Preparation method and application of rhizoma arisaematis processed product influencing miR expression of organism

Abstract

The invention relates to a preparation method of a rhizoma arisaematis processed product influencing miR-124 / 182 expression of an organism, and belongs to the technical field of traditional Chinese medicine processing. The method comprises the following steps: taking tubers of rhizoma arisaematis, adding cold water to infiltrate, crushing, homogenizing and filtering to obtain filter residues I and filtrate I; centrifuging the filtrate I, performing low-temperature alcohol extraction on the centrifugal precipitate and the filter residue I, and filtering to obtain filtrate II; recovering ethanol from the filtrate II, concentrating to obtain extract, merging the extract into centrifugate, performing high-temperature hydrolysis treatment, refrigerating, filtering to obtain filter residue III and filtrate III, concentrating the filtrate III, merging the filtrate III with the filter residue III, drying, and crushing to obtain a rhizoma arisaematis processed product. Through a series of technical combinations, an active substance group capable of influencing the expression of microRNA (miRNA), especially miR-124 / 182, of an organism is created and prepared by taking rhizoma arisaematis as a raw material, and the application further has a huge prospect of developing drugs for influencing neuron damage repair, neuron apoptosis, nerve center excitability abnormality network stability and the like.

Description

Technical Field

[0001] The present invention is a method for creating and preparing an aroid processed product that affects the expression of miR-124 / 182 in an organism, belonging to the technical field of traditional Chinese medicine extraction; the present invention also provides the use of the aroid processed product prepared by the aforementioned method. Background Art

[0002] Arisaematis Rhizoma is a plant of the Araceae family. Arisaema erubescens (Wall.) Schott.〕、Arisaema heterophylla Arisaema heterophyllum Bl.) or Arisaema mandshurica ( Arisaema from Amur Maxim.) Dried tuber. The 2020 edition of the Chinese Pharmacopoeia records that Arisaema can dissipate lumps and reduce swelling. It can be used externally for carbuncle, snake and insect bites.

[0003] The body's miR-124 / 182 are two types of microRNAs in the body. Their differential expression in the blood and central nervous system organs has a significant impact on neuronal damage repair, neuronal apoptosis, abnormal excitability of central nervous system networks, etc.; at the same time, they play a certain role in antiviral and tumorigenesis mechanisms.

[0004] The prior art has not disclosed methods and products for using Arisaema products to influence the expression of miR-124 / 182 in the body. Summary of the invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies of the prior art and provide a method for preparing a processed product of Arisaema consanguineum that affects the expression of miR-124 / 182 in the body. The method uses a series of technical combinations to create and prepare an active substance group that can affect the expression of microRNA (miRNA) in the body, especially miR-124 / 182, using Arisaema consanguineum as a raw material.

[0006] The technical problem to be solved by the present invention is achieved by the following technical solution. The present invention is a method for preparing a product of Arisaema cinereae that affects the expression of miR-124 / 182 in the body, which is characterized by taking fresh Arisaema cinereae and processing it according to the following method: (1) Take the plant Arisaema confusa of the Araceae family. Arisaema erubescens (Wall.) Schott.〕and / or Arisaema heterophylla ( Arisaema heterophyllum Bl.) and / or Arisaema mandshurica ( Arisaema amurense Maxim.) tubers, remove the fibrous roots and skin, and soak them in water; The soaked Arisaema and the remaining water are crushed and homogenized until the tissue cells are crushed, kept warm at 25°C to 45°C, filtered, and the filtrate is set aside. The filter residue is taken and treated 1 to 3 times according to the above crushing, homogenizing, keeping warm and filtering steps, and 1 to 2 times of water is added to the filter residue according to the weight volume ratio before each homogenization; after the treatment, the filter residue I is set aside, and the filtrates are combined to obtain filtrate I; (2) Take filtrate I, centrifuge, set aside the centrifuge liquid, and combine the centrifugal precipitate with the filter residue I; take the combined centrifugal precipitate and filter residue I, add ethanol solution with a concentration of more than 50%, perform ultrasonic extraction, filter, set aside the filtrate, treat the filter residue 1 to 3 times according to the above method to obtain filter residue II, set aside, and combine the filtrates to obtain filtrate II; (3) taking filtrate II, recovering ethanol, concentrating, adding the concentrate to the centrifuge of step (2), stirring, adjusting the pH value to 8-10, and then boiling at 100°C under dilute oxygen conditions for 2-3 times, each time for 1 hour to 6 hours, cooling to room temperature between each treatment; or treating at 90-103 kPa and 100-120°C under dilute oxygen conditions for 2-3 times, each time for 1 hour to 3 hours, cooling to room temperature between each treatment; then refrigerating, filtering, obtaining filter residue III and filtrate III, adjusting the pH value of filtrate III to 7-8, desalting, and then concentrating into a paste, combining with filter residue III, drying, and pulverizing; thus, obtaining a processed arisaema product.

