CN111513155B - Composition for preventing and treating lipid metabolism disorder - Google Patents

Composition for preventing and treating lipid metabolism disorder Download PDF

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CN111513155B
CN111513155B CN202010436353.1A CN202010436353A CN111513155B CN 111513155 B CN111513155 B CN 111513155B CN 202010436353 A CN202010436353 A CN 202010436353A CN 111513155 B CN111513155 B CN 111513155B
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group
composition
liver
tea
rats
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CN111513155A (en
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杨兴鑫
俞捷
李静平
穆健康
顾雯
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Yunnan University of Traditional Chinese Medicine TCM
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Yunnan University of Traditional Chinese Medicine TCM
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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23FCOFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00Tea; Tea substitutes; Preparations thereof
    • A23F3/06Treating tea before extraction; Preparations produced thereby
    • A23F3/14Tea preparations, e.g. using additives
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06Fungi, e.g. yeasts
    • A61K36/062Ascomycota
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06Fungi, e.g. yeasts
    • A61K36/07Basidiomycota, e.g. Cryptococcus
    • A61K36/076Poria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/23Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/232Angelica
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/25Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258Panax (ginseng)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/28Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/285Aucklandia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/82Theaceae (Tea family), e.g. camellia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/88Liliopsida (monocotyledons)
    • A61K36/896Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8969Polygonatum (Solomon's seal)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/88Liliopsida (monocotyledons)
    • A61K36/899Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/06Antihyperlipidemics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/10Preparation or pretreatment of starting material
    • A61K2236/19Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/30Extraction of the material
    • A61K2236/33Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/30Extraction of the material
    • A61K2236/39Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/51Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation 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
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/53Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Abstract

The invention provides a health tea for preventing lipid metabolism disorder, which is prepared from the following raw materials: 1-20g of rhizoma polygonati, 1-20g of angelica, 1-20g of pseudo-ginseng, 1-10g of elecampane, 1-10g of poria cocos, 1-10g of red yeast rice and 1-20g of Pu' er tea. The health tea has reasonable compatibility, and can effectively prevent and treat symptoms related to lipid metabolism disorder.

Description

Composition for preventing and treating lipid metabolism disorder
Technical Field
The application relates to health-care tea, in particular to a composition for preventing and treating abnormal lipid metabolism.
Background
Abnormal lipid metabolism is usually associated with excessive appetite, fatty taste, fatigue, emotional disorder, congenital deficiency, and other diseases, which are caused by liver depression, spleen deficiency, kidney qi deficiency, phlegm stagnation and blood stasis. The disease is localized in the liver, involving the spleen, stomach, kidney and other viscera, and is marked by deficiency of the origin, deficiency of the spleen and kidney, and deficiency of the spleen and kidney, with turbid phlegm and blood stasis as the targets (the journal of Chinese medicine, 2016, 34 (11): 2586-2589).
Although similar compositions exist in the prior art, for example, CN200810058332 discloses a pseudo-ginseng flower and leaf Pu' er tea, which comprises 50-80% by weight of tea leaves, 10% by weight of pseudo-ginseng flowers and 10-40% by weight of pseudo-ginseng flower and leaves, and has good metabolism effects on fat removal, weight reduction, alcoholic liver and fatty liver. CN201310597025 discloses a composition made of the following raw materials by weight: corydalis saxicola bunting, rhizoma picrorhizae, pseudo-ginseng, salvia miltiorrhiza, radix puerariae, rhizoma atractylodis macrocephalae, radix paeoniae alba, poria cocos, radix bupleuri, angelica sinensis, lotus leaves, semen coicis, semen benincasae, rhizoma phragmitis, platycodon grandiflorum, semen lepidii, folium eriobotryae, fritillaria cirrhosa, fructus lycii, semen cassiae, rhizoma alismatis, peach kernels and folium mori, the fat deposition of liver cells is relieved, the liver function is improved, and the liver index is reduced, so that the liver cancer treating composition has an obvious treatment effect on non-alcoholic fatty liver disease rats. However, the prior art composition has unsatisfactory effect or slightly excessive medicinal taste, is not suitable for practical application in production and life, and thus is not widely used.
Disclosure of Invention
In the invention, the formula consists of 7 substances of rhizoma polygonati, angelica, pseudo-ginseng, elecampane, poria cocos, red yeast rice and Pu' er tea, and the substances are both homologous in medicine and food (can be used as medicinal materials and food). In the formula, the Chinese rhubarb has the effects of tonifying qi and yin, strengthening spleen, moistening lung and tonifying kidney, and enters spleen, lung and kidney channels; the angelica has the functions of enriching blood, activating blood, regulating menstruation, relieving pain and relaxing bowel, is an essential medicine for enriching blood, activating blood and removing stasis, and belongs to the liver, heart and spleen channels, the pseudo-ginseng has the functions of removing blood stasis, stopping bleeding, activating blood and relieving pain and belongs to the liver and stomach channels, and the functions of enriching blood, activating blood and removing stasis are realized by the compatibility of the angelica and the stomach; aucklandia root, radix aucklandiae has the actions of moving qi and soothing liver, regulating middle energizer and relieving stagnation, and enters spleen, stomach, liver and lung meridians; the tuckahoe has the effects of promoting the subsidence of swelling by water, excreting dampness, strengthening the spleen and calming the heart, and enters the channels of the heart, the lung, the spleen and the kidney, and the tuckahoe matched with the formula is beneficial to eliminating turbid phlegm and damp evil out of the body. Therefore, the substances in the formula can effectively aim at the pathogenesis of lipid metabolism disorder. In addition, modern researches show that the red yeast rice and the Pu ' er tea have pharmacological activity for obviously regulating lipid metabolism disorder (doctor's academic paper of Zhejiang university, 2012; master's academic paper of Anhui medical university, 2017), and the two substances are combined in the formula, so that the function of the formula for regulating lipid metabolism disorder can be further enhanced. Therefore, by means of soothing liver, strengthening spleen, eliminating dampness and removing blood stasis, the composition can effectively prevent and treat symptoms related to lipid metabolism disorder, and can be used for developing medicaments, health-care products and functional foods for preventing and treating lipid metabolism disorder.
Specifically, the invention provides health-care tea for preventing lipid metabolism disorder, which is prepared from the following raw materials: 1-20g of rhizoma polygonati, 1-20g of angelica, 1-20g of pseudo-ginseng, 1-10g of elecampane, 1-10g of poria cocos, 1-10g of red yeast rice and 1-20g of Pu' er tea.
The health tea is prepared from the following raw materials: 1-10g of rhizoma polygonati, 1-10g of angelica, 1-10g of pseudo-ginseng, 1-5g of elecampane, 1-5g of poria cocos, 1-5g of red yeast rice and 1-10g of Pu' er tea.
