Method for regulating synthesis and degradation of hyaluronic acid by fibroblast

Abstract

A new method for regulating synthesis and degradation of hyaluronic acid by a fibroblast, comprising using at least one selected from nerol, carvacrol, geraniol, an agarwood essential oil, and a sandalwood essential oil.

Description

A method for regulating the synthesis and degradation of hyaluronic acid by fibroblasts Technical Field

[0001] This invention relates to a method for regulating the synthesis and degradation of hyaluronic acid by fibroblasts, and a method for combating skin aging. Background Technology

[0002] Skin aging is the result of both natural aging and photoaging, manifesting as decreased skin elasticity, deepened wrinkles, and the formation of age spots. Fibroblasts, also known as fibroblasts, are the main cellular components of loose connective tissue, differentiated from mesenchymal cells during embryonic development. These cells account for more than 95% of all cells in the dermis. In other words, the structure and function of the entire dermis are maintained by fibroblasts, the mainstay of skin health, thus effectively maintaining skin firmness, elasticity, and hydration.

[0003] Hyaluronic acid (HA) is an important biomolecule found in the skin, possessing various biological functions such as maintaining skin moisture, promoting cell proliferation and migration, and regulating inflammatory responses. The synthesis and degradation of hyaluronic acid are influenced by multiple factors, among which hyaluronic acid synthetase (HAS) and hyaluronic acid depolymerizing enzyme (hyaluronic acid binding protein involved in hyaluronic acid depolymerization, HYBID) play crucial roles. There are three HAS subtypes in mammals: HA synthetase 1 (HAS1), HA synthetase 2 (HAS2), and HA synthetase 3 (HAS3). In human skin dermis, HA degradation is primarily mediated by HYBID. Increased expression of HYBID and decreased expression of HAS1 and HAS2 were detected in photoaged skin samples, indicating that the imbalance between HYBID-mediated HA degradation and HAS-mediated HA synthesis may lead to enhanced HA catabolism in photoaged skin, resulting in reduced HA in the papillary dermis and ultimately causing wrinkles and sagging skin.

[0004] It has been reported that dermal fibroblasts produce 12-60 pg / cell / 24h of hyaluronic acid, approximately 10 times that of keratinocytes. In normal human skin fibroblasts, hyaluronic acid synthesis is mainly controlled by HAS1 and HAS2, with HAS2 being the predominant HAS isoform expressed in fibroblasts. Generally, the amount of hyaluronic acid synthesized is consistent with the mRNA expression level of HAS2. Growth factors such as TGF-β1, PDGF-BB, EGF, and bFGF can all increase hyaluronic acid synthesis by upregulating HAS in normal human skin fibroblasts. TGF-β exhibits differentiated regulation of HAS isoform expression in fibroblasts and keratinocytes. TGF-β upregulates the expression of both HAS1 and HAS2 in fibroblasts, while in keratinocytes, TGF-β only induces HAS1 expression without affecting HAS2, suggesting that dermal and epidermal hyaluronic acid are independently regulated.

[0005] Cellular-level studies have revealed a close correlation between cellular senescence and reduced HA synthesis. Compared to young and non-senescent fibroblasts, senescent and aged fibroblasts showed a significant decrease in their ability to synthesize HA, with a marked reduction in HAS2 expression levels. By comparing the expression of HAS, hyaluronidase, and HA secretion capacity of fibroblasts isolated from skin samples of people of different ages, it was found that HA secretion was reduced in elderly fibroblasts, with downregulated expression of HAS, especially HAS2, while hyaluronidase expression remained largely unchanged. This further suggests that the lack of HA in the dermis of the elderly may be due to insufficient HA secretion by fibroblasts, and the reduced HA secretion capacity is mainly related to the downregulation of HAS2 expression, but not to hyaluronidase expression levels.

[0006] Supplementing with exogenous HA has certain therapeutic effects on improving skin appearance and anti-aging. Exogenous HA can compensate for the deficiency of endogenous HA in aging skin, not only providing moisturizing and physical filling effects, but also exhibiting biological effects such as strengthening the skin barrier, promoting repair, stimulating regeneration, inducing collagen production, and combating oxidative stress. Numerous studies have proven its skin-delaying efficacy, and it is widely used. However, large-molecule HA has poor transdermal absorption when applied topically, and exogenous HA is rapidly cleared from skin tissue, which greatly limits its skincare benefits. Therefore, regulating the level of endogenous HA in the skin has become a uniquely advantageous strategy for delaying skin aging.

[0007] There are two possible strategies for directly regulating skin HA levels through topical application of small molecule compounds or plant extracts: promoting HA synthesis or downregulating HA-degrading enzymes. Increasing HAS mRNA expression levels or improving HA production has become one of the most common indicators for evaluating the efficacy of anti-aging cosmetic ingredients; therefore, many compounds or extracts that affect this pathway have been reported. Furthermore, numerous growth factors and cytokines regulate HAS expression. Growth factors that generally promote skin cell proliferation and migration, such as EGF and KGF, have strong stimulatory activity on HAS2 and HAS3 expression, and their anti-aging efficacy has been confirmed by multiple clinical trials.

[0008] Existing technology literature 1 discloses that trans-retinoic acid is currently the most thoroughly studied drug with a clear anti-skin aging effect. HAS is one of the important targets of this class of drugs. The HAS gene promoter region contains a response regulatory element of the retinoic acid receptor (RAR), and therefore is directly regulated by retinoic acid. Existing technology literature 2 discloses some small molecule compounds with anti-aging effects, such as β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and astaxanthin, which can upregulate the expression of the HAS3 gene in keratinocytes by activating the retinoic acid receptor (RAR) and promote HA synthesis in keratinocytes. Supplementing skin cells with precursor compounds of UDP-Glucuronic acid and UDP-GlcNAc, which are raw materials for HA synthesis, can also regulate HA synthesis. Existing technology document 3 discloses a newly developed HA synthesis inducer, β-NAG2 (1-ethyl-β-N-acetylglucosaminide). β-NAG2 can be metabolized intracellularly into UDP-GlcNAc, a substrate for HA synthesis, thereby inducing HA synthesis in skin cells. Studies have found that β-NAG2 can increase the production of HA in cultured skin tissue and three-dimensional skin substitutes, promote the proliferation of basal cells, increase the expression of differentiation markers such as TGM1 and FLG, and promote epidermal stratification. These results indicate that increasing epidermal HA production can accelerate keratinocyte proliferation and differentiation, thus playing a key role in epidermal morphogenesis and homeostasis maintenance. The latest research in existing technology document 4 further demonstrates that β-NAG2 has anti-wrinkle and anti-aging effects.

[0009] Encouraging progress has also been made in research on inhibiting HA degradation. Sodium copper chlorophyllin, a commonly used cosmeceutical ingredient, is used as a cosmetic colorant, antioxidant, and antibacterial agent. Studies have shown that it can inhibit hyaluronidase (existing technology literature 5). Clinical studies have shown that topical application of sodium copper chlorophyllin can improve skin photoaging and increase skin HA levels, suggesting that its mechanism of action is related to the inhibition of hyaluronidase (existing technology literature 6, 7). Given the key role of HYBID in dermal HA degradation, HYBID has become a target for anti-skin aging research. Certain plant extracts, such as Geranium thunbergii extract and Sanguisorba officinalis root extract, have been found to completely inhibit HA degradation in HYBID-stable HEK293 cells and normal human skin fibroblasts. These extracts not only inhibit HYBID-mediated HA degradation activity but also downregulate HYBID gene expression, leading to an increase in high molecular weight HA. Small-scale clinical trials have shown that these extracts have anti-wrinkle effects (existing technical literature 8, 9). The above studies fully demonstrate that using small molecule compounds or plant extracts to promote skin HA synthesis and / or reduce HA degradation can produce significant and beneficial anti-aging effects, representing a highly promising solution for the development of anti-aging skincare products.

[0010] Existing technical documents

[0011] Non-patent literature:

[0012] 1.Kafi R,Kwak HSR,Schumacher WE,et al.Improvement of naturally aged skin with vitamin A(retinol)[J].Archives of Dermatology,2007,143(5):606-612.

[0013] 2. Sayo T, Sugiyama Y, Inoue S. Lutein, a Nonprovitamin A, Activates the Retinoic Acid Receptor to Induce HAS3-Dependent Hyaluronan Synthesis in Keratinocytes [J]. Bioscience Biotechnology and Biochemistry, 2013, 77(6): 1282-1286.

[0014] 3.Endo Y,Yoshida H,Ota Y,et al.Accelerated human epidermal turnover driven by increased hyaluronan production[J].Journal of Dermatological Science,2021,101(2):123-133.

[0015] 4.Endo Y,Yoshida H,Akazawa Y,et al.Antiwrinkle efficacy of 1-ethyl-β-N-acetylglucosaminide,an inducer of epidermal hyaluronan production[J].Skin Research and Technology,2022,28(1):58-65.

[0016] 5.Mccook J P,Dorogi P L,Vasily D B,et al.In vitro inhibition of hyaluronidase by sodium copper chlorophyllin complex and chlorophyllin analogs[J].Clinical,cosmetic and investigational dermatology,2015,8:443-8.

[0017] 6.Mccook J P,Stephens T J,Jiang L I,et al.Ability of sodium copper chlorophyllin complex to repair photoaged skin by stimulation of biomarkers in human extracellular matrix[J].Clinical,cosmetic and investigational dermatology,2016,9:167-74.

[0018] 7.Sigler ML,Stephens T J.Assessment of the Safety and Efficacy of Topical Copper Chlorophyllin in Women With Photodamaged Facial Skin[J].Journal of Drugs in Dermatology,2015,14(4):401-404.

[0019] 8. YOSHIDA H, YAMAZAKI K, KOMIYA A, et al. Inhibition of HYBID(KIAA1199)-mediated hyaluronan degradation and anti-wrinkle effect of Geranium thunbergii extract[J]. Journal of Cosmetic Dermatology, 2019, 18(4): 1052-1060.

