CN106519054B - A kind of radix pseudostellariae homogeneous polysaccharide and application thereof promoting Islet Cells Insulin secretion - Google Patents
A kind of radix pseudostellariae homogeneous polysaccharide and application thereof promoting Islet Cells Insulin secretion Download PDFInfo
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Abstract
The invention discloses a kind of radix pseudostellariae homogeneous polysaccharides and application thereof for promoting Islet Cells Insulin secretion.Radix pseudostellariae homogeneous polysaccharide of the invention is acidic polysaccharose, molecular weight 4.8 × 104 Da, specific rotatory power [α]D 25+ 174.5o (c 0.485, H2O)；Molecular formula is made of tri- kinds of elements of C, H, O, C:H:O=1:2.1:1.4；It is made of monosaccharides galactose aldehydic acid, rhamnose, galactolipin and arabinose, galactouronic acid is the main monosaccharide composition of polysaccharide, and α-Isosorbide-5-Nitrae-connection galacturonic acid constitutes the main chain of polysaccharide.Part galacturonic acid C6Esterification, C occur for-OH2And C3On hydroxylic moiety be acetylation, there are a small amount of rhamnoses on main chain；The arabinose of 1,5- connection constitutes branch.It can promote Ins-1 cell insulin secretion, provide foundation for the application in the future in the fields such as exploitation antidiabetic medicine, health care product.
Description
Technical field
The present invention relates to a kind of radix pseudostellariae homogeneous polysaccharides and application thereof for promoting Islet Cells Insulin secretion.
Background technique
Diabetes are the metabolic diseases using Persistent hyperglycemia as main feature.With the improvement of people ' s living standards,
Obvious ascendant trend is presented in the increase of living-pattern preservation, operating pressure, diabetes morbidity, becomes that seriously threaten the mankind strong
One of disease of health.Diabetes are divided into two types, type 1 diabetes (type1 diabetes mellitus, T1DM) belong to insulin according to
Rely type, diabetes B (type2 diabetes mellitus, T2DM) is non-insulin-depending type, and T2DM accounts for diabetic
90% or more of sum, hypoinsulinism in various degree and be main pathogenic mechanism with insulin resistance (IR).China
" Chinese chronic disease monitoring and diabetes special survey " that health education center is announced is the results show that there are about maturity-onset diabetes in China
97,000,000 people of patient, it has also become the first in the world diabetes big country, illness rate 9.7% surpass the world average level 6.4%.
The drug therapy of diabetes, substantially to control blood glucose as main means, antidiabetic drug includes biguanides, sulphur at present
Ureas, insulin three categories.In recent years, some new oral antidiabetic drugs also start in clinical use, as alpha-glucosidase inhibitor,
Insulin sensitizer, blood sugar regulator used during user having meals etc..Chemical Antidiabetic Drug curative effect in terms of controlling blood glucose is to affirm very much, but be also in
Reveal secondary failure after limitation and adverse reaction, such as hypoglycemia, lactic acidosis, long-time service.Diabetes, the course of disease it is long and
Complexity cannot substantially eradicate, and eventually lead to the multi visceras complication such as nephrosis；Patient generally requires long-term even lifelong use
Medicine, damage of the side effect to human body are difficult to overcome because of accumulation.Therefore exploitation blood sugar reducing function is mild, steady, lasting, poison is secondary makees
Have great importance with lesser drug.Sight is increasingly turned to traditional Chinese medicine and natural drug by researcher.
The scope that diabetes spp is quenched one's thirst in Chinese medicine.The name quenched one's thirst, head see Huangdi's Internal Classics；The famous doctor's Zhang Zhongjing of the Eastern Han Dynasty
To quench one's thirst in " Synopsis Golden Chamber " and be divided into three types, thirsty and more drink person be above disappear, rapid digestion of food and polyorexia person be in disappear, thirsty, urine
Disappear under being such as cream person；By the clinical research of existing new Chinese medicine treatment diabetes, diabetes are divided into: extreme heat due to deficiency of yin type, damp and hot tired
5 kinds of spleen type, type of deficiency of both QI and YIN, blood stasis causing water retention type and blood stasis train of thought type card types.TCM treatment of diabetes focuses on entirety and improves, is dialectical
By controlling, improves lipid-metabolism and energetic supersession disorder again while hypoglycemic, improves microcirculation, protection islet cell function
Deng；Have accumulated many effective recipe.Especially to complication such as control nephrosis, diabetes, retinopathy
Occur, development has a clear superiority.
