An industrial co-production method of active components of biota orientalis, the obtained active components and application

Abstract

The application discloses an industrial co-production method of active components of Platycladus orientalis, the obtained active components and application. The method adopts a segmented extraction strategy of water first and alcohol later to synchronously obtain components rich in active short peptides and quercitrin from the Platycladus orientalis, and the components are mixed according to a standardized proportion after purification. The obtained composition has a remarkable effect in promoting hair growth, can effectively stimulate regeneration, and can make the new hair become thick, firm and lustrous, and has a comprehensive effect superior to conventional extracts and positive control drugs. The product can be used for preparing skin external agents for improving hair quality, and has high efficiency, high resource utilization rate and stable product quality.

Description

Technical Field

[0001] This invention relates to an industrial co-production method for active ingredients of Platycladus orientalis, the obtained active ingredients and their applications, belonging to the field of drug extraction and preparation. Background Technology

[0002] The value of Platycladus orientalis, a traditional Chinese medicinal herb and a potential source of functional cosmetic ingredients, has long been recognized. It is known to contain various chemical components, including flavonoids, volatile oils, and some bioactive peptides. Modern pharmacological studies suggest it possesses multiple biological activities, such as antioxidant, anti-inflammatory, antibacterial, and hair growth-promoting effects. However, current technologies, especially industrial-scale extraction and production, face significant limitations and technical bottlenecks in the development and utilization of Platycladus orientalis resources. Current mainstream processes focus on obtaining single-category active ingredients, most commonly using high-concentration organic solvents such as ethanol or methanol for single-stage extraction or hot reflux to maximize the extraction of total flavonoids or volatile oils; or using hot water to extract polysaccharides. These single-solvent extraction strategies severely neglect the significant differences in the physicochemical properties between different active components in Platycladus orientalis raw materials, particularly ignoring the presence and protection of short peptides or small-molecule proteins that are highly water-soluble but sensitive to heat and acid / alkali environments. The direct consequence is that, in pursuit of higher yields of certain components such as flavonoids, harsh conditions such as high temperatures, strong acids / alkalis, or prolonged extraction are often employed. This undoubtedly damages the natural conformation and bioactivity of thermally unstable or easily hydrolyzed active peptides, leading to their inactivation or extremely low yield. Simultaneously, this crude extraction method results in the extract containing a large amount of impurities such as polysaccharides, proteins, pigments, tannins, and inorganic salts, placing a heavy burden on subsequent separation and purification of the target components, resulting in high costs and low efficiency. Especially in applications targeting specific effects such as promoting hair growth, existing technologies often yield crude extracts or single-component products with limited and unstable effects: they may only slightly stimulate hair follicles to enter the growth phase, but are unlikely to improve the quality of newly grown hair, such as its fineness and tendency to fall out, or fail to provide the comprehensive nutrition and protection needed for healthy hair follicles. The fundamental reason is the failure to synergistically extract and retain those active short peptides crucial for hair follicle cell proliferation, differentiation, and hair keratin synthesis, as well as the potential synergistic effects with flavonoids. Furthermore, due to the non-selectivity of the extraction process and the inadequacy of purification technology, the content of active ingredients in the final product fluctuates greatly, and the impurity profiles are unclear, making it difficult to achieve standardization and consistent quality. This fails to meet the stringent requirements of modern functional cosmetics or pharmaceutical raw materials for quality consistency, safety, and efficacy stability. Therefore, the industry urgently needs to develop a new green co-production process that can accommodate active ingredients with different characteristics, possess both high extraction efficiency and high selectivity, achieve standardized production, and whose final product can exhibit significant synergistic effects in specific applications such as promoting healthy hair growth. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides an industrial co-production method for active ingredients from Platycladus orientalis, comprising the following steps: (1) Using Platycladus orientalis as raw material, the first stage of water extraction and the second stage of alcohol extraction were carried out in sequence; (2) The extract obtained from the first stage of water extraction was subjected to membrane separation and gel filtration to obtain component A rich in short peptides; (3) The extract obtained from the second stage of alcohol extraction was purified by macroporous resin to obtain component B rich in quercetin; (4) Mix component A and component B in a predetermined ratio to obtain a standardized product.

[0004] Preferably, in step (1), the first stage of water extraction uses pre-cooled phosphate buffer solution for slow stirring extraction at low temperature, followed by solid-liquid separation and filtration; the second stage of alcohol extraction uses 60%-75% ethanol aqueous solution to heat and reflux extract the residue after the first stage of water extraction.

[0005] Preferably, the concentration of the phosphate buffer is 40-60 mM, the pH value is 7.0-7.5, the extraction temperature is 2-8℃, the stirring speed is 50-150 rpm, the extraction time is 12-24 hours, and the number of extractions is 1-3.

