A method for extracting cypress leaf exosomes based on dynamic interface regulation and application of the exosomes in hair care

Abstract

The application discloses a method for extracting cypress leaf exosomes based on dynamic interface regulation and application of the exosomes in hair care. The application uses a specific biocompatible dynamic interface modifier (BDIM) to construct a dynamic interface system through temperature / pH synergistic regulation, realizes mild dissociation of cypress leaf oil exosome complex, can significantly reduce the oil residue of cypress leaf exosomes, can simultaneously complete stabilization of the exosomes, can improve the extraction efficiency and product stability of the cypress leaf exosomes, and can improve the retention rate of active ingredients of the exosomes, thereby avoiding the problems of physical shearing or chemical residue in traditional methods. The cypress leaf exosomes prepared by the method have high yield and extraction effect, are significantly better than traditional extraction methods, can be used for industrialization and large-scale production, and can be used in hair care products to better reduce sebum secretion and hair loss, and provide a new source of active ingredients for upgrading of hair care products.

Description

Technical Field

[0001] This invention relates to the field of bioactive ingredient extraction and cosmetic technology, and more specifically, to a method for extracting exosomes from Platycladus orientalis leaves based on dynamic interface regulation and the application of exosomes in hair care. Background Technology

[0002] As a traditional medicinal plant, the leaves of Platycladus orientalis contain flavonoids, terpenes, and other active ingredients with anti-inflammatory, antioxidant, and hair growth-promoting effects, showing significant application potential in the hair care field. Exosomes, as nanoscale vesicles secreted by cells, can efficiently load active ingredients and achieve targeted delivery. Hair care products prepared using Platycladus orientalis leaf exosomes exhibit better activity and improved bioavailability. However, due to the high oil viscosity of Platycladus orientalis leaf tissue, containing 0.26% volatile oil (mainly α-thujone, thujone, etc.) and various lipid components (stearic acid, palmitic acid, etc.), a large amount of oil will be encapsulated, forming an oil-exosome complex during exosome preparation. This structure poses a significant challenge to the efficient extraction and utilization of exosomes.

[0003] Existing methods for exosome extraction mainly include ultracentrifugation, PEG precipitation, and membrane filtration. However, these methods have several inherent drawbacks when applied to the extraction of exosomes from Platycladus orientalis leaves: Ultracentrifugation relies on the mechanical force generated by high-speed centrifugation for separation, which is not only energy-intensive and cumbersome, but also prone to causing exosome membrane rupture, loss of active ingredients, and ineffective removal of lipid encapsulation; PEG precipitation, while simple to operate, carries the risk of PEG residue, affecting product safety, and the precipitation process can easily lead to exosome aggregation, reducing extraction purity; Membrane filtration often suffers from membrane clogging due to the viscosity of Platycladus orientalis leaf oil, reducing filtration efficiency, and the filtration pressure is difficult to control, easily causing exosome damage. Therefore, due to the high oil viscosity of Platycladus orientalis leaves, existing conventional extraction methods are difficult to efficiently extract and prepare exosomes from Platycladus orientalis leaves.

[0004] Currently, no efficient extraction method for exosomes from Platycladus orientalis leaves, which exhibit high oil viscosity, has been reported. Existing extraction technologies have not solved the problem of the gentle dissociation of oil-exosome complexes. Current improvements are merely simple combinations of various traditional separation methods (such as ultracentrifugation + PEG precipitation, membrane filtration + density gradient centrifugation, etc.), failing to fundamentally overcome the technical bottleneck of oil encapsulation. This results in persistent problems such as low extraction efficiency, poor exosome activity, and difficulty in industrialization of Platycladus orientalis leaf exosomes. Therefore, developing a method that can achieve gentle dissociation of oil-exosome complexes and efficient extraction of Platycladus orientalis leaf exosomes has become an urgent technical need in this field. Hence, this invention application is filed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the problems of low extraction efficiency, poor exosome activity, risk of chemical residue, and difficulty in industrialization caused by the high oil viscosity of Platycladus orientalis leaves in the prior art. The present invention provides a method for extracting Platycladus orientalis exosomes based on dynamic interface regulation and the application of exosomes in hair care.

[0006] The first objective of this invention is to provide a method for efficient extraction of exosomes from Platycladus orientalis leaves based on dynamic interface regulation.

[0007] A second objective of this invention is to provide an exosome from Platycladus orientalis leaves.

[0008] A third objective of this invention is to provide the application of Platycladus orientalis leaf exosomes.

[0009] The fourth objective of this invention is to provide a hair care product.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention discloses a method for efficient extraction of exosomes from Platycladus orientalis leaves based on dynamic interface regulation, comprising the following steps: (1) Construction of dynamic interface regulation system: After pulverizing the leaves of Platycladus orientalis, disperse them in buffer solution, add BDIM with a final concentration of 1.5-2.0 mg / mL, mix well, and adjust the pH of the system to 6.5-7.0 to construct a mixed system; the BDIM is a phosphatidylcholine-polyethylene glycol-cholesterol block copolymer with a molecular weight of 2000-5000 Da, wherein the molar ratio of phosphatidylcholine, polyethylene glycol and cholesterol is (4-7):(2-5):(1-2); (2) Gradient heating and dynamic equilibrium: The mixture is heated to 36-37℃ at a rate of 0.8-1.0℃ / min and maintained in dynamic equilibrium for 30-40min; (3) Separation and purification: Filter the balanced system to remove residues and impurities to obtain crude extract; (4) Concentration and preservation: The crude extract is concentrated by freezing to obtain Platycladus orientalis leaf exosomes.

[0011] This invention utilizes the amphiphilic properties of a biocompatible dynamic interface modulator (BDIM) to construct a dynamic interface system through synergistic temperature / pH regulation. This system enables the gentle dissociation of the Platycladus orientalis leaf oil-exosome complex, significantly reducing the residual oil content in Platycladus orientalis leaf exosomes. Further stabilization of the exosomes improves extraction efficiency and product stability, and increases the retention rate of active ingredients, avoiding the physical shearing or chemical residue problems of traditional methods. The essential difference between the method provided by this invention and traditional methods lies in "achieving gentle dissociation through dynamic interface regulation, rather than forced physical / chemical separation." The use of a specific BDIM achieves highly efficient separation of Platycladus orientalis leaf oil without affecting subsequent exosome extraction and preparation, significantly improving extraction efficiency. Furthermore, after treatment using the method of this invention, other conventional extraction methods in the art can still be used for extraction and preparation, achieving an extraction rate of 6.2 × 10⁻⁶ Platycladus orientalis leaf exosomes. 12 -6.8×10 12 The concentration of arborvitae leaves per gram (g) is determined by traditional ultracentrifugation (2.1 × 10⁻⁶). 11 The concentration of (3.5 × 10⁻⁶ cells / g) is 30-32 times that of the PEG precipitation method (3.5 × 10⁻⁶ cells / g). 11 The exosomes prepared by this invention are 18-19 times more potent than those produced by traditional methods (e.g., per g). Furthermore, the method provided by this invention can be scaled up for industrial production, adaptable to large-scale production of 500 kg / batch, enabling high-efficiency, high-volume production. Using the arborvitae leaf exosomes prepared by this invention in hair care products can better reduce scalp oil secretion and hair loss, providing a new source of active ingredients for upgrading hair care products.

[0012] Specifically, the BDIM used in this invention is a biocompatible dynamic interface regulator, which is an amphiphilic block copolymer containing both hydrophilic and hydrophobic ends. BDIM can self-assemble into micelles in solution. As the most preferred embodiment, the BDIM used in this invention is a phosphatidylcholine-polyethylene glycol-cholesterol block copolymer.

