CN106310116A - Rhizoma Zedoariae oil, and application and pharmaceutical preparation thereof - Google Patents
Rhizoma Zedoariae oil, and application and pharmaceutical preparation thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, and discloses a Rhizoma Zedoariae oil, and application and a pharmaceutical preparation thereof. In the Rhizoma Zedoariae oil, the content of Rhizoma Zedoariae sesquiterpenoids is greater than or equal to 40% and less than or equal to 75%, and the Rhizoma Zedoariae sesquiterpenes are furanodiene, curdione, germacrone and curzerene. The research indicates that the obtained Rhizoma Zedoariae oil has better oxidation resistance.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of Oleum Curcumae and application thereof and medicine
Thing preparation.
Oleum Curcumae of the present invention is the Oleum Curcumae obtained for raw material with Curcuma wenyujin, Curcuma wenyujin
Planting base includes but not limited to Hainan Province.
Background technology
Rhizoma Curcumae is zingiberaceous plant, is divided into Guangxi zedoary, Rhizoma Curcumae, Curcuma wenyujin, originate in Guangxi,
Jiangxi, Sichuan, Zhejiang and Hainan etc. save.Oleum Curcumae extracts from Rhizoma Curcumae dry rhizome
Volatile oil, theory of Chinese medical science thinks that Oleum Curcumae has circulation of qi promoting removing blood stasis, effect of removing food stagnancy pain relieving, existing
Show that Oleum Curcumae also has the effect such as antiviral, antibacterial, antitumor for pharmaceutical research.Cowherb
Art oil chemical composition complex, great majority are sesquiterpenoids, boiling point height and
Have under thermal sensitivity, high temperature and be susceptible to oxidation, decomposition or conversion reaction.
Oleum Curcumae is through clinical practice widely, it was demonstrated that it has efficiently, the feature of low toxicity and
Effective to multiple disease.Aromatic turmeric oil preparation is in cervical cancer, hepatocarcinoma and treating cardiovascular disease side
Face all achieves gratifying curative effect.The aromatic turmeric oil preparation of Clinical practice generally comprises Rhizoma Curcumae
Fat injection, Zedoary turmeric oil glucose injection, compound zedoary oil suppository, compound zedoary oil are soft
Capsule etc..
Oleum Curcumae the most gradually causes the world of medicine as traditional blood-activating and stasis-removing
Pay attention to, and carried out system at aspects such as its resource, plant, pharmacology, preparation, clinics and grind
Study carefully, it was demonstrated that Oleum Curcumae be a pharmacologically active strong, efficiently, safe drugs.Tumor and the heart
Angiopathy is current two big difficult medical problem, also brings two simultaneously and has wide expansion sky
Between drug market.The antitumor of Oleum Curcumae and antithrombotic acitivity and definite curative effect clinically,
Add its antiviral and antibacterial activity, the most greatly imply that Oleum Curcumae will be in future
Drug market occupies one seat.Generally speaking, Oleum Curcumae be one rising
Medicine, along with further developmental research to it from now on, Oleum Curcumae will be at the new drug of China
Initiative system yields unusually brilliant results.
Summary of the invention
For these reasons, applicant studies through for many years, obtains a kind of new Oleum Curcumae,
In this Oleum Curcumae, Rhizoma Curcumae sesquiterpenoids effective site content more than or equal to 40% and is less than or equal to
75%, wherein furanodiene content is more than or equal to 8% less than or equal to 18%, and 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content is big
In being less than or equal to 10% equal to 5%, curdione content is less than or equal to more than or equal to 10%
20%；Containing curzerene and eucalyptol in Oleum Curcumae, wherein curzerene content is more than or equal to 15%
Less than or equal to 35%, wherein the content of eucalyptol is less than or equal to 21% more than or equal to 5%；Research
Showing, the Oleum Curcumae that the present invention obtains has more preferable antioxidation；The present invention obtains
New Oleum Curcumae can use as medicine material.
The present invention is achieved through the following technical solutions.
A kind of Oleum Curcumae, Oleum Curcumae contains Rhizoma Curcumae sesquiterpenoids content more than or equal to 40% and little
In equal to 75%, wherein Rhizoma Curcumae sesquiterpene is furanodiene, curdione, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-and cowherb
Art alkene.
