CN103223018A - Osmanthus fragrans polyphenol extract product, and preparation method and uses thereof - Google Patents
Osmanthus fragrans polyphenol extract product, and preparation method and uses thereof Download PDFInfo
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Abstract
The invention relates to a plant polyphenol extract product extracted from Osmanthus fragrans, and a preparation method and uses thereof, and concretely discloses an Osmanthus fragrans polyphenol extract product preparation method. The method comprises the following steps: 1, mixing Osmanthus fragrans with an aqueous solution of an extracting agent according to a solid/liquid ratio of 1g/15-60ml, carrying out microwave extraction of the obtained mixture under a pressure of 200-500KPa and a power of 80-200W for 3-20min, and repeating the above extraction process; and 2, mixing obtained filtrates to obtain a filtrate mixture, and drying the filtrate mixture to obtain an Osmanthus fragrans polyphenol extract product. The Osmanthus fragrans polyphenol extract product can be used for preparing antitumor medicines or healthcare products, and can also be used for inhibiting the activities of 5alpha-reductase.
Description
Technical field
The present invention relates to agricultural byproducts deep processing and comprehensive utilization field.Relate more specifically to a kind of plant polyphenol kind extract that from Flos Osmanthi Fragrantis, extracts and method for making and purposes.
Background technology
Flos Osmanthi Fragrantis Osmanthus fragrans Lour. has another name called sweet-scented osmanthus, Folium Et Cacumen Murrayae, Jin Su, is one of China's ten great tradition famous flowers, is famous spice berry, also is good afforestation seeds, has good ecological benefits, social benefit and economic benefit concurrently.Flos Osmanthi Fragrantis is long at China's cultivation history, in Sichuan, provinces and regions such as Yunnan, Zhejiang, Guangxi, Hubei all have wild; Basin of Huaihe River to various places on the south the downstream, the Yellow River plant at large, to the north of then with potted plant in the majority.The whole world has 40 kinds of osmanthus cultivars approximately, and China has 27 kinds, is the main distribution center of Flos Osmanthi Fragrantis, and germ plasm resource is extremely abundant.
Flos Osmanthi Fragrantis is traditional fragrant flower plant, though flower is little, lasting fragrance is stable.China has long edible Flos Osmanthi Fragrantis tradition, and before 2000, just with Flos Osmanthi Fragrantis system wine, system scented tea, in recent years along with the continuous expansion of Flos Osmanthi Fragrantis cultivated area and developing rapidly of food industry, products such as Flos Osmanthi Fragrantis cake, Flos Osmanthi Fragrantis sauce also obtain large-scale production.
Flos Osmanthi Fragrantis contains rich nutrient substances.Total amino acids content is up to 13.78% in the Flos Osmanthi Fragrantis, and wherein essential amino acid is 6.26%, accounts for 45.55% of total amino acid content, and amino acid whose reasonable mixture ratio, is a kind of high quality protein.Flos Osmanthi Fragrantis contains abundant vitamin, carotene and various trace element, potassium, ferrum, zinc in addition.About the research of Flos Osmanthi Fragrantis secondary metabolite, mainly concentrate on its quintessence oil and extract and the fragrance ingredient analysis.(2009) such as Li-mei Wang have investigated the derived essential oil difference of different flowering phase Flos Osmanthi Fragrantis, find to contain in the osmanthus essential oil multiple kind of the effective ingredient surplus linalool and oxide thereof, α-Zi Luolantong, alpha, beta-lonone, the nerol etc. 20 that comprises.(2010) such as Chun-Di Hu find that further osmanthus essential oil contains up to 73 kinds of compositions.Research is less aspect the other biological active substance.(2012) such as Chien-Ya Hung have carried out isolation identification to phenols component in the Flos Osmanthi Fragrantis, find that it contains 5 kinds of phenolic compounds such as rutin, verbascoside.Flos Osmanthi Fragrantis has biological activitys such as antioxidation, antiinflammatory, and at neuroprotective, suppress to show certain effect aspect the melanin formation, but get in touch rare research between its effect and the composition.
Microwave extraction in fact mainly is that microwave is to extracting the heat effect of solvent and sample.Microwave heating has two kinds of mechanism, and promptly ionic conduction and dipole rotate, and this principle makes microwave can produce moment high temperature, so extraction time is short; Microwave when running into different material, can produce reflection, absorption and penetration phenomenon in transmission course.When microwave energy penetrated medium, promptly microwave was avoided taking place the problem of traditional heating mode from the heating of single position like this, thereby is made microwave heating have the advantage of homogeneous heating the sample interior direct heating.Simultaneously, because the dielectric property of the various piece that material is formed is often different, microwave just presents the characteristic of selecting heating to this material.The high-pressure microwave assisted extraction is to carry out in airtight extraction pot, its internal pressure can reach more than the 1MPa, therefore solvent boiling point has improved many than the solvent boiling point under the normal pressure, use the inaccessible extraction temperature of same solvent under the normal pressure thereby microwave extraction can be reached, not only can improve extraction efficiency but also be unlikely decomposition extract to be measured.
