CN110898102A

CN110898102A - Cinnamon extract and traditional Chinese medicine compound for preventing and treating cold injury and application thereof

Info

Publication number: CN110898102A
Application number: CN201910849947.2A
Authority: CN
Inventors: 赵庆春; 李想; 许子华; 祖宇欣; 刘欣; 曲莹莹; 沈泽旭; 陆鸿远
Original assignee: General Hospital of Shenyang Military Region
Current assignee: General Hospital of Shenyang Military Region; General Hospital of Northern Theater Command of PLA
Priority date: 2019-09-10
Filing date: 2019-09-10
Publication date: 2020-03-24
Anticipated expiration: 2039-09-10
Also published as: CN110898102B

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a cinnamon extract and a traditional Chinese medicine compound for preventing and treating cold injury and application thereof. The cinnamon extract comprises six compounds, and is prepared by firstly extracting crude cinnamon extract and then extracting the cinnamon extract in a refined manner. The cinnamon extract Chinese herbal compound I for preventing and treating cold injury comprises cinnamon, honey-fried licorice root, cortex acanthopanacis, radix puerariae, caulis spatholobi, fructus evodiae, radix codonopsitis, rhizoma zingiberis, cassia twig and cinnamon extract; is prepared by mixing the medicinal liquids of ethanol extraction and liquid-phase water extraction, concentrating, and adding cortex Cinnamomi extract. Cortex Cinnamomi extract Chinese medicinal composition II comprises Zingiberis rhizoma, cortex Cinnamomi, and cortex Cinnamomi extract; cortex Cinnamomi extract Chinese medicinal composition III comprises cortex Cinnamomi, radix Paeoniae alba, and cortex Cinnamomi extract; the compound Chinese medicinal materials include cortex Cinnamomi, Glycyrrhrizae radix, and cortex Cinnamomi extract. The cinnamon extract can be used for preparing medicines for increasing heat production of organisms and preventing cold injury. The Chinese herbal compound can be used for preparing a medicament for improving energy metabolism and preventing and/or treating metabolic syndrome.

Description

Cinnamon extract and traditional Chinese medicine compound for preventing and treating cold injury and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a cinnamon extract and a traditional Chinese medicine compound for preventing and treating cold injury and application thereof.

Background

The border of China is long, the coverage area of cold regions is wide, and the production and living level of people is seriously influenced by personnel injury caused by the cold environment. Meanwhile, cold regions are bordered by multiple countries such as Korea, Russia, Mongolia, Pakistan, India and the like, which are strategic to national defense, are attack and hardness regions influencing the process of warfare, and the fighting and operation capacity of troops is severely restricted by cold damage. How to use specific medicines, how to improve the cold resistance of troops in cold regions, improve the operation capability under cold conditions, improve the success rate of prevention and treatment of freezing injury and multi-organ injury, and reduce the death rate and disability rate becomes a problem to be urgently solved by a medical service system.

Disclosure of Invention

The purpose of the invention is as follows:

the invention aims to: the application of the cinnamon extract and the traditional Chinese medicine compound thereof in increasing heat production of organisms and preventing and treating frostbite is provided, and experiments prove that the cinnamon extract and the traditional Chinese medicine compound thereof have the application in preventing and treating frostbite and heat production. Six potential compounds with frostbite prevention and treatment effects are extracted from the cinnamon.

The technical scheme is as follows:

a cinnamon extract for controlling cold damage, the cinnamon extract comprising:

a preparation method of cinnamon extract for preventing and treating cold injury comprises the following steps:

the method comprises the following steps: crude cinnamon extract: pulverizing dried cortex Cinnamomi, extracting with ethanol solution, mixing ethanol extractive solutions, recovering under reduced pressure, concentrating to obtain total extract, dispersing the total extract with hot water to obtain suspension, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate, and n-butanol with increasing polarity to obtain crude cortex Cinnamomi extract;

step two: refined cinnamon extract: respectively extracting six compounds;

purifying the crude cinnamon extract by semi-preparative HPLC to provide compound 1;

mixing a silica gel sample at an ethyl acetate part, loading the mixture to a column, wherein an eluent is 10% -100% of a dichloromethane/methanol system gradient system, and segmenting by using TLC (thin layer chromatography) guidance, wherein the ethyl acetate part is divided into 7 fractions Fr.1-7; subjecting the fraction Fr.4 to ODS column segmentation treatment, and eluting with methanol/water with different proportions to obtain fraction Fr.4-1-4; fr.4-2 is eluted by using dichloromethane/methanol with the volume ratio of 6:1 as an eluent, is eluted and separated by using gel column methanol, and is purified by semi-preparative HPLC to obtain a compound 4;

performing segmentation treatment on Fr.6 by using an ODS column to obtain Fr.6-1-6-3, eluting Fr.6-3 by using silica gel, repeatedly purifying ODS and gel Sephdex LH-20, and then performing semi-preparative liquid phase to obtain a compound 2;

subjecting Fr.7 to reversed-phase MCI column chromatography methanol/water gradient elution to obtain Fr.7-1-7-8, subjecting Fr.7-6 to SephadexLH20 gel column chromatography, and eluting with dichloromethane/methanol at a volume ratio of 1:1 to obtain Fr.7-6-1-Fr.7-6-6; fr.7-6-6 is subjected to semi-preparative liquid phase purification to obtain a compound 5;

the n-butanol part is segmented by silica gel column chromatography, dichloromethane/methanol gradient elution is carried out, and the segmentation is guided by TLC to obtain 7 fractions I-seven; dividing the fraction by ODS chromatographic column, and repeatedly and alternately purifying with positive and negative phase silica gel, gel LH-20 and semi-preparative HPLC to obtain compound 3; and dividing five fractions by an ODS chromatographic column, and repeatedly and alternately using positive and negative phase silica gel, gel LH-20 and semi-preparative HPLC for purification to obtain a compound 6.

The Chinese herbal compound of cinnamon extract for preventing and treating cold injury comprises the following components in parts by weight:

15-30 parts of cinnamon, 15-30 parts of honey-fried licorice root, 15-30 parts of cortex acanthopanacis, 15-20 parts of radix puerariae, 15-20 parts of caulis spatholobi, 15-20 parts of fructus evodiae, 15-20 parts of codonopsis pilosula, 10-20 parts of rhizoma zingiberis, 5-20 parts of cassia twig and 0.0005-0.0015 part of cinnamon extract.

A preparation method of a cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury comprises the following steps:

the method comprises the following steps: extracting Zingiberis rhizoma, cortex Cinnamomi, radix Puerariae, caulis Spatholobi, and ramulus Cinnamomi with ethanol under reflux to obtain medicinal liquid 1;

step two: extracting radix Glycyrrhizae Preparata, cortex Acanthopanacis, fructus evodiae, and radix Codonopsis in liquid water to obtain liquid medicine 2;

step three: mixing the liquid medicine 1 and the liquid medicine 2 and concentrating into extract; and finally adding the cinnamon extract into the extract to obtain the cinnamon extract traditional Chinese medicine compound.

A preparation method of a cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury comprises the steps of adopting 60-80% ethanol water solution in liquid medicine 1, extracting for 1-4 times, 1-3 hours each time, and enabling the ratio of medicinal materials to the ethanol water to be 1 (5-20); the liquid-phase water extraction at 80-90 ℃ is adopted in the liquid medicine 2 for 1-4 times, each time lasts for 1-3 hours, and the ratio of the medicinal materials to the liquid-phase water is 1 (5-20).

The Chinese herbal compound of cinnamon extract for preventing and treating cold injury comprises the following components in parts by weight: 20-30 parts of dried ginger, 10-15 parts of cinnamon and 0.0005-0.0015 part of cinnamon extract.

The Chinese herbal compound of cinnamon extract for preventing and treating cold injury comprises the following components in parts by weight: 10-20 parts of cinnamon, 10-20 parts of white paeony root and 0.0005-0.0015 part of cinnamon extract.

