Composition based on dendrobium nobile, preparation method of composition and application of composition in preparation of soft capsules, buccal tablets and oral soluble film preparations

Abstract

The invention relates to the technical field of biological medicine, in particular to a composition based on dendrobium nobile, a preparation method of the composition and application of the composition to preparation of soft capsules, buccal tablets and oral soluble film preparations, the composition takes dendrobium nobile extract as a main active ingredient, and can be matched with auxiliary materials such as excipients, disintegrating agents and film-forming agents to be optimized into different dosage forms. The soft capsule has a slow release effect, the buccal tablet is convenient to carry and take, and the oral soluble film can realize rapid oral mucosa absorption. The preparation method disclosed by the invention is stable in process and suitable for industrial production, and the obtained preparation has good bioavailability and stability, can be widely applied to the field of health-care foods or medicines, and is particularly suitable for enhancing immunity, resisting oxidation, moistening lung, promoting salivation and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to a composition based on Dendrobium nobile, a preparation method thereof and application of the composition in preparing soft capsules, buccal tablets and oral dissolving film preparations. BACKGROUND

[0002] Dendrobium nobile Lindl., a perennial herb of Orchidaceae Dendrobium, is a traditional and precious Chinese medicinal material recorded in Chinese Pharmacopoeia. Its main active ingredients are polysaccharides, alkaloids, flavonoids and phenolic acid compounds. Modern pharmacological studies have confirmed that it has multiple biological activities such as enhancing immunity, antioxidant, moistening lung and generating fluid, and reducing blood sugar.

[0003] In recent years, the development of medicinal and health care value of Dendrobium nobile has made much progress. For example, Chinese patent with authorization announcement number CN107569608B discloses a pharmaceutical composition for reducing blood sugar, which realizes the effect of significantly reducing postprandial blood sugar by combining gypenosides with Dendrobium nobile ultrafine powder in a specific ratio. Chinese patent with authorization announcement number CN109394977B discloses a traditional Chinese medicine composition with the function of reducing blood lipids, which solves the problem of limited effect of single component in reducing blood lipids by scientifically compounding gypenoside extract and Dendrobium nobile extract. In the field of skin care, Chinese patent with authorization announcement number CN115737754B discloses a traditional Chinese medicine composition with the dual effects of anti-allergy and anti-inflammation, which is applied to various skin care products by compounding Dendrobium nobile extract with extract of Privet and Artemisia leaf. Chinese patent with authorization announcement number CN114886813B discloses an anti-aging plant extraction composition, which improves the skin aging phenomenon by using the synergistic effect of Dendrobium nobile stem extract and extract of Magnolia liliflora and Gastrodia root.

[0004] However, the existing technology still has the following limitations in the development and application of Dendrobium nobile. Firstly, in the extraction process, most studies use traditional crushing, solvent soaking or simple enzymatic hydrolysis methods. For example, CN107569608B only uses ultrafine powder treatment, and CN109394977B does not optimize the extraction parameters, resulting in low dissolution rate of active ingredients, easy degradation of heat-sensitive components, and restriction of bioavailability. Secondly, the existing combination is mainly for single effect, and there is less exploration of the synergistic effect of Dendrobium nobile and other active ingredients in enhancing immunity and antioxidant.

[0005] Therefore, new solutions are needed to address the technical bottlenecks of existing Dendrobium nobile preparations in terms of extraction efficiency and efficacy synergy. SUMMARY

[0006] To solve the above technical problems, the present application aims to provide a composition based on Dendrobium nobile and a preparation method thereof and application thereof in preparing soft capsules, buccal tablets and oral dissolving film preparations, which realizes efficient reservation and purification of active ingredients through an innovative method; and based on the synergy between the Dendrobium nobile extract and other active ingredients, the antioxidant and immunomodulatory activities can be synergistically enhanced, thereby providing technical support for the diversification and industrial application of Dendrobium nobile in the field of health food and medicine.

[0007] To achieve the above technical effects, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a composition based on Dendrobium nobile, which at least comprises a Dendrobium nobile extract, and the Dendrobium nobile extract is prepared according to the following method:

[0009] S1: After the fresh stem of Dendrobium nobile is rapidly frozen in liquid nitrogen and vacuum freeze-dried, the Dendrobium nobile freeze-dried powder is obtained by crushing;

[0010] S2: The Dendrobium nobile freeze-dried powder is subjected to complex enzymolysis by cellulase-pectinase to obtain an enzymolysis product;

[0011] S3: The enzymolysis product is subjected to stepwise extraction: ultrasonic extraction is first performed with an ethanol solution with a volume concentration of 40-60%, and a first extraction liquid is separated; the residue is then subjected to decoction extraction, and a second extraction liquid is separated; and the two extraction liquids are combined to obtain a mixed extraction liquid;

[0012] S4: The mixed extraction liquid is subjected to membrane separation: large molecular impurities are removed by passing through a 90-100 kDa ultrafiltration membrane in sequence, and then concentrated by passing through a 1-3 kDa nanofiltration membrane to obtain a concentrated liquid;

[0013] S5: The concentrated liquid is dried to obtain the Dendrobium nobile extract.

[0014] Preferably, in S1, the rapid freezing temperature in liquid nitrogen is-196℃±2℃ and maintained for 3-5 min, and the vacuum freeze-drying conditions are-40℃ to-50℃ / 0.05-0.15 mbar for 20-28 h, and the crushing particle size is passed through a 80-100 mesh sieve.

