in parts by weight: 0.01-20 parts by weight of pyrrolyl diaminopyrimidine oxide; 0.01-20 parts by weight of adenosine; 0.01-20 parts by weight of diaminopyrimidine oxide; 0.01-20 parts by weight of niacinamide; 0.01-20 parts by weight of urea; 100-200 parts by weight of solvent; and 0.01-0.1 parts by weight of tripeptide-1 copper.

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

[0010] Chinese invention patent application CN118319801A discloses a composition containing ginseng root extract, its preparation method, and its application. The composition comprises ginseng root extract, adenosine, diaminopyrimidine oxide, and pyrrolidinediaminopyrimidine oxide. The composition provided by this invention uses ginseng root extract as the main active ingredient and adenosine, diaminopyrimidine oxide, and pyrrolidinediaminopyrimidine oxide as antioxidant enhancers, exerting a synergistic effect and significantly improving the antioxidant effect, thus resisting oxidative stress.

[0011] Chinese invention patent application CN118512476A discloses a pharmaceutical composition with comprehensive effects on improving the scalp environment, its preparation method, and its application. The composition contains 0.1-10% hair follicle repair protein composition, 0.001-0.1% caffeine, 0.001-0.1% pyrrolidine diaminopyrimidine oxide, 0.001-0.1% diaminopyrimidine oxide, 0.1-10% plant extracts, 0.001-0.1% amino acids, and a pharmaceutically acceptable carrier. The plant extracts are selected from any one or a combination of Phyllanthus emblica extract, Platycladus orientalis leaf extract, ginseng extract, Arctium lappa root extract, Trifolium repens leaf extract, Artemisia argyi extract, and Zingiber officinale root extract. The amino acids are selected from any one or a combination of arginine, cystine, ornithine, histidine, tyrosine, citrulline, serine, and nicotinamide. This pharmaceutical composition has a significant repair effect and can comprehensively improve the scalp environment of patients.

[0012] Chinese invention patent application CN118512478A discloses a pharmaceutical composition with anti-hair loss efficacy, its preparation method, and its application. The composition contains 0.1-10% of a hair follicle repair protein composition, 0.1-10% of pyrrolidine diaminopyrimidine oxide, 0.1-10% of diaminopyrimidine oxide, 0.1-10% of a traditional Chinese medicine extract, 0.1-10% of amino acids, and a pharmaceutically acceptable carrier. The traditional Chinese medicine extract is selected from any one or a combination of Phyllanthus emblica extract, Platycladus orientalis leaf extract, Zingiber officinale root extract, Tea extract, Asparagus cochinchinensis root extract, Trifolium repens leaf extract, and Artemisia argyi leaf extract. The amino acids are selected from any one or a combination of silk amino acids, arginine, cystine, nicotinamide, tyrosine, histidine, ornithine, and citrulline. This composition effectively prevents hair loss and has advantages such as high absorption rate, no scalp irritation, non-invasiveness, and rapid effectiveness.

[0013] While these innovative combinations offer more efficient and comprehensive solutions for hair loss treatment to some extent, they still cannot fully realize the anti-hair loss efficacy of pyrrolidinyl diaminopyrimidine oxide as the main active ingredient.

[0014] Initially, although pyrrolidinyl diaminopyrimidine oxide was found to have similar effects to minoxidil, drug development was not pursued. Furthermore, because both diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide are crystalline powders with poor water solubility, hair growth solutions prepared from them must contain large amounts of alcohols, easily leading to discomfort such as dry scalp and itching after use. Secondly, due to the skin's natural barrier, active ingredients have difficulty penetrating into hair follicles and cannot directly act on the target site, resulting in low bioavailability. Later, after a series of studies, Zhang Yan et al. disclosed a pyrrolidinyl diaminopyrimidine oxide β-cyclodextrin or / and β-cyclodextrin derivative inclusion complex in Chinese invention patent application CN108210937A, which improved the solubility of the water-poorly soluble anti-hair loss and hair growth pyrrolidinyl diaminopyrimidine oxide agent in aqueous solution. While the water solubility of pyrrolidinyl diaminopyrimidine oxide increased after being encapsulated in cyclodextrin, its large particle size prevented deep penetration into the hair follicle structure, resulting in a single target and suboptimal anti-hair loss effect.

[0015] Chinese invention patent CN109528725B discloses a nanocomposition containing diaminopyrimidine oxide and pyrrolyl diaminopyrimidine oxide, its preparation method, and its application. The nanocomposition comprises the following components in weight percentages: 5-20% diaminopyrimidine oxide, 2.5-10% pyrrolyl diaminopyrimidine 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 being water. The nanocomposition provided by this invention achieves transdermal co-delivery of multi-target anti-hair loss active ingredients, penetrating deep into hair follicles for synergistic effects, and possesses both anti-hair loss and hair growth effects, making it widely applicable in anti-hair loss and hair growth cosmetics. However, the composition of this solution has a large particle size, resulting in poor skin permeability and absorption, and poor targeting of hair follicles.

[0016] The paper "Microneedle Delivery Platform Integrated with Codelivery Nanoliposomes for Effective and Safe Androgenetic Alopecia Treatment" published in ACS Applied Materials & Interfaces describes a microneedle delivery platform integrated with codelivery nanoliposomes for the treatment of androgenetic alopecia. The core anti-hair loss ingredients, kopexil and kopyrrol, are encapsulated in nanoliposomes (KK-NLPs). However, the simultaneous encapsulation of these active ingredients results in a relatively large particle size, leading to limitations in transdermal absorption and hindering the optimal utilization of their anti-hair loss efficacy.