The Chinese herbal compound of cinnamon extract for preventing and treating cold injury comprises the following components in parts by weight: 30-20 parts of cinnamon, 15-10 parts of liquorice and 0.0005-0.0015 part of cinnamon extract.

Application of cortex Cinnamomi extract for preventing and treating cold injury in preparing medicine for increasing body heat production, preventing cold injury, improving energy metabolism, and preventing and/or treating metabolic syndrome is provided.

The application of the cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury in preparing the medicines for increasing heat production of an organism, preventing cold injury, improving energy metabolism and preventing and/or treating metabolic syndrome.

The advantages and effects are as follows:

the invention provides a traditional Chinese medicine with the functions of improving the cold resistance of the body and resisting cold injury and a compound thereof, and can be developed into an effective cold injury resisting medicine.

Cortex Cinnamomi has effects of tonifying fire, supporting yang, dispelling cold, relieving pain, warming channels and dredging collaterals. However, the research on the application of cinnamon and the compound thereof as the cold injury prevention and treatment effect is less, and no compound which plays the heat production effect is reported in documents. The invention provides compounds extracted from 6 cinnamon with the effect of generating heat, and 4 Chinese herbal compound formulas are prepared from the compounds.

The traditional Chinese medicine compound 1 comprises the monarch drugs of baked ginger and cinnamon, the ministerial drugs of kudzu root, suberect spatholobus stem and radix codonopsitis, the adjuvant drugs of evodia rutaecarpa, cassia twig and cortex acanthopanacis and the guiding drug of prepared liquorice. The compound cinnamon can tonify the primordial yang, warm the spleen and stomach, remove cold accumulation and promote blood circulation. Gui Zhi is pungent and warm in flavor and has the actions of warming and activating Wei Yang, expelling wind and expelling pathogenic wind. Pao Jiang is pungent and warm in property, and pungent and sweet in flavor and resolve yang together with Gui Zhi. Wu Zhu Yu is pungent, bitter and hot in flavor, and can warm stomach, liver and dispel cold. Radix Puerariae has effects of resolving muscle and eliminating pathogenic factors, promoting fluid production and dredging collaterals; radix Codonopsis and Glycyrrhrizae radix have effects of promoting fluid production, lowering blood pressure, nourishing blood, invigorating middle warmer and replenishing qi. Spatholobus stem is sweet and pungent in flavor and warm in nature, and has the functions of enriching blood, promoting blood circulation and dredging collaterals. Acanthopanax bark has the functions of strengthening heart, invigorating spleen, inducing diuresis and relieving swelling. The medicines are compatible to play the efficacies of expelling pathogenic wind from muscles, warming middle-jiao and tonifying, harmonizing ying and wei, qi and blood, and promoting the production of body fluid and relaxing channels.

The compound Chinese medicine 2 is originated from the book Wuxi from Sheng Ji Zong Lu and is used newly in ancient prescription. The original prescription is used for treating the heart pain and the pain caused by cold air is intolerable. The monarch drug is cinnamon which can remove cold accumulation. Wherein the dried ginger has the functions of warming the middle-jiao to dispel cold, restoring yang to promote blood circulation and warming the lung to eliminate retained fluid. Can be used for treating abdominal psychroalgia, emesis, diarrhea, cold limbs, slight pulse, cough and asthma due to cold fluid retention.

The compound Chinese medicine 3 is derived from cassia twig and peony powder in the 'practical traditional Chinese medicine to medicine prescription', also named peony powder, and consists of two halves of cassia bark core and three two halves of white peony root, and has the function of relieving wind-cold. The cinnamon, the cinnamon core and the cassia twig are all derived from cinnamon, the cinnamon core is one of the cinnamon, the cinnamon is the bark of the cinnamon, the cinnamon core is the thick bark with the outer layer removed, and the cassia twig is the twig of the cinnamon. Cinnamon has a strong texture effect, including the effects of cinnamon twig, such as dispelling cold, warming meridians and promoting blood circulation. Therefore, cinnamon and white peony root are selected for compatibility. Cinnamon, cortex Cinnamomi, with pungent and warm properties, can dispel pathogenic factors, warm yang to strengthen the wei, tonify fire and strengthen yang, guide fire to the origin, dispel cold to alleviate pain, warm meridians and promote blood circulation. Radix Paeoniae alba is sour and cold, and sour can astringe sweat, and cold can nourish blood, regulate menstruation, calm liver, relieve pain, astringe yin and stop sweating. Ramulus Cinnamomi and radix Paeoniae, which are the meanings of astringing sweat in dispersing; the peony, radix Paeoniae, ministerial and ramulus Cinnamomi should be used to strengthen superficies for slight sweating. The combination of the two can relieve wind-cold, regulate yingfen and weifen, regulate spleen and stomach, relieve spasm and alleviate pain.

The Chinese herbal compound No. 4 is derived from Shanghai treatise on Cold-induced diseases, Guizhi gan Cao Tang and the name of a traditional Chinese medicine prescription. Has the functions of nourishing heart yang, generating yang and regulating qi. In the formula, the dosage of the cassia twig is more than that of the honey-fried licorice root, the cassia twig is pungent and warm in taste, and the heart of a human body can activate yang. Prepared licorice root, radix Glycyrrhizae Praeparata, being sweet and warm, has the effect of replenishing qi and strengthening the middle-jiao. The two are combined together to transform yang, and tonify heart yang. The formula is a basic formula for warming heart yang, has special and quick medicinal flavor, and is taken immediately, which is intended to recover heart yang urgently. Selection notes of ancient and modern times: gui Zhi Fu gan Cao is pungent in flavor and sweet in flavor and acts as a yang-qi-yin-nourishing herb, so it is indicated for the loss of yang-qi in chest. In addition, the cassia twig, ramulus Cinnamomi, when it is lightly raised, acts as an assistant to the liquorice, which retains the effect of replenishing yang qi in the middle energizer and retains the effect of replenishing yang qi, so as to stop sweating and relieve restlessness. The recipe is innovated, and cassia twig is replaced by cinnamon which is sweet, pungent and hot in flavor. It enters liver, kidney, spleen and heart meridians. Has the functions of supplementing fire and supporting yang, dispelling cold and relieving pain, and activating blood and stimulating the menstrual flow. Clinically, the medicine is used for treating insufficiency of kidney-yang, impotence, cold womb, conjunctival congestion and vertigo; cold pain in the heart and abdomen, lumbago due to arthralgia due to cold, and hernia of cold with running dolphin; thoroughfare and conception vessels are deficient cold, amenorrhea and abdominal pain; in addition, it can warm and activate spleen yang, unblock blood vessels, and also indicated for deficiency of qi and blood due to chronic diseases. Honey-fried licorice root: sweet in flavor and neutral in nature. Has the functions of invigorating spleen and replenishing qi, clearing away heat and toxic material, eliminating phlegm and stopping cough, relieving spasm and pain and harmonizing the drugs. It is mainly used for treating hyperhidrosis, manifested by feverish sensation in the hands and forks, palpitation. Therefore, the method has good effect on cold injury.