According to the international director of tumor research council (IARC) of WHO, will rise 50% to the year two thousand twenty whole world tumor incidence, to annual 1500 ten thousand.The clinical treatment method of malignant tumor has developed multiple as excision, chemotherapy, radiotherapy etc. so far, but these Therapeutic Method also can cause great infringement to normal body in the kill tumor cell, and side effect is very big.By the natural active matter assosting effect, reduce clinical medicine dose or operation strength, have great Research Significance.At the limitation of Flos Osmanthi Fragrantis effective ingredient and bioactivity research thereof, expand the research of Flos Osmanthi Fragrantis effective ingredient, and be applied to the control of tumor, have great exploitation meaning.
(5 α-reductase) are the membrane proteolytic enzymes that relies on reducibility coenzyme II (NADPH) to 5, it can be converted into dihydrotestosterone (DHT) by catalysis testosterone (T), T and DHT act on a kind of androgen receptor, but the catalytic activity of DHT is stronger, androgen receptor there is stronger affinity, so the catalyzed conversion effect of 5 can be amplified androgen response, and then may cause pseudohermaphroditism, acne, androgenetic alopecia, symptoms such as female hirsutism, simultaneously also inseparable with the generation of prostatic hyperplasia even carcinoma of prostate, therefore seek efficient 5 inhibitor and have great importance.Researcher finds that the certain plants chemistry is plain as (Matsuda H, 2001 such as ampelopsin, yellow wingceltis phenol, Flos Impatientis extracts; Shirota O, 2003; Ishiguro K, 2000.) 5 that has to a certain degree suppresses active, one of main active of the Chinese patent medicine QIANLIEKANG of domestic treatment prostatic hyperplasia commonly used also is proved to be a kind of poly ether in the Pollen Brassicae campestris, and research prompting natural plants chemistry element is the high-quality source as the 5 inhibitor.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of Flos Osmanthi Fragrantis polyphenol extract and its production and use.
In order to solve the problems of the technologies described above, the invention provides a kind of preparation method of Flos Osmanthi Fragrantis polyphenol extract, comprise step:
1), adopt the high-pressure microwave extraction method:
Flos Osmanthi Fragrantis and extractant aqueous solution are mixed the back under the pressure of 200 ~ 500KPa according to the solid-liquid ratio of 1g/15 ~ 60ml, and 80 ~ 200W power carries out microwave extraction, and extraction time is 3 ~ 20min; Filter, filter cake and filtrate first;
Substitute Flos Osmanthi Fragrantis with filter cake and repeat above-mentioned microwave extraction and filter at least once, get filtrate once more;
The volumetric concentration of extractant is 70% ~ 90% in the described extractant aqueous solution;
2), carry out dried, with the filtrate first of step 1) gained with after filtrate merges once more the Flos Osmanthi Fragrantis polyphenol extract.
Improvement as the preparation method of Flos Osmanthi Fragrantis polyphenol extract of the present invention: extractant is acetone, methanol or ethanol.
Further improvement as the preparation method of Flos Osmanthi Fragrantis polyphenol extract of the present invention: the drying step 2) is at least a in drying under reduced pressure, lyophilization, the film drying.
Further improvement as the preparation method of Flos Osmanthi Fragrantis polyphenol extract of the present invention: microwave extraction pressure is 300～450KPa in the step 1), and power is 80～120W.
Further improvement as the preparation method of Flos Osmanthi Fragrantis polyphenol extract of the present invention: the extractant aqueous solution is the aqueous acetone solution of volumetric concentration 80%, and solid-liquid ratio 1 g/45ml, pressure are 300KPa, 100W microwave extraction 3min.
The present invention also provides method for preparing simultaneously and got the Flos Osmanthi Fragrantis polyphenol extract: the total phenols weight content is 30% ~ 70% in this Flos Osmanthi Fragrantis polyphenol extract.
Improvement as Flos Osmanthi Fragrantis polyphenol extract of the present invention: in the total phenols, the weight content of phenolic acid is 0.5% ~ 2%, and the weight content of phenethyl alcohol glycoside is 50% ~ 80%.
Further improvement as Flos Osmanthi Fragrantis polyphenol extract of the present invention: in the phenolic acid, the weight content of chlorogenic acid is 40% ~ 70%, and the mass ratio of chlorogenic acid, caffeic acid and P-coumaric acid is 25 ~ 50:5 ~ 15:6 ~ 10; In the phenethyl alcohol glycoside, the weight content of verbascoside is 80% ~ 95%.
