CN110559336A - Preparation method and new medical application of liquorice stem and leaf total flavonoids - Google Patents
Preparation method and new medical application of liquorice stem and leaf total flavonoids Download PDFInfo
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- CN110559336A CN110559336A CN201910996513.5A CN201910996513A CN110559336A CN 110559336 A CN110559336 A CN 110559336A CN 201910996513 A CN201910996513 A CN 201910996513A CN 110559336 A CN110559336 A CN 110559336A
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- stem
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- total flavonoids
- liquorice
- licorice
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/484—Glycyrrhiza (licorice)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention relates to a preparation method and new application of total flavonoids in stems and leaves of liquorice (Glycyrrhiza uralensis Fisch.) or Glycyrrhiza glabra L or Glycyrrhiza inflata Bat. Specifically, the invention discovers that the total flavonoids in the stems and leaves of the liquorice have stronger lipase inhibition effect for the first time, and have good prevention and treatment effects on hyperlipidemia and obesity. Can be used for preparing medicine and health food for preventing and treating hyperlipidemia and obesity.
Description
Technical Field
The invention discloses a licorice stem and leaf total flavone with lipase inhibition effect and application thereof in preventing and treating hyperlipidemia and obesity, and also relates to extraction and separation of the total flavone and a pharmaceutical preparation, belonging to the technical field of medicines.
Background
The fat in food is hydrolyzed by lipase into monoacylglycerol and free fatty acid, and then absorbed in intestinal tract, and then synthesized again in body to cause fat accumulation, finally resulting in obesity. And hyperlipidemia is also directly associated with lipase. The lipase inhibitor can inhibit lipase activity, reduce fat entering body, and prevent and treat hyperlipidemia and obesity, and is helpful for preventing and reducing cardiovascular diseases and diabetes caused by obesity.
Although orlistat, a semi-synthetic pancreatic lipase inhibitor drug of rotz corporation, has been widely used in clinical practice as a weight-reducing drug, it has side effects such as liver injury, kidney injury, diarrhea, fatty stool, flatulence and the like after long-term administration [ zon and vigilant orlistat may cause a serious liver injury risk [ J ]. chinese medicine science, 2011, 1 (7): 10.], which brings trouble to patients. Natural compounds derived from plants have the characteristics of structural diversity, low toxicity, wide sources and the like, so that screening of new lipase inhibitors with smaller side effects from plants has been a research hotspot [ Jiangyuanyao, Lu national English, Liyanfei, and the like. 199-202.].
The invention discovers for the first time that the total flavonoids in the licorice stem leaves have the effect of inhibiting lipase and can be applied to the preparation of health-care food and medicines for preventing and treating hyperlipidemia and obesity. Comprehensively utilizes waste resources and has significance for resource and environmental protection.
The total flavonoids in the stems and leaves of the liquorice are compositions taking pinocembrin as a characteristic component, have various composition structures, and avoid the burden of single structural components on the metabolism of liver and kidney.
Before the completion of the invention, the effect of inhibiting lipase of the total flavonoids in the licorice stems and leaves is not found, and the application of the total flavonoids in the licorice stems and leaves in the medicines and health-care products for preventing and treating hyperlipidemia and obesity is not found.
Disclosure of Invention
One of the purposes of the invention is to provide the total flavonoids in the liquorice stems and leaves containing pinocembrin, which has the effect of inhibiting lipase. According to the invention, effective components in the liquorice stem leaves used as medicinal material production waste are screened through experiments, and the liquorice stem leaf total flavonoids extracted and purified by the process have good lipase inhibition activity, so that the liquorice stem leaf total flavonoids containing pinocembrin are obtained through experiments for the first time, and have outstanding contribution and significant technical progress. The invention provides a new medicine resource, can fully utilize waste resources, and is the most prominent contribution and the most remarkable innovation of the invention.
The second purpose of the invention is to provide a preparation method for extracting licorice stem leaf total flavonoids from licorice stem leaves, which comprises the following steps: cutting Glycyrrhrizae radix stem and leaf, extracting with 8-15 times of 50-60 deg.C hot water for 5-10 hr for two times, filtering, and discarding filtrate; extracting the residue with 70-90% ethanol under reflux for 1-3 times, each for 1-3 hr, filtering, mixing filtrates, adding 0.5-2% active carbon, stirring, filtering, collecting filtrate, passing the filtrate through D101 or AB-8 macroporous resin column treated with 70-90% ethanol, collecting ethanol solution passing through macroporous resin, recovering ethanol under reduced pressure, and drying to obtain total flavone of Glycyrrhrizae radix stem and leaf.
