CN101874819A - Application of ligusticum chuanxiong hort or extract thereof in preparing anti-aging products - Google Patents
Application of ligusticum chuanxiong hort or extract thereof in preparing anti-aging products Download PDFInfo
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- CN101874819A CN101874819A CN2009102436993A CN200910243699A CN101874819A CN 101874819 A CN101874819 A CN 101874819A CN 2009102436993 A CN2009102436993 A CN 2009102436993A CN 200910243699 A CN200910243699 A CN 200910243699A CN 101874819 A CN101874819 A CN 101874819A
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Abstract
The invention discloses a new application of ligusticum chuanxiong hort or an extract thereof. The ligusticum chuanxiong hort or the extract thereof has the following four new purposes: (1) application in preparing anti-aging products; (2) application in preparing gene expression products for regulating caloric restriction pathways; (3) application in preparing gene expression products for regulating insulin signaling pathways; and (4) application in preparing caenorhabditis elegans life extenders. In the invention, the caenorhabditis elegans is used as a model organism, the influence of CXE (Ligusticum Chuangxiong Extract) on caenorhabditis elegan life is observed by taking a whole animal experiment as a detection means, the CXEs of a plurality of concentrations are all found to have a role or trend of life prolongation to the caenorhabditis elegan, wherein the concentration of 25mg/L has an optimal life prolongation effect, and the average life and the longest life are both prolonged by about 15 percent. Molecular mechanism research for life prolongation by utilizing the known relevant caenorhabditis elegan aging gene and adopting a qRT-PCR (Quantitative Real Time-Polymerase Chain Reaction) method shows that the CXE prolongs the life through intervening an Insulin/IGF-1 pathway and a feeding control (caloric restriction) pathway, thereby providing new content and direction for the molecular mechanism research of the drug and also providing a new ideal and means for the research of delaying aging by using the traditional Chinese medicine.
Description
Technical field
The present invention relates to the new purposes of Rhizoma Chuanxiong or its extract slow down aging, particularly Rhizoma Chuanxiong or its extract application in the preparation anti-aging products.
Background technology
Along with the aged tendency of population situation is severe day by day, old and feeble mechanism, slow down aging drug research have become the focus and emphasis problem of life sciences.Old and feeble mechanism is complicated, theory is various, up to now, explain nearly kind surplus in the of 200 of the theory of old and feeble mechanism or theory, comprising: genetic control is said, the cell telomere is said, radical damage is said, somatic mutation is said, immunologic derangement is said, (Fossel M.Cell Senescence in HmanAging and Disease.In:Harman D. (Ed.): Increasing healthy lifespan such as mitochondrial injury theory, conventionalmeasures and slowing the innate aging process.ANN N Y ACAD SCI, 2002,959:14～23).Wherein the approach of the slow down aging of comparatively generally acknowledging mainly contains two, the one, insulin signaling pathway (AntebiA.Inside insulin signaling, communication is key to long life[J] .SciAgingKnowledge environ, 2004, (23): pe25); The 2nd, heat restriction approach (Koen Houthoofd, JacquesR.Vanfleteren.The longevity effect of dietary restriction in Caenorhabditis elegans[J] .Experimental Gerontology, 2006 (41): 1026-1031).
Rhizoma Chuanxiong (Ligusticum chuanxiong Hort.) is Chinese medicine commonly used, and the beginning is stated from Shennong's Herbal, and acrid in the mouth warm in nature, ancient Chinese medicine doctor are used for blood-activating and qi-promoting, wind-expelling pain-stopping more.Version in 2005 is " in the 564 kinds of prescribed preparations and single preparation that Chinese pharmacopoeia I portion records, that uses Rhizoma Chuanxiong has 85 kinds, account for pharmacopeia and record 15% of Chinese patent medicine, Rhizoma Chuanxiong has promoted the exploitation and the related mechanism of its new pharmacodynamic feature are studied in clinical extensive use.Although Rhizoma Chuanxiong extensive use and obtain curative effect preferably in the treatment of Senile disease does not appear in the newspapers to the research of its delaying senility function.
Caenorhabditis elegans (Caenorhabditis elegans, be called for short nematicide) be a kind of simple relatively multicellular organism, hermaphroditism, be the model organism (H.Gershon of the old and feeble brilliance of research, D.Gershon.Caenorhabditiselegans-a paradigm for aging research:advantages and limitations.Mechanisms ofAgeing and De_elopment 123 (2002) 261-274), compare with other species and to have the following advantage that hardly matches: 1) life cycle short (average out to 18～21 days), the filial generation number is many; 2) cultivate observation and easy to operate easily; 3) differentiation of cell height and cell lineage are clear; 4) be first animal of finishing genome sequencing, its genetic background is clear, and old and feeble Mechanism Study is clear and definite relatively.5) better with human homology's property, at least 40% has tangible congener in the gene of coding in human genome.
