CN104840525A - Preparation method of melaleuca alternefolia total flavone and detection method thereof - Google Patents
Preparation method of melaleuca alternefolia total flavone and detection method thereof Download PDFInfo
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Abstract
The invention discloses melaleuca alternefolia total flavone extract and a preparation method thereof. The preparation method comprises the following steps of extracting essence oil from the melaleuca alternefolia branch and leave by water vapor, performing ethanol heating and refluxing extraction, reducing the pressure to recycle the ethanol, concentrating to remove the ethanol flavor, separating and purifying by macroporous absorption resin, and drying; measuring the content of total flavone in the melaleuca alternefolia by adopting an ultraviolet-visible spectrophotometry method. The preparation method has the advantages that the method is accurate and stable, the operation is simple, the requirement of industrial production is met, and the quality of the melaleuca alternefolia total flavone extract can be effectively controlled; the content of total flavone in the melaleuca alternefolia total flavone extract can reach more than 50 percent, the toxicity is little, and the pharmacological functions of resisting inflammation, inhibiting bacteria, alleviating pain, resisting allergy, resisting oxidizing and the like are realized; the preparation method is reasonable and stable, the production cycle is short, the preparation method is suitable for industrial production, the important meaning is realized on the deep development and utilization of melaleuca alternefolia resources, and the development prospect is good.
Description
Technical field
The present invention relates to a kind of preparation field of flavone extract, be specifically related to purification and the detection method thereof of a kind of plant Melaleuca Alternifolia total flavones.
Background technology
Natural plant extracts take plant as raw material, according to extraction object and needs, utilizes the extraction separation method of physics and chemistry, and orientation obtains and a certain or plurality of active ingredients concentrated in plant, and the product not changing its effective ingredient structure and formed.Compared with Chinese traditional herbs, Chinese patent medicine, Chinese medicine extract has clear and definite effective ingredient, has the quantitative target of Quality Control, and harmful components or impurity major part are all removed, and safety, effectiveness, controllability get a promotion than traditional plant medicine.In addition, Chinese medicine extract has also merged modern pharmaceutical new technique, belongs to new medicine product, and have wide space, international market, its industrialization has good development prospect, embodies the technological progress of Chinese Medicine Industry, has adapted to the requirement of the modernization of Chinese medicine.
Melaleuca Alternifolia be Myrtaceae (
myrtaceae) Melaleuca (
melaleucaL.) Melaleuca Alternifolia (
melaleuca alternifolia), be the primary raw material extracting natural tea tree oil, oil yield is greatly about 1 ~ 2%.([document] component Analysis of Essential Oil from Melaleuca Leucadendron L [author] Liu Buming, Peng Wei [periodical name] analytical test journal, 1999. 18 (6): 70-72[summaries] Melaleuca Alternifolia branch and leaf, rhizome is all containing volatile oil, its branch and leaf oil-containing 0.8-1.3%, plant Cortex Melaleucae leucadendrae branch and leaf chemical composition of volatile oil is analyzed and researched, be separated 35 compositions confirming wherein, account for that volatile oil chromatograph rushes peak area more than 90%.) Melaleuca Alternifolia branch and leaf carry the plant part after oil usually used as production waste process and abandoned by as garbage after water distillation and extraction quintessence oil, plant fails to be developed fully, seriously wastes herb resource.There are some researches show, separablely in the branch and leaf of Melaleuca Alternifolia obtain Quercetin, the multiple flavonoid reactive compound such as kaempferol (chemical constitution study [author] Liu Buming of [document] Melaleuca Alternifolia, Dong Xiaomin, Huang Yan, Lin Xiao, Lu Wenjie, tooth opens health [periodical name] Chinese herbal medicine, 2011 (07): 1282-1284[summaries] adopt silica gel column chromatography and recrystallization method to carry out separation and purification, 11 compounds are identified according to physicochemical property and spectral data, be respectively: n-tricontyl-4-cinnamate, Quercetin, nimbecetin, betulic acid, gallic acid, progallin A, cupreol, protocatechuic acid, cholesterol, laccerol, Ji reaches acid.), and content