CN113694043B - Rhodiola rosea glycoside patch for treating muscular atrophy - Google Patents

Abstract

The invention discloses a salidroside transdermal absorption patch for treating amyotrophy and a preparation method thereof. The patch is prepared from salidroside, a framework material, a tackifier, a humectant, a cross-linking agent, a pH regulator and the like to obtain a carrier hydrogel matrix, and then the carrier hydrogel matrix is prepared through the steps of coating, cutting, standing and the like. Experiments prove that the salidroside patch can effectively inhibit the functions of the amyotrophic specific factor Atrogin-1 and the expression of fast muscle fibers in the amyotrophic process, thereby preventing and resisting the amyotrophy. Compared with the oral preparation of salidroside, the patch has the targeted therapeutic effect, directly transmits the medicinal active ingredients to diseased muscle tissues, and can slowly release the medicinal active ingredients for a long time. The patch is convenient and comfortable to use, and has no irritation or allergy to skin.

Description

A kind of salidroside patch for treating muscle atrophy

Technical areas:

The invention relates to the field of pharmacy, and specifically to a salidroside transdermal absorption patch for treating muscle atrophy and a preparation method thereof.

Background technique:

Under physiological conditions, the synthesis and degradation of skeletal muscle proteins are in a dynamic balance. However, clinical muscular dystrophy, disuse muscle atrophy (long-term bed rest, traumatic immobilization, weight loss muscle atrophy during space flight, etc.), denervated/denervated muscle atrophy, senile muscle atrophy, malignant diseases (such as advanced cancer) Cachexia, heart failure, renal failure, diabetes, chronic obstructive pulmonary disease, AIDS and long-term use of glucocorticoids and other diseases) late-stage muscle atrophy, etc., can all cause skeletal muscle protein metabolism imbalance, and the common characteristic manifestation is skeletal muscle weight Characteristic changes such as significant weight loss, reduction in muscle fiber cross-sectional area, and selective loss of muscle fiber type-related proteins (i.e., slow-twitch muscle fiber proteins and fast-twitch muscle fiber proteins) lead to severe muscle strength decline, movement disorders, fatigue, and metabolic disorders. Affect people's daily work and life.

It is currently recognized that when muscle atrophy occurs, the increased expression of the myotrophic factor Atrogin-1 and fast muscle fibers (MyHC-IIb) is an important cause of protein degradation and decreased muscle exercise endurance. Therefore, by inhibiting the expression of Atrogin-1 and MyHC-IIb, the occurrence and development of skeletal muscle atrophy can be prevented and treated, and the ability to work and live can be improved.

The inventor has discovered that Rhodiola rosea and its effective monomer component salidroside have the effect of resisting muscle atrophy, and has applied for the following patents:

The Chinese patent (ZL201210236548.7) "Application of Rhodiola Rosea in the Prevention and Treatment of Muscular Atrophy Diseases" discloses that the plant Rhodiola rosea as an active ingredient can be used to prepare drugs for the prevention and treatment of skeletal muscle atrophy diseases.

The Chinese patent (CN102727505A) "Application of salidroside in preventing and treating muscular atrophy" discloses that the extract of Rhodiola rosea, salidroside, has the effect of preventing and resisting the occurrence of muscular atrophy.

In the above patents, the administration route of Rhodiola rosea or salidroside is oral.

However, the inventor found in subsequent research that oral administration has poor targeting properties. Because the drug cannot be delivered directly to atrophic muscle tissue, salidroside is severely attenuated after liver metabolism under the first-pass effect of the liver, resulting in low concentration of active ingredients and a short duration of action in local muscle atrophy.

If the oral dose of salidroside is increased to achieve an effective drug concentration, unpredictable toxicological risks will arise. Especially for special audience groups such as astronauts or clinical patients who are bedridden for a long time, high-dose medication cannot guarantee the safety of medication. Moreover, it is difficult for astronauts working in space or long-term bedridden patients to take oral medications.

Therefore, local administration of salidroside, such as transdermal absorption preparations, is necessary.