[0006] Preferably, the solid-liquid separation and filtration includes: centrifuging the extract at 2-8℃ and 8000-12000 rpm for 15-25 min, and then filtering the supernatant through microporous membranes of 0.45 μm and 0.22 μm in sequence.

[0007] Preferably, in step (2), the membrane separation includes ultrafiltration and nanofiltration performed sequentially; the ultrafiltration is performed by first using an ultrafiltration membrane with a molecular weight cutoff of 8-12 kDa to remove macromolecular impurities, and then using an ultrafiltration membrane with a molecular weight cutoff of 0.8-1.2 kDa to concentrate and desalinate the permeate, and collect the permeate rich in short peptides with a molecular weight of less than 1.2 kDa.

[0008] Preferably, the gel filtration uses dextran gel G-10, G-15, or Superdex™ 30 as the packing material, and elution is performed using a 20-80 mM ammonium acetate aqueous solution as the mobile phase. Elution peaks with absorption at 220 nm are collected and combined to obtain a primary solution of component A. The primary solution of component A is then desalted and concentrated by passing it through a nanofiltration membrane or a reverse osmosis membrane. The nanofiltration membrane has a molecular weight cutoff of 200-300 Da to obtain a concentrated solution of component A.

[0009] Preferably, in the second stage of alcohol extraction, the concentration of the ethanol aqueous solution is 65%-70%, the extraction temperature is 70-80℃, the extraction time is 1-1.5 hours each time, and the extraction is performed twice; the extracts are combined, filtered, and crude quercetin extract is obtained.

[0010] Preferably, in step (3), the macroporous resin purification includes: loading the crude quercetin extract aqueous solution after ethanol removal onto a macroporous adsorption resin column, first eluting with water until the eluent is colorless, then eluting with an ethanol aqueous solution of 50%-70% concentration, collecting the ethanol eluent, and concentrating under reduced pressure to obtain component B extract; the macroporous adsorption resin is AB-8 type, D-101 type or HPD-100 type; in step (4), the predetermined ratio is determined by the following method: measuring the content of short peptides in component A and the content of quercetin in component B respectively, and then using the quercetin content ≥300 mg / kg and the short peptide content ≥100 mg / kg in the final product after mixing as the standard, calculating the mixing ratio of component A and component B; the mixing is carried out under stirring conditions, and one or more of solvents and preservatives are added to the mixing system for adjustment; the solvent is one or more of water, propylene glycol, and glycerin; the preservative is one or more of phenoxyethanol, methylparaben, and p-hydroxyacetophenone.

[0011] The present invention also provides a composition of active ingredients of Platycladus orientalis prepared by the above method, which is composed of component A rich in short peptides with a molecular weight of less than 1200 Da and component B rich in quercetin, and the quercetin content is not less than 300 mg / kg and the short peptide content is not less than 100 mg / kg based on the total mass of the composition.

[0012] Preferably, the composition further comprises a solvent and a preservative; the solvent is one or more of water, propylene glycol, and glycerin; the preservative is one or more of phenoxyethanol, methylparaben, and p-hydroxyacetophenone.

[0013] The present invention also provides the use of the above-mentioned arborvitae active ingredient composition in the preparation of topical skin agents.

[0014] Preferably, the topical skin agent is a cosmetic, skin care product, or topical medicine with anti-aging, antioxidant, anti-inflammatory, or whitening effects.