[0015] Preferably, in S2, the addition amount of the complex enzyme is 2-3% of the mass of the Dendrobium nobile freeze-dried powder, and the mass ratio of cellulase to pectinase in the complex enzyme is 1:1.5-1:3, the cellulase activity is 8000-15000 U / g, the pectinase activity is 15000-25000 U / g, the enzymolysis pH value is 4.8-5.5, the temperature is 45-55℃, and the time is 1.5-2.5 h.

[0016] Most preferably, the cellulase activity is 12000 U / g, and the pectinase activity is 20000 U / g.

[0017] Preferably, the 50% ethanol ultrasonic extraction conditions in S3 are frequency 35-45 kHz, temperature 45-55℃, time 25-35 min, and solid-liquid ratio 1:10-1:20 (w / v), and the hot water extraction conditions are time 0.8-1.2 h, solid-liquid ratio 1:10-1:20 (w / v), and temperature 65-75℃.

[0018] Preferably, the molecular weight cut-off of the ultrafiltration membrane in S4 is 90-100 kDa, and the operating pressure is 0.2-0.5 MPa, and the molecular weight cut-off of the nanofiltration membrane is 1-3 kDa, and the concentration is to 15-25% of the original volume.

[0019] Preferably, the vacuum freeze-drying conditions in S5 are pre-freezing temperature -40℃ to -50℃ / 2 h, sublimation drying -20℃ to 0℃ / 10 Pa, and analytical drying 20℃ to 30℃ / 5 Pa, until the moisture content is ≤5%.

[0020] In a second aspect, the present application provides a composition based on Dendrobium nobile, which is composed of Dendrobium nobile extract and epigallocatechin gallate in a mass ratio of 5:1 to 9:1.

[0021] In addition, the present application further provides a use of the composition provided in the foregoing second aspect in the preparation of food, health products, or drugs.

[0022] Further, the use is to prepare the composition into soft capsules, buccal tablets, or oral film preparations together with pharmaceutical excipients.

[0023] Further, the present application further provides a preparation method of the foregoing oral film product, which is as follows: the oral film is prepared by dissolving hydroxypropyl methyl cellulose 3-5 parts, the composition 8-12 parts, and silicon dioxide 0.3-0.8 parts in water to prepare a film-forming solution, casting a wet film with a thickness of 400-600 μm, drying at 45-55℃ for 8-12 min, and cutting into film pieces.

[0024] Further, the present application further provides a preparation method of the foregoing soft capsules, which is as follows: the composition is mixed with pharmaceutical excipients to prepare the content, which is filled into gelatin-glycerin capsule shells, has good sealing performance and can mask bad odor, is suitable for long-term storage and gastrointestinal target release.

[0025] Preferably, the pharmaceutical excipients are selected from any one or more of soybean oil and beeswax.

[0026] Further, the present application further provides a preparation method of the foregoing buccal tablets, which is as follows: the composition is used as the core, and fillers (mannitol, xylitol), binders (PVP), flavoring agents (menthol), and lubricants (magnesium stearate) are added, and the buccal tablets are prepared through the granulation and tabletting process, can be absorbed through the oral mucosa to quickly take effect, and are suitable for throat health care or whole-body conditioning.

[0027] Preferably, the filler is any one of mannitol, xylitol, the binder is PVP, the flavoring agent is menthol, and the lubricant is magnesium stearate.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] The present application provides a composition based on Dendrobium nobile, a preparation method thereof and application of the composition in preparation of soft capsules, buccal tablets and oral dissolving film preparations. First, the present application realizes the synergistic dissolution of Dendrobium nobile polysaccharides, flavones and dendrobium alkaloids through a stepwise ultrasonic-thermal water combined extraction process, greatly increases the total amount of active ingredients compared with the traditional single water extraction process, and avoids the volatilization of dendrobium alkaloids and the degradation of flavones caused by high-temperature decoction.

[0030] Secondly, the present application provides a composition by compounding the Dendrobium nobile extract obtained by the aforementioned stepwise ultrasonic-thermal water combined extraction process with EGCG, solves the balance problem of EGCG stability and functional synergy, and specifically, the present application solves the contradiction between high stability and high activity by the original extract-EGCG ratio design, through the hydrogen bond protection of flavones, the steric hindrance effect of polysaccharides and the promotion of EGCG transmembrane transport by both of them, the stability of the composition is significantly enhanced compared with single EGCG group, and the antioxidant activity is greatly improved compared with single extract, realizing the double advantages of "protection-activity". In the composition, the pharmacokinetic study shows that the present application can significantly increase the in vivo exposure of EGCG and effectively improve the bioavailability. DETAILED DESCRIPTION

[0031] The embodiments of the technical scheme of the present application will be described in detail below. The following embodiments are only used to more clearly illustrate the technical scheme of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.

[0032] Those skilled in the art should understand that the present application can also be implemented without certain specific details. In some other embodiments, methods, means, apparatuses and steps familiar to those skilled in the art are not described in detail, in order to highlight the main idea of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. Unless otherwise specified, the units used in the specification are international standard units, and the numerical values and numerical ranges appearing in the present application should be understood to include the systematic errors inevitable in industrial production.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials, reagents or instruments used are commercially available reagents and materials, and the specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The source of the raw materials used in the present application is not limited, and the raw materials used in the present application are ordinary commercially available products in the technical field unless otherwise specified. The "ratio" referred to in the following examples means the ratio of the mass parts unless otherwise specified.