There are also few preparations and applications of skin-based preparations for the treatment of muscle atrophy. Existing patents for patches that can combat muscle atrophy, such as the Chinese invention patent "Chinese medicine compositions, Chinese medicine ointments and external hot compress plasters for the treatment of muscle atrophy and weakness" (CN112057501A) discloses a patch for the treatment of muscle atrophy. Traditional Chinese medicine compositions, Chinese medicine ointments and external hot compress plaster patches for myasthenia and preparation methods thereof. However, the preparation method of Chinese medicine ointments is relatively complex and inconvenient to use.

There are currently no similar reports on patents related to a salidroside patch for treating muscle atrophy.

The salidroside transdermal absorption patch prepared by the invention is relatively simple to prepare, more convenient and comfortable to use, and has no irritation or allergy to the skin. It can avoid the first-pass effect of the liver and irritation to the gastrointestinal tract; it can effectively control the drug release rate, maintain stable blood drug concentration for a long time, and significantly reduce the frequency of administration and the incidence of side effects. If side effects occur, the administration can be interrupted in time. medicine, which greatly improves the safety of medication; for astronauts or long-term bedridden patients who are inconvenient to take it orally, the use of patch administration can greatly improve their medication compliance.

Contents of the invention:

The purpose of the present invention is to provide a salidroside patch for muscle atrophy, which solves the liver first-pass effect, short duration of drug action, and poor targeting of existing oral drugs, especially Western drugs, during the treatment of muscle atrophy. , the problem of inaccurate therapeutic effect on muscle atrophy caused by high side effects and inconvenience of taking.

The drug delivery system provided by the invention uses relatively low-cost sodium polyacrylate and polyvinylpyrrolidone as the matrix, and then adds penetration enhancers, antioxidants and active drugs to form a simple structure. The preparation process of the drug delivery system provided by the present invention is relatively simple, and the transdermal drug delivery system can still maintain sufficient stability.

The salidroside patch of the present invention consists of a drug-loaded hydrogel patch matrix and a backing layer, wherein the raw materials and proportions for preparing the drug-loaded hydrogel patch matrix are as follows:

1%~10% salidroside powder;

2% to 15% skeleton material;

5% to 15% tackifier;

30% to 60% moisturizer;

0.05% ~ 0.2% cross-linking agent;

0.1% to 0.5% preservatives;

0.5% ~ 1.5% pH adjuster;

2% to 10% penetration enhancer;

The rest is distilled water,

The above percentages are mass percentages.

The content of salidroside in the salidroside powder is 98.8%.

The skeleton material is selected from one or two types of carbomer, sodium polyacrylate, potassium polyacrylate, and ammonium polyacrylate.

The tackifier is selected from one or two types of polyvinylpyrrolidone, polyvinyl alcohol, and gelatin.

The moisturizing agent is selected from one or two types of glycerin, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, and sorbitol.

The cross-linking agent is selected from one or both of aluminum glycinate or aluminum hydroxide.

The preservative is selected from one or two of ethylparaben, propylparaben or methylparaben.

The pH adjuster is selected from one or both of tartaric acid or citric acid.

The penetration enhancer is selected from one or two types of peppermint oil, azone or borneol.

As a preferred solution, the skeleton material is sodium polyacrylate, the tackifier is polyvinylpyrrolidone, the humectant is glycerin, the cross-linking agent is aluminum glycinate, the preservative is ethyl paraben, and the pH regulator is tartaric acid. The penetration enhancer is peppermint oil, and the weight ratio of the three ingredients: glycerin, sodium polyacrylate, and polyvinylpyrrolidone is 10:3:2.

Pharmacodynamic studies:

Pharmacodynamic research results at the gene level show that local use of the salidroside transdermal patch provided by the present invention can significantly inhibit the increase in the expression of muscle atrophy factor Atrogin-1 and fast muscle fiber MyHC-IIb mRNA during the formation of muscle atrophy. Proven its effective protective and antagonistic effects on muscle atrophy.

The experimental results also show that the salidroside patch provided by the present invention has no irritation to the skin and no allergic phenomenon.

The beneficial effects of the present invention are:

1. The drug has strong targeting ability and effectively inhibits the expression of the muscle atrophy-specific factor Atrogin-1 and fast muscle fibers.