[0020] 9.YOSHIDA H, YAMAZAKI K, KOMIYA A, et al. Inhibitory effects of Sanguisorba officinalis root extract on HYBID(KIAA 1199)-mediated hyaluronan degradation and skin wrinkling[J]. International Journal of Cosmetic Science, 2019, 41(1):12-20. Summary of the Invention

[0021] The technical problem that the invention aims to solve

[0022] Based on the above understanding, the hyaluronic acid content in the dermis is related to the activity of hyaluronic acid synthase (HAS2) and hyaluronic acid degrading enzyme (HYBID). To increase the hyaluronic acid content in the dermis, it is necessary to find new substances that can promote the expression of hyaluronic acid synthase (HAS2) and inhibit the expression of hyaluronic acid degrading enzyme (HYBID). These substances, acting on skin fibroblasts, can increase the hyaluronic acid content in the dermis, thereby achieving anti-aging effects on the skin.

[0023] means of solving technical problems

[0024] In order to find new substances that can promote the expression of hyaluronic acid synthase (HAS2) and substances that can inhibit the expression of hyaluronic acid degrading enzyme (HYBID), the applicant conducted in-depth research and numerous experiments, and unexpectedly discovered that at least one of nerol, carvacrol, or geraniol can promote the expression of hyaluronic acid synthase (HAS2), and at least one of nerol, carvacrol, agarwood essential oil, or sandalwood essential oil can inhibit the expression of hyaluronic acid degrading enzyme (HYBID), thus completing this invention.

[0025] Specifically, the present invention includes the following solutions:

[0026] 1. A method for regulating the synthesis and degradation of hyaluronic acid by fibroblasts, characterized in that it comprises using at least one selected from nerol, carvacrol, geraniol, agarwood oil, and sandalwood oil.

[0027] 2. The method described in 1 above, characterized in that it includes using at least one of a hyaluronic acid synthase (HAS2) expression promoter or a hyaluronic acid degrading enzyme (HYBID) expression inhibitor, wherein the hyaluronic acid synthase (HAS2) expression promoter comprises at least one selected from nerol, carvacrol, or geraniol, and the hyaluronic acid degrading enzyme (HYBID) expression inhibitor comprises at least one selected from nerol, carvacrol, agarwood essential oil, or sandalwood essential oil.

[0028] 3. A method for combating skin aging, comprising applying a cosmetic to the skin containing at least one selected from nerol, carvacrol, geraniol, agarwood essential oil, and sandalwood essential oil.

[0029] 4. The use of at least one of nerol, carvacrol, geraniol, agarwood essential oil, and sandalwood essential oil in the preparation of cosmetics with anti-aging effects.

[0030] 5. Use of nerol, carvacrol or geraniol in the preparation of hyaluronic acid synthase expression promoters.

[0031] 6. Use of nerol, carvacrol, agarwood oil or sandalwood oil in the preparation of hyaluronic acid degrading enzyme expression inhibitors.

[0032] Invention Effects

[0033] The inventors of this invention have discovered for the first time that at least one of nerol, carvacrol, or geraniol can promote the expression of hyaluronic acid synthase (HAS2), and at least one of nerol, carvacrol, agarwood oil, or sandalwood oil can inhibit the expression of hyaluronic acid degrading enzyme (HYBID). Therefore, by using at least one of nerol, carvacrol, geraniol, agarwood oil, or sandalwood oil, the expression of hyaluronic acid synthase (HAS2) or hyaluronic acid degrading enzyme (HYBID) can be promoted or inhibited, thereby increasing the hyaluronic acid content in the dermis. Furthermore, by applying a cosmetic containing at least one of nerol, carvacrol, geraniol, agarwood oil, or sandalwood oil to the skin, the expression of hyaluronic acid synthase (HAS2) in skin fibroblasts can be promoted or the expression of hyaluronic acid degrading enzyme (HYBID) can be inhibited, thus increasing the hyaluronic acid content in the dermis and achieving an anti-aging effect. Attached Figure Description

[0034] Figure 1 shows the effect of different doses of the oxidant tert-butyl hydroperoxide (t-BHP) on the inhibition of human dermal fibroblast (HDF) proliferation.

[0035] Figure 2 shows the cell morphology of human dermal fibroblasts (HDF) after treatment with different doses of the oxidant tert-butyl hydroperoxide (t-BHP).

[0036] Figure 3 shows the effect of different doses of the oxidant tert-butyl hydroperoxide (t-BHP) on the expression of SASP factor in human dermal fibroblasts (HDF).

[0037] Figure 4 shows the effect of the oxidant tert-butyl hydroperoxide (t-BHP) on the expression of SA-β-gal in human dermal fibroblasts (HDF).

[0038] Figure 5 shows the effect of candidate aromatic compounds on the viability of human dermal fibroblasts (HDF).

[0039] Figure 6 shows the effect of candidate aromatic compounds on the inhibition of human dermal fibroblast (HDF) proliferation by the oxidant tert-butyl hydroperoxide (t-BHP).

[0040] Figure 7 shows the effect of nerol on the senescence induced by the oxidant tert-butyl hydroperoxide (t-BHP) in HDF cells.

[0041] Figure 8 shows the effect of geraniol on the senescence of HDF cells induced by the oxidant tert-butyl hydroperoxide (t-BHP).

[0042] Figure 9 shows the effect of carvacrol on the senescence of HDF cells induced by the oxidant tert-butyl hydroperoxide (t-BHP).

[0043] Figure 10 shows the effect of agarwood oil on the senescence induced by the oxidant tert-butyl hydroperoxide (t-BHP) in HDF cells.

[0044] Figure 11 shows the effect of sandalwood oil on the senescence of HDF cells induced by the oxidant tert-butyl hydroperoxide (t-BHP).

[0045] Figure 12 shows the effect of nerol on the expression of SASP factor in senescent HDF cells induced by the oxidant tert-butyl hydroperoxide (t-BHP).

[0046] Figure 13 shows the effect of geraniol on the expression of SASP factor in senescent HDF cells induced by the oxidant tert-butyl hydroperoxide (t-BHP).

[0047] Figure 14 shows the effect of agarwood oil on the expression of SASP factor in senescent HDF cells induced by the oxidant tert-butyl hydroperoxide (t-BHP).

[0048] Figure 15 shows the effect of sandalwood oil on the expression of SASP factor in senescent HDF cells induced by the oxidant tert-butyl hydroperoxide (t-BHP).

[0049] Figure 16 shows the effect of aromatic compounds on the expression of HYBID in HDF cells stimulated by the oxidant tert-butyl hydroperoxide (t-BHP).

[0050] Figure 17 shows the effect of aromatic compounds on HAS2 expression in HDF cells stimulated by the oxidant tert-butyl hydroperoxide (t-BHP).

[0051] Figure 18 shows the effect of aromatic substances on the secretion of HA in senescent HDF cells induced by the oxidant tert-butyl hydroperoxide (t-BHP). Detailed Implementation

[0052] The present invention will now be described in detail. However, the present invention is not limited to the specific embodiments listed below. Various modifications can be made to the present invention without departing from its spirit.

[0053] To achieve the objectives of this invention, and to identify new substances that can promote the expression of hyaluronic acid synthase (HAS2) and inhibit the expression of hyaluronic acid degrading enzyme (HYBID), the inventors selected a variety of aromatic substances as candidate compounds, established cell models, and screened their protective effects against oxidative damage and cell aging, as well as their regulatory effects on HAS expression and HA synthesis, in a t-BHP-induced dermal fibroblast aging model, in order to discover skin care ingredients with anti-aging effects.

[0054] The specific filtering method is as follows:

[0055] (A) Select candidate aromatic substances.

[0056] (B) Establish an in vitro skin aging cell model.

[0057] (C) In the skin aging cell model established above, we studied the protective effects of candidate aromatic substances on t-BHP-induced oxidative damage and cells, as well as their effects on HAS expression and HA synthesis.

[0058] The present invention will now be described in more detail, but the present invention is not limited thereto. Various modifications can be made to the present invention without departing from the concept of the present invention.

[0059] (A) Select candidate aromatic substances.

[0060] In this invention, 12 aromatic substances, including nerol, were selected as candidate compounds, as detailed below.

[0061] 1. Nerol

[0062] Neroli is a monoterpene alcohol naturally found in plant essential oils such as neroli oil, citrus oil, citronella oil, and rose oil. It is an isomer of geraniol. Neroli has a pleasant aroma of rose and orange blossom, with a slight lemony fruitiness. Its aroma is softer and relatively lighter than geraniol, with a fresh and citrus note. Neroli is widely used as a fragrance ingredient in beverages, food, perfumes, and cosmetics. Modern pharmacological studies have found that neroli possesses various pharmacological activities, including anti-inflammatory, antioxidant, analgesic, anticancer, antiarrhythmic, hepatoprotective, antispasmodic, and antibacterial effects. Studies in an oxazolone-induced mouse model of ulcerative colitis have shown that neroli has significant analgesic and anti-inflammatory effects.

