CN102038702A - Anti-ageing application of hesperidin - Google Patents

Abstract

The invention provides the application of hesperidin for preparing a medicine for delaying senility and treating aging diseases. The hesperidin medicine comprises a medicine excipient or a carrier which are pharmaceutically acceptable. The invention also provides the application of the hesperidin for preparing a food additive. A research finds that in a yeast level, the hesperidin regulates Snk7 activity to influence UTH1 gene expression, thereby prolonging yeast service life. In a human cell SH-5YSY level, the hesperidin increases an SIRT1 genetic transcription level, enzymic activity and protein expression and also increases the genetic transcription level and the protein expression of FOXO3a, and the SIRT1 and the FOXO3a play an important role for prolonging service life. The invention expands the application of the hesperidin in medicine preparation and food additives.

Description

A kind of defying age of Hesperidin is used

Technical field

The invention belongs to the application of plant, relate to the application of Hesperidin, relate in particular to Hesperidin in preparation slow down aging and treatment senile disease medicine and the application in the preparation alimentation composition.

Background technology

Along with the improvement of medical condition, human average life is constantly prolonging, and the problem of an aging population is also deepened gradually simultaneously.By the end of the year 2006, national over-65s population 10,419 ten thousand people account for 7.9% of country's total population, have risen 0.2 percentage point than the last year.Expected when the year two thousand forty, population reached peak-peak, China's over-65s aging population are about 3.3 hundred million, approach whole world aging population sum in 1980.

The aging of population brings is exactly that the sickness rate of senile disease constantly increases, as Alzheimer's disease, diabetes, cardiovascular disease or the like.According to statistics, the prevalence of Alzheimer's disease is more than 5%, 80 years old to be 33% among the crowd of global over-65s, and this means in the old people more than 80 years old have 1/3rd people to suffer from this disease.In China, patient's sum of diabetes expects 2010 and will increase sharply to 8,000 ten thousand to 100,000,000 people, accounts for 10% of population.Cardiovascular disease all is human killer all the time, and it is a world's second largest fatal disease.The annual cardiovascular diseases's of China medical expense brings heavy burden for China's medical system up to 1,100 hundred million yuans.These senile diseases have reduced patient's quality of life, have brought serious burden to the household.In order to improve constantly our life quality, alleviate the society and the burden of family, seek the focus that antiaging agent has efficiently become research.Along with to aging research progressively deeply, people also rise to cell and molecular level from the animal level to the understanding of aging.Relevant old and feeble theory also has in progressively perfect, main theory: free radical and oxidative stress are old and feeble theoretical; Telomere is old and feeble theoretical; Gene is old and feeble theoretical; Entropy increases theory etc.The oxidative stress theory is an important old and feeble theory. flavone compound has good antioxidant activity.Flavone compound has four classes: flavanones, flavones, flavonols, and anthocyanins.Flavone compound not only has good The Ecological and Physiological Effect, and good economic benefits are also arranged. and flavone compound is widely used in food and pharmaceuticals industry. and the plant polyphenol resveratrol can prolong yeast, nematicide, the life-span of fruit bat and mice.Some other antidotal chemical compound also is widely studied.Studying these chemical compounds not only can be for antiaging agent screening provides a large amount of data, but also provides the foundation for research defying age theory.

Hesperidin is to be present in Fructus Citri Limoniae, grapefruit, mandarin orange, the bioflavonoids in citron and the orange.Its structure is the grape glycosylated compound that contains hesperetin flavanone nuclear (3 ', 5 ', 5-trihydroxy-4 '-methoxyl group flavanone), the polyglycoside of 6-O-.alpha.-L-rhamnosyl-D-glucose. partly by the hydroxyl on 7 carbon of hesperetin with its covalent bond.Concrete structure is as follows:

Hesperidin can suppress to be derived from the destruction of the chondrocyte substrate of cartilage, therefore is described to cartilage protection medicine (patent application No EP633022); It can be used as the medicine (German patent application No DE19742 025) of prevention cutaneous pigmentation; As HMG-CoA reductase inhibitor (US Patent No 5763414); As CoA-cholesterol-O-inhibitors, macrophage-lipid complex is cumulative inhibitor (international monopoly WO99/21549) on arterial wall; Strengthen capillary tube in addition in addition, reduce permeability, antiplatelet aggregation, antiinflammatory, antiviral and bringing high blood pressure down and the cholesterol isoreactivity.Studies show that Hesperidin can be caught free radical, but concrete mechanism is still indeterminate, and does not still have the research of the concrete mechanism of Hesperidin defying age.