The salidroside patch directly delivers the active ingredient of the drug to atrophied muscle tissue, which can effectively inhibit the expression of the muscle atrophy-specific factor Atrogin-1 and fast muscle fibers in the process of muscle atrophy, thereby preventing and combating the occurrence of muscle atrophy;

2. The drug has long action time and good stability

The patch has a long-acting sustained-release effect, allowing the drug to act for a long time;

3. Easy to use

Just peel off the protective layer and apply it directly to the skin surface of the atrophic area;

4. Ensure safe medication use and avoid drug side effects

If side effects or other discomforts occur during the medication process, the medication can be terminated by tearing off the patch; it can be applied again when necessary, which improves the safety of medication for patients;

The preparation method of the salidroside transdermal patch provided by the invention is:

formula:

1% to 10% salidroside powder, 2% to 15% skeleton material, 5% to 15% tackifier, 30% to 60% humectant, 0.05% to 0.2% cross-linking agent, 0.1 % ~ 0.5% preservatives, 0.5% ~ 1.5% pH regulator, 2% ~ 10% penetration enhancer, 30% ~ 60% distilled water;

The names of each ingredient are as mentioned above, and the content of salidroside in the salidroside powder is 98.8%.

Step 1: Preparation of drug-loaded hydrogel patch matrix

1) Prepare medicated moisturizing phase, water phase and ethanol phase respectively

Add salidroside powder to the moisturizer and stir until uniform; then add framework materials and cross-linking agent respectively, stir uniformly to obtain a moisturizing phase;

Disperse the tackifier in distilled water, stir slowly until completely dissolved, then add pH adjuster and penetration enhancer, stir until dissolved, and obtain a water phase;

The preservative is dissolved in absolute ethanol to obtain the ethanol phase;

3) Add the above-mentioned ethanol phase and water phase to the moisturizing phase in sequence, and stir for 5 to 30 minutes until mixed evenly to obtain a drug-loaded hydrogel patch matrix.

Step 2: Prepare the patch

After the above-mentioned drug-loaded hydrogel patch matrix is degassed, it is evenly coated on the non-woven backing layer, dried, and then laminated with the backing layer. That is, the salidroside patch for treating muscle atrophy of the present invention is obtained.

Salidroside patches can be cut to the desired size as needed.

The degassing treatment can be ultrasonic degassing or high-speed centrifugal degassing;

The drying can be carried out in an oven at 20-60°C, and the time depends on actual needs, usually 5-40 minutes.

Description of the drawings

Figure 1 shows the finished salidroside patch prepared by the present invention, wherein the left picture is the outside of the patch, and the right picture is the medicine-containing inner surface;

Figure 2 is an in vitro cumulative release curve of salidroside from the salidroside patch prepared in Example 1;

Figure 3 is the comparison result of real-time quantitative real time PCR detection of the effect of salidroside patch on the expression of the muscle atrophy-specific factor Atrogin-1 induced by tail suspension;

Figure 4 is the comparison result of real-time quantitative real time PCR detection of the effect of salidroside patch on the expression of MyHC-IIb in fast muscle fibers induced by tail suspension;

Figure 5 is a schematic diagram of the preparation steps of the salidroside patch of the present invention. Wherein, the "drug extract" is salidroside powder.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with examples. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

The following salidroside powder was purchased from Chengdu Pusi Biotechnology Co., Ltd. It is a white powder or crystal with a melting point of 159°C. The content of salidroside is 98.8%.

The invention is a preparation method of salidroside patch for treating muscle atrophy. The preparation method includes the following steps:

Step 1: According to the mass percentage, weigh 1% to 10% salidroside powder, 2% to 15% skeleton material, 5% to 15% thickening agent, 30% to 60% humectant, 0.05% ~0.2% cross-linking agent, 0.1% ~ 0.5% preservative, 0.5% ~ 1.5% pH regulator, 2% ~ 10% penetration enhancer, 30% ~ 60% distilled water, the mass of each component The sum of the percentages is 100%; the skeleton material is one or two of carbomer, sodium polyacrylate, potassium polyacrylate, ammonium polyacrylate, etc.; the tackifier is one or two of polyvinylpyrrolidone, polyvinyl alcohol, gelatin, etc. One or two kinds; the moisturizing agent is one or two kinds of glycerin, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, and sorbitol; The coupling agent is one or both of aluminum glycinate or aluminum hydroxide; the preservative is one or both of ethyl paraben, propyl ester or methyl ester; the pH adjuster is one or both of tartaric acid or citric acid. One or two kinds; the penetration enhancer is one or two kinds of peppermint oil, azone or borneol.

