Beautyberry extract is preparing the application in fat-reducing medicament
Technical field
The invention belongs to natural medicine fields, and in particular to beautyberry extract is preparing the application in fat-reducing medicament.
Background technique
Obesity is the performance of human body energy metabolic disorder, is to lead to type II diabetes, nonalcoholic fatty liver, cardiovascular disease
Disease, senile dementia, a series of even diseases such as tumour major risk factor, seriously threaten the health of the whole mankind.It is defended according to the world
Raw tissue estimation, has more than 1,000,000,000 population overweights (body mass index BMI > 25) at present, and the number of typical obesity patient is super
Cross 300,000,000 (BMI > 30);In the U.S., it is undergoing more than 2/3rds adult overweight or fat;It is high in fat, high in China
Sugar and high protein are the high heat energy diet dietary structure of representative, and allegro work and sedentary, obese people is gradually huge,
According to statistics obese people's ordered series of numbers whole world first in 2018.
Wherein fat has always been considered as being to lead obesogenous " arch-criminal ": increased by body fat cell volume, quantity,
Cause the precipitating that fat is excessive in part, so that the percentage of body fat percentage of liveweight be caused to increase extremely, common pathological characters are main
Including blood glucose rise, dyslipidemia, oxidation resistance reduction and infiltration of inflammatory factor etc..Thus, it could be seen that fat is in difference
Fat related metabolic diseases in also play important role.
But it cannot treat different things as the same for different types of fat, have one kind not but not lead to obesity, also have subtract on the contrary
Effect of fertilizer.All mammals (mankind, mouse etc.) generally have the fat completely different there are two types of function: white rouge in vivo
Fat tissue (White Adipose Tissue, WAT) and brown adipose tissue (Brown Adipose Tissue, BAT).
WAT stores energy in the form of triglycerides (TG), and obesity and related disease are related with their overload, and
Different negative effects is generated to health according to their positions in vivo.Contain a large amount of mitochondria in BAT, is a kind of natural
Energy-dissipating type fat, can not only burn under environmental stimuli and generate heat, but also is responsible for decomposing and causes fat white
The latter is converted to carbon dioxide, water and heat by fat.But BAT only plays a role in the mankind and (makees in children the infancy
It is generally existing for " baby's fat "), with age, brown fat activity gradually decreases, only remaining in final human body
Brown fat cell.Studies have shown that after cold stimulation or the processing of β adrenal hormone receptor stimulating agent, the subcutaneous white rouge of mouse
It will appear the brown sample fat cell being dispersed in, referred to as " cream-coloured fat " (Beige Adipose) in fat tissue --- between two
Between the common fat of kind, functionally close to brown fat, it can be embedded in white adipose tissue, converted chemical energy to by heat production
Thermal dissipation.For the human body, brown class adipose tissue is exactly that " innately lose weight expert " can cope with fat and its related generation
It thanks to disease, plays an important role in maintaining body temperature and energy balance.More and more statistics indicate that, promote white adipose thin
The browning of born of the same parents, formed brown adipose tissue simultaneously activate its function, the energetic supersession of body can be improved, play effectively eliminate it is extra
The effect of fat.Therefore, will realize obesity controlling one of the most effective ways by energy consumption (release, transfer).
For general population diet control and reinforce taking exercise and can be used as the first choice of loss of weight, but have fat genetic constitution,
Overweight increases risk or the crowd of metabolic disease has occurred, then there is an urgent need to find a kind of highly effective and safe
Method of weight-reducing avoids the generation of other metabolic diseases by intervening with treatment obesity.Chinese medicine is passed because of its long history
It holds and safely and effectively clinical data, the characteristics of synergistic effect with its multicomponent and more targeted plays drug effect in vivo, extensively
The prevention and treatment of the related complications such as fat and insulin resistance, II-patients with type Ⅰ DM and nonalcoholic fatty liver are applied to,
Such as Huanglianshangqing soup, Hoisting powder, Sijunzi Tang and gut purge soup.
Callicarpa nudiflora Callicarpa nudiflora Hook.ex Arn. is Verenaceae Callicarpa bodinieri Levl. platymiscium, with drying
Leaf is used as medicine, and is distributed mainly on the province such as the Guangdong, Hainan and Guangxi in China, and using Hainan Mt. Wu-zhi Shan production as top grade.First recorded in Tang's " this
Grass is picked up any lost article from the road ", it is selected in " Chinese Pharmacopoeia " version in 2015 and increases herbal species newly.It is its bitter, micro-pungent, mild-natured;Whole year can harvest, rhizome
Ye Junke is used as medicine, and has hemostasis and anti-inflammation, eliminating stasis to subdue swelling, antibacterial and detoxicating and other effects.It is callicarpa nudiflora with list as clinical standing drug
Taste medicine patent medicine, dosage form have capsule, tablet, powder and electuary (based on oral), are particularly suitable for hemostasis recovery and inflammatory resolution.It is logical
Acute toxicity testing test is crossed, it is safe and non-toxic in Rational Dosage concentration range.Comprehensive literature report, from callicarpa nudiflora middle discovery
Chemical component based on flavonoids, Phenylpropanoid Glycosides, iridoid and triterpenes;Naked pharmacological activity mainly has hemostasis, anti-inflammatory, suppression
Bacterium improves the effects of immune, antitumor;Generally speaking callicarpa nudiflora crude extract activity is obvious compared with monomeric compound, research range
It is wider, but there is not yet in relation to its lower blood-fat and reduce weight and improve its related metabolic diseases (hyperlipidemia, nonalcoholic fatty liver and pancreas islet
Element resist etc.) in terms of report.
Summary of the invention
The first purpose of this invention, which is to provide, callicarpa nudiflora to be prepared fat-reducing medicament, is improving liver function, reduces liver damage
Application in the drug of wound or inhibition inflammation.
It is preferred that it is described it is callicarpa nudiflora be in the form of beautyberry extract.