quite high (Quercetin in [document] hplc simultaneous determination Melaleuca Alternifolia and kaempferol [author] Lin Xiao, Dong Xiaomin, Chen Mingsheng, Huang Yan, Liu Buming [periodical name] chemistry of forest product and industry, 2012, (04): 113-116[makes a summary] with Quercetin and kaempferol content in ground, hplc simultaneous determination Guangxi 5 Melaleuca Alternifolia, determine the Quercetin in Different sources Melaleuca Alternifolia and kaempferol mass fraction, wherein the Quercetin of south, Yongning dawn and the mass fraction of kaempferol the highest, be respectively 5.151 and 2.530 mg/g, the Quercetin of Yongning and the mass fraction of kaempferol are minimum, be respectively 2.386 and 1.658 mg/g.Mensuration [author] Fan Chaojun, Bao Changyu, the Chen Zhanjuan of [document] Cortex Melaleucae leucadendrae branch and leaf flavonoids content, finish peace [periodical name] Hainan Normal University's journal: natural science edition 25 (1): 63-65[is made a summary] be reference substance with rutin, NaNO
2-Al(NO
3) 3-NaOH is Color Appearance System, with ultraviolet spectrophotometry, measures the content of total flavones in Cortex Melaleucae leucadendrae branch and leaf at 498nm wavelength.Measurement result shows, in Cortex Melaleucae leucadendrae branch and leaf, the mass fraction of total flavones is respectively 0.69% and 0.63%.) and its crude extract has obvious antiinflammatory, analgesia, antibacterial, antiallergic, antioxidative pharmacological action.(pharmacological research [author] Li Yanjing of [document] Melaleuca Alternifolia ethanol extract, Zhong Zhengxian, Lin Xiao, Liu Buming [periodical name] Guangxi traditional Chinese medicine, 2013 (03): 77-79[summaries] Melaleuca Alternifolia ethanol extract can suppress Oleum Tiglii to cause mice auricle swelling; The pain threshold of mice thermostimulation pain can be improved; Except bacillus subtilis, Melaleuca Alternifolia ethanol extract all has bacteriostasis to 7 kinds of strains such as staphylococcus aureus, staphylococcus epidermidis, Candida albicans; The passive cutaneous permeability of mice can be suppressed, there is anti-skin allergy effect; The median lethal dose(LD 50) (LD50) of Melaleuca Alternifolia ethanol extract is 51.749g/kg, and 95% is crediblely limited to 44.700 ~ 59.911g/kg.
Conclusion: Melaleuca Alternifolia ethanol extract has the pharmacological actions such as antiinflammatory, analgesia, antibacterial, antiallergic, and toxicity is little.Therefore research and develop a kind of preparation and method of quality control thereof of Melaleuca Alternifolia total flavones, the pharmaceutical route exploitation new to Melaleuca Alternifolia plant has great practical application meaning with comprehensive utilization.
Summary of the invention
The object of the present invention is to provide a kind of method rationally, stable, simple, be applicable to suitability for industrialized production, there is preparation method and the method for quality control of the Melaleuca Alternifolia total flavones of the pharmacological actions such as antiinflammatory, antibacterial, analgesia, antiallergic, antioxidation.
The present invention is achieved in that
The preparation method of Melaleuca Alternifolia total flavones of the present invention, comprises the following steps:
1. alcohol heating reflux extracts
1. learn from else's experience the Melaleuca Alternifolia stem and leaf after extraction by steam distillation quintessence oil, adding 8 ~ 15 liters of concentration by quality every 800-1200 g Melaleuca Alternifolia stem and leaf is the ethanol of 50 ~ 95%;
2. heating and refluxing extraction 3 times, each 1 ~ 3 hour, filters, merging filtrate;
3. decompression recycling ethanol, and be concentrated into without alcohol taste, be diluted with water to the Melaleuca Alternifolia extracting solution that total flavones concentration is 0.2 ~ 0.8 mg/mL, centrifuging and taking supernatant, stand-by.
2. macroporous adsorbent resin separation and purification
1. X-5 or the AB-8 macroporous adsorbent resin of low pole is selected;
2. adsorb: upper prop Melaleuca Alternifolia extracting solution total flavones concentration is 0.2 ~ 0.8 mg/mL, upper prop Melaleuca Alternifolia extracting liquid volume is 2 ~ 5 times that fill macroporous adsorbent resin volume, and upper column flow rate is 0.5 ~ 3 BV/h; [note: BV/h representation unit time (h) flows through the average liquid measure of unit volume resin];
3. eluting: eluant is 50 ~ 80% ethanol, eluting agent is 2 ~ 5 times that fill macroporous adsorbent resin volume, and elution flow rate is 2 ~ 5 BV/h; Collect 2 ~ 5 times of eluents, decompression recycling ethanol, is concentrated into dry, and 60 DEG C of vacuum dryings, obtain Melaleuca Alternifolia extractive of general flavone.