[0034] It should be particularly noted that all the abbreviations involved in the present application and their corresponding full names are as follows: HPLC (high performance liquid chromatography), LC-MS / MS (liquid chromatography-tandem mass spectrometry), DPPH (1,1-diphenyl-2-trinitrophenylhydrazine radical), EC 50 (50% effective concentration), AUC 0-24 (0-24 hour blood concentration-time curve area), C max (peak blood concentration), T max (time to peak), HPMC (hydroxypropyl methyl cellulose), PVP (polyvinyl pyrrolidone), EGCG (epigallocatechin gallate), SD rat (Sprague-Dawley rat), RH (relative humidity), CV value (coefficient of variation), D90 (particle size at which the cumulative distribution reaches 90%); process parameter abbreviations include: U / g (unit / gram), kDa (kilodalton), MPa (megapascal), rpm (revolutions per minute), μm (micrometer), ℃ (Celsius), and all abbreviations conform to the technical specifications in the art.

[0035] Example 1

[0036] The purpose of the present embodiment is to provide a dendrobium candidum extract and a preparation method thereof, which are as follows:

[0037] S1: Fresh stem pretreatment and freeze-dried crushing

[0038] Take 1 kg of fresh stems of dendrobium candidum, place them in a liquid nitrogen environment, and freeze them at -196℃ for 4 minutes, then transfer them to a vacuum freeze dryer, and treat them at -45℃ and 0.1 mbar for 24 hours. After freeze-drying, crush them with a super micro crusher, and pass them through a 100-mesh sieve to obtain dendrobium candidum freeze-dried powder.

[0039] S2: Cellulase-pectinase complex enzymolysis

[0040] The freeze-dried powder of D. nobile Lindl. was dispersed in acetic acid-sodium acetate buffer solution with pH 5.0 at a material-to-liquid ratio of 1:15 (mass-to-volume ratio, unit: g / mL), and a compound enzyme (consisting of cellulase (activity: 12000 U / g) and pectinase (activity: 20000 U / g) with a mass ratio of 1:2) was added, with the amount of the compound enzyme being controlled to be 3% of the mass of the raw material (3 g of the compound enzyme was added for every 100 g of the freeze-dried powder). The enzyme hydrolysis was performed at 50°C in a water bath for 2 hours, and an enzyme hydrolysate was obtained.

[0041] S3: Ladder-type ultrasonic-thermal water combined extraction

[0042] The enzyme hydrolysate was subjected to ladder-type extraction. First, 50% volume concentration of ethanol solution was added (material-to-liquid ratio of 1:15, mass-to-volume ratio, unit: g / mL, i.e., 15 mL of the ethanol solution was added for every 1 g of the enzyme hydrolysate, and the same was applied in other examples and comparative examples), and the extraction was performed at an ultrasonic frequency of 40 kHz and a temperature of 50°C for 30 minutes. The first extraction liquid was separated by filtration through filter cloth. Deionized water was added to the residue (material-to-liquid ratio of 1:15, mass-to-volume ratio, i.e., 15 mL of the deionized water was added for every 1 g of the enzyme hydrolysate, and the same was applied in other examples and comparative examples), and the extraction was performed by boiling in a water bath at 70°C for 1 hour. The second extraction liquid was separated by filtration. The two extraction liquids were combined, and impurities were removed by filtration through 400-mesh filter cloth, and a mixed extraction liquid was obtained.

[0043] S4: Membrane separation and purification

[0044] The mixed extraction liquid was subjected to ultrafiltration through an ultrafiltration membrane with a molecular weight cut-off of 95 kDa (operating pressure: 0.35 MPa), and macromolecular impurities were removed. The ultrafiltration permeate was collected.

[0045] The ultrafiltration permeate was further subjected to nanofiltration through a nanofiltration membrane with a molecular weight cut-off of 2 kDa, and the retentate was collected as a concentrated liquid.

[0046] S5: Vacuum freeze-drying

[0047] The concentrated liquid was subjected to vacuum freeze-drying. The pre-freezing temperature was -45°C for 2 hours, the sublimation drying stage was -10°C for 10 Pa for 12 hours, and the desorption drying stage was 25°C for 5 Pa for 8 hours. Finally, about 7.0 g of D. nobile Lindl. extract powder was obtained, and the moisture content was 4.2%.

[0048] Example 2

[0049] With reference to Example 1, the purpose of the present example is to provide a D. nobile Lindl. extract and a preparation method thereof, which are as follows:

[0050] S1: Fresh stem pretreatment and freeze-drying and crushing

[0051] Take fresh stems of Dendrobium nobile Lindl. 1 kg, and freeze them in liquid nitrogen at -196 ℃ for 4 minutes. Then, transfer them to a vacuum freeze dryer, and treat them at -50 ℃ and 0.05 mbar for 28 hours. After freeze-drying, crush the freeze-dried powder by using a super micro grinder, and pass it through a 80-mesh sieve.

[0052] S2: Enzymatic hydrolysis by cellulase-pectinase complex

[0053] Disperse the freeze-dried powder of Dendrobium nobile Lindl. in a pH 4.8 acetic acid-sodium acetate buffer according to a solid-liquid ratio of 1:20 (mass / volume ratio, g / mL), and add a cellulase-pectinase complex (mass ratio 1:1.5) composed of cellulase (activity 8000 U / g) and pectinase (activity 15000 U / g). The cellulase-pectinase complex is added in an amount of 3% of the mass of the raw material. According to the mass of the freeze-dried powder, 3 g of the cellulase-pectinase complex is added per 100 g of the freeze-dried powder. Perform enzymatic hydrolysis at 45 ℃ for 2.5 hours in a water bath with shaking, and obtain an enzymatic hydrolysate.