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

[0018] One objective of this invention is to provide a supramolecular nanovesicle formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2. The supramolecular nanovesicles composed of glycyrrhizic acid and acetyl tetrapeptide-2 provided by this invention are used to encapsulate pyrrolidinyl diaminopyrimidine oxide. Acetyl tetrapeptide-2 and glycyrrhizic acid, acting as carriers, can function as reservoirs, continuously releasing the anti-hair loss functional factor pyrrolidinyl diaminopyrimidine oxide, maintaining it at an effective concentration for a prolonged period, thus synergistically enhancing its effectiveness in preventing hair loss.

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

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

[0021] The fourth objective of this invention is to provide a method for preparing supramolecular nanovesicles of pyrrolidinyl diaminopyrimidine oxide.

[0022] The fifth objective of this invention is to provide a pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle or a pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle prepared by the aforementioned preparation method, for use in the preparation of anti-hair loss shampoo and conditioner products.

[0023] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a supramolecular nanovesicle formed by the 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 balance being solvent.

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

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

[0026] Secondly, the present invention provides a method for preparing supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2, comprising the following steps: Glycyrrhizic acid, acetyl tetrapeptide-2 and solvent were mixed in the prescribed amounts to obtain supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2.

[0027] Thirdly, the present invention provides a pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle, comprising pyrrolidinyl diaminopyrimidine oxide, and supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 encapsulated outside the pyrrolidinyl diaminopyrimidine oxide, or supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 prepared by the preparation method described above.

[0028] Furthermore, the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles comprise the following components: Pyrrolidinyl diaminopyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, cosolvents and solvents; Preferably, the pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles comprise the following components by mass percentage: 2.0%-2.8% pyrrolyl diaminopyrimidine 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 being solvent; Preferably, the pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles comprise the following components by mass percentage: 2.36% pyrrolyl diaminopyrimidine oxide, 4.87% glycyrrhizic acid, 0.49% acetyl tetrapeptide-2, 19.68% propylene glycol, 9.13% Tween 20, and the balance being solvent.

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

[0030] Fourthly, the present invention provides a method for preparing the supramolecular nanovesicles of pyrrolidinyl diaminopyrimidine oxide, wherein pyrrolidinyl diaminopyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, a co-solvent, and a solvent are mixed in the prescribed amounts to obtain the supramolecular nanovesicles of pyrrolidinyl diaminopyrimidine oxide.

[0031] Furthermore, this includes the following steps: (a) Dissolve the prescribed amount of pyrrolidinyl diaminopyrimidine oxide and co-solvent in a solvent to obtain solution a; (b) Dissolve the prescribed amounts of glycyrrhizic acid and acetyl tetrapeptide-2 in a solvent to obtain glycyrrhizic acid solution and acetyl tetrapeptide-2 solution, and then mix the glycyrrhizic acid solution and the acetyl tetrapeptide-2 solution to obtain solution b; (c) After mixing solution a and solution b in step 1, place them at 60-85℃ and stir for 10-60 minutes to form a transparent liquid; (d) After a transparent liquid is formed, the temperature is lowered by a program and stirring is continued for 6-8 hours; (e) Filtration yields supramolecular nanovesicles of pyrrolidine diaminopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2.

[0032] Fifthly, the present invention provides the application of the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles described above or the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles prepared by the preparation method described above in the preparation of anti-hair loss shampoo and conditioner products.

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

[0034] The supramolecular nanovesicles of pyrrolidinyl diaminopyrimidine oxide provided by this invention encapsulate pyrrolidinyl diaminopyrimidine oxide in supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2. Using glycyrrhizic acid as a carrier and acetyl tetrapeptide-2 as a carrier increases the solubility and bioavailability of pyrrolidinyl diaminopyrimidine oxide, and significantly improves the amount retained in the skin. This also enhances the inhibitory activity of pyrrolidinyl diaminopyrimidine oxide on 5α-reductase, allowing it to exert its own inhibitory effect on 5α-reductase and reduce excessive sebum secretion caused by testosterone. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 Contour plot showing the effect of glycyrrhizic acid dosage and reaction temperature on the encapsulation efficiency of pyrrolidinyldiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinyldiaminopyrimidine oxide nanovesicles in Example 5. Figure 2 Contour plot showing the effect of glycyrrhizic acid dosage and reaction time on the encapsulation efficiency of pyrrolidinyldiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinyldiaminopyrimidine oxide nanovesicles in Example 5. Figure 3 Contour plot showing the effect of reaction temperature and reaction time on the encapsulation efficiency of pyrrolidinyldiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinyldiaminopyrimidine oxide nanovesicles in Example 5. Figure 4 The response surface plot shows the effect of glycyrrhizic acid dosage and reaction temperature on the encapsulation efficiency of pyrrolidinyldiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinyldiaminopyrimidine oxide nanovesicles in Example 5. Figure 5 The response surface plot shows the effect of glycyrrhizic acid dosage and reaction time on the encapsulation efficiency of pyrrolidinyldiaminopyrimidine oxide supramolecular nanovesicles in the process of preparing pyrrolidinyldiaminopyrimidine oxide nanovesicles in Example 5. Figure 6 The response surface plot shows the effect of reaction temperature and reaction time on the encapsulation efficiency of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles provided in Example 5. Figure 7 Image of supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 prepared in Example 1; Figure 8Image of the supramolecular nanovesicle sample of pyrrolidinyl diaminopyrimidine oxide prepared in Example 5; Figure 9 The particle size distribution diagram of the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles prepared in Example 5; Figure 10 The 5α-reductase inhibition rate is shown in Example 5 and Comparative Examples 1-2. Detailed Implementation