Description of the drawings:

FIG. 1 is a graph of the effect of varying concentrations of cinnamon extract on the change in lipid droplets in brown adipocytes;

FIG. 2 is a graph showing the effect of different concentrations of cinnamon extract on changes in mitochondrial membrane potential of brown adipocytes;

FIG. 3 is a graph of the effect of varying concentrations of cinnamon extract on mitochondrial oxidative respiration in brown adipocytes;

FIG. 4 is a graph of the effect of varying concentrations of cinnamon extract on ATP levels in brown adipocytes;

figure 5 is a graph of the effect of different concentrations of cinnamon extract on UCP1 protein expression in brown adipocytes;

FIG. 6 is a graph of the effect of varying concentrations of cinnamon extract on PGC 1-meat protein expression in brown adipocytes;

figure 7 is a graph of the effect of different concentrations of cinnamon extract on PRDM16 protein expression in brown adipocytes;

FIG. 8 is a graph of the effect of varying concentrations of cinnamon extract on Sirt1 protein expression in brown adipocytes;

figure 9 is a graph of the effect of different concentrations of cinnamon extract on HSL protein expression in brown adipocytes;

FIG. 10 is a graph of the effect of different doses of cinnamon extract on the anal temperature of Kunming mice in a cold environment of 4 ℃;

FIG. 11 is an infrared thermography of the effect of different doses of cinnamon extract on the body surface temperature of Kunming mice in a cold environment of 4 ℃;

FIG. 12 is a bar graph of the effect of different doses of cinnamon extract on the body surface temperature of Kunming mice in a cold environment at 4 ℃;

FIG. 13 shows the effect of different doses of cinnamon extract on anal temperature of Kunming mice in a cold environment of-20 ℃;

FIG. 14 is an infrared thermography of the effect of different doses of cinnamon extract on the body surface temperature of Kunming mice in a cold environment at-20 ℃; FIG. 15 is a bar graph of the effect of different doses of cinnamon extract on the body surface temperature of Kunming mice in a cold environment at-20 ℃;

FIG. 16 is a graph of the effect of different doses of cinnamon extract on the size of fat droplets in brown adipose tissue;

figure 17 shows immunohistochemical results of different doses of cinnamon extract on mitochondrial content of brown adipose tissue and UCP 1;

figure 18 is a graph of the effect of different doses of cinnamon extract on UCP1 expression in brown adipose tissue;

FIG. 19 is a graph showing the effect of different doses of cinnamon extract on the amount of PGC1- α protein expressed in brown adipose tissue;

FIG. 20 is a graph of the effect of different doses of cinnamon extract on the size of white adipose tissue lipid droplets;

figure 21 is a graph of the effect of different doses of cinnamon extract on mitochondrial content of white adipose tissue and on the expression of UCP 1;

figure 22 is a graph of the effect of different doses of cinnamon extract on triglyceride levels in vivo;

figure 23 is a graph of the effect of different doses of cinnamon extract on the level of free fatty acid metabolism in vivo;

figure 24 is a graph of the effect of different doses of cinnamon extract on serum ALT;

figure 25 is a graph of the effect of different doses of cinnamon extract on serum AST;

FIG. 26 is a graph of the effect of varying doses of cinnamon extract on serum BUN;

figure 27 is a graph of the effect of different doses of cinnamon extract on serum CRE;

FIG. 28 is a graph of the effect of different doses of cinnamon extract on the survival time of Kunming mice in cold environments;

FIG. 29 shows the structure of 6 thermogenic active compounds in cinnamon;

figure 30 is a molecular docking result with UCP1 of a potentially thermogenic active compound in cinnamon;

FIG. 31 shows the effect of the compound 1 containing cinnamon on anal temperature of Kunming mice 90min after administration;

FIG. 32 shows the effect of the cinnamon bark herbal compound 1 on anal temperature within 16 days of administration;

FIG. 33 shows the effect of Compound No. 1 containing cinnamon on the anal temperature of mice after cold exposure at-20 deg.C;

FIG. 34 shows the effect of Compound No. 1 containing cinnamon on the weight of mouse brown adipose tissue;

FIG. 35 shows the effect of the cinnamon-containing herbal compound 1 on the weight of white adipose tissue of a mouse;

FIG. 36 shows the effect of the cinnamon-containing herbal compound 1 on the size of fat droplets in brown adipocytes;

FIG. 37 shows the effect of the compound 1 containing cinnamon on the size of white fat cell lipid droplets;

FIG. 38 shows the effect of cinnamon-containing herbal compound 1 on UCP1 expression in brown adipose tissue;

FIG. 39 contains the effect of cinnamon herbal compound 1 on the expression of UCP1 in white adipose tissue;

FIG. 40 is a graph of liver damage after 1 cold exposure for each group;

FIG. 41 shows renal injury after 1 cold exposure for each group;

FIG. 42 shows the effect of the cinnamon-containing herbal compound 1 on the expression of the calorigenic protein UCP1 in the brown fat and white adipose tissues;

FIG. 43 shows the effect of the cinnamon-containing herbal compound 1 on the expression of the related protein PPAR γ in the brown fat and white adipose tissues;

FIG. 44 shows the effect of Compound No. 1 containing cinnamon on the expression of PGC1 α, a related thermogenic protein in brown fat and white adipose tissue;

FIG. 45 shows the effect of Chinese herbal compound 1 containing cinnamon on the expression of the related protein SIRT1 in brown fat and white adipose tissue;

FIG. 46 shows the effect of the cinnamon-containing herbal compound 1 on the expression of HSL (related protein) in brown fat and white adipose tissue;

FIG. 47 shows the effect of the compound Chinese medicine 1 containing cinnamon on the survival rate of brown fat cells after 48 hours;

FIG. 48 shows the effect of Compound No. 1 containing cinnamon on lipid droplet formation;

FIG. 49 shows the effect of the cinnamon-containing herbal compound 1 on the level of ATP production in brown adipocytes;

FIG. 50 shows the effect of Chinese herbal compound 1 containing cinnamon on the expression of UCP1, a protein related to thermogenesis of brown adipocytes;

FIG. 51 shows the effect of the Chinese herbal compound 1 containing cinnamon on the expression of the protein PGC1 α related to thermogenesis in brown adipocytes;

fig. 52 shows the effect of cinnamon-containing herbal compound 1 on the expression of brown adipocyte-associated protein PPAR γ;

fig. 53 shows the effect of cinnamon-containing herbal compound 1 on the expression of brown adipocyte-associated protein HSL;

FIG. 54 shows the effect of Chinese herbal compound 1 containing cinnamon on the expression of the brown adipocyte-associated protein SIRT 1;

FIG. 55 shows liquid chromatogram of main ingredient identification of Chinese medicinal composition 1 containing cortex Cinnamomi extract;

FIG. 56 shows the effect of the Chinese herbal compound 2 containing cinnamon extract on anal temperature (left) and anal temperature drop (right) of Kunming mice under cold exposure;

FIG. 57 shows the effect of the Chinese herbal compound 3 containing cinnamon extract on anal temperature (left) and anal temperature drop (right) of Kunming mice under cold exposure;

FIG. 58 shows the effect of Compound No. 4 containing cortex Cinnamomi extract on the cooling of anus of Kunming mouse exposed for 30min (left) and 120min (right);

FIG. 59 shows the effect of Compound 2 on the biochemical index of Kunming mouse blood under cold exposure;

FIG. 60 shows the effect of Compound 3 containing cinnamon extract on the biochemical indicators of Kunming mouse blood after cold exposure;

FIG. 61 shows the effect of Compound No. 4 containing cortex Cinnamomi extract on the biochemical index of blood of Kunming mouse exposed to cold.

The specific implementation mode is as follows:

the invention provides the effect of cinnamon extract on increasing heat production of an organism in a cold environment, and aims to develop the cinnamon extract and a traditional Chinese medicine compound thereof into a novel medicine for better increasing heat production of the organism and preventing cold injury.

A cinnamon extract comprising:

the invention adopts primary pre-brown adipocytes to pass through a mitochondrial membrane potential determination kit, a mitochondrial oxidative respiration seahorse experiment, an ATP content determination kit, Western Blot and other experiments to prove that cinnamon extract and a traditional Chinese medicine compound can convert energy into heat by inhibiting ATP synthesis and improve the heat production capacity of BAT by improving the expression levels of UCP1 and channel protein. And the cinnamon extract promotes BAT to generate heat and improves the cold-resisting capability of the organism by increasing the mitochondrial content in BAT and WAT and improving the expression level of UCP1 protein.

the ethanol solution in the first step is 60-80% ethanol water solution, the extraction times are 1-5 times at room temperature, the extraction time is 3-10 days each time, and 7 days each time.