[0054] S3: Stepwise ultrasonic-thermal water combined extraction

[0055] Extract the enzymatic hydrolysate: first, add 50% (volume concentration) ethanol solution (solid-liquid ratio 1:10, mass / volume ratio, g / mL), and extract at an ultrasonic frequency of 35 kHz and 45 ℃ for 35 minutes. Filter and separate the first extraction liquid. Add deionized water (solid-liquid ratio 1:10, mass / volume ratio, g / mL) to the residue, and boil at 70 ℃ for 1.2 hours in a water bath. Filter and separate the second extraction liquid. Combine the two extraction liquids, filter through a 400-mesh filter cloth to remove impurities, and obtain a mixed extraction liquid.

[0056] S4: Membrane separation and purification

[0057] Pass the mixed extraction liquid through an ultrafiltration membrane with a molecular weight cut-off of 95 kDa (operating pressure 0.35 MPa) to remove macromolecular impurities, and collect the ultrafiltration permeate.

[0058] Then, pass the ultrafiltration permeate through a nanofiltration membrane with a molecular weight cut-off of 2 kDa, and collect the retentate as a concentrated liquid.

[0059] S5: Vacuum freeze-drying

[0060] Vacuum freeze-dry the concentrated liquid: pre-freeze at -40 ℃ for 2 hours, sublimation drying at -20 ℃ and 10 Pa for 10 hours, and desorption drying at 30 ℃ and 5 Pa for 6 hours. Finally, obtain about 6.4 g of Dendrobium nobile Lindl. extract powder, with a moisture content of 4.8%.

[0061] Comparative Example 1

[0062] The purpose of this comparative example is to provide a Dendrobium nobile Lindl. extract and a preparation method thereof, which uses ultrasonic extraction twice, as follows:

[0063] S1-S2 steps are same as Example 1, and the enzymatic product is prepared.

[0064] S3: hot water extraction

[0065] The enzymatic product is only subjected to hot water extraction: deionized water is added (1:15 of solid-liquid ratio, mass-volume ratio, g / mL), and the mixture is boiled in a water bath at 70°C for 1 hour to obtain a first extraction liquid; the residue is again added with deionized water (1:15 of solid-liquid ratio, mass-volume ratio, g / mL), and the mixture is boiled in a water bath at 70°C for 1 hour to obtain a second extraction liquid; the two extraction liquids are combined, and impurities are removed by filtration through a 400-mesh filter cloth to obtain a mixed extraction liquid.

[0066] S4-S5 are same as Example 1, and finally about 5.8 g of D. candidum extract powder is obtained, with a moisture content of 4.4%.

[0067] Comparative Example 2

[0068] The purpose of the present comparative example is to provide a D. candidum extract and a preparation method thereof, which adopts hot water extraction twice at 70°C, and the preparation method is as follows:

[0069] S1-S2 steps are same as Example 1, and the enzymatic product is prepared.

[0070] S3: hot water extraction

[0071] The enzymatic product is only subjected to hot water extraction: deionized water is added (1:15 of solid-liquid ratio, mass-volume ratio, g / mL), and the mixture is boiled in a water bath at 70°C for 1 hour to obtain a first extraction liquid; the residue is again added with deionized water (1:15 of solid-liquid ratio, mass-volume ratio, g / mL), and the mixture is boiled in a water bath at 70°C for 1 hour to obtain a second extraction liquid; the two extraction liquids are combined, and impurities are removed by filtration through a 400-mesh filter cloth to obtain a mixed extraction liquid.

[0072] S4-S5 are same as Example 1, and finally about 5.8 g of D. candidum extract powder is obtained, with a moisture content of 4.4%.

[0073] Comparative Example 3

[0074] S1-S2 steps are same as Example 1, and the enzymatic product is prepared.

[0075] S3: hot water extraction at 80°C

[0076] High-temperature hot water extraction only: deionized water was added (1:15, mass / volume ratio, g / mL) and the mixture was boiled at 80°C for 1 hour. The first extract was obtained by filtration through filter cloth. The residue was again extracted with deionized water (1:15, mass / volume ratio, g / mL) at 80°C for 1 hour, and the second extract was obtained by filtration. The two extracts were combined and filtered through 400-mesh filter cloth to remove impurities to obtain a mixed extract.

[0077] S4-S5 were the same as in Example 1, and about 5.5 g of D. candidum extract powder was finally obtained, with a moisture content of 4.5%.

[0078] Comparative Example 4

[0079] The purpose of this comparative example was to provide a D. candidum extract and a preparation method thereof, which used 100°C decoction extraction twice, as follows:

[0080] S1-S2 were the same as in Example 1, and an enzymatic product was prepared.

[0081] S3: 100°C decoction extraction

[0082] High-temperature hot water extraction only: deionized water was added (1:15, mass / volume ratio, g / mL) and the mixture was boiled at 80°C for 1 hour. The first extract was obtained by filtration through filter cloth. The residue was again extracted with deionized water (1:15, mass / volume ratio, g / mL) at 80°C for 1 hour, and the second extract was obtained by filtration. The two extracts were combined and filtered through 400-mesh filter cloth to remove impurities to obtain a mixed extract.

[0083] S4-S5 were the same as in Example 1, and about 5.5 g of D. candidum extract powder was finally obtained, with a moisture content of 4.5%.