A kind of Oleum Curcumae, Oleum Curcumae contains Rhizoma Curcumae sesquiterpenoids content more than or equal to 50% and little
In equal to 75%, wherein Rhizoma Curcumae sesquiterpene is furanodiene, curdione, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-and cowherb
Art alkene.
Wherein furanodiene content is more than or equal to 8% less than or equal to 18%, and 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content is more than
Equal to 5% less than or equal to 10%, curdione content is less than or equal to 20% more than or equal to 10%.
Wherein in Oleum Curcumae, curzerene content is less than or equal to 35% more than or equal to 15%.
Wherein containing eucalyptol in Oleum Curcumae, the content of eucalyptol is less than or equal to more than or equal to 5%
21%.
In Oleum Curcumae, furanodiene, curdione, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-and curzerene content are more than or equal to
40% is less than or equal to 75%, and wherein furanodiene is less than or equal to 18% more than or equal to 8%, lucky horse
Ketone content is more than or equal to 5% less than or equal to 10%, and curdione content is less than more than or equal to 10%
Equal to 20%；Containing curzerene and eucalyptol in Oleum Curcumae, wherein curzerene content is more than
In 15% less than or equal to 35%, wherein the content of eucalyptol is less than or equal to more than or equal to 5%
21%.
In Oleum Curcumae, furanodiene, curdione, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-and curzerene content are more than or equal to
50% is less than or equal to 75%, and wherein furanodiene content is less than or equal to 18% more than or equal to 8%,
3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content is more than or equal to 5% less than or equal to 10%, and curdione content is more than or equal to 10%
Less than or equal to 20%；Containing curzerene and eucalyptol in Oleum Curcumae, wherein curzerene content is big
In being less than or equal to 30% equal to 15%, wherein the content of eucalyptol is less than or equal to more than or equal to 7%
21%.
The pharmaceutical preparation prepared for raw material with Oleum Curcumae.
Its pharmaceutical formulations include diseases of eye, ear, nose and throat, pudendum, vagina, rectum, crissum, skin,
The external preparation such as mucosa, or the oral formulations such as tablet, capsule, granule, or injection
Agent etc..
Its pharmaceutical formulations includes: eye drop, ear drop, lotion, vaginal suppository, rectum
Suppository, ointment, capsule, soft capsule, tablet, effervescent tablet, granule, oral
The externals such as liquid, gel, membrane, foam or oral formulations or injection.
Its pharmaceutical formulations raw material be Oleum Curcumae be 164 weight portions, Borneolum Syntheticum is 150 weight portions.
Described pharmaceutical preparation is at preparation prevention and/or the medicine for the treatment of human papilloma virus infection
In application.
Described pharmaceutical preparation prevention and/or treat various virus, antibacterial, mycete, chlamydia,
The infection such as mycoplasma, infusorian, diseases of eye, ear, nose and throat infection, vulvitis, various vaginitis, mixing
The infection of infective vaginitis, skin infection, mucosal infections, hemorrhoid, constipation, cervicitis,
Application in the medicines such as adnexitis, CIN I-CINIII, cervical cancer, antioxidation.
The preparation method of Oleum Curcumae described above include but not limited to for:
Take Rhizoma Curcumae, clean, cut into slices, bake water content 40%-60% for 55-90 DEG C, pulverize granulating
Degree is 5-15mm Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through water and steams
Air stripping takes 24-40 hour, collects the volatile oil in oil water separator, adds 3%-10% volatilization
The sodium chloride of weight of oil or anhydrous sodium sulfate, stirring, standing separation, take supernatant, obtain cowherb
Art oil.
The Oleum Curcumae that the present invention is newly obtained, is to use the dry side less than 100 DEG C in pre-treatment
Method, and make Rhizoma Curcumae water content be reduced to 40%-60%, then distill, use after distillation
The method adding salt refining, obtains new Oleum Curcumae.
Oleum Curcumae of the present invention is the Oleum Curcumae obtained for raw material with Curcuma wenyujin, and the place of production is wrapped
Include but be not limited to Hainan Province.
Oleum Curcumae of the present invention has the pharmacologically active of prior art.
Oleum Curcumae of the present invention is " effective site " concept of the field of Chinese medicines.
So-called effective ingredient in Chinese, refers to as in a herb or compound Chinese medicine extract
The content of class or a few class chemical composition reaches more than the 50% of total extract, and a class or several
The known chemical composition of class is considered as effective ingredient, and the mixture of this class or a few constituents is i.e.
