LU509043B1 - Molecular machine and manufacturing process for a Dendrobium officinale polysaccharide extract and its application as an antioxidant and anti-aging agent - Google Patents

Abstract

The present application relates to the technical field of Dendrobium officinale polysaccharide extract and provides a molecular machine and a production method for a Dendrobium officinale polysaccharide extract and its antioxidant and anti-aging applications, wherein the lactic acid-based molecular machine comprises a first functional motif and a second functional motif; and the first functional motif and the second functional motif are connected by hydrogen bonding; the first functional motif is a lactic acid molecule and the second functional motif is at least one of a betaine molecule, a bitartin molecule, or a levulinic acid molecule; wherein the Dendrobium officinale polysaccharide comprises O-acetyl-β-D-glucopyranoside, and the backbone of the O-acetyl-β-D-glucopyranoside consists of D-mannose and D-glucose connected by a β-1,4-glycosidic bond; The lactic acid-based molecular machine is used to drive the recognition binding of the lactic acid-based molecular machine to the Dendrobium officinale polysaccharides, including driving the recognition binding of the lactic acid-based molecular machine to the β-1,4-glycosidic bonds of the Dendrobium officinale polysaccharides through the affinity of hydrogen bonding, which improves the extraction efficiency of the Dendrobium officinale polysaccharides.

Description

Molecular machine and manufacturing process for a Dendrobium officinale LUS09043

Polysaccharide extract and its use as an antioxidant and anti-aging agent

Technical area

The present invention relates to the technical field of Dendrobium officinale

Polysaccharide extract and in particular to a molecular machine and a

Manufacturing process for a Dendrobium officinale polysaccharide extract and its

Used as an antioxidant and anti-aging agent.

Technology in the background

Dendrobium officinale, a herbaceous medicinal plant of the genus Dendrobium in the family Orchidaceae, is mainly found in Anhui, Sichuan, Zhejiang, Yunnan, and Jiangxi in China. Modern research has shown that Dendrobium is rich in polysaccharides,

flavonoids and polyphenols, which have excellent antioxidant, anti-inflammatory, anti-aging and antiviral effects. Therefore, it is of great importance to

To research extraction solvents and methods for the main active ingredient polysaccharide.

Currently, the main extraction solvents for Dendrobium officinale are

Polysaccharides, water, and organic solvents. The water extraction method has low extraction efficiency, impurities, and other problems that affect organic

Solvents used in the organic solvent extraction method are toxic and environmentally harmful, flammable and explosive, subsequent separation is difficult, detection costs are too high, and traditional alcohol solvents for

Polysaccharides have low solubility, used for the extraction of polysaccharides have low efficiency.

The state of the art suffers from the problem of low cost efficiency of

Extraction of polysaccharides from Dendrobium officinale.

Content of the invention

The present application provides a molecular machine and a manufacturing process for a Dendrobium officinale polysaccharide extract and its antioxidant and anti-aging

Applications ready to solve the problem of high efficiency and low cost of

Dendrobium officinale polysaccharide extract to dissolve.

In a first aspect, embodiments of the present application provide a lactic acid-based molecular machine comprising a first functional base element and a second functional base element;

The first functional base element is connected to the second functional base element by

connected by hydrogen bonds;

The first functional base element is a lactic acid molecule, and the second functional

Base element is at least one of a betaine molecule, a picric acid molecule or a levulinic acid molecule;

Wherein the Dendrobium officinale polysaccharide comprises O-acetyl-B-D-glucan, wherein the

Main chain of O-acetyl-B-D-glucan comprises D-mannose and D-glucose linked by a B-1,4-glycosidic bond;

The lactic acid-based molecular machinery for driving the lactic acid-based molecular machinery to form a recognition binding to a Dendrobium officinale

Polysaccharide by the affinity of hydrogen bonding, comprising driving the lactic acid-based molecular machinery to form a recognition bond with the 3-1,4-glycosidic bond of the Dendrobium officinale polysaccharide by the affinity of

hydrogen bond; LUS09043 where, when the second functional group element is the betaine molecule, the molecular

Machine has the molecular structural formula CH3CH(OH)COO-H-OOCCH2N(CH3)3.

In a second aspect, embodiments of the present application provide

A method for producing a lactic acid-based molecular machine as described in the first aspect, comprising the following steps: mixing at least one of the

Elements betaine, picloram or levulin with the lactic acid to obtain a mixture.

Heat treatment of the mixture until the viscosity of the mixture is 200 mPas~500 mPaes to obtain a lactic acid-based molecular machine; the heat treatment is carried out at a temperature of 30 °C to 60 °C.

In a third aspect, embodiments of the present application provide

A process for the preparation of a Dendrobium officinale polysaccharide extract is provided, comprising the following steps:

Step S100, mixing Dendrobium powder with a solution of a molecular

Lactic acid-based machine followed by ultrasonic enhancement treatment to obtain the Dendrobium extract;

Step S200, centrifuging the Dendrobium extract, taking the upper layer of the clear liquid after centrifugation and filtering it to obtain the Dendrobium officinale

to obtain polysaccharide extract;

Where the solute in the lactic acid-based molecular machine solution is a

A lactic acid-based molecular machine as described in the content of the first aspect and/or a lactic acid-based molecular machine manufactured by the manufacturing method as described in the content of the second aspect.

In a fourth aspect, embodiments of the present application provide an antioxidant and anti-aging application based on a Dendrobium officinale

Polysaccharide extract obtained by a manufacturing process as described in any of the third

aspects described, and which comprises a skin care product made from the

Dendrobium officinale polysaccharide extract was prepared as raw material used for antioxidant and anti-aging repair of human skin.

Compared with the state of the art, the advantageous effect of the present

Application:

In a first aspect of the present application, a molecular machine is

Lactic acid base comprising a first functional unit and a second functional

unit; the first functional unit and the second functional unit are separated by

hydrogen bond; the first functional unit is a lactic acid molecule, and the second functional unit is at least one of a betaine molecule, a

Absinthin molecule or a levulinic acid molecule; The Dendrobium officinale polysaccharide comprises an O-acetyl-B-D-glucan, the main chain of the O-acetyl-B-D-glucan being D-

Mannose and D-glucose, which are connected by a B-1,4-glycosidic bond;

Lactic acid-based molecular machinery to drive a recognizable binding of the

Lactic acid-based molecular machinery to Dendrobium officinale polysaccharides by hydrogen bond affinity, comprising driving a detectable binding of the lactic acid-based molecular machinery to the B-1,4-glycosidic bonds of the

Dendrobium officinale polysaccharides through the affinity of hydrogen bonds. The present application forms a lactic acid-based molecular machinery through

Hydrogen bonds of intermolecular interactions between einek509043

Lactic acid molecule and at least one betaine molecule, one bitartin molecule or one

Levulinic acid molecule The lactic acid-based molecular machine of the present

Application when extracting Dendrobium officinale polysaccharides, drives the lactic acid-based molecular machine to form a recognition compound with Dendrobium officinale

polysaccharides by the affinity of hydrogen bonds, comprising

Driving the lactic acid-based molecular machine to form a recognition compound with the P-1,4-glycosidic bonds of the Dendrobium officinale polysaccharides by the

affinity of hydrogen bonds. It improves the transfer rate of the

active ingredient in Dendrobium, and at the same time, it can maintain the activity of the active ingredient and improve the stability of the active ingredient within Dendrobium, and it can also dissolve and dissociate the cellulose in the cell wall of Dendrobium, which improves the permeability of the cell wall of Dendrobium, and the solubility of the active component within the cell of

Dendrobium improved, and thus the efficiency of the extraction of Dendrobium officinale

Polysaccharides improved.

The second aspect of the present application provides a method for producing a lactic acid-based molecular machine as in the content of the first aspect, comprising the steps of: mixing at least one of betaine, picloram, or levulin with lactic acid to obtain a mixture. The mixture is subjected to a heating treatment until the viscosity of the mixture is 200 mPas~500 mPas to

Lactic acid-based machine; wherein the temperature of the heating treatment is 30 °C to 60 °C; By synthesizing the lactic acid-based molecular machine with a predetermined viscosity by the heating and stirring method that controls the temperature and the duration of the heating treatment, the molecular machine of the present invention can be

Application at room temperature remain stable, clarified, transparent and homogeneous, and there is no

Separation in the subsequent process is required, which makes the manufacturing process of the

lactic acid-based molecular machine is simplified; In addition, the

Synthesis process of the lactic acid-based molecular machines of the present

100% atom-economical, green and economical, biocompatible, safe and non-toxic, and has a three-dimensional ordered structure that facilitates the effective inclusion of the active substances through intermolecular forces.

In a third aspect of the present application, a process for producing a Dendrobium officinale polysaccharide extract is provided, comprising the following steps: Step S100, mixing a Dendrobium officinale powder with a molecular

Lactic acid-based machine solution and subsequent implementation of a

Intensification treatment with ultrasound to obtain the Dendrobium officinale extract;

Step S200, centrifuging the Dendrobium officinale extract, taking out the upper layer of the clear liquid after the centrifugation treatment and filtering it, and obtaining the Dendrobium officinale polysaccharide extract; wherein the solute in the lactic acid-based molecular machine solution is the lactic acid-based molecular machine in the content of the first aspect, and/or the lactic acid-based molecular machine manufactured by the manufacturing method in the content of the second aspect; Compared with the prior art, since the lactic acid-based molecular machine is used to extract the Dendrobium officinale polysaccharide without using an enzyme for the

To extract catalysis, the lactic acid-based molecular machine drives the

Lactic acid-based molecular machine to form a recognition compound with deh}509043

Dendrobium officinale polysaccharide through the affinity of hydrogen bonding, which improves the rate of transfer of the active ingredient in Dendrobium officinale.