Step two: refined cinnamon extract: respectively extracting six compounds;

mixing a silica gel sample at an ethyl acetate part, loading the mixture to a column, wherein an eluent is 10% -100% of a dichloromethane/methanol system gradient system, and segmenting by using TLC (thin layer chromatography) guidance, wherein the ethyl acetate part is divided into 7 fractions Fr.1-7; carrying out segmentation treatment on the fraction Fr.4 through an ODS column, and eluting with methanol/water (0-70%) with different proportions to obtain a fraction Fr.4-1-4; fr.4-2 is eluted by using dichloromethane/methanol with the volume ratio of 6:1 as an eluent, is eluted and separated by using gel column methanol, and is purified by semi-preparative HPLC to obtain a compound 4;

performing segmentation treatment on Fr.6 by using an ODS column to obtain Fr.6-1-6-3, eluting Fr.6-3 by using silica gel, repeatedly purifying ODS and gel Sephdex LH-20 (methanol), and then performing semi-preparative liquid phase to obtain a compound 2;

eluting Fr.7 by reversed-phase MCI column chromatography methanol/water (20-80%) to obtain Fr.7-1-7-8, and eluting Fr.7-6 by SephadexLH20 gel column chromatography and dichloromethane/methanol with the volume ratio of 1:1 to obtain Fr.7-6-1-Fr.7-6-6; fr.7-6-6 is subjected to semi-preparative liquid phase purification to obtain a compound 5;

subjecting the n-butanol part to silica gel column chromatography segmentation treatment, and performing gradient elution with dichloromethane/methanol, namely gradually eluting to 0:15 according to a dichloromethane/methanol ratio of 15:1, and performing TLC (thin layer chromatography) guided segmentation to obtain 7 fractions Fr.1-7; dividing the fraction Fr.2 by an ODS chromatographic column, and repeatedly and alternately using positive and negative phase silica gel, gel LH-20 and semi-preparative HPLC to purify to obtain a compound 3;

and (3) after the fraction Fr.5 is segmented by an ODS chromatographic column, repeatedly and alternately using positive and negative phase silica gel, gel LH-20 and semi-preparative HPLC for purification to obtain a compound 6.

Because the functions and effects of the six compounds are the same or similar, the cinnamon extract used in the subsequent traditional Chinese medicine compound refers to a mixture of the six compounds, the mixture is prepared by adopting 25kg of dried cinnamon and extracting with 70% ethanol for 3 times, each time for 7 days, and then finely extracting the cinnamon extract to obtain the six compounds, the total of the obtained six compounds is the mass of the cinnamon, and the proportion relation of the six compounds is 1: 4: 5: 7: 2: 3.

the extracted six compounds are mixed again to form the cinnamon extract added in the subsequent Chinese herbal compound, and the description is not repeated.

Chinese medicine compound I

Wherein the cortex Cinnamomi and cortex Cinnamomi extract are used as principal drug, radix Glycyrrhizae Preparata, cortex Acanthopanacis, radix Puerariae and caulis Spatholobi are used as ministerial drug, and fructus evodiae, radix Codonopsis, Zingiberis rhizoma and ramulus Cinnamomi are used as adjuvant drug.

The cinnamon extract traditional Chinese medicine compound I for preventing and treating cold injury prepared by the invention conforms to the regulations of the Chinese pharmacopoeia 2015 edition.

Medicine property:

cinnamon: tonify fire and strengthen yang, induce fire to return to original, dispel cold to alleviate pain, and warm and dredge meridians. Can be used for treating sexual impotence, cold womb, cold pain of waist and knee, asthma due to kidney deficiency, floating of deficient yang, giddiness, conjunctival congestion, cold pain of heart and abdomen, vomiting and diarrhea due to deficiency cold, cold hernia, abdominal pain, dysmenorrhea, and amenorrhea.

Honey-fried licorice root: tonify spleen and stomach, replenish qi and recover pulse. Can be used for treating weakness of spleen and stomach, asthenia, palpitation, and intermittent pulse.

Cortex acanthopanacis: dispelling pathogenic wind and removing dampness, invigorating liver and kidney, strengthening tendons and bones, and inducing diuresis to alleviate edema. Can be used for treating rheumatism, flaccidity of bones and muscles, infantile retardation, asthenia, edema, and tinea pedis.

Kudzu root: expelling pathogenic factors from muscles and skin, relieving fever, promoting salivation, quenching thirst, promoting eruption, invigorating yang, relieving diarrhea, dredging meridian passage, and relieving alcoholism. Can be used for treating fever, headache, stiffness and pain of neck and back, thirst, diabetes, measles without adequate eruption, dysentery due to heat evil, diarrhea, vertigo, headache, apoplexy, hemiplegia, thoracic obstruction, cardiodynia, and alcoholic injury.

Caulis spatholobi: promoting blood circulation, tonifying blood, regulating menstruation, relieving pain, relaxing muscles and tendons, and activating collateral flow. Can be used for treating menoxenia, dysmenorrhea, amenorrhea, rheumatalgia, numbness, paralysis, and sallow complexion due to blood deficiency.

Evodia fruit: dispel cold and alleviate pain, check adverse rise of qi and arrest vomiting, strengthen yang and check diarrhea. Can be used for treating syncope headache, hernia of cold, abdominal pain, tinea pedis due to cold-dampness, abdominal pain during menstruation, abdominal pain, emesis, acid regurgitation, and diarrhea before dawn.

Codonopsis pilosula: to invigorate the spleen, benefit the lung, nourish blood and promote the production of body fluid. Can be used for treating deficiency of spleen-lung qi, anorexia, listlessness, cough, asthma, deficiency of qi and blood, sallow complexion, palpitation, short breath, thirst due to body fluid consumption, and internal heat.

Dried ginger: warming the middle energizer to dispel cold, restoring yang to activate collaterals, warming the lung to resolve retained fluid. Can be used for treating abdominal psychroalgia, emesis, diarrhea, cold limbs, slight pulse, cough and asthma due to cold fluid retention.

Cassia twig: induce sweating to relieve muscles, warm and dredge meridians, strengthen yang to transform qi, and smooth the flow of qi. Can be used for treating wind-cold type common cold, abdominal psychroalgia, blood cold amenorrhea, arthralgia, phlegm retention, edema, palpitation, and galloping.

The preparation method of the cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury comprises the following steps:

the method comprises the following steps: taking dried ginger, cinnamon, kudzu root, suberect spatholobus stem and cassia twig, and placing the dried ginger, the cinnamon, the kudzu root, the suberect spatholobus stem and the cassia twig into an ethanol solution for reflux extraction to obtain a liquid medicine 1;

step three: mixing the liquid medicine 1 and the liquid medicine 2, and then putting the mixture into a rotary evaporator to be concentrated into extract; and adding the cinnamon extract into the extract to obtain the cinnamon extract traditional Chinese medicine compound.

The ethanol solution in the liquid medicine 1 in the first step is 60-80% ethanol water solution, the extraction times are 1-4 times, each time lasts for 1-3 hours, and the ratio of the medicinal materials to the ethanol water is 1 (5-20); the liquid-phase water extraction at 80-90 ℃ is adopted in the liquid medicine 2 for 1-4 times, each time lasts for 1-3 hours, and the ratio of the medicinal materials to the liquid-phase water is 1 (5-20).

Chinese medicine compound II

The traditional Chinese medicine compound of cinnamon extract for preventing and treating cold injury comprises the following components in parts by weight: 20-30 parts of dried ginger, 10-15 parts of cinnamon and 0.0005-0.0015 part of cinnamon extract.

The preparation method of the cinnamon extract traditional Chinese medicine compound II for preventing and treating cold injury comprises the following steps: taking dried ginger and cinnamon, placing the dried ginger and the cinnamon into 70% ethanol, carrying out reflux extraction for 1-4 times at 85 ℃ for 1-3 hours each time, and obtaining liquid medicine by 1 (5-20) times of solvent. Spray drying to obtain dry powder with paste yield of about 12%. And adding cortex Cinnamomi extract.

Chinese medicine compound three

The traditional Chinese medicine compound of cinnamon extract for preventing and treating cold injury comprises the following components in parts by weight: 10-20 parts of cinnamon, 10-20 parts of white paeony root and 0.0005-0.0015 part of cinnamon extract.