The health tea is prepared from the following raw materials: 1-5g of rhizoma polygonati, 1-5g of angelica, 1-5g of pseudo-ginseng, 1-4g of elecampane, 1-4g of poria cocos, 1-4g of red yeast rice and 1-5g of Pu' er tea.
The health tea is prepared from the following raw materials: 2-4g of rhizoma polygonati, 2-4g of angelica, 2-4g of pseudo-ginseng, 1-3g of elecampane, 1-3g of poria cocos, 1-3g of red yeast rice and 2-4g of Pu' er tea.
The health tea is prepared from the following raw materials: 3g of rhizoma polygonati, 3g of angelica, 3g of pseudo-ginseng, 2g of elecampane, 2g of poria cocos, 2g of red yeast rice and 3g of Pu' er tea.
The invention also provides a composition for preventing and treating lipid metabolism disorder, which comprises the following raw material medicines: 1-20g of rhizoma polygonati, 1-20g of angelica, 1-20g of pseudo-ginseng, 1-10g of elecampane, 1-10g of poria cocos, 1-10g of red yeast rice and 1-20g of Pu' er tea.
Further, a composition for treating lipid metabolism disorder is provided, and the raw material medicines of the composition comprise: 1-10g of rhizoma polygonati, 1-10g of angelica, 1-10g of pseudo-ginseng, 1-5g of elecampane, 1-5g of poria cocos, 1-5g of red yeast rice and 1-10g of Pu' er tea.
Further, a composition for treating lipid metabolism disorder is provided, and the raw material medicines comprise: 1-5g of rhizoma polygonati, 1-5g of angelica, 1-5g of pseudo-ginseng, 1-4g of elecampane, 1-4g of poria cocos, 1-4g of red yeast rice and 1-5g of Pu' er tea.
Further, a composition for treating lipid metabolism disorder is provided, and the raw material medicines comprise: 2-4g of rhizoma polygonati, 2-4g of angelica, 2-4g of pseudo-ginseng, 1-3g of elecampane, 1-3g of poria cocos, 1-3g of red yeast rice and 2-4g of Pu' er tea.
Further, a composition for treating lipid metabolism disorder is provided, and the raw material medicines of the composition comprise: 3g of rhizoma polygonati, 3g of angelica, 3g of pseudo-ginseng, 2g of elecampane, 2g of poria cocos, 2g of red yeast rice and 3g of Pu' er tea.
The preparation method of the health tea comprises the following steps:
pulverizing the raw materials into fine powder, reflux-extracting with 12 times of 95% ethanol at 90 deg.C, reflux-extracting with 12 times of 70% ethanol for 2 hr each time, mixing the extractive solutions, concentrating at 65 deg.C and 100 deg.C under reduced pressure, recovering ethanol, and making into lyophilized powder. It is administered orally.
The preparation method of the composition comprises the following steps:
pulverizing the raw materials into fine powder, performing reflux extraction with 12 times of 95% ethanol at 90 deg.C, performing reflux extraction with 12 times of 70% ethanol for 2 hr each time, mixing the two extractive solutions, concentrating at 65 deg.C and 100 deg.C under reduced pressure, recovering ethanol, and making into pharmaceutically acceptable preparation by conventional method.
The preparation is selected from decoction, tablet, capsule, pill, dripping pill, granule, and powder.
Drawings
FIG. 1 food intake of rats in each group during the experiment (. Prime.P <0.05,. Prime.P <0.01,. Prime.P <0.001, compared to normal group)
FIG. 2 weight growth tendency of rats in each group during the experiment
FIG. 3 Effect of the composition on serum TC, TG levels in rats with lipid metabolism disorders (. SP <0.05,. SP <0.01,. SP <0.001, compared to the model group)
FIG. 4 liver histomorphology (oil Red O staining, X200) of rats in each group
A, normal group B, model group C, low dose group D, medium dose group E, high dose group F, resveratrol group
FIG. 5 immunohistochemical analysis of rat hepatocytes (tune,. Times.200)
A, normal group B, model group C, low dose group D, medium dose group E, high dose group F, resveratrol group
FIG. 6 Effect of the composition on high fat-induced lipid metabolism disorder rat liver TG, TC, LDL-C and HDL-C
P <0.05 compared to model group; * P < 0.01; * P <0.001
FIG. 7 Effect of the composition on MDA and GSH in rat liver with high fat-induced lipid metabolism disorder (P < 0.05;. P < 0.01;. P <0.001, compared to model group)
FIG. 8 Effect of the composition on the levels of mitochondria Complex I, camplex II, ATP in rat liver with high lipid-induced lipid metabolism disorders (P < 0.05;. P < 0.01;. P <0.001, compared to model group)
FIG. 9 Effect of the composition on the levels of GSH, SOD and MDA in liver mitochondria of rats with high lipid-induced lipid metabolism disorders (P < 0.05; P < 0.01; P <0.001; in comparison with the model group)
FIG. 10 food intake of rats in each group during the experiment
FIG. 11 weight growth trend of rats in each group during the experiment
FIG. 12 Effect of composition, red Rice and Pu her tea on serum TG and TC content in NAFLD rats (P <0.05, P <0.01, P <0.001; P <0.05, # P <0.01, # P <0.001; composition group, # P <0.05, # P <0.01, # P < 0.001)
FIG. 13 liver histomorphology (oil red O staining, X200) of rats in each group
A, normal group; b, model group; c, resveratrol group; d, red yeast rice group; e, pu' er tea group; f, composition group
FIG. 14 immunohistochemical analysis of rat hepatocytes (tune, X200)
A, normal group; b, model group; c, resveratrol group; d, a red yeast group; e, pu' er tea group; f, composition group
FIG. 15 Effect of composition, red Rice and Pu her tea on NAFLD rat liver TG, TC, LDL-C and HDL-C (P <0.05, P <0.01, P <0.001 for model group; P <0.05, # P <0.01, # P <0.001 for composition group)
FIG. 16 Effect of the composition, red Rice and Pu' er tea on high fat induced NAFLD rat liver MDA, GSH (P <0.05, P <0.01, P <0.001 for the model group; P <0.05, # P <0.01, P <0.001 for the composition group)
FIG. 17 effects of composition, red Rice and Pu' er tea on high lipid-induced NAFLD rat liver mitochondria complexI, complexII, ATP levels (P <0.05, # P <0.01, # P <0.001; composition group, # P <0.05, # P <0.01, # P < 0.001)
Detailed Description
EXAMPLE 1 decoction
(1) Prescription
3g of rhizoma polygonati, 3g of angelica, 3g of pseudo-ginseng, 2g of elecampane, 2g of poria cocos, 2g of red yeast rice and 3g of Pu' er tea.