[0063] 2. Geraniol

[0064] Geraniol is a natural monoterpene compound found in many fruits, vegetables, and traditional Chinese medicines, including lavender, lemongrass, and other medicinal and aromatic plants. Geraniol has a pleasant rose aroma and is a key ingredient in rose-based fragrances. It is an indispensable flavoring ingredient in various floral fragrances and is widely used in the cosmetics and food industries. Geraniol possesses a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, antibacterial, and neuroprotective activities. Geraniol exhibits strong antioxidant activity, scavenging oxygen free radicals and activating the Nrf2 antioxidant signaling pathway in various oxidative stress cell and animal models, increasing the expression and activity of antioxidant enzymes. Geraniol prophylactic treatment has a protective effect against doxorubicin-induced kidney injury in rats, reducing doxorubicin-induced kidney damage and lipid peroxidation, increasing the expression of catalase, reduced glutathione, glutathione peroxidase, and superoxide dismutase, and preventing doxorubicin-mediated renal dysfunction. Dietary supplementation with geraniol can improve age-related neuroinflammation and oxidative stress damage in rats. Recent studies have found that geraniol has an antipruritic effect; intraperitoneal injection of geraniol in mice effectively reduced acute pruritus induced by chloroquine and compound 48 / 80; intrathecal administration of geraniol inhibited pruritus induced by the GABAA receptor inhibitor bicuculline, GRP-induced pruritus, and GABAergic neuron inhibition. Furthermore, geraniol can attenuate chronic pruritus induced by diphenylcyclopropenone (DCP) and acetone-ether-water (AEW) application. Geraniol has shown good clinical efficacy in treating chronic bronchitis, not only improving lung ventilation and reducing airway resistance but also enhancing the body's immune function. Geraniol soft capsules are currently available for the prevention and treatment of tracheal and bronchial inflammation.

[0065] 3. Thymol

[0066] Thymol, also known as thymol or thymic phenol, is a natural monoterpenoid phenolic compound widely found in plants of the Lamiaceae family (thyme, basil, oregano, and mint) and other plants (such as those of the Verbenaceae, Scrophulariaceae, Ranunculaceae, and Apiaceae families). Thymol is the main component of thyme essential oil, with a content that can be as high as 50%. Thymol possesses various biological activities, including antibacterial, antiviral, antioxidant, anti-inflammatory, analgesic, wound-healing, and anticancer properties. Thymol and thyme essential oil are widely used in medicine, cosmetics, and food, and in traditional medicine as an expectorant for the treatment of upper respiratory tract diseases.

[0067] 4. Carvacrol

[0068] Carvacrol is a monoterpene phenol, an isomer of thymol, and is commonly found in the volatile oils of plants such as thyme, oregano, winter peppermint, and chamomile. Carvacrol has a pleasant thymol aroma and is often used as a food additive and flavoring ingredient in food, cosmetics, and other daily necessities. It is also used as an antibacterial agent and preservative for food preservation. Carvacrol has no significant toxic side effects on humans and does not pollute the environment; the European Union has rated its safety as "generally recognized as safe (GRAS)". Carvacrol possesses strong pharmacological activity and health benefits, including anti-inflammatory, antioxidant, analgesic, blood sugar regulating, cardiovascular protective, hypotensive, neuroprotective, hepatoprotective, wound healing, antibacterial, and antiparasitic activities.

[0069] 5. Cyclamen aldehyde

[0070] Cyclamenaldehyde, also known as cyclamen aldehyde, is an important synthetic flavoring agent. It is a food flavoring permitted for use under my country's GB2760-96 "Hygienic Standard for the Use of Food Additives." It possesses a unique, sweet aroma of fresh fruit and can be blended with many types of flavorings. It can be used to formulate melon and citrus flavorings, as well as floral-scented daily chemical fragrances, and is widely used in cosmetics, food, and various detergents. Currently, research on the bioactivity of cyclamen aldehyde is extremely limited; however, some studies have found that it is a natural component of rose essential oil.

[0071] 6. Hedione (methyl dihydrojasmonic acid)

[0072] Methyl dihydrojasmonate (MDJ) is obtained by hydrogenation of methyl jasmonate extracted from jasmine essential oil, and is one of the important synthetic jasmine fragrances. Due to its elegant and soft jasmine scent, long-lasting fragrance, and good perfumery properties, it is now widely used in the formulation of synthetic jasmine essential oil, tuberose essential oil, etc., and is also routinely used in the formulation of perfumes and daily cleaning products.

[0073] 7. Octadecaldehyde (Aldehyde C18)

[0074] Octadecaldehyde, also known as γ-nonalactone and coconut aldehyde, possesses characteristic aromas of coconut, peach, and milk, with a mellow fragrance. It is widely used in the blending of various floral and fruity flavorings. In my country, GB2760-86 specifies it as a permitted edible flavoring for the formulation of coconut, milk, and cream-based flavorings. γ-nonalactone can activate TRPA1, acting as a TRPV1 agonist or antagonist at different concentrations. It can inhibit capsaicin-mediated TRPV1 responses, indicating that lactones in food not only impart aroma but also regulate the spiciness and irritation of food by acting on TRPV1 and TRPA1.

[0075] 8. Aldehyde (C14)

[0076] Tetradecanoic acid, also known as peach aldehyde and γ-undecalactone, has a peach aroma and is used to formulate fruity and floral fragrances such as peach, plum, cherry, apricot, and coconut.

[0077] 9. Dihydromyrcenol

[0078] Dihydromyrcene alcohol has a white lemon and cologne-like aroma. Due to its unique aroma quality, dihydromyrcene alcohol has been widely used in fragrance formulations. A concentration of 0.1%-0.5% in fragrances can produce a fresh, powerful, and ethereal scent, and its aroma exhibits good stability in soaps and detergents.

[0079] 10. Atlantic Cedarwood Oil Atlas

[0080] Atlantic cedarwood oil, also known as cedarwood oil, is derived from cedar (also called Himalayan cedar, Himalayan fir, or fragrant cedar). Cedarwood is an important aromatic medicinal plant in the cedar genus, traditionally used to treat convulsions, fever, cough, bronchitis, tuberculosis, joint pain, skin diseases, and ulcers. Cedarwood oil is extracted from the xylem of cedar trees, primarily from Atlantic cedar (Cedrus atlantica). It mainly contains α-cedrene, β-cedrene, α-pinene, β-pinene, cedrol, and atlantone, possessing various pharmacological activities including anti-inflammatory, analgesic, antioxidant, invigorating, antibacterial, antimalarial, and anthelmintic properties. The main components, cedrol and α-cedrene, can improve obesity and metabolic abnormalities induced by a high-fat diet in mice. Cedarwood oil also has pore-tightening properties and antibacterial activity, potentially beneficial for oily skin and improving acne. Furthermore, cedarwood oil is claimed to stimulate hair follicles by increasing scalp blood circulation, promoting hair growth and slowing hair loss, potentially aiding in the treatment of various types of hair loss. However, there is a lack of experimental evidence to support these skincare benefits of cedarwood oil.

[0081] 11. Agarwood Essential Oil

[0082] Agarwood is the resinous woody part of plants belonging to the genus *Aquilaria* of the family Thymelaeaceae, formed under natural or artificial conditions. It has an aromatic fragrance, and the process of resin formation is called resin formation. Only when *Aquilaria* plants are subjected to natural factors (lightning strikes, fire, microbial invasion, etc.) or human factors (cutting, drilling, inoculation, etc.) can they produce various secondary metabolites at the site of injury. These products, after a complex resin formation process, are deposited in the xylem to form agarwood. Currently, the most common types of agarwood on the market include agarwood formed using the whole-stem breaking method (referred to as "board agarwood"), agarwood formed using the whole-body resin formation technique (referred to as "whole-body agarwood"), and agarwood formed using the drilling method (referred to as "Qinan agarwood"). Agarwood has the effects of promoting qi circulation and relieving pain, warming the stomach and stopping vomiting, and calming asthma. It is widely used to treat arthritis, asthma, rheumatism, and gout, and is a valuable traditional Chinese medicine.

[0083] Agarwood essential oil is the most valuable bioactive substance in agarwood, and it has the effects of promoting sleep, calming, anti-oxidation, anti-inflammation, lowering blood lipids, antibacterial and anti-tumor properties.

[0084] 12. Sandalwood album oil (SAO)

[0085] Sandalwood is the dried heartwood of the sandalwood tree (Santalum album L.), a plant belonging to the genus Santalum of the Santalaceae family. It is believed to have effects such as promoting qi circulation, relieving pain, dispelling cold, and regulating the middle jiao (digestive system). Sandalwood album oil (SAO), also known as East Indian sandalwood oil, is an essential oil extracted from the sandalwood tree. It mainly contains sesquiterpenoid compounds such as santalol (α-santalol and β-santalol). There are more than a dozen species of sandalwood trees worldwide, most of which can be used to make essential oils. However, the International Organization for Standardization (ISO) has only published standards for two species: sandalwood (Santalum album) and Western Australian sandalwood (Santalum spicatum). The essential oil produced from Santalum album contains a higher concentration of α- and β-santalol.

[0086] Sandalwood essential oil possesses calming, antioxidant, anti-inflammatory, anti-psoriatic, anti-skin tumor, and keratinocyte proliferation-inhibiting activities. In aromatherapy, topical application of sandalwood essential oil can treat allergies, eczema, abscesses, and dermatitis caused by dry, cracked skin. Sandalwood essential oil mediates its anti-inflammatory and antioxidant effects through multiple mechanisms; it also exhibits antibacterial, antiviral, and anti-skin tumor activity.

[0087] (B) Establishing a skin aging cell model

[0088] Oxidative stress plays a crucial role in both endogenous and exogenous skin aging. Oxidative stress induced by the accumulation of reactive oxygen species (ROS) can lead to damage to lipids, proteins, nucleic acids, and organelles, resulting in cellular senescence, which is one of the core mechanisms mediating skin aging. Tert-butyl hydroperoxide (t-BHP) is an organic peroxide that is relatively stable in vitro. Once inside cells, it is metabolized by cytochrome P450 to produce hydrogen peroxide and alkoxy radicals, leading to rapid oxidative depletion of intracellular glutathione (GSH) and ultimately oxidative damage to cells. Therefore, t-BHP is commonly used in experimental studies as an exogenous oxidant to induce oxidative stress damage and cellular senescence in cells and tissues. Several previous studies have also used t-BHP to induce senescence in dermal fibroblasts.

