CN101933916B - Application of gallic acid in preparing anti-HPV medicine - Google Patents
Application of gallic acid in preparing anti-HPV medicine Download PDFInfo
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Abstract
The invention provides an application of gallic acid in preparing anti-HPV medicine.
Description
Technical field
The invention provides the application of a kind of gallic acid in the anti-HPV medicine of preparation treatment.
Background technology
The property infection that human papilloma virus (HPV) causes is very general, singly be the U.S. just have 20,000,000 people dye this disease.HPV (human papillomavirus) is a kind of incorrigible sexually transmitted disease (STD) after " century incurable disease " AIDS, no matter the men and women might infect HPV, the women catches an illness back even can cause cervical cancer.Because HPV directly infects through skin, cover safe in utilization also can't prevent.
The whole world has 400,014,000 population to receive the infection of HPV, has at least every year 500000 women by diagnosis cervical cancer to be arranged, and these are most of, and countries under development take place.HPV causes cervical cancer and genital wart, is commonly called as the arch-criminal of " cauliflower ", and in the middle of the HPV of kind more than 100 type, having known has 37 kinds to lean on sexual transmission, can cause sexually transmitted disease (STD).They extensively are present in adult group, approach without casting a shadow and leave without leaving a trace, and have no symptom.The the 6th and the 11st type can cause the amphoteric genital wart of men and women.This wart itself is not a cancer, but no words can adequately describe for pain that it causes and psychic trauma.
But, make a circulation to have infected high risk HPV type and just can cause cervical cancer, other anogenital cancer or anus cancer.Nearly 19 kinds more than of these high risk types, wherein common with the 16th and the 18th type that causes cervical cancer.
Cervical cancer is an infectious disease, and it is to prevent, and can treat, cures and eliminate.Cervical cancer is owing to human papilloma virus infection causes.If can prevent HPV to infect, just we can say and to prevent cervical cancer; If do not have HPV to infect, not we can say to get cervical cancer.This is existing final conclusion, and this at the meeting gains public acceptance.Here the cervical cancer of saying just is meant Cervical infiltrating carcinoma, is called for short CC.In general, cervical cancer is exactly an infiltrating carcinoma; Other precancerous lesion just is CIN, comprises the cancer in situ of cervix uteri, returns it in the CIN3 the inside.If infiltrating carcinoma, with regard to the whistle cervical cancer.
Gallic acid, the IUPAC name: 3,4,5-trihydroxybenzoic acid, gallic acid, English name Gallic acid, another name 3,4,5-Trihydroxy benzoic acid; Gallic acid; 3,4,5-trihydroxy-Benzoic acid; 3,4,5-trihydroxybenzoic acid; 3,4,5-trihydroxy-benzoic acid, molecular formula C7H605; Molecular weight 170.12; Physical property: acicular crystal (absolute methanol or chloroform), 235～240 ° of fusing points (decomposition), its skeleton symbol is following:
Belong to organic acid and phenols; Be used for 1. anti-bacteria and anti-virus: external have inhibitory action to staphylococcus aureus, sarcina, α-type streptococcus, Neisseria gonorrhoeae, bacillus pyocyaneus, bacterium flexneri, Bacillus typhi, Salmonella paratyphi A etc.; External; Under 3% concentration, 17 kinds of funguses there is bacteriostasis, influenza virus is also had certain inhibitory action; 2. antitumor: the mouse lung adenoma that morpholine is added due to the sodium nitrite has strong inhibitory action.
Human papillomavirus (Human Papillomavirus; Be called for short HPV) be a kind of epithelium sexually transmitted disease (STD) poison of having a liking for; The specificity that height is arranged; For a long time, known HPV can cause human benign tumor and wart, as is grown near the genitals human verruca vulgaris, the condyloma acuminatum on the skin and mucosa and is grown in the papilloma on the mucosa.As hepatitis B virus, HPV also is a kind of DNA viruses.It is former to be a member of polyoma vacuolating virus section, and cancellation polyoma vacuolating virus section of ICTV (ICTV) in 1999 replaces Papillomaviridae, and therefore, HPV just belongs to this student.
Summary of the invention
The present inventor is unexpected to find that gallic acid has the effect of anti-HPV.
Therefore, the present invention provides the application of gallic acid in the anti-HPV medicine of preparation.
The present invention also provides the application of gallic acid in the medicine of preparation prevention or treatment HPV relevant disease.
HPV relevant disease of the present invention includes but not limited to cervical cancer, other anogenital cancer or anus cancer or genital wart.
Gallic acid of the present invention can be the water extract that contains the Fructus Bruceae of gallic acid.
Gallic acid of the present invention can prepare as follows:
The dry fruit of getting Fructus Bruceae is broken, and with the pure water heating and refluxing extraction, the water extract concentrates the back with ethyl acetate extraction, extract concentrate extractum; Separate with silica gel column chromatography, adopt wet method dress post, appearance on the dry method, with dichloromethane, dichloromethane: 100: 1～1: 1 gradient elution of methanol, collect each component; Collect each component through thin layer chromatography analysis, merge the elution fraction of identical composition; Each component of silica gel column chromatography merges the elution fraction of identical composition through thin layer chromatography analysis, chooses the component that composition is more single and content is higher.