Summary of the invention

The purpose of this invention is to provide the application of Hesperidin in preparation slow down aging and treatment senile disease medicine.The Hesperidin medicine of the present invention's preparation comprises preparation allowable pharmaceutical excipients or carrier.

Described Hesperidin medicine can be made the dosage form of intestinal or non-intestinal combination medicine by this formulation art known method.Dosage form mainly comprises liquid preparation, granule, tablet, electuary, soft gelatin capsule, capsule, drop pill or injection.The form of medication of preparation mainly comprises oral administration or drug administration by injection.

UTH1 is the yeast aging gene, discovering has many startups site with can and prolonging yeast life-span UTH1 promoter to anti-oxidation stress after the UTH1 gene elmination, thereby many transcription factor combination with it, regulate the UTH1 expression of gene. studies show that Mot3, Yap1 and Skn7 are relevant with oxidative stress, and plant polyphenol can be regulated the expression of UTH1 by Skn7.

The present invention discovers that further Hesperidin can (SH-SY5Y) increase SIRT1 mRNA transcriptional level on the human body cell level, the proteic expression of SIRT1 enzymatic activity and SIRT1, and can increase FOXO3a gene and proteic expression.

SIRT1 plays an important role delaying the mammalian cell aging; nearest intercensal study shows that FOXO3a and mankind aging have closely and gets in touch; FOXO3a is an important protein on the insulin/IGF1 signal path; can be by the SIRT1 deacetylation, the more important thing is that the increase of FOXO3a enzyme activity can play the effect of neuroprotective unit cell.

Another object of the present invention provides Hesperidin as the application of active component in the preparation food additive.The food additive that contains Hesperidin is meant and contains one of Hesperidin or derivatives thereof and constitute food compositions or dietary supplement compositions that the purposes of this alimentation composition is a slow down aging, prevention or treatment senile disease.

Is that the alimentation composition of the present invention of feature can use with numerous food compositions and drink form with the inclusion compound Hesperidin as active component, comprise juice, as fruit juice, yogurt, ice cream, cheese, the food made from flour, image planes bag, cookies, cake, cookie, noodles etc.; Milk product, dessert, confectionery articles, food seasoning, fruit salad, stewed fruit to boil water, vitamin complex etc.

This alimentation composition can be liquid, solid or powder.

Usefulness of the present invention is by the research of Hesperidin to yeast life-time dilatation and defying age mechanism, and proposing the Hesperidin activity of fighting against senium first is by Uth1 (yeast level), and SIRT1 and FOXO3A (human body cell level) realize.The present invention discovers that Hesperidin can the significant prolongation yeast life-span, Hesperidin suppresses the UTH1 expression of gene by regulating transcripton Skn7, can be in the application in preparation slow down aging and the treatment senile disease medicine, also can be in the application in the preparation food additive.

Description of drawings

Fig. 1 Hesperidin prolongs the yeast replicability life-span.

Fig. 2 Hesperidin prolongs yeast activity by suppressing UTH1.

Fig. 3 Hesperidin suppresses the UTH1 expression of gene.

Fig. 4 Skn7 deletion back adding Hesperidin does not influence yeast and duplicates the life-span.

Fig. 5 Hesperidin increases SIRT1 mRNA expresses.

Fig. 6 Hesperidin increases the SRIT1 enzymatic activity.

Fig. 7 Hesperidin increases SIRT1 protein expression (dosage activity research).

Fig. 8 Hesperidin increases SIRT1 protein expression (time activity research).

Fig. 9 Hesperidin increases FOXO3a mRNA expresses.

Figure 10 Hesperidin increases the proteic expression of FOXO3a (research of dosage activity relationship).

Figure 11 Hesperidin increases the proteic expression of FOXO3a (time activity relationship research).

The specific embodiment

Below again foregoing of the present invention is described in further detail by embodiment and the accompanying drawing that such some particular compound is prepared example, but this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example, all technology that realizes based on foregoing of the present invention all belong to scope of the present invention.

Embodiment 1 Hesperidin prolongs the yeast life-span.