Extraction effect of the polysaccharides in the Dendrobium officinale extract of the present

Application is 2.35 times more effective than that of aqueous extraction and 1.86 times more effective than that of enzymatic cellulose digestion; In the lactic acid-based molecular machine

System, the polysaccharide compounds can exist stably for 4 months without decomposition, the stability is better Lactic acid-based molecular machine can effectively inhibit the

Growth of microorganisms and prevent the polysaccharide extracts from

Deterioration, the polysaccharide extracts are stored in the room temperature and natural light for 4 months, the microbial content of individual microorganisms does not exceed the norm;

At the same time, the coupled ultrasound amplification can facilitate the dissolution and dissociation of the

Cellulose in the cell wall of Dendrobium officinale can be accelerated, effectively increasing the content of polysaccharides but also reducing the amount of solvents,

Impurities are reduced, the transfer rate of the active ingredient is improved, the

Solving the problem of using organic solvents and residual problems to achieve high-quality and stable production and improve the utilization rate of the

active ingredient and to improve value creation.

A fourth aspect of the present application is an antioxidant and anti-aging

Application of a Dendrobium officinale polysaccharide extract based on a

Preparation method according to any one of the contents of the third aspect, which comprises a skin care product prepared from the Dendrobium officinale polysaccharide extract as a raw material for the antioxidant and anti-aging repair of human skin. Since the extracted Dendrobium officinale polysaccharide extract does not need to be separated, the lactic acid-based molecular machinery in the polysaccharide extract synergizes with the polysaccharide compounds applied to the skin care products to

To exert effects of antioxidant, anti-aging, exfoliating and moisturizing on human skin.

Description of the attached drawings

In order to more clearly illustrate the technical solutions in the embodiments or the prior art of the present invention, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it will be obvious that the accompanying drawings in the following description are some of the embodiments of the present invention, and that other accompanying

Drawings can be obtained based on these drawings without doing any creative work.

Figure 1 is a flow diagram of a process for producing a Dendrobium officinale

polysaccharide extract provided in the present application;

Figure 2 is an infrared spectrogram of the molecular machinery and the synthetic

Monomers of the lactobetaine of the present application;

Figure 3 is a schematic diagram of the thermogravimetric curve of the lactobetaine

molecular machine and the synthetic monomer of the present application;

Figure 4 is a diagram of the quantum chemical calculation analysis of the lactobetaine

Molecular machine A1 of the present application, wherein (a) is a diagram of the

Structural optimization of the lactobetaine molecular machine; (b) is a diagram of the electrostatic surface potential analysis of the lactobetaine molecular machine; and (c) is a

Diagram of the analysis of the indicator function of the interaction area of the lactobetaine

Molecular machine; 5 Figure 5 shows a schematic diagram of the change in the polysaccharide content of the

Polysaccharide extract of Dendrobium officinale of the present application at different placement times under natural light at room temperature;

Figure 6 shows a schematic diagram of the DPPH radical scavenging assay of the Dendrobium officinale polysaccharide extract (lactobetaine molecular machine) of the present application;

Figure 7 shows a schematic diagram of the hydroxyl radical scavenging test of the

Polysaccharide extract of Dendrobium officinale (lactobetaine molecular machine) of the present application;

Figure 8 shows a schematic diagram of the effect of Dendrobium officinale

Polysaccharide extract (lactobetaine molecular machine) of the present application on the

Activity of fibroblasts in the human skin layer;

Figure 9 shows a schematic diagram of the effect of the polysaccharide extract from

Dendrobium officinale (lactobetaine molecular machine) of the present application to the

Human type I collagen content;

Figure 10 shows a schematic diagram of the effect of the polysaccharide extract of

Dendrobium officinale (lactobetaine molecular machine) of the present application on the relative content of MMP-1;

Figure 11 shows a schematic diagram of the effect of the polysaccharide extract of

Dendrobium officinale (lactobetaine molecular machine) of the present application on the relative content of MMP-3.

Detailed description

To make the technical problems, technical solutions, and advantageous effects to be solved by the present application clearer and more understandable, the present application is described in more detail below in combination with exemplary embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the present application.

In the present application, the term “and/or” means a

Association relationship between the associated objects describes that three types of

Relationships can exist, e.g., A and/or B, which can mean the presence of A alone, the presence of both A and B, and the presence of B alone. A and B can be singular or plural. The symbol "/" generally indicates that the objects connected before and after are in an "or" relationship.

In the present application, "at least one" means one or more, and "more than one" means two or more. The expression "at least one of the following" or similar

Expressions refer to any combination of these elements, including any combination of single (one) or multiple (one) elements. For example, "at least one of a, b, or c" or "at least one of a, b, and c" may be expressed as: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, where a, b, and c may each be single or multiple.

It should be understood that in various embodiments of the present

Application the size of the serial number of each of the above processes not dt&/509043

Order of execution is implied, and some or all of the steps may be executed in parallel or sequentially, and the order of execution of each of the processes should be determined by its function and inherent logic, without constraining the process of

implementation of the provisions of this application. The provisions of the

The terms used in the present application are intended solely for the purpose of

Description of the respective embodiment of the regulations and are intended to

The singular forms of "a," "said," and "the" used in the embodiments of the present application and the appended claims also include the plural forms, unless the context clearly indicates otherwise.

The weights of the relevant components mentioned in the specification of the embodiments of the present application may not only refer to the specific contents of the components, but may also refer to the proportional relationship of the weights of the

Components, and therefore, any enlargement or reduction of the contents of the relevant components in accordance with the specification of the embodiments of the present application is within the scope of the disclosure of the specification of the

Embodiments of the present application. In particular, the invention described in the description of

Embodiments of the present application described mass may be given in µg, mg, g, kg and other mass units known in the chemical industry.

The terms “first” and “second” are used for descriptive purposes only, to

They are intended to distinguish between substances for different purposes, such as substances, and are not to be understood as indicating or implying a relative importance or implicitly determining the number of technical characteristics specified. For example, without departing from the scope of the Regulations, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, a characteristic defined as 'first' or 'second' may explicitly or implicitly encompass one or more such characteristics.

In order to facilitate the understanding of the present invention, a more comprehensive and detailed description of the present invention is given below in conjunction with the accompanying drawings of the description and the preferred embodiments, but the scope of the present invention is not limited to the following specific

limited embodiments.

Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise stated, the various raw materials, reagents,

Instruments and equipment, etc., used in the present invention are purchased on the market or manufactured by existing methods.

A molecular machine, a machine composed of molecular substances capable of performing a specific function, has the characteristics of small size, diversity,

Self-assembly, self-adaptation, wide range of polarity and strong

Designability, etc. A molecular machine combines the above-mentioned components through intermolecular interactions (hydrogen bonds, van der Waals forces, electrostatic gravitational forces, etc.) and has a well-defined microstructure and macroscopic properties. LUS09043

A first aspect of an embodiment of the present application provides a molecular

A lactic acid-based machine comprising a first functional unit and a second functional unit; the first functional unit and the second functional unit are connected by hydrogen bonding; the first functional unit is a

Lactic acid molecule and the second functional unit is at least one of a

betaine molecule, a picosidine molecule or a levulinic acid molecule; wherein the Dendrobium officinale polysaccharide comprises an O-acetyl-B-D-glucan, wherein the main chain of the O-acetyl-

B-D-glucan comprises D-mannose and D-glucose linked by a ß-1,4-glycosidic bond; The lactic acid-based molecular machinery is used to drive the lactic acid-based molecular machinery to establish a recognition bond with the Dendrobium officinale polysaccharide, including driving the

Lactic acid-based molecular machinery to bind to the ß-1,4-glycosidic bond of the

Dendrobium officinale polysaccharide through hydrogen bond affinity.

If the second functional group element is a betaine molecule, the molecular machine has the molecular structural formula CH3CH(OH)COO-H-OOCCH:2N(CH3)s.

The present application forms a lactic acid-based molecular machine through hydrogen bonding of a specific bond length of an intermolecular

Interaction between a lactic acid molecule and at least one of a

betaine molecule, a picosidine molecule or a levulinic acid molecule. In the lactic acid-based molecular machine of the present application, the hydroxyl group of the

lactic acid molecule and the carbonyl group of at least one of the betaine molecules, the

picric acid molecule or the levulinic acid molecule hydrogen bonds with

Bond lengths that are smaller than the van der Waals radius, and therefore the stability of the

Lactic acid-based molecular machine is relatively high. When the lactic acid-based molecular machine of the present application extracts polysaccharides from Dendrobium officinale, the lactic acid-based molecular machine drives the lactic acid-based molecular machine to form a recognition compound with Dendrobium officinale

Polysaccharides through the affinity of hydrogen bonding, which allows

It improves the transfer rate of active ingredients in Dendrobium officinale, while maintaining the activity of the active ingredients and improving the stability of the active ingredients within Dendrobium officinale. It can also dissolve and dissociate the cellulose in the cell wall of Dendrobium officinale, improving the permeability of the cell wall of Dendrobium officinale, improving the dissolution of the active components in the cell wall of Dendrobium officinale, and thus improving the extraction efficiency of polysaccharides from Dendrobium officinale.