Preferably: cutting the stem and leaf of licorice into segments, leaching twice with hot water of 55 ℃ in an amount which is 10 times that of the stem and leaf of licorice for 6 hours each time, filtering, and discarding the filtrate; extracting the residue with 85% ethanol under reflux for 2 times, each for 2 hr, filtering, mixing filtrates, adding activated carbon 1.0% of the volume of the medicinal liquid, stirring, filtering, collecting filtrate, passing the filtrate through AB-8 macroporous resin column treated with 85% ethanol, collecting ethanol solution passing through macroporous resin, recovering ethanol under reduced pressure, and drying to obtain total flavone of Glycyrrhrizae radix stem and leaf.
The preparation method of the invention does not use organic reagent, ensures the use safety of the total flavonoids in the liquorice stems and leaves, has simple operation and strong industrialization, and is another innovation point of the invention.
The total flavonoids in the stem and leaves of the liquorice are characterized in that: the content of total flavonoids in the composition is not less than 80%.
In order to realize the technical scheme, the content of the total flavonoids in the liquorice stem leaves is determined by the following method:
Preparation of reference solution rutin reference 10mg is precisely weighed, placed in a 100ml measuring flask, dissolved by methanol and diluted to scale, shaken up, 5ml is precisely weighed, placed in a 10ml measuring flask, diluted to scale by methanol and shaken up, thus obtaining the rutin reference.
Preparing a test solution by weighing flavone powder 50mg precisely, adding an appropriate amount of methanol into a 50ml volumetric flask, carrying out ultrasonic treatment for 20min, taking out, standing at room temperature, adding methanol to a constant volume to a scale, shaking up, taking out 2ml to 25ml volumetric flask, and adding methanol to a constant volume to a scale.
The determination method comprises respectively taking reference solution, test solution, and accompanying blank. The absorbance was measured at a wavelength of 340nm by spectrophotometry (the general rule of the "Chinese pharmacopoeia" 2015 edition), and the absorbance was calculated.
The inhibition effect of the total flavonoids in the licorice stems and leaves on lipase has the effect of preventing and treating hyperlipidemia and obesity, and the pharmacological effects are proved by the following pharmacodynamic test examples.
1. Inhibition of lipase by licorice stem and leaf total flavone
1.1 measurement of Lipase inhibitory Activity
5mL of 0.025mol/L phosphate buffer (pH 7.4) and 4mL of polyvinyl alcohol triolein substrate emulsion (0.229g/mL) were added to a 100mL Erlenmeyer flask, incubated in a 37 ℃ water bath for 10min, followed by addition of 1mL of enzyme solution (2mg/mL) and reaction for 15min, and then stopped by addition of 15mL of 95% ethanol. Add phenolphthalein 2 drops and titrate to reddish with 0.025mol/L NaOH standard solution. The enzyme solution of the blank experiment was added after the reaction was terminated.
1.2 Positive control orlistat inhibition of Lipase
5mL of 0.025mol/L phosphate buffer (pH 7.4) and 4mL of polyvinyl alcohol triolein substrate emulsion (0.229g/mL) were added to a 100mL Erlenmeyer flask, 1mL of a positive control at a concentration of 8.0, 4.0, 2.0, 1.0, 0.5, 0.2, 0.1mg/mL was added, the mixture was incubated in a 37 ℃ water bath for 10min, 1mL of an enzyme solution (2mg/mL) was added, the reaction was then carried out for 15min, and 15mL of 95% ethanol was added to terminate the enzyme reaction. Add phenolphthalein 2 drops and titrate to reddish with 0.025mol/L NaOH standard solution.
1.3 Liquorice root stem and leaf Total Flavonoids have lipase inhibiting effect
5mL of 0.025mol/L phosphate buffer (pH 7.4) and 4mL of polyvinyl alcohol triolein substrate emulsion (0.229g/mL) were added to a 100mL Erlenmeyer flask, 1mL of licorice stem and leaf total flavonoids at concentrations of 5.0, 2.5, 1.0, 0.5, 0.25, 0.1, and 0.05mg/mL were added, the mixture was incubated in a 37 ℃ water bath for 10min, 1mL of enzyme solution (2mg/mL) was added, the reaction was continued for 15min, and 15mL of 95% ethanol was added to terminate the enzyme reaction. Add phenolphthalein 2 drops and titrate to reddish with 0.025mol/L NaOH standard solution. The results are shown in Table 1.
