A light walnut branch and leaf extract, and a preparation method and application thereof

Abstract

This invention belongs to the field of plant extract preparation technology and bioactive material application technology. Specifically, it discloses an extract from the branches and leaves of *Juglans regia* (a type of walnut), its preparation method, and its applications. Using waste branches and leaves from *Juglans regia* as raw material, the process involves pretreatment, two-stage reflux extraction with 65% ethanol, vacuum concentration, fractional purification with AB-8 macroporous resin at 15% / 65%, and drying to obtain a high-flavonoid extract with total flavonoids ≥35% and total monomers of rutin, hyperoside, and astragaloside ≥4%. This invention employs a two-stage ethanol elution process to precisely remove chlorophyll and polysaccharide impurities, significantly improving product purity. The ethanol in this process is recyclable and suitable for industrial mass production, achieving high-value utilization of waste branches and leaves from *Juglans regia*. The extract can be added to after-sun repair, anti-photoaging cosmetics, and topical skin repair preparations, showing broad market application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant extract preparation technology and bioactive material application technology. It relates to an extract of walnut branches and leaves, its preparation method and application. Specifically, it is a high-flavonoid extract prepared by using waste walnut branches and leaves as raw materials, through alcohol extraction and segmental purification with macroporous resin. The extract has UVB photoaging protection, post-sun anti-inflammatory and soothing, and skin barrier repair activities. The industrial preparation process is also disclosed, as well as its application in sun protection and repair skin care products, anti-aging cosmetics, and topical skin medicine preparations. Background Technology

[0002] With increasing ultraviolet radiation and growing consumer awareness of skin health, the demand for natural plant extracts with photoaging protection and after-sun soothing effects has surged in the cosmetics and dermatology fields. Photoaging is skin damage caused by long-term ultraviolet radiation (especially UVB), manifested as excessive production of reactive oxygen species (ROS), activation of matrix metalloproteinases (MMPs), collagen degradation, and inflammatory responses; after-sun soothing requires raw materials that can quickly inhibit the release of UV-induced inflammatory factors, relieve erythema and burning, and repair the skin barrier.

[0003] The smooth walnut (Prunus mira Koehne) is a deciduous tree belonging to the genus Prunus in the Rosaceae family. It is mainly distributed in the Qinghai-Tibet Plateau and Hengduan Mountains of my country, including Tibet, Sichuan, and Yunnan provinces. It is a unique plant with ecological, economic, and ethnic medicinal value. Currently, the industry's development and utilization of the smooth walnut focuses heavily on its fruit and kernels, primarily for consumption or oil extraction, and the technology is relatively mature. However, the branches and leaves of the smooth walnut, as an important part of the plant, are often directly discarded after the fruit is harvested, resulting not only in a serious waste of plant resources but also potentially adding to the environmental burden.

[0004] Existing research confirms that the branches and leaves of *Juglans regia* are rich in flavonoid active substances such as rutin, hyperoside, and astragaloside. These components have significant potential for antioxidant, collagen protection, and anti-inflammatory effects, possessing core value for developing raw materials for sun protection and repair. A search reveals that currently available technologies only focus on the processing of *Juglans regia* kernels and pulp; no systematic research and application plans for the targeted extraction and purification of *Juglans regia* branches and leaves, or for their use in repairing photodamage to the skin, have been found.

[0005] The current extraction of flavonoids from walnut branches and leaves faces specific technical challenges:

[0006] The leaves and branches of the walnut tree differ from those of other plants (such as ginkgo leaves and hawthorn leaves) in that they have an extremely high chlorophyll content, and the chlorophyll and flavonoids exhibit a "synchronous" dissolution characteristic—when the ethanol concentration reaches 65%, flavonoids and chlorophyll dissolve in large quantities simultaneously, resulting in a chlorophyll to flavonoid ratio in the extract that is much higher than that of other plant leaves.

[0007] This characteristic makes:

[0008] (1) Although reducing the ethanol concentration can inhibit chlorophyll dissolution, the flavonoid dissolution rate also drops sharply, so it is impossible to achieve both goals simultaneously;

[0009] (2) Increasing the ethanol concentration can increase the dissolution of flavonoids, but the amount of chlorophyll co-soluble increases exponentially, and subsequent purification steps cannot effectively remove it;

[0010] (3) Conventional macroporous resin single-concentration elution cannot separate chlorophyll and flavonoids because the adsorption behavior of the two on the resin is similar, and single-concentration elution will inevitably lead to the simultaneous elution of chlorophyll and flavonoids.

[0011] Therefore, developing a set of extraction and purification processes for flavonoids from walnut branches and leaves that are suitable for industrialization, retain high activity, and enrich high purity will enable the high-value utilization of waste branches and leaves, fill the technological gap in the field of raw materials for skin repair, and have significant industrial value. Summary of the Invention

[0012] To address the shortcomings of existing technologies, such as the waste of walnut branches and leaves, crude extraction and purification processes, low flavonoid purity, easy degradation of active ingredients, and lack of clear application solutions for photoaging repair, this invention provides a walnut branch and leaf extract, its preparation method, and its application, solving the following technical problems:

[0013] (1) Resource utilization of discarded walnut branches and leaves to solve the waste of resources and environmental burden caused by discarding branches and leaves after harvesting;

[0014] (2) Provide a set of extraction and purification processes with precise parameters, scalable mass production, and maximum retention of flavonoid active monomers;

[0015] (3) A natural extract with a total flavonoid content of ≥35% and a total content of characteristic flavonoid monomers of ≥4% was prepared. It has the triple effects of anti-photoaging, anti-inflammatory after sun exposure, and barrier repair, and is non-irritating and non-allergenic.

[0016] (4) Expand the application of this extract in sunscreen, post-sun repair, anti-aging cosmetics and topical skin repair preparations.

[0017] The first objective of this invention is to provide a method for preparing an extract from the branches and leaves of *Juglans regia*, comprising the following steps:

[0018] S1: Raw material pretreatment

[0019] Select fresh walnut branches and leaves, remove impurities, dead branches and rotten parts, rinse with deionized water 3-5 times to remove surface dust and residual dirt; place in a cool and ventilated place to dry to remove some yellow leaves, debris and most of the moisture, then place in a 60-70℃ constant temperature drying oven to dry to constant weight (moisture content ≤8%); use a universal pulverizer to pulverize, pass through a 40-80 mesh sieve, collect the sieve-through powder, seal and store for later use (avoid moisture and oxidation of active ingredients).

[0020] S2: Alcohol extraction: 65% ethanol aqueous solution was used as the extraction solvent (volume fraction). The 65% ethanol concentration is at the critical concentration point for co-dissolution of chlorophyll in the branches and leaves of *Juglans regia*. At a 65% ethanol concentration, chlorophyll and flavonoids dissolve simultaneously, necessitating precise separation of chlorophyll and flavonoids in subsequent purification steps through specific gradient elution. Two reflux extractions were employed.

[0021] S21: First extraction

[0022] Mix the solids and liquids at a ratio of 1:(8-9) g / mL (mass of walnut branch and leaf powder: volume of ethanol aqueous solution), place them in a constant temperature water bath reflux device, and extract by constant temperature reflux at 80-85℃ for 2 h;

[0023] S22: Second extraction

[0024] Add 65% ethanol to the solid, with a solid-liquid ratio of 1:(6-8) g / mL, and reflux at 80-85℃ for 2 h.

[0025] S23: After each extraction, filter with multi-layer gauze or centrifuge (3000-4000 r / min) for 10-15 min, collect the extract, combine the two extracts, and discard the precipitate (impurities).

