A mulberry root-bark extract and a method for preparing the same

Abstract

The present application relates to the technical field of natural product extraction, and provides mulberry root bark extract and a preparation method thereof.The extract contains 3-8 characteristic components among eight characteristic components of mulberry flavone C, morusin, cyclo-morusin, cyclo-moracen, mulberroside G, morusin M, mulberroside C and mulberry flavone H.Further, the content relationship of four key components, the proportion of 300-800 Da components, the proportion of >1000 Da components, the content of fat-soluble resin impurities and the proportion of chromatographic peak area are coupled and limited by stability index S and synergy index T.The preparation method comprises the following steps: acid buffer infiltration, ethanol ultrasonic reflux extraction, concentration and adjustment of polarity, sedimentation separation, supercritical carbon dioxide impurity removal, resin column narrow window chromatographic separation, microfiltration-ultrafiltration-nanofiltration separation and drying.The obtained extract can regulate the expression of inflammation-related factors and promote the recovery of intracellular signal molecule level, thereby showing good soothing and skin barrier repair effects at the cell level.

Description

Technical Field

[0001] This invention relates to the technical field of mulberry bark extraction, and in particular to a mulberry root bark extract and its preparation method. Background Technology

[0002] Mulberry bark, the dried root bark of the mulberry tree (Morus alba), has a long history of traditional use and natural product research. Existing studies have shown that mulberry bark contains various types of natural products, including polyphenolic compounds, flavonoid derivatives, and other secondary metabolites, and may also contain certain proportions of sugars, proteins, and resins. These components exist in the plant tissue as a complex mixture, and during extraction, they typically enter the extract together, forming a plant extract system composed of compounds with various structural types.

[0003] In the study of natural products from mulberry bark, researchers have discovered that some isopentenyl flavonoids and polyphenolic molecules possess strong hydrogen-donating or electron-transfer capabilities, enabling them to play a role in free radical scavenging and antioxidant reactions. However, the content of these compounds in plant tissues is usually low, and different structural molecules often coexist in the same extraction system. Their role in antioxidant and cellular regulation processes is not determined by a single component, but may be closely related to the combination and proportion of multiple structural molecules.

[0004] In existing technologies, the extraction of mulberry bark typically employs solvent extraction, reflux extraction, or maceration, combined with concentration, drying, or certain separation and purification steps to obtain the extract. However, because plant raw materials themselves contain various compounds with similar structures but different properties, and these components differ in polarity, hydrophobicity, and intermolecular interaction mechanisms, the distribution and relative proportions of various components in the system during extraction and post-processing are easily affected by factors such as the source of the raw material, extraction conditions, and purification methods. This results in variations in the chemical composition of the extract between different batches.

[0005] Furthermore, due to the similar structures and polarities of various flavonoids and polyphenols in mulberry bark, selective enrichment is often difficult to achieve under conventional extraction and purification conditions. Some active characteristic components are easily carried out or partially lost during extraction or purification, making it difficult to maintain a stable ratio between different components. At the same time, plant raw materials may also contain a certain proportion of high-molecular impurities or inactive components. These substances coexist with small-molecule active ingredients in the extraction system and may also affect the overall composition and structure of the extract.

[0006] Meanwhile, related studies have found that the effects of plant extracts at the cellular level are often closely related to the combined state of various small molecule components in the system. For example, under inflammatory stimulation, the expression levels of various inflammation-related factors and the activity of related signaling pathways in cells change, and the synergistic effects between natural products of different structural types may affect these biological processes. In addition, the levels of intracellular second messengers and the expression status of some structural protein-related genes may also be affected by changes in the composition and structure of the extract.

[0007] However, under current technological conditions, the proportions and distribution of various chemical components in plant extracts are difficult to control stably. When the proportions of different structural components in the extract system change, their regulatory effects at the cellular level may also change. Therefore, in the field of related technologies, how to rationally control the composition and structure of extracts in complex plant extraction systems to enable them to exhibit more stable effects in regulating the expression of cellular inflammatory factors, changes in related signaling pathways, and the expression of genes related to cellular function remains a technical problem that requires further research. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned problems existing in the prior art and provide a mulberry root bark extract and its preparation method. By controlling the compositional relationship and content distribution of key characteristic components in the extract, the chemical composition of the obtained extract is made more stable, thereby making it exhibit a more stable and reproducible effect in regulating the expression of cell inflammation-related factors, changes in related signaling pathways, and expression of cell function-related genes.

[0009] To achieve the above objectives, the first aspect of the present invention provides a mulberry root bark extract, wherein the extract contains 3 to 8 of the following eight characteristic components: morinone C, morinol, cyclomorin, cyclomorinone, morinone G, morinol M, morinone C, and morinone H, and satisfies the following conditions: The total content of the characteristic components accounts for 5% to 30% of the total weight of the extract; The mass ratio of mulberry ketone C: morin: cyclomorin: cyclomorin is 1:4-25:0.2-0.6:0.01-0.08.

[0010] First, Kuwanon C is a Diels-Alder type polyphenol adduct unique to the genus *Morus*. Its molecule consists of two aromatic structural units that form a complex polycyclic skeleton through a cycloaddition reaction, containing multiple phenolic hydroxyl groups and a large π-conjugated system. This structure endows Kuwanon C with strong electron delocalization and free radical stabilization capabilities. When reactive oxygen species or other free radicals are present in the system, its phenolic hydroxyl groups can donate hydrogen atoms, converting the free radical into a stable molecule. The generated phenoxy free radical can then remain stable through electron delocalization via the intramolecular conjugated structure, thereby blocking free radical chain reactions. Furthermore, its polycyclic stereostructure allows it to bind to biomolecules at multiple points through hydrogen bonding, π-π interactions, and hydrophobic interactions. Therefore, it not only possesses antioxidant capabilities but also regulates inflammation-related signaling pathways, thus forming a core active structural unit in the entire extract system.

[0011] Secondly, Morusin belongs to the isopentenylated flavonoid class of compounds. Its molecular structure is based on a flavonoid skeleton and contains isopentenyl side chains and pyran ring structures, significantly enhancing the overall hydrophobicity of the molecule. This structural feature makes Morusin more easily enter the lipid environment, such as the cell membrane or lipid microdomains. In the lipid environment, lipid peroxidation often propagates continuously through lipid free radical chain reactions. The phenolic hydroxyl groups in Morusin can donate hydrogen atoms to lipid free radicals, converting them into stable lipid molecules. At the same time, the generated phenoxy free radicals can be stabilized through molecular conjugation, thereby terminating the lipid oxidation chain reaction. In addition, several isopentenyl flavonoid components with similar structures to Morusin exist in mulberry root bark extract, such as Morusin M, Morus root ketone C, Morus senna H, and Morus senna G. These components also have flavonoid skeletons and isopentenyl substitution structures. In the system, they can form an isopentenyl flavonoid structural group together with Morusin, thereby synergistically exerting free radical scavenging and lipid peroxidation inhibition effects in the lipid environment.