[0013] The method provided by this invention mainly includes the following mechanisms of action: 1. Amphiphilic design of BDIM and dynamic interaction mechanism of lipid-exosome interface: BDIM has both hydrophilic and hydrophobic ends. The hydrophobic end can be specifically inserted into the lipid layer of lipid-exosome complex, while the hydrophilic end forms hydrogen bonds with phospholipid groups on the surface of exosome membrane, thereby constructing dynamic interaction sites at the lipid-exosome interface, providing a basis for the mild dissociation of the complex.

[0014] 2. Temperature / pH synergistic regulation of reversible micelle formation and dissociation: By precisely controlling the system temperature and pH, BDIM forms reversible micelles at the lipid-exosome interface. The formation of micelles can further disrupt the lipid layer structure and promote exosome release. Moreover, this process is reversible, avoiding damage to the exosome membrane structure.

[0015] 3. Integrated process for simultaneous extraction and stabilization: During the dynamic interface-controlled extraction process, biocompatible stabilizers are added to achieve simultaneous exosome release and stabilization, eliminating the need for subsequent additional stabilization treatment, simplifying the process flow, and improving the exosome activity retention rate.

[0016] Preferably, in step (1), disease-free arborvitae leaves from the current year are selected and pulverized at -5~0℃ under inert gas protection to form arborvitae leaf powder with a particle size of 0.3-0.5mm.

[0017] Preferably, in step (1), the ratio of arborvitae leaves to buffer solution is 1:15-25 g / mL; the buffer solution is selected from one or more of phosphate buffer (PBS buffer), Tris-HCl buffer, HEPES buffer, and citrate-phosphate buffer.

[0018] Preferably, the ratio of arborvitae leaves to buffer solution is 1:25 g / mL.

[0019] More preferably, the buffer is 0.01-0.05 mol / L PBS buffer; most preferably, it is 0.01 mol / L PBS with pH 6.5-7.0.

[0020] Preferably, the molar ratio of phosphatidylcholine, polyethylene glycol, and cholesterol in BDIM is (5-7):(2-3):(1-2).

[0021] More preferably, the molar ratio of phosphatidylcholine, polyethylene glycol, and cholesterol in BDIM is 5:3:2.

[0022] Preferably, the concentration of BDIM is 1.5-1.75 mg / mL.

[0023] The BDIM provided by this invention is a block copolymer formed by covalently linking phosphatidylcholine, polyethylene glycol, and cholesterol (e.g., through esterification, amidation, or click chemistry), with a molecular weight of 2000-5000 Da. This substance can be synthesized using conventional methods in the art, such as condensing carboxylated PEG sequentially with cholesterol and phosphatidylcholine via a conventional esterification reaction, followed by purification by column chromatography or dialysis to obtain the target product. Alternatively, it can be obtained through custom synthesis services, provided that the molar ratio and molecular weight range provided by this invention are met.

[0024] As a preferred embodiment, the present invention provides a method for synthesizing a phosphatidylcholine-polyethylene glycol-cholesterol block copolymer: Polyethylene glycol (PEG, molecular weight 1000 Da) is dissolved in anhydrous dichloromethane, and 1.2 molar amounts of succinic anhydride and a catalytic amount of 4-dimethylaminopyridine (DMAP) are added. The reaction is carried out at room temperature for 24 h to obtain carboxylated PEG (PEG-COOH). Cholesterol is dissolved in anhydrous dichloromethane, and 1.1 molar amounts of N,N'-dicyclohexylcarbodiimide (DCC) and 0.1 molar amounts of DMAP are added. After activation for 2 h, an equimolar amount of PEG-COOH is added, and the reaction is carried out at room temperature for 48 h to obtain a cholesterol-PEG copolymer (Chol-PEG). Finally, phosphatidylcholine (PC) is similarly carboxylated and reacted with the terminal hydroxyl groups of Chol-PEG in the presence of EDC / NHS. The reaction is purified by dialysis (molecular weight cutoff 2000 Da) and lyophilized to obtain the PC-PEG-Chol block copolymer (BDIM). Furthermore, FT-IR and GPC characterization confirmed that the obtained product was the target block copolymer with a molecular weight of 3000 Da, PDI ≤ 1.3, and a molar ratio of 5:3:2.

[0025] Preferably, in step (1), the mixture is stirred at a speed of 30-70 r / min.

[0026] Preferably, step (3) uses a 0.22μm polyethersulfone membrane for filtration, with a filtration pressure of 0.05-0.15MPa.

[0027] More preferably, the pH value in step (1) is 6.8, the heating rate in step (2) is 0.8℃ / min, and the dynamic equilibrium time is 35min.

[0028] Further, after separation and purification in step (3), in-situ stabilization is performed before concentration and preservation; the in-situ stabilization is: adding 3%-7% w / v stabilizer to the crude extract and incubating at 0-5℃ for 10-30 min.

[0029] Preferably, the stabilizer is selected from one or more of trehalose, sucrose, mannitol, sorbitol, betaine, and glycerol.

[0030] The optimal stabilizer is 5% trehalose.

[0031] Preferably, the freezing and concentration parameters in step (4) are: temperature -15~-25℃, vacuum degree 3-8Pa, and concentration to 1 / 4-1 / 6 of the original volume.

[0032] As the preferred extraction method in this embodiment, the specific preparation steps of Platycladus orientalis leaf exosomes are as follows: (1) Raw material pretreatment: Select disease-free arborvitae leaves from the current year, pulverize them at low temperature (temperature -5-0℃, inert gas protection), and screen to obtain arborvitae leaf powder with a particle size of 0.3mm; (2) Construction of dynamic interface regulation system: The powder of Platycladus orientalis leaves was mixed with 0.01 mol / L PBS buffer at a ratio of 1:20 (g / mL), and BDIM (phosphatidylcholine-polyethylene glycol-cholesterol block copolymer, molar ratio 5:3:2, molecular weight 3000 Da) was added to make the final concentration of BDIM 1.75 mg / mL. The mixture was stirred at 50 r / min until homogeneous, and the pH of the system was adjusted to 6.8. (3) Gradient heating and dynamic equilibrium: The temperature was increased from 4℃ to 37℃ at a rate of 0.8℃ / min, and the dynamic equilibrium was maintained for 35min; (4) Separation and purification: 0.22 μm polyethersulfone membrane was used for filtration at a pressure of 0.1 MPa to remove residues and impurities and obtain crude extract; (5) In-situ stabilization: Add trehalose to the crude extract to a final concentration of 5% (w / v) and incubate at 4°C for 20 min; (6) Concentration and preservation: The stabilized solution was placed in a freeze concentration device and concentrated to 1 / 5 of the original volume at -20℃ and 5Pa to obtain high-concentration Platycladus orientalis leaf exosomes, which were then stored at 4℃.

[0033] This invention provides an exosome of Platycladus orientalis leaf prepared by the above method.

[0034] This invention provides the application of the above method in reducing the residual oil content of Platycladus orientalis leaf exosomes, improving the extraction rate and the retention effect of active ingredients.

[0035] This invention provides the use of Platycladus orientalis leaf exosomes in hair care or in the preparation of hair care products.

[0036] This invention provides a hair care product containing arborvitae leaf exosomes extracted by the above method.

[0037] Preferably, the hair care product is a scalp repair serum.

[0038] More preferably, the amount of Platycladus orientalis exosomes added to the product is 5%-15% (v / v), preferably 10% (v / v).

[0039] More preferably, the product further contains cosmetically acceptable formulations or excipients.

[0040] This invention provides a hair care product that can be used to repair seborrheic alopecia or improve frizzy hair, and can regulate scalp sebum secretion, inhibit scalp inflammation, and promote the proliferation of hair papilla cells.