[0026] S3: Concentration Process

[0027] The combined extracts were placed in a rotary distillation flask and subjected to vacuum rotary distillation under reduced pressure (vacuum degree above 0.06 MPa, temperature 70-80℃) to distill off the ethanol (which can be recovered and used in step S4). The distillation was concentrated to a thick slurry-like paste, with a volume of approximately 600-1000 mL (per 800 g of raw material). The solution was poured into a beaker and refrigerated. After cooling, it became a paste, which could be measured and sampled for later use.

[0028] S4: Resin purification treatment: Segmented purification was performed using AB-8 type macroporous adsorption resin.

[0029] A two-stage gradient elution strategy of "pre-elution with 15% ethanol to remove chlorophyll - elution with 65% ethanol to enrich flavonoids" was adopted. The 15% ethanol elution step and the 65% ethanol extraction step work synergistically: the 15% ethanol can just elute the co-soluble chlorophyll from the resin, while the target flavonoids are retained and directionally enriched in the 65% ethanol elution step.

[0030] S41: Adsorption Washing

[0031] Prepare a plastic reaction box. First, add the calculated amount of resin (containing water, measured by net dry weight), the amount of resin being 1.5-2.0 times the amount of raw material. Then add the extract, dilute with water (the total volume of extract and water should be 3 times the corresponding raw material), add a stir bar, and stir for the first time on a magnetic stirrer for 30 minutes to ensure uniform adsorption and washing. Then filter through a 60-mesh sieve and discard the filtrate. Transfer the filter cake back to the reaction box, add water (the total amount of water added and washing water should be 3 times the corresponding raw material), wash and stir for the second time for 10 minutes, then filter through a 60-mesh sieve again, add about 300 mL of washing water, wash the filter cake and reaction box together, filter until dry, and discard the filtrate.

[0032] S42: Segmented gradient elution

[0033] Deionized water elution: Elute with deionized water, the total amount of which is 4 times the amount of raw material, poured in in batches. This elution section is the impurity section and should be discarded and not incorporated into the target product.

[0034] Elution with 15% ethanol: Prepare a 15% concentration using the recovered ethanol from step S3 at a volume ratio, with the total amount being four times the amount of raw material. Add the ethanol in portions, keeping the valve under the column open to allow the liquid to drip out. Once the amount of dripped liquid is equivalent to the amount of water added to the column, and 5-10 minutes after adding the 15% ethanol, begin collecting the initial section of the 15% ethanol eluent. Add 15% ethanol to the resin layer as needed based on the dripping rate until all eluent is added; the 15% ethanol eluent section is considered an impurity section and should be discarded or used for other purposes, not incorporated into the target product.

[0035] Elution with 65% ethanol: Prepare a 65% concentration using the recovered ethanol from step S3 at a volume ratio, with the total volume being 6 times the amount of raw material. When the 15% ethanol has been used up and only a very thin liquid layer remains on the resin surface, add 65% ethanol in portions and collect the eluent in segments (collect 800 mL from each of the first, middle, and last segments, or convert 500 mL per segment).

[0036] Specifically, the sample is first eluted with deionized water, then with 15% ethanol, and the eluent is discarded. The volume of the 15% ethanol elution is 4 times the amount of the raw material, and it is used to precisely remove chlorophyll impurities that are cosoluble with flavonoids. Then, 65% ethanol is used for elution to selectively enrich flavonoid target compounds. The volume of the 65% ethanol elution is 6 times the amount of the raw material, and all the 65% ethanol eluent is collected in segments.

[0037] S43: Filtering

[0038] The collected 65% ethanol eluent (target enrichment fraction) was filtered through a 0.45 μm filter membrane to remove minute impurities and resin debris.

[0039] S5: Drying process

[0040] The filtered 65% ethanol eluent was placed again in a rotary evaporator and concentrated under reduced pressure (vacuum degree 0.06-0.08 MPa, temperature 50-60℃) until no alcohol odor was detected, to obtain a concentrated paste; the concentrated paste was then vacuum dried or spray dried to obtain a powder of walnut branch and leaf extract.

[0041] Preferably, resin pretreatment is required before step S41, specifically: soaking AB-8 type macroporous adsorption resin in 95% ethanol for 24 h to allow it to fully swell, and then rinsing it with 95% ethanol and deionized water in sequence until the eluent has no alcohol odor, and then setting it aside for later use.

[0042] Preferably, a column loading operation is required before step S42, specifically: the filter cake obtained in step S41 is wet-loaded onto the column (the column includes a glass quartz column and a self-made plastic bottle column, with wire mesh and cotton support at the bottom), 500-600 mL of water is added, the screen is washed and the resin is soaked, the resin is stirred appropriately, and air bubbles are removed. There is a valve at the bottom of the column; opening it allows the column washing water to drip out until the water layer on the top of the resin is very thin, at which point elution begins.

[0043] Preferably, the spray drying conditions in step S5 are: inlet air temperature 180-200℃, outlet air temperature 80-90℃, feed rate 10-15 mL / min, and atomization pressure 0.2-0.3 MPa.

[0044] A second objective of this invention is to provide a *Juglans regia* branch and leaf extract prepared by the above method, wherein the *Juglans regia* branch and leaf extract is a pale yellow powder and satisfies the following combination of characteristics:

[0045] Total flavonoid content ≥35wt%; total content of the three characteristic monomers rutin, hyperoside, and astragaloside ≥4wt%; molar ratio of rutin, hyperoside, and astragaloside is 1:(1.2-1.8):(0.5-0.8); in the HPLC fingerprint, at a detection wavelength of 280nm, three characteristic peaks appear in the retention time range of 12-18min, and the sum of the peak areas accounts for 25%-35% of the total peak area; pH of 1% aqueous solution is 5.5-6.5; chlorophyll residue is... The value is ≤0.05.

[0046] A third objective of this invention is to provide the application of the above-mentioned walnut leaf and branch extract in the preparation of photoaging protection and post-sun exposure soothing and repair products.

[0047] Preferably, the application in the preparation of photoaging protection and post-sun soothing repair products with triple effects of UVB photoaging protection, post-sun anti-inflammatory and soothing, and skin barrier repair is based on the synergistic activity of the extract at a concentration of 0.2% simultaneously achieving: HSF cell UVB protection rate ≥87%, TNF-α inhibition rate ≥89%, and FLG upregulation ≥188%.

[0048] The beneficial effects of this invention are:

[0049] (1) Advantages of resource utilization: This invention uses the discarded branches and leaves of the walnut tree in traditional development as the only raw material, realizing the high-value utilization of the whole plant of the walnut tree, reducing resource waste and environmental burden, while the raw materials are widely available and inexpensive.

[0050] (2) Process advantages: The optimized "ethanol extraction-AB-8 resin segmented purification-drying" process is simple in steps, clear in parameters, and highly controllable, making it suitable for large-scale industrial production. By revealing the "critical separation point" of 65% ethanol, the "quantitative-mass balance point" of 80℃ / 2 h, and the synergistic impurity removal-enrichment effect of "15%→65% two-stage elution", it is proven that the combination of process parameters in this invention is non-obvious. This invention removes most of the chlorophyll precipitate through adsorption washing, and removes highly polar impurities such as inorganic salts, monosaccharides, water-soluble organic phenolic acids, water-soluble pigments, and hydrophilic tannins through deionized water washing and 15% ethanol elution steps. The product has a high content of active ingredients (total flavonoid content ≥35%, and can reach more than 43.5% in preferred embodiments) and high purity (rutin + hyperoside + astragaloside ≥4%, and can reach more than 4.92% in preferred embodiments), and the impurities are thoroughly removed.

[0051] (3) Efficacy advantages:

[0052] Existing plant extracts of the same type have only been verified for basic antioxidant effects. This invention establishes HSF, HaCaT, and RAW264.7 multi-cell models to fully verify the triple effects of anti-photoaging, anti-inflammatory and soothing, and barrier repair. The efficacy targets are clear and the data are quantified.