[0084] Comparative Example 5

[0085] The purpose of this comparative example was to provide a D. candidum extract and a preparation method thereof, which used 60°C decoction extraction twice, as follows:

[0086] S1-S2 were the same as in Example 1, and an enzymatic product was prepared.

[0087] S3: 60°C decoction extraction

[0088] Low-temperature hot water extraction only: deionized water was added (1:15, mass / volume ratio, g / mL) and the mixture was boiled at 60°C for 1 hour. The first extract was obtained by filtration through filter cloth. The residue was again extracted with deionized water (1:15, mass / volume ratio, g / mL) at 60°C for 1 hour, and the second extract was obtained by filtration. The two extracts were combined and filtered through 400-mesh filter cloth to remove impurities to obtain a mixed extract.

[0089] S4-S5 Same as Example 1, finally obtained about 5.0 g of D. candidum extract powder, moisture content 4.5%.

[0090] Test Example 1

[0091] The purpose of this test example is to observe the effects of different extraction processes on the retention of active ingredients, EGCG stability and bioavailability of D. candidum extract, specifically:

[0092] 1.1 Determination of active ingredient content

[0093] Sample source: D. candidum extract powder of Examples 1-2 (ladder type ultrasonic- hot water combined extraction), Comparative Examples 1-5 (single extraction process).

[0094] Detection index: polysaccharide (phenol-sulfuric acid method), total flavonoids (aluminum salt colorimetric method), and shihua alkali (HPLC method).

[0095] Experimental repetition: 3 parallel experiments for each group, the results are expressed as "mean ± standard deviation", and the experimental results are shown in Table 1:

[0096] Table 1 Comparison of active ingredient content of extracts from different extraction processes (mass percentage, %)

[0097] Group Polysaccharide content Total flavonoid content Shihualine content Example 1 48.7±1.2 18.3±0.8 2.8±0.1 Example 2 45.2±1.1 16.5±0.7 2.5±0.2 Comparative Example 1 35.2±0.9 15.1±0.6 2.1±0.1 Comparative Example 2 42.6±1.0 8.7±0.4 0.9±0.1 Comparative Example 3 36.4±1.1 9.2±0.4 1.3±0.1 Comparative Example 4 25.3±1.2 2.1±0.3 0.5±0.1 Comparative Example 5 39.8±0.8 7.3±0.5 0.7±0.1

[0098] The above experimental results show that different extraction processes have significant differences in the retention effect of active ingredients in D. candidum extract. The stepwise ultrasonic- hot water combined extraction process realizes the synergistic retention of active ingredients in D. candidum through stepwise optimization of ethanol phase (50%) and water phase (70°C). The polysaccharide (48.7%), total flavonoids (18.3%) and shihua alkali (2.8%) contents of Example 1 are significantly higher than those of the single extraction process group, and this result is due to the complementary dissolution capacity of the two-phase solvent for different polarity components: the ethanol phase can efficiently dissolve fat-soluble flavonoids and alkaloids, while the 70°C water phase can fully extract water-soluble polysaccharides. Experimental data further reveals the key influence of temperature control on component retention: the flavonoid content of Comparative Example 2 (70°C water extraction) is only 48% of Example 1, the flavonoid loss rate of Comparative Example 3 (80°C water extraction) is more than 50%, and the shihua alkali content of Comparative Example 4 (100°C decoction) is the lowest due to high temperature. In contrast, the stepwise process controls the degradation rate of heat-sensitive components (such as flavonoids and shihua alkali) to within 15% by strictly controlling the ethanol phase temperature ≤55°C and the water phase temperature ≤70°C, while avoiding the polysaccharide deficiency (35.2% vs 48.7%) caused by the lack of hot water dissolution step in single ultrasonic extraction (Comparative Example 1). This process not only solves the component imbalance problem of traditional single solvent extraction, but also further destroys the plant cell wall structure through the pretreatment steps of ultrafine grinding (100 mesh) and composite enzymolysis (cellulase-pectinase mass ratio 1:2), which improves the dissolution efficiency of active ingredients and lays a foundation for the subsequent membrane separation and purification and freeze-drying process of high-quality raw materials.

[0099] 1.2 EGCG stability and antioxidant synergistic effect experiment

[0100] Sample treatment: Mix each extract (including Examples 1-2, Comparative Examples 1-5) with EGCG (commercial product, purity ≥98%) according to a mass ratio of 7:3 to prepare a composite, and at the same time, add:

[0101] Single control group 1 (only D. candidum extract of Example 1, without commercial EGCG);

[0102] Single control group 2 (only commercial EGCG, without D. candidum extract);

[0103] Proportion adjustment group 1 (according to mass fraction, D. candidum extract (Example 1): EGCG = 1:9);

[0104] Proportion adjustment group 2 (according to mass fraction, D. candidum extract (Example 1): EGCG = 3:7)

[0105] Ratio adjustment group 3 (Dendrobium candidum extract (Example 1): EGCG = 5:5 by mass fraction)

[0106] Experimental conditions: Accelerated test conditions were used: 40°C, relative humidity 75% environment for 30 days.

[0107] Detection index: EGCG retention rate (HPLC method), DPPH free radical scavenging activity (EC 50 value), and the experimental results are shown in Table 2.