[0041] The present invention has the following beneficial effects: This invention provides a highly efficient extraction method for exosomes from Platycladus orientalis leaves based on dynamic interface regulation. Utilizing the amphiphilic characteristics of a specific biocompatible dynamic interface regulator (BDIM), a dynamic interface system is constructed through synergistic temperature / pH regulation. This achieves the gentle dissociation of the Platycladus orientalis leaf oil-exosome complex, significantly reducing the oil residue in the exosomes. Simultaneously, the method stabilizes the exosomes while improving extraction efficiency and product stability, and also enhances the retention rate of active ingredients in the exosomes, avoiding the physical shearing or chemical residue problems of traditional methods. The Platycladus orientalis leaf exosomes obtained by this method have a high extraction rate, with significantly better extraction effects than traditional methods, and can be scaled up for large-scale, high-efficiency production. Using the Platycladus orientalis leaf exosomes prepared by this invention in hair care products can better reduce scalp oil secretion and hair loss, providing a new source of active ingredients for the upgrading of hair care products.

[0042] It can also achieve the following significant effects: (1) Significantly improved extraction efficiency: The extraction rate of the method of this invention reaches 6.2 × 10⁻⁶. 12 -6.8×10 12 The concentration of arborvitae leaves per gram (g) is 2.1 × 10⁻⁶ using traditional ultracentrifugation. 11 The concentration per gram is 30-32 times that of the PEG precipitation method (3.5 × 10⁻⁶). 11 18-19 times that of the number / g; (2) Excellent exosome activity and stability: The exosomes extracted by this invention have a membrane structure integrity rate of ≥96.3%, with the best group reaching 97.8%, a flavonoid component retention rate of ≥92.1%, and an activity retention rate of ≥89.5% after 120 days of storage at 4℃, which is significantly better than the traditional method (membrane integrity rate ≤78.6%, activity retention rate ≤52.3%). (3) High product safety: Biocompatible materials and mild extraction conditions are used throughout the process, with no chemical reagent residues, avoiding the residual risk of PEG precipitation method (0.8 mg / mL of PEG residue was detected). (4) The process is simplified and easy to industrialize: extraction and stabilization are completed simultaneously, without the need for additional stabilization steps. The number of process steps is reduced by 30% compared with traditional methods. At the same time, the effective range and optimal value of each parameter are clearly defined. Combined with the industrial scale-up criteria, it can be adapted to large-scale production of 500 kg / batch. (5) Significant hair care effects: The exosomes extracted from the arborvitae leaves of this invention can significantly promote the proliferation of hair papilla cells, improve seborrheic alopecia and frizzy hair, and significantly enhance the activity of the arborvitae leaf exosomes. Attached Figure Description

[0043] Figure 1The figure shows the GPC gel permeation chromatogram of BDIM and the mixture of various monomer raw materials (the raw material physical mixture (Mix, black curve), BDIM product (red curve), the horizontal axis is the elution volume (mL), and the vertical axis is the differential refractive index detector response value (RI Response (au); the Mix is ​​a physical mixture of PC, PEG, and cholesterol).

[0044] Figure 2 Fourier transform infrared spectra of PC, PEG, cholesterol monomers, and BDIM target products (phosphatidylcholine (PC, black curve), polyethylene glycol (PEG, red curve), cholesterol (Chol, blue curve), BDIM block copolymer (green curve), with the horizontal axis representing wavenumber (cm²). -1 (4000~600 reverse arrangement), the vertical axis is the transmittance (%).

[0045] Figure 3 Images of Platycladus orientalis leaf exosomes extracted by the method of the present invention (BDIM) for transmission electron microscopy (TEM). Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0047] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0048] In the examples, BDIM was characterized by Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC); the extraction rate was detected by nanoparticle tracking analysis (NTA); the membrane structure integrity was observed and statistically analyzed by transmission electron microscopy (TEM); the retention rate of flavonoid components was detected by high performance liquid chromatography (HPLC); the stability was evaluated by the activity retention rate after storage at 4°C for 120 days; the micelle formation efficiency was determined by the fluorescent probe method (Nile Red); the residual oil content was detected by organic solvent extraction and weighing method; all experiments were independently repeated 3 times, and the average value was taken.

[0049] Example 1: Extraction of exosomes from Platycladus orientalis leaves 1. Raw material pretreatment: Select disease-free and pest-free Chinese arborvitae leaves from the current year, pulverize them at low temperature (temperature -5~0℃, inert gas protection), and screen to obtain Chinese arborvitae leaf powder with a particle size of 0.4mm.

[0050] 2. Preparation of biocompatible dynamic interface regulator (BDIM): The BDIM used is a block copolymer formed by covalently linking phosphatidylcholine, polyethylene glycol, and cholesterol (molar ratio of 5:3:2) (or block copolymers can be prepared by referring to existing technologies: Shi Z, Fengbo W, Bo L, et al. Synthesis, Characterization, and Evaluation of a Novel Amphiphilic Polymer RGD-PEG-Chol for Target Drug Delivery System[J]. The Scientific World Journal, 2014, 2014:546176.DOI:10.1155 / 2014 / 546176.).

[0051] The specific synthesis method of BDIM is as follows: Polyethylene glycol (PEG, molecular weight 1000 Da) is dissolved in anhydrous dichloromethane, and 1.2 molar amounts of succinic anhydride and a catalytic amount of 4-dimethylaminopyridine (DMAP) are added. The reaction is carried out at room temperature for 24 h to obtain carboxylated PEG (PEG-COOH). Cholesterol is dissolved in anhydrous dichloromethane, and 1.1 molar amounts of N,N'-dicyclohexylcarbodiimide (DCC) and 0.1 molar amounts of DMAP are added. After activation for 2 h, an equimolar amount of PEG-COOH is added, and the reaction is carried out at room temperature for 48 h to obtain cholesterol-PEG copolymer (Chol-PEG). Finally, phosphatidylcholine (PC) is also carboxylated and reacted with the terminal hydroxyl groups of Chol-PEG in the presence of EDC / NHS. After dialysis purification (molecular weight cutoff 2000 Da) and lyophilization, PC-PEG-Chol block copolymer (BDIM) is obtained. By adjusting the feed ratio to control the molar ratio at 5:3:2, the final product has a molecular weight of approximately 3000 Da.

[0052] The prepared BDIM was structurally characterized by gel permeation chromatography (GPC) and Fourier transform infrared spectroscopy (FT-IR) to verify the formation of its covalent bonds. The determination confirmed that the obtained product was the target block copolymer with a molecular weight of 3000 Da, PDI ≤ 1.3, and a molar ratio conforming to 5:3:2. The determination results are as follows: Figure 1 and Figure 2As shown, the physical mixture of PC, PEG, and cholesterol exhibits multiple dispersed peaks, corresponding to significant differences in molecular weight among the three components and indicating only physical mixing without chemical bonding. The BDIM product shows a single symmetrical main peak at 14.25 mL, with a calibrated number-average molecular weight (Mn) of 3012 Da and a molecular weight distribution coefficient (PDI) of 1.22 (<1.3). The product spectrum lacks characteristic peaks of the starting material, indicating extremely low residual free monomer content and high conversion rate in the esterification coupling reaction, successfully preparing the narrow-distribution target block polymer BDIM. FT-IR results show a peak at 3380 cm⁻¹. -1 The peak at 2928 cm⁻¹ is a broad absorption peak characteristic of the stretching of the hydroxyl group (-OH) in the starting material. All three monomers exhibit high absorption intensity at this position, while the absorption of the BDIM product shows a significant decrease at this location, confirming that the terminal hydroxyl group of the starting material is largely consumed by succinate esterification. -1 2854cm -1 The peak represents the stretching vibration of saturated CH, and all four spectral lines retain this characteristic, proving that the main carbon chain backbone of PC, PEG, and cholesterol was not destroyed during the esterification reaction; 1732 cm⁻¹ -1 This is a unique ester carbonyl characteristic absorption peak found in this invention. The three raw materials showed no such absorption, only BDIM exhibited a characteristic absorption, proving that a covalent ester bond is formed through succinic acid bridging; 1240 cm⁻¹ -1 1098cm -1 Attributable to the stretching vibration of the COC ether bond in the PEG chain segment, BDIM fully retains the above-mentioned characteristic absorption; fingerprint region 820-960 cm⁻¹ -1 The characteristic absorption of the cholesterol steroid ring is retained. The above infrared data confirm that the three monomers are covalently coupled via succinate bonds to generate the target BDIM.