[0053] (4) Safety advantages: After purification, irritating wax and a large amount of pigment impurities are removed. It has been proven to be mild and safe through skin irritation, eye irritation and sensitization tests. It is suitable for sensitive skin and fragile skin after sun exposure.

[0054] (5) Application advantages: This extract has a wide range of applications. It can be used directly or in combination with other raw materials to prepare photoaging protection and sun-soothing cosmetics (such as essence, face cream, face mask, toner) and topical pharmaceutical preparations (such as sun-soothing repair gel, photodamage repair cream), filling the application gap of walnut branch and leaf extract in related fields. Attached Figure Description

[0055] Figure 1 This is a high-performance liquid chromatogram of the product of Example 1 of the present invention.

[0056] Figure 2 This is a high-performance liquid chromatogram of the product of Comparative Example 1 of the present invention.

[0057] Figure 3 This is a high-performance liquid chromatogram of the product of Comparative Example 2 of the present invention.

[0058] Figure 4 This is a high-performance liquid chromatogram of the product of Comparative Example 3 of the present invention.

[0059] Figure 5 This is a high-performance liquid chromatogram of the product of Comparative Example 4 of the present invention.

[0060] Figure 6 This is a high-performance liquid chromatogram of the mixed standard of the present invention. Detailed Implementation

[0061] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0062] This invention uses the branches and leaves of *Juglans regia* as the sole raw material. The preferred raw materials are fresh young branches and leaves of *Juglans regia* (harvested annually from June to September), which are pre-treated before being used as extraction raw materials. The main active components of the extract are flavonoids. The total flavonoid content is ≥35% as determined by ultraviolet spectrophotometry, and the total content of rutin, hyperoside, and astragaloside is ≥4% as determined by high-performance liquid chromatography (HPLC). The HPLC chromatogram of the mixed standard is shown below. Figure 6 As shown.

[0063] The technical solution adopted in this invention is as follows:

[0064] A method for preparing an extract from the branches and leaves of *Juglans regia* includes the following steps:

[0065] S1: Raw material pretreatment

[0066] Select fresh walnut branches and leaves, remove impurities, dead branches and rotten parts, rinse with deionized water 3-5 times to remove surface dust and residual dirt; place in a cool and ventilated place to dry to remove some yellow leaves, debris and most of the moisture, then place in a 60-70℃ constant temperature drying oven to dry to constant weight (moisture content ≤8%); use a universal pulverizer to pulverize, pass through a 40-80 mesh sieve, collect the sieve-through powder, seal and store for later use (avoid moisture and oxidation of active ingredients).

[0067] S2: Alcohol extraction: Using 65% ethanol aqueous solution as the extraction solvent (volume fraction), two reflux extractions were performed.

[0068] S21: First extraction

[0069] Mix the solids and liquids at a ratio of 1:(8-9) g / mL (mass of walnut branch and leaf powder: volume of ethanol aqueous solution), place them in a constant temperature water bath reflux device, and extract by constant temperature reflux at 80-85℃ for 2 h;

[0070] S22: Second extraction

[0071] Add 65% ethanol to the solid, with a solid-liquid ratio of 1:(6-8) g / mL, and reflux at 80-85℃ for 2 h.

[0072] S23: After each extraction, filter with multi-layer gauze or centrifuge (3000-4000 r / min) for 10-15 min, collect the extract, combine the two extracts, and discard the precipitate (impurities).

[0073] S3: Concentration Process

[0074] The combined extracts were placed in a rotary distillation flask and subjected to vacuum rotary distillation under reduced pressure (vacuum degree above 0.06 MPa, temperature 70-80℃) to distill off the ethanol (which can be recovered and used in step S4). The distillation was concentrated to a thick slurry-like paste, with a volume of approximately 600-1000 mL (per 800 g of raw material). The solution was poured into a beaker and refrigerated. After cooling, it became a paste, which could be measured and sampled for later use.

[0075] S4: Resin purification treatment: Segmented purification was performed using AB-8 type macroporous adsorption resin.

[0076] S41: Adsorption Washing

[0077] Prepare a plastic reaction box. First, add the calculated amount of resin (containing water, measured by net dry weight), the amount of resin being 1.5-2.0 times the amount of raw material. Then add the extract, dilute with water (the total volume of extract and water should be 3 times the corresponding raw material), add a stir bar, and stir for the first time on a magnetic stirrer for 30 minutes to ensure uniform adsorption and washing. Then filter through a 60-mesh sieve and discard the filtrate. Transfer the filter cake back to the reaction box, add water (the total amount of water added and washing water should be 3 times the corresponding raw material), wash and stir for the second time for 10 minutes, then filter through a 60-mesh sieve again, add about 300 mL of washing water, wash the filter cake and reaction box together, filter until dry, and discard the filtrate.

[0078] S42: Segmented gradient elution

[0079] Deionized water elution: Elute with deionized water, the total amount of which is 4 times the amount of raw material, poured in in batches. This elution section is the impurity section and should be discarded and not incorporated into the target product.

[0080] Elution with 15% ethanol: Prepare a 15% concentration using the recovered ethanol from step S3 at a volume ratio, with the total amount being four times the amount of raw material. Add the ethanol in portions, keeping the valve under the column open to allow the liquid to drip out. Once the amount of dripped liquid is equivalent to the amount of water added to the column, and 5-10 minutes after adding the 15% ethanol, begin collecting the initial section of the 15% ethanol eluent. Add 15% ethanol to the resin layer as needed based on the dripping rate until all eluent is added; the 15% ethanol eluent section is considered an impurity section and should be discarded or used for other purposes, not incorporated into the target product.

[0081] Elution with 65% ethanol: Prepare a 65% concentration using the recovered ethanol from step S3 at a volume ratio, with the total volume being 6 times the amount of raw material. When the 15% ethanol has been used up and only a very thin liquid layer remains on the resin surface, add 65% ethanol in portions and collect the eluent in segments (collect 800 mL from each of the first, middle, and last segments, or convert 500 mL per segment).

[0082] S43: Filtering

[0083] The collected 65% ethanol eluent (target enrichment fraction) was filtered through a 0.45 μm filter membrane to remove minute impurities and resin debris.

[0084] S5: Drying process

[0085] The filtered 65% ethanol eluent was placed again in a rotary evaporator and concentrated under reduced pressure (vacuum degree 0.06-0.08 MPa, temperature 50-60℃) until no alcohol odor was detected, to obtain a concentrated paste; the concentrated paste was then vacuum dried or spray dried to obtain a powder of walnut branch and leaf extract.

[0086] In some embodiments, resin pretreatment is required before step S41, specifically: AB-8 type macroporous adsorption resin is soaked in 95% ethanol for 24 h to allow it to fully swell, and then rinsed with 95% ethanol and deionized water in sequence until the eluent has no alcohol odor, and then set aside.

[0087] In some embodiments, a column loading operation is required before step S42, specifically: the filter cake obtained in step S41 is wet-loaded onto a column (the column includes a glass quartz column and a self-made plastic bottle column, with wire mesh and cotton support at the bottom), 500-600 mL of water is added, the screen is washed and the resin is soaked, the resin is stirred appropriately, and air bubbles are removed. There is a valve at the bottom of the column; opening it allows the column washing water to drip out until the water layer on the top of the resin is extremely thin, at which point elution begins.

[0088] In some embodiments, the spray drying conditions in step S5 are: inlet air temperature 180-200℃, outlet air temperature 80-90℃, feed rate 10-15 mL / min, and atomization pressure 0.2-0.3 MPa.

[0089] The extract of walnut branches and leaves prepared by the above method is a light yellow powder with a total flavonoid content ≥35wt%, a total content of three characteristic monomers, rutin, hyperoside, and astragaloside, ≥4wt%, and a pH of 5.5-6.5 in a 1% aqueous solution.

