CN103623029A - Watermelon vine extract with inflammation-diminishing and pain-relieving effects, as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a watermelon vine extract with inflammation-diminishing and pain-relieving effects, which is a petroleum ether part prepared by alcohol precipitation and water extraction on watermelon vine. Researches discover that the extract has an excellent activity of diminishing inflammation and relieving pain, and can be applied to clinical treatment of rheumatic arthritis, rheumatoid arthritis, scapulohumeral periarthritis, bone and joint pain, cervical and lumbar pain, soft tissue pain, sprain pain and other diseases. According to the watermelon vine extract, the rich resource of the watermelon vine which is the crop waste can be sufficiently utilized, a powerful theoretical basis is provided to further research and development of medical values and clinical significance in the future, and a novel modern traditional Chinese medicinal preparation with excellent inflammation-diminishing and pain-relieving effects is expected to be provided to clinical application.

Description

There is watermelon vine extract of anti-inflammatory and analgesic effect and its preparation method and application

Technical field

The invention belongs to Chinese medicine extract technical field, relate in particular to a kind of watermelon vine extract with anti-inflammatory and analgesic effect and its preparation method and application.

Background technology

Rheumatism is the systemic disease of a class serious harm human health, except arthralgia, also can involve other positions of health, the lighter's pain, movable difficulty, severe one joint deformity function limitation, consequently can not normally learn, works and live, even threat to life, to patient, bring huge physiology and mental anguish ，Ye Gei family and society to bring heavy financial burden.Nearest investigation data show, global rheumatisant approximately has 400,000,000 people, is Yi Ge disease group the hugest in medical domain.In China, there is people more than 200,000,000 to suffer to some extent rheumatic arthritis or rheumatoid arthritis, wherein there are 8,000 ten thousand patients with severe symptoms.In 30 years old later crowds, the people over 80% has rheumatic ostalgia medical history, and with advancing age, incidence rate can increase gradually, therefore, the treatment of rheumatism and prevention is seemed to particularly important.Common rheumatism mainly comprises rheumatic arthritis, rheumatoid arthritis, scapulohumeral periarthritis, osteoarthrosis pain, cervical vertebra pain, soft tissue pain, sprains pain etc.Because its cause of disease is complicated, at present the treatment of such disease is mainly to improve symptom, poor to serious symptom curative effect.And Chinese medicine has been shown important prospect in this regard, and be valued by the people gradually.

Watermelon vine, is the rattan of cucurbitaceous plant Citrullus vulgaris (Citrullus lanatus), and it is as a kind of agricultural waste material, and resource is very abundant, all has output all over the world.Proved that at present Citrullus vulgaris has the effect of clearing away heat-damp and promoting diuresis, cured mainly watery diarrhea, dysentery, scalds the diseases such as atrophic rhinitis.Domestic scholars has also been done a large amount of reports to flesh of Pulp Citrulli, Exocarpium Citrulli clinical efficacy aspect, shows that it has sterilization antiinflammatory action, can treat the diseases such as uremia, pyelonephritis, infantile thrush, chronic diarrhea.Up to now, there is not yet research report and the patent of closing watermelon vine treatment rheumatic arthritis, rheumatoid arthritis, scapulohumeral periarthritis, osteoarthrosis pain, cervical vertebra pain, soft tissue pain, spraining the disease effective sites such as pain.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of watermelon vine extract with anti-inflammatory and analgesic effect and its preparation method and application, to make full use of agricultural waste material---the affluent resources of watermelon vine, be convenient to further investigate from now on its medical value of exploitation and clinical meaning.

For solving the problems of the technologies described above, the present invention by the following technical solutions: have the watermelon vine extract of anti-inflammatory and analgesic effect, this extract is from the petroleum ether part of watermelon vine precipitate with ethanol water extraction.

This extract mainly comprises following ten kinds of compounds: cupreol (β-sitosterol), daucosterol (daucosterol), stigmasterol (stigmasterol), myristin (monomyristin), hexadecanoic acid (palmitic acid), tripalmitin (Monopalmitin), docosane acid glyceride (monobehenin), ursolic acid (ursolic acid), heneicosane acid glyceride (monoheneicosanoin), stearic acid (stearic acid).

The above-mentioned preparation method with the watermelon vine extract of anti-inflammatory and analgesic effect, comprise the following steps: get watermelon vine and shine dry grinding, the ultrasonic 2h of alcoholic solution of volumetric concentration 80% for volume ratio 5:1 by weight, then soak 10d, filter merging filtrate, concentrating under reduced pressure, obtain blackish green extractum, lyophilization obtains green powder; Volume ratio 3:50 is scattered in green powder in deionized water by weight, standing over night, the centrifugal sticky solid suspension of water-fast green and the aqueous solution of separating; The petroleum ether extraction sample of 3 times of volumes for water intaking solution, extract concentrating under reduced pressure, reclaims solvent, and evaporate to dryness, obtains petroleum ether part.