[0053] 3. Construction of dynamic interface control system: The powdered arborvitae leaves were mixed with 0.01 mol / L PBS buffer at a ratio of 1:20 (g / mL). BDIM (phosphatidylcholine-polyethylene glycol-cholesterol block copolymer, molecular weight 3000 Da, molar ratio 5:3:2) was added to make the final concentration of BDIM 1.8 mg / mL. The mixture was stirred at 50 r / min until homogeneous. The pH of the system was adjusted to 6.8 to obtain the arborvitae leaf-BDIM mixed system.

[0054] 4. Gradient heating and dynamic equilibrium: The Platycladus orientalis leaf-BDIM mixture was heated from 4℃ to 37℃ at a rate of 0.8℃ / min and maintained in dynamic equilibrium for 35min.

[0055] 5. Separation and purification: The system was filtered using a 0.22μm polyethersulfone membrane at a pressure of 0.1MPa to remove residues and impurities, yielding a crude extract.

[0056] 6. In-situ stabilization: Add trehalose to the crude extract to a final concentration of 5% (w / v) and incubate at 4°C for 20 min.

[0057] 7. Concentration and preservation: The stabilized solution is placed in a freeze concentration device and concentrated to 1 / 5 of the original volume at -20℃ and 5 Pa to obtain high-concentration Platycladus orientalis leaf exosomes, which are then stored at 4℃.

[0058] The prepared Platycladus orientalis exosomes, such as Figure 3 As shown in the figure, the detection results indicate an extraction rate of 6.8 × 10⁻⁶. 12 The exosomes were collected in 30-145 nm (average 85 nm) per g of Platycladus orientalis leaf, with a membrane structure integrity rate of 97.6%, a flavonoid retention rate of 94.5%, an activity retention rate of 92.1% after 120 days of storage at 4℃, and an oil residue of 0.1 mg / mL. There were no chemical residues, no aggregation, and no rupture.

[0059] Example 2: Selection of Dynamic Interface Control System This invention uses biocompatible dynamic interface modulator (BDIM) to achieve the gentle dissociation of lipid-exosome complexes. Different BDIM components were used to compare their effects on the extraction of exosomes from Platycladus orientalis leaves. The specific preparation method is the same as in Example 1, except that the BDIM used is different. The following settings were used: (1) only phosphatidylcholine (PC); (2) only polyethylene glycol (PEG); (3) only cholesterol (Chol); (4) a combination of PC and PEG (molar ratio of 5:3); (5) a combination of PC and Chol (molar ratio of 5:2); (6) a combination of PEG and Chol (molar ratio of 3:2).

[0060] The extraction rate, membrane integrity, and oil residue effect of the extracted Platycladus orientalis leaf exosomes were determined. The results are shown in Table 1. It shows that the extraction rate of exosomes obtained by incubating Platycladus orientalis leaves with PC, PEG, and Chol alone, which have amphiphilic characteristics, is low and the oil residue is high, which cannot effectively remove the oil components of Platycladus orientalis leaves. However, the combination of PC with PEG and Chol can further reduce the oil residue and improve the membrane integrity of exosomes, but the extraction rate of exosomes is still low. When PC, PEG, and Chol are combined in a specific ratio, the phosphatidylcholine-polyethylene glycol-cholesterol block copolymer can significantly improve the extraction rate of Platycladus orientalis leaf exosomes, with an oil residue as low as 0.1 mg / mL and a membrane integrity of 97.6%, which has a significant extraction effect, proving that there is a significant synergistic effect among the three components.

[0061] Table 1. Extraction effects of different biocompatible dynamic interface modulators (BDIMs)

[0062] Example 3: Selection of BDIM Proportion Based on the results of Example 2, it was shown that the combination of PC+PEG+Chol in treating Platycladus orientalis leaves could effectively reduce the residual oil content (mg / mL). On this basis, the ratio of phosphatidylcholine-polyethylene glycol-cholesterol block copolymer was further optimized and screened. Different combinations of PC+PEG+Chol molar ratios were used for extraction, and the effect of the ratio on the extraction effect was compared. The specific preparation method was the same as in Example 1, except that the molar ratio of BDIM used was different. The ratios were set as follows: (1) phosphatidylcholine:polyethylene glycol:cholesterol = 3:3:4; (2) phosphatidylcholine:polyethylene glycol:cholesterol = 5:3:2; (3) phosphatidylcholine:polyethylene glycol:cholesterol = 7:2:1; (4) phosphatidylcholine:polyethylene glycol:cholesterol = 4:5:1.

[0063] The extraction rate, membrane integrity rate, flavonoid retention rate, and activity retention rate of the extracted Platycladus orientalis leaf exosomes were determined. The results are shown in Table 2. It was found that when PC+PEG+Chol=3:3:4, the residual lipid content was 0.6 mg / mL, indicating insufficient exosome release. When the molar ratio of phosphatidylcholine:polyethylene glycol:cholesterol was 5:3:2, the hydrophobic end of BDIM synergistically bested with the hydrogen bonding efficiency between the hydrophilic end and the exosome membrane, resulting in the most complete dissociation of the lipid-exosome complex. Both the extraction rate and exosome activity reached their peak values, representing the optimal composition ratio for BDIM.

[0064] Table 2 Extraction effect of different BDIM molar ratios

[0065] Furthermore, the study found that BDIM micelle formation is related to extraction rate and oil residue. If the molecular weight of BDIM is too small (below 1500 Da), stable micelles cannot be formed, resulting in incomplete oil dissociation, high oil residue, and a sharp drop in extraction rate. If the molecular weight is too large (above 8000 Da), its hydrophilic-hydrophobic imbalance occurs, leading to excessively large micelles that adhere to exosomes, resulting in exosome aggregation, membrane damage, and decreased activity. This indicates that only within a specific molecular weight range (2000-5000 Da) can appropriately sized, reversible, and mild dynamic micelles be formed, achieving the effect of "oil removal + exosome preservation + high extraction rate" after extraction. The preferred molecular weights used in the raw materials are: phosphatidylcholine (PC): 700-1200 Da; polyethylene glycol (PEG): 1000-2000 Da; cholesterol terminus: 386 Da.

[0066] Example 4: Determination of pH / temperature synergistic regulation conditions BDIM was used to treat Platycladus orientalis leaves, and the oil-exosome complex was gently dissociated by adjusting the environmental conditions such as temperature and pH during treatment. In this example, different pH and temperature conditions were set for treatment to compare the effects of different conditions on the extraction of exosomes from Platycladus orientalis leaves. The specific preparation method was the same as in Example 1, the only difference being the setting of different temperature and pH conditions, which were: (1) no pH adjustment (keeping the system in the natural pH range of ≈5.2); (2) pH adjustment to 8.0 (alkaline system); (3) 4℃ throughout (no temperature rise program); (4) one-step temperature rise to 37℃ (no gradient temperature rise).

[0067] The extraction rate, membrane integrity rate, and BDIM micelle formation efficiency of the extracted Platycladus orientalis exosomes were determined (using the same method as in Test Example 1). The results are shown in Table 3. The results show that pH and gradient temperature control significantly affect the BDIM micelle formation efficiency and extraction effect. Only by simultaneously controlling specific pH / temperature conditions can BDIM micelles be formed to gently dissociate Platycladus orientalis leaves, thereby improving the extraction efficiency.