[0108] Table 2: Stability and antioxidant activity of extract-EGCG compound

[0109]

[0110]

[0111] The above experimental results show that:

[0112] The experimental data reveals a clear positive correlation between the extract ratio and the stability of EGCG: the 7:3 ratio of extract to EGCG (Example 1) exhibits the optimal balance of "stability-activity", and the core mechanism lies in the multi-dimensional protective network formed by the active ingredients of Dendrobium candidum. The experimental data shows that the EGCG retention rate increases linearly with the increase of the extract ratio (R 2 = 0.97): when the extract ratio increases from 10% (1:9) to 90% (9:1), the retention rate increases from 70.2% to 95.1%, which is due to the phenolic hydroxyl donor function of flavonoids - flavonoids form intermolecular hydrogen bonds with EGCG, shielding the oxidation-sensitive sites of its ortho-diphenol hydroxyl groups, while the steric hindrance effect of polysaccharides reduces the contact probability of EGCG with oxygen molecules. However, the antioxidant activity shows the opposite trend: as the concentration of EGCG decreases, the DPPH scavenging EC 50 value increases from 3.0 μg / mL (1:9 ratio) to 9.0 μg / mL (9:1 ratio), indicating that high concentration of EGCG is the basis for maintaining strong antioxidant activity. It is worth noting that the 7:3 ratio (Example 1) achieves a precise balance between the two: the EGCG retention rate is 91.7% (close to 95.1% of the 9:1 group), and the DPPH scavenging activity (EC 50 = 5.3 μg / mL) is nearly 1 times higher than that of the single extract (10.5 μg / mL). The comparative experiment further confirms that the high-temperature extraction group (such as Comparative Example 3, 80°C water extraction) has a flavonoid content of only 9.2% (50% of Example 1), and its EGCG retention rate (55.0%) and antioxidant activity (EC 50= 20.5 pg / mL) significantly decreased, verifying the necessity of the ladder process for flavone retention. In addition, the retention rate of the single control group 2 (only EGCG) was only 65.0%, directly proving that the D. candidum extract is a key protective factor for EGCG stability, and the 7:3 ratio is the optimal choice that takes into account "protection efficiency" and "activity contribution".

[0113] 1.3 Bioavailability experiment

[0114] Animal model: SD rats (male, 200 ± 20 g), 6 in each group.

[0115] Group setting: including Example 1 (D. candidum extract: EGCG = 7:3 by mass fraction), single control group 2 (only commercial EGCG, without D. candidum extract), and:

[0116] Proportion adjustment group 1 (D. candidum extract (Example 1): EGCG = 1:9 by mass fraction);

[0117] Proportion adjustment group 2 (D. candidum extract (Example 1): EGCG = 3:7 by mass fraction)

[0118] Proportion adjustment group 3 (D. candidum extract (Example 1): EGCG = 5:5 by mass fraction) Proportion adjustment group 4 (D. candidum extract (Example 1): EGCG = 9:1 by mass fraction);

[0119] Sample solution preparation: accurately weigh each composition (containing 100 mg of total EGCG), add 20 mL of 0.5% carboxymethylcellulose sodium (CMC-Na) solution, vortex for 5 min to prepare a sample solution with an EGCG content of 5 mg / mL;

[0120] Dosing method: each group of rats was given sample solution by gavage, the dosing volume was 10 mL / kg, the blood sampling time points were 0.25, 0.5, 1, 2, 4, 8, 12, 24 h, the single blood sampling volume was about 0.3 mL / time, the plasma EGCG concentration (LC-MS / MS method) was determined, and the pharmacokinetic parameters were calculated.

[0121] The experimental results are shown in Table 3:

[0122] Table 3: Pharmacokinetic parameters of rats orally taking extract-EGCG compound

[0123] Group AUC 0-24 (h-μg / mL) C max (μg / mL) T max (h)]]> Example 1 19.3±1.5 3.2±0.2 1.5 Proportion adjustment group 1 8.2±0.6 1.8±0.1 1.2 Proportion adjustment group 2 12.1±0.9 2.5±0.2 1.3 Proportion adjustment group 3 15.7±1.1 2.9±0.2 1.4 Proportion adjustment group 4 18.5±1.3 3.0±0.2 1.6 Single control group 2 6.5±0.5 1.5±0.1 1.0

[0124] The above experimental results show that the rat pharmacokinetic study reveals the synergistic promotion effect of the ladder extraction process and ratio optimization on the in vivo absorption of EGCG. The AUC of Example 1 group (7:3 ratio)0-24 The value (19.3 h pg / mL) was 3 times that of the single EGCG group (6.5 h pg / mL), and the C max (3.2 pg / mL) and T max (1.5 h) was also significantly better than other ratio groups, and this improvement was due to the synergistic effect of three mechanisms: first, the intestinal metabolic enzyme (UGT1A1) activity was specifically inhibited by the water-phase extracted Dendrobium polysaccharides, which prolonged the retention time of EGCG in the body (T max 0.5 h longer than the single EGCG group); second, the ethanol-phase dissolved Dendrobium alkaloids could open the tight junctions of intestinal epithelial cells, increasing the permeability of the mucosa, which reduced the AUC value of Comparative Example 1 (ultrasonic extraction only, Dendrobium alkaloids 2.1%) by 34% compared with Example 1; finally, the complex formed by flavonoids and EGCG could improve the absorption efficiency through the receptor-mediated transmembrane transport pathway (e.g., the AUC value of the 5:5 ratio of Comparative Adjustment Group 3 was 15.7 h pg / mL, which was significantly higher than the 12.1 h pg / mL of the 3:7 ratio). A key phenomenon was observed in the ratio experiment: when the extract ratio increased to 90% (9:1 group), although the AUC value (18.5 h pg / mL) was close to that of Example 1, the C max significantly decreased (3.0 vs 3.2 pg / mL), indicating that the contribution of high-concentration EGCG to rapid absorption could not be replaced. The bioavailability of the comparative groups was generally low due to the absence or destruction of components: the AUC value of Comparative Example 2 (70°C water extraction, flavonoids 8.7%) was only 33% of that of Example 1, and the AUC value of Comparative Example 4 (100°C decoction) was only 5.1 h pg / mL due to the severe degradation of active ingredients, further verifying the necessity of the process temperature threshold (water phase ≤ 70°C) and the ratio optimization (7:3).