It replenishes qi to invigorate the spleen, reinforcing both QI and YIN is one of Chinese traditional treatment diabete important channel.Radix pseudostellariae is Caryophyllaceae radix pseudostellariae category
Perennial herb perennial root plant pseudostellaria heterophyllaPseudostellaria heterophylla (Miq.) Pax ex Pax
The dried root of et Hoffm..There is the effect of nourishing generate fluid, tonifying lung invigorating the spleen, be widely applied in the prescription of all kinds of hypoglycemics.
The induction and conclusions such as Miao Yan rosy clouds have gone out different times and have treated medicine law to the traditional Chinese medicine of diabetes.The utilization side-such as Ni Qing card, medicine-
Demonstrate,prove relational theory, with data digging methods such as Scale-free Networks, the effect of from Chinese medicine, the use frequency of type, simple and
The compatibility relationship of different medicines inquires into TCM Syndrome Features and Medicinal Trait that its T2DM merges metabolic syndrome.Chinese medicine use with
Based on supplementing qi and nourishing yin drug, effect is presented with course of disease length by nourishing yin and clearing heat, arrives supplementing qi and nourishing yin, heat-clearing, then arrive qi and activate blood circulation,
Wen Yang, dispelling dampness and promoting diuresis variation tendency.Radix pseudostellariae is the most frequently used core drug.
Team where inventor, ten Yu Nianlai are devoted for years to produce the research of radix pseudostellariae in Fujian Zherong.It is molten using system
Agent extraction method is activity guiding with T1DM and T2DM animal model, carries out screening system to radix pseudostellariae anti-diabetic activity position；
It was found that the polysaccharide of 50~200kDa molecular weight ranges, can improve T2DM insulin resistance, there is significant hypoglycemic effect.
Summary of the invention
The purpose of the present invention is to provide a kind of radix pseudostellariae homogeneous polysaccharides and its use for promoting Islet Cells Insulin secretion
On the way.
A kind of radix pseudostellariae homogeneous polysaccharide promoting Islet Cells Insulin secretion, chemical structural formula are as follows:
;
Its structure feature are as follows: radix pseudostellariae homogeneous polysaccharide is acidic polysaccharose, molecular weight 4.8 × 104 Da, specific rotatory power [α]D 25+
174.5o (c 0.485, H2O)；Molecular formula is made of tri- kinds of elements of C, H, O, C:H:O=1:2.1:1.4；By monosaccharides galactose aldehyde
Acid, rhamnose, galactolipin and arabinose composition, galactouronic acid are the main monosaccharide composition of polysaccharide, α-Isosorbide-5-Nitrae-connection half
Lactobionic acid constitutes the main chain of polysaccharide.Part galacturonic acid C6Esterification, C occur for-OH2And C3On hydroxylic moiety by second
Acylated, there are a small amount of rhamnoses on main chain；The arabinose of 1,5- connection constitutes branch.
The preparation method of the radix pseudostellariae homogeneous polysaccharide, the specific steps are as follows:
(1) the anti-T2DM polysaccharide active component of radix pseudostellariae extracts:
(1) radix pseudostellariae dry product is taken, coarse powder is ground into, crosses 40 meshes；With 90% ethyl alcohol continuous circumfluence extraction 3 times of 8 times of amounts,
2 hours every time, filtering, the dregs of a decoction added appropriate distilled water continuous circumfluence extraction 3 times, 2 hours every time, filtered merging filtrate, were concentrated into
In right amount, spare；
(2) water extract obtained by step (1) is removed into deproteinized with Sevage method, medical fluid: chloroform: n-butanol=25:5:1(v :v), 5min, stratification are shaken, 4000r/min is centrifuged 5min, collects supernatant；Supernatant 90% ethanol precipitation of final concentration,
It stands for 24 hours, is centrifuged 5min, precipitation and separation obtains Thick many candies, is freeze-dried spare；
(3) use 40% ethyl alcohol classification of sedimentation, obtain molecular weight 50~200 kDa ranges polysaccharide, it is more for anti-T2DM
Sugared active site (PF40)；
(2) it isolates and purifies:
The anti-T2DM active component polysaccharide PF40 of radix pseudostellariae is taken, H is dissolved in2In O, make 25 ~ 50mg/mL of solution concentration；With
52 type chromatographic column of DEAE-Cellulose after loading, successively uses H2O, 0.1mol/L ~ 1.0mol/L NaCl solution gradient is washed
De-, polyoses content phend-sulphuric acid detecting and tracking in eluent determines polysaccharide point with high productivity computing method (HPGPC)
Son amount；It is isolated and purified repeatedly according to above-mentioned steps, obtains the homogeneous polysaccharide, be freeze-dried, it is spare.
The present invention also protects radix pseudostellariae homogeneous polysaccharide preparing the application in antidiabetic medicine.