The above-mentioned watermelon vine extract with anti-inflammatory and analgesic effect in preparation treatment rheumatic arthritis, rheumatoid arthritis, scapulohumeral periarthritis, osteoarthrosis pain, cervical vertebra pain, soft tissue pain, sprain the application aspect the medicine of pain.

Medicine is capsule, granule, tablet, solution, pill or injection.

Capsule is soft capsule, and granule is dry suspension, and tablet is dispersible tablet, effervescent tablet, chewable tablet, oral cavity disintegration tablet, and solution is syrup, and pill is concentrated pill, drop pill, micropill.

The above-mentioned watermelon vine extract with anti-inflammatory and analgesic effect preparing auxiliary treatment of rheumatic arthritis, rheumatoid arthritis, scapulohumeral periarthritis, osteoarthrosis pain, cervical vertebra pain, soft tissue pain, sprain the application aspect the health food of pain.

Inventor will carry out precipitate with ethanol water extraction in agricultural waste material watermelon vine, then by petroleum ether extraction, is obtained the extract of petroleum ether part.Research discovery, this extract has good anti-inflammatory and antalgic activity, can be applicable to clinical treatment rheumatic arthritis, rheumatoid arthritis, scapulohumeral periarthritis, osteoarthrosis pain, cervical vertebra pain, soft tissue pain, sprains in the diseases such as pain.Inventor utilizes the means of purification such as the isolation technics such as various column chromatographies and recrystallization to carry out separation to the chemical composition of this extract, therefrom obtain 10 compounds, and fully utilize Modern spectroscopy technology (IR, UV, MS, 1D, 2D-NMR) chemical constitution of separating obtained compound is identified, obtain first the information of main chemical composition of the petroleum ether part extract of watermelon vine precipitate with ethanol water extraction.The present invention takes full advantage of agricultural waste material---the affluent resources of watermelon vine, for its medical value of further investigation exploitation and clinical meaning from now on provide strong theoretical basis, to providing a kind of new modern Chinese medicine preparation with good anti-inflammatory and analgesic effect for clinical.

Accompanying drawing explanation

Fig. 1 is preparation technology's flow chart of watermelon vine extract.

Fig. 2 is the compound separation process chart of watermelon vine petroleum ether part.

Fig. 3 is the comparison diagram that affects on the activation nuclear translocation of RAW264.7 cell LPS stimulation model NF-κ B.

The specific embodiment

One, the preparation of watermelon vine extract

As shown in Figure 1, get the watermelon vine 10kg that shines dry grinding, the ultrasonic 2h of alcoholic solution of volumetric concentration 80% for volume ratio 5:1, then soaks 10d by weight, filter, and merging filtrate, concentrating under reduced pressure, obtains blackish green extractum, and lyophilization obtains 300g green powder; Volume ratio 3:50 is scattered in green powder in 5000mL deionized water by weight, standing over night, centrifugal (3000rpmmin ^-1) separate the sticky solid suspension of water-fast green and aqueous solution; Water intaking solution is used petroleum ether extraction, ethyl acetate, the n-butanol extraction sample of 3 times of volumes successively, and extract is concentrating under reduced pressure respectively, reclaims solvent, and evaporate to dryness, obtains petroleum ether part (30g), ethyl acetate extract (33g), n-butanol portion (100g).

Two, the compound separation of watermelon vine petroleum ether part

As shown in Figure 2, get petroleum ether partly by MCI, remove pigment and obtain sample 20g, through silica gel (100-200 order) column chromatography, with petroleum ether-acetone (98:2-0:100) (98:2,96:4,95:10,90:20,80:30,1:1,2:3,1:3, acetone) be gradient elution solvent system eluting.Petroleum ether-acetone (98:2) eluting part, 2nd～5 parts, petroleum ether recrystallization, obtains compound 1(0.6g); 7th～8 parts, pyridine recrystallization, compound 2(0.3g); Petroleum ether-acetone (96:4) eluting part, 11st～25 parts, pyridine recrystallization obtains compound 3(0.05g); Petroleum ether-acetone (95:10) eluting part, 2nd～7 parts, Gossypol recrystallized from chloroform obtains compound 4(0.1g); 13rd～18 parts, Gossypol recrystallized from chloroform obtains compound 5(0.04g), petroleum ether-acetone (90:20) eluting part, 17th～27 parts, Gossypol recrystallized from chloroform obtains compound 6(0.03g), 22nd～39 parts, through Sephadex LH-20 sephadex column (chloroform: methanol=1:1), Gossypol recrystallized from chloroform obtains compound 7(0.08g) and compound 8(0.05g).Petroleum ether-acetone (80:30) eluting part, 17th～27 parts, pyridine recrystallization obtains compound 9(0.5g); 28th～37 parts, pyridine recrystallization obtains compound 10(0.06g).