(2) Preparation of
Pulverizing the above seven medicinal materials, decocting with 8 times of water twice, each for 2 hr, and concentrating.
Example 2 granules
(1) Prescription
3g of rhizoma polygonati, 3g of angelica, 3g of pseudo-ginseng, 2g of elecampane, 2g of poria cocos, 2g of red yeast rice and 3g of Pu' er tea.
(2) Preparation of
Pulverizing the raw materials into fine powder, reflux-extracting with 12 times of 95% ethanol at 90 deg.C, reflux-extracting with 12 times of 70% ethanol for 2 hr each time, mixing the extractive solutions, concentrating under reduced pressure at 65 deg.C and 100 deg.C, recovering ethanol, preparing lyophilized powder, adding sucrose and dextrin, mixing, granulating, and drying.
EXAMPLE 3 clinical trials
1. Method of treatment
The method for treating hyperlipemia by soothing liver, strengthening spleen, eliminating dampness and removing blood stasis comprises the following steps: 3g of rhizoma polygonati, 3g of angelica, 3g of pseudo-ginseng, 2g of elecampane, 2g of poria cocos, 2g of red yeast rice and 3g of Pu' er tea. 1 dose daily, decocted in water for oral administration, and taken 2 times in the morning and at night. 10 days is 1 course of treatment, and 6 courses of treatment are continued. Follow-up was for 6 months.
2. Standard of therapeutic effect
The effect is shown: after treatment, the blood fat is reduced to a normal level, and after half a year, the blood fat is not increased after the double-check. The method has the following advantages: after treatment, blood fat is obviously reduced, but the blood fat level is slightly higher than the normal level, and the trend of continuous medication is also reduced. And (4) invalidation: the blood lipid level is slightly reduced, but the blood lipid level tends to increase after half a year of stopping taking the medicine.
3. Results
After being treated by 238 patients with hyperlipidemia and obesity for 6 courses of treatment, the patients with hyperlipidemia have different degrees of curative effect changes in mild, moderate and severe degrees, and the significant efficiency is 67.2%. Meanwhile, the functions of liver and kidney and the routine of hematuria are normal after reexamination, and invalid patients are reexamined after half a year, and the rest blood fat has no phenomenon of rising and rebounding.
TABLE 1 therapeutic Effect of 238 patients (% of cases)
Figure BDA0002502419200000031
Figure BDA0002502419200000041
EXAMPLE 4 intervention of the composition on rats with lipid metabolism disorders induced by high-fat diet
1. Experimental materials and methods
(I) test materials
1 materials of the experiment
All the materials are purchased from the medicinal material wholesale market of Kunming snail bay, and certificate specimens are all stored in the key laboratory for preventing and treating metabolic diseases in the Kunming market.
Positive control resveratrol was purchased from Doppel Biotechnology, inc.
2 Experimental animals
Clean-grade SPraque-Dawley healthy male rats at 8 weeks of age 60, weighing 180 + -20g, purchased from WUDO animals Inc., quality certification number: SCXK (Chuan) 2015-030. The experimental conditions and the method are qualified by the ethical examination of the animal experiment center of the Yunnan college of traditional Chinese medicine, and the animal ethical examination document number is as follows: r-062014012. Prior to the experiment, all rats were acclimatized for 1 week, during which time the animals were fed ad libitum with free access to water. Keeping the feeding room quiet, keeping the indoor temperature at 23-25 ℃, keeping the humidity at 55-75%, illuminating by a fluorescent lamp, and keeping the illumination for 12 hours and the darkness for 12 hours in 24 hours every day.
3 Experimental reagent
Experimental animal feed (Suzhou double lion experimental animal feed science and technology limited)
Resveratrol (Chengdu Philide biotechnology limited)
Low-density lipoprotein (LDL-C) test box (Nanjing institute of bioengineering)
Malondialdehyde (MDA) test box (Nanjing institute for bioengineering)
Superoxide dismutase (SOD) test box (Nanjing institute of bioengineering)
Triglyceride (TG) testing box (Nanjing institute for bioengineering)
Total cholesterol (T-CHO) test box (Nanjing institute of biological engineering)
High density lipoprotein (HDL-C) test box (Nanjing institute for biological engineering)
Glutathione peroxidase (GSH) test kit (Nanjing institute of biological engineering)
ATP enzyme test box (Nanjing institute of bioengineering)
BCA protein concentration determination kit (Biyuntian biotechnology research institute)
Rat mitochondrial respiratory chain complex I test kit (Shanghai Jiwei Biotech Co., ltd.)
Rat mitochondrial respiratory chain complex II test box (Shanghai Jiwei Biotech Co., ltd.)
Sucrose (Tianjin City wind boat chemical reagent science and technology Co., ltd.)
Tris (Beijing Bootouda science and technology Limited)
Mannitol (Biosharp science and technology Co., ltd.)
EDTA (Biosharp technologies Co., ltd.)
EGTA (Biosharp technologies Co., ltd.)
Absolute ethyl alcohol (national medicine group chemical reagent Co., ltd.)
4 laboratory instruments
BS224 electronic balance (d =0.0001 g) (Beijing Saillis Instrument systems, inc.)
Vortex oscillator (LAB-BLOGEN Beijie science and technology VTX-E)
JJ1000 type precision electronic balance (d =0.02 g) (double Jie test instruments and instruments factory, well-known)
T-5000 type electronic balance (d =1 g) (ever-maturing Shuangjie testing instrument factory)
DY 89-II type electric glass homogenizer (Ningbo Xinzhi Biotechnology GmbH)
Enzyme mark analyzer (DNM-9602G) (Beijing Pulang New technology Co., ltd.)
Ultraviolet spectrophotometer (756 type) (Shanghai spectrometer Co., ltd.)
High speed desk type refrigerated centrifuge (TJL-20M) (Changshan instrument centrifuge Co., ltd.)
OSB-2100 type water-bath (Shanghai Ailang apparatus Co., ltd.)
N-1100 type rotary evaporator (Shanghai Ailang instrument Co., ltd.)
SHZ-DIII type circulating water vacuum pump (Steuer City Zenhua Instrument Limited liability company)
DL-5-B type centrifuge (Shanghai' an pavilion scientific instrument factory)
Water purifier (Milli-Q Academic A10 ultrapure water system) (MilliPore company, USA)
Ultra pure water system (Milli-Q Reference) (MilliPore, USA)
Ultra-low temperature refrigerator (American Thermo company)
SX-500 type steam sterilizer (TOMY KOGYO CO LTD Co., japan)
HHS type electric constant temperature water bath (Shanghai Boxun industry Co., ltd. Medical equipment factory)
Ultrasonic cleaning machine (14-1275) (Ningbo Xinzhi Biotechnology Co., ltd.)