The detection method of Melaleuca Alternifolia total flavones of the present invention, comprises the following steps:
1. the preparation precision of reference substance storing solution takes control substance of Rutin in right amount, adds methanol and makes the solution of every 1 mL containing 0.20 mg, obtain control substance of Rutin storing solution;
2. the preparation precision amount of standard curve draws control substance of Rutin storing solution 0.5,1,2,3,4,5 mL, puts respectively in 10 mL measuring bottles, adds the NaNO that mass fraction is 5% respectively
2solution 0.5 mL, after shaking up placement 8 min, adds the Al (NO that mass fraction is 10% respectively
3)
3solution 0.5 mL, shakes up placement 8 min, then adds NaOH solution 4.0 mL that mass fraction is 4% respectively, adds methanol dilution to scale, shakes up after placing l5 min, at the absorbance A that 498 nm wavelength places measure solution, carries out blank simultaneously.With concentration (μ g/mL) for abscissa, absorbance (A) is vertical coordinate drawing standard curve;
3. algoscopy: get Melaleuca Alternifolia extractive of general flavone about 20 mg prepared, accurately weighed, put in 100 mL measuring bottles, add methanol make dissolving in right amount and be diluted to scale, shake up, precision measures 2 mL, put in 10 mL measuring bottles, add the Al (NO that mass fraction is 10%
3)
3solution 0.5 mL, shakes up placement 8 min, then adds NaOH solution 4.0 mL that mass fraction is 4% respectively, adds methanol dilution to scale, shakes up after placing l5 min, at the absorbance A that 498 nm wavelength places measure solution, carries out blank simultaneously.This product general flavone content is with rutin (C
27h
30o
16) calculate be no less than 50. 0%;
4. result: adopt preparation of the present invention and method of quality control, empirical tests is tested, and the Melaleuca Alternifolia general flavone content of preparation, in rutin, can reach more than 50%.
Compared with prior art, the substantive distinguishing features that the present invention gives prominence to significant progress is:
1, the present invention prepares the extractive of general flavone of content more than 50% first from Melaleuca Alternifolia branch and leaf, making full use of and improving the foundation that its using value provides science for herb resource.
2, the present invention is reasonable in design, and preparation method is stable, feasible, energy consumption is low, utilizes alcohol aqueous solvent to extract, and can obtain the higher Melaleuca Alternifolia extractive of general flavone of content through a macroporous adsorptive resins separation and purification, method is simple.
3, extracting and developing speed of the present invention is fast, with short production cycle, is applicable to suitability for industrialized production, has good application prospect.
4, the present invention adopts ultraviolet visible spectrophotometry to measure Melaleuca Alternifolia general flavone content, and method is simple, accurate, can adapt to industrial needs, effectively can control the quality of Melaleuca Alternifolia extractive of general flavone.
5, by studying Melaleuca Alternifolia total flavones, establish a kind of preparation and method of quality control of Melaleuca Alternifolia total flavones, be conducive to the further exploitation of Melaleuca Alternifolia Related product, potential and immeasurable social benefit and economic benefit will be produced.
6, Melaleuca Alternifolia total flavones of the present invention is applied in antiinflammatory, antibacterial, analgesia, antiallergic, antioxidative medicine.
Accompanying drawing explanation
Fig. 1 is preparation technology's flow chart of Melaleuca Alternifolia total flavones;
Fig. 2 is that Melaleuca Alternifolia extract joins the change of the absorbance in DPPH solution with the response time.