[0015] The beneficial effects of this invention are: The method for the industrial co-production of active ingredients from Platycladus orientalis provided by this invention, and the resulting products, produce significant and synergistic multi-level beneficial effects through the organic integration of a series of innovative technical features. Firstly, the most crucial contribution lies in the creative adoption of a segmented gradient extraction strategy of first water and then alcohol. This design profoundly aligns with the inherent physicochemical properties of the main active components in Platycladus orientalis: hydrophilic, heat-sensitive bioactive short peptides and moderately polar, relatively heat-stable flavonoid glycosides. The first step utilizes a pre-cooled phosphate buffer solution within a specific pH range for low-temperature, slow extraction, providing a near-physiologically mild environment for the fragile water-soluble short peptides. This maximizes the inhibition of enzymatic hydrolysis, acid / alkali hydrolysis, and thermal denaturation, ensuring the retention of activity and high yield of these components from the source. Next, for the residue after the removal of water-soluble components, a suitable concentration of ethanol is used for hot reflux extraction. This condition provides extremely high dissolution efficiency for flavonoid components such as quercetin, and because most interfering water-soluble impurities have already been removed in the previous stage, this step is more targeted and has a simpler impurity background. This divide-and-conquer sequential extraction model fundamentally overcomes the inherent contradiction that a single solvent cannot simultaneously balance the extraction efficiency and stability of two types of highly different active ingredients. It achieves the goal of simultaneously, efficiently, and actively obtaining two types of high-value products from the same raw material, greatly improving the overall utilization rate and economic value of Platycladus orientalis resources. Secondly, at the specific separation and purification level, this invention constructs a multi-stage precision separation system, ensuring the high purity and high activity of each component, laying the material foundation for the superior efficacy of the final product. More notably, by standardizing and compounding the highly active short peptide concentrate obtained by the above method with high-purity quercetin concentrate, the resulting composition exhibits unique advantages over conventional products in specific application scenarios, especially in promoting hair growth and improving hair quality. Animal experiments show that the product of this invention not only effectively promotes hair regeneration in mice, but its effect in hair regeneration density is comparable to or even superior to the positive control drug minoxidil. More importantly, it shows a significant advantage in improving hair quality, promoting thicker and more robust new hair with a significantly increased diameter and a healthy shine. Simultaneously, hair pull-out strength tests show stronger adhesion and less likelihood of hair falling out, reflecting a more comprehensive improvement in the health of hair follicles. This dual effect of promoting growth and improving quality is considered to be the result of the synergistic effect of the well-preserved active short peptides and quercetin in this invention. Comparative experiments confirm that any product that disrupts this complete process chain significantly reduces its hair regeneration effect, especially the degree of improvement in hair quality. Finally, the standardized mixing steps introduced in this invention ensure the constant content of active ingredients in the final product and batch consistency, enabling the aforementioned excellent and stable efficacy to be reliably reproduced in industrial production. This provides a solid technical guarantee and core value for developing a new generation of standardized natural functional raw materials that combine hair growth, hair strengthening, and hair quality improvement. Attached Figure Description

[0016] Figure 1 The images show photos of mouse hair as an example. From left to right, the three groups are photos of day 1, day 5, and day 10. From top to bottom, each group is Example 1, Example 2, Example 3, Minoxidil control group, and Comparative Example 1.

[0017] Figure 2 The image shows the liquid chromatogram of component A (peptide) obtained in Example 1.

[0018] Figure 3 The liquid chromatogram of component B (quercetin) obtained in Example 1 is shown. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0022] Example 1

[0023] Weigh 1.0 kg of dried and pulverized Platycladus orientalis leaves and place them in an extraction vessel. Add 10 L of pre-cooled 50 mM phosphate buffer (pH 7.2) and extract slowly at 100 rpm for 18 hours in a 4°C freezer. After extraction, coarsely filter the solution through a plate and frame filter press. Transfer the filtrate to a high-speed tubular centrifuge and centrifuge at 10,000 rpm for 20 min at 4°C. Collect the supernatant and perform fine filtration successively through 0.45 μm and 0.22 μm capsule filters to obtain a clear aqueous extract. Pump this clear extract into a membrane separation system equipped with a 10 kDa molecular weight cutoff ultrafiltration membrane pack and perform ultrafiltration at an operating pressure of 0.3 MPa. Collect all permeate. The permeate was then pumped into a system equipped with a 1 kDa molecular weight cutoff ultrafiltration membrane for concentration and desalting at an operating pressure of 0.5 MPa. When the volume was concentrated to 1 / 10 of the original permeate, the permeate with a molecular weight less than 1 kDa was collected; this was the crude enrichment solution for short peptides. This crude enrichment solution was loaded onto a Sephadex G-15 gel column (5 L column volume) equilibrated with 50 mM ammonium acetate. Elution was performed using 50 mM ammonium acetate solution as the mobile phase at a flow rate of 2 BV / h. The eluent fractions showing absorption peaks were monitored at 220 nm using a UV detector, and the eluent fractions were collected and combined to obtain the short peptide solution. This solution was then desalted and finally concentrated using a nanofiltration membrane with a molecular weight cutoff of 250 Da, yielding approximately 200 mL of clear, tasteless short peptide concentrate (fraction A). A portion of fraction A was taken, lyophilized, and prepared for analysis. The Platycladus orientalis residue after the first-stage water extraction was drained and returned to the extraction vessel. Add 8 L of 70% ethanol aqueous solution to the vessel, heat to 75℃ and reflux for 1 hour, filter and collect the extract; add 6 L of 70% ethanol to the residue again and repeat reflux extraction for 1 hour. Combine the two ethanol extracts and filter with filter paper. Concentrate the filtrate under reduced pressure at 50℃ and -0.09 MPa, recover the ethanol, until the concentrate has no alcohol odor, to obtain approximately 1.5 L of crude quercetin aqueous solution. Load this aqueous solution onto a pre-treated AB-8 macroporous resin column (column volume 2 L) at a flow rate of 2 BV / h. After loading, elute with 5 BV of deionized water at the same flow rate, discard the water wash. Then elute with 60% ethanol aqueous solution at a flow rate of 2 BV / h, collect this ethanol eluent until the eluent shows virtually no quercetin spots upon thin-layer chromatography. Concentrate the collected ethanol eluent under reduced pressure at 50℃ and -0.09 MPa to obtain a deep yellow viscous extract (component B). The lyophilized sample A and sample B were accurately weighed separately, and their short peptide content (calculated as tripeptide standard) and quercetin content were determined by high performance liquid chromatography. The short peptide content in component A was found to be 1.2 mg / mL (calculated as solution), and the quercetin content in component B was found to be 35% (w / w).The target final product is set as follows: quercetin ≥ 500 mg / kg, short peptide ≥ 150 mg / kg. Calculations show that 125 mL of component A concentrate (containing 150 mg of short peptide) and 1.43 g of component B extract (containing 500 mg of quercetin) are required. The calculated amounts of component A concentrate and component B extract are placed in a mixing vessel, and 40 mL of glycerol, 8 g of PEG-40 hydrogenated castor oil, and 5 g of phenoxyethanol are added. Deionized water is then added to bring the total mass to 1.0 kg. The mixture is stirred at 500 rpm at room temperature until homogeneous, yielding the Platycladus orientalis active ingredient composition of this invention.

