CN103623038A - Preparation method and purpose of total phenolic acid in pithecellobium clypearia - Google Patents
Preparation method and purpose of total phenolic acid in pithecellobium clypearia Download PDFInfo
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- CN103623038A CN103623038A CN201310666211.4A CN201310666211A CN103623038A CN 103623038 A CN103623038 A CN 103623038A CN 201310666211 A CN201310666211 A CN 201310666211A CN 103623038 A CN103623038 A CN 103623038A
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Abstract
The invention provides a preparation method of total phenolic acid in a plant pithecellobium clypearia. The preparation method comprises the steps of crushing a pithecellobium clypearia raw material, extracting with pure water, condensing to form concrete, suspending with water, then putting on macroporous resin, eluting with acid water, removing water-soluble impurities, allowing phenolic acid substances to be maintained on the macroporous resin, eluting with a water-alcohol system to form a total phenolic acid extract which is also an effective part with pharmacological activity in the pithecellobium clypearia. The total phenolic acid in the pithecellobium clypearia has obvious antineoplastic activity, the pithecellobium clypearia, as traditional Chinese medicine, has lower toxic and side effects, and a new anti-tumor drug can be developed. The total phenolic acid in the pithecellobium clypearia can be prepared into various oral preparations. The method is reasonable in design and has the characteristics of simple technical process, low cost, low energy consumption and low pollution in a production process.
Description
Technical field
The present invention relates to plant medicine technical field, relate in particular to the preparation method of total phenolic acid (extract) in plant Ramulus Et Folium Pithecellobii Lucidi and the new application in preparing antitumor drug.
Background technology
Malignant tumor is the principal disease of harm humans health and lives.The whole world approximately has 5,000,000 people to die from malignant tumor every year according to estimates, is the second largest lethal cause of disease after cardiovascular disease.According to foreign data introduction, hundreds of kind PTS has been developed in the whole world, wherein much belongs to natural plant extracts.Nearly 130～150 kinds of the cancer therapy drug of current global various countries approved listing.Nearly 1300～1500 kinds of the various anti-cancer drug preparations that are mixed with these medicines.In addition the whole world studying among but not yet get the Green Light listing PTS approximately have kind more than 800, wherein belong in the treatment of approximately 400 kinds of cancers of micromolecular chemical anticarcinogenic drug, Drug therapy is a very important link, the effectively use of cancer therapy drug, can help patient to obtain longer life span, have the hope surviving, the most common cancer therapy drug has chemotherapeutics, Chinese medicine, bio-pharmaceuticals, targeted drug etc. at present.In numerous antineoplastic agents, the proportion maximum that natural plant kind antineoplastic agent is shared, has occupied 27.0% share, is secondly anti-metabolism tumor medicine, accounts for 26.1%.In the antineoplastic agent of first 10 of single product rank, plant antineoplastic agent has occupied two seats, is paclitaxel and docetaxel, has occupied respectively front two.Therefore, from plant, develop the antitumor drug making new advances, also become medicine scholars' research emphasis.
Ramulus Et Folium Pithecellobii Lucidi Pithecellobium clypearia (Jack) Benth. is Mimosaceae Ramulus Et Folium Pithecellobii Lucidi platymiscium, has another name called bib tree, flood dragon wood etc., is distributed widely in south China each province, has the effect of anti-inflammation, blood circulation promoting and dispersing pathogen accumulation.The single preparation of being made by the water extracted immersing paste of Ramulus Et Folium Pithecellobii Lucidi leaf and twig---Ramulus Et Folium Pithecellobii Lucidi antiimflammatory tablet (capsule) is used widely as antiinflammatory and treatment respiratory tract infection medicine, in 2005, be incorporated into Chinese Pharmacopoeia, there is clinically heat-clearing and toxic substances removing, removing heat from blood detumescence, the effect of antidiarrheal.For upper respiratory tract infection, acute pharyngolaryngitis, acute tonsillitis, acute gastroenteritis.Research before thinks, the active component of Ramulus Et Folium Pithecellobii Lucidi is mainly flavone and Flavane compound wherein.The patent of Ramulus Et Folium Pithecellobii Lucidi (publication number CN1850146A) has reported that Ramulus Et Folium Pithecellobii Lucidi extract has antiviral activity.Meanwhile, overwhelming majority research all concentrates on antiinflammatory, the anti-virus aspect of Ramulus Et Folium Pithecellobii Lucidi, also there is no the report of anti-tumor activity.