Not only can further excavate the value of Chinese medicine based on the Chinese medicine of this model organism exploitation slow down aging of nematicide, and can also make contributions prolonging the human life-span.
Summary of the invention
The new purposes that the purpose of this invention is to provide Rhizoma Chuanxiong or its extract.
The new purposes of Rhizoma Chuanxiong provided by the present invention or its extract has following four:
1) Rhizoma Chuanxiong or the Rhizoma Chuanxiong extract application in the preparation anti-aging products;
2) Rhizoma Chuanxiong or the Rhizoma Chuanxiong extract application in preparation adjusting heat restriction pathway gene expression product;
3) Rhizoma Chuanxiong or the Rhizoma Chuanxiong extract application in preparation adjusting insulin signaling pathway gene expression product;
4) Rhizoma Chuanxiong or the Rhizoma Chuanxiong extract application in preparation nematicide life extender.
Above-mentioned Rhizoma Chuanxiong extract can be the Rhizoma Chuanxiong water extract.Wherein, the Rhizoma Chuanxiong water extract can obtain from commercial channels.The Rhizoma Chuanxiong water extract also can be prepared according to conventional method, obtain as available following method: choose medical material Rhizoma Chuanxiong (dry rhizome of samphire Rhizoma Chuanxiong Ligusticum chuanxiong Hort.) fragment, add 5～10 times of pure water, heating, boil the back and keep slight boiling condition to continue heating 0.5～1 hour, filter then and collect filtrate 1; Then add 5～10 times of pure water in filtering residue again, heating is boiled the back and is kept slight boiling condition to continue heating 0.5～1 hour, filters then and collects filtrate 2; With filtrate 1 and filtrate 2 mixings, drying obtains the Rhizoma Chuanxiong water extract.
Above-mentioned 1), 2) and 3) three kinds of products all can be medicine, health product or food additive etc.
Described adjusting heat restriction pathway gene is expressed and can be realized by raising the skn-1 expression of gene; The gene expression of described adjusting insulin signaling pathway can be by raising the hsp-70 expression of gene, and downward modulation akt-2 and/or tub-1 expression of gene realize.
Wherein said nematicide specifically can be Caenorhabditis elegans (Caenorhabditis elegans).
When needing, in the said goods, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc.
Slow down aging medicine, health product with the Rhizoma Chuanxiong extract preparation can be made various ways such as injection, tablet, powder, granule, capsule, oral liquid, unguentum, cream.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
The present invention adopts this model organism of nematicide, as detection means, observation CXE finds that to the influence in nematicide life-span a plurality of concentration C XE all have the lengthen the life effect or the trend of lengthening the life to nematicide with the whole animal experiment, the best results of lengthening the life of 25mg/L wherein, average life and maximum life all prolong about 15%.Utilize known nematicide SAG, the Study on Molecular Mechanism that adopts the qRT-PCR method to lengthen the life, show that CXE is by intervening the Insulin/IGF-1 path and limitting the food path and life-saving, for the Study on Molecular Mechanism of this medicine provides new content and direction, also new thinking and means are provided for the research of Chinese medicine slow down aging.
Description of drawings
Fig. 1 is the nematicide survival curve figure among the embodiment 1.
Fig. 2 is the influence that Rhizoma Chuanxiong extract is expressed the nematicide SAG among the embodiment 2, compares with act-1,
1)P＜0.05,
2)P＜0.01.
Fig. 3 influences the sketch map that the nematicide SAG is expressed for Rhizoma Chuanxiong extract.
The specific embodiment
Experimental technique described in the following embodiment if no special instructions, is conventional method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels.
1 material
1.1 bacterial strain, nematicide
Bacterial strain E.coli OP50, wild type Caenorhabditis elegans Bristol (N
2) all available from nematicide heredity center (CGC).
1.2 medicine and reagent
Rhizoma Chuanxiong extract is prepared according to following method: choose medical material Rhizoma Chuanxiong (dry rhizome of samphire Rhizoma Chuanxiong Ligusticumchuanxiong Hort.) fragment, add 6 times of pure water, heating is boiled the back and is kept slight boiling condition to continue heating 40 minutes, filters then and collects filtrate 1; Then add 6 times of pure water in filtering residue again, heating is boiled the back and is kept slight boiling condition to continue heating 40 minutes, filters then and collects filtrate 2; With filtrate 1 and filtrate 2 mixings, the powder that drying obtains is the Rhizoma Chuanxiong water extract.According to this method, 20 gram crude drugs prepare 1 gram Rhizoma Chuanxiong water extract.