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

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

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

[0041] Based on the total mass of the supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 as 100%, the amount of glycyrrhizic acid added is 10.0%-20.0%, for example, it can be 10.0%, 15.0%, 20.0%, etc. Based on the total mass of the supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 as 100%, the amount of acetyl tetrapeptide-2 added is 1.0%-2.0%, for example, it can be 1.0%, 1.5%, 2.0%, etc.

[0042] In some preferred embodiments, the supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 comprise 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 supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 have a particle size of 2-20 nm, preferably 2-10 nm.

[0044] In this invention, the particle size of the product after subsequent encapsulation with pyrrolidinyl diaminopyrimidine oxide is also 2-20 nm.

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

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

[0047] (3) Slowly add the solution (glycyrrhizic acid solution) from step (2) to the solution (acetyl tetrapeptide-2 solution) from step (1) while stirring. (4) Continue stirring for 6-8 h to obtain supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2.

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

[0049] The pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles provided by this invention are based on glycyrrhizic acid and acetyl tetrapeptide-2 encapsulated supramolecular nanovesicles of pyrrolidinyl diaminopyrimidine oxide.

[0050] The pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles comprise the following components: Pyrrolidinyl diaminopyrimidine 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 components are included by weight percentage as follows: 2.0%-2.8% pyrrolidine diaminopyrimidine 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 pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles being 100%, the amount of pyrrolidinyl diaminopyrimidine oxide added is 2.0%-2.8%, for example, it can be 2.0%, 2.4%, 2.8%, etc.; Based on the total mass of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles as 100%, the amount of glycyrrhizic acid added is 2.0%-5.0%, for example, it can be 2.0%, 3.5%, 5.0%, etc.; Based on the total mass of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles as 100%, the amount of acetyl tetrapeptide-2 added is 0.2%-0.5%, for example, it can be 0.2%, 0.3%, 0.4%, 0.5%, etc.; Based on the total mass of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles as 100%, the amount of propylene glycol added is 10.1%-25.3%, for example, it can be 10.1%, 15%, 20%, 25%, 25.3%, etc.; Based on the total mass of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles as 100%, the amount of Tween 20 added is 5.2%-9.3%, for example, it can be 5.2%, 6%, 7%, 8%, 9.3%, etc.

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

[0055] In this invention, glycyrrhizic acid is used as one of the raw materials. Glycyrrhizic acid is one of the main active ingredients of the traditional Chinese medicine licorice. It is mainly composed of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid. Due to its polymerization behavior and its ability to form micelles due to its amphiphilic structure, glycyrrhizic acid is used as an absorption promoter and delivery carrier to improve the transdermal absorption of the active ingredients. Glycyrrhizic acid can reduce excessive sebum secretion caused by testosterone by inhibiting 5α-reductase, thus helping to control hair loss; Acetyl Tetrapeptide-2 is a follicle-targeting membrane-penetrating peptide that can penetrate the skin barrier and act directly on the hair follicle. It has the effects of promoting hair growth and preventing hair loss. Acetyl Tetrapeptide-2 can prevent hair loss. Its main mechanisms of action are as follows: (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, thus helping to promote healthy hair growth; (2) Inhibiting 5α-reductase activity: This polypeptide can reduce the activity of 5α-reductase and reduce the production of dihydrotestosterone (DHT). DHT is an important factor leading to androgenetic alopecia, so inhibiting its production can effectively slow down the hair loss process; (3) Antioxidant protection: Acetyl tetrapeptide-2 also has antioxidant properties, which can neutralize free radicals and protect hair follicles from oxidative stress damage; (4) Regulation of immune response: In some cases, abnormal immune system responses 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 employs supramolecular technology, which can significantly improve the targeting of hair follicles. Supramolecular technology can recombine different molecules into new supramolecular structures. These structures not only retain the functions of the original molecules but also enable the molecules in the new structure to synergize, thereby enhancing efficacy and properties. Supramolecular technology can improve the product's penetration efficiency and absorption rate, reduce the loss of active ingredients, and enhance the stability and gentleness of the ingredients.

[0057] In this invention, glycyrrhizic acid and acetyl tetrapeptide-2 form a supramolecular structure through intermolecular interactions such as hydrogen bonds and van der Waals forces. The resulting supramolecular structure may possess novel anti-hair loss bioactivity or physicochemical properties, providing new materials or candidate molecules for fields such as cosmetic anti-hair loss treatments.

[0058] The fourth aspect of the present invention provides a method for preparing the supramolecular nanovesicles of the pyrrolidinyl diaminopyrimidine oxide, wherein the pyrrolidinyl diaminopyrimidine oxide, glycyrrhizic acid, acetyl tetrapeptide-2, co-solvent and solvent are mixed in the prescribed amounts to obtain the supramolecular nanovesicles of the pyrrolidinyl diaminopyrimidine oxide.