Saccharomyces cerevisiae is used for the research of eukaryotic cell program for a long time, repairs and cell cycle as DNA.Just be widely used in the research of aging course recent years.Yeast has a lot of advantages with research on anti-senescence, and it is first eucaryotic organism that has complete genome group data.The life cycle that saccharomyces cerevisiae is used for research on anti-senescence has two kinds: replicability life-span and time lifetime.The replicability life-span be meant a yeast cells before death can division growth maximum times, the maximum numbers of daughter cell that just can produce.Time lifetime is meant the time that a group yeast cells that is in non-splitting status after nutritional labeling is withdrawn from is survived and can be continued, and judges whether also to survive, and can add nutrition in culture medium, sees whether it can recover mitosis.Used the zymic replicability life-span to do antidotal research in this experiment. utilize a kind of this special yeast in the experiment--the daughter cell of this bacterial strain of K6001--can not produce the offspring in dextrose culture-medium, have only blast cell can produce the offspring, thus thereby definite zymic replicability life-span of number that can go out daughter cell at the microscopically number.

1, experimental technique:

(1)-30 yeast strains of spending preservation is washed three times with the 5ml sterilized water, remove glycerol wherein, add the 1ml sterilized water, piping and druming makes its suspension, joins 10ml fluid medium (1% yeast powder, 2% peptone, 3% galactose) in, put it into shaking table, 28 degree jolting culture yeasts cells 48 hours make it recover energy for growth.

(2) from shaking table, take out cultured yeast,, remove fluid medium wherein, count with blood counting chamber with 5ml sterilized water washing three times.

(3) solid medium (1% yeast powder, 2% peptone, 2% the glucose of adding 5ml in culture dish, 2% agar powder), treat culture medium solidifying after, to wherein adding the good sample of dissolving, add 4000 yeast again, put into constant incubator 28 degree constant temperature culture 48 hours.

(3) the microscopically number goes out the daughter cell number that blast cell produces, and mapping analysis, positive control wherein are resveratrol.

2, experimental result:

After adding the Hesperidin of 5M and 10M, compare zymic life-span of significant prolongation (P＜0.05), its active and positive control resveratrol suitable (referring to Fig. 1) with negative control.

Embodiment 2: thus Hesperidin is regulated UTH1 gene expression prolongation yeast activity by the activity that changes Skn7

1, experimental technique

(1) yeast strain activity experiment: under the background of K6001,, do the yeast activity experiment, experimental technique such as embodiment 1 with Skn7 and UTH1 gene elmination.

(2) variation of UTH1 gene behind the usefulness real-time quantitative PCR analytical technology mensuration adding Hesperidin: the wild-type yeast bacterial strain was cultivated in dextrose culture-medium 48 hours, make it recover growth. in containing the dextrose culture-medium of 25ml, add 0 then, 5,10, the Hesperidin of 50 μ M, adding the zymic culture medium .28 degree concussion of containing of 300ul again cultivated 12 hours, when yeast is in exponential phase, collect yeast, then with hot extract with phenol total RNA. wherein RNeasy Mini Kit (Qiagen, MD, USA) RNA is carried out purification, measure RNA concentration. with AMV first-strand cDNA synthesis kit (Invitrogen, California, USA) RNA is carried out reverse transcription and (add the total RNA of 5g at every turn, oligo (dT) does primer). with LightCycler1.5 (MasterCycler epqradient realplex4 eppendorf, Germany) and SYBRPremix EX TaqTM (TaKaRa, Otsu, Japan) synthetic CDNA is carried out the real-time quantitative PCR analysis, condition is as follows: 40 circulations, 94 ℃ 15 seconds, 60 ℃ 25 seconds, 72 ℃ of 10 seconds .UTH1 primer sequences of and are as follows: positive-sense strand 5 '-CGC CTC TTC TTC CTC CTC TT-3 ', antisense strand 5 '-ACCATC GGA AGG TTG TTC AG-3 ' .TUB1 does house-keeping gene, positive-sense strand is sense 5 '-CCAAGG GCT ATT TAC GTG GA-3 ', antisense strand is 5 '-GGT GTA ATG GCC TCT TGCAT-3 '. final result with TUB1 with the standardization of UTH1 gene.

2, experimental result

(1) doing biological activity test after with UTH1 gene elmination on the K6001 basis, the yeast of finding deletion UTH1 gene is longer than the zymic average replicability life-span of K6001, but the yeast of deletion UTH1 gene adds 10 μ M Hesperidins again, its identical (see figure 2) with the negative control that does not add sample of average replicability life-span illustrates that it is by suppressing the UTH1 expression of gene that Hesperidin prolongs the yeast life-span;

(2) doing biological activity test after with Snk7 gene elmination on the K6001 basis, the yeast of finding deletion Snk7 is after adding 10 μ M Hesperidins, its average replicability life-span no longer prolongs (see figure 4), illustrates that it is to realize by the active change of the Snk7 of its upstream that Hesperidin suppresses the UTH1 expression of gene;

(3) utilize real-time quantitative PC further to verify to add Hesperidin after the UTH1 gene be suppressed. after adding the Hesperidin of 10,50 μ M, the UTH1 gene is obviously suppressed (see figure 3).