Further preferably, the beautyberry extract is prepared by the following method: being taken dry callicarpa nudiflora
Leaf is extracted using methanol or ethyl alcohol or its aqueous solution, and after extracting solution removes insoluble matter, freeze-drying obtains callicarpa nudiflora extraction
Object.It can be, the extracting solution removal insoluble matter is, by extracting solution refrigerated centrifuge, to stand by after extracting solution cooling, take supernatant
Liquid crosses miillpore filter, and filtrate is freeze-dried, and obtains beautyberry extract.
Specifically, the beautyberry extract is prepared by the following method: taking the callicarpa nudiflora of fresh dried
Leaf, natural air drying crushed 80 meshes, and by solid-liquid ratio 2g/30mL, callicarpa nudiflora leaf powder and methanol are mixed, super in 40 DEG C
Sound extracts 30min, after ice water cooling, shakes up and takes extracting solution 1.5mL, in 10 DEG C, 12000rpm refrigerated centrifuge 10min, stands 1
~2min takes 500 μ L of supernatant to cross 0.22 μm of miillpore filter, filtrate is freeze-dried, beautyberry extract is obtained.
The fat-reducing medicament is the drug of obesity caused by preventing high fat diet.
A second object of the present invention is to provide a kind of fat-reducing medicament, improves liver function, reduces hepatic injury or inhibit inflammation
Drug, which is characterized in that containing it is a effective amount of it is callicarpa nudiflora be active constituent and pharmaceutically acceptable auxiliary material.
The dosage form of the drug is powder, capsule, granule or tablet.
Compared with prior art, novelty of the invention and beneficial effect are:
The invention demonstrates that callicarpa nudiflora (LHZZ) LHZZ may be by promoting the fatty acid oxidation in fat cell, mention
The energetic supersession of high-fat cell can enhance mouse to glucose to play and reduce the effect LHZZ of intracellular fat content
Tolerance improves mouse to the sensibility of insulin, significantly increases mouse to the metabolic capability of glucose, LHZZ can prevent high in fat
Obesity caused by diet.It is thin to reduce fat by the expression of lipolysis gene HSL, MGLL in promotion mouse IWAT by LHZZ
Born of the same parents' size promotes the energy of WAT to disappear by promoting the expression of fatty acid oxidation gene, heat production gene, oxidative phosphorylation gene
Consumption promotes the brown stain LHZZ of WAT to reduce fat cell by the expression of lipolysis gene HSL, MGLL in promotion mouse BAT
Size, LHZZ can promote the expression of fatty acid oxidation gene, heat production gene, oxidative phosphorylation gene in BAT, restore BAT function.
LHZZ can reduce TG, ALT in mice serum, AST, IL-1, IL-6, TNF-α level, have lipid-loweringing, improve liver function, reduce
Hepatic injury inhibits the effect of inflammation, and the expression of factor S REBP-1C, FAS, ACC are generated by reducing fat, inhibits in liver
The biosynthesis of fatty acid, LHZZ are prevented by reducing expression and the anti-oxidant and anti-inflammatory symptom of improvement of the fat generation factor
The metabolic impairment of high fat fed conditions induction.It can be seen that beautyberry extract no matter is tested in vitro or mouse is in body
Test all has apparent lower blood-fat and reduce weight effect, can be applied to prepare fat-reducing medicament, improves fat related metabolic diseases (hyperlipemia
Disease, nonalcoholic fatty liver and insulin resistance etc.) symptom, it has a good application prospect, and small toxicity, it is highly-safe, do not have
There is cytotoxic effect.
Detailed description of the invention
Fig. 1 is the lipid-lowering effect that LHZZ acts on fat cell generation.(A) LHZZ cytotoxic activity is carried out for CCK-8 method
Measurement result, (B) are that oil red O stain carries out 3T3L1 fat cell rouge assay as a result, (C) is to be metabolized phase in fat cell
Correlation gene expression testing result.
Fig. 2 is the preventive effect that LHZZ causes mouse obesity disease to high fat diet.It (A) is high fat diet control group (HFD
CON), high fat diet LHZZ treatment group (HFD LHZZ), normal diet controls group (CW CON), normal diet LHZZ treatment group
(CW LHZZ) 8 weeks changes of weight of mouse record.(B, D) be HFD CON, HFD LHZZ, CW CON, CW LHZZ mouse Portugal
Grape sugar tolerance tests (GTT) result.(C, E) be HFD CON, HFD LHZZ, CW CON, CW LHZZ mouse insulin tolerance
Test (ITT) result.
Fig. 3 is influence of the LHZZ to mouse white adipose tissue (WAT).(A) for each test group (CW CON, CW LHZZ,
HFD CON, HFD LHZZ) mouse groin white tissues (IWAT) percentage of liveweight ratio.(B) it is dyed for hematoxylin-eosin (HE)
Observe fat cell size in the IWAT of each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse (scale bar:
100μm).(C, D) is that Western blot detects each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse
The expression of subcutaneous fat and energetic supersession GAP-associated protein GAP AMPK, PGC-1 α and phosphorylation LIPE (p-LiPE).
Fig. 4 is that q-PCR analyzes energy in the IWAT of each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse
Measure the expression of metabolism related gene.It (A) is q-PCR analysis in high fat diet, in the IWAT of HFD CON and HFD LHZZ mouse
The expression of energy metabolism related genes.It (B) is q-PCR analysis in normal diet, in the IWAT of CW CON and CW LHZZ mouse
The expression of mRNA energy metabolism related genes.
Fig. 5 is influence of the LHZZ to mouse brown adipose tissue (BAT).(A, B) is each test group (CW CON, CW
LHZZ, HFD CON, HFD LHZZ) Temperature changing of the mouse in cold stimulation experiment.It (C) is each test group (CW CON, CW
LHZZ, HFD CON, HFD LHZZ) mouse brown fat (BAT) percentage of liveweight ratio.(D) it is seen for hematoxylin-eosin (HE) dyeing
Examine the variation (ratio of fat cell size in the BAT of each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse
Ruler: 100 μm).(E, F) is the BAT that q-PCR analyzes each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse
The expression of middle energy metabolism related genes.
Fig. 6 is influence of the LHZZ to lipid synthesis in mouse liver and inflammatory reaction.It (A) is each test group (CW CON, CW
LHZZ, HFD CON, HFD LHZZ) the Concentration Testing result of TC, TG, ALT, AST in mice serum.It (B) is each test group (CW
CON, CW LHZZ, HFD CON, HFD LHZZ) inflammatory factor IL-1, IL-6 in mice serum, TNF-α Concentration Testing result.