Agar powder (Nippon Union K. K), 5-fluorouracil (5-Fluorouracil, 5-Fu) (Sigma), peptone (peptone), tryptone (OXOID), SYBR Green Realtime PCR Master Mix (Toyobo), TRIzol reagent (Invitrogen), reverse transcription test kit (Promega), all the other reagent are homemade.
1.3 instrument
MIR-553 constant incubator (Japanese SANYO company), S6E stereomicroscope (German Leica company), XTS30-D stereomicroscope (Tyke, Beijing Instr Ltd.), Rotergene 3000 quantitative real time PCR Instruments (Australian Corbett company) grads PCR instrument (German Eppendorf company), desk type high speed refrigerated centrifuger (German Eppendorf company), G:BOX full automatic gel imager (Britain Syngene company), MLS-3780 high-pressure sterilizing pot (Japanese SANYO company) etc.
2 methods
2.1 test grouping
Be made as matched group and 4 CXE medication groups, medicine is water-soluble, and being made into from low to high, concentration is followed successively by: 12.5,25,50 and the drug solution of 100mg/L.
2.2 the routine of nematicide is cultivated
Hermaphroditism nematicide N2, be inoculated in coating E.Coli OP50 the standard nematode culture medium (nematode growthmedium, NGM), in 20 ℃ of constant temperature culture.Every liter of culture medium contains 3g sodium chloride, 20g agar powder, 2.5g peptone, 1ml cholesterol alcoholic solution (concentration is 5mg/ml), 1ml CaCl
2Solution (1mol/L), 1ml MgSO
4Solution (1mol/L) and 25ml KPO
4Solution (pH=6).
2.3 life experiment
(solute of every liter of M9 solution is Na 2.3.1 synchronization is with M9 buffer solution
2HPO
45.8g, KH
2PO
43.0g, NaCl 0.5g, NH
4Cl 1.0g) young adult is washed till in the aseptic EP pipe, add an amount of lysate (0.5% sodium hypochlorite, 0.mol/L cracking nematicide sodium hydroxide), put that centrifugal on the low speed centrifuge (3000rpm 1min), abandons supernatant, reuse M9 buffer solution flushing nematicide 2 times, centrifugal abandon behind the supernatant drawn EP pipe bottom nematicide with liquid-transfering gun and dripped aseptic area in NGM, and the intravital germ cell of cracked nematicide develops into L4 phase larva substantially after about 48 hours, finishes synchronization and is used for life experiment.
2.3.2 contain 12.5mg/L 5-Fu in the medium preparation matched group culture medium, contain the CXE of 12.5mg/L 5-Fu and variable concentrations in the medicine group culture medium.
2.3.3 the synchronized L4 nematicide of life experiment picking every group average 80～110, has 15 approximately in each plate in each group culture medium.Constantly begin to calculate nematicide survival natural law from shifting, shift and be designated as life experiment the 0th day (day 0) same day.Check nematicide every day, record verge of death borer population is rejected unexpected death, escape and capsule line-transect worm.The dead criterion of nematicide: touch polypide with platinum filament, do not have any reaction, promptly be judged as death.More than experiment all repeats twice.
2.4 statistical analysis
Adopt the ANOVA variance test to analyze the life experiment result, quantitative data is represented with X ± S; Adopt two sample t method of inspection comparative analysis genes of interest differential expressions.
3 results
Compare with matched group, in the 1st experiment, 12.5,25,50,100mg/L CXE all can the significant prolongation nematicide average life (be respectively 15.4%, 29.7%, 23.3%, 17.1%, P＜0.01), the maximum life (9.4% of 25mg/L CXE energy significant prolongation nematicide, P＜0.01), 50,100mg/L CXE also has similar effect (1.8%, 4.8%); The 2nd time experimental result is similar to the 1st time, sees Table 1.Comprehensive twice experimental result, 25,50,100mg/L CXE all can the significant prolongation nematicide average life (being respectively 15.7%, 9.1%, 6.2%, P＜0.01) and maximum life (being respectively 15.0%, 6.8%, 6.6%, P＜0.01).
Table 1 variable concentrations Rhizoma Chuanxiong extract is to the influence in nematicide life-span (X ± S)
Annotate: maximum life is the average life of the last existence 15% of colony; Compare with matched group,
1)P＜0.05,
2)P＜0.01
Comprehensive twice experimental result done the survival curve (see figure 1) to the omnidistance survival condition of nematicide and shown, and 25,50,100mg/L CXE all can make the survival curve of nematicide move to right, and is particularly remarkable with 25mg/L.