In some embodiments, the hydrogen bond has a bond length of 1 A~2 A, where the hydrogen bond length is the bond length between the hydrogen (H)

atoms and the oxygen (O) atoms in the betaine molecule, and the shorter bond length ensures that the first functional base element and the second functional base element have a greater interaction force and are not easily destroyed, and the molecular

machinery can remain stable, thereby facilitating the molecular machinery to associate with the Dendrobium officinale polysaccharide.

In some embodiments, the molar ratio of the first functional

base element to the second functional base element 1:(0.25~4), and a molar ratio in

Range of 1:(0.25~4) improves the effect of the molecular machine on lactic acid bast$/509043 in the extraction of Dendrobium officinale polysaccharide. Furthermore, the

Lactic acid based molecular machine formed with a molar ratio of 1:1 between the first functional group element and the second functional group element, the highest yield of Dendrobium officinale polysaccharide extract.

In some embodiments, the Dendrobium officinale polysaccharide is a

Polysaccharide structure comprising a variety of monosaccharides including glucose, mannose and galactose linked by glycosidic bonds and based on

Lactic acid-based molecular machine is driven by the affinity of

Hydrogen bonds driven to bind with the various monosaccharides of the

Dendrobium officinale polysaccharide. This increases the transmission rate of the

active ingredient in Dendrobium officinale increases the yield of Dendrobium officinale

Polysaccharide extract prepared on the basis of lactic acid-based molecular machine is increased, and thus the extraction efficiency of Dendrobium officinale polysaccharide is increased.

A second aspect of embodiments of the present application provides a method for producing a lactic acid-based molecular machine as in the first aspect of the content, comprising the following steps: mixing at least one of betaine, picric acid, or levulinic acid with lactic acid to obtain a mixture. The mixture is subjected to a

Heat treatment is carried out until the viscosity of the mixture is 200 mPass~500 mPaes, with the heat treatment temperature being 30 °C to 60 °C.

Understandably, the molar ratio of the first functional group element to the second functional group element is 1:(0.25~4), and the internal structure of the molecular

Machine that is driven by the different molar ratios of the first functional

The molecular structure of the first group element and the second functional group element is different. Accordingly, the effect of the lactic acid-based molecular machine, which drives the lactic acid-based molecular machine to form a recognition compound with the Dendrobium officinale polysaccharide through the affinity of hydrogen bonding, is different, and the effect of its polysaccharide yield to produce the Dendrobium officinale polysaccharide extract is also different. For example, the molar ratio of betaine to lactic acid used for mixing is one of 1:1, 1:2, 1:3, 1:4, and the produced lactic acid-based molecular machine improves the stability of the active ingredient within the Dendrobium and also improves the permeability of the Dendrobium officinale.

Cell wall. The solubility of the active ingredients within the Dendrobium officinale cell is improved and the transfer rate of the active ingredients in the Dendrobium officinale is increased, thereby

The efficiency of the extraction of Dendrobium officinale polysaccharide is increased and the costs of the extraction of Dendrobium officinale polysaccharide are also reduced, since no enzymes are required for

Catalysis of the extraction. Preferably, the molar ratio of betaine to

Lactic acid used for mixing, 1: 1. In this preferred embodiment, the prepared lactic acid-based molecular machine further improves the solubility of the intracellular active components of Dendrobium officinale, further improves the

Transfer rate of the active ingredients in Dendrobium officinale, thereby increasing the efficiency of

Extraction of Dendrobium officinale polysaccharides is further improved and reduces the

Cost of extracting Dendrobium officinale polysaccharides. Therefore, the polysaccharide

Extract used to produce Dendrobium officinale polysaccharide extract,

a higher polysaccharide yield and is used for the extraction of Dendrobium officinat&509043

Polysaccharide with better performance is used.

The present application synthesizes the lactic acid-based molecular

Machine through a heating and stirring process that controls the temperature and duration of the

Heating treatment controls, and the molecular machine of the present application can remain stable, clarified, transparent, and homogeneous at room temperature without the need for subsequent separation, thereby simplifying the preparation process of the lactic acid-based molecular machine; Furthermore, the synthesis process of the lactic acid-based molecular machine of the present application is 100% atom-economical, green and economical, biocompatible, safe and non-toxic, and possesses a three-dimensional ordered structure, which facilitates the effective capture of the drug by intermolecular forces.

In some embodiments, at least one of betaine, picloram or levulin is combined with

Lactic acid and then heated to obtain the lactic acid-based molecular machine comprising: mixing lactic acid and at least one of betaine, picloram or

Levulin to obtain a mixture. The mixture is heated to 30 °C~60 °C and the

Stirring of the mixture is carried out at 30 °C~60 °C for 3 h~8 h until the viscosity of the

Mixture is 200 mPa~500 mPa to obtain the lactic acid-based molecular machine; The method for preparing the lactic acid-based molecular machines was optimized by adding water to the lactic acid-based molecular machines for dilution to obtain the lactic acid-based molecular machine solution.

As shown in Figure 1, a third aspect of the embodiments of the present

Application provides a process for the preparation of a polysaccharide extract of Dendrobium officinale, which comprises the following steps:

Step S100, Mixing the Dendrobium officinale powder with the molecular

Lactic acid-based machine solution and subsequent strengthening treatment by

Ultrasound to obtain the Dendrobium officinale extract;

Step S200, Centrifuge the Dendrobium officinale extract, remove the upper clear

Liquid after centrifugation and filtration to obtain the Dendrobium officinale polysaccharide

to obtain extract;

Where the solute in the lactic acid-based molecular machine solution contains the

Lactic acid-based molecular machine according to the content of the first aspect and/or the lactic acid-based molecular machine produced by the manufacturing method according to the

Content of the second aspect was produced.

Compared with the state of the art, since the lactic acid-based molecular machine is used to extract the polysaccharides from Dendrobium officinale, there is no

Need to use an enzyme for catalysis, and the lactic acid-based molecular

Machine drives the lactic acid-based molecular machine with the polysaccharides of

Dendrobium officinale through the affinity of hydrogen bonding for recognition, which improves the rate of transfer of the active ingredient into Dendrobium officinale.

Extraction effect of the polysaccharides in the Dendrobium officinale extract of the present

Application is 2.35 times more effective than that of aqueous extraction and 1.86 times more effective than that of enzymatic cellulose digestion; In the lactic acid-based molecular machine

System, the polysaccharide compounds can exist stably for 4 months without decomposition, the stability is better Lactic acid-based molecular machine can effectively inhibit the

Growth of microorganisms and prevent the polysaccharide extracts vdr/909043

Deterioration, the polysaccharide extracts are stored in the room temperature and natural light for 4 months, the microbial content of individual microorganisms does not exceed the norm;

At the same time, the coupled ultrasound enhancement can accelerate the dissolution and dissociation of cellulose in the cell wall of Dendrobium officinale, thereby

Polysaccharide content is effectively increased, but also the amount of solvents is reduced,

Impurities are reduced, the transfer rate of the active ingredients is improved, the

Solving the problem of using organic solvents and residues, achieving high-quality and stable production, improving the utilization rate of active ingredients and added value.

In some embodiments, the pretreatment of the Dendrobium to obtain the Dendrobium powder comprises: washing the Dendrobium with water and freeze-drying the

Dendrobiums at a given drying temperature; The dried Dendrobium officinale is crushed and sieved and preserved at low temperature, whereby the

Freeze-drying time is 1h~6h, the preset drying temperature is -20°C~70°C and the crushing and sieving is a 60-mesh sieve. The particle size of the

Seven obtained Dendrobium officinale powder is not larger than 0.3 mm, and the temperature of low-temperature preservation is -10°C~20°C, which optimizes the pretreatment process of Dendrobium officinale and preserves the active substances or ingredients in

Dendrobium officinale are preserved. The particle size of the Dendrobium officinale powder is not larger than 0.3 mm, i.e. if the particle size of the Dendrobium powder is less than or equal to 0.3 mm, it can have a larger contact area with the Dendrobium officinale officinale

polysaccharides, which improves the extraction effect of Dendrobium officinale polysaccharides.

In some embodiments, the mass-to-volume ratio of the Dendrobium officinale

powder and the molecular machine solution based on lactic acid in step S100 1:5 g/mL~70 g/mL; i.e. 1 g of the Dendrobium officinale powder is mixed with 5 mL~70 mL of the molecular

Lactic acid-based machine solution, which is effective for the extraction of polysaccharides from Dendrobium officinale. Fine lactic acid-based molecular

Machine solution of less than 5 ml is not able to sufficiently extract the polysaccharide in Dendrobium officinale within a certain period of time, while one based on

Lactic acid-based molecular machine solution of more than 70 ml no significant

Effect on increasing the efficiency of the polysaccharide in Dendrobium officinale;

In some embodiments, the concentration of the lactic acid-based molecular machine solution in step S100 is 5 wt% to 80 wt%, i.e., the concentration of the lactic acid-based molecular machine solution after dilution with water is 5 wt% to 80 wt%, in order to improve the efficiency of extraction of polysaccharides from the

Dendrobium officinale. In addition, the water is pure water or ultrapure

Water to reduce the impact of impurities on extraction.