Preferably, gallic acid of the present invention can prepare as follows:
The dry fruit of getting Fructus Bruceae is broken, and with the pure water heating and refluxing extraction, the water extract concentrates the back with ethyl acetate extraction, extract concentrate extractum;
Separate with silica gel column chromatography, adopt wet method dress post, appearance on the dry method; With dichloromethane, dichloromethane: 100: 1～1: 1 gradient elution of methanol, every 500mL collects a, collects 113 parts altogether; Collect each component through thin layer chromatography analysis, merge the elution fraction of identical composition;
Silicagel column is proceeded the gel filtration chromatography separation for isolating the 80th～83 part, and with methanol-eluted fractions, every 8mL collects 1 part, collects 65 parts altogether, and 17～21 parts have yellow crystal to separate out;
Get gel column and carry out the reverse column chromatography for separation of ODS, purification for isolating the 19th part, with 20% methanol-eluted fractions, every 50mL is a, collects 6 parts altogether, and the 3rd, 4 part of adularescent acicular crystal is separated out, and is gallic acid.
For the anti-HPV effect of gallic acid of the present invention is described, the invention provides following experiment:
The PCR of the anti-HPV-DNA of gallic acid detects
The gallic acid of this experimentation derives from the extract of the dry mature fruit of quassia Fructus Bruceae Brucea javanica (L.) Merr, and concrete research contents is following:
Medical material is identified:
The evaluation of Fructus Bruceae
It is often very not detailed and accurate to the record of Chinese medicine morphological characteristic to occupy the successive dynasties " book on Chinese herbal medicine ".Cause the kind confusion phenomena of Chinese medicine quite outstanding, homonym, synonyms etc. fail to clarify all the time.This production to Chinese medicine, scientific research all have obstruction.In addition; The Chinese medicine market phenomenon of adulterating, mix the spurious with the genuine is also more general; In order to control the credibility of Chinese medicine quality, assurance Chinese medicine research, the discriminating of Chinese crude drug has crucial meaning, for this reason; We are according to " related content of first middle Fructus Bruceae of Chinese pharmacopoeia version in 2005 is carried out cultivar identification to Fructus Bruceae used in this subject study.
1 experiment medical material
Fructus Bruceae (20080410) Guangdong Kangmei Pharmaceutical Co., Ltd
2 experimental techniques
2.1 authentication method
According to " the related content regulation of first middle Fructus Bruceae of Chinese pharmacopoeia version in 2005, the cultivar identification of Fructus Bruceae mainly is to differentiate two aspects from character with other material.Concrete standard, method and result see table 1.
The evaluation of table 1 Fructus Bruceae
2.2 qualification result
Meet that " about the regulation of Fructus Bruceae, this medical material is the dry mature fruit of quassia Fructus Bruceae Brucea javanica (L.) Merr in first one of the Chinese pharmacopoeia version in 2005.
3 conclusions
These article meet " in first one of the Chinese pharmacopoeia version in 2005 about the regulation of Fructus Bruceae fully.
Below the used Fructus Bruceae of all experimentatioies be the kind of identifying through this authentication method, be: the dry mature fruit of quassia Fructus Bruceae Brucea javanica (L.) Merr.
The PCR detection method of Fructus Bruceae anti-human papilloma virus (anti-HPV)
At present, the pharmacodynamic study method mainly comprises pharmacodynamics and external pharmacodynamic experiment in the body, and pharmacodynamic experiment mainly is to realize through the animal model of generally acknowledging in the body.We find up-to-date progress comprehensive both domestic and external; The people is unique natural reservoir (of bird flu viruses) of human papillomavirus; Condyloma acuminatum does not still have animal model, can't carry out pharmacodynamic experiment in the body, and the FQ-PCR technology is as a kind of amplification in vitro specific nucleic acid technology; Be widely applied to Clinical detection and scientific research field, it has high susceptibility, specificity, characteristics such as easy and simple to handle, quick.
This experiment is extracted, isolation and purification the time, is used this technology condyloma acuminatum is done external pharmacodynamic experiment to follow the trail of the active substance of its anti-HPV-DNA Fructus Bruceae water extract.
1 material and and reagent
ABI 7700 quantitative real time PCR Instrument American AB I companies
Human papillomavirus HPV (6/11 type) nucleic acid amplification fluorescent detection kit (20080310) Shenzhen various extraction separation of basic company Fructus Bruceae and this laboratory of purifying substance provide
2 experimental techniques
2.1 the preparation of condyloma acuminatum wart body BIAO and BEN
Collection of specimens is collected the typical fresh condyloma acuminatum wart body BIAO and BEN that betides the pudendum position in No.2 Hospital Attached to Guangzhou Traditional Chinese Medicial Univ dermatopathy and venereal disease special outpatient clinic.After accurately claiming its weight, add small amount of physiological saline, homogenizer homogenate; Process wart body suspension; With normal saline it is diluted to different ratios, extracts HPV-DNA respectively, with fluorescence quantitative polymerase chain reaction (FQ-PCR) appearance amplification HPV-DNA; According to amplification, selecting the HPV-DNA copy number for use is 10
6The time wart body suspension as wart body BIAO and BEN.