Utilize Modern spectroscopy technology (IR, UV, MS, 1D, 2D-NMR) chemical constitution of separating obtained compound is identified, confirm that ten kinds of compounds of watermelon vine petroleum ether part are respectively: cupreol (compound 1), daucosterol (compound 2), stigmasterol (compound 3), myristin (compound 4), hexadecanoic acid (compound 5), tripalmitin (compound 6), docosane acid glyceride (compound 7), ursolic acid (compound 8), heneicosane acid glyceride (compound 9), stearic acid (compound 10).

Three, pharmacodynamic experiment: the screening of watermelon vine different parts extract anti-inflammatory activity and mechanism research thereof

1 material and instrument

1.1 cell derived

RAW264.7 mouse macrophage is purchased from Chinese Academy of Sciences's cell bank.

1.2 experiment material

DMEM high glucose medium (Beijing Hyclone); Hyclone (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims); 24 holes, 96 porocyte culture plates (U.S. Corning); Nitric oxide test kit (N0, the green skies, Jiangsu); 5 watermelon vine different parts extracts (water-soluble position, 50% ethanol extraction position, n-butanol extraction position, Ethyl acetate fraction, petroleum ether extraction position); Lipopolysaccharide (1ipopolysaccaride ，LPS， U.S. Sigma-Aldrich), Methlthiazoletrazolium (MTT， U.S. Sigma-Aldrich); Dimethyl sulfoxide (DMSO, analytical pure, Chengdu section dragon); The ELISA test kit of il-1 β (IL-1 β), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), heme oxygenase-1 (HO-1), nitric oxide synthetase (iNOS), COX-2 (COX-2), purchased from Bio-Swamp; Nuclear transcription factor-kappa B (NF-κ B) activation-nuclear translocation detection kit (the green skies, Jiangsu); Fluorescent quantitation eight connecting legs (American AB I); Cultured cell/antibacterial total RNA extraction reagent box (sky, Beijing root); Beta-mercaptoethanol (U.S. Amresco); CDNA the first chain synthetic agent box (sky, Beijing root); Fluorescent quantitation premix test kit enhanced edition (sky, SYBR Green Beijing root).

1.3 experimental apparatus

Inverted fluorescence microscope (DMI4000B, German Leica); CO ₂incubator (BC-J80S50, the rich news in Shanghai); Autoclave (YXQ-LS-100SII, the rich news in Shanghai); Biohazard Safety Equipment (BHC-1100-II A2, Altay); Multi-functional microplate reader (Infinite F200pro, Switzerland Tecan); Desktop electric centrifuge (Changzhou state China); PCR instrument (TGradient, German Biometra); Real-time fluorescence quantitative PCR instrument (7500, American AB I); Refrigerated centrifuger (3k30, German Sartorius); Ultraviolet-visible spectrophotometer (Cary50, U.S. Varian); Ultrasonic cell disruption instrument (JYD-65, Shanghai is accurate).

1.4 solution preparation

PBS buffer: get the PBS powder of purchase, add after the dissolving of 2L ultra-pure water, subpackage, after autoclaving, 4 ℃ save backup.

MTT solution: take MTT pressed powder 250mg, add the PBS buffer of 50mL, after warm water assist in dissolving, with subpackage after 0.22 μ m membrane filtration degerming ,-20 ℃ of preservations.

Medicine (5 different parts extracts of watermelon vine): each medicine all accurately takes 100mg, adds after 1mL DMSO, ultrasonic assist in dissolving, and-20 ℃ of preservations, use is thawed, and is diluted to the concentration needing with complete medium.