[0012] Third, cyclomulberrin belongs to the cyclopentenyl flavonoid class of compounds, and its molecule contains a prominent ortho-phenolic hydroxyl structural unit. The ortho-diphenol structure has a strong metal ion complexing ability, and can bind with Fe²⁺, Fe³⁺, or Cu. 2+ Cyclomorin forms stable complexes with transition metal ions. In biological systems, these metal ions often catalyze the decomposition of peroxides to generate hydroxyl radicals through Fenton reactions or similar reactions. Hydroxyl radicals are among the most reactive free radicals in oxidative stress systems. Cyclomorin reduces the catalytic activity of these metal ions by chelating them, thereby decreasing the generation of hydroxyl radicals. Therefore, it mainly plays a role in inhibiting the source of free radical generation in the system.

[0013] Fourth, cyclomorcanthone (Cyclomulberrochromene / Cyclomorusin) belongs to the cyclopentenyl flavonoid derivatives with a chrome ring structure. The chrome ring structure belongs to the benzopyran system and possesses strong π-electron conjugation ability, which can stabilize free radical intermediates. Furthermore, the cyclization of the isopentenyl group enhances the overall hydrophobicity of the molecule, making it easier to accumulate in the lipid membrane environment. During lipid oxidation, lipid free radicals and lipid peroxide free radicals are often generated and propagated in the cell membrane or lipid microdomains. Cyclomorcanthone can stabilize free radicals in these microenvironments through its conjugated system, thereby reducing their reactivity and inhibiting the further propagation of lipid oxidation. Therefore, it mainly plays a role in stabilizing free radicals in the lipid environment and protecting the membrane structure.

[0014] In the above structural system, mulberry flavonoid C mainly participates in free radical scavenging and signal regulation processes, isopentenyl flavonoids (including mulberry cinnamic acid and its structural analogs) mainly participate in the termination of lipid free radical chain reactions, cyclomoronin inhibits free radical generation by complexing metal ions, and cyclomoronin stabilizes lipid free radicals in the lipid membrane environment. Therefore, these molecules correspond to different stages in their action pathways, such as free radical generation, free radical scavenging, chain reaction propagation, and lipid oxidation.

[0015] When mulberry senna C, morin, cyclomorin, and cyclomorin are present in the aforementioned defined proportions, the quantitative relationships of various structures in the system reach a relative equilibrium, allowing different mechanisms of action to function simultaneously. Simultaneously, structurally similar isopentenyl flavonoids such as morin M, morin root ketone C, mulberry senna H, and mulberry senna G, which can be optionally present in the system, can further participate in this structural network, supplementing the number of isopentenyl flavonoid structural units without altering the core structural proportions. If the proportion of morin is too low, the lipid free radical scavenging ability is insufficient; if the proportion is too high, the system's hydrophobicity increases, potentially affecting the dispersion of other polyphenolic components. If the proportion of cyclomorin is too low, it is difficult to effectively inhibit free radicals generated by metal ion catalysis; if the proportion is too high, molecular aggregates easily form between polyphenol structures. When the proportion of cyclomorin is too low, its inhibitory effect on lipid oxidation is limited; when the proportion is too high, it may alter the system's polarity and affect overall stability.

[0016] Therefore, by limiting the above-mentioned core structural components to a specific ratio range and allowing the presence of several structurally similar isopentenyl flavonoid supplementary components in the system, flavonoids of different structural types can form a stable spatial distribution in the system, enabling multiple reaction steps such as free radical generation, free radical scavenging, and lipid oxidation inhibition to be synergistically regulated, thereby making the overall structure of mulberry root bark extract more stable and exhibiting more stable and significant bioactive effects.

[0017] Furthermore, the extract satisfies the following function: ,and ; Where m1 represents the content of mulberry senna C; m2 represents the total content of isopentenyl flavonoids. In this application, the isopentenyl flavonoids refer to compounds that have a flavonoid skeleton and contain isopentenyl substitution structures, including but not limited to morin, morin M, morin C, morin D, and morin G.

[0018] m3 represents the content of cyclomolarin; m4 represents the content of cyclomoronene; w 300-800 The proportion of components with molecular weights in the range of 300–800 Da to the total weight of the extract; w >1000 The proportion of components with a molecular weight greater than 1000 Da to the total weight of the extract; w res The percentage of fat-soluble resin impurities in the total weight of the extract; A4 represents the ratio of the sum of the peak areas of the four characteristic components (Morus alba ketone C, Morus alba ketone, cyclomorusin, and cyclomorusin) to the total peak area, or the ratio of the sum of the contents of these four characteristic components to the total content of substances, when detected by high performance liquid chromatography.

[0019] By constructing a stability index S, mulberry senna C forms a synergistic structural unit with isopentenyl flavonoids composed of morin, morin M, morin root ketone C, mulberry senna H, and mulberry senna G, and maintains a specific proportional relationship with cyclized flavonoid structural units composed of cyclomorin and cyclomorin. Combined with the internal proportional relationships among these characteristic components, the spatial distribution of polyphenol structures in the system can be made more balanced. When the above structural proportions reach the range defined by the stability index, the above characteristic components form a relatively stable molecular network structure in the system, ensuring a high degree of dispersion of flavonoids with isopentenyl side chains and cyclized flavonoids in the same system, thereby reducing the activity decay caused by local enrichment or intermolecular hydrophobic aggregation.

[0020] Furthermore, by incorporating the molecular weight distribution parameter into the function, the main components in the system are concentrated in the 300–800 Da range, while suppressing the proportion of high molecular weight components with a molecular weight greater than 1000 Da. This can significantly reduce the adsorption or encapsulation of the above-mentioned small molecule flavonoid active ingredients by polysaccharides, proteins, and polymeric phenols, thereby improving the effective exposure of isopentenyl flavonoid structural units and cyclized flavonoid structural units in the system.

[0021] Meanwhile, by coupling the content of lipid-soluble resin impurities with the ratio of the main chromatographic peak, the hydrophobic aggregation effect of non-polar impurities on the isopentenyl side chain structure in the system can be suppressed, thereby making the dispersion state of the above-mentioned characteristic components in the solution system more stable and maintaining their synergistic structural relationship in the system.