Step 2: Add the salidroside powder weighed in step 1 to the moisturizer and stir until uniform; then add the skeleton material and cross-linking agent respectively and stir uniformly to obtain a moisturizing phase; disperse the tackifier in distilled water and slow down. Stir slowly until completely dissolved, then add pH adjuster and penetration enhancer, stir until dissolved, and obtain the water phase; dissolve the preservative with absolute ethanol to obtain the ethanol phase;

Step 3: Add the ethanol phase and the water phase obtained in Step 2 to the moisturizing agent phase obtained in Step 2 in sequence, and stir for 5 to 30 minutes until mixed evenly to obtain a drug-loaded hydrogel patch matrix;

Step 4: After the drug-loaded hydrogel patch matrix obtained in step 3 is degassed by ultrasonic degassing or high-speed centrifugation, it is evenly coated on the non-woven backing layer and dried in an oven at 20-60°C for 5 seconds. -40 minutes, and then laminated with the backing layer and cut into the required size to obtain the salidroside patch for treating muscle atrophy of the present invention.

Below, the present invention is described in detail through six embodiments.

Example 1

Add 3g of salidroside to 30ml of glycerin and stir until uniform; then add 6g of sodium polyacrylate and 0.25g of aluminum glycinate respectively and stir to obtain a moisturizing phase; disperse 5g of polyvinylpyrrolidone in 50ml of distilled water and stir slowly until Completely dissolve, then add 0.5g of citric acid and 5ml of peppermint oil, stir until dissolved to obtain the water phase; dissolve 0.1g of ethyl paraben with absolute ethanol to obtain the ethanol phase; add the ethanol phase and water phase to the moisturizer phase in sequence , stir for 5 to 30 minutes until mixed evenly to obtain a drug-loaded hydrogel patch matrix; degas the hydrogel patch matrix through ultrasonic degassing or high-speed centrifugation, and then evenly coat it on the non-woven backing layer. Dry in an oven at 20° C. for 40 minutes, then laminate with the backing layer, and cut into required sizes to obtain the salidroside patch for treating muscle atrophy of the present invention.

Example 2

Add 4g salidroside to 40ml glycerin and stir until uniform; then add 4g sodium polyacrylate and 0.15g aluminum hydroxide respectively, stir uniformly to obtain a moisturizing phase; disperse 8g polyvinylpyrrolidone in 40ml distilled water and stir slowly Until completely dissolved, add 1.0g of tartaric acid and 3g of borneol, stir until dissolved, and obtain the water phase; dissolve 0.1g of ethyl paraben with absolute ethanol to obtain the ethanol phase; add the ethanol phase and the water phase to the moisturizer phase in sequence, Stir for 5 to 30 minutes until mixed evenly to obtain a drug-loaded hydrogel patch matrix; degas the hydrogel patch matrix through ultrasonic degassing or high-speed centrifugation, and then evenly coat it on the non-woven backing layer. Dry in an oven at 30°C for 30 minutes, then laminate with the backing layer, and cut into required sizes to obtain the salidroside patch for treating muscle atrophy of the present invention.