2 experimental techniques

2.1 treat the preparation of test agent

Watermelon vine picks up from Chongzuo, Guangxi, and through Medicinal Plants of Guangxi garden professor Ma little Jun, is accredited as the rattan of Cucurbitaceae (Cucurbitaceae) plant Citrullus vulgaris (Citrullus Lanatus).Get the watermelon vine 5kg that shines dry grinding, with 10 times, 8 times water gagings, boil and extract 2h, 1.5h respectively, merge extractive liquid,, concentrate drying, obtains water extraction extractum (1.2kg) (every gram of extractum is equivalent to 4.2g crude drug).Get the watermelon vine 5kg that shines dry grinding, add 5 times of amount ethanol, reflux, extract, 2 times, each 1h, filters, merging filtrate is concentrated, in 60 ℃ dry, weigh, obtain 50% ethanol extraction extractum (1.5kg) (every gram of extractum is equivalent to 5.25g crude drug).

With reference to one, watermelon vine extract prepare petroleum ether part, ethyl acetate extract, n-butanol portion for following experiment.

2.2 cell culture

RAW264.7 cell is at 37 ℃, 5%CO ₂under condition, with the DMEM high glucose medium containing 10% hyclone, penicillin (100U/mL) and streptomycin (50 μ g/mL), cultivate, treat that Growth of Cells is to logarithmic (log) phase.

2.3 impacts on RAW264.7 cell viability

Get the cell in the RAW264.7 of exponential phase, trypsinization, adjusting concentration of cell suspension is 10 ⁵individual/mL, be inoculated in (the every hole of 100L) in 96 well culture plates, cultivate after 24h administration, blank group only adds 10% serum DMEM culture fluid, continue to put into incubator and cultivate 24h, take out culture plate, each hole adds 20 μ L MTT (5mg/mL) to put into incubator to hatch 4h, abandon supernatant, every hole adds DMSO150 μ L, and 96 well culture plates are placed in after microplate reader is shaken 10min and at 492nm place, measure the light absorption value in each hole, calculates cell relative inhibition.Cell relative inhibition (%)=(1-experimental group OD average/matched group OD average) * 100%.

The structure of 2.4 inflammatory cell models

RAW264.7 cell is seeded on 96 porocyte culture plates and is grown after 24h, and growth is merged to 70% time, is divided into 4 groups: blank group, and LPS low concentration group 0.5 μ g/mL, middle concentration group 1 μ g/mL, high concentration group 5 μ g/mL.After dosing, continue to cultivate 24h, get medium supernatant and measure nitric oxide (NO) level (being undertaken by test kit description).Culture plate is abandoned after culture medium, adds 100 μ L MTT solution (1mg/mL), continues to cultivate 4h, carefully remove supernatant, every hole adds DMSO150 μ L, and 96 well culture plates are placed in after microplate reader is shaken 10min and at 492nm place, measure the light absorption value in each hole, calculates cell relative inhibition.

2.5 anti-inflammatory activity fraction screenings

Cell culture, to exponential phase, is used 0.25% trypsinization, and complete medium is diluted to 10 ⁵individual/mL, be inoculated in (the 100 every hole of μ L) in 96 well culture plates, cultivate after 24h, (final concentration is respectively 50 to add the DMEM culture medium of pastille (respectively extracting position), 100,200 μ g/mL, the multiple hole of each dosage group is 8), and add LPS (final concentration is 1 μ g/mL), set blank group (adding culture medium) simultaneously, model group (adding culture medium and LPS) continues to hatch 24h, the supernatant that collecting cell is cultivated, measures nitric oxide (NO) level (being undertaken by test kit description).

2.6 active fractions Anti-inflammatory Mechanisms

2.6.1ELISA method is measured the variation of proinflammatory cytokine

RAW264.7 cell is seeded on 24 porocyte culture plates grows after 24h, be divided into blank group, model group (LPS0.5 μ g/mL), high dose group (LPS0.5 μ g/mL+ petroleum ether fraction 200 μ g/mL), middle dosage group (LPS0.5 μ g/mL+ petroleum ether fraction 100 μ g/mL), low dose group (LPS0.5 μ g/mL+ petroleum ether fraction 50 μ g/mL), positive drug group (LPS0.5 μ g/mL+ celecoxib 2 μ g/mL), after administration 24h, collect supernatant, press ELISA test kit description time-and-motion study TNF-α, IL-6, the concentration of IL-1 β.

2.6.2 real-time fluorescence quantitative PCR detects IL-1 β, IL-6, TNF-α, COX-2, iNOS and HO-1mRNA level.

2.6.2.1RNA extract

RAW264.7 cell is seeded on 24 porocyte culture plates grows after 24h, be divided into blank group, model group (LPS0.5 μ g/mL), high dose group (LPS0.5 μ g/mL+ petroleum ether fraction 200 μ g/mL), middle dosage group (LPS0.5 μ g/mL+ petroleum ether fraction 100 μ g/mL), low dose group (LPS0.5 μ g/mL+ petroleum ether fraction 50 μ g/mL), positive drug group (LPS0.5 μ g/mL+ celecoxib 2 μ g/mL), after the intervention of 6h.Follow these steps to extract cell total rna.