The remarks explanation: phenolic acid of the present invention comprises chlorogenic acid, caffeic acid and P-coumaric acid, and described phenethyl alcohol glycoside comprises verbascoside.
The present invention also provide method for preparing simultaneously and the purposes of Flos Osmanthi Fragrantis polyphenol extract, be used for the application of anti-tumor drug or health product in preparation.
The present invention also provide method for preparing simultaneously and the another kind of purposes of Flos Osmanthi Fragrantis polyphenol extract, be used to suppress 5 activity (that is the application in preparation 5 activity inhibitor).
Flos Osmanthi Fragrantis polyphenol extract of the present invention can be made into compositions, it is (better to contain 0.01wt%～99.9wt% in the said composition, 0.1 Flos Osmanthi Fragrantis polyphenol extract of the present invention wt %-90wt%), and contain acceptable carrier (comprising acceptable carrier in medicine, food or the cosmetics).
The inventor is through further investigation, the Flos Osmanthi Fragrantis effective ingredient has been carried out extraction separation, but having obtained a kind of polyphenol extract of effective antitumor---Flos Osmanthi Fragrantis polyphenol extract, to studies show that of this extract, this extract mainly contains compositions such as chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside.
As used herein, Flos Osmanthi Fragrantis is meant the flower of Oleaceae osmanthus Flos Osmanthi Fragrantis.The Flos Osmanthi Fragrantis that can be used as raw material of the present invention can be osmanthus flowers, Flos Osmanthi Fragrantis dry flower, osmanthus flower tea reaches with the Flos Osmanthi Fragrantis is other products of raw material or the Flos Osmanthi Fragrantis residue behind distillation extraction.Preferred feedstock is osmanthus flowers, Flos Osmanthi Fragrantis dry flower.
The raw material that the present invention adopts can carry out pretreatment, also can be without pretreatment.A kind of preferred feedstock is the Flos Osmanthi Fragrantis of powder type.For example, collect fresh Flos Osmanthi Fragrantis or add other products of Flos Osmanthi Fragrantis, be dried to moisture (weight) below 10%, be crushed to about 20～40 purpose powder.
Can be used for the osmanthus cultivars of the present invention and the place of production and be not particularly limited, can be the Flos Osmanthi Fragrantis of different cultivars, as Osmanthus fragrans var.thunbergii., silver-colored osmanthus etc., and also can be from the different places of production such as Guangxi, Zhejiang etc.The Osmanthus fragrans var.thunbergii. flower of preferred aromatic flavor.
Flos Osmanthi Fragrantis polyphenol extract of the present invention should adopt the high-pressure microwave technology to extract, though also available additive method extracts.
A kind of preferable methods be adopt the high-pressure microwave extractive technique from Flos Osmanthi Fragrantis and Related product thereof or the Flos Osmanthi Fragrantis slag extract polyphenol compound.In a preference, extractant is the 80%(volumetric concentration) acetone, extraction pressure is 200～500KPa, solid-liquid ratio 1:15～1:60(g/ml), microwave extraction 3～20min.
In more preferably example, will about 20-40 order Flos Osmanthi Fragrantis powder (moisture weight content≤10%) be incorporated in the interior cup that the polytetrafluoroethylmaterial material of intelligent microwave counteract appearance makes, extractant is selected the 80%(volumetric concentration for use) acetone, solid-liquid ratio 1:15-1:45(g/ml), controlled pressure 200～500KPa, 80～200W microwave treatment, 3～20min, centrifugal, repeat to extract more than 2 times, filter and merge supernatant (promptly, filtrate), supernatant concentrates through rotary evaporation in vacuo, gets the Flos Osmanthi Fragrantis polyphenol extract after the cold drying.
Flos Osmanthi Fragrantis polyphenol extract of the present invention is a kind of polyphenol mixture based on verbascoside.Detecting with forint phenol method, is standard substance with the chlorogenic acid, records that total phenol content is 30% ~ 70% in the Flos Osmanthi Fragrantis polyphenol extract, better 40%～60%; With phenethyl alcohol glycoside mass fraction in the determined by ultraviolet spectrophotometry total phenols is 50%～80%, better 60%～75%; Analyze the composition of phenolic acid and phenethyl alcohol glycoside with HPLC, wherein chlorogenic acid content is 40% ~ 70% in the phenolic acid, better 45% ~ 65%, chlorogenic acid, caffeic acid and P-coumaric acid mass ratio are 25 ~ 50:5 ~ 15:6 ~ 10,25 better ~ 45:5 ~ 12:7 ~ 9, verbascoside content is 80%～95% in the phenethyl alcohol glycoside, better 82%～92%.