[0022] Furthermore, the extract satisfies the following function: And 1000≤T≤2000.

[0023] Further introducing a synergistic index T further limits the relationship between the synergistic effect strength between isopentenyl flavonoid structural units and cyclized flavonoid structural units in the system and the degree of impurity interference, ensuring that the active structures in the extract system maintain a high level of synergistic effect even in the presence of impurity background. Moritake C and the isopentenyl flavonoid components composed of morin, morin M, morin C, morin H, and morin G all contain isopentenyl substituents and polyphenol structural units. These structures can participate in different types of free radical reaction processes in the system. Moritake C, as the core structural unit, provides stable phenolic hydroxyl hydrogen donation sites, while the isopentenyl flavonoid components participate in free radical capture and electron transfer processes through their side chain structures. By introducing the product relationship between the above structural units into the function, the degree of synergistic contribution between the core polyphenol structure and the isopentenyl flavonoid structure in the system can be reflected. When these components are simultaneously within an appropriate content range in the system, a complementary free radical stabilization mechanism can be formed, resulting in an overall antioxidant capacity that exhibits a significantly enhanced effect than the effect of a single component.

[0024] Meanwhile, the cyclized flavonoid structures of cyclomoronin and cyclomoronochrome significantly influence the molecular arrangement in the system. When the content of these cyclized structures is too high, π–π stacking interactions easily occur between the cyclized flavonoids, thereby altering the dispersion state of isopentenyl flavonoid molecules in the system. By adjusting the proportion of cyclized structures in a square form within the function, the aggregation tendency of these structures can be restricted, allowing the cyclized flavonoid structures to primarily play a spatial regulating role in the system without forming excessive aggregation, thus maintaining a more uniform spatial distribution among flavonoid compounds of different structural types.

[0025] Furthermore, by simultaneously incorporating molecular weight distribution parameters and impurity content into the synergy index, a direct functional relationship is established between the synergistic effect strength between active structures in the system and the interaction between polymeric and resinous impurities. This ensures that the polyphenolic network composed of isopentenyl flavonoid structural units and cyclized flavonoid structural units maintains its dominance even in the presence of impurities. When the synergy index falls within a defined range, it indicates that the polyphenolic network composed of the aforementioned characteristic components occupies a dominant structural state overall. Even the presence of small amounts of polymeric components or lipid-soluble resinous impurities will not disrupt the stability of this structural network, thus enabling the extract to maintain a stable chemical structure and bioactivity under different production batches and storage conditions.

[0026] Furthermore, the content of components with a molecular weight greater than 1000 Da in the extract is less than 5%.

[0027] The main purpose of controlling the content of components with a molecular weight greater than 1000 Da in the extract to below 5% is to reduce the proportion of high-molecular-weight impurities in the extract. During the conventional extraction process of mulberry bark, a certain amount of polysaccharides, proteins, polymeric phenols, and some high-molecular-weight condensation products are often present. These substances typically have large molecular weights and easily form aggregates with small-molecule polyphenolic compounds in the system through hydrogen bonding or hydrophobic interactions. When the content of these high-molecular-weight components is high, they can easily encapsulate or adsorb small-molecule components such as mulberry flavonoid C, morin, cyclomorin, and cyclomorin, altering their dispersion state in the system and thus reducing their effective activity. Controlling the content of components with a molecular weight greater than 1000 Da at a low level can significantly reduce the non-specific binding of these high-molecular-weight substances to small-molecule active ingredients, allowing the four characteristic components to maintain a relatively free dispersion state in the system. This enables them to participate more fully in reactions such as free radical scavenging, metal ion complexation, and lipid oxidation inhibition, resulting in more stable overall activity.

[0028] Furthermore, when the extract is detected by high performance liquid chromatography, the sum of the peak areas of the four characteristic components—morrhagic senna C, morin, cyclomorrhagic senna, and cyclomorrhagic senna—accounts for no less than 8% of the total peak area.

[0029] By ensuring that the sum of the peak areas of the four characteristic components in the extract, as detected by high-performance liquid chromatography (HPLC), accounts for no less than 40% of the total peak area, it can be guaranteed that the chemical composition of the extract is dominated by linalool C, morin, cyclomorin, and cyclomorin. Plant extracts typically contain a large number of small molecules with different structural types. If the proportion of impurities in the system is too high, even if the content of the four characteristic components reaches a certain level, the presence of other small molecule impurities may interfere with their intermolecular interactions, thus affecting the overall effect. When the peak area ratio of the above four characteristic components reaches more than 40%, it indicates that the main chemical composition of the system is dominated by these four structural types, and the proportion of other impurities is relatively low. Under this structural condition, it is easier for the four characteristic components to form stable intermolecular interactions, making their synergistic effect in the system more obvious.

[0030] Therefore, by simultaneously controlling the content of polymeric components and the proportion of the main chromatographic peak, the compositional state of the extract can be defined from both the molecular weight structure and chemical composition structure levels. On the one hand, this reduces the interference of polymeric impurities on small molecule active ingredients; on the other hand, it ensures that the main active ingredients dominate the system, allowing the four characteristic components to form a more stable combination structure in the system, thereby improving the stability and consistency of the overall activity performance of the extract.

[0031] A second aspect of the present invention provides a method for preparing the mulberry root bark extract as described above, comprising the following steps: S1: After crushing the mulberry bark raw material, it is soaked in an acidic buffer solution to make the pH of the system 4.5-5.5; S2: The mulberry bark treated in step S1 was subjected to ultrasonic reflux extraction in a 65%–75% (v / v) ethanol solution to obtain an extract. S3: The extract is concentrated under reduced pressure, and some resin impurities and high molecular weight components are separated by adjusting the solvent polarity to obtain a pretreated extract. S4: The pretreated extract is subjected to supercritical carbon dioxide treatment to remove lipid-soluble resin impurities; S5: The material treated in step S4 is reconstituted and separated by chromatography using a macroporous adsorption resin column. Only the elution fraction with an ethanol volume fraction of 63% to 68% is collected as the target fraction. S6: The target fraction is sequentially separated by microfiltration, ultrafiltration and nanofiltration, wherein ultrafiltration removes components with a molecular weight greater than 1000 Da and nanofiltration enriches components with a molecular weight range of 300 to 800 Da. S7: The retentate obtained by nanofiltration is concentrated and dried to obtain the mulberry root bark extract.

