阅读说明：本技术 防治寒冷损伤的肉桂提取物和中药复方及其应用 (Cinnamon extract and traditional Chinese medicine compound for preventing and treating cold injury and application thereof ) 是由 赵庆春 李想 许子华 祖宇欣 刘欣 曲莹莹 沈泽旭 陆鸿远 于 2019-09-10 设计创作，主要内容包括：本发明属生物制药领域,具体涉及防治寒冷损伤的肉桂提取物和中药复方及其应用。肉桂提取物包括六种化合物,制备方法是先进行粗制肉桂提取物,再精提肉桂提取物。防治寒冷损伤的肉桂提取物中药复方一包括肉桂、炙甘草、五加皮、葛根、鸡血藤、吴茱萸、党参、干姜、桂枝、肉桂提取物；制备方法是将乙醇提取和液相水提取的药液混合浓缩,再加入肉桂提取物。肉桂提取物中药复方二包括干姜、肉桂、肉桂提取物；肉桂提取物中药复方三包括肉桂、白芍、肉桂提取物；中药复方四包括肉桂、甘草、肉桂提取物。本发明的的肉桂提取物能够制备增加机体产热、预防寒冷损伤的药物中。中药复方能够在制备改善能量代谢、预防和/或治疗代谢综合征的药物中。(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.)

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:

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.

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:

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.

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:

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.

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.

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.

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;

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;

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; 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

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.

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 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 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.