Flos Osmanthi Fragrantis polyphenol extract of the present invention has antitumor and suppresses the biological activity of 5 vigor, therefore can be used for preparing antitumor or has the inhibiting medicine of 5, health product and food.Therefore, the present invention also provides a kind of compositions (comprising pharmaceutical composition, Halth-care composition, food compositions and cosmetic composition etc.) that contains the Flos Osmanthi Fragrantis polyphenol extract as active ingredient, and these compositionss can be used for prevention, treatment or adjuvant therapy of tumors cell proliferation.
After obtaining the Flos Osmanthi Fragrantis polyphenol extract, available conventional method with its with pharmaceutically, acceptable carrier, excipient or diluent mix mutually on the bromatology or on health product or the cosmetics, form pharmaceutical composition of the present invention, food compositions or Halth-care composition.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.
With pharmaceutical composition or Halth-care composition is example, and they can be solid-state (as granule, tablet, lyophilized powder, suppository, capsule, sublingual lozenge) or liquid (as oral liquid) or other suitable shapes.The content of Flos Osmanthi Fragrantis polyphenol extract of the present invention is generally 1%～99% of composition weight, preferably is 2%～96%, more preferably is 5%～90%, and best 15%～80%.
Halth-care composition can be single agent or multi-agent form.By application dosage, contain 10～2000mg/ agent usually, preferably about 20～1000mg/ agent, more preferably 50～500mg/ agent.
Halth-care composition of the present invention can carry out administration by conventional route, and optimal way is oral.Pharmaceutical preparation should be complementary with administering mode.The amount of application of health product of the present invention is pressed active substance and is calculated, and is generally about Flos Osmanthi Fragrantis polyphenol extract 0.1～500mg every day/kg body weight, preferably about 0.5～50mg/kg body weight.In addition, preparation of the present invention also can use with other auxiliary agents, and for example, other auxiliary agents comprise various existing anti-cancer active matters or anti-oxidation active substance.
The present invention has following advantage:
(a) provide a kind of originate wide, form distinct, best in quality, quality controllable, of many uses and have the Flos Osmanthi Fragrantis polyphenol extract of multiple physiology and pharmacologically active.
(b) realized the high efficiency extraction of polyphenol in Flos Osmanthi Fragrantis and the Related product thereof by the high-pressure microwave extractive technique, operating procedure is easy.
In sum, polyphenol is a compounds that contains the polyhydric phenols hydroxyl, and is very extensive in distributed in nature.Plant polyphenol is of a great variety, mainly comprises phenolic acid, flavone etc., studies show that multiple physiologically actives such as having the free radical of removing, antioxidation, alleviation atherosclerosis antibacterial antitumor.Contain a large amount of polyphenols in the edible flower, and Flos Osmanthi Fragrantis is wide in variety as one of common edible flower, potentiality to be exploited is big, therefore obtains polyphenol extract and expand its health care to have important exploitation meaning from Flos Osmanthi Fragrantis.
The present invention has realized the high efficiency extraction of polyphenol in Flos Osmanthi Fragrantis and the Related product thereof by the ultrasound wave auxiliary extraction technology, and operating procedure is easy, and finds that this extract has good anticancer function, has wide practical use in food, health product, medicine.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside reference substance and Flos Osmanthi Fragrantis sample HPLC separating resulting collection of illustrative plates, collection of illustrative plates shows, 1, be chlorogenic acid about 10.1min, 2, be caffeic acid about 12.8min, 3, about 19.3min be P-coumaric acid, 4, be verbascoside about 30.7min.
Fig. 2 is a Flos Osmanthi Fragrantis Flos Osmanthi Fragrantis polyphenol extract HPLC separating resulting collection of illustrative plates, and wherein B figure is 8-22min amplification effect figure among the A figure.Corresponding with the reference substance collection of illustrative plates, 1, be chlorogenic acid about 10.1min, 2, be caffeic acid about 12.8min, 3, be P-coumaric acid about 19.3min, 4, be verbascoside about 30.7min;
Fig. 3 is respectively the structure chart of chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The detection universal method is:
1), total phenol content is measured in the Flos Osmanthi Fragrantis polyphenol extract---forint phenol method
With the chlorogenic acid is contrast, adopts forint phenol method to measure total phenols.Get 500ul sample (dilution certain multiple, be the Flos Osmanthi Fragrantis polyphenol extract that contains 2mg in every ml sample) or titer (concentration is the chlorogenic acid methanol solution of 10-150 μ g/ml), add the forint phenol of 50ul 2M, mixing reaction 5-8min, the NaOH that adds 1ml 1M, water is settled to 6ml, and the 760nm wavelength is measured absorbance behind the normal-temperature reaction 90min, and calculates the chlorogenic acid equivalent concentration of total phenols.