[0007] The above-mentioned preparation method, its further preferred technical scheme is: In step (1), 1 to 4 times the volume of water is added to the Arisaema consanguineum by weight to volume ratio or by weight to volume ratio after drying, and the Arisaema consanguineum is soaked in cold water in an environment of 0 to 4°C for 24 to 48 hours.

[0008] In step (1), the soaked Arisaema is homogenized and then kept warm at 35°C to 40°C for 30 to 60 minutes.

[0009] In step (1), 1 to 2 times the volume of water is added to the filter residue according to the weight-to-volume ratio, and the filter residue is treated 2 to 3 more times according to the above steps.

[0010] In step (2), after the centrifugal precipitate and the filter residue are combined, 2 to 3 times of 65% to 85% ethanol solution is added according to the weight volume ratio, and ultrasonic extraction is carried out at 35 to 40° C. for 30 to 60 minutes, filtered, the filtrate is set aside, and the filter residue is treated 1 to 2 more times according to the above conditions.

[0011] In step (3), the refrigeration refers to refrigerating the treated liquid at 2-6°C for 24-48 hours and then filtering it.

[0012] The present invention also discloses a method for preparing aranaeum processing byproducts that affect the expression of miR-124 / 182 in the body, which is characterized by taking the filter residue II of step (2) in the above technical scheme, evaporating the ethanol, baking or frying or steaming; cooling to room temperature; drying, and crushing to obtain the aranaeum processing byproducts.

[0013] The preferred baking or frying temperature is 80°C to 90°C until the powder particles have a light yellow appearance. The preferred steaming method is: take the filter residue II, steam it at 100°C to 120°C for 60 to 90 minutes; cool it to room temperature; dry it, and crush it to obtain aranaesthetic processing byproducts.

[0014] The present invention also provides a composition of processed araceae products that affect the expression of miR-124 / 182 in the body, which is characterized by taking the processed araceae products described in any one of the above technical schemes and the araceae processing by-products described in any one of the above technical schemes as raw materials, with the weight ratio of the former to the latter being 1:8~9, mixing, drying, crushing and mixing to obtain the processed araceae product composition.

[0015] The present invention also discloses the use of the processed araceae product or the processed araceae product composition prepared by the preparation method described in the above technical scheme, and its characteristic is that the processed araceae product or the processed araceae product composition is used as a raw material to prepare a drug that affects the expression of miR-124 / 182 in the body.

[0016] The present invention also discloses the use of the byproduct of arisaema processing obtained by any one of the preparation methods in the above technical solutions, which is characterized in that the byproduct is used as a dispersant or carrier for the arisaema processing product.

[0017] Through the research of the present invention, technicians used a series of technical combinations such as cold water infiltration, homogenization, cell disruption, temperature control and insulation, high-speed centrifugation, temperature-controlled ultrasonic extraction of ethanol at a specific concentration, high-temperature hydrolysis under oxygen-controlled conditions, refrigerated separation and desalting, and innovatively separated special active components from Arisaema consanguineum first, and then hydrolyzed them, creating and preparing Arisaema consanguineum processed products that can affect the body's microRNA (miRNA), especially the expression of miR-124 / 182. At the same time, Arisaema consanguineum processing by-products were also prepared and mixed with Arisaema consanguineum processed products to form Arisaema consanguineum processed product compositions.

[0018] In the technical solution of the present invention, the most water-soluble components of Arisaema are collected at a suitable temperature by cold soaking Arisaema and then homogenizing to break the tissue cells. Arisaema is rich in starch polysaccharides. The temperature is controlled within 45 degrees Celsius in the present invention to avoid gelatinization of the homogenate, which is more conducive to solid-liquid separation. The main component of the filtrate I obtained by the technical solution of the present invention through step (1) is water-soluble substances. In the present invention, the filter residue II is a by-product of the method of the present invention, and is most suitable for use as a dispersant or carrier for Arisaema processed products.

[0019] The purpose of centrifuging the filtrate I in the present invention is to separate a small amount of fine solids mixed in the filtrate. Then, the filter residue produced in the previous step and part of the low-polarity substances in the centrifuged solids are extracted by ultrasound at a suitable temperature using ethanol liquid. The main effective components of the filtrate II in the present invention are low-polarity components.