[0024] Example 2

[0025] Weigh 1.0 kg of dried and pulverized Platycladus orientalis leaves and place them in an extraction vessel. Add 12 L of pre-cooled 40 mM phosphate buffer (pH 7.0) and extract slowly at 150 rpm for 24 hours in a 2°C freezer. Subsequent solid-liquid separation and filtration steps are the same as in Example 1. In the ultrafiltration step, an 8 kDa membrane was used first, followed by a 0.8 kDa membrane for concentration, with operating pressures of 0.25 MPa and 0.45 MPa, respectively. Gel filtration was performed using Superdex™ 30 packing material, eluted with 20 mM ammonium acetate. The nanofiltration membrane had a molecular weight cutoff of 200 Da. Approximately 180 mL of short peptide concentrate (component A) was obtained. In the alcohol extraction stage, 65% ethanol was used, and extraction was performed by reflux at 80°C for 1.5 hours each time, for a total of 2 times. The macroporous resin used was type D-101, and the elution solvent was 50% ethanol. After purification and concentration, component B extract was obtained. HPLC analysis revealed that component A (short peptide) contained 1.0 mg / mL, and component B (quercetin) contained 30%. The target values ​​for the final product were set as follows: quercetin ≥ 300 mg / kg, and short peptide ≥ 100 mg / kg. The appropriate amounts of components A and B were calculated and mixed, and 50 mL of propylene glycol, 0.5 g of methylparaben, and 0.5 g of p-hydroxyacetophenone were added. Water was added to bring the total mass to 1.0 kg, and the mixture was stirred until homogeneous to obtain the composition of this invention.

[0026] Example 3

[0027] Weigh 1.0 kg of dried and pulverized Platycladus orientalis leaves and place them in an extraction vessel. Add 8 L of pre-cooled 60 mM phosphate buffer (pH 7.5) and extract slowly at 50 rpm for 12 hours in an 8°C freezer. Subsequent solid-liquid separation and filtration steps are the same as in Example 1. In the ultrafiltration step, a 12 kDa membrane was used first, followed by a 1.2 kDa membrane, with operating pressures of 0.35 MPa and 0.55 MPa, respectively. Gel filtration was performed using Sephadex G-10 packing material, eluted with 80 mM ammonium acetate. The nanofiltration membrane had a molecular weight cutoff of 300 Da. Approximately 220 mL of short peptide concentrate (component A) was obtained. In the alcohol extraction stage, 75% ethanol was used, and extraction was performed by reflux at 70°C for 1 hour each time, for a total of 3 times. The macroporous resin used was HPD-100, and the elution solvent was 70% ethanol. After purification and concentration, component B extract was obtained. HPLC analysis revealed that component A (short peptide) contained 1.5 mg / mL, and component B (quercetin) contained 40%. The target values ​​for the final product were set as follows: quercetin ≥ 400 mg / kg, and short peptide ≥ 200 mg / kg. The appropriate amounts of components A and B were calculated and mixed, and 30 mL of glycerol, 20 mL of propylene glycol, and 3 g of phenoxyethanol were added. Water was added to a total mass of 1.0 kg, and the mixture was stirred until homogeneous to obtain the composition of this invention.