2), Flos Osmanthi Fragrantis polyphenol extract kind phenethyl alcohol glycoside assay---ultraviolet spectrophotometry
With the verbascoside is contrast, adopts the total ethanol glycosides of determined by ultraviolet spectrophotometry.A certain amount of sample (the Flos Osmanthi Fragrantis polyphenol extract that contains 8mg in every ml sample) is used successively the water and the 20%(v/v of 25 times of sample volumes by the D-101 macroporous resin adsorption) ethanol elution, discard eluent, the back is with 50 times of volume 70% ethanol (v/v) eluting, collect eluent, evaporate to dryness, methanol constant volume.The 330nm wavelength is measured absorbance down, and calculates verbascoside equivalent concentration.
3), the analysis of effective component of Flos Osmanthi Fragrantis polyphenol extract---high performance liquid chromatography (HPLC)
By the HPLC partition method Flos Osmanthi Fragrantis polyphenol extract is carried out component separating and analysis.
HPLC separation method: adopt Phenomenex Synergi 4 μ hydro-RP 80A (250*4.6mm) chromatographic columns; Mobile phase: A (acetic acid aqueous solution, pH2.74), B (acetonitrile), 38 ℃ of column temperatures, flow velocity 0.8ml/min; Carry out UV scanning between 200～600nm, the gradient elution program is as follows:
Table 1 gradient elution program
| Time/min | A% | B% |
| 0 | 95 | 5 |
| 2 | 92 | 8 |
| 15 | 84 | 16 |
| 35 | 75 | 25 |
| 50 | 5 | 95 |
| 51 | 0 | 100 |
| 55 | 0 | 100 |
| 56 | 95 | 5 |
| 60 | 95 | 5 |
Flos Osmanthi Fragrantis powder described in following examples is meant that fresh Flos Osmanthi Fragrantis is dried to moisture (weight) below 10%, is crushed to the powder of about 20～40 order gained, and weight is 100g.
About 20-40 order Flos Osmanthi Fragrantis powder is added in the interior cup that the polytetrafluoroethylmaterial material of intelligent microwave counteract appearance makes, extractant is selected 80% acetone (being that volumetric concentration is 80% aqueous acetone solution) for use, solid-liquid ratio 1:15(g/ml), controlled pressure 200KPa, 80W microwave treatment 3min, filter, repeat to extract 2 times again (promptly, substitute the Flos Osmanthi Fragrantis powder with filtering residue, repeat above-mentioned microwave treatment successively and filter 2 times), merges the filtrate of 3 extraction gained, 45 ℃ of rotary evaporation in vacuo of filtrate concentrate, concentrated solution to constant weight, gets the Flos Osmanthi Fragrantis polyphenol extract through the lyophilization of-80 ℃ of pre-freeze final vacuums, and yield is 15.1%.
Extract is carried out composition measurement and analysis, and the result as shown in Figure 1, 2.Fig. 1,2 shows that this extract contains chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside.Colorimetry and HPLC method are analyzed the component content of Flos Osmanthi Fragrantis polyphenol extract, and its total phenol content is 30.5 ± 1.3%.In the total phenols, chlorogenic acid content is 0.25 ± 0.01%, and chlorogenic acid, caffeic acid and P-coumaric acid mass ratio are 30:5:6; Phenethyl alcohol glycoside content is 65.2 ± 3.2%, and verbascoside content is 87.6 ± 1.1% in the phenethyl alcohol glycoside.
About 20-40 order Flos Osmanthi Fragrantis powder is added in the interior cup that the polytetrafluoroethylmaterial material of intelligent microwave counteract appearance makes, extractant is selected 80% acetone for use, solid-liquid ratio 1:25(g/ml), controlled pressure 200KPa, 80W microwave treatment 5min, filter, repeat to extract 2 times again (promptly, substitute the Flos Osmanthi Fragrantis powder with filtering residue, repeat above-mentioned microwave treatment successively and filter 2 times), merges the filtrate of 3 extraction gained, 45 ℃ of rotary evaporation in vacuo of filtrate concentrate, concentrated solution to constant weight, gets the Flos Osmanthi Fragrantis polyphenol extract through the lyophilization of-80 ℃ of pre-freeze final vacuums, and yield is 23.5%.
Extract is carried out composition measurement and analysis, and this extract contains chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside.Colorimetry and HPLC method are analyzed the component content of Flos Osmanthi Fragrantis polyphenol extract, and its total phenol content is 59.7 ± 1.1%.In the total phenols, chlorogenic acid content is 0.4 ± 0.015%, and chlorogenic acid, caffeic acid and P-coumaric acid mass ratio are 35:15:6; Phenethyl alcohol glycoside content is 71.5 ± 1.9%, and verbascoside content is 85.7 ± 2.3% in the phenethyl alcohol glycoside.