[0090] The above-mentioned application of the extracts from the branches and leaves of the walnut tree is in the preparation of products for photoaging protection and post-sun exposure soothing and repair.

[0091] Example 1

[0092] A method for preparing an extract from the branches and leaves of the walnut tree is as follows:

[0093] S1: Raw material pretreatment

[0094] Select 5 kg of fresh walnut branches and leaves, remove impurities and dead branches, rinse 4 times with deionized water, place in a 65℃ constant temperature drying oven to dry to constant weight (moisture content 7%), grind with a universal grinder and pass through a 60-mesh sieve (standard sieve, aperture about 0.25 mm), collect the sieve-passing powder, seal and store for later use.

[0095] S2: Alcohol extraction treatment: Take 800 g of the above powder and place it in a 10 L three-necked flask.

[0096] First extraction: Add about 6400 mL of 65% ethanol aqueous solution (solid-liquid ratio 1:8 g / mL), place in a constant temperature water bath reflux device, and extract at 80℃ for 2 h; filter with multi-layer gauze, invert as much as possible to pour out most of the liquid, and transfer the solid back to the three-necked flask.

[0097] Second extraction: Add approximately 4800 mL of 65% ethanol aqueous solution (solid-liquid ratio 1:6 g / mL), and reflux at 80℃ for 2 h; after slightly cooling, pour out the liquid and filter with gauze.

[0098] The two extracts were combined, cooled, measured, and sampled for testing.

[0099] S3: Concentration Process

[0100] The combined extracts were placed in a rotary distillation flask and distilled under reduced pressure at 0.07 MPa and 75°C. The ethanol was recovered and reused. The distillation was concentrated to a thick paste-like consistency, approximately 800 mL in volume. This was poured into a beaker and refrigerated to obtain a concentrated extract.

[0101] S4: Resin purification treatment

[0102] S41: Adsorption Washing

[0103] Pretreatment of AB-8 macroporous adsorption resin: Take AB-8 resin (net dry weight of about 1200 g, which is 1.5 times the amount of raw material), soak it in 95% ethanol for 24 h, and rinse it with 95% ethanol and deionized water in sequence until the eluent has no alcohol odor.

[0104] Adsorption and Washing: Prepare a 3 L plastic reaction box and add pretreated AB-8 resin. Divide the above extract into two equal portions, each equivalent to 400 g of raw material, and purify them separately. Add the above concentrated extract (equivalent to 400 g of raw material), and dilute with water to a total volume of 1200 g (3 times the amount of raw material). Add a stir bar and magnetically stir for 30 min, filter through a 60-mesh sieve, and discard the filtrate. Transfer the filter cake back to the reaction box, add 900 mL of water (the total amount of water added and washing water is 3 times the amount of raw material), wash and stir for 10 min, filter through a 60-mesh sieve, add about 300 mL of washing water, wash the filter cake and reaction box together, filter until dry, and discard the filtrate.

[0105] Column loading: After washing, the filter cake is wet-loaded onto the column (using a homemade plastic bottle column with wire mesh and cotton for support at the bottom). Add 500mL of water, wash the screen and wet the resin, and stir appropriately to remove air bubbles. Open the valve at the bottom of the column to drip out the washing water. When the water layer on the top of the resin is very thin, begin elution.

[0106] S42: Segmented gradient elution

[0107] Deionized water elution: Elute with deionized water, total volume 1600 mL (4 times the amount of raw material), poured in in portions. This elution section is the impurity section and should be discarded.

[0108] Elution with 15% ethanol: Prepare a 15% concentration using recycled ethanol, totaling 1600 mL (4 times the amount of raw material). Add in portions, and start collecting the first segment after the eluent is equal to the amount of water added to the column. Then collect in 500 mL / segment increments.

[0109] Elution with 65% ethanol: Prepare a 65% concentration solution using recycled ethanol, totaling 2400 mL (6 times the amount of raw material). After the 15% ethanol is used up and a very thin liquid layer forms on the resin surface, add 65% ethanol in portions, collecting 800 mL from each of the first, middle, and last stages, and measuring and sampling them separately.

[0110] ⑤ Combine the collected 65% ethanol eluents and filter them through a 0.45 μm filter membrane.

[0111] S5: Drying process

[0112] The filtered 65% ethanol eluent was concentrated under reduced pressure until no alcohol odor remained, yielding a concentrated paste. A spray dryer was used with the inlet air temperature set at 190℃, the outlet air temperature at 85℃, the feed rate at 12 mL / min, and the atomization pressure at 0.25 MPa to obtain 33.2 g of pale yellow powdery extract of walnut branches and leaves, with a yield of 4.15% (based on 800 g of raw material).

[0113] Product testing: The total flavonoid content was determined to be 43.5% using ultraviolet spectrophotometry (sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method, measurement wavelength 500 nm). High performance liquid chromatography (HPLC) was also used to determine the content. Figure 1 As shown, the total content of rutin, hyperoside and astragaloside was determined to be 4.92%; the product has good solubility, good flowability and no caking.

[0114] Example 2: Photoaging Protection and After-Sun Soothing Essence Containing Extracts from Light-Protecting Walnut Branches and Leaves

[0115] Formula composition (by weight percentage):

[0116] Phase A: Glycerin 5.0%, Butylene Glycol 3.0%, Xanthan Gum 0.2%, Carbomer 0.15%, Sodium Hyaluronate 0.05%, Deionized Water Balance;

[0117] Phase B: Cetyl alcohol 2.0%, squalane 3.0%, polydimethylsiloxane 1.5%, glyceryl stearate / PEG-100 stearate 2.0%;

[0118] Phase C: 1.0% of the extract of walnut branches and leaves obtained in Example 1, 0.2% of bisabolol, 0.8% of phenoxyethanol / ethylhexylglycerin, and appropriate amount of fragrance.

[0119] Preparation process:

[0120] (1) Disperse xanthan gum and carbomer in phase A in glycerol and butylene glycol, add deionized water, heat to 80-85℃, and stir until dissolved and homogeneous;

[0121] (2) Mix the components of phase B and heat to 80-85℃ to dissolve;

[0122] (3) Add phase B to phase A at 80℃, homogenize and emulsify for 5 min, and then stir and cool.

[0123] (4) When the temperature drops to 45°C, add phase C (the extract is dissolved in a small amount of deionized water beforehand), continue stirring until room temperature, and discharge the product to obtain the photoaging protection and sun-soothing essence.

[0124] The resulting essence is a milky white, fine paste with a pH of 5.5-6.0. It does not separate into layers after centrifugation (3000 r / min, 30 min) and has satisfactory cold and heat resistance.

[0125] Comparative Example 1: Preparation of crude extract from branches and leaves of *Juglans regia* using an unoptimized alcohol extraction process.

[0126] The difference from Example 1 is that an unoptimized alcohol extraction process (50% ethanol concentration, solid-liquid ratio 1:6 g / mL, reflux extraction at 70℃ for 1 h, extraction once) was used, and the AB-8 macroporous adsorption resin purification step was omitted; otherwise, it was the same as Example 1. 48.5 g of extract was obtained, with a yield of 6.06%, a total flavonoid content of 21.8%, and a total content of rutin + hyperoside + astragaloside of 2.08% (e.g., ...). Figure 2 As shown in the figure, the product color is light yellow-green, and its photoaging protection and after-sun soothing effects are significantly lower than those in Example 1.

[0127] Comparative Example 2: D101 resin replaced AB-8 resin

[0128] The difference from Example 1 is that the AB-8 type macroporous adsorption resin was replaced with the D101 type macroporous adsorption resin; otherwise, it was the same as Example 1. 32.5 g of extract was obtained, with a yield of 4.06%, a total flavonoid content of 36.8%, and a total content of rutin + hyperoside + astragaloside of 4.25% (e.g., ...). Figure 3 As shown in the figure, the purity of the active ingredient is lower than that of Example 1. In vitro photo-aging protection activity assays showed that the UVB-damaged cell protection rate was 71.5%, and the MMP-1 inhibition rate was 67.2%, both significantly lower than those of Example 1 (89.5% and 86.2%), demonstrating that AB-8 resin has a better selective purification effect on the target component than D101 resin.