[0032] By sequentially introducing acid activation treatment, polar window chromatography, and membrane separation to regulate molecular weight distribution during the extraction process, isopentenyl flavonoids and cyclized flavonoids in the mulberry bark extract system are simultaneously enriched within the same separation range. Molecular weight screening further reduces the adsorption or encapsulation of small flavonoids by high-molecular-weight substances such as polysaccharides, proteins, and polymeric polyphenols, thus allowing for simultaneous control of the proportions and molecular weight distributions of the four characteristic components in the system. Through this multi-stage separation and structural screening process, the main chemical composition of the extract gradually shifts towards a structural system dominated by mulberry flavonoid C, morin, cyclomorin, and cyclomorin, forming a more stable combination relationship between flavonoid compounds of different structural types and improving the overall chemical stability of the extract.

[0033] Furthermore, the acidic buffer solution mentioned in step S1 is a citrate buffer solution, and the mass fraction of the buffer solution is 0.05% to 0.20%.

[0034] Pre-extraction soaking of mulberry bark in a citric acid buffer solution maintains a stable, weakly acidic environment. This partially protonates the phenolic hydroxyl groups in the polyphenolic structures, reducing hydrogen bonding between them and plant cell wall polysaccharides and proteins. This facilitates the release of flavonoids from the plant tissue into the extraction system. Simultaneously, this mild acidic condition prevents significant hydrolysis or degradation of flavonoids, thus promoting the release of active ingredients while inhibiting the dissolution of some high-molecular-weight impurities, resulting in a relatively higher proportion of small-molecule flavonoids in the extract. Furthermore, the ultrafiltration separation in step S6 uses an ultrafiltration membrane with a molecular weight cutoff of 900-1200 Da, and the permeate is collected as nanofiltration feed.

[0035] When using an ultrafiltration membrane with a molecular weight cutoff of 900–1200 Da for separation, large molecular components such as polysaccharides, proteins, and some polymeric phenols in the extraction system can be effectively retained, while smaller molecular weight flavonoids such as linalool C, morin, cyclomorin, and cyclomorin can pass through the membrane smoothly into the subsequent separation process. This process can significantly reduce the proportion of large molecular weight impurities in the extract, thereby reducing the adsorption or embedding effect of these large molecules on the small flavonoid structure in the solution system, making the dispersion state of the small molecule active structure in the system more stable.

[0036] Furthermore, the nanofiltration separation in step S6 employs a nanofiltration membrane with a molecular weight cutoff of 150–300 Da, and controls the membrane flux to be 8–12 L·m⁻¹. -2 ·h -1 .

[0037] Using a nanofiltration membrane with a molecular weight cutoff of 350–450 Da in the membrane separation process, and controlling the membrane flux within the range of 8–12 L·m⁻²·h⁻¹, allows lower molecular weight impurities to preferentially permeate through the membrane and be removed. Simultaneously, it achieves high retention efficiency for flavonoids with molecular weights in the 300–800 Da range, allowing these medium-molecular-weight flavonoids to gradually accumulate in the retentate. By controlling the membrane flux, the concentration polarization phenomenon in the nanofiltration process can be kept relatively stable, thereby improving the selectivity of membrane separation and resulting in a more stable distribution of flavonoids with different structural types within the molecular weight range.

[0038] A third aspect of the present invention provides the use of the above-mentioned mulberry root bark extract in the preparation of compositions for soothing the skin and promoting skin barrier repair.

[0039] The present invention, by adopting the above technical solution, has the following beneficial effects: (1) This invention limits the total content of characteristic components and allows one or more of mulberry senna G, morin M, morin C, and mulberry senna H to exist as structurally similar supplementary components, thereby enabling the main active structures in the extract to form a stable combination system. Among them, mulberry senna C, as the core polyphenol structural unit, mainly participates in free radical scavenging and signal regulation processes; morin and its structurally similar isopentenyl flavonoid components mainly participate in the termination of lipid free radical chain reactions; cyclomorin and cyclomorin, which are cyclic flavonoid structural units, participate in the inhibition of free radical generation and the regulation of lipid oxidation reactions through metal ion complexation and lipid free radical stabilization, respectively. By limiting the four core structural components to a specific ratio range and allowing structurally similar supplementary components to participate in the system without disrupting the core ratio relationship, the active structures corresponding to different reaction pathways can be kept in a relatively balanced state, thereby forming a continuous regulatory effect in multiple reaction stages such as free radical generation, propagation, and termination, making the overall structure of the extract more coordinated and exhibiting more stable biological activity.

[0040] (2) This invention constructs a stability index S to couple and limit the content of mulberry senna C, the total content of isopentenyl flavonoids, the content relationship between cyclomocarpine and cyclomocarpine, as well as the molecular weight distribution, the proportion of the main chromatographic peaks of the eight characteristic components, and the content of lipid-soluble resin impurities, so that the chemical composition of the extract forms a unified constraint relationship from multiple dimensions. This function not only reflects the proportional relationship between the core polyphenol structural unit and the isopentenyl flavonoid structural unit and the cyclized flavonoid structural unit, but also, by introducing the proportion of components in the 300-800 Da molecular weight range and the proportion of components with a molecular weight greater than 1000 Da, it synergistically regulates the molecular weight structure of the extract, thereby reducing the adsorption or embedding effect of high molecular weight impurities on small molecule flavonoid active ingredients, so that the isopentenyl flavonoid structural units and cyclized flavonoid structural units in the system maintain a relatively stable spatial distribution state, and improve their effective exposure in the system.

[0041] (3) This invention further limits the relationship between the synergistic effect strength among the core polyphenol structural units, isopentenyl flavonoid structural units, and cyclized flavonoid structural units and the degree of interference from impurities in the system by setting a synergistic index T. This allows the active structural network in the extract to maintain a high level of synergistic effect even in the presence of small amounts of polymeric components and lipid-soluble resin impurities. By functionalizing the quantitative relationship between linalool C and isopentenyl flavonoid components and constraining the proportion of cyclized flavonoid structures, while introducing molecular weight distribution and impurity content parameters, the polyphenol structural network in the system is made to dominate overall, thereby reducing the destructive effect of impurity background on the active structural network. This makes it easier for the extract to maintain a stable chemical composition and better bioactivity output under different preparation conditions.