Example 3

Add 5g salidroside to 50ml glycerol and stir until uniform; then add 2g sodium polyacrylate and 0.05g aluminum glycinate respectively, stir uniformly to obtain a moisturizing phase; disperse 12g polyvinylpyrrolidone in 30ml distilled water, stir slowly until Completely dissolve, then add 1.5g of citric acid and 2ml of peppermint oil, stir until dissolved to obtain the water phase; dissolve 0.1g of ethyl paraben with absolute ethanol to obtain the ethanol phase; add the ethanol phase and water phase to the moisturizer phase in sequence , stir for 5 to 30 minutes until mixed evenly to obtain a drug-loaded hydrogel patch matrix; degas the hydrogel patch matrix through ultrasonic degassing or high-speed centrifugation, and then evenly coat it on the non-woven backing layer. Dry in an oven at 40°C for 20 minutes, then laminate with the backing layer, and cut into required sizes to obtain the salidroside patch for treating muscle atrophy of the present invention.

Example 4

Add 3g salidroside to the mixture of 30ml glycerin and propylene glycol, stir until uniform; then add 6g carbomer and 0.25g aluminum hydroxide respectively, stir uniformly to obtain a moisturizing phase; disperse 5g polyvinylpyrrolidone in 50ml distilled water , stir slowly until completely dissolved, then add 0.5g of tartaric acid and 5g of borneol, stir until dissolved to obtain a water phase; dissolve 0.1g of ethyl paraben with absolute ethanol to obtain an ethanol phase; add the ethanol phase and water phase in turn to moisturize In the agent phase, stir for 5 to 30 minutes until mixed evenly to obtain a drug-loaded hydrogel patch matrix; degas the hydrogel patch matrix through ultrasonic degassing or high-speed centrifugation, and then evenly coat it on the non-woven backing The layer is dried in an oven at 50° C. for 10 minutes, then laminated with the backing layer, and cut into the required size to obtain the salidroside patch of the present invention for treating muscle atrophy.

Example 5

Add 4g of salidroside to the mixture of 40ml of glycerin and propylene glycol and stir until uniform; then add 4g of carbomer and 0.15g of aluminum glycinate respectively, stir uniformly to obtain a moisturizing phase; disperse 8g of polyvinylpyrrolidone in 40ml of distilled water. Stir slowly until completely dissolved, then add 1.0g of citric acid and 3ml of peppermint oil, stir until dissolved to obtain a water phase; dissolve 0.1g of ethyl paraben with absolute ethanol to obtain an ethanol phase; add the ethanol phase and water phase in sequence In the moisturizer phase, stir for 5 to 30 minutes until mixed evenly to obtain a drug-loaded hydrogel patch matrix; degas the hydrogel patch matrix through ultrasonic degassing or high-speed centrifugation, and then evenly coat it on the non-woven backing On the lining layer, dry it in an oven at 60°C for 5 minutes, then laminate it with the backing layer, and cut it into the required size to obtain the salidroside patch for treating muscle atrophy of the present invention.

Example 6

Add 5g of salidroside to the mixture of 50ml of glycerol and propylene glycol, stir until uniform; then add 2g of carbomer and 0.05g of aluminum hydroxide respectively, stir uniformly to obtain a moisturizing phase; disperse 12g of polyvinylpyrrolidone in 30ml of distilled water , stir slowly until completely dissolved, then add 1.5g of tartaric acid and 2g of borneol, stir until dissolved to obtain a water phase; dissolve 0.1g of ethyl paraben with absolute ethanol to obtain an ethanol phase; add the ethanol phase and water phase in sequence to moisturize In the agent phase, stir for 5 to 30 minutes until mixed evenly to obtain a drug-loaded hydrogel patch matrix; degas the hydrogel patch matrix through ultrasonic degassing or high-speed centrifugation, and then evenly coat it on the non-woven backing The layer is dried in an oven at 20° C. for 40 minutes, then laminated with the backing layer, and cut into the required size to obtain the salidroside patch for treating muscle atrophy of the present invention.

As shown in Figure 1, the salidroside patch for treating muscle atrophy prepared by the present invention has a smooth and flat appearance, even drug distribution, accurate dosage, moderate viscosity, and is easy to use.

Stability experiment of salidroside patches prepared in Examples 1 to 6

Purpose:

In order to detect the stability of the salidroside patch in each embodiment of the present invention, the following stability test research was conducted.

method:

The patches prepared in Examples 1 to 6 were subjected to harsh tests (60°C ± 2°C, RH 75% ± 5%) and left for 3 months to observe whether crystals precipitated from the patches.