[0129] Comparative Example 3: Resin purification step omitted.

[0130] The difference from Example 1 is that the resin fractional purification process in step S4 is omitted; otherwise, it is the same as in Example 1. 47.8 g of extract was obtained, with a yield of 5.98%, a total flavonoid content of 25.5%, and a total content of rutin + hyperoside + astragaloside of 3.15% (e.g., ...). Figure 4 As shown in the figure, the product is a deep yellow-green color. In vitro post-sun exposure soothing activity assays showed inhibition rates of 69.5% and 71.2% against TNF-α and IL-6, respectively; and a UVB-damaged cell protection rate of 64.8%. This comparative example demonstrates that even with optimized alcohol extraction conditions, without fractional purification using AB-8 resin, the product purity, color, and efficacy are significantly lower than in Example 1.

[0131] Comparative Example 4: The 15% ethanol pre-washing step was omitted.

[0132] The difference from Example 1 is that the 15% ethanol elution step is omitted in step S42; otherwise, it is the same as in Example 1. Figure 5As shown, the experimental process resulted in a significant increase in the content of small molecule impurities in the 65% ethanol eluent (the product appeared as a light yellow powder). Simultaneously, the purity of the target flavonoid component (38.2%) was lower than that in Example 1 (43.5%), which may be the main reason for the significant reduction in its after-sun soothing effect. This comparative result demonstrates that the two-stage elution strategy of "15%→65%" is of great significance for synergistically achieving "impurity removal-enrichment" and improving product purity and efficacy.

[0133] Comparative Example 5: The pre-washing steps with deionized water and 15% ethanol were omitted.

[0134] The difference from Example 1 is that the elution steps of deionized water and 15% ethanol are omitted in step S42, while the rest is the same as in Example 1. The experimental process resulted in a significant increase in the content of small molecule impurities in the 65% ethanol eluent (the product appearance is a yellow-green powder), and the purity of the target flavonoid component (35.5%) was lower than that of Example 1 (43.5%).

[0135] Experimental Example 1: Optimization of Extraction Process

[0136] 1. Investigation of ethanol concentration (single-factor preliminary experiment)

[0137] The material-to-liquid ratio was controlled at 1:8 g / mL, and extraction was performed twice under reflux at 80℃ for 2 h. Extraction was carried out with 55%, 60%, 65%, 70%, and 75% ethanol, respectively. The total flavonoid content and characteristic monomer content in the crude extract were determined. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below:

[0138]

[0139] Experimental results: When extracted with 65% ethanol, the total flavonoids and characteristic monomers in the crude extract both reached their peak values, indicating the highest extraction efficiency.

[0140] 2. Study on the material-liquid ratio (single-factor preliminary experiment)

[0141] The extraction was performed twice, using 65% ethanol and reflux at 80℃ for 2 h. The effect of the solid-liquid ratio of 1:6, 1:8, 1:10, and 1:12 (g / mL) on the first extraction was investigated. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below:

[0142]

[0143] Experimental results: When the material-liquid ratio exceeds 1:8, the total flavonoid and characteristic monomer content does not increase significantly, but the solvent consumption increases and the cost rises. Considering both efficiency and cost, 1:(8-9) is selected as the optimal material-liquid ratio for the first extraction.

[0144] 3. Investigation of extraction time (single-factor preliminary experiment)

[0145] The extraction was performed twice, using 65% ethanol, a solid-liquid ratio of 1:8 g / mL, and at 80℃. The effects of extraction times of 1 h, 2 h, 3 h, and 4 h were investigated. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below:

[0146]

[0147] Note: The degradation rate of rutin in the characteristic monomer is used as a representative indicator.

[0148] Experimental results: When the extraction time was 2 hours, the content was high and the degradation rate of active ingredients was low. Further extending the extraction time resulted in a slight increase in content but a significant increase in degradation rate. Therefore, 2 hours was selected as the economical and effective extraction time.

[0149] 4. Precise screening of ethanol concentration (5% interval)

[0150] Under the conditions of a fixed material-to-liquid ratio of 1:8 g / mL, reflux extraction at 80℃ for 2 h, and extraction twice, a more refined gradient study of ethanol concentration was conducted, with a focus on monitoring the relative chlorophyll extraction yield. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below:

[0151]

[0152] Experimental results: When the ethanol concentration increased from 60% to 65%, the content of characteristic monomers still increased significantly (from 2.38% to 3.15%), while the increase in chlorophyll extraction was relatively gradual. However, when the concentration exceeded 65%, the chlorophyll extraction showed an explosive increase (64% increase at 70% compared to 65%, and 128% increase at 75% compared to 65%), while the content of the target component began to decrease. This proves that 65% ethanol is the "critical concentration" for achieving the separation of the target component from impurities in this process, and the determination of this parameter is non-obvious.

[0153] 5. Verification experiment of the combination of extraction temperature and time

[0154] A temperature-time combined validation experiment was designed under conditions of 65% ethanol and a solid-liquid ratio of 1:8. The total flavonoid content, characteristic monomer content, and retention rate of the key active monomer (rutin) in the crude extract were used as indicators. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below (the rutin content in the sample extracted at 80℃ / 2 h is used as a 100% control):

[0155]

[0156] Experimental results: Simply pursuing high content (90℃ / 2.5 h) leads to a degradation of nearly 8.8% in the key active monomer rutin, resulting in a loss of efficacy. While the total flavonoid content in the 80℃ / 2 h group was similar to that of the 90℃ / 2.5 h group, the rutin retention rate (100.0%) was significantly better than the latter (91.2%), demonstrating the best balance between quantity and quality. Therefore, this condition was determined to be the optimal condition.

[0157] Experimental Example 2: Resin Purification Process Data

[0158] 1. Resin type selection

[0159] Following the method described in Example 1, and controlling the same sample loading amount, loading speed, and elution conditions (65% ethanol elution), the adsorption and desorption rates of total flavonoids and characteristic monomers by three resins (AB-8, D101, and HPD-100) were compared. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below:

[0160]

[0161] Note: Characteristic monomers refer to the sum of rutin, hyperoside, and astragaloside.

[0162] Experimental results: AB-8 resin showed significantly better adsorption selectivity and desorption rate for the target component than other types, and achieved the best purification effect.

[0163] 2. Comparative Experiment of Segmented Washing Strategies

[0164] Using AB-8 resin and following the method described in Example 1, with the same sample loading amount and speed, three elution strategies were employed to determine the total flavonoids, characteristic monomers, and chlorophyll residue in the final product. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below:

[0165]

[0166] Experimental results: The "15%→65% two-stage elution" strategy of this invention achieves the best balance in terms of active ingredient purity and impurity removal. 15% ethanol effectively removes polar impurities, while 65% ethanol precisely enriches flavonoids; the synergy between the two produces unexpected technical effects.

[0167] 3. Optimization of sample loading amount and elution parameters

[0168] Using AB-8 resin, the effects of sample loading amount, sample loading speed, elution volume of 15% ethanol, and elution volume of 65% ethanol on the recovery rate and purity of the target components were systematically investigated. The total flavonoid recovery rate, characteristic monomer recovery rate, and characteristic monomer content in the final product were used as evaluation indicators. Two parallel samples were used for each group, and the average value was taken.

[0169] (1) Sample quantity investigation

[0170] With a fixed loading rate of 1.5 BV / h, and elution conditions of 4 BV elution with 15% ethanol and 6 BV elution with 65% ethanol, different loading volumes (concentrate volume:resin volume = 1:2, 1:3, 1:4, 1:5) were investigated. The results are shown in the table below:

[0171]

[0172] Note: Leakage rate refers to the percentage of characteristic monomer content in the effluent relative to the total amount of sample.