[0089] In previous studies, the inventors discovered that t-BHP could induce skin cell aging in a short time (2h) with good reproducibility. Therefore, the invention also uses t-BHP as an inducer of dermal fibroblast aging to create a cell model of skin aging. Based on this cell model, aromatic compounds that can effectively increase HA synthesis and thus antagonize oxidative stress and cell aging are further screened.

[0090] 1. The inhibitory effect of different doses of t-BHP on the proliferation of human dermal fibroblasts (HDF).

[0091] Loss of proliferative capacity is the most important phenotypic feature of cell senescence. Therefore, the inventors first verified the effect of different doses of t-BHP on inhibiting the proliferation of human dermal fibroblasts in order to determine the experimental conditions for t-BHP-induced oxidative damage and cell senescence in fibroblasts.

[0092] Therefore, human dermal fibroblasts (HDF) were treated with different concentrations of t-BHP for 2 hours, followed by incubation in complete medium without t-BHP for another 24 hours. HDF cell viability was then assessed using the CCK8 (Cell Counting Kit-8) method. The results showed that lower concentrations of t-BHP (below 100 μM) had no significant effect on HDF cell viability after 2 hours, but higher doses (above 150 μM) dose-dependently inhibited cell viability, indicating that higher doses of t-BHP (above 150 μM) can inhibit cell proliferation or exhibit cytotoxicity (Figure 1).

[0093] The morphology of HDF cells treated with t-BHP for 2 hours and then cultured for another 24 hours was observed under an inverted microscope, and photomicrographs were taken. The results are shown in Figure 2. It can be seen that t-BHP concentrations below 100 μM did not significantly affect the cell morphology of HDF cells, but higher doses above 150 μM led to significant changes in cell morphology, with cells becoming flattened and enlarged. In the group with doses above 400 μM, the number of suspended and dead cells increased, indicating that higher doses of t-BHP above 150 μM can inhibit cell proliferation or have cytotoxic effects.

[0094] 2. The role of t-BHP in inducing SASP and SA-β-gal expression in HDF

[0095] Besides proliferation arrest, the continuous secretion of SASP factors such as matrix metalloproteinases (MMPs) and the specific expression of senescence-associated β-galactosidase (SA-β-gal) are two other important phenotypic features of cellular senescence. Therefore, the inventors further investigated the effects of different doses of t-BHP on the expression of SASP factors such as MMP-1, MMP-3, and MMP-9 and SA-β-gal in HDF cells to determine the experimental conditions for t-BHP-induced oxidative damage and cellular senescence in fibroblasts.

[0096] HDF cells were incubated with different concentrations of t-BHP (100, 200, 300 μM) for 2 h, and then incubated for another 12 h with complete medium without t-BHP. The mRNA expression levels of SASP factors (MMP-1, MMP-3, and MMP-9) were detected by qPCR. The results showed that t-BHP could increase the mRNA expression levels of MMP-1, MMP-3, and MMP-9 in HDF cells in the dose range of 100-300 μM, and its effect was strongest at a dose of 200 μM (Figure 3).

[0097] HDF cells were incubated with 200 μM t-BHP for 2 h, and then incubated for another 12 h with complete medium without t-BHP. Cells were stained with SPiDER-βgal (a specific fluorescent substrate for SA-β-gal) and Hoechst (a specific staining agent for cell nuclei) to label SA-β-gal positive senescent cells (green fluorescence) and cell nuclei (blue fluorescence), respectively. The results showed that t-BHP could significantly increase the number of SA-β-gal positive (green fluorescence) senescent cells (Figure 4).

[0098] The above experimental results show that treating HDF cells with 200 μM t-BHP for 2 h followed by culturing in complete medium for another 12 h can significantly increase the number of SA-β-gal positive cells (Figure 4) and induce the expression of SASP factors such as MMP-1, MMP-3 and MMP-9 (Figure 3). Moreover, it can significantly inhibit HDF cell proliferation (Figure 1), indicating that treating HDF cells with 200 μM t-BHP for 2 h can induce cell senescence, and therefore can be used as the optimal experimental condition for constructing an HDF cell senescence model.

[0099] (C) In the skin aging cell model established above, we studied the protective effects of candidate aromatic substances on t-BHP-induced oxidative damage and cells, as well as their effects on HAS expression and HA synthesis.

[0100] After establishing a skin aging cell model as described in (B) above, the effects of the above 12 candidate aromatic substances on t-BHP-induced oxidative damage and cell protection, as well as their effects on HAS expression and HA synthesis, were studied on the established skin aging cell model.

[0101] After screening and verifying through cell models the substances that can promote the expression of hyaluronic acid synthase (HAS2) and the substances that can inhibit the expression of hyaluronic acid degrading enzyme (HYBID), these screened active substances can be used as anti-aging ingredients to prepare topical skin agents to obtain topical skin agents with anti-aging effects.

[0102] The term "topical skin preparation" as used in this invention refers to any preparation applied externally to the skin, including but not limited to cosmetics, such as aqueous solutions like toners, lotions, moisturizing lotions, and repairing lotions; spray preparations like moisturizing sprays, anti-wrinkle sprays, and repairing sprays; lotion preparations like moisturizing lotions, anti-wrinkle lotions, repairing lotions, body lotions, and hand lotions; essence preparations like moisturizing serums, anti-wrinkle serums, and repairing serums; gel preparations like moisturizing gels, anti-wrinkle gels, and repairing gels; creams or ointments like face creams, eye creams, moisturizing creams, anti-wrinkle creams, repairing creams, massage creams, hand creams, and body creams; and powders like setting powder, loose powder, pressed powder, and talcum powder.

[0103] The content of substances that promote the expression of hyaluronic acid synthase (HAS2) and substances that inhibit the expression of hyaluronic acid degrading enzyme (HYBID) in topical skin preparations is generally 0.01% to 3%, more preferably 0.1% to 1.5%, and even more preferably 0.5% to 1.2%.

[0104] In addition to the substances screened above that can promote the expression of hyaluronic acid synthase (HAS2) and the substances that can inhibit the expression of hyaluronic acid degrading enzyme (HYBID) as effective ingredients for skin barrier repair agents, the topical skin preparation of the present invention may contain excipients, carriers and other additives, and may appropriately use ingredients commonly added to cosmetics as needed.

[0105] Commonly added ingredients in cosmetics include, but are not limited to, aqueous solvents, oily components, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, water-soluble polymers, thickeners, emulsifiers, preservatives, UV absorbers, metal ion blocking agents, amino acids, organic amines, polymeric emulsions, pH adjusters, neutralizers, skin nutrients, vitamins, antioxidants, antioxidant auxiliaries, fragrances, etc.

[0106] Examples of aqueous solvents include water, alcohols, humectants, or mixtures thereof.

[0107] As water, the water used in cosmetics, pharmaceuticals, etc., can be used, such as purified water, ion-exchanged water, tap water, etc. Depending on the purpose, the aqueous phase may further contain water-soluble alcohols.

[0108] As a water-soluble alcohol, examples include at least one selected from the following: lower alcohols, polyols, polyol polymers, diol alkyl ethers, diol ether esters, glycerol monoalkyl ethers, sugar alcohols, monosaccharides, oligosaccharides, polysaccharides and their derivatives.

[0109] Examples of lower alcohols include ethanol, propanol, isopropanol, isobutanol, tert-butanol, etc.

[0110] Examples of polyols include: diols (e.g., ethylene glycol, propylene glycol, trimethylene glycol, 1,2-butanediol, 1,3-butanediol, tetramethylenediol, 2,3-butanediol, pentamethylenediol, 2-buten-1,4-diol, hexanediol, octanediol, etc.); triols (e.g., glycerol, trimethylolpropane, etc.); tetraols (e.g., pentaerythritol, etc.); pentaols (e.g., xylitol, etc.); and hexaols (e.g., sorbitol, mannitol, etc.).

[0111] Examples of polyol polymers include diethylene glycol, dipropylene glycol, triethylene glycol, polypropylene glycol, tetraethylene glycol, diglycerol, triglycerol, and tetraglycerol.

[0112] Examples of diol alkyl ethers include, for instance, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, ethylene glycol monophenyl ether, ethylene glycol monohexyl ether, ethylene glycol mono-2-methylhexyl ether, ethylene glycol isopentyl ether, ethylene glycol benzyl ether, ethylene glycol isopropyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol dibutyl ether, etc.; diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, diethylene glycol butyl ether, diethylene glycol methyl ethyl ether, triethylene glycol monomethyl ether, triethylene glycol monoethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol monobutyl ether, propylene glycol isopropyl ether, dipropylene glycol methyl ether, dipropylene glycol ethyl ether, dipropylene glycol butyl ether, etc.

[0113] Examples of diol ether esters include, for example, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether acetate, ethylene glycol monobutyl ether acetate, ethylene glycol monophenyl ether acetate, ethylene glycol diadipate, ethylene glycol disuccinate, diethylene glycol monoethyl ether acetate, diethylene glycol monobutyl ether acetate, propylene glycol monomethyl ether acetate, propylene glycol monoethyl ether acetate, propylene glycol monopropyl ether acetate, and propylene glycol monophenyl ether acetate.

[0114] Examples of glycerol monoalkyl ethers include squalene, squalene, and squalene.

[0115] Examples of sugar alcohols include sorbitol, maltitol, maltotriose, mannitol, sucrose, erythritol, glucose, fructose, starch-degrading sugars, maltose, xylose, starch-degrading sugar reducing alcohols, etc.; tetrahydrofurfuryl alcohol; POE-tetrahydrofurfuryl alcohol; POP-butyl ether; POP·POE-butyl ether; trimeroxypropylene glycerol ether; POP-glycerol ether; POP-glycerol ether phosphate; POP·POE-pentaerythritol ether, etc.