2.2 the preparation of medicine
The material base of the anti-HPV-DNA of Fructus Bruceae is mainly followed the trail of in this experiment by external pharmacodynamic experiment, so the preparation of medicine is following:
The water-soluble substances that the Fructus Bruceae extraction separation obtains is with physiological saline solution (if the preparation variable concentrations is then with normal saline dilution), fully vibration evenly, making medicinal liquid is respectively 50 μ L of no sedimentary water preparation, prepare three groups subsequent use.The concrete process chart of external pharmacodynamics PCR experiment is seen (Fig. 1).
2.3 the interaction of medicine and wart body
2.3.1 the interaction of Fructus Bruceae water-soluble substances and wart body
Take out one group of water solublity medicinal liquid, add condyloma acuminatum wart body BIAO and BEN 50 μ L mixings, place 37 ℃ incubation case, during often shake up, medicine and wart soma are fully acted on, get BIAO and BEN respectively the 1st, 3,5 and 7 day of incubation and carry out the FQ-PCR detection.Operate after the same method for other 2 groups.Do negative control with normal saline.
2.2.4 fluorescence quantitative PCR detection HPV-DNA content
2.2.4.1HPV-DNA extract
Extract HPV-DNA according to the incidental HPV-DNA method for distilling of human papillomavirus HPV (6/11 type) nucleic acid amplification fluorescent detection kit: take out BIAO and BEN, the mixing that slightly vibrates, the centrifugal 15min of 13000 commentaries on classics/min; Abandon supernatant; Add 25 μ LDNA extracting solution 2 again, fully mixing is done for 100 ℃ and is bathed 10min; The centrifugal 10min of 13000 commentaries on classics/min, supernatant is the HPV-DNA template.
2.2.4.2 quantitative criterion curve
According to the test kit requirement, set up the quantitative positive criteria article of HPV-DNA of 4 series concentration, be followed successively by 5 * 10
7Copy number/mL, 5 * 10
6Copy number/mL, 5 * 10
5Copy number/mL, 5 * 10
4Copy number/mL sees, is abscissa with the natural logrithm of initial copy number, and cycle threshold is a vertical coordinate, obtains regression straight line and sees (Fig. 3) and standard curve, and the copy number to sample carries out quantitatively in view of the above.
2.2.4.3HPV-DNA amplification
According to the test kit operating instruction, get the PCR reactant liquor 37.6 μ L in the detection kit, TaqDNA polymerase 0.4 μ L, UNG0.03 μ L add HPV-DNA template 2 μ L again in the PCR reaction tube, and button strict control lid places quantitative FQ-PCR appearance cocycle amplification.HPV-DNA temperature in high-temperature denatured, process annealing reach is extended and is carried out cyclic amplification, and cyclic program is set to: 37 ℃, and 5min; 94 ℃, 1min; 95 ℃, 5s; 60 ℃, 30s does 40 circulations altogether.Reaction system is made as 20 μ L.
2.2.4.4 interpretation of result condition enactment
In fluorescent quantitative PCR technique, experimental result is calculated according to the CT value, and C represents Cycle, and T has represented Threshold, and the implication of CT value is: the period that the fluorescence signal in each reaction tube is experienced when arriving the thresholding of setting.Research shows that there is linear relationship in the logarithm of the CT value of each template and the initial copy number of this template, and initial copy number is many more, and the CT value is more little.Utilize the standard substance of known initial copy number can make standard curve, wherein abscissa is represented the logarithm of initial copy number, and vertical coordinate is represented the CT value.Therefore, as long as obtain the CT value of unknown sample, can calculate the initial copy number of this sample from standard curve.According to the test kit description; When using the ABI7700 quantitative real time PCR Instrument to carry out interpretation of result; Baseline (baseline) is taken as 1-10 or 1-15 circulation fluorescence signal, and threshold value (threshold) setting principle is as the criterion with the peak of threshold line just above normal negative control article amplification curve (random noise line).Mark standard substance copy number in setting before the amplification beginning or after finishing simultaneously.
3 experimental results
When the positive (index of copy number>=10 of the amplification of HPV-DNA
3) time, the S type curve that obtains standard is seen figure (Fig. 3); Negative (the index of copy number＜10 of amplification of HPV-DNA in table
3The time), obtain irregular curve and see (Fig. 4).Negative (the index of copy number＜10 of amplification of HPV-DNA in table
3) time, explain that medicine can suppress the HPV-DNA amplification; Positive (the index of copy number>=10 of the amplification of HPV-DNA
3But＜10
6) time, explain that medicine is inversely proportional to HPV-DNA inhibitory action and its index variation, heal when big when the index of the copy number of HPV-DNA, inhibitory action is littler, and promptly drug effect is littler; When the index of the copy number of HPV-DNA more hour, inhibitory action is bigger, promptly drug effect is bigger.