[0020] In the technical solution of the present invention, in a pH 8-9 system, the small molecules in the system are hydrolyzed by high temperature treatment under dilute oxygen conditions and converted into substances that can affect the expression of miR-124 / 182 in the body. Then, components with different water solubility and temperature sensitivity are separated by refrigeration, and then combined and dried after being treated separately to obtain the processed product of the present invention.

[0021] Compared with the prior art, the present invention has the following beneficial results: The present invention uses a series of modern technology combinations to create and prepare active substance groups that can affect the expression of microRNA (miRNA) in the body, especially miR-124 / 182, using Arisaema as raw material, and further has great prospects for developing drugs that affect neuronal damage repair, neuronal apoptosis, abnormal excitability of nerve centers, and network stability, and plays a huge clinical value. The Arisaema processed product prepared by the method of the present invention does not exist as a whole in the natural state. DETAILED DESCRIPTION

[0022] The specific technical solutions of the present invention are further described below to help those skilled in the art further understand the present invention without limiting the rights thereof.

[0023] Example 1, a method for preparing a product of Arisaema consanguineum that affects the expression of miR-124 / 182 in the body, the steps are as follows: 1. Take the plant of Araceae, Arisaema Arisaema erubescens (Wall.) Schott.〕and / or Arisaema heterophylla ( Arisaema heterophyllum Bl.) and / or Arisaema mandshurica ( Arisaema amurenseMaxim.), remove the fibrous roots and skin, soak in 4°C cold water for 24 hours; take the soaked Arisaema and the remaining water, crush and homogenize until the tissue cells are crushed, keep the temperature at 40°C for 60 minutes, filter, set the filtrate aside, take the filter residue, and treat it twice according to the above crushing and homogenizing, temperature control and keeping, and filtering steps, adding 1 time of water to the filter residue according to the weight volume ratio before each homogenization; after the treatment, the filter residue I is set aside, and the filtrates are combined to obtain filtrate I; 2. Take filtrate I, centrifuge, set aside the centrifuge, and combine the centrifugal precipitate with the filter residue I; take the combined centrifugal precipitate and filter residue I, add 70% ethanol solution, ultrasonically extract at 40°C for 60 minutes, filter, set aside the filtrate, treat the filter residue once more according to the above method, and combine the filtrates to obtain filtrate II; 3. Take the filtrate II, recover the ethanol under reduced pressure, concentrate, take the extract, add the extract to the centrifuge in step (2), stir, adjust the pH value to 9, and perform boiling hydrolysis treatment at 100°C at normal pressure twice under dilute oxygen conditions and under the condition of ensuring that the liquid volume remains unchanged, each treatment time is 2 hours, and cool to room temperature between each treatment. Then refrigerate at 4°C for 48 hours, filter, obtain filter residue III and filtrate III, adjust the pH value of filtrate III to 7-8, desalinate by reverse osmosis, concentrate into a paste, combine with filter residue III, dry, and crush; the processed product of Arisaema consanguineum is obtained.

[0024] Example 2, a method for preparing a product of Arisaema consanguineum that affects the expression of miR-124 / 182 in the body, the steps are as follows: 1. Take the plant of Araceae, Arisaema Arisaema erubescens (Wall.) Schott.〕and / or Arisaema heterophylla ( Arisaema heterophyllum Bl.) and / or Arisaema mandshurica ( Arisaema amurense Maxim.), remove the fibrous roots and skin, soak in 4°C cold water for 24 hours; take the soaked Arisaema and the remaining water, crush and homogenize until the tissue cells are crushed, keep the temperature at 40°C for 60 minutes, filter, set the filtrate aside, take the filter residue, and treat it twice according to the above crushing and homogenizing, temperature control and keeping, and filtering steps, adding 1 time of water to the filter residue according to the weight volume ratio before each homogenization; after the treatment, the filter residue I is set aside, and the filtrates are combined to obtain filtrate I; 2. Take filtrate I, centrifuge, set aside the centrifuge, and combine the centrifugal precipitate with the filter residue I; take the combined centrifugal precipitate and filter residue I, add 70% ethanol solution, ultrasonically extract at 40°C for 60 minutes, filter, set aside the filtrate, treat the filter residue once more according to the above method, and combine the filtrates to obtain filtrate II; 3. Take filtrate II, recover ethanol under reduced pressure, concentrate, take extract, add the extract to the centrifuge of step (2), stir, adjust the pH value to 9, and hydrolyze 3 times under dilute oxygen conditions at high pressure of 90-103 kPa and high temperature of 100-120°C, each treatment time is 1 hour, and cool to room temperature between each treatment. Then refrigerate at 4°C for 48 hours, filter, and obtain filter residue III and filtrate III. Adjust the pH value of filtrate III to 7-8, desalt by ion exchange resin, concentrate into a paste, combine with filter residue III, dry, and crush; the processed product of Arisaema is obtained. The absorbance is measured at a wavelength of 400nm by ultraviolet-visible spectrophotometry, and its concentration-absorbance data is similar to the detection data of Example 1.