In some embodiments, the parameters of the ultrasonic enhancement treatment in step S100 are: the temperature of the ultrasonic enhancement treatment is 30 °C~80 °C, the

Duration of ultrasonic intensification treatment is 0.5 h~2 h, and the ultrasonic energy is 100 W~500 W; The assumption of the parameters of intensive ultrasonic treatment in this

This embodiment can carry out the extraction of Dendrobium officinale polysaccharides more efficiently, and due to the strong vibration caused by the ultrasonic waves of the

Ultrasonic extraction process can reduce the extraction time, while the structure and biological activity of the active ingredients can be maintained unchanged, and bioactive substances can be extracted efficiently and cost-effectively without contamination, thus realizing a green and highly efficient extraction of the active ingredients of Dendrobium.

In some embodiments, after step S100 and before step S200, the pH

Value of the Dendrobium officinale extract adjusted to a predetermined pH value; the

Adjusting the pH of the Dendrobium officinale extract to the predetermined pH allows the separation of the lactic acid-based molecular machinery from the

Dendrobium officinale polysaccharide and improves the purity of Dendrobium officinale

polysaccharide. Furthermore, the predetermined pH value is greater than 6.5 and less than 7.0; the predetermined pH value is within the scope of the present embodiment and can

Lactic acid-based molecular machinery more effectively from the Dendrobium officinale

Separate polysaccharides and further improve the purity of Dendrobium officinale polysaccharides.

In some embodiments, the parameters of the centrifugal treatment in step S200 are: the rotation speed of the centrifugal treatment is 6000 r/min ~ 12000 r/min, the

Duration of centrifugal treatment is 5 min ~ 15 min, the filtration membrane of the

Centrifugal treatment is a filtration membrane for the organic phase, and the pore size of the filtration membrane for the organic phase is not larger than 0.45 µm; The

Centrifugal treatment using the centrifugal treatment parameters in this

Embodiment can effectively remove impurities and reduce the concentration of

Dendrobium officinale polysaccharide extract.

A fourth aspect of the embodiment of the present application provides an antioxidant and anti-aging application of a Dendrobium officinale polysaccharide extract based on a manufacturing process as in any of the fourth aspects, comprising a

Skin care product made from Dendrobium officinale polysaccharide extract as a raw material to be used for antioxidant and anti-aging repair of human skin.

The Dendrobium officinale polysaccharide extracts of the present application for antioxidant and anti-aging applications do not need to be isolated, and the lactic acid-based molecular machines in the polysaccharide extracts synergize with

Polysaccharides in skin care applications with antioxidant, antioxidative, exfoliating and moisturizing effects on human skin.

In some forms, Dendrobium officinale polysaccharide extract is also used in cytotoxicity tests, skin patch tests, or conventional nine tests for cosmetics, thus expanding the application scenarios of Dendrobium officinale polysaccharide extract.

In order to make the above implementation details and operations of the present application clearly understandable for the technical personnel in the field, as well as the molecular

Machinery of the embodiments of the present application, the preparation method of the

extract and the progressive performance of the antioxidant and anti-aging applications, the following is an example to illustrate the above technical solutions by means of a number of embodiments.

Manufacturing example 1

A method for producing a lactic acid-based molecular machine, wherein Betaht/909043 is mixed with lactic acid in a molar ratio of 1:1 to obtain a mixture; the

mixture is subjected to a heat treatment until the viscosity of the mixture is 325 mPa-s to obtain a viscous liquid lactic acid-based molecular machine A1;

Where the temperature of the heat treatment is 50 °C and the duration of the heat treatment is 6

hours.

Manufacturing example 2

A method for producing a lactic acid-based molecular machine, wherein the

The molar ratio of betaine to lactic acid was set to 1:2, the viscosity of the mixture was 308 mPa-s and the remaining steps and parameters were the same as in

Preparation Example 1 to obtain a lactic acid-based molecular machine A2.

Manufacturing example 3

A method for producing a lactic acid-based molecular machine, in which the

The molar ratio of betaine to lactic acid was set to 1:3, the viscosity of the mixture was 280 mPa-s and the remaining steps and parameters were the same as those of

Preparation Example 1 to prepare a lactic acid-based molecular machine A3.

Manufacturing example 4

A method for producing a lactic acid-based molecular machine, wherein the

The molar ratio of betaine to lactic acid was set to 1:4, the viscosity of the mixture was 271 mPaes and the remaining steps and parameters were the same as those of the

Preparation Example 1, and a lactic acid-based molecular machine A4 was prepared.

Manufacturing example 5

A method for producing a lactic acid-based molecular machine, wherein

Betaine is replaced by picloram and the remaining steps and parameters are the same as in

Preparation Example 1 is to prepare a lactic acid-based AS molecular machine.

Manufacturing example 6

A method for producing a lactic acid-based molecular machine by

Replacing betaine with levulinic acid, the remaining steps and parameters being the same as in Preparation Example 1, to prepare a lactic acid-based molecular machine A6.

Comparison with manufacturing example 1

A method for producing a lactic acid-based molecular machine, wherein betaine is replaced with choline chloride and the remaining parameters are the same as in Preparation Example 1 to produce a lactic acid-based molecular machine DA1.

Test example 1: Infrared spectral analysis of lactobetaine molecular machines

Whether the lactic acid-based molecular machine was successfully synthesized was determined by analyzing the shifts and broadenings of the main functional groups of the

Lactic acid-betaine molecular machines A1 to A4 and the synthesized monomers, betaine and

Lactic acid, determined using an infrared spectroscopy analyzer.

Specifically, the samples to be examined were the lactic acid-based molecular

Machines A1 to A4, the betaines and lactic acid were mixed and pressed with KBr in a ratio of 1:50, and the wavelength scanning range was 500 cm™'~4000 cm’. As shown in Figure 2, Figure 2 shows the infrared spectra of the lactic acid-betaine molecular machines A1 to

A4, the synthetic monomers betaine and lactic acid, with 1:1 the lactic acid-betaine

Molecular machine A1, 1:2 the lactic acid betaine molecular machine A2, 1:3 the lactic acid 509043

Betaine molecular machine A3 and 1:4 the lactic acid betaine molecular machine A4.

In lactic acid, the broader absorption peak around 3450 cm”! is due to the hydroxyl group at the

carboxyl group in lactic acid, and the broader absorption peak at about 1680 cm"! is due to the carbonyl group in lactic acid. For betaine, two absorption peaks appeared at 3300 cm"! and 1620 cm"!, indicating the presence of hydroxyl and

Confirm the presence of amino groups in betaine. For lactic acid betaines A1 to A4, three strong and broadened absorption peaks at approximately 3635 cm² ~ 3097 cm², 1784 cm² ~ 1682 cm², and 1677 cm² ~ 1525 cm², which are shifted from the respective characteristic peaks of the monomers, indicate the presence of -COOH---NH hydrogen bonds. The formation of

Hydrogen bonding leads to a decrease in the vibrational frequency and a broadening of the spectral band, which results in a shift, broadening and even overlap of the characteristic peaks of the two components.

Test example 2: Thermogravimetric curve analysis of molecular machines on

Lactic acid base

A Mettler TGA2 thermogravimetric analyzer was used to analyze the thermal stability and thermal decomposition of lactic acid-betaine molecular machines A1 to A4 and the synthetic monomers betaine and lactic acid. The test samples of lactic acid-betaine molecular machines A1 to A4, betaine, and lactic acid were protected by nitrogen, and the temperature elevation rate was set at 10 °C/min. The temperature was increased from room temperature (25 °C) to 600 °C. Thermogravimetric analysis curves (TGA) were plotted to obtain the decomposition temperatures of the corresponding samples.

Curves of the lactobetaine molecular machines A1 to A4, the synthetic monomers betaine and

Lactic acid are shown schematically in Figure 3, where 1:1 the lactobetaine molecular machine

A1, 1:2 the lactobetaine molecular machine A2, 1:3 the lactobetaine molecular machine A3 and 1:4 the lactobetaine molecular machine A4. As can be seen in Figure 3, the decomposition temperatures of the lactobetaine molecular machines A1 to A4 slow down the

Weight reduction gradients between their pure raw material components. The thermal

Stability of the lactobetaine molecular machine is slightly better compared to monomeric lactic acid.

Test Example 3: Quantum chemical computational analysis of a lactobetaine-based molecular machine

Figure 4 shows the quantum chemical calculation analysis of the molecular machine A1 of

Lactobetaine, as shown in Figure 4. (a) is the diagram of the structure optimization; (b) is the

Electrostatic surface potential analysis diagram (Electrostatic surface potential,

ESP); (c) the analysis diagram of the interaction region indicator function (IRI);

As shown in (a) in Figure 4, the geometry optimization and the

Frequency calculation of the lactic acid betaine molecular machine and no imaginary

frequency, which indicates that the current structure lies at the local minima of the potential energy surface and can exist stably. There is an interacting

Hydrogen bond between the hydroxyl group of lactic acid and the carbonyl group of

Betaine, and the hydrogen bond length in this embodiment is 1.41046 À, and the group distance between lactic acid and betaine in the lactic acid-betaine

The molecular machine is smaller than the van der Waals radius. Based on the optimized structure of the lactic acid-betaine molecular machine, the interaction groups between betaine and lactic acid are mutually confirmed by infrared spectral analysis.