Detailed description of the invention
embodiment one:
Get Melaleuca Alternifolia branch and leaf 1000g, add water 3L, heats 70 minutes, extract volatile oil, after receiving oil, discard the aqueous solution in container, add 12L 75% ethanol, heating and refluxing extraction 3 times, each 2 hours, merge extractive liquid, filter, filtrate decompression is recycled to without ethanol taste, is dissolved in water, and is made into the Melaleuca Alternifolia extracting solution that total flavones concentration is 0.5 mg/mL.Get processed good X-5 macroporous adsorbent resin 80 g and (be equivalent to dried resin 30 g, column volume 200 mL), wet method dress post, 600 mL Melaleuca Alternifolia extracting solution are adsorbed with the upper column flow rate of 3 BV/h, substantially clarify to eluent with the water elution of 3 BV, water elution liquid discards, with 4BV 75% ethanol with the flow velocity eluting of 2BV/h, collect eluent, decompression recycling ethanol, be concentrated into dry, 60 DEG C of vacuum dryings, obtain Melaleuca Alternifolia extractive of general flavone, measure, calculate, general flavone content is 63. 5%.
embodiment two:
Get Melaleuca Alternifolia branch and leaf 1200g, add water 4L, heats 70 minutes, extract volatile oil, after receiving oil, discard the aqueous solution in container, add 15L 50% ethanol, heating and refluxing extraction 3 times, each 3 hours, merge extractive liquid, filter, filtrate decompression is recycled to without ethanol taste, is dissolved in water, and is made into the Melaleuca Alternifolia extracting solution that total flavones concentration is 0.8 mg/mL.Get processed good X-5 macroporous adsorbent resin 60g and (be equivalent to dried resin 20g, column volume 150mL), wet method dress post, 750mL Melaleuca Alternifolia extracting solution is adsorbed with the upper column flow rate of 2BV/h, substantially clarify to eluent with the water elution of 4BV, water elution liquid discards, with 5BV 50% ethanol with the flow velocity eluting of 4BV/h, collect eluent, decompression recycling ethanol, be concentrated into dry, 60 DEG C of vacuum dryings, obtain Melaleuca Alternifolia extractive of general flavone, measure, calculate, general flavone content is 58.9%.
embodiment three:
Get Melaleuca Alternifolia branch and leaf 800g, add water 2L, heats 70 minutes, extract volatile oil, after receiving oil, discard the aqueous solution in container, add 8L 95% ethanol, heating and refluxing extraction 3 times, each 1 hour, merge extractive liquid, filter, filtrate decompression is recycled to without ethanol taste, is dissolved in water, and is made into the Melaleuca Alternifolia extracting solution that total flavones concentration is 0.2 mg/mL.Get processed good AB-8 macroporous adsorbent resin 50g and (be equivalent to dried resin 20g, column volume 150mL), wet method dress post, 450mL Melaleuca Alternifolia extracting solution is adsorbed with the upper column flow rate of 0.5BV/h, substantially clarify to eluent with the water elution of 4BV, water elution liquid discards, with 3BV 80% ethanol with the flow velocity eluting of 5BV/h, collect eluent, decompression recycling ethanol, be concentrated into dry, 60 DEG C of vacuum dryings, obtain Melaleuca Alternifolia extractive of general flavone, measure, calculate, general flavone content is 61.2%.
the detection method of Melaleuca Alternifolia total flavones
The assay of total flavones
It is appropriate that precision takes control substance of Rutin, adds methanol and make the solution of every 1 mL containing 0.20 mg rutin, obtain control substance of Rutin storing solution.Separately get Melaleuca Alternifolia extractive of general flavone about 20 mg prepared, accurately weighed, put in 100 mL measuring bottles, add methanol and make dissolving in right amount and be diluted to scale, shake up, precision measures 2 mL, puts in 10 mL measuring bottles, adds the Al (NO that mass fraction is 10%
3)
3solution 0.5 mL, shakes up placement 8 min, then adds NaOH solution 4.0 mL that mass fraction is 4% respectively, adds methanol dilution to scale, shakes up after placing l5 min, at the absorbance A that 498 nm wavelength places measure solution, carries out blank simultaneously.This product general flavone content calculates with rutin and is no less than 50. 0%.
Content assaying method test is as follows:
1. the preparation precision amount of standard curve draws control substance of Rutin storing solution 0.5,1,2,3,4,5 mL, puts respectively in 10 mL measuring bottles, adds the NaNO that mass fraction is 5% respectively
2solution 0.5 mL, after shaking up placement 8 min, adds the Al (NO that mass fraction is 10% respectively
3)
3solution 0.5 mL, shakes up placement 8 min, then adds NaOH solution 4.0 mL that mass fraction is 4% respectively, adds methanol dilution to scale, shakes up after placing l5 min, at the absorbance A that 498 nm wavelength places measure solution, carries out blank simultaneously.With concentration (μ g/mL) for abscissa, absorbance (A) is vertical coordinate drawing standard curve, obtains regression equation Y=11.85X+0.0277, r=0.9998, and when showing within the scope of 10 ~ 100 μ g/mL, rutin linear relationship is good.