It (C) is the liver weight of each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse.(D, E) is q-PCR analysis
The table of glycolipid metabolism related gene in each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse liver tissue
It reaches.(F) it is cut for hematoxylin-eosin (HE) dyeing observation each group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) Mouse Liver
Tissue pathologies change's (scale bar: 100 μm) of piece.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
The preparation of 1 beautyberry extract of embodiment
Beautyberry extract (LHZZ) is prepared by the following method: the callicarpa nudiflora leaf of fresh dried is taken, it is natural
It air-dries, crushed 80 meshes, by solid-liquid ratio 2g/30mL, callicarpa nudiflora leaf powder and methanol are mixed, in 40 DEG C of ultrasound extractions
30min shakes up after ice water cooling and takes extracting solution 1.5mL, in 10 DEG C, 12000rpm refrigerated centrifuge 10min, standing 1~
2min takes 500 μ L of supernatant to cross 0.22 μm of miillpore filter, filtrate is freeze-dried, beautyberry extract is obtained.
External effect for reducing fat of 2 beautyberry extract of embodiment to fat cell
1.1 reagents and instrument
Fetal calf serum (Serana), penicillin, streptomysin, DMEM culture medium, insulin, isobutyl methylxanthine
(Sigma), dexamethasone (Sigma), Cell counting Kit -8 (ccK-8) (Jian Cheng Bioengineering Research Institute, Nanjing of China),
Electronic balance (Sai Duolisi), chloroform, isopropanol, dehydrated alcohol, QPCR Reverse Transcriptase kit, PCR instrument (Bio-rid) fluorescence
Quantitative PCR apparatus (Thermo Fisher, USA)
1.2 cell culture and drug treatment
(1) cell culture: by mouse 3T3-L1 (CL-173, ATCC) PECTORAL LIMB SKELETON to be supplemented with 10%v/v new life small
It is cultivated in the DMEM of cow's serum (Serana), benzyl penicillin (100IU/mL) and streptomysin (100 μ g/mL).By in 10%v/v
In the presence of fetal calf serum (FBS), with 10 μ g/mL insulin, 500 μM of isobutyl methylxanthines (Sigma) and 1 μM of dexamethasone
(Sigma) the Adipogenesis mixture formed handles cell, in 37 DEG C of 5%CO2Cell differentiation inducing activity in environment.After 2 days, with benefit
Culture medium filled with 10 μ g/mL insulin replaces the culture medium containing Adipogenesis mixture, and cultivates 2 days.The training of replacement in every 2 days
Base is supported 9 days after differentiation starts, obtains the 3T3-L1 cell for drug treatment.
(2) drug treatment:
(1) CCK-8 method carries out determination of cytotoxic activity: respectively adding to beautyberry extract (LHZZ) thin containing 3T3-L1
Make its concentration 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.6mg/mL, 3.2mg/mL in 96 orifice plates of born of the same parents,
48h is cultivated, and not add the processing of LHZZ as blank control.(city biology work is built using Cell counting Kit -8 (ccK-8)
Journey research institute, Nanjing of China) measurement processing after cell survival rate, assess the cytotoxicity of LHZZ;All steps are according to system
Quotient is made to illustrate to carry out.
(2) oil red O stain carries out 3T3L1 fat cell rouge assay: being handled using the LHZZ that concentration is 0.8mg/mL
3T3-L1 cell 2 days, not add the processing of LHZZ as a control group.Then carefully light and slow to remove culture solution, with the light and slow drift of PBS
It washes, adds the fixed 30min of 10% neutral formalin, dilution oil red saves liquid, oil red: deionized water=3:2 volume ratio, filter paper filtering,
It is placed at room temperature for 10min, dyeing 10min or so, the volume covering added lives board bottom.75% isopropyl alcohol of decoloration, removes
Extra dyestuff, is redyed, light brazilwood dyeing 1/5min, PBS rinsing.Glycerin gelatine mounting, microscopically observation.
(3) Metabolism-Related Genes Expression situation detects in fat cell: being handled using the LHZZ that concentration is 0.8mg/mL
3T3-L1 cell 2 days, not add the processing of LHZZ as a control group.Then the DNA for extracting 3T3-L1 cell, using fluorescent quantitation
PCR detect respectively fatty acid oxidation index of correlation PPARa, Acadvl, Acadl, Acadm, Acads, Cpt2, Cpt1b, Fabp3,
The mRNA expression of HSL, Mgll, Ucp1.
1.3 experimental result
Beautyberry extract (LHZZ) acts on the lipid-lowering effect of fat cell generation as shown in Figure 1.Fig. 1 (A) is
CCK-8 method carries out LHZZ determination of cytotoxic activity as a result, Fig. 1 (B) is that oil red O stain carries out 3T3L1 fat cell rouge containing measurement
Determine as a result, Fig. 1 (C) is Metabolism-Related Genes Expression situation testing result in fat cell.
By Fig. 1 (A) it is found that the LHZZ of various concentration acts on fat cell, it is proliferated and is all had no significant effect, explanation
LHZZ is safe and non-toxic in setting concentration range, acts on the normal growth unrestraint of cell.
By Fig. 1 (B) it is found that occurring the small fat drips of annular to differ in size after oil red O stain into the cell, with control group (CON)
It compares, the fat content in the fat cell of LHZZ (0.8mg/mL) processing group significantly reduces.
By Fig. 1 (C) it is found that the fatty acid oxidation related gene PPARa, Acadvl of LHZZ (0.8mg/mL) processing group,
The expression of Acadm and Cpt1b significantly rise (*P < 0.05,**p<0.01).Wherein PPARa is peroxisome proliferator-activated
Receptor is primarily involved in the regulation of transcription factor expression, in cell differentiation, development and metabolism (sugar, rouge, protein) and height
Deng biology tumour occur in play critical, especially intraor extracellular lipid metabolism destination gene expression.Therefore through upper
Results presumption is stated, LHZZ may be by promoting the fatty acid oxidation in fat cell, the energetic supersession of fat cell is improved, from
And play the effect for reducing intracellular fat content.