[0059] In some preferred embodiments, the method includes the following steps: (a) Dissolve the prescribed amount of pyrrolidinyl diaminopyrimidine oxide and co-solvent in a solvent to obtain solution a; (b) Dissolve the prescribed amounts of glycyrrhizic acid and acetyl tetrapeptide-2 in a solvent to obtain glycyrrhizic acid solution and acetyl tetrapeptide-2 solution, and then add the glycyrrhizic acid solution to the acetyl tetrapeptide-2 solution for mixing to obtain solution b; (c) After mixing solution a and solution b in step 1, place them at 60-85℃ and stir for 10-60 minutes to form a transparent liquid; (d) After a transparent liquid is formed, the temperature is lowered by a program and stirring is continued for 6-8 hours; (e) Filtration yields supramolecular nanovesicles of pyrrolyl diaminopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2.

[0060] Specifically, the preparation process of the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles includes: (1) Accurately weigh the formula amount of pyrrolidinyl diaminopyrimidine oxide, propylene glycol, Tween 20 and deionized water, put them in a beaker and stir evenly; (2) Weigh the amount of glycyrrhizic acid in the formula and dissolve it in a certain amount of deionized water to obtain a glycyrrhizic acid solution; weigh the amount of acetyl tetrapeptide-2 in the formula and add a certain amount of deionized water to dissolve it completely to obtain an acetyl tetrapeptide-2 solution; slowly add the glycyrrhizic acid solution to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6-8 h, and then slowly add it to the mixed solution in step (1) while stirring. (3) Mix the solutions from steps (1) and (2) thoroughly, place them at 80°C, and continue stirring for 30 minutes to form a transparent liquid; (4) After a transparent liquid is formed, the temperature is lowered to 25°C and stirring is continued for 6-8 hours; (5) The supramolecular nanovesicles of pyrrolidine diaminopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration with a 0.22 µm microporous membrane.

[0061] The present invention provides a method for preparing supramolecular nanovesicles of pyrrolidinyl diaminopyrimidine oxide. This method is simple, provides accurate dosage, and is suitable for large-scale production. The preparation process meets the requirements of pyrrolidinyl diaminopyrimidine oxide as a novel anti-hair loss raw material, possessing not only mild and safe properties but also excellent water solubility and no side effects. Furthermore, it lays the foundation for the research and development of nano-preparations for traditional Chinese medicine.

[0062] The fifth aspect of this invention provides the application of the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles described above, or the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles prepared by the preparation method described above, in the preparation of anti-hair loss shampoo and conditioner products.

[0063] This invention relates to the application of glycyrrhizic acid and acetyl tetrapeptide-2-encapsulated pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles in anti-hair loss shampoo and conditioner products. These glycyrrhizic acid and acetyl tetrapeptide-2-encapsulated pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles can be directly applied to various anti-hair loss shampoo and conditioner products, such as anti-hair loss shampoos, anti-hair loss lotions, anti-hair loss creams, and anti-hair loss serums. Through further scientific formulation, the anti-hair loss effect of pyrrolyl diaminopyrimidine oxide can be maximized in these products.

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

[0065] 2. By utilizing traditional Chinese medicine nano-assembly technology (using traditional Chinese medicine nano-supramolecular self-assembly technology), the unique characteristics and advantages of "drug and adjuvant integration" are brought into play. Acetyl tetrapeptide-2 and glycyrrhizic acid, as carriers, can play the role of storage, continuously releasing the anti-hair loss functional factor pyrrolidinyl diaminopyrimidine oxide, maintaining it at an effective concentration for a longer period of time, synergistically enhancing its effect and enabling it to exert a highly effective anti-hair loss effect.

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

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

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

[0069] 6. The pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles of the present invention have high safety. The preparation method does not use toxic organic solvents, there are no organic solvent residues, and they have little skin irritation and high safety.

[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 will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

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

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

[0074] The formulation is shown in Table 1: Table 1. Proportions of Acetyl Tetrapeptide-2 and Glycyrrhizic Acid in the Supramolecular Nanovesicle System

[0075] Preparation method: Using the corresponding proportions of the components in formulations 1-6 above, supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 are prepared. The specific preparation process includes: (1) Accurately weigh the amount of acetyl tetrapeptide-2 in the formula and dissolve it in a certain amount of deionized water; (2) Weigh out the amount of glycyrrhizic acid specified in the formula and add a certain amount of deionized water to dissolve it completely; (3) Slowly add the solution (glycyrrhizic acid solution) from step (2) to the solution (acetyl tetrapeptide-2 solution) from step (1) while stirring. (4) Continue stirring for 6-8 h to obtain supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2. Figure 7 In Example 1, the vesicle particle sizes obtained from each formulation were all in the range of 2-20 nm.

[0076] Particle size, zeta potential, and polydispersity index (PDI) were measured using a nanoparticle size analyzer, and 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, when the ratio of glycyrrhizic acid to acetyl tetrapeptide-2 is 10:1 (corresponding product image is shown below) Figure 7 At this point, the supramolecular nanovesicles had a small particle size of 5.46 nm, and small zeta potential and PDI. The zeta potential was -4.12 mV and the PDI was 0.239. Therefore, the final ratio of glycyrrhizic acid to acetyl tetrapeptide-2 was chosen to be 10:1 for subsequent experiments.