Results and conclusions:

As shown in Table 1, the patch prepared according to the present invention has high stability and can be stably stored for a long time.

Table 1 Stability test results

Measurement of cumulative release of salidroside patches prepared in Examples 1 to 6

Purpose:

The skin penetration effect of the salidroside patch of the present invention is detected.

method:

The patch prepared in Example 1 was subjected to the following cumulative release measurement test of salidroside. The specific method is as follows:

The TP-6 intelligent transdermal tester is used. The upper chamber is the diffusion chamber and the lower chamber is the receiving chamber. The receiving pool has a volume of 15mL, an effective diffusion area of 2.01cm ² , and is heated by a constant temperature water bath at a temperature of (37+0.2)C.

During the test, the receiving chamber is filled with 20% ethanol-physiological saline solution as the receiving solution, a 0.8 μm microporous filter membrane is attached to the receiving chamber, and the patch is affixed to the filter membrane to create a backing layer. Towards the diffusion chamber, secure it between the two chambers to exclude air bubbles. The magnetic stirring speed is 300r/min.

At the set time points of 0.5, 1, 2, 3, 4, 6, 12, and 24 hours, accurately pipette 1 mL of the receiving solution, add the same volume of fresh receiving solution, and filter for HPLC measurement.

Results and conclusions:

The cumulative release results of salidroside are shown in Figure 2.

The patch prepared by the invention has a high drug utilization rate, can continuously and effectively penetrate the skin, improves the clinical application of the drug, and avoids the phenomenon that the drug cannot achieve drug efficacy due to low penetration amount.

Pharmacodynamic experiment of salidroside patch in treating muscular atrophy

1. Experimental purpose:

The effect of the salidroside patch provided by the present invention on the expression of muscle atrophy-specific factor Atrogin-1 and fast muscle fiber MyHC-IIb mRNA is detected to determine the efficacy of the preparation in treating muscle atrophy.

1. Experimental animals, experimental materials and instruments

Experimental animals: Male C57 mice were purchased from Beijing Weitonglihua Animal Center, production license number:

SCXK (Beijing) 2020-0001; Experimental Animal Quality Certificate Number: 0261086.

Salidroside patch: prepared according to the method of Example 1.

Other sources of materials:

Trizol and reverse transcriptase were purchased from Invitrogen;

SYBR was purchased from ABI;

Main instruments and equipment: ABI Step-One Plus real time PCR instrument, electronic balance, and tail cage.

3. Experimental methods:

Tail suspension for 7 days was used to induce skeletal muscle atrophy in mice. By comparing the RNA of the dorsal soleus muscle of the hindlimbs of the mice in the ground control group, the tail suspension group, and the tail suspension and salidroside patch treatment groups, real-time quantification was performed. PCR method was used to detect the effect of salidroside patch on the expression of muscle atrophy-specific factor Atrogin-1 and fast muscle fiber MyHC-IIb during the development of muscle atrophy induced by tail suspension.

3.1 Preparation of tail-suspension mouse muscular atrophy model

Eight-week-old male C57BL/6 mice were used as experimental subjects. According to the principle of uniform body weight (that is, there is no significant difference in the average weight of mice in each group), they were divided into a ground control group, a tail-suspension group for 7 days, and a tail-suspension group for 7 days at the same time. The mice in the Tiansi patch treatment group and the tail suspension group followed the internationally recognized mouse tail suspension method (Gregory R.Adams, et al. J ApplPhysiol 95:2185–2201, 2003), so that the hind limbs of the mice were just off the ground. , the tilt angle between the body and the ground is approximately 30°. The mice can freely move around, eat and drink in the cage with their heads down. The animals were kept in individual cages, with room temperature maintained at 23±2°C and 12 hours of light per day. Muscle atrophy was induced by tail suspension for 7 days.

3.2 Salidroside patch treatment of mice

The dorsal side of the mouse's hind limbs was depilated, and the salidroside patch was cut into a size of 5 mm × 5 mm and applied to the most elevated part of the muscle belly on the dorsal side of the hind limbs, that is, the skin lateral to the gastrocnemius muscle. The dorsal sides of the hind limbs of mice in the simple tail suspension group were treated with blank control patches. During tail suspension, the patch was replaced once a day.