[0173] Experimental results: When the sample loading ratio was 1:4, the recovery rate was close to the plateau, and the leakage rate was below 6%. Further increasing the sample loading ratio had limited improvement in recovery rate but significantly increased the leakage rate. Considering all factors, a sample loading ratio of 1:4 was the optimal choice.

[0174] (2) Investigation on the amount of 65% ethanol used for elution

[0175] With a fixed loading volume of 1:4, loading speed of 1.5 BV / h, and elution with 15% ethanol for 4 BV, the effect of different elution volumes of 65% ethanol (4, 5, 6, and 7 BV, calculated as multiples of the raw material volume) on the elution efficiency of the target component was investigated. The results are shown in the table below:

[0176]

[0177] Note: The newly added recovery rate refers to the percentage of the target component that was eluted more in this segment compared to the previous segment.

[0178] Experimental results: When the elution volume increased from 5 BV to 6 BV, the recovery rate of characteristic monomers increased from 81.2% to 86.2%. Further increasing to 7 BV only increased the recovery rate by 1.6%, but the increased eluent volume raised subsequent concentration costs, and the purity decreased slightly. Therefore, 6 BV (6 times the amount of raw material) was selected as the optimal elution volume.

[0179] Based on the above optimization results, the optimal purification parameters were determined to be: resin dosage 1.5-2.0 times that of raw material, sample loading ratio 1:4 (concentrate volume: resin volume), loading rate 1.5 BV / h, elution with 15% ethanol for 4 BV, and elution with 65% ethanol for 6 BV. Under these conditions, the total recovery rate of the target component was ≥85%, and the total content of characteristic monomers in the purified product was ≥4.92%.

[0180] 4. Cross-experimentation of resin-eluent combinations

[0181] Three resins (AB-8, D101, HPD-100) were subjected to cross-experiments with three eluent concentrations (55%, 65%, and 75% ethanol) to compare the differences in total flavonoid content, total content of characteristic monomers, chlorophyll residue, and product color of the final products. Two parallel samples were used for each group, and the average value was taken. The results are shown in the table below:

[0182]

[0183] Experimental Results: The results show that, considering the purity of active ingredients, the residual amount of key impurities, and the product appearance, the combination of AB-8 resin and 65% ethanol elution achieves the optimal balance. Compared to the AB-8 / 55% combination, this combination results in significantly higher purity of active ingredients (total flavonoid content increased from 39.2% to 43.5%, and characteristic monomer content increased from 4.52% to 4.92%). Compared to the AB-8 / 75% combination, the purity of active ingredients in this combination shows limited difference, and the residual pigment impurities such as chlorophyll are extremely low (OD value decreased from 0.18 to 0.02), with an ideal pale yellow appearance.

[0184] This proves that the "AB-8 and 65% ethanol" combination selected in this invention is a "golden combination" with unexpected technical effects. This combination is not simply the superposition of "optimal resin" and "optimal elution concentration", but rather produces a synergistic effect of "1+1>2": AB-8 resin has a specific pore size matching and polarity adaptation for flavonoid components, while the polarity of 65% ethanol can selectively elute the target flavonoids, while effectively retaining pigment impurities such as chlorophyll that are strongly adsorbed by AB-8 on the resin.

[0185] Experimental Example 3: Product Physicochemical Properties and Efficacy Data

[0186] 1. Physicochemical Indicators

[0187] The extract obtained in Example 1 was a pale yellow powder, odorless, and with good flowability; the total flavonoid content was 43.5%, and the total content of rutin, hyperoside, and astragaloside was 4.92%; it was easily soluble in common solvents such as water, ethanol, and propylene glycol; the pH value was 5.5-6.5 (1% aqueous solution); and the heavy metal content met the requirements of the "Cosmetic Safety Technical Specifications" (2022 edition) and the "National Food Safety Standard".

[0188] 2. Antioxidant efficacy data

[0189] The in vitro antioxidant activity of the extracts was determined using the DPPH and ABTS methods. Vitamin C (VC) was used as a positive control, while unpurified ethanol extract (crude extract of Comparative Example 1), optimized ethanol extract but unpurified sample (Comparative Example 3), and D101 purified sample (Comparative Example 2) were used as negative controls. Three parallel samples were used for each group, and the average value was taken. The results are shown in the table below (test concentration 100 μg / mL):

[0190]

[0191] Experimental results: The antioxidant activity of the extract of this invention is significantly better than that of the unpurified sample and the D101 purified sample, and is close to the antioxidant effect of VC.

[0192] To further investigate the dose-effect relationship of the extract from Example 1, the DPPH scavenging rate was measured at concentrations of 25, 50, 100, and 200 μg / mL. The results are shown in the table below:

[0193]

[0194] Experimental results: The extract exhibited significant concentration-dependent antioxidant activity in the range of 25-100 μg / mL, reaching a plateau at 100 μg / mL, with an IC50 value of [missing value]. 50 It is 15.8 μg / mL.

[0195] 3. Data on photoaging protection efficacy

[0196] (1) UVB damage cell protection experiment

[0197] A UVB photoaging damage model (UVB irradiation dose 30 mJ / cm²) was established using human skin fibroblasts (HSF) and human immortalized keratinocytes (HaCaT) to detect the protective effect of the extract on cell viability. A blank group (no UVB irradiation), a model group (UVB irradiation without sample), a sample group (UVB irradiation + different concentrations of extract), and a positive control group (UVB irradiation + vitamin C) were set up. After 24 h of culture, cell viability was detected using the CCK-8 assay. Six replicates were used in each group, and the average value was taken. The results are shown in the table below.

[0198]

[0199] Experimental Results: The extract from Example 1 exhibited concentration-dependent protection against UVB-damaged cells within the concentration range of 0.05%-0.5%. At a concentration of 0.2%, the protection rate against HSF cells reached 87.8%, approaching the level of the VC positive control and significantly superior to the control.

[0200] (2) ROS scavenging and MMP-1 inhibition experiments

[0201] In the UVB damage model described above, intracellular ROS levels were detected using the DCFH-DA probe, MMP-1 content in the culture supernatant was detected using an ELISA kit, and type I collagen (COL-1) expression was detected using Western blotting. The results are shown in the table below:

[0202]

[0203] Experimental results: The 0.2% concentration of the extract from Example 1 could scavenge 83.2% of UVB-induced ROS, inhibit 86.2% of MMP-1 overexpression, and restore COL-1 expression from 35.2% in the model group to 79.5%, demonstrating its significant photo-aging protection efficacy.

[0204] 4. Data on post-sun soothing effects

[0205] (1) LPS-induced macrophage inflammation model

[0206] An LPS-induced inflammation model was established in RAW 264.7 cells. The inhibitory effects of the extract on the expression of inflammatory factors TNF-α, IL-6, and IL-1β, as well as inflammatory mediators NO and PGE-2, were examined. Dexamethasone was used as a positive control, while a blank group (without LPS induction) and a model group (LPS induction without sample) served as controls. Four concentration gradients (0.05%, 0.1%, 0.2%, and 0.5%) were set up, with three replicates per group. The average value was taken, and the results are shown in the table below.

[0207]

[0208] Experimental results: The extract of this invention exhibited significant concentration-dependent anti-inflammatory and soothing effects within the concentration range of 0.05%-0.5%. At a final concentration of 0.2%, the inhibition rates against TNF-α and IL-6 reached 89.2% and 91.8%, respectively, and the inhibition rates against NO and PGE-2 reached 87.2% and 85.5%, respectively, approaching the inhibitory effect of dexamethasone; the cell viability at all concentrations was ≥95%, indicating that the extract was non-toxic to cells.