-80 ℃ refrigerator 8920 (834747-328) (Thermo scientific)
DY 89-II type electric glass homogenizer (Ningbo Xinzhi Biotechnology GmbH)
SPectrama Plus plate reader (Molecular Devices, USA)
(II) Experimental method
1 preparing the extract of the composition
Grinding the composition into fine powder, and mixing the components in a ratio of 3:3:3:2:2:2:3, mixing, then carrying out reflux extraction with 12 times of 95% ethanol at 90 ℃, carrying out reflux extraction with 12 times of 70% ethanol for 2h each time, combining the two extracting solutions, carrying out concentration at 65 ℃ and 100 ℃ under reduced pressure, recovering ethanol, and preparing the freeze-dried powder.
2 replication, grouping and administration of lipid metabolism disorder rat model
The 60 clean grade SPraque-Dawley healthy SD male rats were randomly divided into 6 groups of 10, i.e. normal (saline), model (saline), positive (resveratrol), low, medium and high dose groups of composition extracts (grouping and dosing details are shown in table 2); each group of rats was gavaged with 1mL of 250g body weight for 12 weeks 1 time per day. Except for the normal group, each group was fed with high fat diet (basal diet 79%, lard 10%, egg yolk 10%, cholesterol 1%), and during the experiment, the food intake of rats was recorded once a day, the body weight of rats was recorded once a week, and the food intake and body weight of rats per week were calculated for each group.
TABLE 2 rat grouping and dosing
Figure BDA0002502419200000051
Figure BDA0002502419200000061
3 sample Collection
After the last administration at 12 weeks, the experimental rats were fasted for 12h, anesthetized with sodium pentobarbital, bled, immediately centrifuged (3500 r. Min. -1, 10 min) to separate the serum, and the desired organs were stored in a refrigerator at-80 ℃ for further use.
4 histopathological Observation
Dyeing with oil red O: the liver is taken, and morphological changes are observed after fixing, dehydration, paraffin embedding, flaking and oil red O staining.
TUNEL detection of hepatocyte apoptosis: reference methods, immunohistochemistry was performed to detect apoptosis.
5 serum Biochemical index determination
Detecting the content of total cholesterol TC and triglyceride TG in serum according to the specific steps of the kit instruction.
Index of 6 organs
Each organ (liver, spleen, kidney) was dissected and weighed, and the organ index was calculated as organ index = [ organ weight g/body weight g ] × 100%.
7 Biochemical index determination of liver tissue
0.1g of rat liver was minced in a homogenizer tube (ice bath procedure), 0.9mL of precooled raw material was addedHomogenizing with saline 10 times, transferring to 1.5mL centrifuge tube, fermenting at 3000rpm at 4 deg.C for min -1 Centrifuging for 10min, and collecting supernatant. And (3) determining the protein content in the supernatant by using a BCA protein determination kit, adding the reagents in sequence according to the operation of a kit specification, oscillating, fully mixing uniformly, and determining the contents of TC, TG, HDL-C, LDL, MDA and GSH in the supernatant.
8 indexes related to liver cell mitochondria
(1) Hepatocyte mitochondrial isolation
Taking 0.1g of rat liver, adding 1mL of precooled mitochondrial separation fluid (0.21M mannitol, 0.07M sucrose, 10mM Tris base, 1mM EDTA, 0.5mM EGTA, pH to 7.4), and homogenizing in an ice water bath; transferring the tissue homogenate to a centrifuge tube, centrifuging at 4 ℃ and 1000 Xg for 10min, discarding the precipitate, transferring the supernatant to another centrifuge tube, centrifuging at 4 ℃ and 10000 Xg for 10min, and obtaining the precipitate, namely the hepatocyte mitochondria.
(2) Determination of MDA, SOD, GSH, ATP synthase and complete I, II in liver cell mitochondria
Taking liver cell mitochondria, adding physiological saline, mixing uniformly to form a suspension, and determining the protein concentration of the mitochondria by using a BCA protein concentration determination kit; 100 mu L of mitochondrial suspension is taken respectively, and according to the operation of the kit specification, SOD activity is determined by an hydroxylamine method, MDA content is determined by a thiobarbituric acid method, GSH and ATP synthase activity is determined by a colorimetric method, and Complex I and II activity is determined by a double-antibody sandwich enzyme-linked immunosorbent assay.
9 statistical processing
Mean ± sd of each group
Figure BDA0002502419200000062
Data were analyzed using SPSS 21.0 statistical software, and between-group comparisons were performed using analysis of variance (One Way-ANOVA), and between groups were performed using the LSD-t test, where P <0.05 was considered as a statistical difference.
2. Results of the experiment
Effect of composition on weight gain of rats with lipid metabolism disorder induced by high fat
In the experimental process, rats in each group are not dead, normally eat and drink water, move freely, have good fur glossiness and quickly respond to external stimulation. From the beginning of the experiment to the end of the experiment at 12 weeks, the results show that the average daily food intake of the rats in each group has no significant difference (figure 1), the rats in each group have a positive growth trend (figure 2), and the weight is normally increased.
(II) influence of composition on TC and TG levels in serum of rat with high fat induced lipid metabolism disorder
As shown in FIG. 3, TG content was significantly increased in the model group (P < 0.001) compared to the normal group, indicating that the induction of high-fat diet is very likely to cause serum lipid disorders in rats. Compared with the model group, at the end of the 12 th week experiment, the content of TC and TG in the serum of the resveratrol group rat is reduced remarkably (P <0.01, P < -0.001), the content of TG in the high-medium low-dose group is reduced remarkably (P < 0.001), and the content of TC in the high-dose group is reduced remarkably (P < 0.05) without significant difference. The result shows that the composition extract can regulate the dyslipidemia of rats with lipid metabolism disorder induced by high-fat feed.
(III) Effect of composition on organ index of rat with lipid metabolism disorder induced by high fat
After 12 weeks of experiment, the liver, kidney and spleen of each rat were weighed, and the liver index, spleen index and kidney index of each group were calculated (table 2). The results show that the liver indexes of other groups are significantly different (P is less than 0.05, P is less than 0.01, P is less than 0.001) compared with the liver indexes of the normal group, and the results show that the liver is enlarged after the high-fat feed is fed for 12 weeks; compared with the normal group, the spleen index and the kidney index have no significant difference (P < 0.05), and the spleen index and the kidney index are remarkably lower in the model group than in other groups, which indicates that high fat diet may damage the spleen and the kidney to a certain extent, and the damage can be improved after the drug alcohol extract is administrated. Compared with the model group, except the high-dose group, the liver swelling degree of other administration groups can be reduced, and the compound extract can improve the liver injury of the rat with the high fat-induced lipid metabolism disorder.