It is considered as effective site.
Furanodiene: No. CAS: 19912-61-9；3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-: No. CAS: 6902-91-6；
Curdione: No. CAS: 13657-68-6；Curzerene: No. CAS: 17910-09-7；Eucalyptus globulus
Olein: No. CAS: 470-82-6.
Oleum Curcumae effective site described above, can be according to the vapor distillation of the present invention
Method prepares, it is also possible to meet standards of pharmacopoeia Oleum Curcumae as raw material, according to preparation
The method of liquid phase prepares:
Liquid phase chromatogram condition: with octadecylsilane chemically bonded silica as filler；With acetonitrile for stream
Dynamic phase A, water is Mobile phase B, carries out gradient elution, and gradient is: 0～20min is geometric ratio
Example A60% and B40% is transformed into A95% and B5%, 21～35min maintain A95%
And B5%；Detection wavelength is 216nm.
The following test of the present invention, is in repeatedly creative experimental basis, for institute of the present invention
The concluding test that technical scheme to be protected is carried out.
One, antioxidation
Trial drug:
Test 1 group: Oleum Curcumae: wherein furanodiene content 17.8%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content
6.4%, curdione content 15.7%；Containing curzerene and eucalyptol in Oleum Curcumae, wherein
Curzerene content 9.4%, the wherein content 4.6% of eucalyptol.
Preparation method: take Rhizoma Curcumae, cleans, cuts into slices, bake water content 7.8%, powder for 55 DEG C
Being broken into granularity is 10mm Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through
Vapor extraction 26 hours, collects the volatile oil in oil water separator, obtains Oleum Curcumae.
Test 2 groups: Oleum Curcumae: wherein furanodiene content 15.9%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content
7.5%, curdione content 15.6%；Containing curzerene and eucalyptol in Oleum Curcumae, wherein
Curzerene content 11.7%, the wherein content 4.9% of eucalyptol.
Preparation method: take Rhizoma Curcumae, cleans, cuts into slices 65 DEG C and bake water content 37.4%, powder
Being broken into granularity is 10mm Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through
Vapor extraction 28 hours, collects the volatile oil in oil water separator, obtains Oleum Curcumae.
Test 3 groups: Oleum Curcumae: furanodiene content 14.1%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content 7.6%,
Curdione content 16.5%；Containing curzerene and eucalyptol, wherein curzerene in Oleum Curcumae
Content 15.1%, the wherein content 5.0% of eucalyptol.
Preparation method: take Rhizoma Curcumae, cleans, cuts into slices, bake water content 40%, powder for 70 DEG C
Being broken into granularity is 10mm Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through
Vapor extraction 32 hours, collects the volatile oil in oil water separator, adds 3% volatile oil
The sodium chloride of weight, stirring, standing separation, take supernatant, obtain Oleum Curcumae.
Test 4 groups: Oleum Curcumae: furanodiene content 13.7%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content 8.9%,
Curdione content 18.9%；Containing curzerene and eucalyptol, wherein curzerene in Oleum Curcumae
Content 25.3%, the wherein content 14.7% of eucalyptol.
Preparation method: take Rhizoma Curcumae, cleans, cuts into slices, bake water content 50%, powder for 85 DEG C
Being broken into granularity is 10mm Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through
Vapor extraction 38 hours, collects the volatile oil in oil water separator, adds 6% volatile oil
The sodium chloride of weight, stirring, standing separation, take supernatant, obtain Oleum Curcumae.
Test 5 groups: Oleum Curcumae: wherein furanodiene content 11.8%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content 9.9%,
Curdione content 19.5%；Containing curzerene and eucalyptol, wherein curzerene in Oleum Curcumae
Content 27.8%, the wherein content 20.4% of eucalyptol.
Taking Rhizoma Curcumae, clean, cut into slices, bake water content 60% for 90 DEG C, being ground into granularity is
15mm Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through vapor extraction
40 hours, collect the volatile oil in oil water separator, add the 10% anhydrous sulfur of volatile oil weight
Acid sodium, stirring, standing separation, take supernatant, obtain Oleum Curcumae.
Detection method of content: (Chinese Pharmacopoeia one annex VI D of version in 2010) measures.