[0068] Table 3 Extraction effects under different pH / temperature conditions

[0069] Example 5: Stabilization treatment of exosomes After dynamic interface control, preliminary separation and purification are performed to remove residues and impurities from the system. Further, a stabilizer (trehalose) is added to stabilize the system. This embodiment adds a stabilizer at different stages of the treatment to compare the effect of stabilizer addition on the extraction efficiency of Platycladus orientalis leaf exosomes. The specific preparation method is the same as in Example 1, the only difference being the addition of a stabilizer at different extraction stages. The following groups are set up: Group 1 (no stabilizer): no trehalose is added throughout the process, and the remaining steps are the same as in Example 1.

[0070] Group 2 (added in advance): Trehalose (final concentration 5%) was added during the construction stage of the dynamic interface control system (at the same time as BDIM was added), and the subsequent steps were the same as in Example 1.

[0071] Group 3 (method of the present invention): Following step 5 of Example 1, trehalose (final concentration 5%) is added to the crude extract after separation and purification.

[0072] The extraction rate, activity retention rate, and particle size change of the extracted Platycladus orientalis leaf exosomes were determined. The particle size change after lyophilization refers to the relative change rate of the average hydrated particle size of the exosome sample before and after undergoing "freeze-drying → reconstitution". Dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA) is typically used to determine the average particle size before lyophilization (freshly prepared) and after lyophilization and reconstitution, respectively, and the result is calculated using the following formula: Change in particle size after freeze-drying (%) = (average particle size after freeze-drying and rehydration) (Average particle size before freeze-drying) / Average particle size before freeze-drying × 100%; A positive result indicates that the particle size increases after freeze-drying, suggesting that exosomes aggregated or fused during the freeze-drying process; A result close to 0 indicates that the particle size remains basically unchanged, indicating that the exosomes remain monodisperse after freeze-drying and rehydration, and have good structural integrity.

[0073] The results are shown in Table 4. Group 1 had the lowest activity retention rate and the largest change in particle size after freeze-drying. Group 2 may have experienced a decrease in extraction rate or activity due to the premature presence of trehalose interfering with the micelle formation or filtration process of BDIM. Group 3 (the method of this invention) had a high activity retention rate, minimal change in particle size after freeze-drying, and no impact on the extraction rate. This indicates that adding trehalose after extraction and filtration can significantly improve the long-term stability of exosomes.

[0074] Table 4 Results of exosome stabilization treatment

[0075] Example 6: Optimization of extraction conditions for exosomes from Platycladus orientalis leaves 1. Optimization of conditional parameters To further optimize the extraction method, this embodiment also optimized the final concentration of BDIM, heating rate, dynamic equilibrium time, particle size, pH value, stirring speed, and other parameters. Finally, the results were verified by statistical methods such as orthogonal experiments, range analysis, and variance analysis. The same preparation method as in Example 1 was used, but different parameter conditions were set for extraction: (1) the final concentration of BDIM was adjusted to 1.5 mg / mL; (2) the final concentration of BDIM was adjusted to 1.75 mg / mL; (3) the final concentration of BDIM was adjusted to 3.0 mg / mL; (4) the arborvitae leaf powder was mixed with 0.01 mol / L... PBS buffer material-to-liquid ratio (1:15); (5) Material-to-liquid ratio (1:25); (6) Material-to-liquid ratio (1:35); (7) Heating rate adjusted to 1.0℃ / min; (8) Heating rate adjusted to 2.0℃ / min; (9) Dynamic equilibrium time 15min; (10) Dynamic equilibrium time 40min; (11) Trehalose concentration 7%; (12) Particle size 0.3mm; (13) Particle size 0.5mm; (14) Particle size 1.2mm; (15) pH value 6.0; (16) pH value 6.5; (17) pH value 7.0; (18) pH value 7.5; (19) Stirring speed 20r / min; (20) Stirring speed 70r / min; (21) Stirring speed 100r / min; (22) Filtration pressure (0.15MPa); (23) Filtration pressure (0.2MPa).

[0076] After extraction under the conditions described above, the extraction rate, average particle size, membrane structure integrity, flavonoid retention rate, and activity retention rate after 120 days of storage at 4℃ were measured for each exosome. The results are shown in Table 5. It is evident that when the final BDIM concentration reached 3.0 mg / mL, the extraction rate decreased, the exosome particle size increased, and the membrane structure integrity, flavonoid retention rate, and activity retention rate after 120 days of storage at 4℃ decreased, with an aggregation rate of 28%. Increasing the heating rate (2.0℃ / min) significantly reduced all measured indicators, with an exosome rupture rate of 32%. Adjusting the dynamic equilibrium time too low (15 min) resulted in a decreased extraction rate, increased exosome particle size, reduced membrane structure integrity, flavonoid retention rate, and activity retention rate after 120 days of storage at 4℃, and an oil residue of 0.5 mg / mL. Adjusting the particle size to 1.2 mm resulted in an oil residue of 0.8 mg / mL and insufficient exosome release. Adjusting the pH to 6.0 resulted in a 40% decrease in BDIM micelle formation efficiency and incomplete oil dissociation. When the pH value was too high (7.5), the exosome membranes showed slight rupture, leading to increased leakage of active ingredients. When the stirring speed was adjusted too quickly (100 r / min), the exosome rupture rate reached 18%, resulting in significant mechanical shear damage. When the stirring speed was too slow (20 r / min), BDIM and Platycladus orientalis leaf powder were not mixed evenly, resulting in localized undissociated oils and residual oil levels of 0.6 mg / mL. At a filtration pressure of 0.2 MPa, the exosome compression damage rate reached 15%, and a small amount of exosome membrane fragments were detected in the filtrate. Therefore, it is shown that the best extraction effect can only be achieved within the appropriate condition range. The extraction method has a good extraction effect within the following conditions: final BDIM concentration of 1.5-2.0 mg / mL, heating rate of 0.8-1.0℃ / min, dynamic equilibrium time of 30-40 min, particle size of 0.3-0.5 mm, pH value of 6.5-7.0, stirring speed of 30-70 r / min, and filtration pressure of 0.05-0.15 MPa. It can keep the exosomes without residue, aggregation, or rupture.

[0077] Table 5 Results of optimized measurements under different conditions

[0078] 2. Statistical verification (1) The optimal combination of multiple parameters under synergistic effect was further verified by orthogonal verification experiments to eliminate the interaction interference between parameters. Based on the above single-factor optimization results and reverse verification conclusions, five core influencing parameters (final BDIM concentration, heating rate, dynamic equilibrium time, particle size, and pH value) were selected for orthogonal experimental design. Finally, the optimal parameter combination was determined to be: final BDIM concentration 1.75 mg / mL, heating rate 0.8 ℃ / min, dynamic equilibrium time 35 min, particle size 0.3 mm, and pH 6.8.

[0079] Following the optimized method and conditions described above, the exudate from Platycladus orientalis leaves was extracted and prepared, and the results showed an extraction rate of 6.7 × 10⁻⁶. 12 The exosomes were collected in 1 g of Platycladus orientalis leaves. The average particle size of the exosomes was 76 nm. The membrane structure integrity rate was 97.8%. The flavonoid retention rate was 94.2%. After being stored at 4℃ for 120 days, the activity retention rate was 92.3%. There were no residues, no aggregation, and a small amount of rupture.

[0080] (2) Results of range analysis and variance analysis: By calculating the mean of the comprehensive scores of each factor at different levels, the range (R) data were obtained. The results showed that the order of significance of the effect on the extraction effect was: D (particle size) > A (BDIM concentration) > E (pH value) > B (heating rate) > C (equilibrium time), which is consistent with the above results.