TABLE 1 inhibition of Lipase by Total Flavonoids of Licorice Stem and leaf
The result shows that the orlistat has good dose-effect relationship on the inhibition effect of lipase. The licorice stem leaf total flavonoids have good dose-effect relationship on the inhibition effect of lipase under the conditions that the concentration is 8.0, 4.0, 2.0, 1.0, 0.5, 0.2 and 0.1, and the inhibition effect of the licorice stem leaf total flavonoids on the lipase activity is weaker than that of the positive drug orlistat.
2. Influence of preventive administration of total flavonoids in licorice stems and leaves on blood lipid content of experimental hyperlipidemic rat
After the animals were purchased, they were first kept and observed to acclimatize for 1 week. Then, cutting the tail of each animal to take blood, and measuring TC; TG; HDL-cho and LDL-cho contents, randomly classified according to blood lipid levels as: a normal control group; a group of high lipid models; positive medicine blood fat recovery capsule control group; glycyrrhrizae radix stem and leaf total flavone 800mg/kg, 400mg/kg and 200 mg/kg; each group had 10. Except group 1 animals were fed regular diet as normal control; the rest 2-6 animals are fed with high cholesterol feed (based on 87.8% basic feed, add2% cholesterol, 10% lard and 0.2% propyl thiouracil) were added, and the feed intake was: 25 g/mouse/day. Each group of animals is dosed with ig once a day, wherein the volume of ig is 10 ml/kg; normal control and high fat model group animals had ig equal volume of distilled water per day; the administration was continued for 4 weeks. After the last 1 administration, all animals were fasted (without water) for 16 hours, blood was drawn by clipping tails, serum was separated, and Total Cholesterol (TC) in serum was measured; triglyceride (TG); high density lipoprotein cholesterol (HDL-cho) and low density lipoprotein cholesterol (LDL-cho). Comparing the difference of blood lipid level between groups, and adding or subtracting standard deviation from the mean value of the listed dataRepresents; statistical treatment methods used an interclass t-test.
TABLE 2 detection results of blood lipid content of rats in each group before preventive administration of total flavonoids from licorice stems and leaves
Note: the four indexes of the blood fat of each group of animals before administration are balanced and consistent, and the comparison among the groups is P > 0.05.
TABLE 3 Effect of Glycyrrhiza glabra stem and leaf Total Flavonoids on the blood lipid content of experimental hyperlipidemic rat by 4 weeks of prophylactic administration
Note: in comparison with the set of models,*P<0.05；**P<0.01；***P<0.001. Compared with the Xuezhikang capsule group★P<0.05。
As can be seen from the data listed in tables 2 and 3, before the experiment, the blood lipid levels of the animals in each group are relatively uniform, and the distribution among the groups has no obvious difference. After continuously feeding the high-fat feed for 4 weeks, the serum total cholesterol content and the low-density lipoprotein cholesterol content of the model animals are obviously increased compared with those of a normal control group (P < 0.001). This suggests that an animal model for hyperlipidemia (high cholesterol and high low density lipoprotein) has been developed. In three administration dose groups set by the licorice stem and leaf total flavonoids, the serum total cholesterol content and the low density lipoprotein content of animals in two dose groups of 800mg/kg and 400mg/kg are obviously lower than those in a high-fat model group (P <0.001 and P <0.05 respectively). In addition, the 800mg/kg administration dose group of the liquorice stem leaf total flavone has obviously better reducing effect on the serum total cholesterol and low-density lipoprotein cholesterol of high-fat model animals than that of the positive control medicament Xuezhikang (compared with Xuezhikang capsule group, P is less than 0.05). The experimental results show that the liquorice stem and leaf total flavonoids can obviously inhibit the formation of experimental rat hyperlipidemia after being administered for 4 weeks, and mainly can obviously reduce the contents of serum total cholesterol and low-density lipoprotein cholesterol. The ratio of the total cholesterol content of serum to the cholesterol content of high density lipoprotein (TC/HDL-cho) is reduced.
3. Influence of licorice stem and leaf total flavonoids on blood rheological property of experimental hyperlipidemia rat
after the experimental animals are purchased, the animals are fed regularly and are adapted to the environment for 1 week, and after the animals are purchased, the animals are firstly raised and observed to be adapted to the environment for 1 week. Then randomly dividing into: a normal control group; a group of high lipid models; positive control drug Xuezhikang capsule group; glycyrrhrizae radix stem and leaf total flavone 800mg/kg, 400mg/kg and 200 mg/kg; each group had 10. Except group 1 animals were fed regular diet as normal control; the other 2-6 groups of animals are fed with high cholesterol feed (2% of cholesterol, 10% of lard and 0.2% of propyl thiouracil are added on the basis of 87.8% of basic feed), and the feed intake is as follows: 25 g/mouse/day. Each group of animals is dosed with ig once a day, wherein the volume of ig is 10 ml/kg; normal control and high fat model group animals had ig equal volume of distilled water per day; the administration was continued for 14 d. After the last 1 administration, animals were fasted for 12 hours without water deprivation, anesthetized with 10% chloral hydrate (4mL/kg) ip, 3.5mL of blood was taken via the abdominal aorta, and heparin anticoagulated. The blood rheology was measured.