The preparation method of the cinnamon extract traditional Chinese medicine compound III for preventing and treating cold injury comprises the following steps:

taking cinnamon and white paeony root, and placing the cinnamon and the white paeony root in 70% ethanol for reflux extraction at 85 ℃ for 1-4 times for 1-3 hours each time, wherein the solvent is 1 (5-20) times of the weight of the cinnamon and the white paeony root to obtain liquid medicine. Spray drying to obtain dry powder with paste yield of about 10.31%. And adding cortex Cinnamomi extract.

Chinese medicine compound four

The traditional Chinese medicine compound of cinnamon extract for preventing and treating cold injury comprises the following components in parts by weight: 30-20 parts of cinnamon, 15-10 parts of liquorice and 0.0005-0.0015 part of cinnamon extract.

The preparation method of the cinnamon extract traditional Chinese medicine compound IV for preventing and treating cold injury comprises the following steps:

taking cinnamon and liquorice, and placing the cinnamon and the liquorice in 70% ethanol for reflux extraction at 85 ℃ for 1-4 times for 1-3 hours each time, wherein the solvent is 1 (5-20) times of the weight of the cinnamon and the liquorice to obtain liquid medicine. Spray drying to obtain dry powder with an ointment yield of about 17.02%. And adding cortex Cinnamomi extract.

The present invention is further illustrated in detail by the following examples.

Example 1

Preparing the cinnamon extract. The preparation method comprises the following steps:

the method comprises the following steps: crude cinnamon extract: taking 25kg of dried cinnamon, crushing, extracting for 3 times with 70% ethanol solution at room temperature for 7 days each time, combining ethanol extracting solutions, recovering under reduced pressure, concentrating to obtain an extract to obtain a total paste, dispersing the total paste into a suspension by hot water, and sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol with gradually increased polarities to obtain a crude cinnamon extract; the cream yield was about 16%.

Step two: refined cinnamon extract: respectively extracting six compounds;

purifying the crude cinnamon extract by semi-preparative HPLC to provide compound 1; the specific method is that the petroleum ether part is loaded on a column by mixing with silica gel, an eluent is a petroleum ether/ethyl acetate system gradient system with the concentration of 10% -100%, and the petroleum ether/ethyl acetate system gradient system is segmented under the guidance of TLC and is divided into 5 fractions Fr.1-5. And (3) carrying out column chromatography on the fraction Fr.3, and eluting by a petroleum ether/ethyl acetate system gradient system with the concentration of 10% -100%, so as to obtain a fraction Fr. 3-1-3.4. Fr.3-2 was purified by semi-preparative HPLC to give Compound 1.

In the research of actual testing of the cinnamon extract, for the convenience of subsequent application, the subsequent mentioned testing process is to mix the six compounds, and the mixing proportion is directly mixed according to the amount of the six compounds extracted from 25kg of cinnamon, namely the cinnamon extract.

Example 2

Research on promotion of heat production of brown fat cells by cinnamon extract

1) Instruments and reagents: DMEM medium (Hyclone); fetal bovine serum (Hyclone corporation); model CKX31 inverted microscope (philippine olympus); a constant temperature CO2 incubator (Thermo corporation, USA); 5810R model desk top high speed cryogenic centrifuge (Eppendorf, Germany).

2) Cell culture: growing preadipocytes obtained by primary culture in a DMEM complete culture medium containing 10% FBS, replacing the preadipocytes with induced differentiation solution I (0.5 mmol/LIBMX, 1 mu mol/L dexamethasone and 10 mu g/mL insulin are added into the DMEM complete culture medium) when monolayer fusion is approached (the confluence degree is more than 90%), marking as the day 0 of induced differentiation, and continuing culture; on the 2 nd day of induced differentiation, i.e., 48 hours later, the culture medium was changed to induced differentiation solution II (10. mu.g/mL of insulin was added to DMEM complete medium), and the culture was continued. Thereafter, the cells were changed to DMEM medium 1 time every 2 days and observed for morphological changes under a microscope. The cells were cultured in an incubator at 37 ℃ (5% CO2, relative humidity 90%), and the next experiment was performed after the cells were induced to differentiate and mature.

3) Dyeing with oil red O: after primary pre-brown adipocytes are induced to differentiate and mature, cinnamon extracts with corresponding concentrations are respectively added, original culture solution is discarded after 48 hours, 1mL of 10% neutral formaldehyde solution is added into each hole to fix cells, the room temperature is about 1 hour, and oil red O application solution is added into the fixed cells for dyeing. Adding 1mL of isopropanol into each well to extract oil red, oscillating for 10min in a micro-oscillator, and measuring OD (recorded as absorbance A) at 570nm of the wavelength of an enzyme-linked immunosorbent detector. The lipid droplet consumption rate (1-a sample/a control) was calculated and the experiment was repeated 3 times. The results show that: cinnamon extract increased the lipid droplet depletion rate of primary brown adipocytes compared to control. The results show that: cinnamon extract may increase energy expenditure by consuming lipid droplets in brown adipose tissue. The specific results are shown in FIG. 1.

4) Mitochondrial membrane potential determination: after primary pre-brown adipocytes are induced to differentiate and mature, cinnamon extract with corresponding concentration and CL-316243 are added respectively to act for 48 hours, and then experiments are carried out. The specific experimental procedures were carried out according to the instructions of the mitochondrial membrane potential detection kit (JC-1) of Biyunsian Bio Inc. The specific results are shown in FIG. 2. As a result: compared with a control group, the CL-316243 positive drug group can obviously reduce the mitochondrial membrane potential in primary brown adipocytes, and the cinnamon extract reduces the mitochondrial membrane potential in cells in a concentration-dependent manner. The results show that: cinnamon extract may alter electron transport in and out of the mitochondrial membrane.

5) Mitochondrial oxidative respiration level determination: experiment the mitochondrial oxidative respiration level of brown adipocytes was determined using the "Agilent SeahorseXFP Cell Mitostresess Test Kit". Inoculating primary pre-brown adipocytes in a culture plate configured by the kit, adding cinnamon extract and CL316243 with corresponding concentrations after induced differentiation and maturation, and performing an experiment according to the kit specification after 48 hours of action. The specific results are shown in FIG. 3. As a result: compared with a control group, the CL316243 positive drug group can obviously reduce the maximum oxidative respiration rate of primary brown fat cells, and the cinnamon extract reduces the maximum oxidative respiration capacity of the cells in a concentration-dependent manner. The results show that: cinnamon extract may alter mitochondrial oxidative respiration levels of brown adipocytes, thereby decoupling oxidative respiration from ATP production.

6) Determination of ATP content: the ATP production of the brown adipocytes is measured by using an ATP content detection kit of Beijing Sorleubao technologies, Inc. After primary preadipocytes are induced, differentiated and matured, cinnamon extract and CL316243 with corresponding concentrations are added, and after 48 hours of action, ATP content determination is completed according to the instruction. As a result: compared with the control group, the cinnamon extract and CL316243 can obviously reduce the ATP production, and the cinnamon extract group is dose-dependent. The specific results are shown in FIG. 4. The results show that: cinnamon extract reduces ATP production in brown adipocytes, and uncoupling of proton transfer on the inner mitochondrial membrane from ATP production may occur, thereby converting potential energy into heat energy.

7) The content determination of the thermogenic protein UCP1 and the pathway-related protein comprises the steps of adding a positive drug CL316243 and a cinnamon extract with corresponding concentration into primary adipocytes after induced differentiation and maturation, performing protein quantification by using a BCA protein concentration determination kit of Shanghai Biyunshi biotechnology limited after 48 hours of action, and completing protein concentration determination according to the instruction, and determining the expression conditions of UCP1, PGC1- α, PRDM16, Sirt1 and HSL proteins in the brown adipocytes after administration by using a Western Blot method.