Summary of the invention
One of object of the present invention is to provide the preparation method of total phenolic acid of a kind of plant Ramulus Et Folium Pithecellobii Lucidi, the method is to be broken into coarse powder by getting Ramulus Et Folium Pithecellobii Lucidi raw material powder, with Aqua pure extract, normal pressure is concentrated into 25 ℃ and surveys the extractum that relative density is 1.25-1.30, and extractum is directly gone up macroporous resin and obtained total phenolic acid with water alcohol system eluting.The method adopts macroporous resin chromatography one step can obtain effective site, specifically by following steps, realizes:
1. get the Aqua pure extract 2～3 times that Ramulus Et Folium Pithecellobii Lucidi raw material doubly measures with 8-10, extract and adopt heating extraction or supersound extraction;
2. after extracting solution being merged, normal heating is concentrated into 25 ℃ and surveys the extractum that relative density is 1.25-1.30.
3. for gained extractum, after the water suspendible of 3 times of volumes, go up macroporous resin, after first using 3-5 column volume of sour water (PH:3-4) eluting, use again 40%-60% ethanol elution, collect ethanol elution, decompression recycling ethanol, spraying is dry obtains dried powder, and total yield is approximately more than 8%.The macroporous resin adopting is DM301, NKA-9, AB-8, D101, DM130, DM-18, D312 type low pole or Semi-polarity macroporous resin.
The dried powder of preparation is adopted to visible spectrophotometry, is reference substance with gallic acid (self-control, purity 97%), 0.6%FeCl
3-0.9%K
3[Fe (CN)
6] (1:0.9) mixed solution be developer, detect wavelength 720nm.Measure its total phenolic content.
Two of object of the present invention is to provide the new purposes of total phenolic acid in preparing antitumor drug in Ramulus Et Folium Pithecellobii Lucidi.The excipient preparation that in described plant Ramulus Et Folium Pithecellobii Lucidi, total phenolic acid adds pharmaceutics to allow is prepared into pharmaceutical preparation, and dosage form is various oral formulations.Described oral formulations comprises tablet, capsule, pill or granule.
The present invention has taked the process route different from former studies, directly uses water extraction, concentrated rear macroporous resin eluting.The tracking that the fraction that macroporous resin is eluted has carried out under anti-tumor activity guidance is separated, has screened and the new effective site with anti-tumor activity, and this effective site mainly contains phenolic acid and glucosides thereof, and the compound structure that wherein content is higher is as follows:
Ramulus Et Folium Pithecellobii Lucidi total phenolic acid extract has good in vitro and in vivo anti-tumor activity, and its toxic and side effects of chemical anti-tumor drugs is less relatively.The present invention is simultaneously also that Chinese medicine has been carried out to useful secondary development, for the development and utilization of Ramulus Et Folium Pithecellobii Lucidi provides more broad market prospect.
Usefulness of the present invention is: (1) has adopted in the past different extraction processes, the new total phenolic acid of effective site that utilized macroporous resin enrichment, and utilize the total phenolic content of spectrophotometry, its content reaches more than 80%, and be convenient to make various preparations, yield reaches more than 8%.(2) effective site obtaining by this method, has significant pharmacological action, can suppress the propagation of OPM, SGC-7901, SMMC-7721, RPMI-8226 and S180 tumor cell.(3) the total phenolic acid obtaining can be made various oral formulations, and administration is simple, and drug utilization degree is high.(4) the inventive method is reasonable in design, has that technique is simple, cost is low, energy consumption is low and production process is polluted low feature.
The specific embodiment
The present invention is further described in conjunction with specific embodiments.