The internal functions of lowering blood-fat and reducing weight of 3 beautyberry extract of embodiment
1.1 reagents and instrument
Gastric perfusion needle, feed, padding, blood sugar test paper, blood glucose meter (ACON BIOTECH, China), clinical thermometer, ice machine, -80
DEG C ultra low temperature freezer, automatic biochemistry analyzer (Hitachi 7080, Japan)
1.2 animal models, grouping administration, fasting blood-glucose and the detection of empty stomach sugar tolerance
(1) animal model and grouping are administered: Male wild-type C57BL/6J (WT) mouse of 6 week old is purchased from Guangzhou traditional Chinese medicine
University's (GuangZhou, China).Mouse is maintained in the animal housing under 20-22 DEG C of controlled temperature, 12 hours illumination/dark cycles.
C57BL/6 mouse half feeding chow diet (9% fat;Laboratory diet), the other half feeding HFD (60kcal% fat;
Research Diets, USA), it arbitrarily drinks water, until experiment terminates, after three months, 24 C57BL/6 mouse is randomly divided into 4
Group (every group 6), i.e. normal diet controls group (CW CON), normal diet LHZZ treatment group (CW LHZZ, 500mg/kg) are high
Rouge diet control group (HFD CON), high fat diet LHZZ treatment group (HFD LHZZ, 500mg/kg), all control group stomach-fillings are given
Pure water is given, administration group gives 500mg/kg LHZZ through stomach-filling, is administered 8 weeks.The daily work of mouse of each test group during observation administration
Emotionally condition monitors Temperature changing hourly in mouse temperature 24 hours, and timing claims 2 weight daily, once in the morning and once at night, record
During test and at the end of mouse weight situation of change.
(2) after administration 8 weeks, glucose mouse fasting blood-glucose and the detection of empty stomach sugar tolerance: is carried out to the mouse of each test group
Resistance test (GTT), Glucose Tolerance Test (ITT) detect mouse fasting blood-glucose and empty stomach sugar tolerance.For glucose tolerance
It tests (GTT), the mouse of every group of fasting 16 hours receives intraperitoneal injection glucose (1.5g/kg).Insulin tolerance is tried
It tests (ITT), the mouse of every group of fasting 6 hours receives intraperitoneal injection actrapid monotard (2IU/kg).It is received using from tail vein tip
The blood of collection, with the blood glucose level of glucose meter (ACON BIOTECH, China) measurement 0,15,30,60,90 and 120 minute.System
It counts, in terms of milligram/decilitre, and analyzes area under the curve, compare the otherness between every group of mouse.
(3) it tissue sampling, freezen protective and fixation: after each test group mouse completes GTT, ITT detection, weighs in, remembers
Record final weight.Eyeball takes blood, and the neck that breaks is put to death.With alcohol wipe mouse body, dissected.Back of mice brown rouge is taken immediately
Fat and subcutaneous white adipose, epididymal adipose tissues, liver, intestine and small intestine, excrement weigh the weight of adipose tissue and liver, take pictures, and
Have in observation mouse peritoneal without exception.Tissue half is put into liquid nitrogen cryopreservation in EP pipe, and half is put into the EP equipped with 4% paraformaldehyde
It is impregnated in pipe.Entire experiment will be rapidly completed as far as possible, be put into liquid nitrogen group be woven in materials after be transferred to -80 DEG C of refrigerators and save
For use.The mouse blood for taking blood to obtain on eyeball stands half an hour as in 1.5mL EP pipe, blood is collected, at 4 DEG C with 2000
× g is centrifuged 15 minutes and prepares serum, and serum is placed in -80 DEG C of refrigerators and is saved for use.
1.3 experimental result
Beautyberry extract (LHZZ) causes the preventive effect of mouse obesity disease as shown in Figure 2 to high fat diet.Fig. 2
It (A) is high fat diet control group (HFD CON), high fat diet LHZZ treatment group (HFD LHZZ), normal diet controls group (CW
CON), normal diet LHZZ treatment group (CW LHZZ) 8 weeks changes of weight of mouse record.Fig. 2 (B, D) is HFD CON, HFD
LHZZ, CW CON, CW LHZZ mouse glucose tolerance test (GTT) result.Fig. 2 (C, E) be HFD CON, HFD LHZZ,
CW CON, CW LHZZ mouse insulin tolerance test (ITT) result.
By Fig. 2 (A) it is found that compared with CW CON, the extremely significant increase of the weight of HFD CON mouse (***p<0.001);In height
It is given compared to stomach-filling under fatty (HFD) and normal (CW) eating condition with the weight of the mouse of LHZZ (n=6) processing 8 weeks pure
The control group of water decreases.In HFD condition, gives the 4th week mouse weight of LHZZ and begin to be substantially reduced, with HFD CON
Compare, the weight of HFD LHZZ mouse significantly reduce (*p<0.05).In CW condition, the weight for giving LHZZ mouse is also dropped
It is low, compared with CW CON, the weight loss of CW LHZZ mouse.About groin white adipose tissue (IWAT), epididymis white rouge
Fat tissue (EWAT) and liver weight, it is observed that the weight of tissue is reduced after giving LHZZ.
By Fig. 2 (B, D) it is found that in GTT, compared with HFD CON, HFD LHZZ can obviously inhibit the rising of mouse blood sugar
And blood glucose is kept to be in reduced steady state, metabolic capability of the enhancing mouse to sugar.And the blood glucose level of CW LHZZ mouse
Slightly below CW CON.
By Fig. 2 (C, E) it is found that in ITT, the blood glucose level of HFD LHZZ mouse is substantially less than HFD CON.And CW
The blood glucose level of LHZZ mouse is also slightly below CW CON.
By the AUC in Fig. 2 (B-E) it is found that LHZZ has extremely significant enhancing mouse sugar patience and improves insulin sensitivity
Positive effect (*P < 0.05,**P < 0.01,***P < 0.001), the above result shows that, LHZZ can enhance mouse to the resistance to of glucose
By property, improves mouse to the sensibility of insulin, significantly increase mouse to the metabolic capability of glucose, LHZZ can prevent drink high in fat
Obesity caused by eating.