The active substance screening technology of Fructus Bruceae water extract and anti-HPV testing result
Effect experiment result according to extraction of front Fructus Bruceae system and extract; For keeping maximum working substance quality; The further separation and purification of acetic acid ethyl ester extract to Fructus Bruceae water extract such as the method for will utilizing column chromatography is tested in this part; Monitor with HPLC simultaneously, filter out the wherein active substance of anti-HPV with the external effect experiment of PCR, effective monomer combines nuclear-magnetism to carry out the structure evaluation.
Idiographic flow is seen Fig. 5.
1 silica gel column chromatography
The column chromatography for separation technology is modern a kind of physical chemistry separation means; The separation principle of silica gel chromatography is to separate according to different the obtaining of the absorption affinity of material on silica gel; The material that polarity is bigger generally speaking is prone to by silica gel adsorption; The more weak material of polarity is difficult for by silica gel adsorption, and whole chromatography process promptly is absorption, desorbing, absorption again, desorption process again.
1.1 column chromatography condition
Fructus Bruceae (20080410) Guangdong Kangmei Pharmaceutical Co., Ltd
The grand experimental facilities company limited of Nereid on the DK-S24 type electric-heated thermostatic water bath
Ethyl acetate (20090215) analytical pure Guangzhou Chemical Reagent Factory
Subsidiary factory of column chromatography silica gel (0090015) SILVER REAGENT Haiyang Chemical Plant, Qingdao
Dichloromethane (200910080) analytical pure Guangzhou Chemical Reagent Factory
Methanol (20091004-2) analytical pure Guangzhou Chemical Reagent Factory
Eluent: dichloromethane, methanol gradient elution; Flow velocity: 8mL/min
1.2 operational approach
The dry fruit 4kg that gets Fructus Bruceae is broken, use the pure water heating and refluxing extraction, concentrates the back with ethyl acetate extraction, extract concentrated extractum.
Adopt wet method dress post: get column chromatography silica gel 700g stir with dichloromethane and drain bubble after pour in the post, treat its natural subsidence with eluent methylene chloride; Appearance on the dry method: after the 40g extractum of ethyl acetate extraction fully dissolved with the 100mL ethyl acetate, add in the 100g silica gel, stir, wait to volatilize the top layer that the powder that obtains after the ethyl acetate carefully is added to pillar again; Eluting: with dichloromethane (CH2Cl2), dichloromethane: methanol (Meth) (100: 1～1: 1) gradient elution, every 500mL collects a, collects 113 parts altogether.
1.3 monitoring method
1.3.1 silicon thin-layer chromatography
Thin layer chromatography (Thin Layer Chromatography) TLC commonly used representes; Be the thin plate chromatography again; Being a kind of in the chromatography, is a kind of very important experimental technique of sharp separation and qualitative analysis small amount of matter, belongs to the solid-liquid adsorption chromatography; It has had both the advantage of column chromatography and paper chromatography, is applicable to the separation of small amount of sample on the one hand; When making lamellae, strengthen the adsorption layer thickening on the other hand, therefore, can be used to refining sample again, this method is specially adapted to that the less or higher temperature of volatility is prone to change and the material that can not use gas chromatographic analysis.In addition, thin layer chromatography also can be used to follow the tracks of organic reaction and carries out column chromatography a kind of " prerun " before.
Each component of silica gel column chromatography merges the elution fraction of identical composition through thin layer chromatography analysis.
1.3.2 HPLC
1.3.2.1 instrument and reagent
The high performance liquid chromatograph Waters2695 U.S.
The PDA detector Waters2998 U.S.
High speed centrifuge BACKMAN COULTER
Electronic balance U.S. Denver
Methanol (071700) chromatographically pure U.S. fisher company
Formic acid (960404) analytical pure Guangzhou Chemical Reagent Factory
1.3.2.2 chromatographic condition
Chromatographic column: waters symmetry post (150mm * 3.9mm, 5 μ m); Mobile phase: A, 0.1% formic acid (pH:2.9); B, methanol.Gradient elution: 0～23min, B (%) 10～27; 23～29min, B (%) 27～30; 29～30min, B (%) 30～37; 30～50min, B (%) 37～50.Full wavelength scanner; Volume flow: 1.0mL/min; Sample size: 10 μ L.
1.3.2.3 sample monitoring
The medicine of getting acetic acid ethyl ester extract, vanillic acid standard substance, luteolin 7-0-β-D-glucopyanoside standard substance and each component of silica gel column chromatography fully dissolves with 70% methanol in right amount, 10 μ L sample introductions, and the record chromatogram is seen 6-11.