In the interior cup that the polytetrafluoroethylmaterial material that about 20-40 order Flos Osmanthi Fragrantis powder is incorporated in the intelligent microwave counteract appearance is made, extractant is selected 80% acetone for use, solid-liquid ratio 1:35(g/ml), controlled pressure 300KPa, 80W microwave treatment 8min, filter, repeat to extract 2 times again (promptly, substitute the Flos Osmanthi Fragrantis powder with filtering residue, repeat above-mentioned microwave treatment successively and filter 2 times), merges the filtrate of 3 extraction gained, 45 ℃ of rotary evaporation in vacuo of filtrate concentrate, concentrated solution to constant weight, gets the Flos Osmanthi Fragrantis polyphenol extract through the lyophilization of-80 ℃ of pre-freeze final vacuums, and yield is 25.1%.
Extract is carried out composition measurement and analysis, and this extract contains chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside.Colorimetry and HPLC method are analyzed the component content of Flos Osmanthi Fragrantis polyphenol extract, and its total phenol content is 60.8 ± 2.1%.In the total phenols, chlorogenic acid content is 0.4 ± 0.01%, and chlorogenic acid, caffeic acid and P-coumaric acid mass ratio are 40:15:7; Phenethyl alcohol glycoside content is 61.6 ± 0.9%, and verbascoside content is 87.1 ± 2.5% in the phenethyl alcohol glycoside.
In the interior cup that the polytetrafluoroethylmaterial material that about 20-40 order Flos Osmanthi Fragrantis powder is incorporated in the intelligent microwave counteract appearance is made, extractant is selected 80% acetone for use, solid-liquid ratio 1:40(g/ml), controlled pressure 400KPa, 100W microwave treatment 8min, filter, repeat to extract 2 times again (promptly, substitute the Flos Osmanthi Fragrantis powder with filtering residue, repeat above-mentioned microwave treatment successively and filter 2 times), merges the filtrate of 3 extraction gained, 45 ℃ of rotary evaporation in vacuo of filtrate concentrate, concentrated solution to constant weight, gets the Flos Osmanthi Fragrantis polyphenol extract through the lyophilization of-80 ℃ of pre-freeze final vacuums, and yield is 28.0%.
Extract is carried out composition measurement and analysis, and this extract contains chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside.Colorimetry and HPLC method are analyzed the component content of Flos Osmanthi Fragrantis polyphenol extract, and its total phenol content is 51.5 ± 1.3%.In the total phenols, chlorogenic acid content is 0.8 ± 0.01%, and chlorogenic acid, caffeic acid and P-coumaric acid mass ratio are 45:12:6; Phenethyl alcohol glycoside content is 68.8 ± 1.7%, and verbascoside content is 88.5 ± 2.1% in the phenethyl alcohol glycoside.
Embodiment 5
In the interior cup that the polytetrafluoroethylmaterial material that about 20-40 order Flos Osmanthi Fragrantis powder is incorporated in the intelligent microwave counteract appearance is made, extractant is selected 80% acetone for use, solid-liquid ratio 1:45(g/ml), controlled pressure 300KPa, 100W microwave treatment 3min, filter, repeat to extract 2 times again (promptly, substitute the Flos Osmanthi Fragrantis powder with filtering residue, repeat above-mentioned microwave treatment successively and filter 2 times), merges the filtrate of 3 extraction gained, 45 ℃ of rotary evaporation in vacuo of filtrate concentrate, concentrated solution to constant weight, gets the Flos Osmanthi Fragrantis polyphenol extract through the lyophilization of-80 ℃ of pre-freeze final vacuums, and yield is 30.4%.
Extract is carried out composition measurement and analysis, and this extract contains chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside.Colorimetry and HPLC method are analyzed the component content of Flos Osmanthi Fragrantis polyphenol extract, and its total phenol content is 63.6 ± 3.2%.In the total phenols, chlorogenic acid content is 0.25 ± 0.01%, and chlorogenic acid, caffeic acid and P-coumaric acid mass ratio are 50:15:10; Phenethyl alcohol glycoside content is 60.1 ± 1.5%, and verbascoside content is 89.3 ± 1.3% in the phenethyl alcohol glycoside.
These embodiment 5 gained total phenol contents are the highest, reach 19.3% of Flos Osmanthi Fragrantis dry flower.
In order fully to prove technical advantage of the present invention place, the inventor has also carried out following Comparative Examples:
Ratio 1-1 ~ 1-Comparative Examples 1-4, only make extractant into 60% acetone, 100% acetone, 80% methanol, 80% ethanol respectively by 80% acetone; All the other all are equal to embodiment 5.
Extract is carried out composition measurement and analysis, and this extract contains chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside.Concrete content is as described in Table 2.
Table 2
Ratio 2-1, make controlled pressure 300KPa, 100W microwave treatment 3min into " controlled pressure 200KPa, 100W microwave treatment 3min ", all the other all are equal to embodiment 5.