Chromatographic condition and system suitability are with octadecylsilane chemically bonded silica for filling
Agent, with acetonitrile as mobile phase A, water is Mobile phase B, and the regulation according to the form below carries out gradient
Eluting；Detection wavelength is 216nm.Number of theoretical plate is calculated should be not less than by curdione peak
5000。
The preparation of reference substance solution take curdione, furanodiene, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, curzerene,
Eucalyptol reference substance is appropriate, accurately weighed, adds dehydrated alcohol and makes every 1ml containing curdione
35 μ g, furanodiene 50 μ g, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-20 μ g, curzerene 20 μ g, eucalyptol 10 μ g
Mixed solution, to obtain final product.
The preparation of need testing solution takes this product 0.1g, accurately weighed, puts in 50ml measuring bottle,
Adding dehydrated alcohol to scale, shake up, precision measures 5ml, puts in 25ml measuring bottle, adds anhydrous
Ethanol, to scale, shakes up, and filters, takes subsequent filtrate, to obtain final product.
Algoscopy precision respectively draws reference substance solution and each 5 μ 1 of need testing solution, injects
Chromatograph of liquid, measures, to obtain final product.
Experimental animal: BALB/C mice, male and female half and half, 18-22g.
Medicine is prepared: take above-mentioned Oleum Curcumae, adds edible salad oil, is configured to
5mg/10mL, standby.
Test method: take mice, random packet (blank group, commercially available Rhizoma Curcumae line of oils,
Test 1 group to test 5 groups), often group 20, male and female half and half, respectively organize gastric infusion, give
Dose is 1ml (5mg/kg), and once a day, blank group gives normal saline 1ml,
Successive administration 13 days, broken end takes blood, measures indices, and assay method presses test kit explanation
Carry out；Statistical procedures: use t inspection.
Result of the test: be shown in Table 1.
The different Oleum Curcumae Oxidation Resistance Test Results of table 1
Note: compare * * P ＜ 0.01 with blank group；##P ＜ 0.01 is compared with testing 2 groups,
#P ＜ 0.05.
Conclusion (of pressure testing): above-mentioned test shows, along with curzerene and the increasing of the content of eucalyptol
Adding, its antioxidant effect increases, and curzerene content more than 15%, eucalyptol content is more than
Its antioxidant effect of the Oleum Curcumae of 5% dramatically increases, and tests 2 groups there were significant differences or be the most aobvious
Write difference, illustrate that the new Oleum Curcumae of the present invention is significant.
Two, other pharmacology tests
Oleum Curcumae: furanodiene content 14.1%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content 7.6%, curdione contains
Amount 16.5%；Containing curzerene and eucalyptol, wherein curzerene content 15.1% in Oleum Curcumae,
The wherein content 5.0% of eucalyptol.
Preparation method: take Rhizoma Curcumae, cleans, cuts into slices, bake water content 40%, powder for 70 DEG C
Being broken into granularity is 10mm Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through
Vapor extraction 40 hours, collects the volatile oil in oil water separator, adds 3% volatile oil
The sodium chloride of weight, stirring, standing separation, take supernatant, obtain Oleum Curcumae.
Investigational agent is 1.: the Oleum Curcumae that said method obtains.
Investigational agent is 2.: said method obtains Oleum Curcumae and suppository (every piece of bolt that Borneolum Syntheticum is made
Agent 164mg Han Oleum Curcumae, Borneolum Syntheticum 150mg)
(1) anti-human papilloma virus (anti-HPV) test
Investigational agent: the suppository that 2. said method is obtained Oleum Curcumae by investigational agent and Borneolum Syntheticum is made
(every piece of suppository 164mg Han Oleum Curcumae, Borneolum Syntheticum 150mg)
Cell line
CaSki cell line, the human cervical carcinoma cell lines that HPV16 is positive, H8 cell line, people
Cervical squamous intraepithelial immortalized cell line, by Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
Biophysics room provides.
Experimental technique
1, cell is cultivated
CaSki cell is in the DMEM culture medium containing 5% hyclone, and H8 cell is containing
In the DMEM culture medium of 2% hyclone, investigational agent 37 DEG C, 5%CO contained by it2Saturated
Cultivate under humidity environment.