1.3.2.4 result
The HPLC atlas analysis is chosen the component that composition is more single and content is higher, sees Figure 11: (215.6, composition 271.3nm) is main to 80～83 duplicate samples with 3.27min; (259.5, composition 293.9nm) is main to 60～66 duplicate samples, and (226, composition 278.4nm) takes second place 40.4min with 6.5min; 45 duplicate samples are main with the composition of 10.31min (254.7nm), and the composition of 28.6min (277.3nm) takes second place; (244, composition 280.8nm) is main to 33～36 duplicate samples, and the composition of 46.4min (278.4nm) takes second place with 40.1min; (218.1,260.7, composition 291.5nm) is to lead (same with vanillic acid) to 29 duplicate samples with 14.227min; (229.9,278.4, composition 309.4nm) is main to 21～22 duplicate samples with 17.54min.
Component above-mentioned composition is more single and that content is higher is screened according to the method for the external effect experiment of PCR, and drug level is 2.5mg/mL, and with the medicine for external use " You Tuoxin " of commercially available treatment condyloma acuminatum as positive control, the result sees table 2.
Table 2 silica gel column chromatography composition is than one-component PCR testing result
1.3.2.5 discuss
Can find out that by the drug effect result isolating 80～83 duplicate samples of silica gel column chromatography have the effect of anti-HPV-DNA, can continue the medicine of this component is separated and the drug effect tracking; According to the separation principle of silica gel column chromatography, the material that polarity is bigger generally speaking is prone to by silica gel adsorption, and the time of staying of 80～83 duplicate samples in silicagel column is longer, and then the composition of this component should be the bigger material of polarity.
" You Tuoxin " can suppress HPV and infect epithelial division and the hypertrophy that institute causes excipuliform propagation as the present conventional external used medicine of clinical treatment CA, makes it to necrose, come off, thereby plays the effect of treating CA.Can find out that through the external effect experiment result of this PCR " You Tuoxin " directly do not destroy the effect of HPV-DNA; The mechanism of action of the anti-HPV of active substance that follows the trail of with this experimentation is different; These active substance then are through directly acting on HPV-DNA, kill virus and onset.
2 gel filtration chromatographies
Gel filtration (gel filtration) is also referred to as exclusion chromatography (exclusion chromatography), gel chromatography (gel chromatography) or sieve chromatography (molecular sieve chromatofraphy).
Gel is the solid matter that is condensed and formed by colloid solution, and inside has netted sieve aperture, utilizes the sieve aperture in the spherical gel; When making the gel filled excessively tubing string of molecular flow, macromole can't get into the gel sieve aperture, and the hole between gel and tubing string of only flowing through; Can flow out tubing string soon; Less molecule so the time of staying in tubing string is longer, is distinguished the molecule that varies in size because get into the sieve aperture in the gel thus.By the drug effect interpretation of result of front, can isolating 80～83 duplicate samples of silicagel column be proceeded gel filtration chromatography separation and active substance screening.
2.1 column chromatography condition
Glass chromatography column (the Guangzhou glass apparatus company of 100cm * 2cm)
Column chromatography gel (10026532) Sephadex LH-20 GE Healthcare Bio-Sciences AB
Methanol (20091004-2) analytical pure Guangzhou Chemical Reagent Factory
2.2 operational approach
Get column chromatography gel 50g stir with methanol and drain bubble after pour in the post, treat its natural subsidence with methanol drip washing.After 80～83 duplicate samples of again silicagel column being got are fully dissolved with small amount of methanol, carefully be added to the top layer of pillar.With the methanol isocratic elution, flow speed control was at 3 seconds/.Every 8mL collects 1 part, collects 65 parts altogether, and the flow process of separation method is seen Figure 12.
2.3 result
Yellow precipitate is all arranged in the 17th～21 duplicate samples; Each component that gel column is got is through thin layer chromatography; Gel the 3rd～21 duplicate samples is observed under the 360nm ultraviolet has skin dark stain; And gel the 3rd～9 duplicate samples composition is identical, and gel the 11st～15 duplicate samples composition is identical, and gel the 19th～21 duplicate samples composition is identical and more single.Choose gel the 8th, 14,17,19,20,21 increments and carry out the HPLC analysis, see Figure 13,14.Collection of illustrative plates can find out that gel 17,19,20,21 duplicate samples compositions are single.
The sample compound concentration of 8,13, No. 20 components of gel filtration chromatography is 2.5mg/mL, detects according to the external effect experiment of PCR, the result shows that gel 20 components have the effect of anti-HPV, and testing result is seen table 3.
The elution fraction PCR testing result of table 3 gel filtration chromatography
2.4 discuss
All there is the yellow crystal thing to separate out in gel the 17th～21 duplicate samples, analyzes through HPLC in conjunction with 17,19,20,21 increments and can find out that the crystallization of being separated out in these components should be same monomeric compound; And show that through the external effect experiment of PCR this chemical compound has the effect of anti-HPV-DNA, need be further purified in the hope of obtaining purity higher effective monomer it.