Ratio 2-2, make controlled pressure 300KPa, 100W microwave treatment 3min into " controlled pressure 500KPa, 100W microwave treatment 3min ", all the other all are equal to embodiment 5.
Ratio 2-3, make controlled pressure 300KPa, 100W microwave treatment 3min into " controlled pressure 300KPa, 200W microwave treatment 3min ", all the other all are equal to embodiment 5.
Ratio 2-4, make controlled pressure 300KPa, 100W microwave treatment 3min into " controlled pressure 300KPa, 80W microwave treatment 20min ", all the other all are equal to embodiment 5.
Comparative Examples 2-5, with solid-liquid ratio by 1:45(g/ml) make 1:20(g/ml into), all the other all are equal to embodiment 5.
Comparative Examples 2-6, with solid-liquid ratio by 1:45(g/ml) make 1:60(g/ml into), all the other all are equal to embodiment 5.
The extract of Comparative Examples 2-1 ~ Comparative Examples 2-6 gained is carried out composition measurement and analysis, all contain chlorogenic acid, caffeic acid, P-coumaric acid and verbascoside in this extract.Concrete content is as described in Table 3.
Table 3
1. cell strain and condition of culture
Human breast cancer cell (MCF-7, MDA), Human Prostate Cancer Cells (PC-3, LN-CaP), culture medium is RPMI-1640, contains 10% inactivated fetal bovine serum, 37 ℃ of cultivations in the 5% carbon dioxide incubator.
2. the MTT colorimetry detects cell proliferation rate
Collect the logarithmic (log) phase cell, adjusting concentration of cell suspension is 50000/ml, and every hole adds 100ul cell suspension, and cell is put into incubator and cultivated, and treats administration in adherent back second day, and cell is put into incubator and cultivated 24h.After drug effect finished, every hole added 20ulMTT (5mg/ml), cultivated 3-4h, stopped cultivating, and careful the suction removed culture fluid in the hole.Every hole adds 150ulDMSO, 37 ℃ of incubations 10 minutes or shaking table low-speed oscillation 10 minutes.The back is abscissa with absorbance (A) the value record result in each hole of microplate reader detection 490nm with time, and light absorption value is that vertical coordinate is drawn cell growth curve.Be calculated as follows the suppression ratio (IR) of medicine on cell proliferation.IR=[1-(the average A value that average A value/control wells of measuring of medication group is measured)] * 100%, and carry out regression analysis by SPSSl7.0 software, calculate IC
50Value (semilethal inhibition concentration) is estimated the Flos Osmanthi Fragrantis polyphenol extract.
Flos Osmanthi Fragrantis polyphenol extract in table 4, each embodiment and the Comparative Examples is to the IC of each tumor cell
50Value
| IC 50（ug/ml） | MCF-7 | MDA | PC-3 | LN- |
| Embodiment | ||||
| 1 | 690.9 | 503.7 | 250.4 | 318.1 |
| |
630.5 | 490.2 | 189.8 | 220.2 |
| |
604.5 | 450.3 | 140.2 | 193.9 |
| |
653.2 | 499.8 | 177.6 | 211.1 |
| Embodiment 5 | 534.4 | 399.3 | 136.6 | 140.2 |
| Comparative Examples 1-1 | 598.7 | 453.8 | 250.4 | 318.1 |
| Comparative Examples 1-2 | 624.5 | 509.2 | 169.8 | 220.2 |
| Comparative Examples 1-3 | 604.4 | 465.3 | 160.2 | 193.9 |
| Comparative Examples 1-4 | 673.2 | 498.9 | 177.6 | 211.1 |
| Comparative Examples 2-1 | 544.4 | 429.3 | 156.3 | 180.2 |
| Comparative Examples 2-2 | 590.9 | 501.1 | 250.4 | 288.1 |
| Comparative Examples 2-3 | 610.5 | 491.8 | 189.8 | 251.8 |
| Comparative Examples 2-4 | 684.5 | 480.3 | 190.2 | 310.9 |
| Comparative Examples 2-5 | 693.2 | 489.8 | 224.6 | 218.1 |
| Comparative Examples 2-6 | 552.4 | 423.5 | 146.6 | 194.2 |
1. the preparation of rat 5
Get 3 of cleaning level female sd inbred rats, fasting can't help taking off after water spends the night cervical vertebra execution, takes out on the liver ice platform to shred.Buffer with pre-cooling is made 1:5 homogenate at glass homogenizer, and under 4 ℃, centrifugal 15 min under the 10000 g centrifugal force get centrifugal 1 h under the endochylema part 100000 g centrifugal force, outwell supernatant, and precipitation is thick microgranule extract.Add the certain volume buffer, and carry out the homogenate suspension with homogenizer.Packing ,-80 ℃ of refrigerators are preserved.