[0042] (4) This invention simultaneously controls the content of components with a molecular weight greater than 1000 Da and the peak area ratio of the eight characteristic components in high performance liquid chromatography, thereby creating a dual constraint on the molecular weight structure and chemical composition structure of the extract. On the one hand, it reduces the non-specific binding effect of high molecular weight substances such as polysaccharides, proteins and polymeric phenols on small molecule active ingredients. On the other hand, it ensures that the main chemical composition of the system is dominated by characteristic structural units such as linalool C, isopentenyl flavonoids, cyclomolarin and cyclomolarin, thereby forming a more stable intermolecular interaction relationship among various active structures in the system and improving the stability and consistency of the overall activity performance of the extract.

[0043] (5) This invention utilizes a series of steps during the extraction process, including acid activation treatment, ultrasonic reflux extraction, supercritical carbon dioxide selective impurity removal, polar window chromatography, and membrane separation to regulate molecular weight distribution, to hierarchically regulate the chemical composition of the mulberry bark extraction system. Acid activation treatment reduces the hydrogen bond strength between polyphenolic structures and plant cell wall polysaccharides and proteins, thereby promoting the release of flavonoids from plant tissues. Ultrasonic reflux extraction further promotes the entry of isopentenyl flavonoids into the extraction system under relatively mild conditions. Supercritical carbon dioxide treatment preferentially removes lipid-soluble resin impurities and reduces hydrophobic aggregation. Polar window chromatography allows mulberry flavonoid C, morin, cyclomorin, cyclomorin, and some structurally similar isopentenyl flavonoids to be simultaneously enriched within similar polarity ranges. Ultrafiltration and nanofiltration membrane separation remove polysaccharides, proteins, and low-molecular-weight impurities through molecular weight screening, gradually concentrating the main components within the 300–800 Da range. Through the above-mentioned multi-level structural screening and separation process, the flavonoids of different structural types in the extract form a relatively stable combination relationship, and reduce the interference of high molecular weight components and hydrophobic impurities on the small molecule active structure. This makes the chemical composition of the obtained extract more stable, and at the same time improves the consistency of its activity and the reproducibility of the process. Detailed Implementation

[0044] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0045] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0046] The technical solutions 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. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

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

[0048] The present invention will now be described in detail with reference to specific embodiments, which are intended to understand rather than limit the invention.

[0049] In this embodiment, the mulberry bark raw material used is the dried root bark of *Morus alba* L., a plant of the Moraceae family. The raw material is harvested from the roots of 3-5 year old mulberry trees. After harvesting in autumn, the root bark is peeled off and naturally dried to obtain the medicinal material. The mulberry bark raw material is the dried root bark of *Morus alba* L. from the Dabashan Mountains in Wanyuan City, Dazhou City, Sichuan Province. Wanyuan City is located in the heart of the southern foothills of the Dabashan Mountains in northeastern Sichuan Province, belonging to the northern subtropical humid monsoon climate zone. The terrain is mostly low and medium mountains with high altitude, large diurnal temperature range, abundant rainfall, and good sunshine conditions, resulting in an excellent ecological environment. The soil in this area is influenced by the geological structure of the Dabashan Mountains, rich in various minerals and trace elements, and has good water conservation and biodiversity conditions. These ecological conditions are conducive to the growth of mulberry trees and the accumulation of secondary metabolites, resulting in a relatively rich content of flavonoids, polyphenols, and related active ingredients in the mulberry root bark. After harvesting, the mulberry root bark is washed and dried before being used as a medicinal raw material. According to the quality standards of mulberry root bark in Part I of the 2020 edition of the Chinese Pharmacopoeia, its morphological characteristics and thin-layer chromatography were identified to confirm that its source is the dried root bark of the mulberry (Morus alba L.) plant of the Moraceae family.

[0050] Before use, the mulberry bark raw material is first removed of impurities and dried in a 50°C hot air drying oven to a moisture content of 7.0%. Then, it is pulverized by a pulverizer and passed through a 40-mesh sieve to obtain mulberry bark powder with a particle size of about 0.4 mm, which is used for subsequent extraction.

[0051] Example 1 This embodiment discloses a mulberry root bark extract, which is prepared through the following steps: S1: Weigh 10.000 kg of mulberry bark powder, add 40 L of 0.10% citric acid buffer solution to make the pH of the system 5.0, and soak at room temperature for 30 min to reduce the binding strength between polyphenolic compounds and plant cell walls and promote the release of flavonoid structures from plant tissues.

[0052] S2: Add the material obtained in step S1 to the extraction tank, add 100 L of 70% ethanol solution, and perform ultrasonic-assisted reflux extraction at 65℃, wherein the ultrasonic frequency is 25 kHz, the ultrasonic power is 800 W, the reflux is repeated five times, and the extraction time is 90 min; after the extraction is completed, filter to obtain the first extract. S3: The extract obtained in step S2 was concentrated to approximately 12 L under reduced pressure at 55°C and a vacuum of −0.085 MPa, then lyophilized. Pure water was gradually added to adjust the ethanol volume fraction to 50%, and the mixture was allowed to stand at 45°C for 60 min to allow some resin impurities and high-molecular-weight polyphenols to precipitate. After standing, the precipitate was removed by centrifugation at 4000 rpm for 10 min, and the supernatant was lyophilized as a pretreated extract. This provides a lower resin background and a more controllable molecular weight distribution for subsequent supercritical fluid chromatography and narrow-window chromatography.

[0053] S4: Place 2,000 kg of the pretreated extract obtained in step S3 into a supercritical carbon dioxide extraction device and process it under the conditions of 30 MPa pressure, 45 °C temperature, carbon dioxide flow rate of 18 kg / h, and continuous extraction for 120 min to remove lipid-soluble resin impurities and waxy substances in the system.

[0054] S5: Dissolve the material obtained in step S4 in a 40% (v / v) ethanol solution, adjust the pH of the solution to 5.0 ± 0.2, and then load the sample onto a D101 macroporous adsorption resin column for separation. The column diameter is 20 cm, the column height is 100 cm, and the resin packing volume is 30 L. During elution, gradient elution is performed sequentially using 50%, 60%, 70%, and 80% (v / v) ethanol solutions, and the elution process is monitored by a 280 nm UV detection signal. At the same time, the eluent is collected in 1.0 L segments and rapid HPLC spotting is performed to confirm the co-elution range of the four characteristic component peaks. Only the elution segment corresponding to 64%–66% (v / v) ethanol is collected as the target fraction.