TABLE 3 Effect of compositions on organ index of lipid metabolism-disturbed rats: (
Figure BDA0002502419200000071
n=7)
Figure BDA0002502419200000072
* P <0.05, P <0.01, P <0.001, compared to the normal group; # P <0.05, # P <0.01, # P <0.001, compared to the model group
(IV) Effect of the composition on liver histomorphology of rat with high fat induced lipid metabolism disorder
The histomorphosis results of the liver of rats in each group show (figure 4), the area percentage of lipid droplets of rats in the normal group is 4.27%, the area percentage of lipid droplets of rats in the model group is 55.47%, the area percentage of lipid droplets of resveratrol group is 38.68%, and the compound extract is low, and the area percentage of lipid droplets of rats in the medium and high dose groups is 40.82%, 39.21% and 45.17%. The rats in the model group show diffuse hepatocellular edema degeneration, are scattered in a large number of round vacuolated lipid droplets with different sizes and inflammatory cell infiltration in the sink area. The pathological morphology of the liver of the rats interfered by the resveratrol group and the administration group is close to that of the normal control group, a small amount of inflammatory cells infiltrate, and the number of lipid drops is less than that of the model group. The result shows that the composition extract can effectively improve the liver histomorphosis caused by high-fat feed.
The immunohistochemical Tunel apoptosis rate analysis result shows (figure 5), the liver cell apoptosis rates of the normal group, the model group, the resveratrol group, the low metering group, the medium dosage group and the high dosage group are respectively 19.76%, 35.00%, 6.23%, 41.26%, 16.98% and 22.72%, and the results show that the medium dosage group and the high dosage group of the composition extract can relieve cell apoptosis in the process of lipid metabolism disorder and protect liver cells from being damaged.
(V) Effect of the composition on lipid metabolism disorder rat liver TG, TC, LDL-C and HDL-C
The results in FIG. 6 show that the liver tissues of the model group have significantly increased TC, TG and LDL-C contents (P < 0.001) and significantly decreased HDL-C contents (P < 0.001), compared with the normal group, indicating that the liver of the rat induced by high fat has significant lipid metabolism disorder. Compared with a model group, TC and TG in liver tissues of the resveratrol group are remarkably reduced (P is less than 0.01, P is less than 0.001), HDL-C content is remarkably increased (P is less than 0.001), and LDL-C content has a reduction trend but has no remarkable change, so that the resveratrol group can regulate high-fat induced liver lipid abnormality; after the compound extract with different doses is given, the TC and TG contents in the low, medium and high dose administration groups are reduced remarkably (P is less than 0.001); the LDL-C content of the high-dose administration group is reduced remarkably (P is less than 0.001), and meanwhile, the low-dose and medium-dose administration groups can reduce the LDL-C content to a certain extent without remarkable difference; the HDL-C content in the low, medium and high dose administration groups has a certain rising trend, but no significant difference exists. The results show that the compound extract can better improve the liver lipid abnormality induced by high-fat feed.
(VI) Effect of the composition on MDA and GSH of rat liver with lipid metabolism disorder
As shown in the results of FIG. 7, compared with the normal group, the MDA content in liver mitochondria of the model group rats is remarkably increased (P < 0.001), and the GSH activity is remarkably reduced (P < 0.05), which indicates that oxidative stress and lipid peroxidation damage exist in the livers of the rats with lipid metabolism disorder. Compared with a model group, after the compound extract is administered, the MDA content in each dose administration group is reduced extremely remarkably (P is less than 0.001), the GHS content in a medium dose administration group is increased extremely remarkably, and low and high dose administration groups are increased to a certain extent but are not changed remarkably. The composition extract can effectively regulate the oxidative stress and lipid peroxidation damage of rats with lipid metabolism disorder, thereby relieving the occurrence of the lipid metabolism disorder.
(VII) Effect of the composition on ATP synthase and Complex I, II in liver mitochondria of rats with lipid metabolism disorder
As can be seen from the results of FIG. 8, in the liver mitochondria of the model group rat, complex I, complex II, ATP synthase (Na) are present in the liver mitochondria + -K + -ATPase, ca 2+ -Mg 2+ -ATPase) content is extremely reduced (P < 0.001), and the energy metabolism disorder of liver mitochondria of rats with the lipid metabolism disorder is shown. And compared with a model group, the resveratrol remarkably increases Na + -K + -ATPase Activity (P)<0.001 Significant differences in Complex II activity (P)<0.05 To some extent increase Ca 2+ -Mg 2+ The activity of ATPase and Complex II is not significantly different, which shows that the ATPase and the Complex II can improve the energy metabolism of rats with lipid metabolism disorder to a certain extent; for feedingAfter the compound alcohol extract with different doses is added, the Complex II activity in liver mitochondria of low, medium and high dose administration groups is remarkably improved (P is less than 0.001); the low-dose administration group can increase Na to a certain degree + -K + -atpase and complexii activities, but without significant difference; high dosage of Na + -K + -ATPase, ca 2+ -Mg 2+ Trend of ATPase activity, but no significant difference. The result shows that the compound extract can regulate the disturbance of the energy metabolism of the rat with the disorder of the lipid metabolism to a certain extent.
(VIII) influence of composition on MDA, GSH and SOD content in liver mitochondria of rat with lipid metabolism disorder
As shown in the results of FIG. 9, compared with the normal group, the liver mitochondria of the rats in the model group have significantly increased MDA content (P < 0.05), significantly reduced GSH content (P < 0.05), and somewhat reduced SOD and activity (no significant difference), indicating that the liver mitochondria of the rats with lipid metabolism disorder have oxidative stress and lipid peroxidation damage. Compared with a model group, the resveratrol group obviously reduces the MDA content (P < 0.05), obviously improves the GSH (P < 0.05) and improves the SOD activity to a certain extent, but has no obvious change, which indicates that the resveratrol group can improve the oxidative stress capability of rats with lipid metabolism disorder to a certain extent; after the composition extract is administered, the MDA content of a low-dose administration group is remarkably reduced (P is less than 0.01), and the MDA content of a medium-dose administration group and a high-dose administration group is reduced to a certain extent but has no remarkable difference; the SOD and GSH activity of the low-dose administration group has a certain rising trend (no obvious change), and the SOD activity of the medium-dose administration group is obviously raised (P is less than 0.01); the activity of the high-dose SOD and GSH is uniformly increased to a fixed extent (without obvious change); the compound extract is shown to be capable of regulating liver mitochondrial function to a certain extent and improving oxidative stress capability of NALFD rat liver.