[0081] (3) Analysis of variance ( F (Test): The F-test analysis showed that the F-values ​​of all five core parameters were greater than the critical values. F 0.05 (2, 10) = 4.10, P <0.05 indicates that each parameter has a statistically significant effect on the extraction effect, and there is no significant interaction between the parameters (interaction term). F Value < 3.28 P The result was >0.05, which verifies the scientific validity of the parameter settings.

[0082] Comparative Example 1: Traditional Ultracentrifugation The only difference from Example 1 is that BDIM is not added for processing. After the raw material is pretreated, it is directly separated by ultracentrifugation (10,000g centrifugation for 30 min, 100,000g centrifugation for 70 min) and then purified.

[0083] The prepared Platycladus orientalis leaf exosomes were analyzed, and the results showed an extraction rate of 2.1 × 10⁻⁶. 11 The exosomes were 80-200 nm in size (average 120 nm) per g of Platycladus orientalis leaf, with a membrane structure integrity rate of 75.2% and a flavonoid retention rate of 68.3%. After storage at 4℃ for 120 days, the activity retention rate was 45.1%, with a small amount of rupture.

[0084] Comparative Example 2: Traditional PEG Precipitation Method The only difference from Example 1 is that BDIM is not added for treatment. After the raw material is pretreated, PEG6000 precipitation method (final PEG concentration 10%) is used directly. After incubation at 4°C for 12 hours, centrifugation is performed and then subsequent purification is carried out.

[0085] The prepared Platycladus orientalis leaf exosomes were analyzed, and the results showed an extraction rate of 3.5 × 10⁻⁶. 11 The exosomes were 70-180 nm in size (average 105 nm) per g of Platycladus orientalis leaf, with a membrane structure integrity rate of 78.6%, a flavonoid retention rate of 72.5%, an activity retention rate of 52.3% after 120 days of storage at 4℃, PEG residue of 0.8 mg / mL, and a small amount of aggregation.

[0086] Comparative Example 3: Traditional Ultrafiltration The raw material pretreatment was the same as in Example 1. PBS buffer (material-to-liquid ratio 1:20) was added, and the tissue was homogenized. After centrifugation at 4°C and 5000g for 20 min, the supernatant was collected. The supernatant was concentrated by ultrafiltration using a 300kDa ultrafiltration membrane at 4°C and 0.2MPa pressure. The retentate was collected to obtain the exosome solution.

[0087] The extracted Platycladus orientalis leaf exosomes were analyzed, and the results showed an extraction rate of 1.8 × 10⁻⁶. 11 The exosomes were distributed in the range of 28-190 nm (average 112 nm) per gram of Platycladus orientalis leaves. The membrane structure integrity rate was 76.8%, the flavonoid retention rate was 70.2%, the activity retention rate was 48.7% after 120 days of storage at 4℃, the membrane pore blockage rate reached 42% during ultrafiltration, and a small amount of rupture occurred.

[0088] Comparative Example 4: No BDIM processing performed The only difference from Example 1 is that BDIM is not added in step 2, while the other parameters (including feed-to-liquid ratio, pH adjustment, gradient temperature increase, filtration, stabilization, concentration, etc.) are exactly the same as in Example 1.

[0089] The extracted Platycladus orientalis leaf exosomes were analyzed, and the results showed an extraction rate of 1.5 × 10⁻⁶. 12 The exosomes were 1 / g of Platycladus orientalis leaves, with a particle size distribution of 45-210nm (average 125nm), a membrane structure integrity rate of 88.2%, a flavonoid retention rate of 82.6%, an activity retention rate of 70.5% after 120 days of storage at 4℃, and an oil residue of 1.2mg / mL.

[0090] Transmission electron microscopy revealed that a large amount of lipid-exosome complex remained undissociated, resulting in insufficient exosome release. This indicates that without the addition of BDIM, physical methods such as buffer extraction, gradient heating, and filtration alone cannot effectively break down the lipid encapsulation layer in Platycladus orientalis leaf tissue. A significant amount of lipid-exosome complex remained, with an extraction rate only 24% of that achieved with BDIM, and exosome purity and activity were significantly reduced. Therefore, the dynamic interface regulation effect of BDIM is a necessary technical feature for achieving gentle dissociation of the lipid-exosome complex and efficient extraction of Platycladus orientalis leaf exosomes, and cannot be replaced by purely physical methods.

[0091] Comparative Example 5: Using poloxamer F127 as a regulator The only difference from Example 1 is that BDIM is replaced with poloxamer F127 of equal concentration (number average molecular weight of about 12,600 Da), while the other parameters are the same as in Example 1.

[0092] The prepared Platycladus orientalis leaf exosomes were analyzed, and the results showed an extraction rate of 3.2 × 10⁻⁶. 12 The exosomes were 45-180 nm in size (average 115 nm) per g of Platycladus orientalis leaf, with a membrane structure integrity rate of 88.5%, a flavonoid retention rate of 84.3%, an activity retention rate of 75.2% after 120 days of storage at 4℃, an oil residue of 0.9 mg / mL, and a micelle formation efficiency of 65%, which is significantly lower than that of BDIM (98%) in this invention.

[0093] This indicates that although poloxamer F127 is amphiphilic, its specific adsorption capacity at the lipid-exosome interface is weaker than that of BDIM used in this invention, and it cannot achieve gentle dissociation of the lipid layer. The extraction efficiency and activity retention rate are significantly inferior to those of this invention.

[0094] Comparative Example 6: Using chitosan-phospholipid complex as a regulator The only difference from Example 1 is that BDIM is replaced with an equal concentration of chitosan-phospholipid complex (chitosan molecular weight 50 kDa, phosphatidylcholine to chitosan mass ratio 2:1), and the other parameters are the same as in Example 1.

[0095] The extracted Platycladus orientalis leaf exosomes were analyzed, and the results showed an extraction rate of 2.8 × 10⁻⁶. 12 The exosomes were collected in 85.2% of the arborvitae leaves per gram, with a particle size distribution of 50-195 nm (average 120 nm), a membrane structure integrity rate of 85.2%, a flavonoid retention rate of 82.1%, an activity retention rate of 72.8% after 120 days of storage at 4℃, an exosome aggregation rate of 25%, and a lipid residue of 1.1 mg / mL.

[0096] This indicates that although the chitosan-phospholipid complex has positive charge and membrane affinity, its hydrophobic effect is insufficient, making it unable to effectively insert into the oil layer. Furthermore, the positive charge induces exosome aggregation, leading to a decrease in extraction purity and activity, making it unsuitable for exosome extraction from the high-oil-viscosity tissue of Platycladus orientalis leaves.

[0097] Comparative Example 7: No pH or temperature control (natural conditions) The only difference from Example 1 is that the pH of the system was not adjusted during extraction (the natural pH of the mixture of Platycladus orientalis leaf powder and buffer was approximately 5.2), and no gradient temperature increase was performed (the temperature was maintained at 4°C throughout the process). All other parameters were the same as in Example 1.

[0098] The extracted Platycladus orientalis leaf exosomes were analyzed, and the results showed an extraction rate of 1.5 × 10⁻⁶. 12 The exosomes were 45-200 nm in size (average 128 nm) per g of Platycladus orientalis leaf, with a membrane structure integrity rate of 80.3%, a flavonoid retention rate of 75.6%, an activity retention rate of 65.4% after 120 days of storage at 4℃, an oil residue of 1.8 mg / mL, and a BDIM micelle formation efficiency of only 20%.

[0099] The results showed that when the pH deviated from the range of 6.5-7.0, the hydrophilic-hydrophobic balance of BDIM was disrupted, and the micelle formation ability decreased significantly. At the same time, without gradient heating, the reversible formation and dissociation of micelles could not be induced, and the dissociation of the lipid-exosome complex was insufficient. This proves that pH and temperature regulation are indispensable key links in the technical solution of this invention.