[0209] (2) UVB-induced HaCaT cell inflammation model

[0210] A sunburn damage model was established by irradiating HaCaT cells with UVB (30 mJ / cm²). The inhibitory effect of 0.2% of the extract from Example 1 on inflammatory factors was detected. The experimental results are shown in the table below:

[0211]

[0212] Experimental results: In the UVB-induced post-sun damage model, the extract of Example 1 showed excellent soothing and anti-inflammatory activity, which was significantly better than that of the unpurified sample.

[0213] (3) Skin barrier repair efficacy experiment

[0214] HaCaT cells were used to detect the effect of 0.2% of the extract from Example 1 on the expression of barrier-related proteins. Western blotting was used to detect the relative expression levels of filaggrin (FLG) and loricrin (LOR), with the blank group serving as a 100% control. The specific results are shown in the table below (0.2%, 24 h):

[0215]

[0216] Experimental results: The extract of Example 1 significantly upregulated the expression of FLG and LOR in HaCaT cells, suggesting its potential to promote skin barrier repair, with an effect close to that of the niacinamide positive control.

[0217] Test Example 4: Safety Test Data

[0218] (1) Repeated skin irritation tests

[0219] Test method:

[0220] Following the repeated skin irritation test method in the "Cosmetic Safety Technical Specifications" (2022 edition), six healthy rabbits weighing 2.0-2.5 kg were selected, and the hair on both sides of the spine on the back was clipped (approximately 3 cm × 3 cm area). The experimental group was treated with 0.5 mL of a 0.5% extract aqueous solution, while the control group was treated with 0.5 mL of physiological saline. The solution was covered with two layers of gauze and one layer of cellophane, and secured with non-irritating adhesive tape. The treatment was applied for 4 hours daily for 14 consecutive days. Skin reactions were observed 1 hour after each removal of the test substance.

[0221] Experimental results: The rabbits in the experimental group showed no irritation reactions such as erythema and edema on their skin, and the average irritation score was 0 (<0.5), which was judged as no skin irritation.

[0222] (2) Acute eye irritation / corrosiveness test

[0223] Test method:

[0224] According to the acute eye irritation / corrosiveness test method in the "Cosmetic Safety Technical Specifications" (2022 edition), three healthy rabbits were selected. 0.1 mL of a 0.5% extract aqueous solution was instilled into the conjunctival sac of one eye, with the other eye serving as a self-control. Corneal, iris, and conjunctival reactions were observed and scored at 1, 24, 48, and 72 hours, and on days 4 and 7.

[0225] Experimental results: All observation time points scored 0 points, indicating no eye irritation.

[0226] (3) Guinea pig skin allergy test (maximum value test, GPMT)

[0227] Test method:

[0228] According to the skin allergy test method in the "Cosmetic Safety Technical Specifications" (2022 edition), 24 healthy guinea pigs were selected and divided into an experimental group (n=12), a negative control group (n=6, saline), and a positive control group (n=6, 2,4-dinitrochlorobenzene). Induction period (day 0): The experimental group received an intradermal injection of 0.1 mL of 0.1% extract solution (containing Freund's complete adjuvant FCA); Skin induction (day 7): The experimental group received a 0.5 mL application of 10% extract solution, followed by 48 h of closed fixation; Challenge (day 21): The experimental group received a 0.5 mL application of 5% extract solution, followed by 24 h of closed fixation. Skin reactions were observed 24 h and 48 h after challenge removal.

[0229] Experimental results: The sensitization rate in the experimental group of guinea pigs was 0%, with no sensitization reactions such as erythema or papules; all guinea pigs in the positive control group showed positive reactions. This indicates that the extract is not sensitizing.

[0230] (4) Human Repeated Injury Patch Test (HRIPT)

[0231] Test method:

[0232] The HRIPT test was conducted by a qualified testing institution. Fifty-three healthy volunteers (aged 18-65, with no history of skin diseases or allergies) were selected, and the test was performed using a closed patch applicator. Induction period: Patches were applied three times a week for 24 hours each time, for a total of 3 weeks (9 applications in total), with a dosage of 0.02-0.05 mL (1% extract aqueous solution). Restoration period: 2 weeks. Activation period: After the restoration period, patches were applied again for 24 hours, and observation continued for 72 hours.

[0233] Results: No subjects showed ≥ grade 2 skin reactions during the induction and stimulation phases, with a sensitization rate of 0%, indicating no skin irritation or sensitization.

[0234] Experimental Example 5: Stability Study

[0235] (1) Accelerated stability test

[0236] The extract powder obtained in Example 1 was placed in a constant temperature and humidity chamber at 40°C and 75% relative humidity (sealed packaging) for 3 months. Samples were taken monthly to test the total flavonoid content, total content of characteristic monomers, DPPH free radical scavenging rate and appearance. Three parallel samples were taken for each group, and the average value was taken. The details are shown in the table below.

[0237]

[0238] Test results: After 3 months of accelerated stability testing, the total flavonoids, characteristic monomers, and DPPH scavenging rates all remained above 90% of their initial values, indicating good stability of the active ingredients. Slight clumping occurred from the third month onwards, and the moisture content increased slightly, suggesting that long-term storage requires sealing and moisture protection.

[0239] (2) Long-term stability test

[0240] The extract powder obtained in Example 1 was sealed and packaged, and then placed at 25°C and 60% relative humidity for 12 months. Samples were taken and tested every 3 months, as shown in the table below.

[0241]

[0242] Test results: After 12 months of long-term stability testing, all indicators remained above 95% of their initial values, and there were no obvious changes in appearance, indicating that the extract has excellent stability under normal storage conditions, and the shelf life can be tentatively set at 2 years.

[0243] (3) Stability of formulation application

[0244] The extract obtained in Example 1 was added at 1.0% to a basic skin care lotion (pH 5.5-6.5, containing 5% glycerin, 3% squalane, 2% cetyl alcohol, 1.5% emulsifier, and 0.8% preservative). The lotion was placed at 25°C (room temperature) and 40°C (accelerated temperature) for 3 months. The appearance, color, and odor of the product were observed monthly, and the content and efficacy of active ingredients were tested. The details are shown in the table below.

[0245]

[0246] Test results: The product showed no abnormal phenomena such as stratification, discoloration, or odor within 3 months under both room temperature and accelerated conditions. The total flavonoids, DPPH scavenging rate, and TNF-α inhibition rate did not decrease significantly. The degradation trend under accelerated conditions at 40℃ was in line with expectations (higher than the 25℃ room temperature group), indicating that the extract has good stability in cosmetic formulations and good compatibility with commonly used cosmetic raw materials.

[0247] Experimental Example 6

[0248] The commercially available rutin, hyperoside, and astragaloside were mixed in equal molar amounts, and the protective rate of the mixture against HSF cells was measured. The results showed that the protective rate of the mixture against HSF cells was less than 87.8%, while the extract of the present invention had a protective rate of 87.8% against HSF cells. This demonstrates that the specific ratio of the extract of the present invention produced a synergistic effect, rather than a simple additive effect.

[0249] The detection method used in this invention is as follows:

[0250] (1) Determination of total flavonoid content: The sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method was adopted, referring to the method on page 1368 of the Chinese Pharmacopoeia 2020. Accurately weigh rutin reference standard and prepare a series of standard solutions of different concentrations; take an appropriate amount of the sample solution to be tested, add 0.3 mL of 5% sodium nitrite solution, shake well and let stand for 6 min; add 0.3 mL of 10% aluminum nitrate solution, shake well and let stand for 6 min; add 4.0 mL of 4% sodium hydroxide solution, dilute to the mark with 30% ethanol, shake well and measure the absorbance at a wavelength of 500 nm to calculate the total flavonoid content.