[0028] Comparative Example 1 (Changing the extraction order) 1.0 kg of Platycladus orientalis leaves were weighed and extracted twice using the alcohol extraction conditions (70% ethanol, reflux at 75°C) as described in Example 1. The extracts were combined and purified according to the method in Example 1 to obtain the quercetin component (component B'). The residue after alcohol extraction was then extracted again using the water extraction conditions (pre-cooled PBS, slow stirring at 4°C) as described in Example 1. The extract was purified according to the method in Example 1 to obtain the short peptide component (component A'). The contents of components A' and B' were determined and mixed according to the target ratio of the final product in Example 1 to prepare comparative composition 1.

[0029] Comparative Example 2 (using pure water instead of buffer solution) Weigh 1.0 kg of Platycladus orientalis leaves. Except for replacing the extraction solvent in the first stage with an equal volume of pre-cooled deionized water (pH approximately 6.0, unadjusted), all other steps (including extraction temperature, time, subsequent purification, and the second-stage alcoholic beverage mixing) were exactly the same as in Example 1. Prepare comparative composition 2.

[0030] Comparative Example 3 (Low-temperature environment missing) Weigh 1.0 kg of Platycladus orientalis leaves. Except for changing the water extraction temperature in the first stage to room temperature (25°C), all other steps are exactly the same as in Example 1. Prepare comparative composition 3.

[0031] Comparative Example 4 (Change in membrane separation sequence) Weigh 1.0 kg of Platycladus orientalis leaves. The first stage of water extraction is the same as in Example 1. After obtaining a clear water extract, concentrate it directly using a 1 kDa ultrafiltration membrane without undergoing 10 kDa ultrafiltration. Subsequent steps are the same as in Example 1. Prepare comparative composition 4.

[0032] Comparative Example 5 (Single Solvent Extraction - Ethanol) 1.0 kg of Platycladus orientalis leaves were weighed and extracted without fractional extraction. The raw material was extracted twice directly using the alcohol extraction conditions of Example 1 (70% ethanol, reflux at 75°C). The extracts were combined, and half of the extract (representing the complete alcohol extract) was concentrated under reduced pressure. Without purification with macroporous resin, it was directly used as the "mixed component" and the amount added was calculated according to the quercetin content in the final product target of Example 1, but the short peptide standard component could not be added. Comparative composition 5 (containing only the complete alcohol extract, without specifically enriched short peptides) was prepared.

[0033] Comparative Example 6 (Single Solvent Extraction - Water) 1.0 kg of Platycladus orientalis leaves were weighed and extracted without fractional extraction. The raw material was directly extracted for 18 hours using the water extraction conditions of Example 1 (pre-cooled PBS, slow stirring at 4°C). The extract was subjected to membrane separation and gel filtration as described in Example 1 to obtain component A (short peptide). Since there was no alcohol extraction step, quercetin-enriched component B could not be obtained. The final product consisted only of component A, and the amount added was calculated based on the target concentration of the short peptide in Example 1. Comparative composition 6 (containing only the short peptide component, without quercetin) was prepared.

[0034] Comparative Example 7 (Missing Standardized Mixture) Component A concentrate and component B extract were prepared according to the method of Example 1, but the content was not determined and they were not mixed in proportion. 100 mL of component A concentrate and 5 g of component B extract were randomly taken and mixed with the same type and amount of excipients as in Example 1, and water was added to 1.0 kg to prepare comparative composition 7.

[0035] The performance of all the above-mentioned embodiments and comparative examples were tested using the following methods, and the results are shown in Table 1.

[0036] Test method: Yield determination: Short peptide yield: Accurately measure the volume of the short peptide concentrate (component A or similar) prepared in each example and comparative example, and freeze-dry a portion of it, weighing the freeze-dried powder. Determine the purity of the short peptides (calculated as tripeptides) in the freeze-dried powder by HPLC. Short peptide yield (mg / kg raw material) = [Concentrate volume (mL) × Short peptide concentration (mg / mL)] / Raw material mass (kg).

[0037] Quercetin yield: The mass of quercetin extract (component B or analogue) prepared in each example and comparative example was accurately weighed, and the purity of quercetin was determined by HPLC. Quercetin yield (mg / kg raw material) = [extract mass (g) × quercetin purity (%) × 10] / raw material mass (kg).

[0038] Composition activity test: DPPH radical scavenging rate: Accurately weigh each final composition sample and prepare a series of test solutions with appropriate solvents to achieve different concentration gradients. Following the DPPH method, take 2 mL of 0.1 mM DPPH ethanol solution, add 2 mL of sample test solutions of different concentrations, mix well, and react in the dark for 30 min. Measure the absorbance (As) at 517 nm. Simultaneously measure the absorbance (Ac) of 2 mL of DPPH solution and 2 mL of solvent, and the absorbance (Aj) of 2 mL of sample test solution and 2 mL of solvent. Scavenging rate (%) = [1 - (As - Aj) / Ac] × 100%. Calculate the half-maximal scavenging concentration (IC50). 50 Value, IC 50 The lower the value, the stronger the antioxidant capacity.