[0055] S6: Adjust the solid content of the target fraction obtained in step S5 to 1.0%, first filter it through a 0.22 μm microfiltration membrane to remove suspended particles, and then introduce it into the ultrafiltration system. The ultrafiltration membrane used has a molecular weight cutoff of 1000 Da, and it is operated at 25°C and 0.50 MPa. The circulation concentration factor is controlled at 5.0 to enhance the removal of components >1000 Da. Subsequently, the ultrafiltration permeate is subjected to an equal-volume washing treatment, that is, an equal volume of 30% ethanol aqueous solution is added to the permeate side, and then it is rapidly passed through the membrane under the same ultrafiltration conditions to reduce the probability of large molecules being dragged into the permeate. Finally, the permeate is collected as nanofiltration feed. The nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 400 Da, and is operated at 25°C and 0.80 MPa, with the membrane flux controlled at 10 L·m -2 ·h -1 Simultaneously, the nanofiltration retentate is washed with an equal volume of water to allow small molecule impurities with a molecular weight below 300 Da to further permeate through the membrane and be removed. S7: The nanofiltration retentate obtained in step S6 was concentrated under reduced pressure at 60°C to make the solid content of the system reach 25%, and then freeze-dried. The pre-freezing temperature was −40°C, the vacuum degree was ≤20 Pa, and the drying time was 32 h. Finally, 0.82 kg of mulberry root bark extract powder was obtained, and this sample was recorded as E1.

[0056] Sample E1 was analyzed by high performance liquid chromatography. The results showed that the contents of mulberry senna C (m1) were 0.82%, mulberry senna (m2) were 12.66%, cyclomocarboxylic acid (m3) were 0.44%, and cyclomocarboxylic acid (m4) were 0.01%. The total contents of the four characteristic components were 13.93%, and the mass ratio was 1:15.51:0.538:0.011.

[0057] Further analysis under the same chromatographic conditions revealed that the sum of the peak areas of the four characteristic components accounted for the proportion of the total peak area, yielding A4 = 0.139. Molecular weight distribution was determined using gel permeation chromatography, resulting in w. 300-800 =0.65, w >1000 =0.02. The proportion of lipid-soluble resin impurities was determined by petroleum ether (boiling range 30–60 °C) extraction-gravimetric method, yielding w res =0.015.

[0058] Substituting the above test results into the following formula yields: =0.17; =1627.

[0059] Example 2 This embodiment discloses a mulberry root bark extract, the preparation method of which is basically the same as that of Example 1, except that the membrane separation conditions in step S6 have been adjusted.

[0060] S6: Adjust the solid content of the target fraction obtained in step S5 to 5.0%, first filter it through a 0.22 μm microfiltration membrane to remove suspended particles, and then pass it into an ultrafiltration system. The ultrafiltration membrane used has a molecular weight cutoff of 1000 Da, and is operated at 25°C and an operating pressure of 0.50 MPa, with the circulation concentration factor controlled at 5.0. Subsequently, the ultrafiltration permeate is washed with 1.5 times its volume, i.e., 1.5 times the volume of 30% ethanol aqueous solution is added to the permeate side before passing it through the membrane under the same ultrafiltration conditions. Then, it enters a nanofiltration system for separation. The nanofiltration membrane has a molecular weight cutoff of 400 Da, and is operated at 25°C with the membrane flux controlled at 9 L·m -2 ·h -1 .

[0061] The final extract sample was E2.

[0062] The test results are as follows: m1=0.68%; m2=10.27%; m3=0.38%; m4=0.02%; A4=0.1135; w 300-800 =0.72; w >1000 = 0.018; w res = 0.014; The calculations yielded: S = 0.145, T = 1566.

[0063] Example 3 This embodiment discloses a mulberry root bark extract, the preparation method of which is basically the same as that of Example 1, except that the supercritical carbon dioxide extraction conditions in step S4 have been adjusted.

[0064] S4: Place 2,000 kg of the pretreated extract obtained in step S3 into a supercritical carbon dioxide extraction device and process it under the conditions of 32 MPa pressure and 45℃ temperature, with a carbon dioxide flow rate of 18 kg / h. Add ethanol as an entrainer with an entrainer flow rate of 1.2 kg / h and extract continuously for 120 min.

[0065] The final extract sample was E3.

[0066] Example 4 This embodiment discloses a mulberry root bark extract, the preparation method of which is basically the same as that of Example 1, except that the chromatography collection window in step S5 has been adjusted.

[0067] S5: During gradient elution, only the elution fraction with an ethanol volume fraction of 63% to 66% is collected as the target fraction.

[0068] The final extract sample was E4.

[0069] Comparative Example 1 The preparation method of this comparative example is basically the same as that of Example 1, except that citrate buffer was not added in step S1.

[0070] S1: Weigh 10.000 kg of mulberry bark powder, add 40 L of pure water and soak for 30 min without adjusting the pH.

[0071] Sample C1 was obtained.

[0072] Comparative Example 2 The preparation method of this comparative example is basically the same as that of Example 1, except that the chromatography window collected in step S5 is enlarged.

[0073] S5: Collect the elution fraction with an ethanol volume fraction of 60% to 70% as the target fraction.

[0074] Sample C2 was obtained.

[0075] Comparative Example 3 The preparation method of this comparative example is basically the same as that of Example 1, except that step S4 is omitted.

[0076] That is, the pretreated extract obtained in step S3 is directly fed into step S5 for chromatography separation.

[0077] Sample C3 was obtained.

[0078] Comparative Example 4 The preparation method of this comparative example is basically the same as that of Example 1, except that the molecular weight cutoff of the ultrafiltration membrane in step S6 has been adjusted.

[0079] S6: Separation is performed using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da.

[0080] Sample C4 was obtained.

[0081] Comparative Example 5 The preparation method of this comparative example is basically the same as that of Example 1, except that the molecular weight cutoff of the nanofiltration membrane in step S6 has been adjusted.

[0082] S6: Separation is performed using a nanofiltration membrane with a molecular weight cutoff of 100 Da.

[0083] Sample C5 was obtained.

[0084] Table 1 The following tests were performed on the above samples: Self-stability test: Alternating between 4℃, 48℃, room temperature light exposure, and -18~48℃, the degree of decrease in total active ingredient A4 (mass percentage) and the precipitation time were observed over one month; the time at which precipitation occurred under any test condition is the precipitation time statistically shown in the table. Solubility test: The sample powder was dissolved in a 10% butanediol-water system at room temperature to a concentration of 100 ppm. A √ indicates clear dissolution, and an × indicates turbidity. A small standard deviation indicates that the data points are more concentrated and have less fluctuation, suggesting high consistency between batches; a large standard deviation indicates that the data is dispersed and has greater fluctuation, suggesting significant differences between individuals or batches. Table 2 Conclusions: ① The 30-day A4 data shows a decrease rate compared to the 0-day data, with the implementation series significantly outperforming the comparative example. ② Longer precipitation times lead to greater stability, with the implementation series showing significantly better performance than the comparative example. ③ The standard deviation of the repeated experimental examples is much smaller than that of the comparative example, indicating good process repeatability.