The preparation of the total phenolic acid of embodiment 1 Ramulus Et Folium Pithecellobii Lucidi
Get about Ramulus Et Folium Pithecellobii Lucidi 200g, add 2000ml water, 80 ℃ of heating extraction 2 hours, filtered filtration residue adds 80 ℃ of heating extraction of 1000ml water 1 hour again, merges filtrate twice, and 110 ℃ of normal pressures are concentrated into 25 ℃ and survey the extractum that relative densities are 1.25-1.30.By upper D101 macroporous resin after 200ml water suspendible for this extractum, first use that with 40% alcohol-water, to be eluted to effluent again after 3 volumes of sour water (PH=3) eluting closely colourless, collect this part eluent, decompression recycling ethanol, the dry dried powder that obtains of spraying, obtain the total phenolic acid of 22.1g, total yield is about 11.1%.Utilize spectrophotometry, its total phenolic content is 83%.
The preparation of the total phenolic acid of embodiment 2 Ramulus Et Folium Pithecellobii Lucidi
Get about Ramulus Et Folium Pithecellobii Lucidi 200g, add 1600ml pure water, 70 ℃ of heating extraction 2 hours, filtered filtration residue adds 70 ℃ of heating extraction of 1000ml pure water 1 hour again, merges filtrate twice, and 110 ℃ of normal pressures are concentrated into 25 ℃ and survey the extractum that relative densities are 1.25-1.30.By upper D312 macroporous resin after 200ml water suspendible for this extractum, first use that with 50% alcohol-water, to be eluted to effluent again after 3 volumes of sour water (PH=4) eluting closely colourless, collect this part eluent, decompression recycling ethanol, the dry dried powder that obtains of spraying, obtain the total phenolic acid of 20.3g, total yield is about 10.2%.Utilize spectrophotometry, its phenolic content is 80%.
The preparation of the total phenolic acid of embodiment 3 Ramulus Et Folium Pithecellobii Lucidi
Get about Ramulus Et Folium Pithecellobii Lucidi 1000g, add 8000ml pure water, 90 ℃ of heating extraction 2 hours, filtered filtration residue adds 90 ℃ of heating extraction of 3000ml pure water 1 hour again, merges filtrate twice, and 105 ℃ of normal pressures are concentrated into 25 ℃ and survey the extractum that relative densities are 1.25-1.30.By upper AB-8 macroporous resin after 1000ml water suspendible for this extractum, first use that with 40% alcohol-water, to be eluted to effluent again after 3 volumes of sour water (PH=3) eluting closely colourless, collect this part eluent, decompression recycling ethanol, the dry dried powder that obtains of spraying, obtain the total phenolic acid of 98g, total yield is about 9.8%.Utilize spectrophotometry, its phenolic content is 85%.
The preparation of the total phenolic acid of embodiment 4 Ramulus Et Folium Pithecellobii Lucidi
Get about Ramulus Et Folium Pithecellobii Lucidi 1000g, add 8000ml80% pure water, 80 ℃ of heating extraction 2 hours, filtered filtration residue adds 80 ℃ of heating extraction of 3000ml pure water 1 hour again, merges filtrate twice, and 110 ℃ of normal pressures are concentrated into 25 ℃ and survey the extractum that relative densities are 1.25-1.30.By upper DM301 macroporous resin after 1000ml water suspendible for this extractum, after first using after 3 volumes of sour water (PH=4) eluting, with 50% alcohol-water, be eluted to effluent more closely colourless, collect this part eluent, decompression recycling ethanol, the dry dried powder that obtains of spraying, obtain the total phenolic acid of 45g, total yield is about 10.5%.Utilize spectrophotometry, its phenolic content is 81%.
Embodiment 5 tablet preparations
Get the total phenolic acid 50g of Ramulus Et Folium Pithecellobii Lucidi that above-mentioned 1～4 arbitrary embodiment method obtains, after pulverizing, cross 80 mesh sieves, 100g mixes with starch, adds 10% starch slurry 10g and makes soft material and add magnesium stearate 1g, after dried starch 10g mixes, makes 1000, both.Every containing the total phenolic acid 50mg of Ramulus Et Folium Pithecellobii Lucidi.