The molecular mechanism that embodiment 2, Rhizoma Chuanxiong extract prolong the Caenorhabditis elegans life-span
1 material, instrument are with embodiment 1.
2 real-time fluorescence quantitative PCRs (qRT-PCR) experiment grouping: be made as matched group and medication group (the strongest group of the effect of lengthening the life in the life experiment, concentration is the CXE solution of 25mg/L).
3 Study on Molecular Mechanism of lengthening the life
3.1 the extraction of the total RNA of nematicide and cDNA are synthetic
About 1000 the L4 phase nematicides of matched group and each picking of medication group are raised after 10 days in culture medium, collect respectively and respectively organize nematicide, with TRIZOL method extracted total RNA, RNase free DNase enzyme action, agarose gel electrophoresis identifies that RNA does not have obvious degradation behind the TRIZOL method purification, and is frozen in-80 ℃.Get the total RNA of 2 μ g, carry out reverse transcription with MML-V reverse transcriptase by specification.
3.2 primer is synthetic
To gene relevant in the known nematicide with aging, sequential design synthetic pcr primer thing (Beijing AudioCodes biotechnology Co., Ltd) according to the wormbase announcement, sequence is as follows: the akt-2 forward primer: 5 '-ATTCAGCGAAGCACGAAC-3 ', downstream primer: 5 '-CACAACCCAAAGTCAGTG-3 '; The hsp-70 forward primer: 5 '-GGGTACTACGTACTCATG-3 ', downstream primer: 5 '-ATCACCAACGAGTCTCTC-3 '; The skn-1 forward primer: 5 '-AGTGACTGCAACAGCTAC-3 ', downstream primer: 5 '-AGAAACGAGACTCGTGAG-3 '; The tub-1 forward primer: 5 '-TCCAATTGCTCAACCACG-3 ', downstream primer: 5 '-AGGGAGTACTTCACTTGG-3 ', and with act-1 as the internal reference thing, its forward primer: 5 '-AAGACCACGTCATCAAGG-3 ', downstream primer: 5 '-TTCTCCATATCATCCCAGTT-3 '.
3.3 real-time fluorescence quantitative PCR detects matched group and medication group destination gene expression difference
Adopt chimeric fluorescence method testing goal gene expression difference.Adopt 20 μ L reaction systems (template cDNA 2 μ g supply 20 μ L systems with deionized water for SYBR GREENMaster Mix 10 μ L, forward primer, each 1 μ L of downstream primer (10 μ mol)).The PCR reaction condition: 94 ℃ of pre-degeneration 5min, 94 ℃ of degeneration 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 40 circulations.Each sample is all done 3 secondary pollutants and is learned repetition.Adopt 2
-△ △ CtMethod is (referring to document: Livak, K.J.; Schmittgen, T.D.Analysis of relative gene expressiondata using real-time quantitative PCR and the 2 (△ △ Ct) method.Methods, 2001,25:402-408) amplification curve is analyzed.
4, statistical analysis
Adopt the ANOVA variance test to analyze the life experiment result, quantitative data is represented with X ± S; Adopt two sample t method of inspection comparative analysis genes of interest differential expressions.
5, Rhizoma Chuanxiong extract is to the influence of SAG expression
25mg/L CXE intervened after 10 days, and SAG is expressed and changed in the nematicide body, saw Table 2, Fig. 2.Act-1 compares with house-keeping gene, and hsp-70, skn-1 gene expression are obviously raised after the medication, and akt-2, tub-1 gene expression significantly reduce.
Each genes of interest Ct of table 2 matched group and medication group and-△ △ Ct value
Annotate: the Ct value refers to the period that the fluorescence signal in each reaction tube is experienced when arriving the thresholding of setting.
Numerical value is 3 wherein 1 results in the repetition in the table.