A. every hole adds PBS to clean after 1 time, adds lysate 350 μ L, vortex concussion 1min.

B. all solution is transferred to Filter column CS upper (Filter column CS is placed in collecting pipe), the centrifugal 2min of 12000rpm, collects filtrate.

C. in filtrate, add 1 times of volume 70% ethanol, proceed to together (adsorption column CR3 puts into collecting pipe) in adsorption column CR3 after mixing, the centrifugal 1min of 12000rpm, outwells the waste liquid in collecting pipe, and adsorption column CR3 is put back in collecting pipe.

D. in adsorption column CR3, add 350 μ L protein liquid removal RW1, the centrifugal 1min of 12000rpm, outwells the waste liquid in collecting pipe, and adsorption column CR3 is put back in collecting pipe.

E. to adsorption column CR3 central authorities, add the DNase I working solution of 80 μ L, room temperature is placed 15min.

DNase I storage liquid preparation: DNase I dry powder (1500U) is dissolved in to 550 μ L RNase-Free dd H ₂in O, softly mix-20 ℃ of storages (can preserve 9 months) after subpackage.

The preparation of DNase I working solution: get 10 μ L DNase I storage liquid and put into new RNase-Free centrifuge tube, add 70 μ L RDD buffer solution, softly mix.

F. in adsorption column CR3, add 350 μ L protein liquid removal RW1, the centrifugal 1min of 12000rpm, outwells the waste liquid in collecting pipe, and adsorption column CR3 is put back in collecting pipe.

G. in adsorption column CR3, add 500 μ L rinsing liquid RW, the centrifugal 1min of 12000rpm after room temperature placement 2min, outwells waste liquid, and adsorption column CR3 is put back in collecting pipe.Repeat 1 time.

H.12000rpm centrifugal 2min, outwells waste liquid.Adsorption column CR3 is placed in to super-clean bench and blows the rinsing liquid that dries adsorbing material remnants.

I. adsorption column CR3 is proceeded in a new RNase-Free centrifuge tube, add 50 μ L RNase-Free dd H ₂o, room temperature is placed 2min, and the centrifugal 2min of 12000rpm, obtains RNA solution.Put-70 ℃ of preservations.

2.6.2.2 measure RNA concentration and purity

Get 10 μ LRNA samples, with the TE (or sterile distilled water) of pH7.4, dilute 100 times.Take TE as blank, with ultraviolet spectrophotometer, measure the OD value of 260nm and 280nm.Rna content (μ g/mL)=OD ₂₆₀value * 40 μ g/mL * extension rates.And according to OD ₂₆₀nm/OD ₂₈₀nm ratio is estimated RNA purity.RNA sample purity is with OD ₂₆₀: OD ₂₈₀between=1.8-2.0, can think that RNA extracting is successful.

2.6.2.3RNA reverse transcription becomes cDNA

Adopt a day root FastQuant cDNA first chain synthetic agent box (removing genome), operation is undertaken by explanation, and step is as follows:

A. sample RNA is thawed on ice; 5 * g DNA Buffer, FQ-RT Primer Mix, 10 * Fast RT Buffer, RNase-Free dd H ₂o thaws in room temperature, after thawing, is placed on ice rapidly.Before using, every kind of solution vortex vibration is mixed, brief centrifugal to collect the liquid that remains in tube wall.

B. following operating procedure is please carried out on ice, according to the removal system preparation mixed liquor of genomic DNA: 5 * g DNA Buffer2 μ L, sample rna 5 μ L, R Nase-Free dd H ₂o supplies 10 μ L.Thoroughly mix.Briefly centrifugal, and be placed in 42 ℃, hatch 3min.Then be placed on ice and place.

C. prepare reverse transcription reaction system and close liquid: 10 * Fast RT Buffer2 μ L, RT Enzyme Mix1 μ L, FQ-RT Primer Mix2 μ L, RNase-Free dd H ₂o supplies 10 μ L.In first prewired 26 parts of this ratios, mix.

D. reverse transcription mixed liquor 10 μ L are added in the reactant liquor of b step, fully mix.

E.42 ℃, hatch 15min.

F.95 ℃, be put on ice after hatching 3min, the cDNA obtaining puts-20 ℃ of preservations.