The mechanism of formation of the molecular machinery of lactic acid betaine was qualitatively analyzed using ESP, as shown in Figure 4(b). The carboxyl region of

Betaine is electronegative, and a large region around the encapsulated nitrogen atom is electropositive. The hydroxyl region of lactic acid is electropositive, and the carboxyl region is electronegative. After the formation of the molecular machinery of betaine lactate, the electropositive region of betaine and the electronegative region of lactic acid are attracted to each other to form a stable molecular machine structure. The indicator function for

Interaction regions (IRI) was used to measure the molecular van der Waals forces, the

Hydrogen bonds and spatial repulsion, as shown in (c) in Figure 4, where the blue color in the interaction relationship represents strong attractive

The red color represents strong spatial resistance, where the stronger the spatial resistance, the harder it is for the atoms of the corresponding two regions to attract each other. The large green region in the transition represents weak van der Waals interaction forces. The large green region of the interaction force between betaine and lactic acid indicates that a van der Waals force exists between betaine and lactic acid, and the small blue disc between the hydroxyl and carboxyl groups indicates that a strong hydrogen bond interaction force also exists between betaine and lactic acid.

Embodiment 1: (1) Lactic acid-based molecular machine solution with a concentration of 10 wt% was prepared using lactic acid-based molecular machine Al as the solvent and ultrapure water as the solvent. (2) Dendrobium officinale powder was added to the lactic acid-based molecular machine

machine solution was added and the extract was ultrasonically

receive intensive treatment.

The mass-volume ratio of Dendrobium officinale powder and lactic acid-based molecular machine solution was 1g:50mL; the parameters of the intensive

Ultrasonic treatment: the extraction temperature was 60°C, the extraction time was 1h, the

Ultrasonic power was 300W; the particle size of the Dendrobium officinale powder was no larger than 0.3mm. (3) Adjust the pH of the Dendrobium officinale extract to 6.6, centrifuge

the above-mentioned Dendrobium officinale extract, the centrifugation parameter was 10000 rpm, centrifuge for 15 minutes, take the upper layer of the

supernatant after centrifugation and filter it, and the filter membrane used for the

Filtration was a 0.45 µm filter membrane for the organic phase to ensure

To obtain Dendrobium officinale polysaccharide extract based on the molecular machinery of lactic acid betaine, it was designated as extraction solution B1.

Embodiment 2:

The manufacturing concentration of the lactobetaine molecular machine solution in step (1) of

Embodiment 1 was adjusted to 20 wt%, the pH of the Dendrobium officinale

extract was set to 6.7, and the remaining steps and parameters were the same as in Embodiment 1, and extract B2 was prepared.

Embodiment 3: LU509043

Adjusting the preparation concentration of the lactobetaine molecular machine solution in

Step (1) of Embodiment 1 to 30 wt%, adjusting the pH of the Dendrobium officinale extract to 6.75, and the remaining steps and parameters were the same as those of Embodiment 1 to prepare the extract B3.

Embodiment 4:

Adjusting the preparation concentration of the lactobetaine molecular machine solution in

Step (1) of Embodiment 1 to 40 wt%, adjusting the pH of the Dendrobium officinale extract to 6.8, and the remaining steps and parameters were the same as those of Embodiment 1 to prepare the extract B4.

Embodiment 5:

The preparation concentration of the lactobetaine molecular machine solution in step (1) in

Embodiment 1 was adjusted to 50 wt%, the pH of the Dendrobium officinale

Extract was set to 6.9, and the remaining steps and parameters were the same as in Embodiment 1, and extract BS was prepared.

Embodiment 6:

Replacing the solute in step (1) of Embodiment 4 from the lactic acid-based molecular machine A1 with the lactic acid-based molecular machine AS, and the remaining steps and parameters were the same as those of Embodiment 4, and the extract solution B6 was prepared.

Embodiment 7:

Replacing the solute in step (1) of embodiment 4 from a

Molecular machine Al based on lactic acid by a molecular machine A6 on

Lactic acid base, and the remaining steps and parameters were the same as in

Embodiment 4 to prepare the extraction solution B7.

Comparative example 1:

The solute in step (1) of Example 4 was transported by a molecular machine to

Lactic acid base A1 is replaced with a lactic acid-based molecular machine DA1, and the remaining

Steps and parameters were the same as those of Example 4 to prepare the extract DBI.

Comparative example 2:

A preparation of Dendrobium officinale polysaccharide extract (water), the specific steps are as follows: (1) Preparation of aqueous extract: Dendrobium officinale powder is converted into ultrapure

water and an enhanced ultrasound treatment is carried out to create an aqueous

Extract, wherein the mass-volume ratio of Dendrobium officinale powder to

Water is 1 g:50 ml and the parameters of the enhanced ultrasound treatment are: the

Extraction temperature is 60 °C, the extraction time is 1 h and the

Ultrasonic power is 300 W; and the particle size of the Dendrobium officinale powder is not larger than 0.3 mm. (2) Extraction: The above extract was centrifuged with a centrifugation parameter of 10,000 rpm for 15 min, and the upper layer of the clear liquid was taken out and filtered. The filter membrane used for filtration was a 0.45 μm filter membrane for the organic phase, and the excess impurities were removed.

Extract DB2 was prepared.

Comparative Example 3: LUS09043

Fine preparation of a Dendrobium officinale polysaccharide extract (cellulase), the individual steps are as follows: (1) Preparation of the enzymatic solution: Cellulase is dissolved in citric acid buffer to obtain an enzymatic solution; the concentration of cellulase and citric acid is 1 mg/ml. (2) Extraction: Dendrobium officinale powder was added to the enzymatic solution for a period of time and then heated to inactivate the enzyme to extract the cellulase.

To obtain the extract, the mass-volume ratio of Dendrobium officinale powder to the enzymatic solution was 1g:50mL, and the enzyme extraction time was 2h, the

The enzyme extraction temperature was 40°C, and the heating temperature for enzyme inactivation was 80°C, and the corresponding time was 25 min. The above extract was centrifuged with a centrifugation parameter of 10,000 rpm for 15 minutes. The upper layer of the clear liquid was collected and filtered. The filter membrane used for filtration was a 0.45 μm filter membrane for the organic phase, and the excess impurities were removed, thus preparing extract DB3.

Test example 4: Test of polysaccharide yield of extracts with different

Concentrations of molecular machine solutions based on lactic acid and various

Types of extracts

Based on each of the above 7 embodiments and the 3 different

Ratios, each of the extracts was tested for the polysaccharide yield of the extracts according to the following method: 50 mg of extracts B1 to B7 of the above 7 embodiments and extracts DB1 to

DB3 of the 3 ratio pairs were weighed into 250 mL glass test tubes and diluted to 250 mL each with distilled water.

Then, 1 mL of the diluted extracts were added to 10 mL glass test tubes and 1 mL of distilled water to 2 mL glass test tubes, and 1 mL of aqueous phenol solution with 5% volumetric concentration and 5 mL of concentrated sulfuric acid with 98% volumetric

concentration were added to each of the 10 mL glass test tubes.

Then, each 10 mL glass tube was shaken well and allowed to stand for 30 minutes, and the absorbance was measured at 490 nm with a UV spectrophotometer and analyzed and calculated using a glucose standard curve to obtain the polysaccharide yield corresponding to each extraction solution. The polysaccharide yield is the ratio between the amount of polysaccharide actually obtained during extraction and the total

Polysaccharide in raw Dendrobium officinale powder and is generally expressed as a percentage.

The calculation formula for the polysaccharide yield is:

Polysaccharide yield = (actually obtained amount of polysaccharide/total input of

raw materials) x 100%.

The actual amount of polysaccharide obtained is the weight of the polysaccharide in the

Dendrobium officinale polysaccharide extract (i.e. the extraction solution), the total amount of

Raw materials are the total weight of the Dendrobium raw material powder introduced into the extraction and the polysaccharide yield is the exploitation index of the extracted polysaccharide.

The polysaccharide yields of the extracts prepared by the seven embodiments or three contrasting ratios are shown in Table 1, and various

Combinations and concentrations of lactobetaine molecular machines extracted/ 909043

Polysaccharides are better than traditional water extraction and enzymatic processes. Among them, the extraction effect of lactobetaine-based molecular machines was the best, and the extraction efficiency was highest when the concentration was as high as 197.6 mg/g, which was 2.35 times higher than that of the water extraction method (84.2 mg/g) and 1.86 times higher than that of the cellulolytic enzyme digestion method (106.2 mg/g), and it has a broad market application prospect. Lactic acid-based molecular machines not only have higher extraction efficiency but are also green solvents with anti-inflammatory, moisturizing, and anti-wrinkle effects, and their

Use can reduce waste, simplify processes and reduce costs.