Example 5 comparative study on the intervention Effect of the composition, red Rice and Pu her tea on rats with high fat diet induced lipid metabolism disorder
In order to further prove that the composition of the invention obtains more excellent effects of reducing blood fat and regulating lipid metabolism disorder compared with red yeast rice and Pu' er tea, action comparison research is carried out.
1. Experimental materials and methods
(I) test materials
1 materials of the experiment
All the materials are purchased from the medicinal material wholesale market of Kunming snail bay, and certificate specimens are all stored in the key laboratory for preventing and treating metabolic diseases in the Kunming market.
Resveratrol, a positive drug, was purchased from Doppel Biotechnology, inc.
2 Experimental animals
8 week old clean grade Spraque-Dawley healthy male rats 60, body weight 180 + -20g, purchased from WUDUDUDUDO animal Co., ltd., quality certification number: SCXK (Chuan) 2015-030. The experimental conditions and the method are qualified by the ethical examination of the animal experiment center of the Yunnan college of traditional Chinese medicine, and the animal ethical examination document number is as follows: r-062014012. Prior to the experiment, all rats were acclimatized for 1 week, during which time the animals were fed ad libitum with free access to water. Keeping the breeding room quiet, keeping the indoor temperature at 23-25 ℃, keeping the humidity at 55-75%, lighting by a fluorescent lamp, keeping the lighting for 12h and dark for 24h every day.
3 Experimental reagent
Experimental animal feed (Suzhou double lion experimental animal feed science and technology Co., ltd.)
Resveratrol (Chengdu Philide biotechnology limited)
Low-density lipoprotein (LDL-C) test box (Nanjing institute of bioengineering)
Malondialdehyde (MDA) test box (Nanjing institute for bioengineering)
Superoxide dismutase (SOD) test box (Nanjing institute of bioengineering)
Triglyceride (TG) test box (Nanjing institute of bioengineering)
Total cholesterol (T-CHO) test box (Nanjing institute for biological engineering)
High density lipoprotein (HDL-C) test box (Nanjing institute of bioengineering)
Glutathione peroxidase (GSH) test box (Nanjing institute of bioengineering)
ATP enzyme testing box (Nanjing institute for biological engineering)
BCA protein concentration determination kit (Biyuntian biotechnology research institute)
Rat mitochondrial respiratory chain complex I test kit (Shanghai Jiwei Biotech Co., ltd.)
Rat mitochondrial respiratory chain complex II test kit (Shanghai Jiwei Biotech Co., ltd.)
Sucrose (Tianjin City wind boat chemical reagent science and technology Co., ltd.)
Tris (Beijing Bootoda science and technology Co., ltd.)
Mannitol (Biosharp science and technology Co., ltd.)
EDTA (Biosharp technologies Co., ltd.)
EGTA (Biosharp technologies Co., ltd.)
Absolute alcohol (national medicine group chemical reagent Co., ltd.)
4 Experimental instrument
BS224 electronic balance (d =0.0001 g) (Beijing Saillis Instrument systems, inc.)
Vortex oscillator (LAB-BLOGEN Beijie science and technology VTX-E)
JJ1000 type precision electronic balance (d =0.02 g) (double Jie test instruments and instruments factory, well-known)
T-5000 type electronic balance (d =1 g) (ever-maturing Shuangjie testing apparatus factory)
DY 89-II type electric glass homogenizer (Ningbo Xinzhi Biotechnology GmbH)
Enzyme mark analyzer (DNM-9602G) (Beijing Pulang New technology Co., ltd.)
Ultraviolet spectrophotometer (756 type) (Shanghai spectrometer Co., ltd.)
High speed desk type refrigerated centrifuge (TJL-20M) (Changshan instrument centrifuge Co., ltd.)
OSB-2100 type water-bath (Shanghai Ailang apparatus Co., ltd.)
N-1100 type rotary evaporator (Shanghai Ailang instrument Co., ltd.)
SHZ-DIII type circulating water vacuum pump (Steuer City Zenhua Instrument Limited liability company)
DL-5-B type centrifuge (Shanghai' an pavilion scientific instrument factory)
Water purifier (Milli-Q Academic A10 ultrapure water system) (Millipore corporation, USA)
Ultra pure water system (Milli-Q Reference) (Millipore corporation, USA)
Ultra-low temperature refrigerator (American Thermo company)
SX-500 type steam sterilizer (TOMY KOGYO CO LTD Co., japan)
HHS type electric heating constant temperature water bath (Shanghai Boxun industry Co., ltd.)
Ultrasonic cleaning machine (14-1275) (Ningbo Xinzhi Biotechnology Co., ltd.)
-80 ℃ refrigerator 8920 (834747-328) (Thermo scientific)
DY 89-II type electric glass homogenizer (Ningbo Xinzhi Biotechnology GmbH)
SpectraMax Plus plate reader (Molecular Devices, USA)
(II) Experimental method
1 preparation of composition, red Rice and Pu-erh tea extract
Composition extract (B): grinding the composition into fine powder, and mixing the components in a ratio of 3:3:3:2:2:2:3, mixing, then carrying out reflux extraction with 12 times of 95% ethanol at 90 ℃, carrying out reflux extraction with 12 times of 70% ethanol for 2h each time, combining the two extracting solutions, carrying out concentration at 65 ℃ and 100 ℃ under reduced pressure, recovering ethanol, and preparing the freeze-dried powder.
Red yeast rice and Pu' er tea extracts: pulverizing red rice and Pu' er tea into fine powder, respectively reflux-extracting with 12 times of 95% ethanol at 90 deg.C, reflux-extracting with 12 times of 70% ethanol for 2 hr each time, mixing the extractive solutions, concentrating at 65 deg.C and 100 deg.C under reduced pressure, recovering ethanol, and making into lyophilized powder.
2NAFLD rat model replication, grouping and dosing
The 60 cleaning grade Spraque-Dawley healthy SD male rats are randomly divided into 6 groups, and 10 rats in each group are a normal group (normal saline), a model group (normal saline), a positive drug group (resveratrol), a composition group, a monascus group and a Pu' er tea group (the grouping and the administration are detailed in Table 4); each group of rats was gavaged with 1mL of 250g body weight for 12 weeks 1 time per day. Except for the normal group, each group was fed with high fat diet (basal diet 79%, lard 10%, egg yolk 10%, cholesterol 1%), and during the experiment, the food intake of rats was recorded once a day, the body weight of rats was recorded once a week, and the food intake and body weight of rats per week were calculated for each group.
TABLE 4 rat grouping and dosing
Figure BDA0002502419200000101
Figure BDA0002502419200000111
3 sample Collection
After the last administration at 12 weeks, the experimental rats were fasted for 12h, anesthetized with sodium pentobarbital, bled, immediately centrifuged (3500 r. Min. -1, 10 min) to separate the serum, and the desired organs were stored in a refrigerator at-80 ℃ for further use.