[0116] As monosaccharides, examples include at least one selected from the following: trioses (e.g., D-glyceraldehyde, dihydroxyacetone, etc.), tetraoses (e.g., D-erythrose, D-erythulose, D-threose, erythritol, etc.), pentoses (e.g., L-arabinose, D-xylose, L-lythose, D-arabinose, D-ribose, D-ribulose, D-xylulose, L-xylulose, etc.), and hexoses (e.g., D-glucose, D-tarose, D-alulose, D-galactose, D-...). Fructose, L-galactose, L-mannose, D-tagatose, etc.), heptoses (e.g., heptanose, heptanose), octoses (e.g., octanose), deoxyglucoses (e.g., 2-deoxy-D-ribose, 6-deoxy-L-galactose, 6-deoxy-L-mannose), aminoglucoses (e.g., D-glucosamine, D-galactosamine, sialic acid, muramic acid, etc.), uronic acids (e.g., D-glucuronic acid, L-guluronic acid, D-galacturonic acid, L-iduronic acid, etc.), etc.

[0117] As oligosaccharides, examples include at least one of the following: sucrose, umbelliferous sugar, lactose, psyllium, α,α-trehalose, etc.

[0118] As polysaccharides, examples include at least one of the following: cellulose, quince seed, chondroitin sulfate, starch, galactan, dermatan sulfate, glycogen, gum arabic, heparan sulfate, hyaluronic acid, tragacanth gum, keratin sulfate, chondroitin, xanthan gum, mucin sulfate, guar gum, dextran, locust bean gum, succinyl polysaccharide, carnosic acid, etc.

[0119] Other polyols include, for example, at least one selected from polyoxyethylene methyl glucoside (Glucam E-10), polyoxypropylene methyl glucoside (Glucam P-10), etc.

[0120] Examples of moisturizers include: propylene glycol, glycerin, 1,3-butanediol, dipropylene glycol, xylitol, sorbitol, maltitol, chondroitin sulfate, hyaluronic acid, mucin sulfate, carboxylic acid, determinate collagen, sodium lactate, bile salts, dl-pyrrolidone carboxylate, epoxide derivatives, short-chain soluble collagen, diglyceride (EO)PO adduct, prickly pear extract, yarrow extract, sweet clover extract, etc.

[0121] As an oily component, examples include liquid fats, solid fats, waxes, hydrocarbons, higher fatty acids, higher alcohols, synthetic ester oils, silicone oils, etc. It should be noted that in this specification, oil and oil-soluble components are also included and referred to as "oily components".

[0122] Examples of liquid oils include: avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, egg yolk oil, sesame oil, peach kernel oil (almond oil), wheat germ oil, camellia oil, castor oil, flaxseed oil, safflower oil, cottonseed oil, perilla seed oil, soybean oil, peanut oil, tea seed oil, torreya nut oil, rice bran oil, Chinese tung oil, Japanese tung oil, jojoba oil, wheat germ oil, triglycerides, etc.

[0123] Examples of solid fats include: cocoa butter, coconut oil, horse oil, hydrogenated coconut oil, palm oil, tallow, mutton tallow, hydrogenated tallow, palm kernel oil, lard, beef bone fat (oil), wood wax kernel oil, hydrogenated oil, beef foot fat, wood wax, hydrogenated castor oil, etc.

[0124] Examples of waxes include: beeswax, candelilla wax, cotton wax, carnauba wax, bayberry wax, insect wax (white wax), whale wax, lignite wax, rice bran wax, lanolin, kapok wax, lanolin acetate, liquid lanolin, sugarcane wax, lanolin fatty acid isopropyl ester, hexyl laurate, reduced lanolin, jojoba wax, hard lanolin, shellac wax, POE lanolin alcohol ether, POE lanolin alcohol acetate, POE cholesterol ether, lanolin fatty acid polyethylene glycol ester, POE hydrogenated lanolin alcohol ether, etc.

[0125] Examples of hydrocarbon oils include: liquid paraffin, ceresin, squalane, pterostilbene, paraffin wax, pure ceresin, squalene, petrolatum, microcrystalline wax, etc.

[0126] Examples of high-grade fatty acids include: lauric acid, myristic acid, palmitic acid, stearic acid, benzolic acid, oleic acid, undecenoic acid, tall acid, isostearic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

[0127] Examples of higher alcohols include: straight-chain alcohols (e.g., lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol, cetearyl alcohol, etc.); branched-chain alcohols (e.g., monostearate glyceryl ether (squalane), 2-decyltetradecanool, lanolin alcohol, cholesterol, phytosterol, hexyldodecanool, isostearyl alcohol, octyldodecanool, etc.).

[0128] Synthetic ester oils include: hydrogenated polydecene, isopropyl myristate, cetyl octanoate, octyl dodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyl decyl dimethyl octanoate, cetyl lactate, myristyl lactate, lanolin acetate, isocetyl stearate, isocetyl isostearate, 12-hydroxystearate cholesterol ester, ethylene glycol di-2-ethylhexanoate, dipentaerythritol fatty acid ester, N-alkyl ethylene glycol monoisostearate, neopentyl glycol didecanoate, diisostearate malate, glyceryl di-2-heptyl undecanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, tetra-2-ethylhexanoate. Pentaerythritol ester, tri-2-ethylhexanoate glyceryl ester, tricaprylic acid glyceryl ester, triisopalmitoate glyceryl ester, trimethylolpropane triisostearate, cetyl ethylhexanoate, 2-ethylhexyl palmitate, trimyristic acid glyceryl ester, tri-2-heptylundecanoate glyceryl ester, castor oil fatty acid methyl ester, oleic acid ester, acetylglycine ester, 2-heptylundecyl palmitate, diisobutyl adipate, N-lauroyl-L-glutamic acid-2-octyldodecyl ester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexyl sebacate, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, diisopropyl sebacate, 2-ethylhexyl succinate, triethyl citrate, etc.

[0129] Examples of silicone oils include: polydimethylsiloxane, methylhydropolysiloxane, methylphenylpolysiloxane, stearoyloxymethylpolysiloxane, polyether-modified organopolysiloxane, fluoroalkyl·polyoxyalkylene co-modified organopolysiloxane, alkyl-modified organopolysiloxane, terminal-modified organopolysiloxane, fluorinated organopolysiloxane, amino-modified organopolysiloxane, PEG-10 polydimethylsiloxane, silicone, acrylic polysiloxane, trimethylsiloxysilicate, organosilicon RTV rubber, and other organosilicon compounds.

[0130] As an oil used in conjunction with hydrogenated polyisobutylene, it is preferable to use an oil that has poor compatibility with hydrogenated polyisobutylene and is volatile. In this way, during application, the volatile oil will evaporate, and due to the poor compatibility, the hydrogenated polyisobutylene will remain on the skin to form a film, bringing a firming sensation.

[0131] Examples of anionic surfactants include: fatty acid soaps (e.g., sodium lauryl sulfate, sodium palmitate, etc.); higher alkyl sulfate salts (e.g., sodium lauryl sulfate, potassium lauryl sulfate, etc.); alkyl ether sulfate salts (e.g., POE-triethanolamine lauryl sulfate, sodium POE-sodium lauryl sulfate, etc.); N-acylsarcosine (e.g., sodium lauroylsarcosine, etc.); higher fatty acid amide sulfonates (e.g., sodium N-myristoyl-N-methyl taurate, sodium coconut oil fatty acid methyl taurate, sodium lauryl methyl taurate, etc.); phosphate salts (POE-oleyl ether phosphate, POE-stearyl ether phosphate, etc.); sulfosuccinates (e.g., sodium di-2-ethylhexyl sulfosuccinate, sodium monolauroyl monoethanolamide polyoxyethylene sulfosuccinate, sodium lauryl polypropylene glycol sulfosuccinate). Sodium succinate, etc.; alkylbenzene sulfonates (e.g., sodium linear dodecylbenzene sulfonate, triethanolamine linear dodecylbenzene sulfonate, linear dodecylbenzene sulfonic acid, etc.); higher fatty acid ester sulfates (e.g., sodium hydrogenated coconut oil fatty acid glyceride sulfate, etc.); N-acylglutamates (e.g., monosodium N-lauroylglutamate, disodium N-stearoylglutamate, monosodium N-myristoyl-L-glutamate, etc.); sulfated oils (e.g., Turkish red oil, etc.); POE-alkyl ether carboxylic acids, POE-alkyl allyl ether carboxylates, α-olefin sulfonates, higher fatty acid ester sulfonates, secondary alcohol sulfates, higher fatty acid alkanolamide sulfates, sodium lauroyl monoethanolamide succinate, N-palmitoyl aspartic acid di-triethanolamine, sodium caseinate, potassium cetyl phosphate, etc.

[0132] Examples of cationic surfactants include: alkyl trimethylammonium salts (e.g., stearyltrimethylammonium chloride, lauryltrimethylammonium chloride, etc.); alkyl pyridinium salts (e.g., cetylpyridinium chloride, etc.); distearate dimethylammonium dialkyl dimethylammonium salt; poly(N,N”-dimethyl-3,5-methylenepiperidinium chloride); alkyl quaternary ammonium salts; alkyl dimethyl benzylammonium salts; alkyl isoquinoline onion salts; dialkyl morpholine onion salts; POE-alkylamines; alkylamine salts; polyamine fatty acid derivatives; pentanol fatty acid ester derivatives; benzalkonium chloride; benzyl chloride, etc.

[0133] Examples of amphoteric surfactants include: imidazoline amphoteric surfactants (e.g., sodium 2-undecyl-N,N,N-(hydroxyethylcarboxymethyl)-2-imidazoline, disodium 2-cocoyl-2-imidazoline hydroxide-1-carboxyethoxy, etc.); betaine surfactants (e.g., 2-heptadecyl-N-carboxymethyl-N-hydroxyethylimidazoline betaine, lauryl dimethylaminoacetic acid betaine, alkyl betaine, amide betaine, sulfobetaine, etc.).