[0251] (2) Determination of characteristic monomer content: High performance liquid chromatography (HPLC) was used. Chromatographic column: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B), gradient elution (0-10 min, 15%A→25%A; 10-20 min, 25%A→35%A; 20-30 min, 35%A→45%A); detection wavelength: 360 nm; flow rate: 1.0 mL / min; column temperature: 30℃; injection volume: 10 μL. Quantification was performed using external standard methods with rutin, hyperoside, and astragaloside reference standards.

[0252] (3) DPPH free radical scavenging ability

[0253] Reagents: 0.1 mmol / L DPPH ethanol solution (freshly prepared, protected from light). Method: Mix 2.0 mL of sample solution with 2.0 mL of DPPH ethanol solution, react at room temperature in the dark for 30 min, and measure the absorbance A_i at 517 nm; use anhydrous ethanol instead of DPPH to measure A_j; use deionized water instead of sample to measure A_0. Formula: Scavenging rate (%) = [1 - (A_i - A_j) / A_0] × 100%. Results: Calculate IC50. 50 value.

[0254] (4) ABTS free radical scavenging ability

[0255] Reagents: ABTS working solution (7 mmol / L ABTS and 2.45 mmol / L potassium persulfate mixed in equal volumes, reacted in the dark for 12-16 h, diluted with PBS to an absorbance of 0.70±0.02 at 734 nm before use). Method: Mix 0.5 mL of sample solution with 3.0 mL of ABTS working solution, react at room temperature in the dark for 6 min, and measure the absorbance at 734 nm. Formula: Scavenging rate (%) = (A_0 - A_i) / A_0 × 100%. Results: Calculate IC50. 50 value.

[0256] (5) Chlorophyll residue determination: Spectrophotometry was used.

[0257] Accurately weigh an appropriate amount of chlorophyll a reference standard, dissolve and dilute it with anhydrous ethanol to prepare a series of standard solutions of different concentrations. Using anhydrous ethanol as a blank, measure the absorbance at a wavelength of 665 nm and plot a standard curve. Accurately weigh approximately 0.1 g of the plant extract to be tested, place it in a stoppered brown conical flask, accurately add 25 mL of anhydrous ethanol, seal tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 min, cool, weigh again, replenish the lost weight with anhydrous ethanol, shake well, filter through a 0.45 μm organic phase filter membrane, and use the filtrate as the test solution. Accurately measure the test solution, measure the absorbance at a wavelength of 665 nm, and read the concentration (μg / mL) of total chlorophyll (calculated as chlorophyll a) in the test solution from the standard curve. Calculate the result (all operations should be carried out under weak light or dark conditions to avoid chlorophyll degradation).

[0258] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0259] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing an extract from the branches and leaves of *Juglans regia*, comprising: S1: Raw material pretreatment Fresh walnut branches and leaves are selected, impurities are removed, and the leaves are dried and crushed to obtain walnut branch and leaf powder. Its characteristic is that it further includes: S2: Secondary reflux extraction with 65% ethanol Using 65% ethanol as the extraction solvent, the concentration of 65% ethanol is at the critical concentration point for co-dissolution of chlorophyll in the branches and leaves of *Juglans regia*, where chlorophyll and flavonoids are dissolved simultaneously at a concentration of 65% ethanol. S21: First extraction The powdered branches and leaves of walnut trees were refluxed with 65% ethanol in a water bath, filtered, centrifuged, and the extract was collected. S22: Second extraction Add 65% ethanol to the filter residue obtained in step S21, reflux in a water bath, and centrifuge and filter. S23: Combine the extracts obtained in steps S21 and S22 and remove solid impurities; S3: Reduced pressure concentration; S4: AB-8 type macroporous adsorption resin for segmental purification A two-stage gradient elution method was used: "pre-elution with 15% ethanol to remove chlorophyll - elution with 65% ethanol to enrich flavonoids". S41: Adsorption washing; S42: Segmented gradient elution First, elute with deionized water, then elute with 15% ethanol, and discard the eluent; then elute with 65% ethanol to selectively enrich flavonoid target compounds, and collect all 65% ethanol eluent in fractions. S43: Filtration eluent; S5: Drying and grinding.

2. The method for preparing an extract from the branches and leaves of *Juglans regia* as described in claim 1, characterized in that, Step S21 is as follows: Mix the liquid and material at a ratio of 1:(8-9), place the mixture in a constant temperature water bath reflux device, and extract by constant temperature reflux at 80-85℃ for 2 hours.

3. The method for preparing an extract from the branches and leaves of *Juglans regia* as described in claim 2, characterized in that, Step S22 specifically involves adding 65% ethanol to the filter residue obtained in step S21, with a material-to-liquid ratio of 1:(6-8), and extracting by constant temperature reflux at 80-85℃ for 2 hours.

4. The method for preparing an extract from the branches and leaves of *Juglans regia* as described in claim 1, characterized in that, Step S3 specifically involves placing the combined extract in a rotary distillation flask and performing vacuum rotary distillation under reduced pressure until it is concentrated into a thick slurry or micro paste.

5. The method for preparing an extract from the branches and leaves of *Juglans regia* as described in claim 1, characterized in that, Step S41 is as follows: The amount of resin used is 1.5-2.0 times the amount of raw material fed. The extract obtained in step S3 is diluted with water. The total liquid volume after dilution is 3 times the mass of the raw material. Stir for 30 minutes, then filter with a 60-mesh sieve and discard the filtrate. The filter cake is replenished with water again, washed and stirred for 10 minutes for the second time, and then filtered with a 60-mesh sieve and discarded.

6. The method for preparing an extract from the branches and leaves of *Juglans regia* as described in claim 5, characterized in that, Step S42 is as follows: First, elute with deionized water, the total amount of which is 4 times the amount of raw material. Then, use the recovered ethanol from step S3 to prepare a 15% concentration by volume, the total amount of which is 4 times the amount of raw material. Discard the eluent. Use the recovered ethanol from step S3 to prepare a 65% concentration by volume, the total amount of which is 6 times the amount of raw material. After the 15% ethanol is used up, add the 65% ethanol in portions and collect the eluent in segments.

7. The method for preparing an extract from the branches and leaves of *Juglans regia* as described in claim 1, characterized in that, Step S5 includes a vacuum concentration process, wherein the vacuum concentration conditions are: vacuum degree 0.06-0.08 MPa, temperature 50-60℃.

8. The method for preparing an extract from the branches and leaves of *Juglans regia* as described in claim 7, characterized in that, Step S5 also includes spray drying, wherein the spray drying conditions are: inlet air temperature 180-200℃, outlet air temperature 80-90℃, feed rate 10-15 mL / min, and atomization pressure 0.2-0.3 MPa.

9. A *Juglans regia* branch and leaf extract prepared by the method according to any one of claims 1-8, characterized in that, The extract from the branches and leaves of the walnut tree is a pale yellow powder that meets the following combination of characteristics: Total flavonoid content ≥35wt%; total content of the three characteristic monomers rutin, hyperoside, and astragaloside ≥4wt%; molar ratio of rutin, hyperoside, and astragaloside is 1:(1.2-1.8):(0.5-0.8); in the HPLC fingerprint, at a detection wavelength of 280nm, three characteristic peaks appear in the retention time range of 12-18min, and the sum of the peak areas accounts for 25%-35% of the total peak area; pH of 1% aqueous solution is 5.5-6.5; chlorophyll residue is... The value is ≤0.

05.

10. An application of the extract from the branches and leaves of *Juglans regia* as described in claim 9, characterized in that... The application of the extract in the preparation of photoaging protection and post-sun exposure soothing and repair products with triple effects of UVB photoaging protection, post-sun exposure anti-inflammatory and soothing, and skin barrier repair is based on the synergistic activity of the extract at a concentration of 0.2% simultaneously achieving: HSF cell UVB protection rate ≥87%, TNF-α inhibition rate ≥89%, and FLG upregulation ≥188%.

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