2, the selection of drug level
1 piece is dissolved in the DMEM culture medium of 44ml serum-free by reagent, contained by it
Investigational agent concentration is 3.95 × 104Mg/L is after solution is clarified, and aperture is the filter of 22 μm
Filtration sterilization, after subpackage, 4 DEG C keep in Dark Place standby.According to trial test result, experimental group is final
The investigational agent concentration added is respectively 19.77mg/L, 39.55mg/L, 79.09mg/L, right
The culture medium of equivalent is then added according to group.
3, cellular morphology is observed
Living cells is taken a picture: the cell of exponential phase is inoculated in 6 orifice plate culture plates, every hole
10×104Individual cell, in 6 well culture plates of each experimental group, a piece of coverslip is placed in every hole,
Form the cover plate with cell, after d2 cell attachment, be replaced by pastille culture medium, cultivate 3d
After, observation of cell form under inverted microscope.
4, the test medicinal liquid of MTT cytotoxicity assay method detection variable concentrations is to CaSki
With H8 cell growth inhibition situation
Cell is inoculated in six plate 96 porocyte culture plates, and 3000, every hole cell, d2 is more
Being changed to pastille culture medium, contained investigational agent concentration is respectively 19.77mg/L, 39.55mg/L,
79.09mg/L, and set normal control, parallel 5 holes of each concentration.37 DEG C, 5%CO2Full
With cultivation 24h under humidity, rear every hole adds 5mg/ml Methyl thiazoly tetrazolium assay (MTT) 22 μ l,
Continuing to cultivate 4h, abandoning supernatant, every hole adds dimethyl sulfoxide 150 μ l, 37 DEG C of cultivations
20min, microplate reader measures OD value, wavelength 492nm, continuous 6d, observes its growth feelings
Condition.
5, cell cycle detection
With the culture medium culturing 5d containing variable concentrations medicine, each medicine in six well culture plates
Parallel 3 holes of concentration, collect cell, prepare single cell suspension, PBS 2 times, pre-cooling
70% ethanol is fixed, and 4 DEG C of preservations are to be checked.RNA is starched through 0.01%RNA enzyme treated cell,
After 50mg/L propidium iodide DNA dyeing 30min, agglomerating carefully through 300 mesh screen removings
Born of the same parents, apply cell percentages and the cell of different phases in flow cytomery cell cycle
Apoptosis rate.Every part of specimen measures 5000 cells.Distinguish residing for cell according to DNA content
Cell Cycle.
6, mrna expression detection
Collecting the cell cultivating d5, TRIZOL extracts RNA after processing, and reverse transcription is
CDNA, expands HPV16E6, E7, expands β-actin as internal reference simultaneously, HPV16E6,
E7 primer (616bp): upstream 5`-TGACTTTGCTTTTCGGGATT3`, downstream:
5`-GAGAACAGATGGGGCACAC-3`.β-actin (552bp) primer sequence: on
Trip: 5`-ATCATGTTTGA-GACCTTCAACACC-3`, downstream: 5`-
CATGGTGGT-GCCGCCGCCAGACAG-3`.Amplification is: meets for 94 DEG C and becomes
Property 5min, 94 DEG C of degeneration 45s, 50 DEG C of renaturation 45s, 72 DEG C extend 1min, expand 30
Circulation；72 DEG C extend 5min；PCR primer 45 DEG C preservation.2% agarose gel electrophoresis is seen
Examine result, voltage 40V.(NIH software carries out electrophoretic band figure to application Scion Image 4.0
Semi-quantitative analysis as gray value.Testing gene electrophoretic band gray level ratio=band gray scale to be measured
Value/with a specimen internal reference β-actin band gray value.Each dosing sample at least detects 3
Secondary.
7, statistical method
Application SPSS 13.0 statistical software carries out one factor analysis of variance (ANOVA), t inspection
Deng.