Embodiment 6 capsule preparations
Get the total phenolic acid 50g of Ramulus Et Folium Pithecellobii Lucidi that above-mentioned 1～4 arbitrary embodiment method obtains, after pulverizing, cross 80 mesh sieves, 100g mixes with starch, add 10% starch slurry 10g and make soft material, after granulating with 14 mesh sieves, put 60 ℃～70 ℃ dry after in 12 mesh sieve granulate, be inserted in No. 1 Capsules.Fill altogether 1000, every capsules is containing the total phenolic acid 50mg of Ramulus Et Folium Pithecellobii Lucidi.
Embodiment 7 pill preparations
Get the total phenolic acid 50g of Ramulus Et Folium Pithecellobii Lucidi that above-mentioned 1～4 arbitrary embodiment method obtains, after pulverizing, cross 80 mesh sieves, proper honey is made ball as adhesive.
Embodiment 8 granule preparations
Get the total phenolic acid 50g of Ramulus Et Folium Pithecellobii Lucidi that above-mentioned 1～4 arbitrary embodiment method obtains, after pulverizing, cross 80 mesh sieves, add 10% starch slurry and make soft material, after granulating with 14 mesh sieves, put 60 ℃～70 ℃ dry after in 12 mesh sieve granulate, subpackage, sealing, packing both obtains.
Embodiment 9
The total phenolic acid of Ramulus Et Folium Pithecellobii Lucidi obtaining with embodiment 1, has obvious inhibitory action to the propagation of human tumor cells human myeloma OPM, gastric cancer SGC-7901, hepatocarcinoma SMMC-7721 and people's bone RPMI-8226.
Adopt improvement mtt assay: OPM, the SGC-7901, SMMC-7721 and the digestion of RPMI-8226 tumor cell that are fused to monolayer, for single-cell suspension liquid, are added in 96 well culture plates to 90 μ L/ holes.At 37 ℃, 5%CO
2in incubator, hatch after 24h, add certain density given the test agent, 10 μ L/ holes.Given the test agent group arranges 3 multiple holes.Negative control is isopyknic PBS, and positive control is cisplatin (DDP), continues to hatch after 48 hours, and every hole adds the MTT solution 5mg/ml of 10 μ l, 10 μ L/ holes, and 37 ℃ of lucifuges are hatched 2-4h; Every hole adds the MTT of 100 μ l to dissolve buffer(20%SDS, 1M HCl), hatch to crystallization for 37 ℃ and dissolve completely; Utilize full-automatic microplate reader to read the absorbance at 570nm and 490nm place, calculate OD(570)-OD(490) numerical value as the OD value in every hole; According to following formula, calculate relative inhibition: the average OD value of cell relative inhibition (%)=100%-(experimental group OD value/matched group) * 100%.IC with each sample of SPSS12.0 computed in software
50value, the results are shown in Table 1.
Table 1 is the inhibitory action of extract of the present invention to human tumor cells propagation
Table 1
In embodiment 10 extract of the present invention, suppress sarcoma S180 transplanted tumor in nude mice proliferation test
Adopt Nude Mouse Model: (6 weeks large, 18-20g) after weighing, with containing 5 * 10 to get 50 BALB/c nude mices
9the single-cell suspension liquid of individual/L SGC-7901 cell is at nude mice right side armpit subcutaneous injection, and 0.1ml/ only.Treat that transplantation tumor volume growth is to 0.5cm
3time, nude mice is divided into 5 groups at random, 10 every group, be respectively the total phenolic acid of the present invention little, in, heavy dose of group (100,500,1000mg/kg), negative control group sterile saline, positive control is cyclophosphamide group (30mg/kg).Set time every day gastric infusion, be 2 weeks experimental period, drug withdrawal next day, weighs, and strips tumor and weigh, and by following method, calculates tumour inhibiting rate, the results are shown in Table 2.