[0025] Example 3, a method for preparing a product of Arisaema consanguineum that affects the expression of miR-124 / 182 in the body, the steps are as follows: 1. Take the plant of Araceae, Arisaema Arisaema erubescens (Wall.) Schott.〕and / or Arisaema heterophylla ( Arisaema heterophyllum Bl.) and / or Arisaema mandshurica ( Arisaema amurense Maxim.), remove the fibrous roots and skin, soak in 4°C cold water for 24 hours; take the soaked Arisaema and the remaining water, crush and homogenize until the tissue cells are crushed, keep the temperature at 40°C for 60 minutes, filter, set the filtrate aside, take the filter residue, and treat it twice according to the above crushing and homogenizing, temperature control and keeping, and filtering steps, adding 1 time of water to the filter residue according to the weight volume ratio before each homogenization; after the treatment, the filter residue I is set aside, and the filtrates are combined to obtain filtrate I; 2. Take filtrate I, centrifuge, set aside the centrifugal liquid, and combine the centrifugal precipitate with the filter residue I; take the combined centrifugal precipitate and filter residue I, add 70% ethanol solution, ultrasonically extract at 40°C for 60 minutes, filter, set aside the filtrate, treat the filter residue once more according to the above method, combine the filtrate to obtain filtrate II and set aside; collect the filter residue at the same time.

[0026] 3. Evaporate the ethanol from the filter residue in the previous step, bake at 90°C until the powder particles have a light yellow appearance, cool to room temperature, dry, and crush to obtain aranaesthetics processing by-products.

[0027] Example 4, preparation of Arisaema confusa processed product composition: Take the Arisaema processed product powder of Example 1 and the Arisaema by-product powder of Example 3, mix them in a weight ratio of 1:9, and mix them thoroughly to obtain an Arisaema processed product composition.

[0028] Example 5: Effect of processed Arisaema ciliata products on the expression of miR-124 / 182 in rat plasma Twelve clean SD rats with a body weight of 280±20 g were taken and divided into a normal group and a drug-treated group by a random number table method, with 6 rats in each group. The normal group was fed normally, and the drug-treated group was fed with the processed product of Arisaema consanguineum prepared in Example 1 on the basis of normal feeding, and gavage was administered at 100 mg / kg body weight for 7 consecutive days. 2 ml of blood was collected from the tail vein, and the expression of miR-124 and miR-182 in rat blood was detected by real-time fluorescence quantitative PCR. Total RNA was extracted by Trizol kit, and the one-step miRNA quantitative reaction system was: 2 × one-step qPCR mix 10 µL, upstream primer (10 µmol), downstream primer (10 µmol), RT prime (10 µmol) each 0.4 µL, RNA template 100 ng, DEPC-H2O supplemented to 20 µL, and internal reference RT primer was replaced by DEPC-H2O. Reaction conditions: reverse transcription cycle 1, temperature 37~42℃ 60 min, deformation cycle 1, 95℃ 10 min, 95℃ 20s, amplification cycle 40, 62℃ 30s, 72℃ 30s. -△△Ct Calculate the relative expression. The results are shown in the following table:

[0029] Note: Compared with the same group before treatment, * P<0.05.

[0030] The experiment showed that after rats were fed with processed arisaema, the expression of miR-124 / 182 in the plasma of the two groups of rats was significantly different from that of rats fed with normal diet (P<0.05), which revealed that processed arisaema can significantly affect the expression of miR-124 / 182 in the body.