Influence of 4 beautyberry extract of embodiment to fat acid decomposition in white adipose tissue and fatty acid oxidation
1.1 test material
Each test group mouse groin white adipose tissue (IWAT) of 3 freezen protective of embodiment;What paraformaldehyde was fixed
Each test group mouse groin white adipose tissue (IWAT).
1.2 experimental method
1.2.1 the effect for reducing fat Mechanism Study of protein expression analysis
Western blot detect mouse white adipose tissue (WAT) in energetic supersession GAP-associated protein GAP AMPK, PGC-1 α and
The expression of phosphorylation LIPE (p-LiPE).
IWAT is cracked in the RIPA buffer with protease inhibitors, after homogenizing, with 12000rpm centrifugation 15
Minute, supernatant is sucked out and with BCA kit quantification.20-40 μ g protein is loaded to 10%SDS- polyacrylamide gel
On, and will be on isolated Protein transfer to pvdf membrane.After electrotransfer, with being dissolved in Tris buffered saline/Tween buffer (pH
=8.0,0.05% polysorbas20) in 5% skimmed milk close membrane 1 hour.Western blot analysis is carried out using specific antibody.
By trace at 4 DEG C with anti-PGC-1 α, anti-2 (phosphate T172) antibody of AMPK α 1 (phosphate T183)+AMPK α, anti-p-LiPE
The primary antibody of antibody (Abcam, USA) is incubated overnight.Then it is washed three times with TBST (0.5% Tween-20 is in TBS), every time 10
Minute.It is further incubated for 80 minutes with the diluted goat anti-rabbit igg secondary antibody of 1:5000, after washing three times, then uses ECL method
(Bio-Rad, USA) development.Image is obtained with ChemiDoc XRS+ (Bio-Rad, United States).
1.2.2 pathological examination
The IWAT of fixed each test group mouse is subjected to pathology section examination, specifically, part IWAT is taken, through physiology salt
Water is cleaned, and IWAT is fixed in 4% paraformaldehyde (PFA), is embedded in paraffin, and is cut into 7 μm of slices.Using hematoxylin-
The variation of fat cell in each test group mouse IWAT is observed in Yihong (HE) dyeing.
1.2.3 the effect for reducing fat Mechanism Study of energetic supersession pathway gene
(1) q-PCR detects the expression of mRNA energy metabolism related genes in mouse subcutaneous fat;
Weighed from mouse IWAT 40mg carry out the careful grinding of low temperature, be added 1mL TRIpure Reagent (Aidlab,
China) crack on ice, be added after the completion 200 μ L pre-cooling chloroform, mixing be placed at room temperature for 5min layering, whirlpool shake 1min into
One step mixes, and uses low-temperature and high-speed centrifuge immediately, and 4 DEG C × 12000rpm/min is centrifuged 15min, takes the general 400 μ L of supernatant, is added
500 μ L isopropanols, whirlpool mix 1min, are placed at room temperature for 10min.4 DEG C × 12000rpm/min is centrifuged 10min, carefully sucks
Clear liquid retains white precipitate.This precipitating blow outstanding washing with the alcohol of no RNA enzyme water prepared 75%, 4 DEG C ×
12000rpm/min is centrifuged 15min.Solution is discarded supernatant, EP tube opening, which is placed in superclean bench, makes alcohol volatilize naturally.With
No 20 μ L of RNA enzyme water dissolves RNA, obtains total serum IgE, and by with 260 He of NanoDrop2000 spectrophotometer measurement
OD at 280nm carrys out the RNA of Quantitative Separation.It is total with 2 μ g according to HiFiScript cDNA synthetic agent box (Cwbio, China)
RNA synthesizes cDNA, is incubated for 15 minutes at 42 DEG C, is incubated for 5 minutes at 85 DEG C.In QuantStudioTM 6Flex system
QPCR is carried out using PowerUpTMSYBRTM Green Master Mix (Thermo Fisher, USA), respectively to fatty group
Knit common tags (Adiponectin, aP2, Pparg, Cebpa), lipolysis (HSL, MGLL), fatty acid oxidation (PPara,
Acadvl, Acadl, Acadm, Acads, Cpt2, Cpt1b, Fabp3), heat production (SIRT6, Ppargc1a, Ucp1, Prdm16,
Adrb3, Dio2, Cidea), oxidative phosphorylation (Aco2, Atp5al, cox5b, Ndufb8, sdnb, Uqcrc2, Uqcrfsl) phase
The expression of correlation gene is detected.
1.3 experimental result
Influence of the beautyberry extract (LHZZ) to mouse white adipose tissue (WAT) is as shown in Fig. 3,4.
Fig. 3 (A) is normal diet controls group (CW CON), normal diet LHZZ processing group (CW LHZZ), high fat diet pair
According to group (HFD CON), groin white tissues (IWAT) percentage of liveweight ratio of high fat diet LHZZ treatment group (HFD LHZZ) mouse.
Fig. 3 (B) is hematoxylin-eosin (HE) dyeing observation each group (CW CON, CW LHZZ, HFD CON, HFD LHZZ)
Fat cell size (scale bar: 100 μm) in the IWAT of mouse.
Fig. 3 (C, D) is that Western blot detects each group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse
The expression of subcutaneous fat and energetic supersession GAP-associated protein GAP AMPK, PGC-1 α and phosphorylation LIPE (p-LiPE).
Fig. 4 is that q-PCR analyzes energy generation in the IWAT of each group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse
Thank to the expression of related gene.Fig. 4 A is that q-PCR analyzes the energy in the IWAT of HFD CON and HFD LHZZ mouse in high fat diet
Measure the expression of metabolism related gene.Fig. 4 B is that q-PCR is analyzed in normal diet, in the IWAT of CW CON and CW LHZZ mouse
The expression of mRNA energy metabolism related genes.