[0085] Performance testing I. Detection of Keratinocyte Inflammation Suppression and Skin Barrier-Related Gene Expression The regulatory effects of samples on inflammation-related genes and skin barrier-related genes were detected using an in vitro cultured human keratinocyte model. Human keratinocytes were cultured, and an inflammation model was established after cell growth stabilized by adding inducing stimuli. A basal control group (BC group), a model control group (NC group), a positive control group, and a sample treatment group were set up. Positive control PC1 used dexamethasone to verify the effectiveness of the detection system for the inflammation-inhibiting marker IFNγ; positive control PC2 used WY14643 to verify the effectiveness of the detection system for skin barrier-related genes LOR and FLG. The sample group consisted of mulberry root bark extract prepared in Example 1, and was added to the cell culture system at concentrations of 0.001953% and 0.003906%, respectively.

[0086] Cell samples were collected and total RNA was extracted after a certain period of drug treatment. cDNA was obtained through reverse transcription, and the target gene was detected using real-time quantitative PCR. -ΔΔCT The method calculated changes in gene expression levels and performed statistical analysis using the t-test method. Significance compared to the BC group was indicated by "#", and significance compared to the NC group was indicated by "#". The value “" indicates that a p-value < 0.05 is considered statistically significant, and a p-value < 0.01 is considered highly statistically significant. The test results are shown in Table 3 below.

[0087] Table 3. Results of IFNγ expression detection in keratinocytes The results showed that, compared with the BC group, the IFNγ gene expression level in the NC group was significantly increased, indicating that the inflammatory stimulation model was successfully established. Compared with the NC group, the IFNγ expression level in the PC1 group was significantly decreased, indicating that the detection system was reliable. The sample groups at concentrations of 0.001953% and 0.003906% significantly reduced IFNγ gene expression, with downregulation rates of 15.42% and 39.30%, respectively, indicating that the mulberry root bark extract has a significant inhibitory effect on the inflammatory response.

[0088] The expression of skin barrier-related genes LOR and FLG was further detected. The results are shown in Tables 4 and 5 below.

[0089] Table 4. Results of LOR expression detection in keratinocytes Table 5. Results of FLG expression detection in keratinocytes The results showed that, compared with the BC group, the expression level of LOR gene was significantly reduced in the NC group, indicating that the stimulation conditions damaged the keratinocyte barrier function. Compared with the NC group, the expression level of LOR in the PC2 positive control group was significantly increased, indicating that the detection system was effective. The sample groups at concentrations of 0.001953% and 0.003906% significantly upregulated the expression level of LOR gene, with increases of 27.50% and 35.00%, respectively.

[0090] In FLG gene detection, compared with the BC group, the FLG expression level in the NC group was significantly decreased, while the PC2 group showed a significant upregulation of FLG expression. Both sample groups at concentrations of 0.001953% and 0.003906% exhibited a promoting effect on FLG gene expression, with upregulation rates of 10.71% and 39.29%, respectively.

[0091] The above results indicate that the mulberry root bark extract prepared in this application can not only significantly inhibit the expression of the inflammation-related factor IFNγ, but also significantly increase the expression levels of skin barrier-related proteins LOR and FLG, indicating that the extract can promote skin barrier repair while inhibiting the inflammatory response.

[0092] II. Macrophage Inflammatory Regulation and Detection of Immune-Related Signals The regulatory effect of samples on the expression of inflammation-related factors was evaluated using an in vitro cultured macrophage model. The experiment included a basal control group (BC group), an inflammation model group (NC group), a positive control group (PC group), and sample treatment groups. The sample groups used mulberry root bark extract prepared in Example 1, and were added to the cell line at concentrations of 0.04% and 0.125%, respectively. After stimulating the cells to establish an inflammation model, sample treatment was performed. After treatment, cellular RNA was extracted and reverse transcribed. The expression levels of various inflammation and immune-related genes were detected by real-time quantitative PCR. -ΔΔCT The results were calculated using the method described above, and statistical analysis was performed using the t-test method. The test results are shown in Table 6-7.

[0093] Table 6 Results of TNF-α expression detection in macrophages In the detection of TNF-α, the expression level of TNF-α in the NC group was significantly increased compared with that in the BC group; the positive control group effectively reduced TNF-α expression. The sample groups showed significant inhibitory effects at concentrations of 0.04% and 0.125%, with downregulation rates of 20.77% and 52.76%, respectively.

[0094] Table 7 Results of IL-6 expression detection in macrophages In the IL-6 assay, compared with the NC group, the sample group reduced IL-6 expression by 66.57% and 81.83% at concentrations of 0.04% and 0.125%, respectively, indicating that the extract can effectively reduce the expression levels of key cytokines in the process of inflammatory signal amplification.

[0095] Secondly, for the samples prepared by comparative examples 1-5, some test results are shown in Tables 8 and 9. It can be seen that the inflammatory suppression ability and skin barrier regulation ability in the same cell model are significantly reduced, that is, the inhibitory effect on inflammatory factors such as IFNγ, TNF-α, and IL-6 is weakened, and the promoting effect on the expression of skin barrier-related genes such as LOR and FLG is also significantly reduced.

[0096] Table 8 Table 9 III. Heavy Metal Resistance Testing (MTT Method) Human immortalized keratinocytes (HaCaT) were used as model cells, and the effect of samples on cell viability under heavy metal stress was evaluated using the MTT assay. Cells were seeded in 96-well plates and cultured overnight at 37°C and 5% CO2. After cell attachment, drug treatment was administered. The experiment included a blank control group, a heavy metal treatment group, and a sample treatment group. Heavy metal treatment used mercuric chloride (Hg) solution at a concentration of 34 μM, and the sample concentration was 0.625%.

[0097] After incubating the cells for 24 h, MTT reagent was added, and after incubation for another 4 h, the supernatant was discarded. DMSO was added to dissolve the formazan crystals generated, and the absorbance was measured at 492 nm. The relative cell viability was then calculated.