2. precision test gets same need testing solution, METHOD FOR CONTINUOUS DETERMINATION 6 times, and result RSD is 1.50%.
3. stability test gets same need testing solution, adding NaOH solution also with after methanol constant volume 15min, respectively in 0 after this, 30,45,60,90,120 min measure A values, result RSD is 1.37%, shows that need testing solution was stablized in 120 minutes.
4. replica test is got same batch sample and is prepared need testing solution 6 parts, and measure by drafting method, result RSD is 2.22%.
5. recovery test gets Melaleuca Alternifolia extractive of general flavone 10 mg of known content, and precision adds control substance of Rutin in right amount respectively, and measure content by drafting method, and calculate the response rate, response rate meansigma methods is 98. 2%, RSD is 2. 4%.
Methodology result of the test shows that the method is accurate, stable, simple to operate, can guarantee the quality of product.
Melaleuca Alternifolia extract toxicity is little, and have the pharmacological actions such as antiinflammatory, antibacterial, analgesia, antipruritic, antioxidation, carry out pharmacodynamics and acute toxicity test with Melaleuca Alternifolia extract, result is as follows simultaneously:
1. anti-inflammation test
Get male mice 50, be divided into blank group, the positive (aspirin 0.2 g/kg) matched group, Melaleuca Alternifolia extract (high concentration dosage: 5.1 g crude drug/kg, middle concentration dose: 2.6 g crude drug/kg, low concentration dosage: 1.3 g crude drug/kg) group at random, often organize 10 mices.Every day gavage successive administration aspirin and Melaleuca Alternifolia extract 1 time, successive administration 7 days.The 7th administration after 1 hour, get 2% Oleum Tiglii 50 μ L trowelling and cause scorching modeling in the auris dextra of mice, within 4 hours, post-tensioning neck puts to death mice, prolong mice left and right auricle 6 mm card punch and take off same area auricle, weigh, and using two auricle quality differences (mg) as swelling index calculate inhibitory rate of intumesce (%).Anti-inflammation test result shows, compared with Normal group, high, medium and low three dosage of Melaleuca Alternifolia extract are to Oleum Tiglii induced mice auricle edema inhibited (P<0.05 or P<0.01), suppression ratio is respectively 36.44%, 29.07% and 30.67%, but have no dose-dependent relationship, prompting Melaleuca Alternifolia extract has certain inhibitory action to Earlier period of inflammation.
2. analgesic test
Select female mice, mice is placed on electric heating constant-temperature water-bath tank pot, adjust the temperature to 55 DEG C, record mice is from dropping into hot plate to occurring that the time (s) of licking metapedes reaction is index, measure the latency of pain response of every mice, test and get average twice, filter out the qualified female mice 50 of latency of pain response in 5 ~ 30 s scopes, qualified mice is divided into Normal group at random, aspirin (0.2 g/kg) group, Melaleuca Alternifolia extract (high concentration dosage: 5.1 g crude drug/kg, middle concentration dose: 2.6 g crude drug/kg, low concentration dosage: 1.3 g crude drug/kg) group, the aspirin of the above dosage of administration group gastric infusion every day and Melaleuca Alternifolia extract 1 time, successive administration 7 days.In last administration after 1 hour, measure the latency of pain response of each mice as stated above in 15min, 30min and 60 min, replication 2 times, exceed the unresponsive mice of 60 s then by 60 s, obtain each cell mean.Analgesic test result shows, compared with Normal group, high, medium and low three the dosage groups of Melaleuca Alternifolia extract all obviously extend the incubation period (P<0.05 or P<0.01) of mice thermostimulation pain within the regular hour, and extend with administration time and change, show that Melaleuca Alternifolia extract has Central Analgesic Effect.