By Fig. 3 (A) it is found that compared with CW CON, the IWAT percentage of liveweight ratio of HFD CON mouse dramatically increase (**p<0.01);
In normal diet, the IWAT percentage of liveweight ratio of CW CON and CW LHZZ mouse does not have notable difference;In high fat diet, with HFD
CON compares, the IWAT percentage of liveweight of HFD LHZZ mouse than significantly reduce (*P < 0.05), show that LHZZ can substantially reduce drink high in fat
The IWAT of mouse in food promotes fat acid decomposition.
Adipose cell size by Fig. 3 (B) it is found that compared with CW CON, in CW LHZZ mouse IWAT;With HFD CON ratio
Compared with, adipose cell size in HFD LHZZ mouse IWAT, the above result shows that, no matter in normal diet or high fat diet,
LHZZ can reduce the fat cell in mouse IWAT, reduce the size of fat cell.
By Fig. 3 (C, D) it is found that in high fat diet, compared with HFD CON, AMPK, PGC- in HFD LHZZ mouse IWAT
The expression of 1 α, p-LiPE obviously increases, wherein AMPK, p-LiPE expression increase have significant difference (*P < 0.05,**p<
0.01);In normal diet, compared with CW CON, the expression of AMPK, p-LiPE increase in CW LHZZ mouse IWAT, wherein
The expression of p-LiPE increase have significant difference (**P < 0.01), the above result shows that LHZZ can promote AMPK in mouse IWAT,
The expression of PGC-1 α, p-LiPE.Under high fat fed conditions, LHZZ processing promotes PGC-1 α and AMPK the protein table of mouse
It reaches.Induction PGC-1 alpha expression is activated by AMPK to adjust fatty acid oxidation and promote the energy of subcutaneous white adipose tissue (WAT)
Amount consumption, promotes the brown stain of WAT.
As shown in Figure 4, in high fat diet, compared with HFD CON, lipolysis dependency basis in HFD LHZZ mouse IWAT
Because the expression of HSL, MGLL dramatically increases (*P < 0.05), fatty acid oxidation related gene PPara, Acads, Cpt2, Cpt1b,
The expression of Fabp3 obviously increases, and PPara, Acads, Cpt2 expression increase have significant difference (*P < 0.05), it produces
The expression of hot related gene SIRT6, Ppargc1a, Ucp1, Prdm16, Adrb3, Dio2, Cidea obviously increase, and Dio2,
The expression of Cidea increase have significant difference (*P < 0.05), oxidative phosphorylation related gene Aco2, Atp5al, cox5b,
The expression of Ndufb8, sdnb, Uqcrc2, Uqcrfsl obviously increase, and the expression increase of Aco2, Atp5al, Ndufb8 have
Significant difference (*p<0.05)。
In normal diet, compared with CW CON, lipolysis related gene HSL, MGLL in the IWAT of CW LHZZ mouse
Expression obviously increase, and HSL expression increase have significant difference (*P < 0.05), fatty acid oxidation related gene
The expression of PPara, Acadvl, Acadl, Acadm, Acads, Cpt2, Cpt1b, Fabp3 obviously increase, and PPara,
The expression of Acadvl, Acadl, Acads, Cpt2 increase have significant difference (*P < 0.05), heat production related gene Ucp1,
The expression of Prdm16, Adrb3, Dio2, Cidea obviously increase, and Cidea expression increase have significant difference (*p<
0.05), the expression of oxidative phosphorylation related gene Aco2, Atp5al, cox5b, Ndufb8, sdnb, Uqcrc2, Uqcrfsl are equal
It obviously increases.
The above result shows that LHZZ reduces rouge by the expression of lipolysis gene HSL, MGLL in promotion mouse IWAT
Fat cell size promotes the energy of WAT by promoting the expression of fatty acid oxidation gene, heat production gene, oxidative phosphorylation gene
Consumption, promotes the brown stain of WAT.
Influence of 5 beautyberry extract of embodiment to the heat production gene expression in brown adipose tissue
1.1 experiment reagents and equipment
Chloroform, isopropanol, dehydrated alcohol, TRIpure Reagent (Aidlab, China), clinical thermometer, NanoDrop
2000 spectrophotometers, HiFiScript cDNA synthetic agent box (Cwbio, China), PowerUpTMSYBRTM Green
Master Mix (Thermo Fisher, USA).
1.2 experimental method
(1) animal model and grouping are administered: Male wild-type C57BL/6J (WT) mouse of 6 week old is purchased from Guangzhou traditional Chinese medicine
University's (GuangZhou, China).Mouse is maintained in the animal housing under 20-22 DEG C of controlled temperature, 12 hours illumination/dark cycles.
C57BL/6 mouse half feeding chow diet (9% fat;Laboratory diet), the other half feeding HFD (60kcal% fat;
Research Diets, USA), it arbitrarily drinks water, until experiment terminates, after three months, 24 C57BL/6 mouse is randomly divided into 4
Group (every group 6), i.e. normal diet controls group (CW CON), normal diet LHZZ treatment group (CW LHZZ, 500mg/kg) are high
Rouge diet control group (HFD CON), high fat diet LHZZ treatment group (HFD LHZZ, 500mg/kg), all control group stomach-fillings are given
Pure water is given, administration group gives 500mg/kg LHZZ through stomach-filling, is administered 8 weeks.
Cold stimulation experiment: only by every mouse single cage list by the mouse of CW CON, CW LHZZ, HFD CON, HFD LHZZ
It is put into low temperature (4 DEG C) environment 6 hours, every cage is put into a small amount of padding, bio-occlusion and food and water, records mouse body per hour
Temperature.
The expression of mRNA energy metabolism related genes in q-PCR detection mouse brown adipose tissue (BAT): cold thorn will be carried out
Each group mouse after swashing experiment is put to death, dissection, is taken out BAT and is detected, specific steps are as follows: take 30mg BAT, use TRIpure
Reagent (Aidlab, China) extracts total serum IgE, and by with 2000 spectrophotometer measurement 260 of NanoDrop and 280nm
The OD at place carrys out the RNA of Quantitative Separation.According to HiFiScript cDNA synthetic agent box (Cwbio, China), closed with 2 μ g total serum IgEs
It at cDNA, is incubated for 15 minutes at 42 DEG C, is incubated for 5 minutes at 85 DEG C.It is used in QuantStudioTM 6Flex system
PowerUpTMSYBRTM Green Master Mix (Thermo Fisher, USA) carries out qPCR, logical to adipose tissue respectively
With label (Adiponectin, aP2, Pparg, Cebpa), lipolysis (HSL, MGLL), fatty acid oxidation (ppara,
Acadvl, Acadl, Acadm, Acads, cpt2, cpt1b, Fabp3), heat production (Sirt6, Prdm16, Ppargc1a, Ucp1,
Adrb3, Dio2, Cidea), oxidative phosphorylation (Aco2, Atp5al, cox5b, Ndufb8, sdnb, Uqcrc2, Uqcrfsl) phase
The expression of correlation gene is detected.