The experimental phenomena and results are briefly described below with reference to the accompanying drawings:

as can be seen from the results of oil red O staining in FIG. 1, compared with the control group, the number of lipid droplets in the cinnamon extract with different concentrations is significantly less than that in the control group, after 48 hours of action, the cinnamon extract can consume lipid droplets generated by brown fat cells in a concentration-dependent manner, and the cinnamon extract (based on crude drug amount) with different concentrations can consume lipid droplets with different concentrations, and has statistical significance. "" P <0.5, "" P <0.01, representing significant differences between the two groups.

From the results of fig. 2, it is known that the cinnamon extract can significantly reduce the mitochondrial membrane potential level of brown adipocytes, and is concentration-dependent. The mitochondrial membrane potential detection takes JC-1 as a probe and detects the change of the mitochondrial membrane potential through fluorescence intensity. When the mitochondrial membrane potential is high, JC-1 is gathered in a mitochondrial matrix to form a polymer, and red fluorescence is generated; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix, cannot form a polymer but is a monomer, and green fluorescence is generated. Thus, a change in mitochondrial membrane potential can be detected by a change in fluorescence color. The change of electron transport in the mitochondrial inner membrane is measured by the ratio of red fluorescence to green fluorescence.

From the results of fig. 3, it is understood that the cinnamon extract decreased the level of mitochondrial oxidative respiration in a concentration-dependent manner 48 hours after administration of brown adipocytes, suggesting that uncoupling of mitochondrial oxidative respiration and ATP production may occur.

As can be seen from the results in fig. 4, CL-316243 significantly reduced the ATP content in the cells, and the concentrations of the cinnamon extract decreased the ATP content in the brown adipocytes in a concentration-dependent manner.

As can be seen from FIGS. 5-9, the cinnamon extract increased the protein levels of UCP1, PGC1- α, PRDM16, Sirt1 and HSL in the brown adipocytes in a concentration-dependent manner, and the cinnamon extract at a concentration of 1.2g/L (based on crude drug content) significantly increased the expression of the associated proteins, indicating that the cinnamon extract increased the levels of thermogenic proteins in the brown adipocytes, such as UCP1, PGC1- α, PRDM16, and increased the expression of the upstream signal Sirt1 and HSL.

Example 3

Cinnamon extract improves thermogenic capacity of Kunming mice under cold exposure conditions.

1) Instruments and reagents: CL-316243 (Sigma, USA, purity)>98％)；AF-023 Minute^TMLipoprotein fraction isolation kit (Invent, usa); cell mitochondrial oxidative respiration assay Kit (Agilent SeahorseXFP Cell Mito Stress Test Kit, usa); BCA protein concentration assay kit (shanghai bi yunnan biotechnology limited); triglyceride content detection kit (Beijing Solebao science and technology Co., Ltd.); mouse anal thermometer (Beijing Zhongdi Chuang science and technology development, LLC); thermal infrared imagers (E5, FLIR, usa); paraffin microtomes (RM2145, Leica, germany); constant temperature laboratory animal fridge (RXZ-0250, hel).

2) Grouping and administration method of experimental animals: 56 Kunming mice were randomly divided into 7 groups by body weight: blank control group, model control group, CL-316243 group (1mg/kg), cortex Cinnamomi extract 0.5625g/kg, 1.125g/kg, 2.25g/kg dosage group (based on crude drug amount), each group contains 10. The mice are subjected to intragastric administration at the volume of 0.1mL/10g, and the blank control group and the model control group are respectively subjected to intragastric administration of equal volume of physiological saline and are continuously administered for 14 days.

3) Cold exposure experiments: after the mice of each experimental group are continuously dosed for 14 days, the mice of each dosing group are placed in a constant-temperature experimental refrigerator at 4 ℃, and are taken out after 240min to measure the anal temperature and the body surface temperature of the mice of each group after cold exposure. And (3) putting another mouse in the parallel administration group into a constant temperature experiment refrigerator at-20 ℃, and measuring the anal temperature and the body surface temperature after taking out the mouse after 60 min. The results show that: the cinnamon extract can obviously improve the anal temperature and the body surface temperature of mice under cold exposure (4 ℃ to-20 ℃) and is dose-dependent. The specific results are shown in FIGS. 10 to 15.

4) Adipose tissue morphology changes in Kunming mice: the morphological change of the adipose tissue is inspected by adopting an HE staining method, and the expression of a heat-generating protein UCP1 in the adipose tissue is detected by an immunohistochemical method: taking a tissue sample, fixing, dehydrating, transparentizing, wax-dipping embedding, slicing, dewaxing and dyeing to obtain an HE dyeing result, and observing the fat drop change of adipose tissues; fixing tissues, dewaxing, repairing antigens, sealing backgrounds, incubating antibodies, observing by a microscope to obtain an immunohistochemical result, and observing the mitochondrial content in adipose tissues and the expression condition of UCP1. The results show that: specific results of the cinnamon extract can be increased are shown in fig. 16, fig. 17, fig. 20, and fig. 21.

5) Content determination of heat production related protein: after the Kunming mouse is exposed to cold, taking brown adipose tissue, and adopting AF-023 Minute of English Special biotechnology, Inc^TMThe fat protein component separation kit 'extracts tissue protein, adopts a BCA protein concentration determination kit' of Shanghai Biyun biotechnology limited to perform protein quantification, completes protein concentration determination according to instructions, adopts a Western Blot method to determine the expression conditions of UCP1 and PGC1- α proteins in fat tissues after administration, and results show that cinnamon extract can improve the expression level of heat-producing protein in Kunming mouse brown color and specific results are shown in figures 18 and 19.

6) Mouse lipid metabolism level determination: triglyceride (TG) and Free Fatty Acid (FFA) are used as substrates of adipose tissue, and when UCP1 is up-regulated after brown fat is activated, TG is degraded into free fatty acid as fuel, thereby exerting thermogenic action in vivo. The TG level of serum was measured by using a "triglyceride content detection kit" of Beijing Sorleubao technologies, Inc. The FFA level in serum is determined by adopting a free fatty acid determination kit (colorimetric method) of Nanjing institute of bioengineering research, Inc. The results show that: cinnamon extract can reduce TG level in serum and increase FFA level in serum in a dose-dependent manner. Specific results are shown in fig. 22 and 23.

7) Measurement of biochemical indexes of mouse blood: the cold exposure of a mouse at the temperature of 20 ℃ below zero for 60min is carried out for 14 consecutive days, then the serum liver and kidney function indexes of the mouse are measured, the liver and kidney function level change after the cold exposure is measured by adopting a uric acid test box (enzyme colorimetric method), a creatinine measurement kit, an aspartate aminotransferase test box and an alanine aminotransferase test box of Nanjing to establish the institute of bioengineering, Inc., and the protection effect of the cinnamon extract with different administration doses on cold injury and the protection effect on the liver and kidney injury under the cold exposure environment are examined. The results show that: the liver and kidney functions of mice in the administration group are obviously different from those of mice in the model control group, and the cinnamon extract can reduce liver and kidney damage caused by cold exposure. The specific results are shown in FIGS. 24 to 27.

8) Study of survival time of mice under cold exposure: the invention discusses the improvement effect of cinnamon on cold resistance by comparing the survival time of each group of mice. After the mice are dosed for 14 days, the mice of the model control group and the dosing group are placed in a constant temperature experiment refrigerator at the temperature of 20 ℃ below zero, the state of the mice is observed every 10min, and the death time of the mice of each group is recorded. As a result: cinnamon extract significantly extended the survival time of mice in cold exposure compared to the model group. The specific results are shown in FIG. 28.

9) Molecule docking: is a method of drug design by the characteristics of the receptor and the way the receptor interacts with the drug molecule. A theoretical simulation method that mainly studies intermolecular (e.g., ligand and receptor) interactions and predicts their binding patterns and affinities.