3. bacteriostatic test
With MH nutrient broth to staphylococcus epidermidis, staphylococcus aureus, Salmonella paratyphi B, Escherichia coli, Pseudomonas aeruginosa, shigella flexneri, bacillus subtilis
,candida albicans 8 strains are inoculated, and cultivate after within 18 hours, bringing back to life strain and test in the bacteriological incubator of 37 DEG C.Getting MH nutrient broth and crude drug content is that each 50 μ L of ethanol extract of 0.5 g/mL add in the 96 each holes of porocyte culture plate the first row, doubling dilution is adopted to be diluted to descending each hole after mixing, get 0.05% TTC sterile solution and Maxwell reduced turbidity bacterium liquid adds in sterile centrifugation tube in right amount, draw 50 μ L after mix homogeneously respectively to add in corresponding micropore, make the medicine of 1 to 7 row be respectively the Concentraton gradient of 125,62.5,31.25,15.63,7.82,3.91,1.95 mg/mL.Meat soup control wells, bacterium liquid control wells and solvent control hole is established at eighth row.Cultivated 24 hours at 37 DEG C in bacteriological incubator by this microwell plate, take out and observe, naked eyes are seen and are looked into meat soup color in orifice plate, with the drug level corresponding to not aobvious red micropore for minimal inhibitory concentration.Result of the test shows, Melaleuca Alternifolia extract has stronger antibacterial action to staphylococcus epidermidis, staphylococcus aureus, Candida albicans; To shigella flexneri, Pseudomonas aeruginosa, Escherichia coli, there is moderate antibacterial action; To Salmonella paratyphi B, there is low antibacterial action; To bacillus subtilis without antibacterial action.
Melaleuca Alternifolia extracts object to the mensuration of 8 strain strain MIC
4. mice passive cutaneous anaphylaxis test
Sero-fast preparation: take medical Radix Trichosanthis 37.5 mg and be dissolved in 15 mL gel aluminum hydroxides.Get healthy mice 10, male and female half-and-half, inject above-mentioned gel aluminum hydroxide suspension 50 μ L in mice two hind paw, inject 100 μ L altogether.After 15 days, mice broken end is got blood, centrifugalize antiserum, and mixing, puts refrigerator for subsequent use.
Passive sensitization of skin and exciting: separately get mice 50, male and female half-and-half, be divided into Normal group at random, hismanal (10 mg/kg), Melaleuca Alternifolia extract (high concentration dosage: 5.1 g crude drug/kg, middle concentration dose: 2.6 g crude drug/kg, low concentration dosage: 1.3 g crude drug/kg) group, every day gastric infusion hismanal and Melaleuca Alternifolia extract 1 time, successive administration 5 days.Within 5th day, get mixed antiserum, be diluted in normal saline by 1:5, in every mice stomach wall intradermal injection two points, often 0.03 mL, and continue gastric infusion 2 days, in last administration after 12 hours, antigen attack is carried out to every mice, every mice is by 0.2 mL/ dosage tail vein injection 2.5 mg/mL Radix Trichosanthis (1% azovan blue-normal saline) only, mice is put to death after 20 min, take off abdominal part locus coeruleus skin graft, be soaked in the test tube containing 5 mL acetone-normal saline solutions (7:3 v/v) after shredding, place 48 hours, centrifuging and taking supernatant, light absorption value is measured at wavelength 610 nm place, compare between organizing, evaluate the anti-allergic effects of medicine.
Skin allergy experimental result shows, compare with blank group, high, medium and low three dosage of Melaleuca Alternifolia extract all can reduce mouse skin locus coeruleus absorbance (P<0.05 or P<0.01), and in dose-dependent relationship, show that Melaleuca Alternifolia extract can suppress mouse skin permeability, there is certain anti-skin allergy effect.
Melaleuca Alternifolia extract on the impact of mice passive cutaneous anaphylaxis, PCA (
± s, n=10)
5. free radical scavenging activity determination experiment
The determination in 5.1 response time
Melaleuca Alternifolia extract under optimal conditions is mixed with certain concentration solution, getting 0.10 mL liquid to be measured, to add 3.90 mL concentration be that 10 mol/L DPPH are by base methanol solution, under 515 nm conditions, time sweep is carried out to it after shaking up rapidly, to determine the suitable response time.As shown in Figure 2, extract adds after in DPPH solution, and front its absorbance of 15 min declines very fast (dropping to 0.12 from 0.968), and substantially tend towards stability after reacting 30 min, therefore this experiment employing 30 min is as the reaction assay time.