[0100] Comparative Example 8: Stabilizer-free and freeze-dried The only difference from Example 1 is that trehalose is not added in step 5, and freeze drying is used instead of freeze concentration in step 6 (pre-freezing temperature -40°C, pre-freezing time 4 h, sublimation temperature -10°C, sublimation time 12 h, resolution temperature 25°C, resolution time 6 h), while the other parameters are the same as in Example 1.

[0101] The exosomes prepared from Platycladus orientalis leaves were analyzed, and the results showed that the extraction rate before freeze-drying was 6.5 × 10⁻⁶. 12 The membrane structure integrity rate was 96.2% for exosomes per gram of Platycladus orientalis leaves. After freeze-drying, the average particle size increased from 82 nm to 125 nm (an increase of 48%), and the membrane structure integrity rate decreased to 85.5%. After reconstitution at 4℃, the activity retention rate was only 62.1%. This indicates that without in-situ stabilization, exosomes are prone to significant aggregation and membrane structure damage during freeze-drying, resulting in a substantial decrease in activity. However, the simultaneous addition of stabilizers during extraction can effectively protect the integrity of exosomes during concentration and drying, demonstrating the necessity of the in-situ stabilization step.

[0102] The results of the comparative examples are shown in Table 6. Comparative Examples 1-3, prepared using traditional extraction methods, exhibited poor extraction rates, low membrane structure integrity, and low flavonoid retention rates, along with high lipid residue. Furthermore, the activity retention rate after 120 days of storage at 4°C was significantly lower than in Example 1. Comparative Examples 4-6, which used the method of Example 1 without BDIM or with its replacement, also showed poor results. Even with BDIM but without temperature control, the extracted exosomes were of poor quality, indicating that BDIM combined with temperature control is crucial for improving extraction efficiency. While the stabilizer and freeze-drying steps do not affect the lipid residue of the exosomes, they do affect the membrane structure integrity, flavonoid retention rate, and activity retention rate after 120 days of storage at 4°C. Therefore, using a stabilizer and freeze-drying further enhances the retention of active ingredients in the exosomes.

[0103] Table 6 Comparison of the results of the above comparative examples

[0104] Application Example 1: Industrialized Production Process (500 kg / batch) This embodiment describes an industrial-scale production process, based on the optimal comprehensive parameter set, adapted to a production scale of 500 kg / batch. The specific process and parameters are as follows: 1. Industrialization process flow diagram Raw material acceptance → continuous low-temperature pulverization → dynamic interface-controlled reaction (continuous) → cross-flow filtration purification → in-situ stabilization → continuous freeze concentration → finished product inspection → freeze drying / cold storage.

[0105] 2. Equipment selection criteria Continuous low-temperature pulverizer unit: Selected model CFS-500, with a capacity of 500kg / h, suitable for processing raw materials of 500kg / batch; adopts inert gas sealed protection, which can control the pulverization temperature from -5 to 0℃, avoids oxidation of arborvitae leaf powder, and meets the low-temperature requirements of raw material pretreatment.

[0106] Continuous dynamic interface controlled reactor: 10m model selected 3 Reactor (with real-time temperature control, pH monitoring and automatic feeding module), volume adapted to 10m³ 3 Reaction system (500kg raw material + 10m 3 The buffer solution module and real-time monitoring module ensure precise control of temperature and pH, while the automatic feeding module is adapted for continuous production.

[0107] Cross-flow filtration system: Selected membrane material is polyethersulfone, pore size is 0.22μm, and filtration area is 5m². 2 Cross-flow velocity of 3 m / s reduces membrane fouling and is compatible with 10m membranes. 3With a filtration efficiency of / h, it meets the requirements of industrial continuous filtration.

[0108] Continuous freeze-thaw concentration equipment: RFC-10 model, capacity 10m³ 3 / h, can achieve precise control of -20℃ and 5Pa, adapt to continuous concentration needs, and reduce energy consumption by 25% compared with batch type.

[0109] 3. Criteria for scaling up process parameters Scale-up criteria for reaction volume: linear scale-up based on a feed-to-liquid ratio of 1:20; 500 kg of raw material corresponds to 10 m³ / kg of reaction volume. 3 Buffer solution, reaction vessel volume selected: 10m³ 3 Reserve 20% expansion space.

[0110] Stirring speed scale-up criteria: Based on the principle of constant power coefficient, the laboratory stirring speed is 50 r / min (paddle diameter 5 cm), and the industrial reactor paddle diameter is 1.5 m. The stirring speed will remain at 50 r / min after scale-up to ensure uniform mixing.

[0111] Filtration parameter scaling criteria: The filtration area is linearly related to the throughput; in the laboratory, a throughput of 1 L / h corresponds to a filtration area of ​​0.01 m². 2 Industrialized processing capacity 10m³ 3 / h corresponds to a filtration area of ​​5m² 2 The filtration pressure remains constant at 0.1 MPa.

[0112] Temperature control parameter scaling criteria: The heating rate is maintained at 0.8℃ / min, achieved by increasing the heating area; the industrial-scale reactor has a heating area of ​​10m². 2 This ensures the accuracy of the gradient heating.

[0113] 4. Specific production steps (1) Raw material acceptance and pretreatment: 500 kg of disease-free arborvitae leaves from the current year were selected. The batch acceptance standards were: moisture content ≤12%, volatile oil content ≥0.22%, and heavy metal content ≤0.1 mg / kg. The CFS-500 continuous low temperature pulverizer was used for pulverization, with nitrogen gas sealing protection, pulverization temperature -5-0℃, and particle size ≤0.3 mm after grading and screening. The pulverized powder was then transported to the reaction vessel through a sealed pipeline. (2) Dynamic interface regulation response: towards 10m 3 Add 10m to the reactor 3Pre-cool (4℃) PBS buffer, add 75L of BDIM solution (concentration 175g / L, phosphatidylcholine: polyethylene glycol: cholesterol = 5:3:2) to ensure a final concentration of 1.75mg / mL; stir at 50r / min, and heat from 4℃ to 37℃ at a rate of 0.8℃ / min. During the heating process, automatically feed the solution to adjust the pH to 6.8±0.1 and maintain dynamic equilibrium for 35min. The reaction solution is continuously discharged into the filtration system. (3) Separation and purification: The cross-flow filtration system is used for filtration, with a filtration pressure of 0.08-0.12MPa and a cross-flow velocity of 3m / s. The filtrate is transported to the stabilization tank through a closed pipeline. (4) In-situ stabilization: Add 500 kg of trehalose (final concentration 5%) to the stabilization tank, stir evenly at 30 r / min, and incubate at 4℃ for 20 min; (5) Continuous freeze concentration: The stabilized solution is fed into the RFC-10 continuous freeze concentration equipment and concentrated to 2m at -20℃ and 5Pa. 3 ; (6) Finished product inspection and storage: After sterile filtration (0.22μm terminal filter), the concentrate is aseptically filled into 20L / barrel and stored at 4℃; or it is sent to an LGJ-1000 freeze dryer for freeze drying (pre-freezing temperature -40℃, pre-freezing time 4h, sublimation temperature -10℃, sublimation time 12h, desorption temperature 25℃, desorption time 6h) to obtain the powdered finished product, which is then sealed and stored at room temperature.

[0114] Results of industrial-scale production testing indicators: Extraction rate 6.5×10 12 The product contains 97.2% of the arborvitae leaf per gram, with a membrane structure integrity rate of 97.2%, a flavonoid retention rate of 93.5%, an activity retention rate of 91.8% after 120 days of storage at 4℃, and a batch-to-batch variation coefficient of ≤3%, meeting the GMP standards for cosmetic raw material production.

[0115] Application Example 2: Hair Care Application Using the arborvitae leaf exosomes obtained in Example 1 as the sole active ingredient, a scalp repair essence was prepared to repair seborrheic alopecia and frizzy hair.