[0125] In summary, the stepwise ultrasonic-thermal water combined extraction process, through the innovative design of "two-phase solvent step-by-step extraction + temperature threshold control", systematically solves the problems of low retention rate of active ingredients and poor functional synergy in traditional extraction methods. In terms of component retention, the step-by-step extraction of ethanol phase (≤ 55°C) and water phase (≤ 70°C) achieves the synergistic dissolution of flavonoids, alkaloids, and polysaccharides, avoiding the selectivity limitations of single solvents and the degradation of heat-sensitive components (e.g., Dendrobium alkaloids were not detected in the 100°C decoction group); in terms of functional synergy, the 7:3 extract-EGCG ratio balances stability (retention rate 91.7%) and antioxidant activity (EC 50= 5.3 pg / mL), the core mechanism is the hydrogen bond protection of flavonoids, the steric hindrance effect of polysaccharides and the promotion of EGCG transmembrane transport; at the level of bioavailability, the extract prepared by the ladder process makes the AUC value of EGCG reach 3 times of the single EGCG group through the triple effect of delaying metabolism, enhancing penetration and optimizing transport, and the rapid dissolution characteristics (2.8 seconds) of the oral dissolving film dosage form meet the clinical needs of oral administration. Based on the experimental results, the core value of this process is reflected in three dimensions: first, the temperature-sensitive threshold (water phase ≤ 70°C) is determined, which provides a key parameter for industrial production; second, the functional ratio of 7:3 is verified, which lays a theoretical foundation for the development of compound preparations; third, the integration optimization of "component retention-function synergy-dosage form adaptation" is achieved, which provides a popular technical paradigm for the efficient use of active ingredients of natural products.

[0126] Example 3

[0127] On the basis of the above examples and comparative examples, the purpose of this example is to provide a preparation method of the aforementioned composition, which is a soft capsule, as follows:

[0128] Raw materials: the content includes the Dendrobium nobile Lindl. extract-EGCG composition (mass ratio 7:3) prepared in Example 1 20 g, soybean oil 70 g, and beeswax 10 g; the capsule shell is composed of gelatin 40 g, glycerol 10 g, pure water 50 g, and titanium dioxide 0.5 g (light shielding agent).

[0129] Preparation method: when preparing the content, melt the soybean oil and beeswax in a 38°C water bath, add the composition powder, stir with a homogenizer at 8000 rpm for 10 minutes, and then treat with vacuum degassing (-0.08 MPa) to obtain a homogeneous suspension. When preparing the capsule shell, mix and swell the gelatin, glycerol and water, then dissolve them by stirring at 65°C, add titanium dioxide and mix evenly, and then keep warm and stand for degassing. Use a rotary soft capsule machine to press the pills and shape them, control the content temperature at 40°C and the capsule shell temperature at 55°C, and press them into pills through a 8 mm aperture mold. The wet pills are first shaped at 25°C and RH 30% for 4 hours, then transferred to a 20°C drying room to balance the moisture content to 6-8%, and finally packaged with aluminum plastic.

[0130] Example 4

[0131] On the basis of the above examples and comparative examples, the purpose of this example is to provide a preparation method of the aforementioned composition, which is a soft capsule, as follows:

[0132] Formulation: contains the core composition prepared in Example 1 30 g, filler mannitol 45 g, xylitol 10 g, adhesive 5% PVP ethanol solution 20 g, flavoring agent menthol 0.5 g, and lubricant magnesium stearate 1 g.

[0133] Preparation method: granulation stage, the composition, mannitol, xylitol was mixed after 80 mesh sieve, the mixture was obtained; 5 g PVP was dissolved in ethanol, and the final volume of the solution was 100 ml, 5% PVP ethanol solution was prepared, and the 5% PVP ethanol solution was used as the binder, and the mixture was mixed with the binder in the fluidized bed granulator (inlet air temperature 50 ℃, atomization pressure 0.2 MPa) to obtain wet granules. The wet granules were dried at 45 ℃ to a moisture content of 2.45 wt%, sieved through a 30 mesh sieve to obtain dry granules. Then menthol was dissolved in 3 ml of ethanol to obtain a flavoring solution, which was then uniformly sprayed onto the surface of the prepared dry granules, and then entered the total mixing stage, at which the formula amount of magnesium stearate was added, mixed with a three-dimensional mixer at 20 rpm for 10 minutes, and then compressed into tablets by a rotary tablet press, with a tablet weight of 500 mg and a hardness of 55±5 N.

[0134] Example 5

[0135] On the basis of the above examples and comparative examples, the purpose of this example is to provide a preparation method of the aforementioned formulation, which is a mouth dissolving film, as follows:

[0136] Formulation: composed of hydroxypropyl methyl cellulose (HPMC E5) 4 g, the composition prepared in Example 1 10 g, silicon dioxide 0.5 g and pure water 85.5 g.