Result
2. investigational agent acts on lower Cellular cycle and apoptosis rate
Investigational agent acts on H8 cell, and G1 phase cell increases, G2 phase slightly Leukopenia, but
It is equal no difference of science of statistics (P ＞ 0.05).Suppository acts on CaSki cell, and G1 phase cell increases
Add (P ＜ 0.05), G2 phase Leukopenia (P ＜ 0.05), S phase Leukopenia (P ＜ 0.05), make cell hinder
Stagnant in the G1 phase.Investigational agent acts on H8 cell, and dosing group apoptosis rate slightly increases, but without system
Meter learns difference (P ＞ 0.05), (being shown in Table 2).Suppository acts on CaSki cell, though dosing group nothing
Significantly apoptotic peak, but have significant difference (P ＜ 0.05) (being shown in Table 3)
2. table 2 investigational agent acts on lower H8 cell cycle distribution and apoptosis rate
2. table 3 investigational agent acts on lower CaSki cell cycle distribution and apoptosis rate
Table 4RT-PCR product electrophoretic band gray value
Test 2: Oleum Curcumae extracorporeal anti-tumor screening test research
Investigational agent: investigational agent 1. (Oleum Curcumae that said method obtains)
Experimental cell:
People's rectum cancer cell strain (HT-29), Human colorectal cancer cells strain (DLD-1): An Pu
Pool Bioisystech Co., Ltd give, source: ATCC (Unite States Standard biology product collecting center).
Human lung carcinoma cell (lymphatic metastasis) NCI-H292, people's height transfer lung carcinoma cell 95-D,
National People's Congress cell lung cancer cell NCI-H460 [H460], Non-small cell lung carcinoma cell
NCI-H1650, Non-small cell lung carcinoma cell A549, human liver cancer cell Hep G2, cervix uteri
Cancerous cell HeLa, human colon cancer cell HCT116, people malignant melanoma cell A-375,
Gastric carcinoma cells (undifferentiated) HGC-27, gastric carcinoma cells MGC80-3: source section of China
Shanghai life science institute of institute.
Experimental technique:
Adjusting cell density is 5 × 105～8 × 105Individual/mL, every hole refinement in 96 well culture plates
Born of the same parents' suspension 100 μ L (every hole about 5 × 104～8 × 104Individual).In 37 DEG C of volume fractions 5%
CO2In saturated humidity incubator.1. investigational agent is dissolved in culture medium, be configured to 7 grades dense
Degree group (makes its final concentration of 6400 μ g/ml, 3200 μ g/ml, 1600 μ g/ml, 800 μ
g/ml、400μg/ml、200μg/ml、100μg/ml).Every hole reagent adds 100 μ L；Cloudy
Property matched group reagent adding not dosing, often group sample sets 6 multiple holes.With medicine effect 48h
After, every hole adds 20 μ L MTT (final concentration 0.5mg/ml) liquid, after continuing to cultivate 4h,
Abandoning supernatant, every hole adds 150 μ L DMSO, and shaking table shakes up and all dissolves to crystallization, and enzyme joins
Instrument is 490nm in measuring wavelength, each additional examination fluid apertures average (T) is compared with control wells value (C),
Percent inhibition [suppression ratio=(1-T/C) × 100 of each concentration group are calculated with following equation
%], by IC50Software for calculation calculates the half-inhibition concentration (IC of cell50)；Experiment repeats
2 times.
Statistical method
All data all input EXCEL2003 and carry out statistical analysis, and each Sets of Measurement data are intended to
CalculateBetween group variable analysis is first carried out before comparing between each experimental group group
(F-test), when between group variable is neat while, compare employing Student-T inspection between group and (non-join
To T inspection) add up, when between group variable is uneven, use correction Student-T inspection
Carry out statistical analysis.
Result of the test is shown in (table 5)
Table 5
Under the conditions of this experimentation, investigational agent to pulmonary carcinoma, hepatocarcinoma, cervical cancer, colon cancer,
Gastric cancer and Humanmachine tumour have the strongest suppression and lethal effect, IC50?
162.2～657.3 μ g/ml, wherein the strongest to cervical cancer cell (Hela) effect, IC50For
162.2 ± 2.56 μ g/ml, and suppression ratio increases along with the increase of drug level, prompting test
Medicine has stronger cytotoxicity and significant concentration dependant, and the inhibitory action to cervical cancer
The strongest.Show that different tumor cells is had certain suppression special by the Oleum Curcumae that this method prepares
Different in nature and different sensitivity.
Test 3: In Vitro Anti mycologic test
Investigational agent: investigational agent 1. (Oleum Curcumae that said method obtains)
Comparison medicine: Clotrimazole vaginal tablets (manufacturer: the red pharmaceutcal corporation, Ltd in the Wanshan Mountain, Guangxi)
Strain
Candida albicans (containing Quality Control strain ACTT10231, totally 30 strain), smooth candida coccus
(30 strain), Quality Control strain is purchased from Guangdong Culture Collection, and clinical strain is by Hainan medical science
Clinical laboratory of Affiliated Hospital of institute and clinical laboratory of Hainan People's Hospital provide.