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

[0079] Example 2 Example 2 provides a pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle. Several formulations are provided in Example 2 to explore the dosage of acetyl tetrapeptide-2 and glycyrrhizic acid in the supramolecular nanovesicle system.

[0080] The formulation is shown in Table 3: Table 3. Proportions of acetyl tetrapeptide-2 and glycyrrhizic acid in supramolecular nanovesicle systems

[0081] Preparation method: Using the corresponding raw materials in the component proportions of formulas 7-12 above, the supramolecular nanovesicles of pyrrolidinyl diaminopyrimidine oxide were prepared. The specific preparation process included: (1) Accurately weigh the formula amount of pyrrolidinyl diaminopyrimidine oxide, propylene glycol, Tween 20 and deionized water, put them in a beaker and stir evenly; (2) Weigh the amount of acetyl tetrapeptide-2 in the formula, dissolve it in a certain amount of deionized water to obtain acetyl tetrapeptide-2 solution; weigh the amount of glycyrrhizic acid in the formula, add a certain amount of deionized water to dissolve it completely to obtain glycyrrhizic acid solution; slowly add the glycyrrhizic acid solution to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6~8 h, and then slowly add it to the mixed solution in step (1) while stirring. (3) Mix the solutions from steps (1) and (2) thoroughly, place them at 80°C, and continue stirring for 30 minutes to form a transparent liquid; (4) After a transparent liquid is formed, the temperature is lowered to 25°C and stirring is continued for 6-8 hours; (5) The supramolecular nanovesicles of pyrrolidine diaminopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration with a 0.22 µm microporous membrane.

[0082] Encapsulation efficiency was calculated using high-performance liquid chromatography (HPLC), and 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 amount of acetyl tetrapeptide-2 was 0.2 parts and the amount of glycyrrhizic acid was 2 parts, the encapsulation efficiency was the highest at 86.39%, the particle size was small at 8.16 nm, and the Zeta potential and PDI were small, with a Zeta potential of -3.78 mV and a PDI of 0.264. Therefore, acetyl tetrapeptide-2 of 0.2 parts and glycyrrhizic acid of 2 parts 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 pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles.

[0085] Example 3 Example 3 provides a pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle. Several formulations are provided in Example 3 to investigate the temperature during stirring and mixing of the supramolecular nanovesicle system.

[0086] The formulation is shown in Table 5: Table 5. Temperature parameters during stirring and mixing of supramolecular nanovesicle systems.

[0087] Preparation method: Using the corresponding raw materials in the component proportions of formulations 13-18 above, the supramolecular nanovesicles of pyrrolidinyl diaminopyrimidine oxide were prepared. The specific preparation process included: (1) Accurately weigh the formula amount of pyrrolidinyl diaminopyrimidine oxide, propylene glycol, Tween 20 and deionized water, put them in a beaker and stir evenly; (2) Weigh the amount of acetyl tetrapeptide-2 in the formula, dissolve it in a certain amount of deionized water to obtain acetyl tetrapeptide-2 solution; weigh the amount of glycyrrhizic acid in the formula, add a certain amount of deionized water to dissolve it completely to obtain g glycyrrhizic acid solution; slowly add the glycyrrhizic acid solution to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6~8 h, and then slowly add it to the mixed solution in step (1) while stirring. (3) Mix the solutions from steps (1) and (2) thoroughly, place them under different reaction temperature conditions, and continue stirring for 30 min to form a transparent liquid; (4) After a transparent liquid is formed, the temperature is lowered to 25°C and stirring is continued for 6-8 hours; (5) The supramolecular nanovesicles of pyrrolidine diaminopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration with a 0.22 µm microporous membrane.

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

[0089] When the reaction temperature is 80℃, the zeta potential is relatively small at -3.59 mV, while the encapsulation efficiency is the highest at 89.23%. The particle size is also small and the PDI is 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-pyrrolyl diaminopyrimidine oxide nanovesicles are relatively stable.

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

[0091] Example 4 Example 4 provides a pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicle. Several formulations are provided in Example 4 to explore the reaction time in the supramolecular nanovesicle system.

[0092] The formulation is shown in Table 7.

[0093] Table 7. Reaction time parameters in supramolecular nanovesicle systems

[0094] Preparation method: Using the corresponding raw materials from the component proportions in formulations 19-24 above, the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles were prepared. The specific preparation process included: (1) Accurately weigh the formula amount of pyrrolidinyl diaminopyrimidine oxide, propylene glycol, Tween 20 and deionized water, put them in a beaker and stir evenly; (2) Weigh the amount of acetyl tetrapeptide-2 in the formula, dissolve it in a certain amount of deionized water to obtain acetyl tetrapeptide-2 solution; weigh the amount of glycyrrhizic acid in the formula, add a certain amount of deionized water to dissolve it completely to obtain glycyrrhizic acid solution; slowly add the glycyrrhizic acid solution to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6~8 h, and then slowly add it to the mixed solution in step (1) while stirring. (3) Mix the solutions from steps (1) and (2) thoroughly, place them at 80°C, and continue stirring for different reaction times to form a transparent liquid; (4) After a transparent liquid is formed, the temperature is lowered to 25°C and stirring is continued for 6-8 hours; (5) The supramolecular nanovesicles of pyrrolidine diaminopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration with a 0.22 µm microporous membrane.