The purification of 3 active substance (the reverse column chromatography of ODS)
The most frequently used " omnipotent post " filler is " C18 ", is called for short " ODS " post, i.e. octadecylsilane chemically bonded silica filler (Octadecylsilyl).Because C18 is a chain alkyl bonding phase, higher carbon content and better hydrophobicity are arranged then.The result of gel filtration chromatography can find out that gel the 17th～21 increment is an active component from the front, and its composition is single, and polarity is bigger, can utilize the ODS post that it is further purified.
3.1 column chromatography condition
Glass chromatography column (the Guangzhou glass apparatus company of 100cm * 2cm)
ODS-A (AA12S50) Sephadex LH-20 Beijing intelligent De Yi Science and Technology Ltd.
Methanol (20091004-2) 12nm S-50 μ m Guangzhou Chemical Reagent Factory
3.2 operational approach
Gel the 17th～21 increment is than single component, gets gel the 19th duplicate samples and carries out the reverse column chromatography of ODS, and with methanol swelling ODS, 70% methanol carries out the transition to 20% methanol balance pillar, 20% methanol-eluted fractions, and every 50mL is a, collects 6 parts altogether.The flow process of separation method is seen Figure 15.
3.3 result
3rd, 4 parts of component adularescent acicular crystals are separated out, and this crystallization according to the external effect experiment method of PCR, is detected after acting on 1,4,7 day respectively with the CA suspension, and its MEC is 0.5mg/mL.The result sees table 4.
The MEC PCR testing result of the white needle of table 4
" figure is set the HPV-DNA BIAO and BEN and the real-time amplification curve of HPV-DNA is seen, when the positive (index of copy number>=10 of the amplification of HPV-DNA according to " the PCR detection method of Fructus Bruceae anti-human papilloma virus (anti-HPV) " the 3rd point
3) time, the S type curve that obtains standard is seen figure (Fig. 3); Negative (the index of copy number＜10 of amplification of HPV-DNA in table
3The time), obtain irregular curve and see (Fig. 4).Negative (the index of copy number＜10 of amplification of HPV-DNA in table
3) time, explain that medicine can suppress the HPV-DNA amplification; Positive (the index of copy number>=10 of the amplification of HPV-DNA
3But＜10
6) time, explain that medicine is inversely proportional to HPV-DNA inhibitory action and its index variation, heal when big when the index of the copy number of HPV-DNA, inhibitory action is littler, and promptly drug effect is littler; When the index of the copy number of HPV-DNA more hour, inhibitory action is bigger, promptly drug effect is bigger." judgement table 4 result.
The structure of effective monomer is identified
1HPLC identification chromatography condition
The high performance liquid chromatograph Waters2695 U.S.
The PDA detector Waters2998 U.S.
Chromatographic column: waters symmetry post (150mm * 3.9mm, 5 μ m); Mobile phase: A, 0.1% formic acid (pH:2.9); B, methanol.Gradient elution: 0～23min, B (%) 10～27; 23～29min, B (%) 27～30; 29～30min, B (%) 30～37; 30～50min, B (%) 37～50.Full wavelength scanner; Volume flow: 1.0mL/min; Sample size: 10 μ L.
This acicular crystal and reference substance gallic acid are carried out the HPLC analysis, see Figure 16,17.Its spectrum is consistent with chromatographic behavior.2NMR identifies that the effective monomer that above-mentioned separation and purification is obtained is numbered YDZ-1, and entrusts pharmaceutical college of Zhejiang University that it is carried out nuclear magnetic resonance spectroscopy.Concrete carbon spectrum, hydrogen spectrum are seen Figure 18,19.Preliminary definite this monomer is a gallic acid.
Conclusion
This experiment is a medical material with the dry mature fruit of quassia Fructus Bruceae Brucea javanica (L.) Merr; Utilize the solvent and the column chromatography methods of various opposed polarities that Fructus Bruceae is carried out extraction separation and purification; Track the active substance of anti-HPV through the external effect experiment of PCR, in separation process, monitor simultaneously with HPLC (HPLC).Obtaining white needle in the last water soluble part, is gallic acid through Preliminary Identification.And the external effect experiment result of PCR shows that its MEC is 0.0005g/mL.
Among the present invention, gallic acid can add the medicine acceptable carrier as required and process pharmaceutical preparation.
Preparation of the present invention is the pharmaceutical dosage forms of UD, and said unit dosage form is meant the unit of preparation, as every of tablet, and capsular every capsules, every bottle of oral liquid, every bag of granule etc.
Preparation of the present invention gallic acid wherein, its shared percentage by weight in preparation can be 0.1-99.9%, all the other are the medicine acceptable carrier.
Preparation of the present invention obtains through gallic acid and medicine acceptable carrier are mixed with.
Gallic acid of the present invention; Its pharmaceutical dosage forms can be any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, preferably injection, more preferably freeze-dried powder.
Gallic acid of the present invention, the preparation of its oral administration can contain excipient commonly used, such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet in case of necessity.
The filler that is suitable for comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, for example sodium starch glycollate.Suitable lubricant comprises, for example magnesium stearate.The acceptable wetting agent of appropriate drug comprises sodium lauryl sulphate.