TABLE 4 Effect of Total Flavonoids of Licorice Stem and leaf on hemorheology of rats with experimental hyperlipidemia
Note: and comparing with high-fat model group, P <0.05 > and P < 0.01.
It can also be seen from the experimental results in table 4 that the whole blood viscosity (low cut, medium cut and high cut) and plasma viscosity of the animals in the high-fat model group are significantly higher than those in the normal control group (P < 0.01). The results in table 4 show that the total flavonoids from the stem and leaf of licorice root can significantly improve the hemorheological properties of rats with experimental hyperlipidemia. Can reduce the viscosity of whole blood (low cut, medium cut and high cut) and blood plasma.
Detailed Description
The present invention is realized (demonstrated) by the following examples, but the technical solution of the present invention is not limited thereto.
EXAMPLE 1 preparation of Total Flavonoids from Liquorice Stem and leaf
Extracting 50kg of liquorice stem and leaf medicinal slices twice with 10 times of hot water at 55 ℃ for 6 hours, filtering, and discarding the filtrate; extracting the stem and leaf of Glycyrrhrizae radix with 85% ethanol under reflux for 2 times (2 hr), filtering, mixing filtrates, adding medicinal activated carbon 1.0% of the volume of the medicinal liquid, stirring, filtering, collecting filtrate, passing the filtrate through pretreated AB-8 macroporous resin column, collecting ethanol solution passing through macroporous resin, recovering ethanol under reduced pressure, and drying to obtain 1622g total flavone of stem and leaf of Glycyrrhrizae radix with total flavone content of 88.1%.
EXAMPLE 2 preparation of capsules
Taking 200g of total flavonoids in the stem and leaf of the liquorice, adding 100g of proper amount of starch, granulating by using 75% ethanol, drying, and encapsulating to prepare 1000 granules.
EXAMPLE 3 preparation of tablets
Taking 200g of total flavonoids in the stem and leaf of liquorice, adding 100g of starch, granulating with 75% ethanol, drying, tabletting and preparing into 1000 tablets.
Claims (5)
1. A preparation method of total flavonoids in liquorice stems and leaves is characterized by comprising the following steps: cutting Glycyrrhrizae radix stem and leaf, extracting with 8-15 times of 50-60 deg.C hot water for 5-10 hr for two times, filtering, and discarding filtrate; extracting the residue with 70-90% ethanol under reflux for 1-3 times, each for 1-3 hr, filtering, mixing filtrates, adding 0.5-2% active carbon, stirring, filtering, collecting filtrate, passing the filtrate through D101 or AB-8 macroporous resin column treated with 70-90% ethanol, collecting ethanol solution passing through macroporous resin, recovering ethanol under reduced pressure, and drying to obtain total flavone of Glycyrrhrizae radix stem and leaf.
2. The method for preparing licorice stem and leaf total flavonoids according to claim 1, wherein the extract comprises: cutting the stem and leaf of licorice into segments, leaching twice with hot water of 55 ℃ in an amount which is 10 times that of the stem and leaf of licorice for 6 hours each time, filtering, and discarding the filtrate; extracting the residue with 85% ethanol under reflux for 2 times, each for 2 hr, filtering, mixing filtrates, adding activated carbon 1.0% of the volume of the medicinal liquid, stirring, filtering, collecting filtrate, passing the filtrate through AB-8 macroporous resin column treated with 85% ethanol, collecting ethanol solution passing through macroporous resin, recovering ethanol under reduced pressure, and drying to obtain total flavone of Glycyrrhrizae radix stem and leaf.
3. the licorice stem and leaf total flavonoids according to claims 1 and 2, characterized in that: the content of total flavone is not less than 80%.
4. The effects and applications of licorice stem and leaf total flavonoids according to any one of claims 1-3, wherein: comprises a preparation which contains the total flavonoids from the liquorice stems and leaves with effective treatment dose and one or more pharmaceutically acceptable medicinal excipients or other medicaments which can be combined with the total flavonoids from the liquorice stems and leaves.
5. The grass stem and leaf total flavonoids according to any one of claims 1-4, having lipase inhibiting effect, can be used for preparing medicines or health foods for preventing and treating obesity.
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