In the Mechanism Study of aging, insulin and insulin-like growth factor-i (Insulin/IGF-1) signal path is a high conservative (comprising nematicide, fruit bat, mice and even primate), the most detailed life-span of research regulation and control link: normal condition rolls off the production line and discharges multiple Insulin/IGF sample peptide in the polypide, the receptors bind of these peptides and cell surface and produce a series of signal cascade reaction in cell finally suppresses it by phosphorylation DAF-16 to enter nucleus; When external environment changes (as hunger, insect population is overstocked) the Insulin/IGF-1 signal path is suppressed, cause DAF-16 to go into nuclear not by phosphorylation, the effect of DAF-16 performance transcription factor in nucleus and promote many resistance expression of gene and bring into play the effect of life-saving, therefore changing with this signal path Expression of Related Genes can influence senescence process, regulation and control life-span length directly or indirectly.The akt-2 encoded protein is the congener of serine/threonine kinase Akt/PKB, and it is positioned at the DAF-16 upstream, and energy phosphorylation DAF-16 suppresses it and enters nucleus, and therefore, the akt-2 down regulation of gene expression can prolong the nematicide life-span.The heat shock protein of hsp-70 coding is a kind of molecular chaperones, helps albumen correctly to be folded into native conformation, to life-span performance positive control.In the online polypide, tub-1 is positioned at the daf-16 upstream, and it is low expresses and having lengthen the life effect and this effect and depend on DAF-16.The albumen of skn-1 gene code is the congener of Nrf2, and it is positioned at Insulin/IGF-1 signal path downstream, this signal path kinases AKT-1, energy phosphorylation SKN-1 such as AKT-2 and directly suppress its expression; SKN-1 can improve resisting stress capability, prolongs the nematicide life-span, and its effect of lengthening the life is independent of DAF-16.Limit food (being the heat restriction) can cause longevity in many species, and can reduce and old and feeble relevant disease incidence.Limit food can promote skn-1 to express, and improves the Repiration of nematicide and prolongs the nematicide life-span.Said gene to the sketch map of nematicide life-span influence as shown in Figure 3.
[0069] among the present invention, CXE is by raising the gene that has positive regulating and controlling effect in the Insulin/IGF-1 signal path to lengthening the life, as hsp-70; Reduce the gene in part negative regulation life-span in this path, bring into play the effect of lengthening the life as akt-2, tub-1; In addition, limit food (heat restriction) is a generally acknowledged fabulous life-prolonging method, and our experiment shows that CXE causes longevity by raising limit food important controlling gene skn-1.Simultaneously, CXE has reflected also that to the multiple regulatory mechanism in life-span Chinese medicine has the characteristics of many target spots, manifold effect to the influence of aging.
Sequence table
<160>10
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
attcagcgaa?gcacgaac 18
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
cacaacccaa?agtcagtg 18
<210>3
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
gggtactacg?tactcatg 18
<210>4
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
atcaccaacg?agtctctc 18
<210>5
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
agtgactgca?acagctac 18
<210>6
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
agaaacgaga?ctcgtgag 18
<210>7
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>7
tccaattgct?caaccacg 18
<210>8
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>8
agggagtact?tcacttgg 18
<210>9
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>9
aagaccacgt?catcaagg 18
<210>10
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>10
ttctccatat?catcccagtt 20
Claims (10)
1. Rhizoma Chuanxiong or the Rhizoma Chuanxiong extract application in the preparation anti-aging products.
2. application according to claim 1 is characterized in that: described product is medicine, health product or food additive.
3. application according to claim 1 and 2 is characterized in that: described Rhizoma Chuanxiong extract is the Rhizoma Chuanxiong water extract.
4. Rhizoma Chuanxiong or the Rhizoma Chuanxiong extract application in the product of restriction pathway gene expression of preparation adjusting heat or the gene expression of adjusting insulin signaling pathway.
5. application according to claim 4 is characterized in that: described product is medicine, health product or food additive.
6. according to claim 4 or 5 described application, it is characterized in that: described adjusting heat restriction pathway gene is expressed by raising the skn-1 expression of gene and is realized; The gene expression of described adjusting insulin signaling pathway is by raising the hsp-70 expression of gene, and downward modulation akt-2 and/or tub-1 expression of gene realize.
7. according to arbitrary described application in the claim 4 to 6, it is characterized in that: described Rhizoma Chuanxiong extract is the Rhizoma Chuanxiong water extract.
8. Rhizoma Chuanxiong or the Rhizoma Chuanxiong extract application in preparation nematicide life extender.
9. application according to claim 8 is characterized in that: described nematicide is a Caenorhabditis elegans.
10. according to Claim 8 or 9 described application, it is characterized in that: described Rhizoma Chuanxiong extract is the Rhizoma Chuanxiong water extract.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105309387A (en) * | 2014-06-04 | 2016-02-10 | 兰州红菌生物技术有限责任公司 | Kit for screening anti-aging drugs and using method thereof |
| CN108849771A (en) * | 2018-08-20 | 2018-11-23 | 扬州大学 | Peony bud is in the application extended in the nematode service life |
-
2009
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105309387A (en) * | 2014-06-04 | 2016-02-10 | 兰州红菌生物技术有限责任公司 | Kit for screening anti-aging drugs and using method thereof |
| CN108849771A (en) * | 2018-08-20 | 2018-11-23 | 扬州大学 | Peony bud is in the application extended in the nematode service life |
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