[0095] Encapsulation efficiency was calculated using high-performance liquid chromatography (HPLC), and particle size, zeta potential, and polydispersity index (PDI) were determined 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 at 84.32%, the PDI was the lowest at 0.261, and the particle size and zeta potential were also small, with a particle size of 6.16 nm and a zeta potential of -4.38 mV.

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

[0098] Example 5 Based on the results of Examples 2-4, 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 evaluation index. The dosage of pyrrolidinyl diaminopyrimidine oxide was fixed at 1 part. The range of each factor for investigation was determined. The Box-Benhken design-response surface methodology was used to optimize the preparation process of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles (Table 9).

[0099] Table 9. Box-Behnken Response Surface Factors and Level Design Table

[0100] Preparation method: (1) Accurately weigh the formula amount of pyrrolidinyl diaminopyrimidine oxide, propylene glycol, Tween 20 and deionized water, put them in a beaker and stir evenly; (2) Weigh the amount of acetyl tetrapeptide-2 in the formula, dissolve it in a certain amount of deionized water to obtain acetyl tetrapeptide-2 solution; weigh the amount of glycyrrhizic acid in the formula, add a certain amount of deionized water to dissolve it completely to obtain glycyrrhizic acid solution; slowly add the glycyrrhizic acid solution to the acetyl tetrapeptide-2 solution while stirring, continue stirring for 6~8 h, and then slowly add it to the mixed solution in step (1) while stirring. (3) Mix the solutions from steps (1) and (2) thoroughly, place them under different reaction temperature conditions, and continue stirring for different reaction times to form a transparent liquid; (4) After a transparent liquid is formed, the temperature is lowered to 25°C and stirring is continued for 6-8 hours; (5) The supramolecular nanovesicles of pyrrolidine diaminopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2 were obtained by filtration with a 0.22 µm microporous membrane.

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

[0102] Table 10. Experimental Design and Results of Box-Benhken Response Surface Methodology

[0103] Table 11. Results of Analysis of Variance

[0104] Input the encapsulation rate data from Table 10 into Design-Expert 8.0.6.1 software to obtain the fitted 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). The significance test shows that X2, X2X3, ... in the fitted model... 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 is not significant ( P A value ≥ 0.05 indicates that the regression model has good predictive ability, and the regression equation fits well across the entire regression region. The order of influence of each factor on the encapsulation rate is as follows: X 2> X3 > X1, meaning reaction temperature > reaction time > glycyrrhizic acid dosage. The effect surface plot and contour plot of each factor were drawn using Design-Expert 8.0.6.1 software. The results are shown below. Figures 1-6 Based on the analysis results, the predicted optimal process conditions for pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles, obtained by Design-Expert 8.6.0.1 analysis, are as follows: glycyrrhizic acid dosage of 1.99 parts, reaction temperature of 80.93℃, reaction time of 31.36 min, and encapsulation efficiency of 86.28%.

[0105] Three batches of pyrrolidinyl diaminopyrimidine oxide nanovesicles were prepared according to the prescribed process conditions (the optimal composition ratio of Example 5: by mass percentage, the components included: 2.36% pyrrolidinyl diaminopyrimidine oxide, 4.87% glycyrrhizic acid, 0.49% acetyl tetrapeptide-2, 19.68% propylene glycol, 9.13% Tween 20, and the balance solvent, prepared using the preparation method in Example 5, wherein the reaction temperature was 80.93℃ and the reaction time was 31.36 min). The encapsulation efficiencies of the three batches of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles were 86.71%, 86.47%, and 86.34%, respectively. The test results were compared with the predicted values ​​of the fitted 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 <5%, indicating that the preferred preparation process is stable and reliable. In addition, the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles are stable in an aqueous environment and are pale 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 pyrrolidinyl diaminopyrimidine oxide solution, the components and their mass percentages as follows: 2.36% pyrrolidine diaminopyrimidine oxide, 19.68% propylene glycol, 9.13% Tween 20 and balance deionized water.

[0107] The preparation method using the formulation of Comparative Example 1 above includes the following steps: (1) Accurately weigh the formula amount of pyrrolidinyl diaminopyrimidine oxide, propylene glycol, Tween 20 and deionized water, put them in a beaker and stir evenly; (2) After mixing thoroughly, place at 80°C and continue stirring for 30 min to form a transparent liquid; (3) After a transparent liquid is formed, the temperature is lowered to 25°C and stirring is continued for 6-8 hours; (4) The pyrrolidinyl diaminopyrimidine oxide solution was obtained by filtration through a 0.22 µm microporous membrane.

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

[0109] The preparation method using the formulation of Comparative Example 2 above includes the following steps: (1) Accurately weigh the formula amount of pyrrolidinyl diaminopyrimidine oxide, propylene glycol, Tween 20 and deionized water, put them in a beaker and stir evenly; (2) Weigh out the amount of glycyrrhizic acid specified in the formula and slowly add it to the mixed solution in step (1) while stirring. (3) Mix the solutions from steps (1) and (2) thoroughly, place them at 80°C, and continue stirring for 30 minutes to form a transparent liquid; (4) After a transparent liquid is formed, the temperature is lowered to 25°C and stirring is continued for 6-8 hours; (5) Pyrrolidinyl diaminopyrimidine oxide nanovesicles (non-targeted) were obtained by filtration with a 0.22 µm microporous membrane.