Can fill through mixing, the method that tabletting etc. are commonly used prepares solid oral composition.Mix repeatedly active substance is distributed in those compositionss of a large amount of filleies of whole use.
The form of oral liquid for example can be aqueous or oily suspensions, solution, Emulsion, syrup or elixir, perhaps can be a kind of available water before use or other suitable composite dry products of carrier.This liquid preparation can contain conventional additive; Such as suspending agent; For example sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenation edible fat; Emulsifying agent, for example lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), for example almond oil, fractionated coconut oil, such as oily ester, propylene glycol or the ethanol of the ester of glycerol; Antiseptic, for example para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if desired, can contain conventional flavouring agent or coloring agent.
For injection, the liquid unit dosage forms of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, can this chemical compound be suspended or dissolving.The preparation of solution is normally through being dissolved in active substance in a kind of carrier filter-sterilized before it is packed into a kind of suitable bottle or ampoule, sealing then.For example a kind of local anesthetic of adjuvant, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, can be after the bottle of packing into that this compositions is freezing, and under vacuum, water is removed.
Gallic acid among the present invention; When being prepared into medicament, optionally add suitable medicine acceptable carrier; Said medicine acceptable carrier is selected from: meglumine, mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDTA disodium, EDTA calcium sodium, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and derivant thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, soil temperature 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
Compositions of the present invention is confirmed usage and dosage according to patient's situation in use.
Description of drawings
Fig. 1 pharmacology PCR experimental technique flow chart
The quantitative regression straight line of Fig. 2 HPV-DNA
Fig. 3 HPV-DNA amplification positive sample is figure as a result
Fig. 4 HPV-DNA negative BIAO and BEN figure as a result that increases
Fig. 5 Fructus Bruceae water extract active substance trace flow figure
Fig. 6 Fructus Bruceae water extract acetic acid ethyl ester extract, vanillic acid standard substance and luteolin 7-0-β-D-glucopyanoside standard substance HPLC collection of illustrative plates
1 represents luteolin 7-0-β-D-glucopyanoside standard substance HPLC collection of illustrative plates, and 2 represent vanillic acid standard substance HPLC collection of illustrative plates, and 3 represent Fructus Bruceae water extract acetic acid ethyl ester extract HPLC collection of illustrative plates
Fig. 7 silica gel column chromatography 75-90 component HPLC collection of illustrative plates
1 represents silica gel column chromatography 88-90 component HPLC collection of illustrative plates, and 2 represent silica gel column chromatography 84-87 component HPLC collection of illustrative plates, and 3 represent silica gel column chromatography 80-83 component HPLC collection of illustrative plates, and 4 represent silica gel column chromatography 75-79 component HPLC collection of illustrative plates
Fig. 8 silica gel column chromatography 57-74 component HPLC collection of illustrative plates
1 represents silica gel column chromatography 70-74 component HPLC collection of illustrative plates, and 2 represent silica gel column chromatography 67-69 component HPLC collection of illustrative plates, and 3 represent silica gel column chromatography 60-66 component HPLC collection of illustrative plates, and 4 represent silica gel column chromatography 57-59 component HPLC collection of illustrative plates
Fig. 9 silica gel column chromatography 37-56 component HPLC collection of illustrative plates
1 represents silica gel column chromatography 54-56 component HPLC collection of illustrative plates; 2 represent silica gel column chromatography 48-53 component HPLC collection of illustrative plates; 3 represent silica gel column chromatography 46-47 component HPLC collection of illustrative plates; 4 represent silica gel column chromatography 45 component HPLC collection of illustrative plates, and 5 represent silica gel column chromatography 44 component HPLC collection of illustrative plates, and 6 represent silica gel column chromatography 37-43 component HPLC collection of illustrative plates
Figure 10 silica gel column chromatography 23-36 component HPLC collection of illustrative plates
1 represents silica gel column chromatography 33-36 component HPLC collection of illustrative plates; 2 represent silica gel column chromatography 31-32 component HPLC collection of illustrative plates, and 3 represent silica gel column chromatography 29-30 component HPLC collection of illustrative plates, and 4 represent silica gel column chromatography 29 component HPLC collection of illustrative plates; 5 represent silica gel column chromatography 23-28 component HPLC collection of illustrative plates
Figure 11 silica gel column chromatography composition is than one-component HPLC collection of illustrative plates
1 represents silica gel column chromatography 80-83 component HPLC collection of illustrative plates; 2 represent silica gel column chromatography 60-66 component HPLC collection of illustrative plates; 3 represent silica gel column chromatography 45 component HPLC collection of illustrative plates; 4 represent silica gel column chromatography 33-36 component HPLC collection of illustrative plates, and 5 represent silica gel column chromatography 29 component HPLC collection of illustrative plates, and 6 represent silica gel column chromatography 21-22 component HPLC collection of illustrative plates
Figure 12 gel filtration chromatography separation process figure
Figure 13 gel filtration chromatography 8,14 component HPLC collection of illustrative plates
1 represents the HPLC collection of illustrative plates of gel filtration chromatography component 8, and 2 represent the HPLC collection of illustrative plates of gel filtration chromatography component 14,
Figure 14 gel filtration chromatography 17,19,20 and 21 component HPLC collection of illustrative plates
1 represents the HPLC collection of illustrative plates of gel filtration chromatography component 17, and 2 represent the HPLC collection of illustrative plates of gel filtration chromatography component 19, and 3 represent the HPLC collection of illustrative plates of gel filtration chromatography component 20, and 4 represent the HPLC collection of illustrative plates of gel filtration chromatography component 21
The separation process figure of the reverse post of Figure 15 ODS
The spectrogram of Figure 16 gallic acid reference substance and extract
The chromatogram of Figure 17 gallic acid reference substance and extract
1 represents the GA reference substance, and 2 represent YDZ-1
The carbon-13 nmr spectra figure of Figure 18 compound I
The hydrogen nuclear magnetic resonance spectrogram of Figure 19 compound I
The specific embodiment
Following examples are for the present invention is further explained, and can not limit the present invention by any way.