The mensuration of 5.2 Melaleuca Alternifolia extract scavenging ability of DPPH free radicals
Accurately weighed 100 mg Melaleuca Alternifolia extracts, ethanol is settled to 100 mL.Compound concentration is the alcoholic solution of 1.0,0.8,0.6,0.4,0.2,0.1 mg/ mL respectively, as experimental group.Prepare the Quercetin of isoconcentration gradient as a control group simultaneously.Adopt kinetic monitoring means to measure the time dependent absorbance A of solution, every 30 s value 1 time, when absorbance is substantially constant, medicine and radical reaction completely, record final absorbance.
Free radical scavenging activity (%)=[1-(A
i-A
j)/A
0] × 100
In formula: A
0for the blank absorbency of non-dosing DPPH solution, A
ifor the absorbance of DPPH solution after dosing, A
jfor the absorbance of sample solution self.
Median effective dose (the EC of 5.3 scavenging free radicals
50) calculating and the evaluation of radical scavenging activity (AE)
According to medicine addition and free radical scavenging activity (%), the relation curve of both making, obtaining by nonlinear regression analysis the consumption that regression equation calculation obtains Melaleuca Alternifolia extract when DPPH free radical original quality concentration is reduced to 50% (stable state) is median effective dose (EC
50), show that Melaleuca Alternifolia extract removes the EC of DPPH free radical
50be 17.48 μ g/mL.
acute toxicity test
Get mice 40, male and female half-and-half, be divided into 4 groups at random, often organize 10, before gavage, 12 h fast can't help water.Administration group is by the Mouse Weight gastric infusion Melaleuca Alternifolia extract 1 time of 0.04 mL/g, matched group gavage gives same volume distilled water, conventional raising after administration, observe the motion of mice, breathing, abdominal part irritation, continuous observation 14 days, and record mouse toxicity reaction, death toll and death condition.Add up the mortality rate of each group, calculate the LD of Melaleuca Alternifolia extract to mouse stomach administration by Bliss method
50be 50.9 g/kg, LD
5095% confidence interval be 44.35 ~ 57.65 g/kg.Show that Melaleuca Alternifolia extract has less toxicity.
Claims (5)
1. a preparation method for Melaleuca Alternifolia total flavones, is characterized in that: the preparation method of described Melaleuca Alternifolia total flavones comprises the following steps:
(1) alcohol heating reflux extracts: the Melaleuca Alternifolia stem and leaf after extraction by steam distillation quintessence oil of 1. learning from else's experience, and adding 8 ~ 15 liters of concentration by quality every 800-1200 g Melaleuca Alternifolia stem and leaf is the ethanol of 50 ~ 95%; 2. heating and refluxing extraction 3 times, each 1 ~ 3 hour, filters, merging filtrate; 3. decompression recycling ethanol, and be concentrated into without alcohol taste, be diluted with water to the Melaleuca Alternifolia extracting solution that total flavones concentration is 0.2 ~ 0.8 mg/mL, centrifuging and taking supernatant, stand-by;
(2) macroporous adsorbent resin separation and purification: 1. select macroporous adsorbent resin; 2. adsorb: in upper prop Melaleuca Alternifolia extracting solution, total flavones concentration is 0.2 ~ 0.8 mg/mL, upper prop Melaleuca Alternifolia extracting liquid volume is 2 ~ 5 times that fill macroporous adsorbent resin volume, and upper column flow rate is 0.5 ~ 3 BV/h; 3. eluting: eluant is 50 ~ 80% ethanol, eluting agent is 2 ~ 5 times that fill macroporous adsorbent resin volume, and elution flow rate is 2 ~ 5BV/h; Collect 2 ~ 5 times of eluents, decompression recycling ethanol, is concentrated into dry, and 60 DEG C of vacuum dryings, obtain Melaleuca Alternifolia extractive of general flavone.
2. according to the preparation method of the Melaleuca Alternifolia total flavones described in claim 1, it is characterized in that: described macroporous adsorbent resin is X-5 or AB-8 macroporous adsorbent resin.
3. the Melaleuca Alternifolia total flavones described in claim 1, is characterized in that: this general flavone content calculates with rutin and is no less than 50. 0%.