[0110] Test case Experimental Example 1: Stability Experiment of Phenolic Diaminopyrimidine Oxide Supramolecular Nanovesicles The pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles prepared by the optimal preparation process in Example 5 (the optimal components of Example 5 include: 2.36% pyrrolidinyl diaminopyrimidine oxide, 4.87% glycyrrhizic acid, 0.49% acetyl tetrapeptide-2, 19.68% propylene glycol, 9.13% Tween 20 and the balance solvent) were stored under conditions of high temperature and high humidity, direct sunlight, and repeated freeze-thaw cycles in a freezing environment. They were taken out at 1, 2, 4, 8, 14 and 21 days, respectively, and their appearance was observed. The particle size, zeta potential, PDI and encapsulation efficiency of the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles were measured.

[0111] The stability test results showed that the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles remained relatively stable after being placed under high temperature and humidity, direct sunlight, and repeated freeze-thaw cycles for 21 days (Tables 12-14), indicating that the pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles had good stability after being placed under high temperature and humidity, direct sunlight, and repeated freeze-thaw cycles for 21 days.

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

[0113] Table 13. Stability of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles under strong direct light conditions.

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

[0115] Experimental Example 2: Assay of 5α-reductase inhibitory activity of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles 1. The test sample stimulates the standard enzyme. (1) Prepare standardized enzymes, dilute them to a concentration of 80 U / L, spread them evenly on cell culture plates, and add 100 μL to each well.

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

[0117] (3) Select a kit to detect enzyme levels.

[0118] 2. Reagent kit testing (1) Adding samples: Add 50 μL of standard and sample to the enzyme-labeled plate. Add the sample to the bottom of the well of the enzyme-labeled plate, trying not to touch the well wall, and gently shake to mix.

[0119] (2) Incubation: After sealing the plate with sealing film, incubate at 37℃ for 30 min.

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

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

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

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

[0124] (7) Washing: Same as (4).

[0125] (8) Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop 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 (at this time, the blue color will immediately turn yellow).

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

[0128] 3. 5α-Reductase Inhibition Rate The formula for calculating the enzyme activity of the sample to be tested is as follows:

[0129] The 5α-reductase inhibition rates obtained in Example 5 (the product prepared using the optimal components of Example 5) and Comparative Examples 1-2 are shown in Table 15 and... Figure 10 As shown, the inhibition of 5α-reductase activity by pyrrolyl diaminopyrimidine oxide was significantly enhanced after encapsulation with acetyl tetrapeptide-2 and glycyrrhizic acid. At low concentrations, the inhibition rate of pyrrolyl diaminopyrimidine oxide solution (Comparative Example 1) was 11.57%, the inhibition rate of glycyrrhizic acid-pyrrolyl diaminopyrimidine oxide nanovesicles (Comparative Example 2) was 4.47%, and the inhibition rate of pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles encapsulated with acetyl tetrapeptide-2 and glycyrrhizic acid (Example 5) was 40.12%, demonstrating a significant improvement in anti-hair loss and oil control efficacy. The results also showed that the inhibitory effect of pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles encapsulated with acetyl tetrapeptide-2 and glycyrrhizic acid on 5α-reductase was not a simple "1+1=2" effect of mixing, but rather a synergistic effect 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 serum containing pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles. The components of the anti-hair loss serum are shown in Table 16 below: Table 16. A hair loss prevention essence containing pyrrolidine diaminopyrimidine oxide supramolecular nanovesicles

[0132] The preparation method, using the formulation in Application Example 1, includes the following steps: (1) Add water of phase A1, and then add the remaining raw materials in sequence while stirring. Heat the mixture to 80~85℃, and keep it at a constant temperature for 15 minutes. After homogenization and uniform dispersion, start stirring and cooling. (2) Cool down to 70~75℃, add the pre-dissolved A2 phase raw material, and continue stirring and cooling; (3) Cool down to 40~45℃, add the pre-dissolved B phase raw material, stir and mix evenly, then add the C phase raw material and stir until completely dissolved; (4) Add the D phase raw material after mixing it evenly in advance, stir it evenly, and then add the E phase raw material in sequence and stir it evenly until it becomes transparent.

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

[0134] Table 17. An anti-hair loss essence containing glycyrrhizic acid-pyrrolidine diaminopyrimidine 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 a hair loss prevention essence containing glycyrrhizic acid-pyrrolyl diaminopyrimidine oxide nanovesicles. The components of the hair loss prevention essence are shown in Table 18 below.

[0137] Table 18. A hair loss prevention essence containing pyrrolidine diaminopyrimidine oxide supramolecular nanovesicles

[0138] The preparation method, using the formulation in Application Example 3, includes the following steps: (1) Add water of phase A1, and then add the remaining raw materials in sequence while stirring. Heat the mixture to 80~85℃, and keep it at a constant temperature for 15 minutes. After homogenization and uniform dispersion, start stirring and cooling. (2) Cool down to 70~75℃, add the pre-dissolved A2 phase raw material, and continue stirring and cooling; (3) Cool down to 40~45℃, add the pre-dissolved B phase raw material, stir and mix evenly, then add the C phase raw material and stir until completely dissolved; (4) Add the D phase raw material after mixing it evenly in advance, stir it evenly, and then add the E phase raw material in sequence and stir it evenly until it becomes transparent.