3.3. Soleus muscle sampling after completion of tail suspension

After tail suspension, all mice were collected according to standard procedures. After pentobarbitona anesthesia, the mice were suddenly killed by cervical dislocation. The mice were placed in the prone position. The dorsal side of the hind limbs was wiped with a 75% alcohol cotton ball, and the skin on the lateral side of the Achilles tendon was cut. Use skin forceps to fully expose the dorsal muscles of the hind limbs, cut the dorsal Achilles tendon, carefully separate the soleus muscle, and cut off excess fat and fascia, and then homogenize the tissue to extract RNA.

3.4 Extraction, detection and quantification of RNA

A. Lysis: Place the left and right soleus muscles of each mouse into a tissue homogenizer, add 1 ml of Trizol, fully grind the tissue homogenate, and allow it to be fully lysed at room temperature for half an hour.

B. Separation of each phase: Add chloroform at the ratio of 0.2ml chloroform/1ml Trizol, cover tightly and mix vigorously for 15 seconds, and leave at room temperature for 2-3 minutes. Centrifuge at 12000g for 15 minutes at 4°C. Then draw the supernatant aqueous phase into another new EP tube.

C. Precipitate RNA: Add isopropyl alcohol to the absorbed water phase at the ratio of 0.5 ml isopropyl alcohol/1 ml Trizol, mix by inverting, and leave at room temperature for 15 minutes. Centrifuge at 12,000 g for 10 minutes at 4°C to obtain RNA precipitation.

D. Rinse RNA: discard the supernatant, add 75% ethanol at the ratio of 1ml 75% ethanol/1ml Trizol to wash the RNA precipitate twice, then centrifuge the precipitation at 7500g at 4°C for 5 minutes, discard the supernatant.

E. Re-dissolve RNA: Dry the RNA precipitate in air at room temperature for 5-10 minutes, dissolve the RNA precipitate with an appropriate amount of DEPC water, and dissolve it in water at 55°C-60°C for 10 minutes.

F. RNA purity detection and quantification: Add 1 μl of RNA solution to 99 μl of DEPC water and mix well. Use a UV spectrophotometer to measure the ratio and concentration of A260/A280.

3.5 Reverse transcription reaction

25μL reaction system operation steps:

aTake a 0.5mL EP tube

b After shaking gently, place at 70°C for 10 minutes, place at -20°C for 3 to 5 minutes, and shake gently.

cAdd in tube a:

d 42℃90min, 95℃10min

eSynthesized cDNA was stored at –20C for later use.

3.6 Primer design

Use DNAMAN software to design primers based on NCBI database sequences, and then select appropriate primers for synthesis based on annealing temperature and sequence specificity (see Table 2).

Table 2 Target gene Atrogin1, MyHC-IIb and internal reference GAPDH primer sequences

3.7 Real time PCR detection of target gene expression

50μL reaction system operation steps:

PCR reaction conditions: denaturation at 94°C for 5 minutes. 94℃ for 40 seconds, 62℃ for 15 seconds, 72℃ for 20 seconds, 40 cycles. Extension at 72°C for 10 minutes.

4. Data analysis methods

Data are expressed as mean ± standard deviation, and data were analyzed using between-group analysis of variance. **P<0.01, *P<0.05 indicates significant statistical difference.

5. Experimental results

As shown in Figure 3 and Figure 4.

Compared with the ground control group, the mRNA expression of Atrogin-1 and fast muscle fiber MyHC-IIb in the soleus muscle of mice suspended for 7 days was increased by 2.7 times (***P<0.001) and 5.65 times (***P<0.001) respectively. , while the up-regulation of Atrogin-1 and MyHC-IIb mRNA expression in the salidroside patch treatment group was smaller;

Compared with mice that were tail-suspended for 7 days, salidroside patch treatment significantly inhibited the muscle atrophy-specific factor Atrogin-1 (***P<0.001) and fast-twitch muscle fiber MyHC-IIb (** P<0.01) mRNA expression increased.