Test method
Minimal inhibitory concentration (MIC) assay method: use coubling dilution to make pastille and put down
Plate, by every kind of tested bacterium solution, instills corresponding flat board, flat board is inverted, then after dry again
Put into cultivation 48h in 35 DEG C of incubators of incubator.Making without each strain culturing of medicine plating
Positive control.
Minimal bactericidal concentration (MBC) assay method: without medicine MH agar plate bottom surface glass
" # " on glass Oil Painting Pen, each flat board is divided into 12 or 16 regions, and (each region will training
Support different strains), and write corresponding strain number.Tested bacterium is cultivated at pastille MH meat soup
After 24h, taking-up shakes up, and draws 10ul with sample loading gun, according to strain number, instills without medicine
In the region that flat board is numbered accordingly, again flat board is inverted after dry, is then placed in 35 DEG C of cultivations
Case is cultivated 48h.Positive control area mark " positive ", inoculation is without bacterial strain in medicine broth tubes.
Result of the test is shown in Table 6, table 7
The MIC measurement result of table 6 In Vitro Anti mycologic test
The MBC measurement result of table 7 In Vitro Anti mycologic test
Under the conditions of this experimentation, investigational agent has obvious anti-candida albicans in vitro
With smooth candida coccus effect, its antifungal activity and Clotrimazole vaginal tablets no significant difference
Preparation embodiment
Embodiment 1
Oleum Curcumae: furanodiene content 14.1%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content 7.6%, curdione contains
Amount 16.5%；Containing curzerene and eucalyptol, wherein curzerene content 15.1% in Oleum Curcumae,
The wherein content 5.0% of eucalyptol.
Preparation method: take Rhizoma Curcumae, cleans, cuts into slices, bake water content 40%, powder for 70 DEG C
Being broken into granularity is 10mm Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through
Vapor extraction 32 hours, collects the volatile oil in oil water separator, adds 3% volatile oil
The sodium chloride of weight, stirring, standing separation, take supernatant, obtain Oleum Curcumae.
Embodiment 2
Oleum Curcumae: furanodiene content 13.7%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content 8.9%, curdione
Content 18.9%；Containing curzerene and eucalyptol, wherein curzerene content 25.3% in Oleum Curcumae,
The wherein content 14.7% of eucalyptol.
Preparation method: take Rhizoma Curcumae, cleans, cuts into slices, bake water content 50%, powder for 85 DEG C
Being broken into granularity is 10mm Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through
Vapor extraction 38 hours, collects the volatile oil in oil water separator, adds 6% volatile oil
The sodium chloride of weight, stirring, standing separation, take supernatant, obtain Oleum Curcumae.
Embodiment 3
Oleum Curcumae: wherein furanodiene content 11.8%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content 9.9%, Rhizoma Curcumae two
Ketone content 19.5%；Containing curzerene and eucalyptol, wherein curzerene content in Oleum Curcumae
27.8%, the wherein content of eucalyptol big 20.4%.
Taking Rhizoma Curcumae, clean, cut into slices, bake water content 60% for 90 DEG C, being ground into granularity is 15mm
Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through vapor extraction 40 hours,
Collect the volatile oil in oil water separator, add 10% volatile oil weight anhydrous sodium sulfate, stir
Mix, standing separation, take supernatant, obtain Oleum Curcumae.
Above-described embodiment Oleum Curcumae has the pharmacologically active of prior art, and than prior art
Oleum Curcumae has more preferable pharmacological action；
Above-described embodiment Oleum Curcumae is " effective site " concept of the field of Chinese medicines.
So-called effective ingredient in Chinese, refers to as in a herb or compound Chinese medicine extract
The content of class or a few class chemical composition reaches more than the 50% of total extract, and a class or several
The known chemical composition of class is considered as effective ingredient, and the mixture of this class or a few constituents is i.e.
It is considered as effective site.
Furanodiene: No. CAS: 19912-61-9；3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-: No. CAS: 6902-91-6；
Curdione: No. CAS: 13657-68-6；Curzerene: No. CAS: 17910-09-7；Eucalyptus globulus
Olein: No. CAS: 470-82-6.