[0098] The test results are shown in Table 10 below: Table 10 The results showed that under the same heavy metal stress conditions, the sample of Example 1 significantly improved cell viability, and its protective effect was significantly better than that of Comparative Example 3. Based on the corresponding parameters, Example 1 simultaneously met the defined ranges for S and T, while Comparative Example 3, due to the lack of supercritical removal of lipid-soluble resin impurities, resulted in lower w... res The significant increase reduces the system's ability to protect cells.

[0099] IV. Indirect Free Radical Scavenging Ability Detection (SOD Method) The activation capacity of the samples for the endogenous antioxidant system was evaluated using an in vitro superoxide dismutase (SOD) activity assay. The samples were added to the reaction system at a mass fraction of 0.5%, and the reaction was carried out at 37 °C for 20 min. The absorbance was measured at 450 nm, and the SOD inhibition rate and SOD activity were calculated.

[0100] The test results are shown in Table 11 below: Table 11 The results showed that the SOD activity of Example 1 was significantly higher than that of Comparative Example 3, indicating that it could more effectively promote the response of the endogenous antioxidant enzyme system. This difference mainly stemmed from the fact that supercritical extraction in the examples reduced the content of lipid-soluble resin impurities, thereby enhancing the availability and synergistic effect of the active ingredients.

[0101] V. Detection of Direct Free Radical Scavenging Ability (DPPH Method) The direct antioxidant capacity of the samples was evaluated using a DPPH free radical scavenging assay. The samples were diluted at different concentration gradients (0.07%–0.17%), mixed with DPPH solution, and reacted in the dark for 30 min. The absorbance was measured at 517 nm, and the free radical scavenging rate and IC50 value were calculated.

[0102] The test results are shown in Table 12 below: Table 12 The results showed that Example 1 exhibited significantly better DPPH free radical scavenging ability than Comparative Example 3, with a lower IC50 and a higher maximum scavenging rate, indicating that the extract possesses strong free radical scavenging capabilities. These results demonstrate that by regulating the composition and distribution of the polyphenolic active structures in the extract, its antioxidant activity can be enhanced, thereby inhibiting free radical accumulation under oxidative stress, helping to maintain skin microenvironment stability and reduce oxidative stimulation.

[0103] VI. Verification of the Influence of Molecular Weight Distribution Control on Activity Example 2 and Comparative Example 4 were selected for comparison, with the main difference between the two being the different molecular weight cutoff of the ultrafiltration membrane.

[0104] The test results are shown in Table 13 below: Table 13 The results showed that when the molecular weight cutoff of ultrafiltration increased, macromolecular components entered the system, which significantly increased w>1000, resulting in a significant decrease in T value. At the same time, the activity index decreased synchronously, indicating that molecular weight distribution control plays an important role in maintaining the activity of the system.

[0105] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A mulberry root bark extract, characterized in that, The extract contains 3 to 8 of the following eight characteristic components: morin citrin C, morinol, cyclomorin, cyclomorinone, morin citrin G, morinol M, morin root ketone C, and morin citrin H, and meets the following conditions: The total content of the characteristic components accounts for 5% to 30% of the total weight of the extract; The mass ratio of mulberry ketone C: morin: cyclomorin: cyclomorin is 1:4-25:0.2-0.6:0.01-0.

08.

2. The mulberry root bark extract according to claim 1, characterized in that, The extract satisfies the following function: ,and 0.5; Where m1 represents the content of mulberry senna C; m2 represents the total content of isopentenyl flavonoids, which include one or more of morinol, morinol M, morinone C, morinone D, and morinone G. m3 represents the content of cyclomolarin; m4 represents the content of cyclomoronene; w 300-800 The proportion of components with molecular weights in the range of 300–800 Da to the total weight of the extract; w >1000 The proportion of components with a molecular weight greater than 1000 Da to the total weight of the extract; w res The percentage of fat-soluble resin impurities in the total weight of the extract; A4 represents the ratio of the sum of the peak areas of the four characteristic components (Morus alba ketone C, Morus alba salicum, cyclomorusin, and cyclomorusin) to the total peak area, or the ratio of the sum of the contents of these four characteristic components to the total content of substances, when detected by high performance liquid chromatography.

3. The mulberry root bark extract according to claim 2, characterized in that, The extract satisfies the following function: And 1000≤T≤2000.

4. The mulberry root bark extract according to claim 1, characterized in that, The content of components with a molecular weight greater than 1000 Da in the extract is less than 5%.

5. The mulberry root bark extract according to claim 1, characterized in that, When the extract is detected by high performance liquid chromatography, the sum of the peak areas of the four characteristic components—morrhagic senna C, morin, cyclomorrhagic senna, and cyclomorrhagic senna—accounts for no less than 8% of the total peak area.

6. A method for preparing the mulberry root bark extract according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1: After crushing the mulberry bark raw material, it is soaked in an acidic buffer solution to make the pH of the system 4.5-5.5; S2: The mulberry bark treated in step S1 was subjected to ultrasonic reflux extraction in a 65%–75% (v / v) ethanol solution to obtain an extract. S3: The extract is concentrated under reduced pressure, and some resin impurities and high molecular weight components are separated by adjusting the solvent polarity to obtain a pretreated extract. S4: The pretreated extract is subjected to supercritical carbon dioxide treatment to remove lipid-soluble resin impurities; S5: The material treated in step S4 is reconstituted and separated by chromatography using a macroporous adsorption resin column. Only the elution fraction with an ethanol volume fraction of 63% to 68% is collected as the target fraction. S6: The target fraction is sequentially separated by microfiltration, ultrafiltration and nanofiltration, wherein ultrafiltration removes components with a molecular weight greater than 1000 Da, and nanofiltration enriches components with a molecular weight range of 300 to 800 Da. S7: The retentate obtained by nanofiltration is concentrated and dried to obtain the mulberry root bark extract.

7. The method for preparing mulberry root bark extract according to claim 6, characterized in that, The acidic buffer solution mentioned in step S1 is a citrate buffer solution with a mass fraction of 0.05% to 0.20%.

8. The method for preparing mulberry root bark extract according to claim 6, characterized in that, The ultrafiltration separation in step S6 uses an ultrafiltration membrane with a molecular weight cutoff of 900-1200 Da, and the permeate is collected as nanofiltration feed.

9. The method for preparing mulberry root bark extract according to claim 6, characterized in that, The nanofiltration separation in step S6 uses a nanofiltration membrane with a molecular weight cutoff of 350–450 Da, and controls the membrane flux to be 8–12 L·m⁻¹. -2 ·h -1 .

10. The use of the mulberry root bark extract according to any one of claims 1 to 5 in the preparation of compositions for soothing the skin and promoting skin barrier repair.

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