Table 1 Polysaccharide yield of extracts B1 to B7 and extracts DB1 to DB3 yield mg/g mg/g

E > 2: 20wt% Carnitine 3: 30wt% 1: Choline chloride 4: 40wt% 2: Water extraction 5: 50wt% 3: Enzymatic

extraction

Embodiment 8:

The concentration of 40 wt% of the lactic acid betaine molecular machine solution from

Embodiment 4 was taken, the extraction temperature of the ultrasonic

Intensification treatment was set at 30°C, and the remaining steps and parameters were the same as those of Embodiment 4, and extract B8 was prepared.

Embodiment 9:

The concentration of 40 wt% betaine lactate molecular machine solution from

Embodiment 4 was taken, and the extraction temperature of the reinforcing

Ultrasonic treatment was set at 40°C, and the remaining steps and parameters were the same as those of Embodiment 4, and extract B9 was prepared.

Embodiment 10:

The concentration of 40 wt% betaine lactate molecular machine solution from

Embodiment 4 was taken, the extraction temperature of the ultrasonic

The strengthening treatment was set at 50°C, and the remaining steps and parameters were the same as those of Embodiment 4, and extract B10 was prepared.

Embodiment 11:

The concentration of 40 wt% betaine lactate molecular machine solution of

Embodiment 4 was taken, and the extraction temperature of the

Ultrasonic enhancement treatment was set to 70°C, and the rest of the steps and

Parameters were the same as those of Embodiment 4, and the extraction solution B11 was prepared.

Test example 5: Test of the polysaccharide yield of the extracts at different

Extraction temperatures of the ultrasonic intensification treatment LU509043

Starting from the extracts B6 prepared in the above embodiments 4, 8 to 11,

B8 to B11, the polysaccharide yield of the extracts was tested, and the polysaccharide yield of each extract prepared at different extraction temperatures is shown in Table 2, which shows that the polysaccharide yield gradually increases with increasing temperature, but slightly decreases at a higher extraction temperature of 70 °C.

Table 2: Polysaccharide yield of the individual extracts obtained at different

extraction temperatures.

Embodiment 12:

The concentration of 40 wt% of the lactobetaine molecular machine solution from

Embodiment 4 was taken and the extraction time of the ultrasonic

The intensification treatment was set to 0.25 h. The remaining steps and parameters were the same as in Embodiment 4, and extraction solution B12 was prepared.

Embodiment 13:

The concentration of 40 wt% betaine lactate molecular machine solution from

Embodiment 4 was taken, and the extraction time of the

Ultrasound enhancement treatment was set to 0.5 h. The remaining steps and

Parameters were the same as in Embodiment 4, and extract B13 was prepared.

Embodiment 14:

The concentration of 40 wt% betaine lactate molecular machine solution from

Embodiment 4 was taken and the extraction time of the ultrasonic

The amplification treatment was set to 1.5 h. The remaining steps and parameters were the same as in Embodiment 4, and extract B14 was prepared.

Embodiment 15:

The concentration of 40 wt% lactobetaine molecular machine solution was taken from

Embodiment 4 and set the extraction time of the ultrasonic intensification treatment to 2.0 h. The remaining steps and parameters were the same as in Embodiment 4, and extract B15 was prepared.

Test example 6: Test of the polysaccharide yield of the extracts with different

Extraction times of ultrasound enhancement treatment

Starting from the extracts prepared in the above embodiments 4, 13 to 15

B6, B13 to B15, the polysaccharide yield of the extracts was tested, and the

Polysaccharide yield of the extracts prepared with different extraction times is in

As shown in Table 3, it can be seen that the polysaccharide yield gradually increased within 1 h, but the polysaccharide yield remained essentially unchanged or even decreased slightly with a longer extraction time. This is due to the fact that the extraction time is too long, the extraction reaches its limits, and as the time increases, the locally high temperature of the ultrasound causes some compounds to begin to decompose.

Table 3: Polysaccharide yield of extracts prepared with different extraction times.

Test example 7: Test of polysaccharide yield from extracts with different

Storage times under natural light at room temperature

The extract B4 obtained from embodiment 4 with a mass concentration of 40

wt% of the lactobetaine molecular machine solution, an extraction temperature of 60°C for the ultrasound-assisted treatment and an extraction time of 1 h for the ultrasound-assisted treatment was placed under natural light at room temperature, and the polysaccharide concentration in the sample of extract B4 was tested after 7, 15, 30, 60 and 120 days of placement, respectively, and the test results are shown in Figure 5: Figure 5 shows a schematic diagram of the changes in the polysaccharide content of extract B4 at different storage times under natural light at room temperature. As the

storage period of 7 days and moved up to 120 days, the

Polysaccharide concentration of extract B4 essentially unchanged with a slight

decrease. This indicates that the polysaccharides in the lactic acid betaine

Molecular system can exist stably and do not decompose within 4 months, whereby the stability is relatively good.

Test example 8: Effect of different storage times under natural light at

Room temperature on the microbiological content of the extracts

The extract B4 prepared in embodiment 4 was stored under natural light at

Room temperature according to the “Microbiological Test Methods” in the “Technical

Specification for the Safety of Cosmetics” (2015 edition). After 7, 15, 30, 60 and 120 days, samples of extract B4 were tested for total bacteria, total molds/yeasts,

Staphylococcus aureus, heat-resistant coliforms and Pseudomonas aeruginosa were examined, and the results are shown in Table 4. The microbiological content of each

Microorganisms complied with the Technical Code for the

safety of cosmetics and did not exceed the standard. This indicates that the

Lactobetaine molecular machine effectively inhibits microbial growth and

can prevent deterioration of the polysaccharide extract.

Table 4 Results of the germ content in extract B4 after different

Exposure time under natural light at room temperature 7 days 15 days 30 days 60 days 120 days

Bacteria TTT LUS09048 . <500CFU/g <500CFU/g <500CFU/g <500CFU/g <500CFU/g mstotal

Molds/Yeasts <100CFU/g <100CFU/g <100CFU/g <100CFU/g <100CFU/g total

Staphylococcus Not Not Not Not Not aureus detected detected detected detected detected

Heat-resistant Not Not Not Not Not

Coliforms detected detected detected detected detected

Pseudomonas Not Not Not Not Not aeruginosa detected detected detected detected detected

Test Example 9: Test for scavenging DPPH radicals in extracts

Extract B4 obtained under the optimal extraction conditions of Embodiment 4 was tested for scavenging of DPPH radicals as follows.

A sample tube (T), a sample background tube (To), a DPPH tube (C) and a solvent background tube (Co) were set up, and three parallel tubes were set up for each tested concentration of the sample tube (T) of the extract B4, to be configured respectively with different tested mass concentrations of the extract solution (0.0625 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml), where the

Solvent of the extract solution is water.

As an example, the DPPH radical scavenging test was carried out with the extract solution in a

mass concentration of 1 mg/ml, using the following sample tube (T), the

Sample background (To), the DPPH tube (C) and the solvent background (Co) were set up:

Sample tube (T): 1 ml extract solution (mass concentration of 1 mg/ml) + 2 ml water + 1 ml DPPH ethanol solution (DPPH concentration of 0.04 mg/ml);

Sample background tube (To): 1 ml extract solution (mass concentration of 1 mg/ml) + 2 ml water + 1 ml 95% ethanol solution;

DPPH tube (C): 3 ml water + 1 ml DPPH ethanol solution (DPPH mass concentration: 0.04 mg/ml);

Solvent background tube (Co): 3 ml water + 1 ml 95% ethanol solution.

The solution in each of the above-mentioned reaction tubes was pipetted into a cuvette and the absorbance values were determined at 517 nm. The DPPH radical scavenging rates of the extracts with different mass concentrations were determined using the DPPH

capture rate formula, and the specific results are shown in Figure 6.

DPPH free radical scavenging rate (%) = (1 — => x 100% where T is the absorbance value of the sample tube, To is the absorbance value of the

sample background tube, C is the absorbance value of the DPPH tube and Co is the

Absorption value of the solvent background tube.

As can be seen in Figure 6, which is a schematic diagram of the DPPH radical scavenging curves for each different mass concentration of extract B4, there was a significant difference in the scavenging of DPPH by the extract at eink}/509043

Mass concentration of 0.5 mg/mL of extract B4 (p < 0.05), indicating that the

Extract B4 has a better capture effect on DPPH. And the DPPH capture rate gradually increased with the increase in the mass concentration of Extract B4.

Test Example 10: Test for scavenging hydroxyl radicals from extracts

Extract B4 obtained under the optimal extraction conditions of Embodiment 4 was tested for the degradation of hydroxyl radicals as follows.

Set up a sample tube (A sample), a loss tube (A loss) and an undamaged tube (A undamaged), whereby for the sample tubes (T) for each

Mass concentration of the extract solution requires setting up three parallel tubes, each configured with a different mass concentration of the extract solution, and the solvent for diluting the extract solution is water.