[0116] 1. Product preparation Formula composition: Platycladus orientalis leaf exosome concentrate (10%), sodium hyaluronate (0.5%), panthenol (2%), glycerin (5%), dipotassium glycyrrhizate (0.2%), sterile ultrapure water (balance).

[0117] Preparation process: Sodium hyaluronate and glycerin are dissolved in ultrapure water and stirred at a constant temperature of 70°C. After cooling to 40°C, Platycladus orientalis leaf exosome concentrate, panthenol, and dipotassium glycyrrhizate are added. After stirring evenly, the mixture is filtered through a 0.22μm filter membrane for sterilization, dispensed into sterile brown bottles, and stored at 4°C.

[0118] 2. Setting different exosome concentrations Based on the exosomes extracted from Platycladus orientalis leaves in Example 1, three groups of scalp repair essences with different exosome concentrations were prepared. The other ingredients in the formula remained unchanged, and the amount of exosome concentrate added was 5%, 10%, and 15%, respectively, which were designated as experimental group 1, experimental group 2, and experimental group 3.

[0119] 3. Test criteria Forty-five patients with seborrheic alopecia (aged 25-45, half male and half female) were randomly divided into three groups (15 people in each group). Each group used one of the three serums three times a week for 12 weeks. The following key efficacy indicators were measured.

[0120] (1) Rate of reduction in scalp sebum secretion (%) Measurement Method: A sebum meter (Sebumeter SM815, Courage+Khazaka) was used to measure a fixed area on the subject's scalp (a 2cm x 2cm area on the occipital side of the top of the head). Before each measurement, the subject sat quietly for 30 minutes in a temperature and humidity controlled room (temperature 22±1℃, humidity 50±5%). After washing the scalp with a standard cleanser, the same area was measured before treatment (week 0) and at week 4 after treatment. Each area was measured three times, and the average value was taken.

[0121] Calculation formula: Sebum secretion reduction rate (%) = (Pre-treatment sebum amount) (Oil amount after treatment) / Oil amount before treatment × 100%.

[0122] (2) Daily hair loss reduction rate (%) Measurement Method: The shampooing test method was used. Subjects used a uniformly provided shampoo (containing no active ingredients) to wash their hair for three consecutive days. Hair loss during each wash (including hair in the shampoo and hair lost during combing after drying) was collected and counted by a designated person. Two 3-day shampooing tests were conducted before treatment (week 0) and again at week 8 after treatment, and the average daily hair loss was calculated (total hair loss over 3 days / 3).

[0123] Calculation formula: Daily hair loss reduction rate (%) = (Daily hair loss before treatment) (Average daily hair loss after treatment) / Average daily hair loss before treatment × 100%.

[0124] (3) Hair follicle density increase rate (%) Measurement method: A dermatoscope (FotoFinder Medicam 800, polarized light mode, magnification ×50) was used. A 1cm mark was made on the fixed area of ​​the subject's scalp (3cm from the hairline on the left temporal region). 2In the observation area, dermoscopic images were taken, and two trained dermatologists independently counted the number of follicular units (each follicular unit contains 1-4 hairs) in the area, and the average value was taken. Measurements were taken before treatment (week 0) and at week 12 after treatment.

[0125] Calculation formula: Hair follicle density increase rate (%) = (hair follicle density after treatment - hair follicle density before treatment) / hair follicle density before treatment × 100%.

[0126] (4) Hair shaft diameter thickening rate (%) Measurement method: Dermoscopy image analysis software (TrichoScience 2.0) was used. Within the above 1cm... 2 Within the observation area, 30 hair shafts were randomly selected, and the transverse diameter of their midsection (approximately 1 cm from the scalp) was measured, with the average value taken. Measurements were taken before treatment (week 0) and at week 12 after treatment.

[0127] Calculation formula: Hair shaft diameter thickening rate (%) = (Hair shaft diameter after treatment - Hair shaft diameter before treatment) / Hair shaft diameter before treatment × 100%.

[0128] (5) Incidence of adverse reactions (%) Measurement method: At each follow-up visit (weeks 2, 4, 8, and 12), a dermatologist will conduct a clinical examination of the subject's scalp, inquire about and record whether there are any local adverse reactions such as erythema, itching, stinging, desquamation, or folliculitis.

[0129] Calculation formula: Adverse reaction incidence (%) = Number of subjects who experienced any adverse reaction / Total number of subjects in the group × 100%.

[0130] (6) Statistical processing All data are expressed as mean ± standard deviation (Mean ± SD). Paired t-tests were used for comparisons within groups before and after treatment, and one-way ANOVA was used for comparisons between groups. P <0.05 was considered statistically significant. Statistical analysis was performed using SPSS 26.0 software. Table 7 shows the mean values ​​of the 15 subjects in each group. The data were found to be normally distributed, and there were no significant differences in baseline values ​​between groups. P >0.05).

[0131] The results, shown in Table 7, indicate that the hair care effect gradually increases with increasing exosome concentration. However, the difference in effect between the 10% and 15% concentration groups was small (difference in sebum secretion reduction rate was 3%, and difference in hair follicle density increase rate was 2%). Considering both cost and effect, a 10% exosome concentration is the optimal application ratio. Safety verification was also conducted; no adverse reactions such as scalp allergies or stinging occurred in any subjects throughout the trial, and skin irritation tests showed the product to be non-irritating.

[0132] Table 7. Hair loss prevention effect

[0133] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for efficient extraction of exosomes from Platycladus orientalis leaves based on dynamic interface regulation, characterized in that, Includes the following steps: (1) Construction of dynamic interface regulation system: After pulverizing the leaves of Platycladus orientalis, disperse them in buffer solution, add BDIM with a final concentration of 1.5-2.0 mg / mL, mix well, and adjust the pH of the system to 6.5-7.0 to construct a mixed system; the BDIM is a phosphatidylcholine-polyethylene glycol-cholesterol block copolymer with a molecular weight of 2000-5000 Da, wherein the molar ratio of phosphatidylcholine, polyethylene glycol and cholesterol is (4-7):(2-5):(1-2); (2) Gradient heating and dynamic equilibrium: The mixture is heated to 36-37℃ at a rate of 0.8-1.0℃ / min and maintained in dynamic equilibrium for 30-40min; (3) Separation and purification: Filter the balanced system to remove residues and impurities to obtain crude extract; (4) Concentration and preservation: The crude extract is concentrated by freezing to obtain Platycladus orientalis leaf exosomes.

2. The method according to claim 1, characterized in that, (1) The leaves of Platycladus orientalis are crushed into micro powder with a particle size of 0.3-0.5 mm.

3. The method according to claim 2, characterized in that, (1) The ratio of Chinese arborvitae leaf to buffer solution is 1:15-25 g / mL; the buffer solution is selected from one or more of phosphate buffer, Tris-HCl buffer, HEPES buffer, and citrate-phosphate buffer.

4. The method according to claim 3, characterized in that, (3) The filtration pressure is 0.05-0.15MPa.

5. The method according to claim 1, characterized in that, After separation and purification in (3), the solution is stabilized in situ and then concentrated and stored. The in situ stabilization is as follows: 3%-7% w / v stabilizer is added to the crude extract and then incubated at 0-5℃ for 10-30 min.

6. The method according to claim 5, characterized in that, The stabilizer is selected from one or more of trehalose, sucrose, mannitol, sorbitol, betaine, and glycerol.

7. A Platycladus orientalis leaf exosome prepared by the method according to any one of claims 1 to 6.

8. The application of the method according to any one of claims 1 to 6 in reducing the residual oil content of Platycladus orientalis exosomes, improving the extraction rate and the retention effect of active ingredients.

9. The use of the Platycladus orientalis leaf exosomes of claim 7 in hair care or in the preparation of hair care products.

10. A hair care product, characterized in that, The arborvitae leaf exosomes extracted by the method according to any one of claims 1 to 6.

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