4. according to the preparation method of the Melaleuca Alternifolia total flavones described in claim 1, it is characterized in that: the Melaleuca Alternifolia total flavones following steps obtained detect:
(1) preparation of reference substance storing solution: it is appropriate that precision takes control substance of Rutin, adds methanol and makes the solution of every 1 mL containing 0. 20 mg rutins, obtain control substance of Rutin storing solution;
(2) preparation of standard curve: accurate amount draws control substance of Rutin storing solution 0.5,1,2,3,4,5 mL, is placed in 10 mL volumetric flasks respectively, adds the NaNO that mass fraction is 5% respectively
2solution 0.5 mL, after shaking up placement 8 min, adds the Al (NO that mass fraction is 10% respectively
3)
3solution 0.5 mL, shakes up placement 8 min, then adds NaOH solution 4.0 mL that mass fraction is 4% respectively, adds methanol dilution to scale, shakes up after placing l5 min, at the absorbance A that 498 ± 2 nm wavelength places measure solution, carries out blank simultaneously; With concentration (μ g/mL) for abscissa, absorbance (A) is vertical coordinate drawing standard curve;
(3) algoscopy: get Melaleuca Alternifolia extractive of general flavone about 20 mg prepared, accurately weighed, put in 100 mL measuring bottles, add methanol make dissolving in right amount and be diluted to scale, shake up, precision measures 2 mL, put in 10 mL measuring bottles, add the Al (NO that mass fraction is 10%
3)
3solution 0.5 mL, shakes up placement 8 min, then adds NaOH solution 4.0 mL that mass fraction is 4% respectively, adds methanol dilution to scale, shakes up after placing l5 min, at the absorbance A that 498 ± 2 nm wavelength places measure solution, carries out blank simultaneously;
This product general flavone content calculates with rutin and is no less than 50. 0%.
5. the Melaleuca Alternifolia total flavones described in claim 1, is characterized in that: this total flavones is applied in be prepared in antiinflammatory, analgesia, antibacterial, antiallergic, antioxidative medicine.
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Cited By (5)
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| CN107669745A (en) * | 2017-08-21 | 2018-02-09 | 广西澳树宝生物科技有限公司 | A kind of continuous method for preparing tea oil and cajeputtree flavones |
| CN107890437A (en) * | 2017-12-05 | 2018-04-10 | 深圳市润昇源生物科技有限公司 | Facial mask and preparation method thereof is repaired in a kind of whitening containing mutual 100,000 layers of essential oil of leaf |
| CN107917886A (en) * | 2016-10-10 | 2018-04-17 | 武汉光谷人福生物医药有限公司 | The method for measuring total sugar content in Desmodium styracifolium general flavone |
| CN110279753A (en) * | 2019-08-13 | 2019-09-27 | 天津科技大学 | A kind of tea tree ferment and its preparation method and application |
| CN107525862B (en) * | 2017-08-03 | 2020-03-17 | 广西壮族自治区中医药研究院 | Quality detection method of total flavonoid extract of kohlrabi fruits |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107917886A (en) * | 2016-10-10 | 2018-04-17 | 武汉光谷人福生物医药有限公司 | The method for measuring total sugar content in Desmodium styracifolium general flavone |
| CN107525862B (en) * | 2017-08-03 | 2020-03-17 | 广西壮族自治区中医药研究院 | Quality detection method of total flavonoid extract of kohlrabi fruits |
| CN107669745A (en) * | 2017-08-21 | 2018-02-09 | 广西澳树宝生物科技有限公司 | A kind of continuous method for preparing tea oil and cajeputtree flavones |
| CN107890437A (en) * | 2017-12-05 | 2018-04-10 | 深圳市润昇源生物科技有限公司 | Facial mask and preparation method thereof is repaired in a kind of whitening containing mutual 100,000 layers of essential oil of leaf |
| CN110279753A (en) * | 2019-08-13 | 2019-09-27 | 天津科技大学 | A kind of tea tree ferment and its preparation method and application |
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Application publication date: 20150819 Assignee: Guangxi Houde Pharmaceutical Co.,Ltd. Assignor: GUANGXI INSTITUTE OF CHINESE MEDICINE & PHARMACEUTICAL SCIENCE Contract record no.: X2022450000405 Denomination of invention: Preparation and determination of total flavonoids from Melaleuca alternifolia Granted publication date: 20180518 License type: Common License Record date: 20221227 |