4 histopathological Observation
Dyeing with oil red O: the liver is taken, and morphological changes are observed after fixation, dehydration, paraffin embedding, flaking and oil red O staining.
TUNEL detection of hepatocyte apoptosis: reference methods, immunohistochemistry was performed to detect apoptosis.
5 serum biochemical index determination
Detecting the content of total cholesterol TC and triglyceride TG in serum according to the specific steps of the kit instruction.
Index of 6 organs
Each organ (liver, spleen, kidney) was dissected and weighed, and the organ index was calculated as = [ organ weight g/body weight g ] × 100%.
7 Biochemical index determination of liver tissue
0.1g of rat liver was minced in a homogenizing tube (ice bath procedure), 0.9mL of precooled physiological saline was added to homogenize 10 times, the homogenate was transferred to a 1.5mL centrifuge tube, centrifuged at 3000 rpm-1 for 10min at 4 ℃, and the supernatant was taken for later use. And (3) determining the protein content in the supernatant by using a BCA protein determination kit, adding the reagents in sequence according to the operation of the kit specification, oscillating, fully mixing uniformly, and determining the contents of TC, TG, HDL-C, LDL, MDA and GSH in the supernatant.
8 indexes related to liver cell mitochondria
(1) Hepatocyte mitochondrial isolation
Taking 0.1g of rat liver, adding 1mL of precooled mitochondrial separation fluid (0.21M mannitol, 0.07M sucrose, 10mM Tris base, 1mM EDTA, 0.5mM EGTA, pH to 7.4), and homogenizing in an ice water bath; transferring the tissue homogenate to a centrifuge tube, centrifuging at 4 ℃ and 1000 Xg for 10min, discarding the precipitate, transferring the supernatant to another centrifuge tube, centrifuging at 4 ℃ and 10000 Xg for 10min, and obtaining the precipitate, namely the hepatocyte mitochondria.
(2) ATP synthase and complete I, II assay in liver cell mitochondria
Taking liver cell mitochondria, adding physiological saline, mixing uniformly to form a suspension, and determining the protein concentration of the mitochondria by using a BCA protein concentration determination kit; 100 mu L of mitochondria suspension is taken respectively, the operation is carried out according to the instruction of the kit, the ATP synthase activity is measured by a colorimetric method, and the Complex I and II activity is measured by a double-antibody sandwich method enzyme-linked immunosorbent assay.
9 statistical processing
Mean plus or minus standard deviation of each group of data
Figure BDA0002502419200000112
Data were analyzed using SPSS 21.0 statistical software, and between-group comparisons were performed using analysis of variance (One Way-ANOVA), and between groups were performed using the LSD-t test, where P <0.05 was considered as a statistical difference.
2. Results of the experiment
(I) influence of composition, red yeast rice and Pu' er tea on weight gain of high fat-induced NAFLD rats
In the experimental process, rats in each group are not dead, normally eat and drink water, move freely, have good fur glossiness and quickly respond to external stimulation. From the beginning of the experiment to the end of the 12-week experiment, the results showed that there was no significant difference in average food intake per week for each group of rats (fig. 10), and each group of rats showed a positive growth trend (fig. 11) and normal weight gain.
(II) influence of composition, red yeast rice and Pu' er tea on serum TC and TG levels of high-fat-induced NAFLD rats
As shown in FIG. 12, TG content was significantly increased (P < 0.001) and TC content was significantly increased (P < 0.05) in the model group compared to the normal group, indicating that high-fat diet induction caused serum lipid disorders in rats. Compared with the model group, after the experiment in the 12 th week, the content of TC and TG in the serum of the resveratrol group rat is obviously reduced (P is less than 0.05 and P is less than 0.001), which shows that the resveratrol group rat can regulate and regulate the high fat induced NAFLD rat dyslipidemia; compared with the model group, the content of TG in the red yeast rice group, the Pu 'er tea group and the composition group is remarkably reduced (P is less than 0.001), and the content level of TG in the composition group is remarkably lower than that in the red yeast rice group and the Pu' er tea group (P is less than 0.05); meanwhile, the TC content of the composition is remarkably reduced (P is less than 0.05), and the TC contents of the red yeast rice group and the composition group have a reduction trend but are not remarkably changed. The results show that the red yeast rice, the Pu 'er tea and the composition can adjust the blood lipid abnormality of the NAFLD rat, and the effect of the composition is obviously superior to that of the red yeast rice or the Pu' er tea.
(III) influence of composition, red yeast rice and Pu' er tea on organ index of rat with high fat induction NAFLD
After 12 weeks of experiment, the liver, kidney and spleen of each group of rats were weighed, and the liver index, spleen index and kidney index of each group were calculated (table 5). The results show that the kidney index and the spleen index of each administration group are not significantly different (P is less than 0.05) compared with the normal group except the spleen index of the monascus group, which indicates that the experimental method does not cause significant damage to the kidney and the spleen of the rat. Compared with the normal group, the liver index difference of other groups is obvious (P is less than 0.05, P is less than 0.01, P is less than 0.001), which indicates that after the high fat induces NAFLD rats for 12 weeks, the liver is enlarged and damaged; compared with the model group, the liver index of each administration group is reduced, the composition group is obviously reduced, and the red yeast rice group and the Pu' er tea group have no obvious difference, which shows that the composition can obviously inhibit the high fat induced liver injury of NAFLD rats.
TABLE 5 Effect of the compositions, red Rice and Pu her tea on the visceral index of NAFLD rats: (
Figure BDA0002502419200000121
n=7)
Figure BDA0002502419200000122
(IV) influence of composition, red yeast rice and Pu' er tea on liver histomorphology of rat subjected to high fat induction of NAFLD
The histomorphometric results of the liver of rats in each group showed (fig. 13) that the area percentage of lipid droplets in the rats in the normal group was 4.27%, the area percentage of lipid droplets in the model group was 55.47%, the area percentage of lipid droplets in the resveratrol group was 38.68%, and the area percentage of lipid droplets in the red yeast, pu' er tea and composition groups was 45.17%, 42.02% and 35.94%. The rats in the model group show diffuse hepatocellular edema degeneration, are scattered in a large number of round vacuolated lipid droplets with different sizes and inflammatory cell infiltration in the sink area. The pathological morphology of the liver of the rats is close to that of a normal control group, the resveratrol group and the administration group are infiltrated by a small amount of inflammatory cells, and the number of lipid drops is less than that of a model group. The results show that the red yeast rice, the Pu 'er tea and the composition extract can effectively improve the liver histomorphosis of a rat subjected to high fat induction of NAFLD, and the effect of the composition extract is better than that of the red yeast rice or the Pu' er tea extract.