[0137] Preparation method: when preparing the film-forming solution, HPMC was dispersed in pure water at 60 ℃ for swelling for 30 minutes, and magnetically stirred (300 rpm) until completely dissolved; the composition and silicon dioxide were added, treated with a homogenizer at 5000 rpm for 5 minutes, and then vacuum degassed (-0.09 MPa) for 30 minutes. In the casting and drying stage, the film-forming solution was cast onto a PET film substrate, the wet film thickness was controlled at 500 μm, and the film was dried at 50 ℃ with hot air at a wind speed of 1 m / s for 10 minutes, and then cut into 2×2 cm squares when the moisture content of the dry film was controlled at 3.76 wt%. Each piece contains 20 mg of the composition, and is stored in an aluminum foil bag in the dark.

[0138] It should be particularly noted that the three formulations all take the product of the stepwise extraction process of Example 1 (Dendrobium candidum extract-EGCG 7:3 compound) as the core ingredient, and realize functional adaptation through the optimization of excipients: the soft capsules focus on gastrointestinal targeted release, the buccal tablets focus on taste and rapid disintegration, and the mouth dissolving film realizes rapid absorption by the oral mucosa, providing differentiated choices for different administration scenarios (such as patients with difficulty swallowing, rapid onset needs).

[0139] The above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the present application, and all should be covered in the scope of the claims of the present application. The technical, shape and structure parts not described in detail in the present application are all known technologies.

Claims

1. A Dendrobium nobile-based composition, characterized in that, At least comprising Dendrobium nobile Lindl extract, the Dendrobium nobile Lindl extract is prepared according to the following method: S1: taking fresh stem of Dendrobium nobile Lindl, freezing in liquid nitrogen, vacuum freeze-drying, and crushing to obtain Dendrobium nobile Lindl freeze-dried powder; S2: the Dendrobium nobile Lindl freeze-dried powder is subjected to enzymolysis by cellulase-pectinase compound enzyme to obtain an enzymolysis product; S3: the enzymolysis product is subjected to step extraction: first ultrasonic extraction with 40-60% (volume concentration) ethanol solution to separate the first extraction liquid; the residue is subjected to decoction extraction to separate the second extraction liquid; and the two extraction liquids are combined to obtain a mixed extraction liquid; S4: the mixed extraction liquid is subjected to membrane separation: sequentially removing macromolecular impurities through 90-100 kDa ultrafiltration membrane, and then concentrating through 1-3 kDa nanofiltration membrane to obtain a concentrated liquid; S5: the concentrated liquid is dried to obtain the Dendrobium nobile Lindl extract.

2. A Dendrobium nobile-based composition as claimed in claim 1, wherein, In the S1, the liquid nitrogen freezing temperature is-196℃±2℃ and maintained for 3-5 min, and the vacuum freeze-drying conditions are-40℃ to-50℃ / 0.05-0.15 mbar for 20-28 h, and the crushing particle size is passed through an 80-100 mesh sieve.

3. The Dendrobium nobile-based composition of claim 1, wherein the Dendrobium nobile-based composition is a composition for improving skin elasticity and skin firmness. In the S2, the cellulase-pectinase compound enzyme is added in an amount of 2-3% of the mass of the Dendrobium nobile Lindl freeze-dried powder, the mass ratio of cellulase to pectinase in the compound enzyme is 1:1.5-1:3, the cellulase activity is 8000-15000 U / g, the pectinase activity is 15000-25000 U / g, the enzymolysis pH value is 4.8-5.5, the temperature is 45-55℃, and the time is 1.5-2.5 h.

4. The Dendrobium nobile-based composition of claim 1, wherein the Dendrobium nobile-based composition is a composition for improving skin elasticity and skin firmness. In the S3, the 50% ethanol ultrasonic extraction conditions are frequency 35-45 kHz, temperature 45-55℃, time 25-35 min, and solid-liquid ratio 1:10-1:20 (w / v), and the 65-75℃ hot water extraction conditions are time 0.8-1.2 h and solid-liquid ratio 1:10-1:20 (w / v).

5. The Dendrobium nobile-based composition of claim 1, wherein the Dendrobium nobile-based composition is a composition for improving skin elasticity and skin firmness. In the S4, the ultrafiltration membrane has a molecular weight cut-off of 90-100 kDa and an operating pressure of 0.2-0.5 MPa, and the nanofiltration membrane has a molecular weight cut-off of 1-3 kDa and is concentrated to 15-25% of the original volume.

6. A Dendrobium nobile-based composition as claimed in claim 1, wherein, In the S5, vacuum freeze-drying conditions are used, and the conditions are: pre-freezing temperature-40℃~-50℃ / 2h, sublimation drying-20℃~0℃ / 10Pa, desorption drying 20℃~30℃ / 5Pa, and water content≤5%.

7. The Dendrobium nobile-based composition of claim 1, wherein the Dendrobium nobile-based composition is a composition for improving skin elasticity. The composition is composed of the Dendrobium nobile Lindl extract and epigallocatechin gallate in a mass ratio of 5:1-9:

1.

8. Use of the Dendrobium nobile Lindl-based composition according to claim 7 in the preparation of food, health products or drugs.

9. Use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The use is to prepare the composition into soft capsules, buccal tablets or oral film preparations with pharmaceutical excipients.

10. The use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The preparation method of the oral film product is: the oral film is prepared by dissolving hydroxypropylmethyl cellulose 3-5 parts, the composition 8-12 parts, and silicon dioxide 0.3-0.8 parts in water to prepare a film-forming solution, casting a wet film with a thickness of 400-600 μm, drying at 45-55℃ for 8-12 min, and cutting into film pieces.

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