Embodiment 3: Hesperidin increases SIRT1 mRNA and expresses enzymatic activity and protein expression.

1, experimental technique:

(1) measure to add the variation of SIRT1 gene behind the Hesperidin with the real-time quantitative PCR analytical technology: in diameter is the capsule of 3.5cm, insert 1 * 106 cell, cultivate and made it adherent in 24 hours. again to wherein adding 0,10,30, the Hesperidin of 100 μ M, cultivated 24 hours. take out cell, with RNeasy Mini Kit (Qiagen, MD, USA) RNA is carried out purification, measure RNA concentration. with AMV first-strand cDNAsynthesis kit (Invitrogen, California, USA) RNA is carried out reverse transcription and (add the total RNA of 2.5 μ g at every turn, oligo (dT) does primer). with Light Cycler1.5 (MasterCycler epqradient realplex4 eppendorf, Germany) and SYBR Premix EX TaqTM (TaKaRa, Otsu, Japan) synthetic CDNA is carried out the real-time quantitative PCR analysis, condition is as follows: 40 circulations, 94 ℃ 15 seconds, 60 ℃ 25 seconds, 72 ℃ of and 10 seconds. primer sequence is as follows: SIT1 positive-sense strand 5 '-TGG CAA AGG AGCAGA TTA GTA GG-3 ', antisense strand 5 '-CTG CCA CAA GAA CTA GAG GAT AAGA-3 ' .18S ribosomal RNA does house-keeping gene, positive-sense strand is sense 5 '-GTA ACC CGT TGA ACCCCA TT-3 ',, antisense strand is 5 '-CCA TCC AAT CGG TAG TAG CG-3 '. final result with the 18S ribosomal RNA with the standardization of SIRT1 gene.

(2) increase of SIRT1 enzyme activity: utilize SIRT1/Sir2 deacetylase fluorometric assay kit, according to workbook wherein, add an amount of Hesperidin, room temperature was cultivated 30 minutes. and at exciting light is 340nm, absorbing light is under the 440nm condition, measures the fluorescent absorption value.

(3) increase of SIRT1 expressing quantity: detect the variation of SIRT1 expressing quantity with western blotting. in being the capsule of 3.5cm, diameter inserts 1 * 106 cell, cultivate and made it adherent in 24 hours. again to wherein adding 0,10,30, the Hesperidin of 100 μ M, cultivated 24 hours. with lysate (1%triton X-100,0.5%sodium deoxycholate, 0.1%SDS, and 2mM EDTA) collecting cell. with SDS-PAGE with Protein Separation, then albumen is forwarded on the pvdf membrane, incubate one anti-ly, and then incubate and be connected with the two anti-of cured peroxidase. with test kit (Amersham, GE Healthcare, Tokyo Japan) develops.

2, experimental result:

(1) after the SH-5YSY cell dropped into the Hesperidin of 10,30,100 μ M, SIRT1 mRNA transcriptional level obviously raise, and showed that SIRT1 gene expression increases. (see figure 5)

(2) utilize SIRT1/Sir2 deacetylase fluorometric assay kit, finding can obviously increase the SIRT1 enzyme activity behind the Hesperidin that drops into 5,10 μ M. (see figure 6)

(3) utilize western blotting, the dosage activity relationship is discovered: drop into 10 in the SH-5YSY cell, 30, behind the Hesperidin of 100 μ M, can obviously increase the proteic expression (see figure 7) of SIRT1. the time activity relationship is discovered, drop into Hesperidin two days later, the proteic expression of SIRT1 increases (see figure 8) to some extent.