The immunohistochemical Tunel apoptosis rate analysis result (figure 14) shows that the liver cell apoptosis rates of the normal group, the model group, the resveratrol group, the red yeast rice group, the Pu 'er tea group and the composition group are respectively 19.76%, 35.00%, 6.23%, 11.45%, 47.98% and 11.87%, which indicates that the three extracts can relieve cell apoptosis in the NAFLD process and protect liver cells from being damaged, and the composition and the red yeast rice have better effects than the Pu' er tea.
(V) influence of composition, red yeast rice and Pu' er tea on TG, TC, LDL-C and HDL-C of liver of NAFLD rat
The results in FIG. 15 show that the liver tissues of the model group have significantly increased TC, TG and LDL-C contents (P <0.01, P < 0.001) and decreased HDL-C contents without significant difference compared with the normal group, indicating that the liver of the high-fat-induced NAFLD rat has significant lipid metabolism disorder. Compared with a model group, the resveratrol group has significant differences of TC, TG and LDL-C in liver tissues (P is less than 0.05, P is less than 0.01, and P is less than 0.001), which indicates that the resveratrol group can regulate the high fat induced liver lipid abnormality of NAFLD rats; after different extracts are given, TC and TG contents in a monascus group, a Pu' er tea group and a composition group are obviously reduced (P is less than 0.01 and P is less than 0.001), LDL-C contents in the monascus group and the composition group are obviously reduced (P is less than 0.05), and HDL-C in three extract administration groups has a rising and falling trend but has no obvious difference; compared with the composition group, the TC of the monascus group and the Pu' er tea group is remarkably increased (P is less than 0.05), and TG also has a tendency to be increased. The results show that the three extracts can better improve the high fat induced liver lipid abnormality of the NAFLD rat, and the effect of the composition group is obviously better than that of the monascus group or the Pu' er tea group.
(VI) influence of composition, red yeast rice and Pu' er tea on liver MDA and GSH-PX of NAFLD rat
As shown in the results of FIG. 16, the GSH-PX activity in the liver of the model group rat is remarkably reduced (P < 0.001) and the MDA content is remarkably increased (P < 0.001) compared with the liver of the normal group, which indicates that the liver of the NAFLD rat has oxidative stress and lipid peroxidation injury. Compared with a model group, the resveratrol group GSH-PX has obviously improved activity (P is less than 0.05) and obviously reduced MDA content (P is less than 0.01), which shows that resveratrol can reduce the oxidative stress and lipid peroxidation injury of the liver of a NAFLD rat; compared with a model group, the activity of GSH-PX in the composition group is obviously improved (P is less than 0.001), the red yeast rice group and the Pu' er tea group have the trend of increasing but have no obvious difference, and simultaneously, the MDA content of the three extracts is obviously reduced (P is less than 0.05, P is less than 0.01, P is less than 0.001); compared with the composition group, the activity of GSH-PX in the Pu 'er tea group is obviously reduced, the red yeast rice group also has a reduction trend without obvious difference, and simultaneously, the MDA content of the Pu' er tea and the red yeast rice group is obviously increased (P is less than 0.05, P is less than 0.01). The results show that the three extracts can regulate the oxidative stress and lipid peroxidation injury of the liver of a rat NAFLD, so that the occurrence of non-alcoholic fatty liver is relieved, and the effect of the composition is obviously superior to that of red yeast rice and Pu' er tea.
(V) influence of the composition, red yeast rice and Pu' er tea on ATP synthase, complexI and ComplexII in liver mitochondria of NAFLD rats shown in the result of figure 17, compared with the normal group, the activities of ComplexI, complexII and ATP synthase (Na + -K + -ATP ase and Ca2+ -Mg2+ -ATP ase) in liver mitochondria of model rats are obviously reduced (P is less than 0.01, P is less than 0.001), which indicates that high fat induces energy metabolism disorder of liver mitochondria of NAFLD rats. Compared with a model group, the enzyme activities of Na + -K + -ATP ase and complexII enzyme in the resveratrol administration group are significantly different (P is less than 0.05 and P is less than 0.01), the enzyme activities of Ca2+ -Mg2+ -ATP ase and complexI enzyme are improved to a certain extent, but the significant difference is not generated, which shows that the enzyme activities can improve the energy metabolism abnormity of liver mitochondria of NAFLD rats to a certain extent.
Compared with a model group, after different extracts are given, the activities of ATP synthase (Na + -K + -ATP ase, ca2+ -Mg2+ -ATP ase) in liver mitochondria of a Pu 'er tea group and a composition group are obviously improved (P is less than 0.05, P is less than 0.01, P is less than 0.001), the activity of ATP synthase in liver mitochondria of a monascus group is also improved, but no obvious difference exists, meanwhile, the activities of ComplexI and II enzyme of the composition group are obviously improved (P is less than 0.01), and the activities of ComplexI and II enzyme of the monascus group and the Pu' er tea group are improved, but no obvious difference exists; compared with the composition group, the activity of ATP synthase (Na + -K + -ATP ase, ca2+ -Mg2+ -ATP ase) and the activity of ComplexI enzyme in the Pu' er tea group are obviously reduced (P is less than 0.05, P is less than 0.01); the activities of the red yeast ATP synthase and the enzymes of complete I and II are reduced, but no significant difference exists. The results show that the three extracts can regulate the energy metabolism disorder of liver mitochondria of NAFLD rats to a certain extent, and the drug effect of the composition is obviously superior to that of red yeast rice or Pu' er tea.

Claims (4)

1. A composition for preventing and treating lipid metabolism disorder comprises the following raw materials: 1-10g of rhizoma polygonati, 1-10g of angelica, 1-10g of pseudo-ginseng, 1-5g of elecampane, 1-5g of poria cocos, 1-5g of red yeast rice and 1-10g of Pu' er tea.
2. The composition of claim 1, wherein: the preparation is selected from decoction, tablet, capsule, pill, granule, and powder.
3. The composition of claim 1, wherein the extraction process comprises the steps of: pulverizing the raw materials into fine powder, reflux-extracting with 12 times of 95% ethanol at 90 deg.C, reflux-extracting with 12 times of 70% ethanol for 2 hr each time, mixing extractive solutions, concentrating at 65 deg.C and 100 deg.C under reduced pressure, recovering ethanol, and making into lyophilized powder.
4. Use of the composition of any one of claims 1 to 3 for the preparation of a medicament for the prevention and treatment of lipid metabolism disorders.
CN202010436353.1A 2020-05-21 2020-05-21 Composition for preventing and treating lipid metabolism disorder Active CN111513155B (en)

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