2.6.2.4 real-time fluorescence quantitative PCR detects

On amplification instrument, carry out real time PCR, adopt Fast Start DNA Master SYBR Green I kit to detect, result adopts the Δ Δ C in relative quantification _tmethod is calculated mRAN and is expressed, and internal reference is β Actin.Reaction system adopts 25 μ L systems, the cDNA primer of IL-1 β, IL-6, TNF-α, COX-2, iNOS and HO-1 by invitrogen Shanghai company design synthetic primer as following table:

Table 1 primer information

PCR reaction system is as follows:

Quantitative fluorescent PCR condition:

Melt curve analysis is analyzed

Ready reaction plate:

A. label on doing on Sptting plate, guarantees to comprise in each Sptting plate an internal reference.

B. the suitable pcr amplification premix reagent (containing cDNA) of drawing 25 μ L, splashes in each reacting hole of Sptting plate.

C. these Sptting plates are placed on ice, until be ready to be packed in PCR instrument.

PCR Sptting plate distributes and sees the following form.

Table 2PCR Sptting plate distributes

Blank 1

Blank 1

Blank 1

Blank 2

Blank 2

Blank 2

Blank internal reference

Middle dosage internal reference

Blank

3

Blank 3

Blank 3

Blank 4

Blank 4

Blank 4

Blank internal reference

Middle dosage internal reference

Model

1

Model 1

Model 1

Model 2

Model 2

Model 2

Blank internal reference

Middle dosage internal reference

Model

3

Model 3

Model 3

Model 4

Model 4

Model 4

Blank internal reference

Middle dosage internal reference

Low dosage 1

Low dosage 1

Low dosage 1

Low dosage 2

Low dosage 2

Low dosage 2

Model internal reference

High dose internal reference

Low dosage 3

Low dosage 3

Low dosage 3

Low dosage 4

Low dosage 4

Low dosage 4

Model internal reference

High dose internal reference

Middle dosage 1

Middle dosage 1

Middle dosage 1

Middle dosage 2

Middle dosage 2

Middle dosage 2

Model internal reference

High dose internal reference

Middle dosage 3

Middle dosage 3

Middle dosage 3

Middle dosage 4

Middle dosage 4

Middle dosage 4

Model internal reference

High dose internal reference

High dose 1

High dose 1

High dose 1

High dose 2

High dose 2

High dose 2

Low dosage internal reference

Positive internal reference

High dose 3

High dose 3

High dose 3

High dose 4

High dose 4

High dose 4

Low dosage internal reference

Positive internal reference

Positive

1

Positive 1

Positive 1

Positive 2

Positive 2

Positive 2

Low dosage internal reference

Positive internal reference

Positive

3

Positive 3

Positive 3

Positive 4

Positive 4

Positive 4

Low dosage internal reference

Positive internal reference

2.6.3ELISA the content of kit measurement iNOS, HO-1 and COX-2 protein

RAW264.7 cell is seeded on 24 porocyte culture plates grows after 24h, be divided into blank group, model group (LPS0.5 μ g/mL), high dose group (LPS0.5 μ g/mL+ petroleum ether fraction 200 μ g/mL), middle dosage group (LPS0.5 μ g/mL+ petroleum ether fraction 100 μ g/mL), low dose group (LPS0.5 μ g/mL+ petroleum ether fraction 50 μ g/mL), positive drug group (LPS0.5 μ g/mL+ celecoxib 2 μ g/mL), after drug effect 24h, abandon supernatant, PBS cleans 1 time, every hole adds the PBS of 1mL pre-cooling, be placed in ice-water bath, carry out ultrasonic disruption, condition is: 5% power, pulverize 3s, interval 3s, totally 20 times.After broken, solution is transferred in centrifuge tube, 10000rpm/min, 4 ℃ of centrifugal 10min.Get supernatant preserves in-20 ℃.Before measuring, room temperature is thawed, and strictly by test kit description, measures.

2.6.4NF-the activation nuclear translocation of κ B research

Cell spreads after the adherent 24h of 24 orifice plate, after drug effect 24h, abandons supernatant, and PBS cleans 1 time.Follow these steps to operate

A. add 200 μ L fixative, fixedly 10min.

B. absorb fixative, with cleaning mixture washing 3 times, each 5min.

C. add 200 μ L immunostaining confining liquids, room temperature sealing 1h.

D. absorb immunostaining confining liquid, add 100 μ LNF-κ Bp65 antibody, incubated at room 1h.

E. careful sucking-off NF-κ Bp65 antibody is in suitable container, and 4 ℃ of preservations, stay and do use next time.

F. cleaning mixture washing is 3 times, each 7min.

G. add the anti-rabbit Cy3 of 100 μ L, incubated at room 1h.

H. the careful anti-rabbit Cy3 of sucking-off is in suitable container, and 4 ℃ of preservations, stay and do use next time.