[0139] Experimental Example 3 5α-reductase inhibitory activity test of an anti-hair loss essence containing pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles.

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

[0141] Table 19. 5α-Reductase Inhibition Rate of Anti-Hair Loss Essences in Application Examples 1-3

[0142] Analysis of the results in Table 19: From the results of application examples 1-3, the gel prepared with pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles has significant anti-hair loss and oil control effects. In application example 1, when the mass fraction of pyrrolidinyl diaminopyrimidine oxide nanovesicles is 3 wt%, the anti-hair loss and oil control effects are already very obvious. Moreover, as the proportion of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles increases, the 5α-reductase inhibition rate of the anti-hair loss essence also increases accordingly, indicating that pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles can effectively improve the anti-hair loss and oil control effects of the essence.

[0143] Experiment Example 4: Subjective Human Perception Experiment Subjects were not allowed to wash their hair within 48 hours ± 5 hours before each test, and were not allowed to comb their hair on the day of the test. After arriving at the test site and adapting to the environment, the same trained technician used the same comb to comb the 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 shed hair was then collected and counted.

[0144] Thirty healthy men and women aged 18-60 years with mild hair thinning and hair length between 5-40 cm were selected. Those who experienced significant hair loss and had more than 10 hairs lost during the 60-comb test were divided into three groups of 10 each. Each group used hair care products listed in Examples 1-3, twice daily (morning and evening), following the instructions for each application, including washing and massaging the scalp for 1-3 minutes during the washing process. The 60-comb test was performed before use and at 4, 8, and 12 weeks after use, and the number of hairs lost was recorded. The weekly hair loss reduction rate (%) was calculated using the formula: (Number of hairs lost before use - Number of hairs lost at 12 weeks) / Number of hairs lost before use * 100% (Table 20).

[0145] Table 20. 5α-Reductase Inhibition Rate of Anti-Hair Loss Essences in Application Examples 1-3

[0146] The results show that the essence containing pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles prepared using the process defined in this 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, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 of pyrrolidinyl diaminopyrimidine oxide, characterized in that, It includes pyrrolidinyl diaminopyrimidine oxide, and supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 encapsulated outside the pyrrolidinyl diaminopyrimidine oxide; The pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles comprise, by mass percentage: 2.0%-2.8% pyrrolyl diaminopyrimidine 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 being deionized water; The supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 comprise the following components by mass percentage: 10.0%-20.0% glycyrrhizic acid, 1.0%-2.0% acetyl tetrapeptide-2, and the balance being deionized water. The preparation method of the pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles includes the following steps: (a) Dissolve the prescribed amounts of pyrrolidinyl diaminopyrimidine oxide, propylene glycol, and Tween 20 in deionized water to obtain solution a; (b) Dissolve the prescribed amounts of glycyrrhizic acid and acetyl tetrapeptide-2 in deionized water to obtain glycyrrhizic acid solution and acetyl tetrapeptide-2 solution, and then mix the glycyrrhizic acid solution and the acetyl tetrapeptide-2 solution to obtain solution b; (c) After mixing solutions a and b, place them at 60-85℃ and stir for 10-60 minutes to form a transparent liquid; (d) After a transparent liquid is formed, the temperature is lowered by a program and stirring is continued for 6-8 hours; (e) Filtration yields supramolecular nanovesicles of pyrrolidine diaminopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2.

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

3. The pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles according to claim 1, characterized in that, The supramolecular nanovesicles formed by the self-assembly of glycyrrhizic acid and acetyl tetrapeptide-2 have a particle size of 2-20 nm.

4. The pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles according to claim 1, characterized in that, The pyrrolyl diaminopyrimidine oxide supramolecular nanovesicles comprise the following components by mass percentage: 2.36% pyrrolyl diaminopyrimidine oxide, 4.87% glycyrrhizic acid, 0.49% acetyl tetrapeptide-2, 19.68% propylene glycol, 9.13% Tween 20, and the balance being deionized water.

5. The method for preparing pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles according to any one of claims 1-4, characterized in that, Includes the following steps: (a) Dissolve the prescribed amounts of pyrrolidinyl diaminopyrimidine oxide, propylene glycol, and Tween 20 in deionized water to obtain solution a; (b) Dissolve the prescribed amounts of glycyrrhizic acid and acetyl tetrapeptide-2 in deionized water to obtain glycyrrhizic acid solution and acetyl tetrapeptide-2 solution, and then mix the glycyrrhizic acid solution and the acetyl tetrapeptide-2 solution to obtain solution b; (c) After mixing solutions a and b, place them at 60-85℃ and stir for 10-60 minutes to form a transparent liquid; (d) After a transparent liquid is formed, the temperature is lowered by a program and stirring is continued for 6-8 hours; (e) Filtration yields supramolecular nanovesicles of pyrrolidine diaminopyrimidine oxide encapsulated with glycyrrhizic acid and acetyl tetrapeptide-2.

6. The application of pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles as described in any one of claims 1-4 or pyrrolidinyl diaminopyrimidine oxide supramolecular nanovesicles prepared by the preparation method described in claim 5 in the preparation of anti-hair loss shampoo and conditioner products.

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