Embodiment 4: Hesperidin increases FOXO3a mRNA and expresses protein expression

1, experimental technique:

(l) measure to add the variation of FOXO3a gene behind the Hesperidin with the real-time quantitative PCR analytical technology: in diameter is the capsule of 3.5cm, insert 1 * 106 cell, cultivate and made it adherent in 24 hours. again to wherein adding 0,10,30, the Hesperidin of 100 μ M, cultivated 24 hours. take out cell, with RNeasy Mini Kit (Qiagen, MD, USA) RNA is carried out purification, measure RNA concentration. with AMV first-strand cDNAsynthesis kit (Invitrogen, California, USA) RNA is carried out reverse transcription and (add the total RNA of 2.5 μ g at every turn, oligo (dT) does primer). with Light Cycler1.5 (MasterCycler epqradient realplex4eppendorf, Germany) and SYBR Premix EX TaqTM (TaKaRa, Otsu, Japan) synthetic CDNA is carried out the real-time quantitative PCR analysis, condition is as follows: 40 circulations, 94 ℃ 15 seconds, 60 ℃ 25 seconds, 72 ℃ of and 10 seconds. primer sequence is as follows: FOXO3a positive-sense strand 5 '-TGG CAA AGGAGC AGA TTA GTA GG-3 ', antisense strand 5 '-CTG CCA CAA GAA CTA GAG GAT AAGA-3 ' .18S ribosomal RNA does house-keeping gene, positive-sense strand is sense 5 '-GTA ACC CGT TGA ACCCCA TT-3 ',, antisense strand is 5 '-CCA TCC AAT CGG TAG TAG CG-3 '. final result with the 18S ribosomal RNA with the standardization of FOXO3a gene.

(2) increase of FOXO3a expressing quantity: detect the variation of FOXO3a expressing quantity with western blotting. in being the capsule of 3.5cm, diameter inserts 1 * 106 cell, cultivate and made it adherent in 24 hours. again to wherein adding 0,10,30, the Hesperidin of 100 μ M, cultivated 24 hours. with lysate (1%triton X-100,0.5%sodium deoxycholate, 0.1%SDS, and 2mM EDTA) collecting cell. with SDS-PAGE with Protein Separation, then albumen is forwarded on the pvdf membrane, incubate one anti-ly, and then incubate and be connected with the two anti-of cured peroxidase. with test kit (Amersham, GE Healthcare, Tokyo Japan) develops.

2, experimental result:

(1) after the SH-5YSY cell dropped into the Hesperidin of 10,30,100 μ M, FOXO3a gene expression obviously increased (see figure 9).

(2) utilize western blotting, the dosage activity relationship is discovered: drop into 10 in the SH-5YSY cell, 30, behind the Hesperidin of 100 μ M, can obviously increase the proteic expression (see figure 10) of FOXO3a. the time activity relationship is discovered, drop into Hesperidin two days later, the proteic expression of FOXO3a increases (seeing Figure 11) to some extent.

The sequence that the present invention relates to

＜110〉Zhejiang University

＜120〉a kind of defying age of Hesperidin is used

<160>10

<210>1

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the specificity forward primer of yeast UTH1

<440>1

cgc?ctc?ttc?ttc?ctc?ctc?tt

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the specificity downstream primer of yeast UTH1

<440>2

acc?atc?gga?agg?ttg?ttc?ag

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the specificity forward primer of yeast TUB1

<440>3

cca?agg?gct?att?tac?gtg?ga

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉yeast TUB1 specificity downstream primer

<440>4

ggt?gta?atg?gcc?tct?tgc?at

<210>5

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the specificity forward primer of people's SIRT1

<440>5

tgg?caa?agg?agc?aga?tta?gta?gg

210>6

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the specificity downstream primer of people's SIRT1

<440>6

ctg?cca?caa?gaa?cta?gag?gat?aag?a

<210>7

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the specificity forward primer of people's 18S ribosomal RNA

<440>7

gta?acc?cgt?tga?acc?cca?tt

<210>8

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the specificity downstream primer of people's 18S ribosomal RNA

<440>8

cca?tcc?aat?cgg?tag?tag?cg

<210>9

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the specificity forward primer of people's FOXO3a

<440>9

tgg?caa?agg?agc?aga?tta?gta?gg

<210>10

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the specificity downstream primer of people's FOXO3a

<440>10

ctg?cca?caa?gaa?cta?gag?gat?aag?a

Claims (4)

1. the application of Hesperidin in preparation treatment senile disease medicine, this Hesperidin has following structure:

Described medicine comprises preparation allowable pharmaceutical excipients or carrier.

2. the application of Hesperidin in the medicine of preparation slow down aging, described medicine comprises preparation allowable pharmaceutical excipients or carrier.

3. the application of Hesperidin according to claim 1 and 2 is characterized in that, the dosage form of described medicine is solid preparation or liquid preparation.

4. the application of Hesperidin in the preparation food additive, the form of described food additive is liquid, solid or powder.

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