2. the determination of activity of 5
A. the determination of activity system of 5: 0.5mL 20 μ M phosphate buffers (pH=6.5), 0.1 mL50% ethanol (reaction tube) or sample solution (sample cell), 150 μ L 0.5mg/mL testosterones, 250 μ L 0.8mg/mL NADPH add that 0.5mL concentration is followed successively by 0,0.96,1.92,2.88,3.84, the 4.80mg/mL enzyme extract.Reaction is after 30 minutes down at 37 ℃, and adding 2.5mL dichloromethane stops reaction, and the 100 μ g/mL propyl group hydroxybenzoates that add 0.25mL then rock 60s as interior mark, centrifugal 10min under 400g centrifugal force.
B. HPLC measures enzymatic activity: shift out about 1ml organic facies and evaporate to dryness, residue is dissolved in 1.5ml methanol, gets 10 μ L high performance liquid chromatogram sample introductions and measures the ratio of residual testosterone and interior mark peak area and be designated as the r reaction, the low explanation of r response value enzymatic activity height.Chromatographic condition: post (4.6 * 250mm, 5 μ m) luna C18(2); Column temperature: 40 ℃; Sample size: 10 μ L; Flow velocity: 1mL/min; Mobile phase: 65% methanol, detect wavelength: 242nm.
Sample is to 5 suppression ratio=(r
Sample-r
Reaction)/(r
Blank-r
Reaction) * 100%;
Annotate: r
Blank--add dichloromethane before-reaction starts and make enzyme deactivation, measure the blank pipe value of enzyme;
C. identical method is that contrast is tested with the finasteride, calculates the finasteride equivalent of Flos Osmanthi Fragrantis polyphenol extract.
Flos Osmanthi Fragrantis polyphenol extract in table 5, each embodiment and the Comparative Examples is to the 5 inhibitory action
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (10)
1. the preparation method of Flos Osmanthi Fragrantis polyphenol extract is characterized in that comprising step:
1), adopt the high-pressure microwave extraction method:
Flos Osmanthi Fragrantis and extractant aqueous solution are mixed the back under the pressure of 200 ~ 500KPa according to the solid-liquid ratio of 1g/15 ~ 60ml, and 80 ~ 200W power carries out microwave extraction, and extraction time is 3 ~ 20min; Filter, filter cake and filtrate first;
Substitute Flos Osmanthi Fragrantis with filter cake and repeat above-mentioned microwave extraction and filter at least once, get filtrate once more;
The volumetric concentration of extractant is 70% ~ 90% in the described extractant aqueous solution;
2), carry out dried, with the filtrate first of step 1) gained with after filtrate merges once more the Flos Osmanthi Fragrantis polyphenol extract.
2. the preparation method of Flos Osmanthi Fragrantis polyphenol extract according to claim 1 is characterized in that: described extractant is acetone, methanol or ethanol.
3. the preparation method of Flos Osmanthi Fragrantis polyphenol extract according to claim 2 is characterized in that: the drying described step 2) is at least a in drying under reduced pressure, lyophilization, the film drying.
4. according to the preparation method of claim 2 or 3 described Flos Osmanthi Fragrantis polyphenol extracts, it is characterized in that: microwave extraction pressure is 300～450KPa in the described step 1), and power is 80～120W.
5. the preparation method of Flos Osmanthi Fragrantis polyphenol extract according to claim 4 is characterized in that: the extractant aqueous solution is the aqueous acetone solution of volumetric concentration 80%, and solid-liquid ratio 1 g/45ml, pressure are 300KPa, 100W microwave extraction 3min.
6. get the Flos Osmanthi Fragrantis polyphenol extract as either party's method preparation in the claim 1 ~ 5, it is characterized in that: the total phenols weight content is 30% ~ 70% in this Flos Osmanthi Fragrantis polyphenol extract.
7. Flos Osmanthi Fragrantis polyphenol extract according to claim 6 is characterized in that: in the described total phenols, the weight content of phenolic acid is 0.5% ~ 2%, and the weight content of phenethyl alcohol glycoside is 50% ~ 80%.
8. Flos Osmanthi Fragrantis polyphenol extract according to claim 7 is characterized in that: in the described phenolic acid, the weight content of chlorogenic acid is 40% ~ 70%, and the mass ratio of chlorogenic acid, caffeic acid and P-coumaric acid is 25 ~ 50:5 ~ 15:6 ~ 10; In the phenethyl alcohol glycoside, the weight content of verbascoside is 80% ~ 95%.
9. get the purposes of Flos Osmanthi Fragrantis polyphenol extract as either party's method preparation in the claim 1 ~ 5, it is characterized in that: be used for the application of anti-tumor drug or health product in preparation.
10. get the purposes of Flos Osmanthi Fragrantis polyphenol extract as either party's method preparation in the claim 1 ~ 5, it is characterized in that: be used to suppress the 5 activity.
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