Tumour inhibiting rate=(1-experimental group tumor weight average number/matched group tumor weight average number) * 100%
Table 2 is total phenolic acid inhibitory action to sarcoma S180 transplanted tumor in nude mice propagation
Table 2
Claims (10)
1. the preparation method of total phenolic acid in a kind of plant Ramulus Et Folium Pithecellobii Lucidi, is characterized in that: by following steps, realize:
(1) get Ramulus Et Folium Pithecellobii Lucidi raw material water extraction;
(2) extracting solution is merged to the rear concentrated extractum that to obtain;
(3) upper macroporous resin eluting after gained extractum water suspendible, first uses sour water eluting, then is 40%-60% ethanol elution by volumetric concentration, collects ethanol elution, decompression recycling ethanol, the dry dried powder that obtains of spraying.
2. the preparation method of total phenolic acid in a kind of plant Ramulus Et Folium Pithecellobii Lucidi according to claim 1, is characterized in that, the 8-10 that the water in step (1) is raw material doubly measures, and extracts and adopts heating or supersound extraction, extracts 2-3 time.
3. the preparation method of total phenolic acid in a kind of plant Ramulus Et Folium Pithecellobii Lucidi according to claim 2, is characterized in that: the described heating extraction temperature of step (1) is 70 ℃-90 ℃.
4. the preparation method of total phenolic acid in a kind of plant Ramulus Et Folium Pithecellobii Lucidi according to claim 1, is characterized in that: in step (2), adopt normal heating concentrated, temperature is 105 ℃-115 ℃.
5. the preparation method of total phenolic acid in a kind of plant Ramulus Et Folium Pithecellobii Lucidi according to claim 1, is characterized in that: the described macroporous resin of step (3) is DM301, NKA-9 type, AB-8, D101, DM130, DM-18, D312 type low pole or Semi-polarity macroporous resin.
6. the preparation method of total phenolic acid in a kind of plant Ramulus Et Folium Pithecellobii Lucidi according to claim 1, is characterized in that: the described sour water pH value of step (3) is 3-4.
7. the clinical acceptable pharmaceutical preparation that in any one plant Ramulus Et Folium Pithecellobii Lucidi of preparing of claim 1-6 prepared by total phenolic acid and pharmaceutically acceptable carrier.
8. pharmaceutical preparation according to claim 7, is characterized in that, described pharmaceutical preparation is selected from tablet, capsule or granule.
9. total phenolic acid or the application of pharmaceutical preparation claimed in claim 7 in preparing antitumor drug in any one plant Ramulus Et Folium Pithecellobii Lucidi preparing of claim 1-6.
10. application according to claim 9, is characterized in that, described tumor is human myeloma, sarcoma, gastric cancer or hepatocarcinoma.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108159108A (en) * | 2018-02-26 | 2018-06-15 | 辽宁昱众合医药科技有限公司 | The preparation of pithecellobium clypearia total phenolics and its application in antimicrobial antiphlogistic drug |
| CN115919921A (en) * | 2022-08-04 | 2023-04-07 | 广州白云山花城药业有限公司 | Application of pithecellobium clypearia extract in preparation of drugs for preventing coronavirus proliferation |
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| CN1765894A (en) * | 2004-10-29 | 2006-05-03 | 广州莱泰制药有限公司 | Novel flavane derivative and its preparation method and uses |
| CN101596229A (en) * | 2009-04-15 | 2009-12-09 | 广州莱泰制药有限公司 | The method of quality control of Ramulus Et Folium Pithecellobii Lucidi extract and preparation thereof |
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| CN1765894A (en) * | 2004-10-29 | 2006-05-03 | 广州莱泰制药有限公司 | Novel flavane derivative and its preparation method and uses |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108159108A (en) * | 2018-02-26 | 2018-06-15 | 辽宁昱众合医药科技有限公司 | The preparation of pithecellobium clypearia total phenolics and its application in antimicrobial antiphlogistic drug |
| CN108159108B (en) * | 2018-02-26 | 2021-08-10 | 辽宁昱众合医药科技有限公司 | Preparation of pithecellobium clypearia total phenolic acid and application of pithecellobium clypearia total phenolic acid in antibacterial and anti-inflammatory drugs |
| CN115919921A (en) * | 2022-08-04 | 2023-04-07 | 广州白云山花城药业有限公司 | Application of pithecellobium clypearia extract in preparation of drugs for preventing coronavirus proliferation |
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Application publication date: 20140312 |