[0134] Examples of lipophilic nonionic surfactants include: sorbitol fatty acid esters (e.g., sorbitol monooleate, sorbitol monoisostearate, sorbitol monolaurate, sorbitol monopalmitate, sorbitol monostearate, sorbitol sesquioleate, sorbitol trioleate, sorbitol pent-2-ethylhexanoate diglyceride, sorbitol tetra-2-ethylhexanoate diglyceride, etc.); polyglycerol fatty acids. Glyceryl esters (e.g., glyceryl monostearate, glyceryl monoerucate, glyceryl sesquioleate, glyceryl monostearate, glyceryl pyroglutamic acid ester, glyceryl malate monostearate, etc.); propylene glycol fatty acid esters (e.g., propylene glycol monostearate, etc.); hydrogenated castor oil derivatives; glyceryl alkyl ethers; PEG-10 polydimethylsiloxane, PEG-5 glyceryl stearate, PPG-13-decyltetradecyl alcohol polyether-24, PEG-5 glyceryl stearate, etc.

[0135] Examples of hydrophilic nonionic surfactants include: POE-sorbitol fatty acid esters (e.g., POE-sorbitol monooleate, POE-sorbitol monostearate, POE-sorbitol monooleate, POE-sorbitol tetraoleate, etc.); POE-sorbitol fatty acid esters (e.g., POE-sorbitol monolaurate, POE-sorbitol monooleate, POE-sorbitol pentaoleate, POE-sorbitol monostearate, etc.); and POE-glycerol fatty acid esters (e.g., POE-glycerol monostearate). POE-glycerol monoisostearate, POE-glycerol triisostearate, etc.; POE-fatty acid esters (e.g., POE-distearate, POE-monodioleate, ethylene distearate, etc.); POE-alkyl ethers (e.g., POE-lauryl ether, POE-oleyl ether, POE-stearyl ether, POE-betaine ether, POE-2-octyldodecyl ether, POE-cholestanol ether, etc.); Pluronic type (e.g., Pluronic, etc.); POE·POP-alkyl ethers (e.g., POE·POP- Cetyl ether, POE·POP-2-decyltetradecyl ether, POE·POP-monobutyl ether, POE·POP-hydrogenated lanolin, POE·POP-glyceryl ether, etc.; tetra-POE·tetra-POP-ethylenediamine condensate (e.g., Tetronic, etc.); POE-caster oil hydrogenated castor oil derivatives (e.g., POE-caster oil, POE-hydrogenated castor oil, POE-hydrogenated castor oil monoisostearate, POE-hydrogenated castor oil triisostearate, POE-hydrogenated castor oil monopyroglutamic acid monoisostearate diester, POE-hydrogenated castor oil maleate). (etc.); POE-beeswax and lanolin derivatives (e.g., POE-sorbitol beeswax, etc.); alkanolamides (e.g., coconut oil fatty acid diethanolamide, lauric acid monoethanolamide, fatty acid isopropanolamide, etc.); POE-propylene glycol fatty acid esters; POE-alkylamines; POE-fatty acid amides; sucrose fatty acid esters; alkylethoxydimethylamine oxide; trioleyl phosphate; oleyl alcohol polyether-10, PEG-100 stearate, methoxy PEG / PPG-25 / 4 cetyl ethylhexanoate, polysorbate-60, PEG-40 stearate, sucrose stearate. Examples of natural water-soluble polymers include: plant-based polymers (e.g., gum arabic, tragacanth, galactomannan, guar gum, carob gum, ebony gum, carrageenan, pectin, agar, quince seeds, algal colloids (brown algae extracts), starch (rice, corn, potato, wheat), glycyrrhizic acid); microbial polymers (e.g., xanthan gum, dextran, succinyl dextran, succinyl polysaccharide, etc.); and animal-based polymers (e.g., collagen, casein, albumin, gelatin, etc.).

[0136] Examples of semi-synthetic water-soluble polymers include: starch polymers (e.g., carboxymethyl starch, methyl hydroxypropyl starch, etc.); cellulose polymers (methyl cellulose, ethyl cellulose, methyl hydroxypropyl cellulose, hydroxyethyl cellulose, sodium cellulose sulfate, hydroxypropyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, cellulose powder, etc.); alginate polymers (e.g., sodium alginate, propylene glycol alginate, etc.); PEG-240 / HDI copolymer bis-decyltetradecyl alcohol polyether-20; acrylate / C10-30 alkanol acrylate crosspolymers, etc.

[0137] Examples of thickeners include potassium hydroxide, acrylate / C10-30 alkanol acrylate crosspolymers, acrylate / C10-30 alkanol acrylate crosspolymers, sodium acrylate / sodium acryloyl dimethyl taurate copolymers, acrylate / hexadecyl ethoxy (20) itaconic acid ester copolymers, acrylate / hexadecyl ethoxy (20) methacrylate copolymers, acrylate / tetradecyl ethoxy (25) acrylate copolymers, acrylate / octadecyl ethoxy (20) itaconic acid ester copolymers, acrylate / octadecyl ethoxy (20) methacrylate copolymers, acrylate / octadecyl ethoxy (50) acrylate copolymers, acrylate / VA crosspolymers, PAA (polyacrylic acid), sodium acrylate / vinyl isodecanoate crosspolymers, Carbomer (polyacrylic acid) and its sodium salts, etc., which are polyacrylic acid thickeners.

[0138] As an emulsifier, PEG-240 / HDI copolymer bis-decyltetradecyl alcohol polyether-20 can be cited as an example.

[0139] Examples of preservatives include phenoxyethanol, benzyl alcohol, methyl paraben (a hydroxyl thickener), and aromatic preservatives such as p-hydroxyacetophenone.

[0140] Examples of UV absorbers include: benzoic acid-based UV absorbers (e.g., p-aminobenzoic acid (hereinafter abbreviated as PABA), PABA monoglyceride, N,N-dipropoxy PABA ethyl ester, N,N-diethoxy PABA ethyl ester, N,N-dimethyl PABA ethyl ester, N,N-dimethyl PABA butyl ester, N,N-dimethyl PABA ethyl ester, etc.); aminoanisic acid-based UV absorbers (e.g., N-acetylaminoanisic acid methyl ester, etc.); and salicylic acid-based UV absorbers (e.g., amyl salicylate, menthyl salicylate, methyl salicylate, octyl salicylate, phenyl salicylate, benzyl salicylate, p-isopropanol phenyl salicylate, etc.). ); Cinnamic acid-based ultraviolet absorbers (e.g., octyl methoxycinnamate, ethyl 4-isopropylcinnamate, methyl 2,5-diisopropylcinnamate, ethyl 2,4-diisopropylcinnamate, methyl 2,4-diisopropylcinnamate, propyl p-methoxycinnamate, isopropyl p-methoxycinnamate, isopentyl p-methoxycinnamate, octyl p-methoxycinnamate (2-ethylhexyl p-methoxycinnamate), 2-ethoxyethyl p-methoxycinnamate, cyclohexyl p-methoxycinnamate, ethyl α-cyano-β-phenylcinnamate, 2-ethylhexyl α-cyano-β-phenylcinnamate, mono-2-ethylhexanoyl-di-p-methoxycinnamate, etc.); Benzophenone-based ultraviolet absorbers (e.g., 2,4-dihydroxybenzophenone, 2,2”-dihydroxy-4-methoxybenzophenone, 2,2”-dihydroxy-4,4”-dimethoxybenzophenone, 2,2”,4,4”-tetrahydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4”-methylbenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonate, 4-phenylbenzophenone, 2-ethylhexyl-4”-phenyl-benzophenone-2-carboxylate, 2-hydroxy-4-n-octyloxybenzophenone, 4-hydroxy-3-carboxybenzophenone, etc.); 3-(4”-methylbenzylene)-d,l-camphor, 3-... Benzyl-d,l-camphor; 2-phenyl-5-methylbenzoxazole; 2,2”-hydroxy-5-methylphenylbenzotriazole; 2-(2”-hydroxy-5”-tert-octylphenyl)benzotriazole; 2-(2”-hydroxy-5”-methylphenylbenzotriazole; dibenzylhydrazine; anisole methane; 4-methoxy-4”-tert-butyldibenzoylmethane; 5-(3,3-dimethyl-2-norbornyl)-3-pentane-2-one, dimorpholinopyridazinone; 2-ethylhexyl-2-cyano-3,3-diphenylacrylate; 2,4-bis-{[4-(2-ethylhexyloxy)-2-hydroxy]-phenyl}-6-(4-methoxyphenyl)-(1,3,5)-triazine, etc.

[0141] Examples of metal ion blocking agents include: 1-hydroxyethane-1,1-diphosphonic acid, tetrasodium 1-hydroxyethane-1,1-diphosphonic acid, disodium edetate (EDTA2 sodium), trisodium edetate (EDTA3 sodium), tetrasodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, phosphoric acid, citric acid, ascorbic acid, succinic acid, edetate, and trisodium ethylenediamine hydroxyethyltriacetate.

[0142] Examples of amino acids include: neutral amino acids (e.g., threonine, cysteine, etc.); basic amino acids (e.g., hydroxylysine, etc.). Additionally, examples of amino acid derivatives include: sodium lauroyl sarcosinate (sodium lauroyl sarcosinate), acyl glutamate, sodium acyl β-alanine, glutathione, and pyrrolidone carboxylic acid.

[0143] Examples of organic amines include: monoethanolamine, diethanolamine, triethanolamine, morpholine, triisopropanolamine, 2-amino-2-methyl-1,3-propanediol, 2-amino-2-methyl-1-propanol, etc.

[0144] Examples of polymeric emulsions include: acrylic resin emulsions, ethyl polyacrylate emulsions, acrylic resin solutions, alkyl polyacrylate emulsions, polyvinyl acetate resin emulsions, and natural rubber latex.

[0145] Examples of pH adjusters include: lactic acid-sodium lactate, citric acid-sodium citrate, succinic acid-sodium succinate, and other buffers.