Claims

1. A method for preparing a product of Arisaema cinereae that affects the expression of miR-124 / 182 in the body, characterized in that: Take fresh Arisaema and process it according to the following method: (1) Take the plant Arisaema confusa of the Araceae family. Arisaema erubescens (Wall.) Schott.〕and / or Arisaema heterophylla ( Arisaema heterophyllum Bl.) and / or Arisaema mandshurica ( Arisaema amurense Maxim.) tubers, remove the fibrous roots and skin, and soak in cold water; The soaked Arisaema and residual water are taken, crushed and homogenized until the tissue cells are crushed, kept warm at 25°C-45°C, filtered, and the filtrate is set aside, and the filter residue is taken, and the crushing, homogenization, temperature control, insulation and filtering steps are repeated for 1-3 times, and 1-2 times of water is added to the filter residue according to the weight volume ratio before each homogenization; after the treatment, the filter residue I is set aside, and the filtrates are combined to obtain filtrate I; (2) taking filtrate I, centrifuging, setting aside the centrifugal liquid, and combining the centrifugal precipitate with the filter residue I; taking the combined centrifugal precipitate and filter residue I, adding ethanol solution with a concentration of more than 50%, performing temperature-controlled ultrasonic extraction, filtering, setting aside the filtrate, and treating the filter residue 1 to 3 times according to the above method to obtain filter residue II, which is set aside for processing arisaema by-products, and combining the filtrates to obtain filtrate II; (3) taking filtrate II, recovering ethanol, concentrating, adding the concentrate to the centrifuged liquid of step (2), stirring, adjusting the pH value to 8-10, and subjecting the mixture to boiling hydrolysis treatment at 100°C at normal pressure under dilute oxygen conditions and while ensuring that the liquid volume remains unchanged for 2-3 times, each treatment time being 1 hour to 6 hours, and cooling to room temperature between each treatment; Alternatively, the product is hydrolyzed under dilute oxygen conditions at high pressure of 90-103 kPa and high temperature of 100-120°C for 2-3 times, with each treatment lasting 1-3 hours, and cooled to room temperature between each treatment; then refrigerated and filtered to obtain filter residue III and filtrate III, the filtrate III is adjusted to a pH value of 7-8, desalted, concentrated into a paste, combined with filter residue III, dried, and crushed; the processed product of Arisaema is obtained.

2. The preparation method according to claim 1, characterized in that: In step (1), 1 to 4 times the volume of water is added to the Arisaema consanguineum by weight to volume ratio or by weight to volume ratio after drying, and the Arisaema consanguineum is soaked in cold water in an environment of 0 to 4°C for 24 to 48 hours.

3. The preparation method according to claim 1, characterized in that: In step (1), the soaked Arisaema is homogenized and then kept at 35°C to 40°C for 30 to 60 minutes.

4. The preparation method according to claim 1, characterized in that: In step (1), 1 to 2 times the volume of water is added to the filter residue according to the weight-to-volume ratio, and the filter residue is treated 2 to 3 more times according to the above steps.

5. The preparation method according to claim 1, characterized in that: In step (2), after the centrifugal precipitate and the filter residue are combined, 2 to 3 times of 65% to 85% ethanol solution is added in a weight-to-volume ratio, and ultrasonic treatment is performed at 35 to 40° C. for 30 to 60 minutes, filtered, the filtrate is set aside, and the filter residue is treated 1 to 2 more times according to the above conditions.

6. The preparation method according to claim 1, characterized in that: In step (3), the refrigeration refers to refrigerating the treated liquid at 2-6°C for 24-48 hours.

7. A method for preparing a by-product of processing Arisaema, characterized in that: Take the filter residue II of step (2) in claim 1, evaporate the ethanol, bake, fry or steam; cool to room temperature; dry and crush to obtain arisaema processing by-product.

8. The preparation method according to claim 7, characterized in that: The baking or frying temperature is 80°C to 90°C, until the powder particles have a light yellow appearance, thereby obtaining aranea processing by-product.

9. The preparation method according to claim 7, characterized in that: The steaming method is as follows: take the filter residue II, steam it at 100°C-120°C for 60-90 minutes; cool it to room temperature; dry it, and crush it to obtain the arisaema processing by-product.

10. A processed product composition of Arisaema consanguineum that affects the expression of miR-124 / 182 in the body, characterized in that: Take the processed aroid product described in any one of claims 1 to 6 and the processed aroid by-product described in any one of claims 7 to 9, with the weight ratio of the former to the latter being 1:8-9, mix, dry, crush and blend to obtain the processed aroid product composition.

11. Use of the processed aroid product obtained by the preparation method according to any one of claims 1 to 6 or the processed aroid product composition according to claim 10, characterized in that: The use is to use the processed products of Arisaema consanguineum or the processed products combination of Arisaema consanguineum as raw materials to prepare drugs that affect the expression of miR-124 / 182 in the body.

12. Use of the byproduct of Arisaema consanguineum processing obtained by any one of the preparation methods of claims 7 to 9, characterized in that: The use is to use it as a dispersant or carrier for arisaema processing products.

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