[0039] Tyrosinase inhibition rate: L-DOPA was used as the substrate. In a 96-well plate, 40 μL of phosphate buffer (pH 6.8), 40 μL of sample test solutions of different concentrations, and 40 μL of tyrosinase solution (250 U / mL) were added sequentially. After mixing, the plate was incubated at 37°C for 10 min. Then, 40 μL of 2.5 mM L-DOPA solution was added, and the absorbance was immediately monitored at 490 nm over time to calculate the reaction rate. Wells without samples served as controls (100% enzyme activity). Inhibition rate (%) = (1 - reaction rate of sample group / reaction rate of control group) × 100%. The half-maximal inhibitory concentration (IC50) was calculated. 50 value;

[0040] Examples 1-3 of this invention demonstrate that a segmented extraction and co-production process using water (low-temperature buffer) followed by alcohol can stably yield 100-200 mg of short peptides and 300-580 mg of quercetin per kilogram of Platycladus orientalis raw material, with the final composition precisely meeting the preset standards, indicating a stable and controllable process. Comparative Example 1 shows that while the quercetin yield was comparable with alcohol extraction followed by water extraction, the short peptide yield plummeted to less than one-third of that in Example 1, proving that the segmented extraction sequence—first with mild water extraction to protect the short peptides, then with alcohol extraction of the residue—is crucial for preserving the heat-sensitive active ingredients. Comparative Examples 2 and 3 show that while using pure water slightly reduced the yield, the reduction was not significant, but the IC50 value was high. 50The values ​​were slightly lower than in Example 1, suggesting that a neutral phosphate buffer environment may be more conducive to protecting the active conformation of certain peptides. Increasing the water extraction temperature led to a significant decrease in both the yield and activity of short peptides, demonstrating a synergistic effect between low temperature and the buffer system in protecting the activity of short peptides. Comparative Example 4 shows that skipping the 10 kDa ultrafiltration and directly using a 1 kDa membrane resulted in short peptide yields and activities similar to Example 1. However, the main contribution of this step was the pre-removal of a large amount of macromolecular impurities such as polysaccharides and proteins, reducing subsequent 1 kDa membrane fouling and gel column burden. This has significant value for flux and membrane lifetime maintenance in continuous industrial operation. Comparative Examples 5, 6, and 7 show that single-solvent extraction can only obtain one type of main active ingredient, and the combined activity (IC50) is limited. 50 The significantly inferior composite product compared to the examples demonstrates that the specific components obtained under the preparation conditions of this invention, consisting of short peptides and quercetin, possess synergistic antioxidant and whitening activities. Comparative Example 7 shows that without content determination and standardized mixing, the product activity fluctuates greatly between batches, failing to guarantee stable end-efficacy, highlighting the importance of the standardized steps in this invention for product quality control. This invention, through a series of combinations including water-to-alcohol fractional extraction, low-temperature buffer system protective extraction, multi-stage membrane separation and gel filtration purification, selective enrichment with macroporous resin, and standardized mixing, synergistically solves the technical challenge of efficiently, highly active, and standardized co-production of two different types of active ingredients from Platycladus orientalis, achieving significantly better results than existing single extraction or non-optimized co-production methods.

[0041] Application example: Evaluation experiment on the effect of promoting hair regeneration in mice. To evaluate the effects of the arborvitae active ingredient composition obtained in this invention on promoting hair regeneration and improving hair quality, the following animal experiments were designed.

[0042] Experimental animals and grouping: Seventy-two healthy, 6-week-old male C57BL / 6J mice were selected (this strain of mice, after being shaved during the resting phase, can simultaneously enter the growth phase, making it a classic model for studying hair growth). After one week of acclimatization, they were randomly divided into 12 groups of 6 mice each: blank control group (applied matrix), positive control group (5% minoxidil tincture), and the example and comparative groups.

[0043] Hair removal and drug administration: Hair was shaved from both sides of the spine on the backs of mice in each group using an electric shaver, covering an area of ​​approximately 2cm × 3cm. Drug administration began the following day. The final products from the examples and comparative examples, as well as 5% minoxidil solutions and blank substrates prepared with the same matrix (water, glycerol, and phenoxyethanol ratios as in Example 1), were used as the test substances for each group. Every morning at a fixed time, 50 μL of the corresponding test substance was evenly applied to the shaved area of ​​the mice using a micropipette, gently massaging to promote absorption. An equal volume of blank substrate was applied to the blank control group. Drug administration continued for 10 consecutive days.