1.3 experimental result
Influence of the beautyberry extract (LHZZ) to mouse brown adipose tissue (BAT) is as shown in Figure 5.
Fig. 5 (A, B) is normal diet controls group (CW CON), normal diet LHZZ processing group (CW LHZZ), high fat diet
The Temperature changing of control group (HFD CON), high fat diet LHZZ treatment group (HFD LHZZ) mouse in cold stimulation experiment.
Fig. 5 (C) is the brown fat (BAT) of each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse
Percentage of liveweight ratio.
Fig. 5 (D) is that each test group (CW CON, CW LHZZ, HFD CON, HFD are observed in hematoxylin-eosin (HE) dyeing
LHZZ) in the BAT of mouse fat cell size variation (scale bar: 100 μm).
Fig. 5 (E, F) is the BAT that q-PCR analyzes each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse
The expression of middle energy metabolism related genes.
By Fig. 5 (A, B) it is found that in high fat diet, the body temperature of HFD LHZZ mouse is higher than HFD CON, in normal diet
In, the body temperature of CW LHZZ mouse is lower than CW CON, the above result shows that, under high fat fed conditions, LHZZ can promote mouse production
Raw higher heat.
By Fig. 5 (C) it is found that in normal diet, compared with CW CON, the BAT percentage of liveweight of CW LHZZ mouse is than increasing;
In high fat diet, compared with HFD CON, the BAT percentage of liveweight ratio of HFD LHZZ mouse dramatically increase (*P < 0.05), the above knot
Fruit shows that no matter in normal diet or high fat diet, LHZZ can increase the BAT of mouse.
Adipose cell size by Fig. 5 (D) it is found that compared with CW CON, in CW LHZZ mouse BAT;With HFD CON ratio
Compared with, adipose cell size in HFD LHZZ mouse BAT, the above result shows that, no matter in normal diet or high fat diet,
LHZZ can reduce the fat cell in mouse BAT, reduce the size of fat cell.
By Fig. 5 (E, F) it is found that in high fat diet, compared with HFD CON, lipolysis in the BAT of HFD LHZZ mouse
The expression of related gene HSL, MGLL obviously increase, and HSL expression increase have significant difference (*P < 0.05), fatty acid
The expression of oxidation related gene ppara, Acadvl, Acadm, Acads, cpt2, cpt1b, Fabp3 obviously increase, heat production phase
The expression of correlation gene Sirt6, Prdm16, Ppargc1a, Ucp1, Adrb3, Dio2, Cidea obviously increase, and Sirt6,
The expression of Prdm16, Ppargc1a, Ucp1 increase have significant difference (*P < 0.05), oxidative phosphorylation related gene
The expression of Atp5al, cox5b, Ndufb8, Uqcrc2, Uqcrfsl obviously increase.
Just producing in diet, compared with CW CON, the expression of lipolysis related gene HSL in the BAT of CW LHZZ mouse
Increasing, the expression of fatty acid oxidation related gene Acadvl, Acadm, Acads, cpt2 increase, heat production related gene Sirt6,
The expression of Prdm16, Ppargc1a, Ucp1, Adrb3, Dio2, Cidea increase, oxidative phosphorylation related gene Aco2, cox5b,
The expression of Ndufb8, sdhb increase.
The above result shows that LHZZ reduces fat by the expression of lipolysis gene HSL, MGLL in promotion mouse BAT
Cell size, LHZZ can promote the expression of fatty acid oxidation gene, heat production gene, oxidative phosphorylation gene in BAT, restore BAT
Function.
Influence of 6 beautyberry extract of embodiment to lipid synthesis in liver and inflammation
1.1 test material
Each test group murine liver tissue of 3 freezen protective of embodiment, serum.The fixed each test group Mouse Liver of paraformaldehyde
Tissue.
1.2 experimental method
1.2.1 q-PCR detection is in high fat diet, glycolipid metabolism phase in HFD CON and HFD LHZZ mouse liver tissue
The expression of correlation gene: taking 20mg mouse liver tissue, extracts total serum IgE, note using TRIpure Reagent (Aidlab, China)
Meaning is without enzyme process.By with the OD at 2000 spectrophotometer measurement 260 of NanoDrop and 280nm come the RNA of Quantitative Separation.
According to HiFiScript cDNA synthetic agent box (Cwbio, China), cDNA is synthesized with 2 μ g total serum IgEs, is incubated for 15 at 42 DEG C
Minute, it is incubated for 5 minutes at 85 DEG C.Using PowerUpTMSYBRTM Green Master Mix (Thermo Fisher,
USA qPCR) is carried out in QuantStudioTM 6Flex system, respectively to glycolipid synthesis related gene (SREBP- in liver
1C, FAS, ACC, PGC-1 α, PEPCK, G6pase, Sirt6) expression detected.
1.2.2 blood biochemical index of correlation detects: detecting normal diet controls group (CW CON), normal diet LHZZ respectively
Processing group (CW LHZZ), high fat diet control group (HFD CON), high fat diet LHZZ treatment group (HFD LHZZ) mice serum
In every biochemical indicator, with evaluate LHZZ to lipid of mice level, liver function, inflammatory reaction influence, index of correlation are as follows: 1)
Blood lipid is related: triglycerides (TG), total cholesterol (TC);2) liver function is related: plasma alanine transaminase (ALT), millet straw turn
Adnosine deaminase (AST);3) inflammatory cytokine is related: interleukin 1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor
Sub- α (TNF-α).