The preparation of lyophilized formulations:
(1) with the gallic acid of embodiment 1, adds meglumine; 100 ℃ were heated 1 hour, and cold preservation is spent the night, and added mannitol, filtered standardize solution;
(2) activated carbon decolorizing;
(3) moisturizing;
(4) aseptic filtration;
(5) fill;
(6) lyophilizing.
Get gallic acid and the Polyethylene Glycol-6000 of embodiment 1, water-bath is dissolved, and after change is even, adds volatile oil of Lignum Dalbergiae Odoriferae, and mixing moves in the drop pill machine, processes 1000 drop pill.
Gallic acid, sucrose and the dextrin of getting embodiment 1 processed 200 in tablet by weight 1: 3: 1 ratio according to conventional method.
Gallic acid, sucrose and the dextrin of getting embodiment 1 processed 125 bags of granules in 1: 3: 1 ratio according to conventional method.
Gallic acid, sucrose and the dextrin of getting embodiment 1 processed granule in 1: 3: 1 ratio according to conventional method, records capsule according to conventional method.
Claims (2)
1. the method for preparing of a gallic acid comprises the steps:
The dry fruit of getting Fructus Bruceae is broken, and with the pure water heating and refluxing extraction, the water extract concentrates the back with ethyl acetate extraction, extract concentrate extractum; Separate with silica gel column chromatography, adopt wet dress post, appearance on the dry method, with dichloromethane, dichloromethane: 100: 1～1: 1 gradient elution of methanol, collect each component; Collect each component through thin layer chromatography analysis, merge the elution fraction of identical composition; Each component of silica gel column chromatography merges the elution fraction of identical composition through thin layer chromatography analysis, chooses the component that composition is more single and content is higher.
2. method as claimed in claim 1 comprises the steps:
The dry fruit of getting Fructus Bruceae is broken, and with the pure water heating and refluxing extraction, the water extract concentrates the back with ethyl acetate extraction, extract concentrate extractum;
Separate with silica gel column chromatography, adopt wet dress post, appearance on the dry method; With dichloromethane, dichloromethane: 100: 1～1: 1 gradient elution of methanol, every 500mL collects a, collects 113 parts altogether; Collect each component through thin layer chromatography analysis, merge the elution fraction of identical composition;
Silicagel column is proceeded the gel filtration chromatography separation for isolating the 80th～83 part, and with methanol-eluted fractions, every 8mL collects 1 part, collects 65 parts altogether, and 17～21 parts have yellow crystal to separate out;
Get gel column and carry out the reverse column chromatography for separation of ODS, purification for isolating the 19th part, with 20% methanol-eluted fractions, every 50mL is a, collects 6 parts altogether, and the 3rd, 4 part of adularescent acicular crystal is separated out, and is gallic acid.
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| CN2010102947544A CN101933916B (en) | 2010-09-28 | 2010-09-28 | Application of gallic acid in preparing anti-HPV medicine |
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| CN2010102947544A CN101933916B (en) | 2010-09-28 | 2010-09-28 | Application of gallic acid in preparing anti-HPV medicine |
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| CN114748460B (en) * | 2022-05-09 | 2023-09-15 | 中国农业科学院兰州畜牧与兽药研究所 | Medicine for treating calf cryptosporidium infection and application thereof |
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| TW200936152A (en) * | 2007-11-26 | 2009-09-01 | Modetex Biomedical Materials & Thchnology Ind Corp | Plant derived compounds containing the same for the treatment of cervical cancer |
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| TW200936152A (en) * | 2007-11-26 | 2009-09-01 | Modetex Biomedical Materials & Thchnology Ind Corp | Plant derived compounds containing the same for the treatment of cervical cancer |
Non-Patent Citations (2)
| Title |
|---|
| 代敏等.人乳头瘤病毒及其疫苗研究进展.《科学通报》.2009,第54卷(第19期), * |
| 罗小秀等.鸦胆子治疗宫颈癌及其作用机制的研究进展.《中国现代医生》.2010,第48卷(第9期), * |
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