I. cleaning mixture washing is 2 times, each 7min.

J. add 100 μ L nucleus dyeing liquid (DAPI), room temperature dyeing 5min.

K. absorb nucleus dyeing liquid, with cleaning mixture washing 3 times, each 5min.

L. drip the anti-fluorescent quenching mounting liquid of appropriate amount, fluorescence microscopy Microscopic observation after coverslip mounting.The dyeing of NF-κ B is red fluorescence, and nuclear DAPI dyeing is blue-fluorescence.

3 results

3.1 impacts on RAW264.7 cell viability

As can be seen from Table 3, within the scope of experimental concentration 200 μ g/mL, watermelon vine different parts extract does not all have remarkable toxicity to RAW264.7 cell.At this, below concentration range, can carry out anti-inflammatory activity screening experiment.

Table 3 is on the impact of RAW264.7 cell viability (n=8)

3.2RAW264.7 cell LPS stimulates mould to set up

LPS all can significant stimulation RAW264.7 cell NO emission levels in 5 μ g/mL, 1 μ g/mL, 0.5 μ g/mL concentration range, and to cytoactive without remarkable inhibitory action.Therefore choose LPS low dosage in follow-up study as the modeling concentration of experiment, in Table 4.

Table 4 variable concentrations LPS stimulates the impact (n=8) on RAW264.7 cell NO release and cell survival

3.3 pairs of effects that RAW264.7 cell LPS stimulates model NO to discharge

5 kinds of extracts all discharge and have certain inhibitory action the NO of RAW264.7 cell, wherein remarkable with petroleum ether group.Therefore assert the anti-inflammatory activity position that petroleum ether part is watermelon vine, in Table 5.

The impact (n=8) that table 5 stimulates model NO to discharge on RAW264.7LPS

3.4 RAW264.7 cell LPS is stimulated to the effect of model proinflammatory cytokine

Under the stimulation of LPS, the pro-inflammatory mediator concentration of model group significantly raises, and shows that cell exists serious inflammatory reaction.Each concentration administration effect of petroleum ether 24h, the release of pro-inflammatory mediator IL-1 β, IL-6, TNF-α is suppressed significantly, shows that it can suppress the release of pro-inflammatory mediator.Positive drug group celecoxib also has significant inhibitory action to proinflammatory cytokine, in Table 6.

The impact of table 6 petroleum ether part on proinflammatory cytokine

3.5 couples of RAW264.7 cell LPS stimulate model iNOS, COX-2, the effect of HO-1

Under the stimulation of LPS, model group HO-1 concentration significantly raises, and shows that cell exists serious inflammatory reaction.Each concentration administration effect of petroleum ether 24h, to iNOS, COX-2 all has remarkable inhibitory action, and the downstream product HO-1 of COX-1 is expressed without impact.Show that its antiinflammatory action is the result that selectivity suppresses COX-2.Positive drug group celecoxib has also shown same trend, in Table 7.

Table 7 petroleum ether part is to iNOS, COX-2, the impact that HO-1 expresses

3.6 RAW264.7 cell LPS is stimulated to the effect of model mRNA

RNA concentration and Purity: OD ₂₆₀be 0.25, OD ₂₈₀be 0.13.OD ₂₆₀nm/OD ₂₈₀nm=1.92, purity meets the requirement of RNA experiment.Concentration=OD of RNA ₂₆₀nm * extension rate * 40, calculate the concentration of mRNA between between 30-40 μ g/mL, and content is between between 1.5-2 μ g.

The relative expression of mRNA the results are shown in Table 8 and Fig. 3, by result, can be found out, under the stimulation of LPS, the iNOS of macrophage, COX-2, HO-1, IL-1 β, IL-6, TNF-α mRNA relative expression quantity is all multiplied, after increasing pharmaceutical intervention, the mrna expression of all the other albumen except HO-1 is all decreased significantly, and this is consistent with the result that ELISA method is measured protein expression.

Table 8 couple RAW264.7 cell LPS stimulates model iNOS, COX-2, HO-1, IL-1 β, IL-6, the impact of TNF-α mRNA relative expression quantity

3.7 stimulate the impact of the activation nuclear translocation of model NF-κ B to RAW264.7 cell LPS

From immunofluorescence microscopy, under the induction of LPS, the increasing expression of NF-κ B also all shifts in core, and after administration, red fluorescence reduces, and occurs significantly reducing in core.Show that watermelon vine petroleum ether part has the effect that suppresses NF-κ B p65 activation nuclear translocation, sees Fig. 3.