6. Experimental conclusion

The tested salidroside patch could significantly inhibit the expression of the muscle atrophy-specific factor Atrogin-1 induced by tail suspension;

The tested salidroside patch could significantly inhibit the expression of MyHC-IIb mRNA in fast-twitch muscle fibers;

Therefore, the salidroside patch of the present invention can be used to combat the development of muscle atrophy.

Claims (5)

1. A transdermal absorption agent that inhibits the expression of the muscle atrophy-specific factor Atrogin-1 and inhibits the expression of fast muscle fibers, consisting of a drug-loaded hydrogel patch matrix and a backing layer; wherein, the raw materials for preparing the drug-loaded hydrogel patch matrix Contains 1% to 10% salidroside powder, and the percentage is the mass percentage; The preparation method of the drug-loaded hydrogel patch matrix is characterized by: 1) Prepare medicated moisturizing phase, water phase, and ethanol phase respectively: Mix salidroside powder, moisturizer, framework material, and cross-linking agent to obtain a moisturizing phase; Mix and dissolve the tackifier, distilled water, pH regulator, and penetration enhancer to obtain a water phase; Dissolve the preservative with absolute ethanol to obtain the ethanol phase; 2) Add the above ethanol phase and water phase to the moisturizing phase in sequence to obtain a drug-loaded hydrogel patch matrix; The raw material proportions for preparing the drug-loaded hydrogel patch matrix are as follows: 1%~10% salidroside powder; 2% to 15% skeleton material; 5% to 15% tackifier; 30% to 55% moisturizer; 0.05% ~ 0.2% cross-linking agent; 0.1% to 0.5% preservatives; 0.5% to 1.5% pH adjuster; 2% to 10% penetration enhancer; Appropriate amount of ethanol; The rest is distilled water; The above percentages are mass percentages; The content of salidroside in the salidroside powder is 98.8%. 2. The transdermal absorbent agent according to claim 1, wherein the skeleton material is selected from one or two types of carbomer, sodium polyacrylate, potassium polyacrylate, and ammonium polyacrylate; The tackifier is selected from one or two types of polyvinylpyrrolidone, polyvinyl alcohol, and gelatin; The moisturizing agent is selected from one or two types of glycerol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, and sorbitol; The cross-linking agent is selected from one or both of aluminum glycinate or aluminum hydroxide; The penetration enhancer is selected from one or two types of peppermint oil, azone or borneol. 3. The transdermal absorbent according to claim 1 or 2, the preservative is selected from one or two of ethyl paraben, propyl ester or methyl ester; the pH regulator is selected from tartaric acid or lemon. One or both acids. 4. The transdermal absorbent according to claim 1, wherein the skeleton material is sodium polyacrylate; the thickening agent is polyvinylpyrrolidone; the humectant is glycerin; the cross-linking agent is aluminum glycinate; and the preservative is ethyl paraben. ;The pH adjuster is tartaric acid; The penetration enhancer is peppermint oil; and the weight ratio of the three ingredients: glycerin, sodium polyacrylate, and polyvinylpyrrolidone is 10:3:2. 5. The preparation method of the transdermal absorbent agent according to any one of claims 1-4, according to the following steps: Step 1 Preparation of drug-loaded hydrogel patch matrix 1) Prepare medicated moisturizing phase, water phase, and ethanol phase respectively. Add salidroside powder to the moisturizer and stir until uniform; then add framework materials and cross-linking agent respectively, stir uniformly to obtain a moisturizing phase; Disperse the tackifier in distilled water, stir slowly until completely dissolved, then add pH adjuster and penetration enhancer, stir until dissolved, and obtain a water phase; The preservative is dissolved in absolute ethanol to obtain the ethanol phase; 3) Add the above ethanol phase and water phase to the moisturizing phase in sequence, and stir for 5 to 30 minutes until mixed evenly to obtain a drug-loaded hydrogel patch matrix; Step 2: Prepare the patch After the above-mentioned drug-loaded hydrogel patch matrix is degassed, it is evenly coated on the non-woven backing layer, dried, and then laminated with the backing layer to obtain a patch; it is cut into the required size.