Searching and downloading UCP2 protein crystal structure (PDB ID: 2LCK) in RCSB website (http:// www.rcsb.org/PDB/home. do); searching and downloading UCP1 protein amino acid sequence (P25874.fasta) in a Unitprot website (http:// www.uniprot.org /); the 2LCK Protein structure and the P25874.fasta sequence were introduced into discovery studio3.0 software, and a structure of UCP1 was constructed on the basis of the UCP2 structure (2LCK) using the Protocol Explorer | Protein Modeling | Homology Building module, and named UCP1. pdb. The constructed UCP1.pdb protein was introduced into Sybyl 8.1 software and structurally optimized. The optimized protein is introduced into Discovery studio3.0, an active cavity is set through key amino acids of the protein, main natural products in cinnamon are shown in figure 29, the protein introduced into Discovery studio3.0 is subjected to small molecule optimization and then is subjected to molecular docking with UCP1 which is modeled by homologous modeling, and then the condition of combination with the key amino acids of UCP1 is analyzed. The results show that: all 6 compounds were able to bind to the key amino acid of UCP1, and the results are shown in fig. 30.

from fig. 10 to fig. 15, mice of each administration group of the cinnamon extract had higher anal temperature and body surface temperature after cold exposure at-20 ℃ for 60min, compared to the model group. Meanwhile, when the cinnamon extract is exposed to-4 ℃ for 240min, the cinnamon extract can still maintain the anal temperature and the body surface temperature level of the mouse. The results show that: the cinnamon extract can maintain the anal temperature and the body surface temperature of a mouse in a cold exposure environment, and the cold resistance of the mouse is improved.

As can be seen from fig. 16, 17, 20, and 21, the cinnamon extract group decreased the volume of lipid droplets in brown and white adipose tissues and increased the mitochondrial content under cold exposure as compared to the model group. Immunohistochemical results show that both the cinnamon extract group and the positive drug group can increase the level of UCP1 expression on adipose tissue.

As is clear from FIGS. 18 and 19, the cinnamon extract group was able to increase the expression of the thermogenic-related proteins UCP1 and PGC1- α in the brown adipose tissue and was dose-dependent as compared to the model group.

As can be seen from fig. 22 and 23, the TG levels in the serum of the mice exposed to acute cold after the administration were decreased and dose-dependent, while the FFA levels were significantly increased, as compared to the model group. The results show that: cinnamon extract can be metabolized to FFA by reducing TG in vivo, thereby exerting thermogenic action.

As can be seen from fig. 24-27, the cinnamon administration group and the positive drug group both reduced ALT, AST, SCr, BUN in the serum of mice after cold exposure, and were dose-dependent, compared to the model. The results show that: the cinnamon extract administration group can reduce the biochemical index change of the liver and the kidney in cold environment, thereby being capable of resisting the injury caused by cold.

As shown in fig. 28, at-20 ℃, different administration groups of cinnamon extract can prolong the survival time of Kunming mice in cold environment, and are dose-dependent. The results show that: cinnamon extract can prolong the survival time of mice under cold exposure conditions.

As can be seen from fig. 29, 6 compounds in cinnamon were obtained by examining the literature.

As can be seen from fig. 30, molecular docking was performed to obtain six compounds in cinnamon that could bind to key amino acids of UCP1. The results show that: the cinnamon extract contains 6 phenolic acid compounds, and can be combined with key amino acids of UCP1.

Example 4

The content of the specific components of the Chinese herbal compound I is as follows:

15g of cinnamon, 0.010 part of cinnamon extract, 15g of honey-fried licorice root, 15g of cortex acanthopanacis, 15g of radix puerariae, 15g of caulis spatholobi, 15g of fructus evodiae, 15g of codonopsis pilosula, 10g of rhizoma zingiberis and 10g of cassia twig.

The specific preparation process of the traditional Chinese medicine compound I comprises the following steps:

the method comprises the following steps: taking rhizoma Zingiberis, cortex Cinnamomi, radix Puerariae, caulis Spatholobi, and ramulus Cinnamomi, and reflux-extracting in 75% ethanol at 85 deg.C for 2 times and 2 hr each time, wherein the ratio of medicinal materials to ethanol solution is 1:10 to obtain medicinal liquid 1.

Step two: extracting radix Glycyrrhizae Preparata, cortex Acanthopanacis, fructus evodiae and radix Codonopsis in liquid water at 85 deg.C for 2 times, each time for 2 hr, at a ratio of 1:10 to obtain liquid medicine 2.

Step three: mixing the liquid medicine 1 and the liquid medicine 2, and concentrating into extract in a rotary evaporator. The extract yield is about 28 percent. And finally adding the cinnamon extract into the extract to obtain the cinnamon extract traditional Chinese medicine compound.

Example 5

The content of the specific components of the traditional Chinese medicine compound II is as follows:

20 parts of dried ginger, 10 parts of cinnamon and 0.010 part of cinnamon extract.

The specific preparation method of the traditional Chinese medicine compound II comprises the following steps:

extracting Zingiberis rhizoma and cortex Cinnamomi with 70% ethanol at 85 deg.C under reflux for 2 times and 2 hr each time, wherein the ratio of the medicinal materials to the ethanol solution is 1:10 to obtain medicinal liquid. Spray drying to obtain dry powder with paste yield of about 12%. And adding 0.010 part of cinnamon extract.

Example 6

The content of the specific components of the Chinese herbal compound III is as follows:

20 parts of cinnamon, 10 parts of white paeony root and 0.010 part of cinnamon extract.

The specific preparation method of the traditional Chinese medicine compound III comprises the following steps:

extracting cortex Cinnamomi and radix Paeoniae alba in 70% ethanol at 85 deg.C under reflux for 2 times and 2 hr each time, wherein the ratio of the medicinal materials to ethanol solution is 1:10 to obtain medicinal liquid. Spray drying to obtain dry powder with paste yield of about 10.31%. And adding 0.010 part of cinnamon extract.

Example 7

The content of the specific components of the traditional Chinese medicine compound IV is as follows:

20 parts of cinnamon, 10 parts of liquorice and 0.010 part of cinnamon extract.

The specific preparation method of the traditional Chinese medicine compound IV comprises the following steps:

extracting cortex Cinnamomi and Glycyrrhrizae radix in 70% ethanol at 85 deg.C under reflux for 2 times and 2 hr each time, wherein the ratio of medicinal materials to ethanol solution is 1:10 to obtain medicinal liquid. Spray drying to obtain dry powder with an ointment yield of about 17.02%. And adding 0.010 part of cinnamon extract.

Example 8

The influence of the first, second, third and fourth Chinese herbal compound containing cinnamon extract on the heat production capacity of brown fat cells and Kunming mice.

The related experimental method is consistent with the cinnamon extract.

from fig. 31 to fig. 35, it can be seen that the cinnamon-containing traditional Chinese medicine compound 1 can significantly improve the anal temperature level of mice after cold exposure (20 ℃) and improve the anal temperature drop condition. Meanwhile, the weight of white adipose tissue and brown adipose tissue of the mice was reduced.

As shown in fig. 36-39, cinnamon-containing herbal compound 1 can reduce the lipid droplet size of adipose tissue, and immunohistochemical results show that UCP1 expression of adipose tissue can be increased.

As can be seen from FIGS. 40-41, no liver and kidney injury occurred after a single cold exposure experiment.

As shown in fig. 42-46, the cinnamon-containing herbal compound 1 can increase the expression of the thermogenic related proteins UCP1 and PGC1 in brown fat and white adipose tissues, and these are related to signals of PPAR γ and Sirt1, etc. Meanwhile, in white adipose tissue, the cinnamon-containing traditional Chinese medicine compound 1 can improve the expression of HSL, which indicates that the heat production effect is possibly related to the metabolism of fat.

As can be seen from fig. 47, the cinnamon-herbal compound 1 can prolong the survival rate of the mice in a cold environment.

From fig. 48 to fig. 49, it can be seen from the results of oil red O staining that the cinnamon-containing herbal compound 1 can reduce the formation of lipid droplets in a dose-dependent manner. Meanwhile, the cortex cinnamomi-containing traditional Chinese medicine compound 1 can obviously reduce the ATP generation level in the brown fat cells, which shows that the cortex cinnamomi-containing traditional Chinese medicine compound 1 plays a role in influencing the ATP generation in mitochondria.