4 discuss

NO is inflammatory reaction and immunoregulatory effector, is also crucial regulatory factor.NO participates in the conduction of inflammation signal, with inflammation factor interaction.NO has generation at the every one-phase of inflammatory reaction process, and therefore, the content that can measure NO in body is evaluated the anti-inflammatory activity of a certain anti-inflammatory drug.In addition, because being excessively created in the developing of inflammation, tumor, infection, cardiovascular disease, autoimmune disease etc. of NO played crucial regulating action.Therefore, NO not only becomes the new therapeutic strategies such as inflammation, autoimmune disease, and has become the important target spot of new drug development.

Cyclooxygenase-1 (COX-1) plays an important role in inflammatory reaction with COX-2 (COX-2).Recent studies have found that, in the synovial tissue of rheumatoid arthritis patients, can measure COX-1 and COX-2, the rheumatoid synovial cell who cultivates in vitro show hugely bite, fiber is female, endothelium and single core inflammatory cell are all expressed two kinds of enzymes.The Mus of pounding out COX-1 can alleviate arachidonic inflammatory reaction, and the Mus of pounding out COX-2 can occur inflammatory reaction equally through inflammatory stimulus, and its extent of reaction with contrast Mus no significant difference.

RAW264.7 mouse macrophage model is that the huge born of the same parents of biting that are widely used in extracorporeal anti-inflammatory screening experiment are, this model is subject to after LPS stimulates expressing and to put cytokine profiles and free radical (comprising NO, reactive oxygen species (ROS), IL-1 and TNF-α etc.), the inflammatory mediator that these are important plays an important role in the generation of inflammatory reaction and development.

The present invention studies discovery, the anti-inflammatory activity position that petroleum ether part is watermelon vine.Its macrophage inflammatory reaction to LPS induction has remarkable inhibitory action, its mechanism of action may be by selectivity, to suppress the activity of COX-2, suppressing NF-κ B nuclear translocation activates, thereby pro-inflammatory mediator IL-1 β, the IL-6, the TNF-α that suppress LPS induction discharge and the activity of iNOS, reduce the release of NO, whether watermelon vine petroleum ether part antiinflammatory action also exists other target spots to need further research.

Claims (7)

1. a watermelon vine extract with anti-inflammatory and analgesic effect, is characterized in that this extract is from the petroleum ether part of watermelon vine precipitate with ethanol water extraction.

2. the watermelon vine extract with anti-inflammatory and analgesic effect according to claim 1, is characterized in that this extract mainly comprises following ten kinds of compounds: cupreol, daucosterol, stigmasterol, myristin, hexadecanoic acid, tripalmitin, docosane acid glyceride, ursolic acid, heneicosane acid glyceride, stearic acid.

3. the preparation method described in claim 1 with the watermelon vine extract of anti-inflammatory and analgesic effect, it is characterized in that comprising the following steps: get watermelon vine and shine dry grinding, the ultrasonic 2h of alcoholic solution of volumetric concentration 80% for volume ratio 5:1 by weight, then soak 10d, filter merging filtrate, concentrating under reduced pressure, obtain blackish green extractum, lyophilization obtains green powder; Volume ratio 3:50 is scattered in green powder in deionized water by weight, standing over night, the centrifugal sticky solid suspension of water-fast green and the aqueous solution of separating; The petroleum ether extraction sample of 3 times of volumes for water intaking solution, extract concentrating under reduced pressure, reclaims solvent, and evaporate to dryness, obtains petroleum ether part.

4. the watermelon vine extract described in claim 1 with anti-inflammatory and analgesic effect in preparation treatment rheumatic arthritis, rheumatoid arthritis, scapulohumeral periarthritis, osteoarthrosis pain, cervical vertebra pain, soft tissue pain, sprain the application aspect the medicine of pain.

5. application according to claim 4, is characterized in that: described medicine is capsule, granule, tablet, solution, pill or injection.

6. application according to claim 5, it is characterized in that: described capsule is soft capsule, described granule is dry suspension, and described tablet is dispersible tablet, effervescent tablet, chewable tablet, oral cavity disintegration tablet, described solution is syrup, and described pill is concentrated pill, drop pill, micropill.

7. the watermelon vine extract described in claim 1 with anti-inflammatory and analgesic effect preparing auxiliary treatment of rheumatic arthritis, rheumatoid arthritis, scapulohumeral periarthritis, osteoarthrosis pain, cervical vertebra pain, soft tissue pain, sprain the application aspect the health food of pain.

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