A Sample (i.e. sample tube): 1 mL o-diazophene ethanol solution (concentration 5 mmol/L) + 2 mL phosphate buffer (concentration 0.2 mol/L) + 1 mL extract solution + 1 mL

Ferrous sulfate solution (5 mmol/L) + 1 mL H2O2 solution (concentration 0.1% v/v);

A Loss (i.e. lost tube): 1 mL o-diazophene ethanol solution (concentration 5 mmol/L) + 2 mL phosphate buffer (concentration 0.2 mol/L) + 1 mL ultrapure water + 1 mL

Ferrous sulfate solution (5 mmol/L) + 1 mL H2O2 solution (concentration 0.1% v/v);

A undamaged (i.e. no tube damaged): 1 mL o-diazophene ethanol solution (concentration 5 mmol/L) + 2 mL phosphate buffer (concentration 0.2 mol/L) + 1 mL

Ferrous sulfate solution (5 mmol/L) + 2 mL ultrapure water.

The above sample tube (A sample), the loss tube (A loss) and the undamaged

Tubes (A undamaged) were placed in a water bath at 37 °C for 1 h, and the absorbance of the solutions contained therein was determined at a wavelength of 536 nm to determine A sample, A

Loss or A to be preserved undamaged.

A Sample À Vertust

Hydroxyl radical scavenging capacity (*%) = —— —m— X 100%

À undamaged” DS Verbast where A sample is the absorbance of the solution in the sample tube, À wasted is the absorbance of the solution in the waste tube and A undamaged is the absorbance of the solution in the undamaged tube.

As shown in Figure 7, which is a schematic diagram of the hydroxyl radical capture curves for

Each different mass concentration of extract B4 represents a significant

Difference in the capture of hydroxyl radicals by extract B4 at 0.5 mg/mL (p < 0.05), indicating that extract B4 has a better capture effect on

hydroxyl radicals, and the catching ability of hydroxyl radicals was increased with the increase of

Mass concentration of extract B4 gradually increased.

Test Example 11: Cytotoxicity test of extracts

An application of a lactic acid-based molecular machine for Dendrobium officinale polysaccharide extract: A lactic acid-based molecular machine for

Dendrobium officinale polysaccharide extract (lactobetaine molecular machine) was used to

Used to conduct cytotoxicity tests.

Extract B4, obtained under the optimal extraction conditions of embodiment 4, was tested for cytotoxicity against fibroblasts of the human dermis.

Principle: Cell viability is determined by metabolic activity. MTH/509043 (Thiazolyl Blue) is metabolically degraded in living cells, forming blue-violet, insoluble

Methyl sludge is formed, and the number of living cells correlates with the color of the

Methyl sludge, which is measured with a photometer after it has been dissolved in alcohols.

Steps: (1) Human keratinocytes and human skin fibroblasts in 96-well plates with

culture medium, 100 uL per well, and incubate for 24 h. (2) After the cells had attached to the wall for 24 h, the medium was discarded, and 100 uL of different mass concentrations of the B4 extract solution (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL, 8 mg/mL, 16 mg/mL) were added to each well of the

sample group. In addition, a blank group (no B4 extract solution, 100 μl

culture medium) and a negative group (no B4 extract solution, add 100 μl cell suspension with culture medium), with each mass concentration of the sample group, the

Blank group and negative group each comprised 3 wells, so that a total of 54

wells were formed. (3) After the cells were incubated and cultured for 24 hours, 10 μl of MTT (thiazolyl blue) solution was added to each well, and the cells were incubated for 4 hours in the

Incubator. (4) Discard the culture medium, add 150 μl of DMSO (dimethyl sulfoxide) to each well, and measure the absorbance at 490 nm (OD490 nm) after shaking for 10 minutes. Then compare with the negative group that was not treated with the B4 solution of the test substance extract and calculate the results.

Relative cell survival rate (%) = TestODagonm, x 100%

NegOD4gonm

Where TestOD4oonm is the average OD490 nm absorbance of the B4 extract solution of the test substance; NegOD4oonm is the average OD490 nm absorbance of the negative

Control group.

As shown in Figure 8, Figure 8 is a schematic diagram of the trend of relative

Survival rate of human dermal layer fibroblasts in cytotoxicity tests with different mass concentrations of the extract B4 solution of the present application.

If the relative cell survival rate is more than 90% or more, it can be concluded that the B4 extract solution at the corresponding concentration has no biotoxic effect and has good biocompatibility. In human

In skin fibroblasts, the relative survival rate of extract B4 at a concentration of 2 mg/mL was over 90% and it showed good biocompatibility. Therefore, the

Test concentration of 2 mg/mL for the subsequent anti-aging test on human

Skin fibroblasts were selected.

Test Example 12: Test of the effect of the extract on the content of human type I collagen

Extract B4 prepared in embodiment 4 was tested for its effect on human type I collagen content:

Principle: Starting from fibroblasts of the human skin layer, the firming and wrinkle-preventing effect of the samples to be tested is determined by detecting changes in the

synthesis content of human type I collagen; type I collagen is one of the

Main components of the extracellular matrix of the dermis, which is synthesized intracellularly by dermal fibroblaster 909043 through the synthesis of extracellularly secreted type I collagen and then, after cleavage of the telopeptides, by the action of preterminal collagen peptidase to

Collagen fibers are polymerized. Therefore, the firming and anti-wrinkle effect of the samples under test can be assessed based on the amount of type I collagen secreted by the fibroblasts after administration.

Steps: (1) Spreading of a plate culture of human skin fibroblasts in 24-well plates. (2) Preparation of the solution: Preparation of working solutions with different

Mass concentrations of extract B4, the positive control group (i.e., PC group) and the negative control group (i.e., NC group).

Negative control group: culture medium;

Positive control group: 100 mg/mL concentration of Vc (vitamin C);

Sample group 1: 0.5 mg/ml concentration of extract B4 with water as solvent;

Sample group 2: 1.0 mg/mL concentration of extract B4 with water as solvent;

Sample group 3: 2.0 mg/ml concentration of extract B4, solvent is water; (3) Dosage: According to the above test protocol, a negative control group, a positive control group, a total of five groups of different mass concentrations of

Extract B4 solution, each group of three complex wells total 15 wells, to 24-

Well plate, when the fusion rate of human dermatofibroblasts reached 40% to 60%, the group to add the drug, each well was added to the corresponding 250 pL of the working solution, and the effect of adding the drug for 72 h. The

Drug was used to determine the content of human type I collagen using ELISA kits. (4) The ELISA kit was used to determine the content of human type I collagen in

Determine cell supernatant of each group.

As shown in Figure 9, Figure 9 is a schematic diagram of the effects of different mass concentrations of the extract B4 solution and the control group on the

Content of human type I collagen, i.e. the relative content of human type I

Collagen produced by different mass concentrations of the extract B4 solution and the

Control group was tested in this application.

Conclusions of the test: (1) Compared to the NC group, the upregulation of human type I

Collagen content in the PC group was significant (P < 0.05), indicating that the

positive control of this test. (2) Compared to the NC group, the content of human type I collagen in the

B4 extract solution at the test concentrations of 0.5 mg/mL, 1 mg/mL and 2 mg/mL (P < 0.05), and with increasing concentration, the content of human type I collagen increased.

This means that the B4 extract solution has the ability to stimulate the synthesis of human type-

I-Collagen at concentrations of 0.5 mg/mL, 1 mg/mL and 2 mg/mL, and that it has anti-wrinkle and firming effects in each of the above concentration ranges.

Test Example 13: Testing MMP-1 release from extracts of human dermal fibroblasts

Extract B4 prepared in embodiment 4 was tested for the release of MMP-1 from human dermal layer fibroblasts.

Principle: The test kit uses the double antibody sandwich enzyme immunoassay / 509043

Technology. The specific anti-human MMP-1 antibody is labeled on an enzyme-labeled

Plate pre-coated with high affinity. Standard solutions, samples to be tested and biotinylated detection antibodies are added to the wells of the plate. After

Incubation, the MMP-1 present in the samples binds to the solid phase antibody and the

Detection antibody. After washing to remove unbound material,

Horseradish peroxidase-labeled streptavidin (streptavidin-HRP) was added. After

After washing, the chromogenic substrate TMB is added, and the color is developed under exclusion of light; the hue of the color reaction is proportional to the concentration of MMP-1 in the sample.

Steps: (1) Spreading culture of human skin fibroblasts was performed in 24-well plates for 18 to 24 hours. (2) Wash the cells twice with PBS buffer and prepare the maintenance medium with different mass concentrations of extract B4, the positive control group (i.e.,

PC group) and the negative control group (i.e. NC group).

Negative control group: culture medium;

Positive control group: 100 mg/mL concentration of Vc (vitamin C);

Sample group 1: 0.5 mg/ml concentration of extract B4 with water as solvent;

Sample group 2: 1.0 mg/mL concentration of extract B4 with water as solvent;

Sample group 3: 2.0 mg/ml concentration of extract B4, solvent is water; (3) Drug addition: according to the above test protocol, a negative control group, a positive control group, a total of five groups of different mass concentrations of

Extract B4 solution, each group of three complex wells total 15 wells, to 24-

Well plate, when the fusion rate of human dermatofibroblasts reached 40% to 60%, the group for the addition of the drug, respectively, each well was added to the corresponding working solution of 250 uL, the addition of the drug worked for 72 h. The

Drug was added to the working solution, and the working solution was added to each

wells, and the drug worked for 72 hours. (4) The ELISA kit was used to determine the content of MMP-1 in the cell supernatant of each

group to determine.

As shown in Figure 10, Fig. 10 shows the schematic diagram of the test of the relative

MMP-1 content for the MMP-1 release test of human dermal fibroblasts with different mass concentrations of the B4 extract solution and the control group.