[0146] Examples of neutralizing agents include behenic acid, sodium hydroxide, aminomethylpropanol, and arginine.

[0147] Examples of vitamins include: vitamins A, B1, B2, B6, C, E and their derivatives, pantothenic acid and its derivatives, biotin, etc.

[0148] Examples of antioxidants include: tocopherols, butylated hydroxytoluene, butylated hydroxyanisole, and gallic acid esters.

[0149] Examples of antioxidant auxiliaries include: phosphoric acid, citric acid, ascorbic acid, maleic acid, malonic acid, succinic acid, fumaric acid, phospholipids, hexametaphosphate (ester), phytic acid, and ethylenediaminetetraacetic acid.

[0150] The following test examples and embodiments further demonstrate the effectiveness of the present invention, but the present invention is not limited to the following test examples and embodiments.

[0151] Test case

[0152] Experimental Example 1. Experiment on the effect of candidate aromatic compounds on the proliferative activity of human dermal fibroblasts (HDF).

[0153] The CCK8 assay was used to detect the effects of candidate aromatic substances on HDF cell viability and proliferation over a wide dose range (0-1000 μM) to determine the safe dose range of the candidate aromatic substances and to identify their screening dose range. HDF cells were cultured at a concentration of 1 × 10⁶ cells / year. 4 Cells were seeded at a density of [number] cells / well in 96-well plates. After culturing for 24 hours, different concentrations of candidate aromatic substances (0-1000 μM) were added. A control group (wells without sample cells) and a blank group (wells without sample cells) were set up, with three replicates in each group. After culturing for 24 hours, the cells were washed twice with PBS, and CCK-8 working solution was added. After further culturing for 2 hours, the absorbance at 450 nm was measured in each well using a microplate reader. Relative cell viability was calculated using the following formula: Cell viability (%) = (OD value of sample group - OD value of blank group) / (OD value of control group - OD value of blank group). The experimental results are shown in Figure 5.

[0154] The results in Figure 5 show that most candidate aromatic compounds did not significantly inhibit HDF cell viability or exhibit significant cytotoxicity within the dose range of 15.63–1000 μM. However, some aromatic compounds showed cytotoxicity at higher doses, including geraniol at doses above 250 μM, hydroxycitronellal at doses above 125 μM, thymol at doses above 500 μM, and CEDAR WOOD OIL ATLAS at doses above 0.25%, which either exhibited cytotoxicity or inhibited cell proliferation. Interestingly, some aromatic compounds promoted HDF cell proliferation within certain dose ranges, including carvacrol at doses of 31.25–250 μM and cyclamen aldehyde at doses of 31.25–1000 μM, which promoted HDF cell proliferation in a dose-dependent manner.

[0155] Experimental Example 2. Experiment on the effect of candidate aromatic compounds on the inhibition of HDF cell proliferation by t-BHP.

[0156] Studies on establishing a skin aging cell model have demonstrated that treatment of HDF cells with 200 μM t-BHP for 2 hours significantly inhibits HDF cell proliferation, indicating that 200 μM t-BHP treatment for 2 hours is sufficient to induce oxidative stress damage in HDF cells, thereby inhibiting cell proliferation. This experiment further investigates the effect of candidate aromatic compounds on the inhibitory effect of t-BHP on HDF cell proliferation, aiming to screen aromatic compounds with protective effects against t-BHP-induced oxidative damage to HDF cells.

[0157] Given that aromatic compounds may exert oxidative damage protection through two mechanisms—direct scavenging of oxygen free radicals and induction of endogenous antioxidants (including antioxidant enzymes and endogenous HA synthesis)—we hypothesized that pretreatment with aromatic compounds for a period of time might be more beneficial for their oxidative damage protection, since the induction of endogenous antioxidants requires a certain amount of time. Therefore, in a screening experiment, the inventors compared the protective effects of aromatic compounds pretreatment for 6 hours and 0.5 hours against oxidative damage induced by 2 hours of t-BHP stimulation under two experimental conditions. Specifically, HDF cells were first pre-incubated with different concentrations of aromatic compounds for 6 hours or 0.5 hours, then 200 μM t-BHP was added, and the cells were stimulated for 2 hours. The cells were then washed once with PBS, added to a culture medium containing the same concentration of aromatic compounds, and cultured for another 24 hours. Cell viability in each experimental group was detected using the CCK8 assay. The results are shown in Figure 6.

[0158] The results in Figure 6 show that stimulation of HDF cells with 200 μM tBHP for 2 h significantly reduced cell viability, indicating that tBHP induced oxidative damage in HDF cells. Meanwhile, nerol, geraniol, carvacrol, agarwood oil, and sandalwood oil dose-dependently attenuated the inhibitory effect of t-BHP on HDF cell proliferation, suggesting that these aromatic compounds have a protective effect against tBHP-induced oxidative damage. More notably, pretreatment with aromatic compounds for 6 h provided a stronger protective effect on HDF cells than pretreatment for 0.5 h, suggesting that these compounds may exert their protective effect against oxidative damage by inducing endogenous antioxidants.

[0159] Example 3. Experiment on the effect of aromatic compounds on t-BHP-induced senescence of HDF cells.

[0160] Aromatic compounds attenuated the inhibitory effect of t-BHP on HDF cell proliferation, indicating not only their protective effect against t-BHP-induced oxidative damage but also suggesting a potential protective effect against t-BHP-induced cellular senescence, as cell arrest is one of the most important characteristics of cellular senescence. Besides proliferation arrest, the specific expression of senescence-associated β-galactosidase (SA-β-gal) is also an important phenotypic feature of cellular senescence. This study investigated the effect of aromatic compounds with oxidative damage protection on SA-β-gal activity in t-BHP-induced HDF cells to further verify their protective effect against t-BHP-induced HDF cell senescence.

[0161] To this end, HDF cells were pre-incubated with different concentrations of aromatic compounds for 6 hours, followed by the addition of 200 μM t-BHP to stimulate the cells for 2 hours. Then, t-BHP was removed with PBS, and the cells were cultured for another 12 hours in a medium containing the same concentration of aromatic compounds. After culture, cells were stained with SPiDER-βgal (a specific fluorescent substrate for SA-β-gal) and Hoechst to label SA-β-gal positive senescent cells (green fluorescence) and cell nuclei (blue fluorescence), respectively. The cells were observed and photographed under an inverted fluorescence microscope, and the percentage of senescent cells in each experimental group was counted, i.e., the percentage of green fluorescent positive cells to the total number of blue fluorescent cells. The results are shown in Figures 7 to 11. The results showed that t-BHP alone significantly increased the percentage of SA-β-gal positive (green fluorescence) senescent cells (the percentage of green fluorescence positive cells out of the total number of blue fluorescence cells), while pretreatment with aromatic compounds such as nerol, geraniol, carvacrol, agarwood oil, and sandalwood oil significantly reduced the percentage of t-BHP-induced senescent cells, indicating that these aromatic compounds have a protective effect against t-BHP-induced senescence of HDF cells.

[0162] Experimental Example 4. Experiment on the effect of aromatic compounds on the expression of SASP factor in t-BHP-induced senescent HDF cells.

[0163] In addition to specifically expressing SA-β-gal, senescent cells continuously secrete a series of senescence-associated secretory phenotype (SASP) factors. In particular, senescent skin fibroblasts continuously secrete various matrix metalloproteinases (MMPs) and other SASP factors, degrading collagen, elastin, and fibronectin, leading to decreased skin elasticity. This is the main pathological basis for the sagging and wrinkle formation of aging skin.

[0164] This study further examined the effect of active aromatic compounds that significantly reduced the number of t-BHP-induced senescent cells on the mRNA expression levels of SASP factors (MMP-1, MMP-3, and MMP-9) in senescent HDF cells, in order to further verify the anti-aging effects of these aromatic compounds.

[0165] To this end, HDF cells were pre-incubated with different concentrations of aromatic compounds for 6 h, and then 200 μM t-BHP was added to stimulate the cells for 2 h. The t-BHP was then removed with PBS, and the cells were cultured in a medium containing the same concentration of aromatic compounds for another 12 h. After the culture was completed, the mRNA expression levels of SASP factors (MMP-1, MMP-3 and MMP-9) were detected by qPCR. The results are shown in Figures 12 to 15.

[0166] The results in Figures 12-15 show that t-BHP alone significantly increased the expression levels of MMP-1, MMP-3, and MMP-9 in cells, while pretreatment with aromatic compounds such as nerol, geraniol, agarwood oil, and sandalwood oil significantly downregulated the expression levels of MMP-1, MMP-3, and MMP-9 in t-BHP-induced senescent cells, indicating that these aromatic compounds can inhibit the production of SASP factor in senescent HDF cells.

[0167] The results in Figures 6-15 show that aromatic compounds such as nerol, geraniol, carvacrol, agarwood oil, and sandalwood oil can effectively attenuate the inhibitory effect of t-BHP on HDF cell viability and significantly reduce the number of t-BHP-induced senescent cells. At the same time, they significantly downregulated the expression levels of SASP factors such as MMP-1, MMP-3, and MMP-9 in t-BHP-induced senescent cells, indicating that these aromatic compounds have antioxidant and anti-cellular senescence effects.

[0168] Experimental Example 5. Effect of aromatic compounds on the expression of hyaluronic acid-degrading enzyme HYBID in t-BHP-stimulated HDF cells.

[0169] It is known that HA degradation in human skin fibroblasts is mainly mediated by HYBID (hyaluronan binding protein involved in hyaluronan depolymerization) (also known as KIAA1199 / CEMIP). Increased expression of HYBID can be detected in photoaged skin samples, indicating that HYBID-mediated HA degradation may lead to enhanced HA catabolism in aging skin, reduced HA in the papillary dermis, and ultimately, skin wrinkles and sagging.