[0044] Macroscopic morphological observation and scoring: On days 1, 5, and 10 after drug administration, the hair-loss areas of mice were photographed using a high-resolution digital camera under the same light conditions. Three observers, unaware of their group assignments, independently scored the mice according to the Bogdanska hair regeneration scoring criteria (0 points: no hair regeneration; 1 point: sparse, fine downy hair; 2 points: moderate hair regeneration, fine hair; 3 points: near-complete hair regeneration, coarse hair; 4 points: complete regeneration, dense, coarse, and shiny hair). The average score was taken.

[0045] Hair weighing and diameter measurement: After photographing on day 10, all newly grown hair in the bald area on the back of each mouse was carefully cut with surgical scissors and weighed (mg) using an electronic balance. Twenty hairs were randomly selected from each group, and their diameter (μm) was measured under a stereomicroscope using a micrometer. The average value was calculated.

[0046] Hair Plucking Force Test (Day 10): Using a digital pull tester, a small clump of hair (about 50 hairs) was plucked vertically from the center of the hair removal area at a constant rate. The maximum force (g) when the hair was pulled away from the skin was recorded to indirectly assess the strength of the hair follicle and the hair growth cycle.

[0047] Macroscopic observation and scoring results: On day 1, all groups had bald skin and a score of 0. On day 5, visible black hair spots (signs of early hair growth) began to appear in the positive control group (minoxidil) and all example groups, with a score of approximately 1.5-2.0; hair spots appeared later or were sparser in the blank control group and comparative groups (especially comparatives 5 and 6), with a score of approximately 0.5-1.0. On day 10, the differences were significant: Examples 1-3: Hair completely regenerated and densely covered. The hair was jet black with a noticeable sheen, and stood upright and was thick. The score was 3.8-4.0.

[0048] Positive control group (minoxidil): Hair regrowth was complete, with coverage density similar to the control group. However, the hair color was relatively dull, the luster was poor, and the hair was mostly curly or flat, appearing finer and softer. The score was 3.5.

[0049] Comparative Example 1 (with altered extraction order): Hair regrowth was good, but the density and thickness were slightly inferior to the Example 1 group, and the luster was average. The score was approximately 3.2 points.

[0050] Comparative Example 5 (single ethanol extract): Hair regeneration, but with lower density, fine and soft hair, and no luster. Score approximately 2.5 points.

[0051] Comparative Group 6 (single water-extracted short peptide): Hair regeneration was the worst, with only sparse vellus hair growing. The score was approximately 1.8.

[0052] Blank control group: Only a small amount of fine, soft downy hairs were observed. The score was approximately 1.2 points.

[0053] Hair pull-out force test (day 10): The hair pull-out force of the positive control group (minoxidil) was 52.3 ± 6.1 g. The hair pull-out force of the Example 1 group was significantly higher, at 78.5 ± 7.8 g. The comparative example 1 group had a pull-out force of 60.2 ± 5.9 g. The comparative example 5 group had a pull-out force of 48.7 ± 5.5 g. The comparative example 6 group and the blank control group had the lowest pull-out forces, at 30.4 ± 4.2 g and 28.1 ± 4.0 g, respectively.

[0054] Application results show that the compositions obtained in the embodiments of the present invention exhibit comprehensive superiority over minoxidil and all comparative examples in promoting hair regeneration and improving hair quality in mice: Regarding growth promotion, the weight, length, and number of new hair follicles in Examples 1-3 were significantly higher than those in the positive control group (P<0.05), while the indicators in Comparative Examples 1-7 were lower than those in the Example groups to varying degrees. Specifically, the hair weight and number of hair follicles in Comparative Examples 3 and 5 were significantly reduced, indicating that low-temperature protection and segmented extraction are crucial for preserving the active ingredients. Regarding hair quality improvement, the diameter and dermal thickness of new hair in the Example groups were significantly better than those in the minoxidil group (P<0.01), and macroscopic observation showed that the hair was thicker and shinier, reflecting a better state of hair follicle health. Although Comparative Examples 2 and 4 showed some improvement... While the indicators were close to those of the Example Group, overall differences remained. The hair diameter and dermal thickness of Comparative Examples 1 and 7 were significantly lower than those of the Example Group, demonstrating the crucial impact of extraction order and standardized compounding on the final product quality. Regarding enhanced hair follicle firmness, the hair pull-out force of the Example Group was significantly higher than that of the positive control group and all comparative groups, indicating that the hair was less likely to fall out. Simultaneously, the Example Group significantly upregulated the expression of positive hair follicle growth factors IGF-1 and VEGF, with effects significantly superior to the minoxidil group and all comparative groups. In contrast, Comparative Examples 6 and 5 showed much lower expression of both factors than the Example Group, strongly demonstrating that only the specific components A and B obtained through the method of this invention can produce a significant synergistic effect, achieving a comprehensive improvement in the quantity, quality, and firmness of hair regeneration.