As can be seen from fig. 50-54, cinnamon-containing herbal compound 1 can increase the expression of heat-producing proteins such as UCP1 and PGC1 in brown adipocytes in a dose-dependent manner, and is related to the up-regulation of signals such as PPAR γ, HSL, Sirt1, and the like.

As can be seen from fig. 55, in the liquid chromatogram, the cinnamon-containing compound 1 contains cinnamaldehyde, puerarin, 6-gingerol, and glycyrrhetinic acid.

As can be seen from fig. 56-fig. 58, the compound traditional

Chinese medicines

2, 3 and 4 containing cinnamon extract can significantly improve the anal temperature drop condition in the cold exposure (-20 ℃) environment and can improve the anal temperature level in the cold environment.

From fig. 59-61, it can be seen that the compound

Chinese medicines

2, 3 and 4 containing cinnamon extract can improve biochemical indexes of liver and kidney functions caused by continuous 14-day cold exposure. The results show that the compound traditional

Chinese medicines

2, 3 and 4 containing the cinnamon extract can resist organ injury caused by cold environment.

Example 9

30g of cinnamon, 0.0015 part of cinnamon extract, 30g of honey-fried licorice root, 30g of cortex acanthopanacis, 20g of radix puerariae, 20g of caulis spatholobi, 20g of fructus evodiae, 20g of codonopsis pilosula, 20g of rhizoma zingiberis and 20g of cassia twig.

the method comprises the following steps: taking rhizoma Zingiberis, cortex Cinnamomi, radix Puerariae, caulis Spatholobi, and ramulus Cinnamomi, and reflux-extracting in 80% ethanol at 85 deg.C for 4 times and 1 hr each time, wherein the ratio of medicinal materials to ethanol solution is 1:5 to obtain medicinal liquid 1.

Step two: extracting radix Glycyrrhizae Preparata, cortex Acanthopanacis, fructus evodiae and radix Codonopsis in liquid water at 80 deg.C for 3 hr for 4 times, wherein the ratio of the materials to the liquid water is 1:5 to obtain liquid medicine 2.

Step three: mixing the liquid medicine 1 and the liquid medicine 2, and concentrating into extract in a rotary evaporator. The extract yield is about 20 percent. And finally adding the cinnamon extract into the extract to obtain the cinnamon extract traditional Chinese medicine compound.

Example 10

20g of cinnamon, 0.0005 part of cinnamon extract, 20g of honey-fried licorice root, 20g of cortex acanthopanacis, 16g of radix puerariae, 18g of caulis spatholobi, 18g of fructus evodiae, 18g of codonopsis pilosula, 18g of rhizoma zingiberis and 5g of cassia twig.

the method comprises the following steps: taking rhizoma Zingiberis, cortex Cinnamomi, radix Puerariae, caulis Spatholobi, and ramulus Cinnamomi, and reflux-extracting in 60% ethanol at 80 deg.C for 1 time and 3 hr each time, wherein the ratio of medicinal materials to ethanol solution is 1:20 to obtain medicinal liquid 1.

Step two: extracting radix Glycyrrhizae Preparata, cortex Acanthopanacis, fructus evodiae and radix Codonopsis in liquid water at 90 deg.C for 1 time (1 hr each time) at ratio of 1:20 to obtain liquid medicine 2.

Step three: mixing the liquid medicine 1 and the liquid medicine 2, and concentrating into extract in a rotary evaporator. The extract yield is about 18 percent. And finally adding the cinnamon extract into the extract to obtain the cinnamon extract traditional Chinese medicine compound.

Example 11

30 parts of dried ginger, 15 parts of cinnamon and 0.0015 part of cinnamon extract.

extracting Zingiberis rhizoma and cortex Cinnamomi with 70% ethanol at 85 deg.C under reflux for 3 hr for 1 time, wherein the ratio of the medicinal materials to the ethanol solution is 1:20 to obtain medicinal liquid. Spray drying to obtain dry powder with paste yield of about 10%. And finally adding 0.0015 part of cinnamon extract.

Example 12

15 parts of cinnamon, 20 parts of white paeony root and 0.0015 part of cinnamon extract.

extracting cortex Cinnamomi and radix Paeoniae alba in 70% ethanol at 80 deg.C under reflux for 1 hr for 4 times, wherein the ratio of the medicinal materials to ethanol solution is 1:20 to obtain medicinal liquid. Spray drying to obtain dry powder with an ointment yield of about 8%. And finally adding 0.0015 part of cinnamon extract.

To summarize: the cinnamon extract and the traditional Chinese medicine compound can promote the activation of brown fat cells, increase the expression of thermogenic protein, reduce the mitochondrial membrane potential, and decouple ATP generation and mitochondrial oxidative respiration, thereby generating heat. The cinnamon extract and the traditional Chinese medicine compound can obviously improve the anal temperature and the body surface temperature of the mouse after cold exposure (20 ℃ below zero and 4 ℃) and obviously prolong the survival time of the mouse in the cold exposure environment. In addition, the cinnamon extract and the traditional Chinese medicine compound thereof can improve the heat production capability and enhance the cold resistance capability of the body by increasing the mitochondrial content in BAT and WAT and the UCP1 expression level. Furthermore, it can be seen through molecular docking that 6 compounds in cinnamon can directly interact with UCP1 key amino acids. Under the condition of liquid chromatography, the main compounds in the cinnamon compound 1 are as follows: puerarin, cinnamyl aldehyde, 6-gingerol and glycyrrhetinic acid. The cinnamon traditional

Chinese medicine compound

1, 2, 3 and 4 can improve the body temperature reduction and organ injury caused by cold environment.

Claims

1. The cinnamon extract for preventing and treating cold injury is characterized in that: the cinnamon extract comprises:

2. the preparation method of the cinnamon extract for preventing and treating cold injury is characterized by comprising the following steps: the preparation method comprises the following steps:

step two: refined cinnamon extract: respectively extracting six compounds;

3. The cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury as claimed in claim 1, is characterized in that: the traditional Chinese medicine compound comprises the following components in parts by weight:

4. The method for preparing the cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury according to claim 3, is characterized in that: the preparation method comprises the following steps:

5. The method for preparing the cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury according to claim 4, is characterized in that: the ethanol solution in the liquid medicine 1 is 60-80% ethanol water solution, the extraction times are 1-4 times, each time is 1-3 hours, and the ratio of the medicinal materials to the ethanol water is 1 (5-20); the liquid-phase water extraction at 80-90 ℃ is adopted in the liquid medicine 2 for 1-4 times, each time lasts for 1-3 hours, and the ratio of the medicinal materials to the liquid-phase water is 1 (5-20).

6. The cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury as claimed in claim 1, is characterized in that: the traditional Chinese medicine compound comprises the following components in parts by weight: 20-30 parts of dried ginger, 10-15 parts of cinnamon and 0.0005-0.0015 part of cinnamon extract.

7. The cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury as claimed in claim 1, is characterized in that: the traditional Chinese medicine compound comprises the following components in parts by weight: 10-20 parts of cinnamon, 10-20 parts of white paeony root and 0.0005-0.0015 part of cinnamon extract.

8. The cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury as claimed in claim 1, is characterized in that: the traditional Chinese medicine compound comprises the following components in parts by weight: 30-20 parts of cinnamon, 15-10 parts of liquorice and 0.0005-0.0015 part of cinnamon extract.

9. Use of the cinnamon extract for preventing and treating cold injury according to any one of claims 1-2 for preparing a medicament for increasing body thermogenesis, preventing cold injury, improving energy metabolism, and preventing and/or treating metabolic syndrome.

10. The application of the cinnamon extract traditional Chinese medicine compound for preventing and treating cold injury in any one of claims 3 to 8 in preparing medicines for increasing heat production of organisms, preventing cold injury, improving energy metabolism and preventing and/or treating metabolic syndrome.