Above by the description of detailed description of the invention, the invention will be further described, but this
Being not limitation of the present invention, those skilled in the art are according to the basic think of of the present invention
Thinking, various modifications may be made or improves, but basic without departing from the present invention
Thought, the most within the scope of the present invention.
Claims (12)
1. an Oleum Curcumae, it is characterised in that Oleum Curcumae contain Rhizoma Curcumae sesquiterpenoids content more than or equal to 40% and
Less than or equal to 75%, wherein Rhizoma Curcumae sesquiterpene is furanodiene, curdione, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-and curzerene.
A kind of Oleum Curcumae the most according to claim 1, wherein furanodiene content is little more than or equal to 8%
In equal to 18%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content is more than or equal to 5% less than or equal to 10%, and curdione content is more than or equal to
10% is less than or equal to 20%.
A kind of Oleum Curcumae the most according to claim 1, wherein in Oleum Curcumae, curzerene content is more than or equal to
15% is less than or equal to 35%.
4. according to the Oleum Curcumae described in any one of claim 1-3, wherein containing eucalyptol, eucalyptus globulus in Oleum Curcumae
The content of olein is less than or equal to 21% more than or equal to 5%.
5. an Oleum Curcumae, it is characterised in that furanodiene, curdione, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-and Rhizoma Curcumae in Oleum Curcumae
Alkene content is more than or equal to 40% less than or equal to 75%, and wherein furanodiene content is less than or equal to more than or equal to 8%
18%, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content is more than or equal to 5% less than or equal to 10%, and curdione content is less than more than or equal to 10%
Equal to 20%；Containing curzerene and eucalyptol in Oleum Curcumae, wherein curzerene content is less than more than or equal to 15%
Equal to 35%, wherein the content of eucalyptol is less than or equal to 21% more than or equal to 5%.
6. according to a kind of Oleum Curcumae described in any one of claim 1-5, it is characterised in that be former with Oleum Curcumae
The pharmaceutical preparation of material preparation.
A kind of Oleum Curcumae the most according to claim 6, its pharmaceutical formulations include diseases of eye, ear, nose and throat, pudendum,
The external preparation such as vagina, crissum, rectum, skin, mucosa, or tablet, capsule, granule etc. are oral
Preparation, or injection etc..
A kind of Oleum Curcumae the most according to claim 6, its pharmaceutical formulations includes: eye drop, an ear
Agent, lotion, vaginal suppository, rectal suppository, ointment, capsule, soft capsule, tablet, effervescent tablet,
The externals such as granule, oral liquid, gel, membrane, foam or oral formulations or injection.
9. according to a kind of Oleum Curcumae described in claim 1-5, its pharmaceutical formulations raw material be Oleum Curcumae be 164
Weight portion, Borneolum Syntheticum is 150 weight portions.
A kind of Oleum Curcumae the most according to claim 8, it is characterised in that described pharmaceutical preparation is in preparation
Application in the medicine of prevention and/or treatment human papilloma virus infection.
11. a kind of Oleum Curcumae according to claim 8, it is characterised in that described pharmaceutical preparation is in prevention
And/or treat infection, the diseases of eye, ear, nose and throat senses such as various virus, antibacterial, mycete, chlamydia, mycoplasma, infusorian
Dye, vulvitis, various vaginitis, mixed infective vaginitis, skin infection, mucosal infections, hemorrhoid sense
Application in the medicines such as dye, constipation, cervicitis, adnexitis, CIN I-CINIII, cervical cancer, antioxidation.
12. according to a kind of Oleum Curcumae described in any one of claim 1-5, it is characterised in that preparation method is:
Taking Rhizoma Curcumae, clean, cut into slices, bake water content 40%-60% for 55-90 DEG C, being ground into granularity is 5-15mm
Rhizoma Curcumae granule；Take Rhizoma Curcumae granule and put in extraction pot, airtight, it is passed through vapor extraction 24-40 hour, collects oil
Volatile oil in water separator, adds sodium chloride or the anhydrous sodium sulfate of 3%-10% volatile oil weight, stirs,
Standing separation, takes supernatant, obtains Oleum Curcumae.
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| CN114149393A (en) * | 2021-12-10 | 2022-03-08 | 海南碧凯药业有限公司 | Sesquiterpene compound and preparation method thereof |
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