[0055] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0056] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. An industrial co-production method for active ingredients from Platycladus orientalis, characterized in that, Includes the following steps: (1) Using Platycladus orientalis as raw material, the first stage of water extraction and the second stage of alcohol extraction were carried out in sequence; (2) The extract obtained from the first stage of water extraction was subjected to membrane separation and gel filtration to obtain component A rich in short peptides; (3) The extract obtained from the second stage of alcohol extraction was purified by macroporous resin to obtain component B rich in quercetin; (4) Mix component A and component B in a predetermined ratio to obtain a standardized product.

2. The method according to claim 1, characterized in that, In step (1), the first stage of water extraction uses pre-cooled phosphate buffer solution for slow stirring extraction at low temperature, followed by solid-liquid separation and filtration; the second stage of alcohol extraction uses 60%-75% ethanol aqueous solution to heat and reflux extract the residue after the first stage of water extraction.

3. The method according to claim 2, characterized in that, The phosphate buffer solution has a concentration of 40-60 mM and a pH of 7.0-7.5; the extraction temperature is 2-8℃, the stirring speed is 50-150 rpm, the extraction time is 12-24 hours, and the extraction is performed 1-3 times.

4. The method according to claim 2, characterized in that, The solid-liquid separation and filtration process includes: centrifuging the extract at 2-8℃ and 8000-12000 rpm for 15-25 min, and then filtering the supernatant through microporous membranes of 0.45 μm and 0.22 μm.

5. The method according to claim 1 or 4, characterized in that, In step (2), the membrane separation includes ultrafiltration and nanofiltration performed sequentially; the ultrafiltration is to first use an ultrafiltration membrane with a molecular weight cutoff of 8-12 kDa to remove macromolecular impurities, and then use an ultrafiltration membrane with a molecular weight cutoff of 0.8-1.2 kDa to concentrate and desalinate the permeate, and collect the permeate rich in short peptides with a molecular weight of less than 1.2 kDa.

6. The method according to claim 5, characterized in that, The gel filtration process uses dextran gel G-10, G-15, or Superdex™ 30 as the packing material, and eluents with a 20-80 mM ammonium acetate aqueous solution as the mobile phase. Elution peaks with absorption at 220 nm are collected and combined to obtain a primary solution of component A. The primary solution of component A is then desalted and concentrated by passing it through a nanofiltration membrane or a reverse osmosis membrane. The nanofiltration membrane has a molecular weight cutoff of 200-300 Da to obtain a concentrated solution of component A.

7. The method according to claim 2, characterized in that, In the second stage of alcohol extraction, the concentration of the ethanol aqueous solution is 65%-70%, the extraction temperature is 70-80℃, the extraction time is 1-1.5 hours each time, and the extraction is performed twice. The extracts are combined, filtered, and crude quercetin extract is obtained.

8. The method according to claim 1, characterized in that, In step (3), the macroporous resin purification includes: loading the crude quercetin extract aqueous solution after ethanol removal onto a macroporous adsorption resin column, first eluting with water until the eluent is colorless, then eluting with an ethanol aqueous solution of 50%-70% concentration, collecting the ethanol eluent, and concentrating under reduced pressure to obtain component B extract; the macroporous adsorption resin is AB-8 type, D-101 type or HPD-100 type; in step (4), the predetermined ratio is determined by the following method: measuring the content of short peptides in component A and the content of quercetin in component B respectively, and then using the quercetin content ≥300 mg / kg and the short peptide content ≥100 mg / kg in the final product after mixing as the standard, calculating the mixing ratio of component A and component B; the mixing is carried out under stirring conditions, and one or more of solvents and preservatives are added to the mixing system for adjustment; the solvent is one or more of water, propylene glycol, and glycerin; the preservative is one or more of phenoxyethanol, methylparaben, and p-hydroxyacetophenone.

9. A composition of active ingredients of Platycladus orientalis prepared by the method according to any one of claims 1-8, characterized in that, It consists of component A, which is rich in short peptides with a molecular weight of less than 1200 Da, and component B, which is rich in quercetin. The quercetin content is not less than 300 mg / kg and the short peptide content is not less than 100 mg / kg based on the total mass of the composition.

10. The use of the Platycladus orientalis active ingredient composition according to claim 9 in the preparation of a topical skin preparation.

Images