The beneficial effects of the present invention are: the present invention relates to from the anti-effective portion of diabetes B polysaccharide of Chinese medicine radix pseudostellariae
The preparation and its chemical structure of the homogeneous polysaccharide of 1 isolated structure novel, can promote Ins-1 cell insulin in position
Secretion provides foundation for the application in the future in the fields such as exploitation antidiabetic medicine, health care product.
Detailed description of the invention
Fig. 1 is the IR spectrogram of radix pseudostellariae homogeneous polysaccharide；
Fig. 2 is radix pseudostellariae homogeneous polysaccharide1H NMR spectrum；
Fig. 3 is the polysaccharide nmr analysis of radix pseudostellariae homogeneous polysaccharide13C-NMR spectrum；
Fig. 4 is that the H-H COSY of radix pseudostellariae homogeneous polysaccharide is composed；
Fig. 5 is the hsqc spectrum of radix pseudostellariae homogeneous polysaccharide；
Fig. 6 is that the HMBC of radix pseudostellariae homogeneous polysaccharide is composed.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not limited only to these embodiments.
One, embodiment
A kind of preparation method for the radix pseudostellariae homogeneous polysaccharide promoting Islet Cells Insulin secretion, the specific steps are as follows:
(1) the anti-T2DM polysaccharide active component of radix pseudostellariae extracts:
(1) radix pseudostellariae dry product is taken, coarse powder is ground into, crosses 40 meshes；With 90% ethyl alcohol continuous circumfluence extraction 3 times of 8 times of amounts,
2 hours every time, filtering, the dregs of a decoction added appropriate distilled water continuous circumfluence extraction 3 times, 2 hours every time, filtered merging filtrate, were concentrated into
In right amount, spare；
(2) water extract obtained by step (1) is removed into deproteinized with Sevage method, medical fluid: chloroform: n-butanol=25:5:1(v :v), 5min, stratification are shaken, 4000r/min is centrifuged 5min, collects supernatant；Supernatant 90% ethanol precipitation of final concentration,
It stands for 24 hours, is centrifuged 5min, precipitation and separation obtains Thick many candies, is freeze-dried spare；
(3) use 40% ethyl alcohol classification of sedimentation, obtain molecular weight 50~200 kDa ranges polysaccharide, it is more for anti-T2DM
Sugared active site (PF40)；
(2) it isolates and purifies:
The anti-T2DM active component polysaccharide PF40 of radix pseudostellariae is taken, H is dissolved in2In O, make 25 ~ 50mg/mL of solution concentration；With
52 type chromatographic column of DEAE-Cellulose after loading, successively uses H2O, 0.1mol/L ~ 1.0mol/L NaCl solution gradient is washed
De-, polyoses content phend-sulphuric acid detecting and tracking in eluent determines polysaccharide point with high productivity computing method (HPGPC)
Son amount；It is isolated and purified repeatedly according to above-mentioned steps, obtains the homogeneous polysaccharide, be freeze-dried, it is spare.
The structural formula of radix pseudostellariae homogeneous polysaccharide made from above-described embodiment:
Two, the chemical structure characterization of the radix pseudostellariae homogeneous polysaccharide
It is 4.8 × 10 that HPGPC, which measures molecular weight,4 Da, specific rotatory power [α]D 25+ 174.5o (c 0.485, H2O)；Before PMP column
Derivatization method measures monosaccharide composition, and homogeneous polysaccharide is made of galacturonic acid, rhamnose, galactolipin and arabinose；Element point
It analyses polysaccharide and is free of N and S element, be mainly made of tri- kinds of elements of C, H, O, C:H:O=1:2.1:1.4.
Infrared spectroscopy (IR) shows typical polysaccharide Absorption Characteristics peak, 3426.69,2930.25,1418.51,
1021.35cm-1Strong absworption peak, be respectively from the flexible vibration of C-O in the outer C-O of hydroxyl-OH, C-H, ring and saccharide ring；
1745.20cm-1There is carboxyl absorption peak, infers the homogeneous polysaccharide in conjunction with monosaccharide composition analysis derived from the carboxyl of uronic acid
For acidic polysaccharose, Fig. 1 is seen.