Conclusion of the test: (1) There was a significant decrease (P<0.05) in MMP-1 content in the PC group in

Compared to the NC group, indicating that the positive control was effective for this test. (2) Compared to the NC group, a significant decrease (P<0.05) in MMP-1

The concentration of the B4 extract solution was found to be significantly different at the tested concentrations of 0.5 mg/mL, 1 mg/mL, and 2 mg/mL, with the MMP-1 content decreasing with increasing concentration. This means that the B4 extract solution is capable of inhibiting the secretion of MMP-1 at

concentrations of 0.5 mg/mL, 1 mg/mL and 2 mg/mL, and that in each of these

Concentration ranges have an anti-wrinkle and firming effect.

Test Example 14: Testing of MMP-3 release from extracts from human

Skin layer fibroblasts LU509043

Extract B4 prepared in embodiment 4 was tested for the release of MMP-3 from human dermal layer fibroblasts.

Principle: Irradiation of cultured fibroblasts with UVA (long-wave ultraviolet

Radiation) leads to high mRNA expression of MMP-3 (matrix metalloproteinase), and the extent of degradation of the extracellular matrix is closely related to MMP-3. If

When skin cells overexpress MMPs, the structure of the extracellular matrix of the dermis is severely damaged, especially the normal structure of collagen fibers and elastic fibers.

An in vitro model of premature aging of human skin fibroblasts was used to induce premature aging with UV light. The anti-wrinkle effect of extract B4 was evaluated by determining the level of matrix metalloproteinase-3 (MMP-3) in the extracellular matrix.

Steps: (1) Spreading culture of human skin fibroblasts in 24-well plates was carried out for 18 to 24 hours. (2) Liquid preparation: Preparation of working solutions with different

Mass concentrations of extract B4, the positive control group (i.e., PC group) and the negative control group (i.e., NC group).

Negative control group: culture medium;

Positive control group: 100 mg/mL concentration of Ve (vitamin C);

Sample group 1: 0.5 mg/ml concentration of extract B4 with water as solvent;

Sample group 2: 1.0 mg/ml concentration of extract B4 with water as solvent;

Sample group 3: 2.0 mg/ml concentration of extract B4, solvent is water; (3) Drug addition: according to the above test protocol, a negative control group, a positive control group, a total of 5 groups of different quality concentrations of extract B4 solution, each group of 3 replicate wells for a total of 15 wells to 24-well plate, when the fusion rate of human dermatofibroblasts reached 40% to 60%, the group was added to the drug, and each well was added to the corresponding 250 μl of the working solution, and the effect of adding the

drug for 72 h. The group was irradiated with fresh medium for 4 hours, and then each well was irradiated with fresh medium. (4) UVA stimulation: After 4 hours of drug administration, UVA

Irradiation was carried out according to the above test protocol for the groups that needed to be stimulated; after completion of the irradiation, the old medium was removed in each

Well replaced with fresh medium and placed in the cell culture incubator to

Cultivation for 20 hours. (5) The ELISA kit was used to determine the MMP-3 content in the cell supernatant of each

group used.

As shown in Figure 11, Figure 11 shows a schematic diagram of the comparison of the relative content of MMP-3 between different mass concentrations of B4-

Extract solution and the control group for the MMP-3 release test on human

Dermal layer fibroblasts.

Conclusion: (1) Compared with the NC group, MMP-3 content was significantly decreased in the PC group, suggesting that the positive control of this test was effective.

(2) Compared with the NC group, the B4 extract solution showed a significant decrease (P<0.05) in MMP-3 content at the tested concentrations of 0.5 mg/mL, 1 mg/mL, and 2 mg/mL, with the MMP-3 content decreasing more with increasing concentration. This means that the B4 extract solution has the ability to inhibit MMP-3 secretion at concentrations of 0.5 mg/mL, 1 mg/mL, and 2 mg/mL, and in each of the above-mentioned concentrations.

Concentration ranges have an anti-wrinkle and firming effect.

In the present invention, the prepared molecular machines were

Lactic acid base in conjunction with an ultrasonic-assisted extraction process is used to extract the polysaccharide compounds of Dendrobium officinale, and the driving

Force for the formation of the molecular machines based on lactic acid was found at the molecular

Level by infrared spectroscopy and quantum chemical calculation analysis; then the optimal extraction conditions were determined by a one-sided experimental design:

Lactobetaine molecular machine concentration of 40 wt%, extraction temperature of 60°C and extraction time of 1 hour.

The yield of polysaccharides in the final Dendrobium officinale polysaccharide

Extract (also called extract) was 197.6 mg/g and was 2.35 times higher than the

Water extraction method (84.2 mg/g). Lactobetaine molecular machines not only have higher extraction efficiency, but are also green solvents with anti-inflammatory, moisturizing, and anti-wrinkle properties, and their use reduces waste, simplifies processing, and reduces costs. The result from the above

Preparation of Dendrobium officinale extract, the main components of which

Polysaccharide compounds, lactic acid-based molecular machines and water, can be used as a new cosmetic raw material, which has excellent antioxidant, anti-wrinkle, moisturizing and other effects.

The foregoing is only a preferred embodiment of the present application and is not intended to limit the present application, and all changes, equivalent

Replacements and improvements that are within the spirit and principles of this

application fall within the scope of protection of the present application.

Claims (10)

Claims LU509043 1. A lactic acid-based molecular machine, characterized in that it comprises a first functional motif and a second functional motif; the first functional base element is linked to the second functional base element by hydrogen bonds; the first functional base element is a lactic acid molecule, and the second functional base element is at least one of a betaine molecule, an absinthin molecule, or a levulinic acid molecule; wherein the Dendrobium officinale polysaccharide comprises O-acetyl-β-D-glucan, the main chain of the O-acetyl-β-D-glucan comprising D-mannose and D-glucose linked by a β-1,4-glycosidic bond; The lactic acid-based molecular machinery for driving the lactic acid-based molecular machinery to establish a recognition bond with a Dendrobium officinale polysaccharide through the affinity of hydrogen bonding, comprising driving the lactic acid-based molecular machinery to establish a recognition bond with the ß-1,4-glycosidic bond of the Dendrobium officinale polysaccharide through the affinity of hydrogen bonding; Wherein, when the second functional group member is the betaine molecule, the molecular machine has the molecular structural formula CH3CH(OH)COO-H-OOCCH2N(CH3)3. 2. Fine lactic acid-based molecular machine according to claim 1, characterized in that the molar ratio of the element of the first functional group to the element of the second functional group is 1:(0.25~4). 3. A method for producing a lactic acid-based molecular machine according to claim 1, characterized in that it comprises the steps of mixing the betaine, picloram, or levulin with the lactic acid to obtain a mixture, heating the mixture until the viscosity of the mixture is 200 mPa·s to 500 mPa·s to obtain a lactic acid-based molecular machine; wherein the temperature of the heating treatment is 30°C to 60°C. 4. A production method for a Dendrobium officinale polysaccharide extract, characterized by comprising the following steps: step S100, mixing the Dendrobium officinale powder with a lactic acid-based molecular machine solution and then subjecting it to an intensification treatment with ultrasonics to obtain a Dendrobium officinale extract; step S200, centrifuging the Dendrobium officinale extract, taking out the upper layer of the clear liquid after centrifugation, and filtering it to obtain a Dendrobium officinale polysaccharide extract; wherein the solute in the lactic acid-based molecular machine solution is a lactic acid-based molecular machine according to claim 1 or 2, and/or a lactic acid-based molecular machine produced by the production method according to claim 3. 5. The manufacturing method according to claim 4, characterized in that the mass-to-volume ratio of the Dendrobium officinale powder and the lactic acid-based molecular machine solution in step S100 is 1:5 g/mL~70 g/mL; and/or the particle size of the Dendrobium officinale powder is not larger than 0.3 mm. 6. The manufacturing method according to claim 4, characterized in that the concentration of the lactic acid-based molecular machine solution in step S100 is 5 wt% to 80 wt%. 7. The manufacturing method according to claim 5, characterized in that the parameters of the ultrasonic amplification treatment in step S100 are: the temperature of the ultrasonic amplification treatment is 30 °C~80 °C, the duration of the ultrasonic amplification treatment is 0.5 h~2 h, and the ultrasonic power is 100 W~500 W. 8. The manufacturing method according to claim 5, characterized in that the parameters of the centrifugal treatment in step S200 are: the rotation speed of the centrifugal treatment is 6000 r/min ~ 12000 r/min, the duration of the centrifugal treatment is 5 min ~ 15 min, and the filtration membrane of the centrifugal treatment is an organic phase filtration membrane, and the pore size of the organic phase filtration membrane is not larger than 0.45 µm. 9. The antioxidant and anti-aging application of a Dendrobium officinale polysaccharide extract based on a preparation process according to any one of claims 4 to 8, characterized in that it comprises a skin care product made from the Dendrobium officinale polysaccharide extract for use in the antioxidant and anti-aging repair of human skin. 10. The antioxidant and anti-aging application of Dendrobium officinale polysaccharide extract according to claim 9, characterized in that the Dendrobium officinale polysaccharide extract is also used for cytotoxicity tests, patch tests of human skin or routine cosmetic tests.