[0170] In their experiments establishing a skin aging cell model, the inventors discovered that t-BHP, when inducing HDF cell aging, not only induces high expression of multiple MMPs but also simultaneously induces HYBID. This suggests that t-BHP induces HA degradation by promoting HYBID expression, thereby depriving cells of HA protection. This may be one of the mechanisms by which t-BHP induces cell aging. This experiment investigated the effects of certain aromatic compounds with anti-aging activity on HYBID expression in t-BHP-stimulated HDF cells to explore the possible mechanisms of the anti-aging effects of aromatic compounds.

[0171] To this end, HDF cells were pre-incubated with different concentrations of aromatic compounds for 6 h, and then 200 μM t-BHP was added to stimulate the cells for 2 h. The t-BHP was then removed with PBS, and the cells were cultured for another 12 h with the same concentration of aromatic compounds. After the culture was completed, the mRNA expression level of HYBID was detected by qPCR. The results are shown in Figure 12.

[0172] Figure 16 shows that t-BHP alone significantly increased the expression level of HYBID in HDF cells, while aromatic compounds such as nerol, agarwood oil, and sandalwood oil significantly downregulated the expression level of HYBID in t-BHP-induced aging cells, indicating that these aromatic compounds may inhibit HA degradation, but geraniol had no effect. (Experiments on the effect of carvacrol are still ongoing.)

[0173] Experimental Example 6. An experiment to investigate the effect of aromatic compounds on the expression of hyaluronic acid synthase HAS2 in t-BHP-stimulated HDF cells.

[0174] Dermal fibroblasts are known to have a strong ability to synthesize HA, with a production rate as high as 12-60 pg / cell / 24h, approximately 10 times that of keratinocytes. In normal human skin fibroblasts, HA synthesis is mainly handled by HAS1 and HAS2, with HAS2 being the predominant HAS isoform expressed in fibroblasts. Overall, the amount of HA synthesized is consistent with the mRNA expression level of HAS2, and during oxidative stress, endogenous HA synthesized through HAS2 provides crucial antioxidant protection for the body's tissues and cells. The inventors discovered in experiments that primary cultured HDF cells expressed HAS1 and HAS3 at extremely low levels. When their mRNA levels were detected by qPCR, the Ct value often reached around 32 (the higher the Ct value, the lower the gene expression level; generally, a Ct value greater than 35 is considered to indicate that the gene is not expressed or cannot be accurately measured), making them almost undetectable. In contrast, the Ct value for HAS2 mRNA expression was around 22, indicating that the expression level of HAS2 in HDF cells is much higher than that of HAS1 and HAS3, and that HAS2 is the main HAS subtype for HA synthesis in HDF cells.

[0175] This experiment further investigated the effect of aromatic compounds on HAS2 expression in t-BHP-stimulated HDF cells to determine whether their protective effect against t-BHP-induced oxidative damage is related to the promotion of endogenous HA synthesis. To this end, HDF cells were pre-incubated with different concentrations of aromatic compounds for 6 h, followed by stimulation with 200 μM t-BHP for 2 h. Cells were then collected, and HAS2 mRNA levels were detected by qPCR. The results are shown in Figure 17.

[0176] The results in Figure 17 show that stimulation of HDF cells with 200 μM tBHP for 2 h did not significantly affect HAS2 expression, while nerol, geraniol, and carvacrol were able to dose-dependently increase the mRNA level of HAS2 in t-BHP-stimulated HDF cells, indicating that these aromatic compounds can promote HAS2 expression in HDF cells. This suggests that the antioxidant and anti-cellular aging effects of these aromatic compounds may be related to the promotion of endogenous HA synthesis.

[0177] Experimental Example 7. Effect of aromatic compounds on t-BHP-induced secretion of hyaluronic acid 2 by senescent human fibroblasts.

[0178] To verify the regulatory effect of the above-mentioned aromatic substances on HA metabolism, the inventors further investigated their effects on HA secretion induced by t-BHP in senescent HDF cells using ELISA experiments.

[0179] HDF cells were pre-incubated with different concentrations of aromatic substances for 6 h, followed by the addition of 200 μM t-BHP for 2 h of stimulation. The t-BHP was then removed with PBS, and the cells were cultured for another 24 h with the same concentration of aromatic substances. After the culture was completed, the cell culture supernatant was collected, and the concentration of HA in the cell culture supernatant was measured using an HA ELISA kit (Beijing Solarbio Science & Technology Co., Ltd.). The results showed that t-BHP alone could significantly reduce the HA level in the HDF cell culture supernatant, while pretreatment with nerol, carvacrol, geraniol, agarwood essential oil, and sandalwood essential oil could significantly increase the HA level in the supernatant of t-BHP-induced senescent HDF cells (Figure 18). This indicates that these aromatic substances can increase the total amount of HA secreted by HDF cells by inhibiting HA degradation and / or promoting HA synthesis.

[0180] According to this invention, the experimental conditions for t-BHP-induced oxidative stress and cellular senescence in HDF cells were first explored. It was found that treatment with 200 μM t-BHP for 2 hours could induce HDF cells to exhibit typical cellular senescence phenotypes after 12-24 hours, including decreased cell proliferation, SA-β-gal positivity, and high expression of SASP factors such as MMP-1, MMP-3, and MMP-9. Thus, a skin aging cell model of t-BHP-induced oxidative stress and cellular senescence in HDF cells was established.

[0181] Further screening of aromatic compounds that can effectively antagonize oxidative stress and cellular senescence using this cell model revealed that nerol, geraniol, carvacrol, agarwood oil, and sandalwood oil, among 12 candidate aromatic compounds, effectively attenuated the inhibitory effect of t-BHP on HDF cell proliferation and significantly reduced the number of t-BHP-induced senescent cells. Simultaneously, these compounds significantly downregulated the expression levels of SASP factors such as MMP-1, MMP-3, and MMP-9 in t-BHP-induced senescent cells, indicating that these aromatic compounds possess antioxidant and anti-cellular senescence effects. Further investigation into the effects of active aromatic compounds on HA metabolism in t-BHP-induced senescent HDF cells revealed that t-BHP alone significantly increased HYBID expression levels in HDF cells, while aromatic compounds such as nerol, agarwood oil, and sandalwood oil significantly downregulated HYBID expression levels in t-BHP-induced senescent cells, suggesting that these aromatic compounds may inhibit HA degradation. Furthermore, nerol, geraniol, and carvacrol dose-dependently increased HAS2 mRNA levels in t-BHP-stimulated HDF cells, indicating that these aromatic compounds can induce HAS2 expression in HDF cells, thereby promoting endogenous HA synthesis. These experimental results suggest that the antioxidant and anti-cellular senescence effects of these aromatic compounds may be related to inhibiting HA degradation and / or promoting endogenous HA synthesis.

[0182] Example

[0183] Example 1. Toner

[0184] The lotion was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0185] Example 2. Toner

[0186] The lotion was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0187] Example 3. Toner

[0188] The lotion was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0189] Example 4. Toner

[0190] The lotion was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0191] Example 5. Toner

[0192] The lotion was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0193] Example 6. Emulsion

[0194] The emulsion was prepared according to conventional methods in the art, in the following proportions. The proportions of each component are % by mass.

[0195] Example 7. Emulsion

[0196] The emulsion was prepared according to conventional methods in the art, in the following proportions. The proportions of each component are % by mass.

[0197] Example 8. Emulsion

[0198] The emulsion was prepared according to conventional methods in the art, in the following proportions. The proportions of each component are % by mass.

[0199] Example 9. Emulsion

[0200] The emulsion was prepared according to conventional methods in the art, in the following proportions. The proportions of each component are % by mass.

[0201] Example 10. Emulsion

[0202] The emulsion was prepared according to conventional methods in the art, in the following proportions. The proportions of each component are % by mass.

[0203] Example 11. Face Cream

[0204] The face cream was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0205] Example 12. Face Cream

[0206] The face cream was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0207] Example 13. Face Cream

[0208] The face cream was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0209] Example 14. Face Cream

[0210] The face cream was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0211] Example 15. Face Cream

[0212] The face cream was prepared according to conventional methods in the art, in the following proportions. The proportions of each ingredient are % by mass.

[0213] Industrial availability

[0214] According to the present invention, a novel method for increasing hyaluronic acid content in the dermis can be provided, characterized by the application of at least one selected from nerol, carvacrol, geraniol, agarwood essential oil, and sandalwood essential oil to the skin. A novel method for skin anti-aging can also be provided, characterized by the application of a cosmetic containing at least one selected from nerol, carvacrol, geraniol, agarwood essential oil, and sandalwood essential oil to the skin.

Claims

1. A method for regulating the synthesis and degradation of hyaluronic acid by fibroblasts, characterized in that, It contains at least one of nerol, carvacrol, geraniol, agarwood oil, and sandalwood oil.

2. The method according to claim 1, characterized in that, The method includes using at least one of a hyaluronic acid synthase (HAS2) expression promoter or a hyaluronic acid degrading enzyme (HYBID) expression inhibitor, wherein the hyaluronic acid synthase (HAS2) expression promoter comprises at least one selected from nerol, carvacrol, or geraniol, and the hyaluronic acid degrading enzyme (HYBID) expression inhibitor comprises at least one selected from nerol, carvacrol, agarwood essential oil, or sandalwood essential oil.

3. A method for combating skin aging, comprising applying a cosmetic to the skin containing at least one selected from nerol, carvacrol, geraniol, agarwood essential oil, and sandalwood essential oil.

4. The use of at least one of nerol, carvacrol, geraniol, agarwood essential oil, and sandalwood essential oil in the preparation of cosmetics with anti-aging effects.

5. Use of nerol, carvacrol or geraniol in the preparation of hyaluronic acid synthase expression promoters.

6. Use of nerol, carvacrol, agarwood oil or sandalwood oil in the preparation of hyaluronic acid degrading enzyme expression inhibitors.

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