Use alanine amino in automatic biochemistry analyzer (Hitachi 7080, Japan) each test group mice serum of measurement
Transferase, aspartate aminotransferase, triglycerides and total cholesterol (ALT, AST, TG and TC) are horizontal.ALT, AST, TC and
The detection method of TG is performance rate method, cholesterol oxidation enzyme process and enzyme process.
Using ELISA kit (ABclonal, China), according to the explanation and guidance of manufacturer, to serum inflammatory cell
The factor (IL-1, IL-6, TNF-α) carries out enzyme-linked immunosorbent assay, sample 1:6 is diluted, as a result with the pg/mL table of each sample
Show.
1.2.3 pathological examination
The hepatic tissue of fixed each test group mouse is subjected to pathology section examination, specifically, part hepatic tissue is taken, through life
It manages salt water to clean, hepatic tissue is fixed in 4% paraformaldehyde (PFA), is embedded in paraffin, and is cut into 7 μm of slices.Using Soviet Union
Tissue pathologies change's (scale bar: 100 μm) of each test group mouse liver slice is observed in H & E (HE) dyeing.
1.3 experimental result
Influence of the beautyberry extract (LHZZ) to lipid synthesis in mouse liver and inflammatory reaction is as shown in Figure 6.
Fig. 6 (A) be each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mice serum in TC, TG, ALT,
The Concentration Testing result of AST.
Fig. 6 (B) is inflammatory factor in each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mice serum
The Concentration Testing result of IL-1, IL-6, TNF-α.
Fig. 6 (C) is the liver weight of each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse.
Fig. 6 (D, E) is that q-PCR analyzes each test group (CW CON, CW LHZZ, HFD CON, HFD LHZZ) mouse liver
The expression of glycolipid metabolism related gene in tissue.
Fig. 6 (F) is hematoxylin-eosin (HE) dyeing observation each group (CW CON, CW LHZZ, HFD CON, HFD LHZZ)
Tissue pathologies change's (scale bar: 100 μm) of mouse liver slice.
By Fig. 6 (A) it is found that compared with CW CON, TC, TG level in HFD CON mice serum increase, especially TC's
Increasing degree it is extremely significant (***P < 0.001), it is horizontal to give the TG that LHZZ treatment can reduce in mice serum;With CW CON ratio
Compared with ALT, AST level increase in HFD CON mice serum, give LHZZ and treat ALT, AST water that can be reduced in mice serum
It is flat;In the case where just producing eating condition, compared with CW CON, AST level in CW LHZZ mice serum significantly reduce (*P < 0.05),
Under high fat fed conditions, compared with HFD CON, ALT, AST level in HFD LHZZ mice serum significantly reduce (*p<
0.05,**p<0.01)。
By Fig. 6 (B) it is found that compared with CW CON, IL-1, IL-6, TNF-α level in HFD CON mice serum increase,
And IL-6, TNF-α level increase have significant difference (*p<0.05);It gives LHZZ and treats the IL- that can be reduced in mice serum
1, IL-6, TNF-α are horizontal, and especially under high fat fed conditions, IL-6, TNF-α level in LHZZ reduction mice serum are more
Obviously, have significant difference (*p<0.05)。
By Fig. 6 (C) it is found that mouse can be reduced by giving LHZZ treatment no matter in normal diet or high fat fed conditions
Liver weight, especially under high fat fed conditions, the effect that LHZZ reduces mouse liver weight is become apparent, and has conspicuousness poor
Different (*p<0.05)。
By Fig. 6 (D, E) it is found that in high fat fed conditions, give LHZZ treatment mouse liver tissue in fat generate because
The expression of sub- SREBP-1C, FAS, ACC significantly reduce (*P < 0.05), the expression of PGC-1 α, PEPCK, Sirt6 increase.?
Normal diet condition, give LHZZ treatment it was similarly observed that mouse liver tissue in fat generate factor S REBP-1C, FAS,
The expression of ACC reduces.
By Fig. 6 (F) it is found that normal diet group, the hepatic tissue pathology of CW CON and CW LHZZ are sliced indistinction.But
High in fat group, HFD CON group is clear that after oil red dyeing, a large amount of vacuole occur, is drawn because of fatty liver
It rises.After giving drug, the hepatic tissue of HFD LHZZ group is obviously repaired, and does not see fatty oil vacuole.Illustrate that LHZZ can be with
Fatty liver is repaired, liver tissue lesions caused by fatty liver are lowered.
The above result shows that LHZZ can reduce TG, ALT in mice serum, AST, IL-1, IL-6, TNF-α level, tool
Have lipid-loweringing, improve liver function, reduce hepatic injury, inhibit the effect of inflammation, and by reduce fat generate factor S REBP-1C,
The expression of FAS, ACC inhibit the biosynthesis of fatty acid in liver, and LHZZ is by reducing the expression of the fat generation factor and changing
It is apt to anti-oxidant and anti-inflammatory symptom to prevent the metabolic impairment of high fat fed conditions induction.
7 formulation method of embodiment
1. powder: fresh callicarpa nudiflora leaf raw material medicinal material is picked, after natural air drying, is crushed, 80 meshes are crossed, it is canned to be
?.
2. capsule: fresh callicarpa nudiflora leaf raw material medicinal material being picked, after natural air drying, crushes, crosses 80 meshes, use
70% alcohol steep is concentrated under reduced pressure leaching liquor, then is freeze-dried, at powdered.Canned capsule.
3. granule: fresh callicarpa nudiflora leaf raw material medicinal material being picked, after natural air drying, crushes, crosses 80 meshes, use
70% alcohol steep is concentrated under reduced pressure leaching liquor, then is freeze-dried, at powdered.Add starch, magnesium stearate uniformly to mix, is made
Particle, it is dry, it is canned to obtain the final product.
4. tablet: fresh callicarpa nudiflora leaf raw material medicinal material being picked, after natural air drying, crushes, 80 meshes is crossed, with 70%
Alcohol steep is concentrated under reduced pressure leaching liquor, then is freeze-dried, at powdered.Add starch, magnesium stearate uniformly to mix, particle be made,
It is dry, tabletting to get.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.