According to1H NMR spectrum (as shown in Figure 2) and polysaccharide nmr analysis13C-NMR spectrum (as shown in Figure 3), δ
Two groups of signals of 176.50ppm and 172.18ppm derive from 6 carbon (C of α-galacturonic acid6), as part galacturonic acid C6-
When esterification occurs for OH, C6 Chemical displacement value to low field migrate；Nearby there is one group of signal according to 54 pmm of nuclear-magnetism rule δ,
δ 176.50ppm signal should belong to C6The galacturonic acid of methyl esters occurs, 172.18ppm signals assignment is not in methyl esters occurs
Galacturonic acid, and δ 54.11ppm signal is attributed to CH3O；Galacturonic acid C2And C3On hydroxyl be easy to be acetylation, so
δ 21.38ppm signals assignment is in CH3CO。
α-Isosorbide-5-Nitrae is derived from according to signal strength and alditol acid polysaccharide NMR parsing rule, δ 100.29ppm anomeric carbon signals letter
Connect the C of galacturonic acid1；δ 108.11ppm and 62.38ppm signal is respectively belonging to the C of arabinose1And C5, and δ
18.04ppm faint signal is derived from the C of rhamnose6.(as shown in Figure 4) and hsqc spectrum (such as Fig. 5 are composed according to H-H COSY
It is shown) and α-Isosorbide-5-Nitrae connection galacturonic acid pertinent literature, α-Isosorbide-5-Nitrae connection other C of galacturonic acid and H signal ownership such as table 1.
δ 100.29ppm anomeric carbon signals and the different head H of δ 5.10ppm have coherent signal in HSQC, illustrate that δ 5.10ppm is α-Isosorbide-5-Nitrae connection half
The different head H of lactobionic acid1Signal.HMBC(is as shown in Figure 6) there are clearly galacturonic acid C4 and H1 coherent signal.Therefore,
Galactouronic acid is the main monosaccharide composition of polysaccharide, and Isosorbide-5-Nitrae-connection galacturonic acid constitutes the main chain of polysaccharide.
1 homogeneous polysaccharide main chain of table13C and1H composes chemical shift
Three, pharmacological experiment (influence of the radix pseudostellariae homogeneous polysaccharide to Ins-1 cells secrete insulin)
1. cell culture and operating process
Rat Langerhans islet oncocyte INS-1 cell, routine culture is in 1640 culture medium of RPMI containing 10% fetal calf serum, separately
Penicillin containing 100U/mL, 100mg/mL streptomysin, 1 mM Sodium Pyruvate, 2 mM L-Glutamines and 50 μM of beta -mercaptoethanols.
Cell culture is in 37 DEG C, 5%CO2It is cultivated in incubator.Cell changes liquid, four days biography generation every other day.
Cultivate fluid exchange: the INS-1 being newly inoculated with is adherent after for 24 hours, and adherent preceding cell is bright, and small volume, refractivity are strong,
The feeler of cell, which stretches out, after adherent is presented irregular polygonal.Culture solution is using preceding needing to heat in 37 DEG C of water-baths, generally every other day
It changes the liquid once.
Cell passage: cell patch is not grown quickly afterwards, and for cell fusion degree up to 80% or more, needing at this time will be thin after 3-4 days
Born of the same parents' passage.Give up original culture solution, 37 DEG C of PBS are rinsed 2 times；0.25% Tripsin-EDTA digestive juice 1mL is added, is placed in
37 DEG C of static digestion l-2min；Add RPMI-1640 culture solution to blow and beat repeatedly, form single cell suspension, is passed on by 1:2-1:3, in
37℃、5%CO2It is cultivated in incubator.
Cell cryopreservation: selection growth conditions are good, the cell of the nearly 80-90% of fusion degree.Microscopically observation, form are full
Full, cell outline understands, obvious karyomorphism is seen under high power lens.0.25% Tripsin-EDTA is added to digest, the list blown and beaten
Cell suspension is centrifuged 3-5min in 1000 rpm.Cell cryopreservation is in the cryopreservation tube of 1.8ml.10% DMSO and 90% FBS is added to match
The frozen stock solution of system is tightened pipe lid, is marked on cryopreservation tube, including cell code name and freezes the date.(4 DEG C of cold compartment of refrigerator
30min) → low temperature refrigerator (- 20 DEG C of 1h) → low temperature refrigerator (- 80 DEG C overnight) → liquid nitrogen.
Cell recovery: an INS-1 cell frozen is taken out, cell cryopreservation tube is constantly shaken in 37 DEG C of water-baths, freezes
Liquid needs melt completely in 1min.After dissolving, 75% cotton ball soaked in alcohol cleaning disinfection of nozzle, frozen stock solution is sucked in centrifuge tube, is added
The piping and druming of 10mL culture solution mixes, and DMSO is removed in 800rpm centrifugation 5min centrifugation.After cell is resuspended, it is inoculated in culture bottle, every bottle
Add 5 mL of culture solution, piping and druming mixes, and is placed in 37 DEG C of 5%CO2In incubator.
2. the preparation of reagent
It accurately weighs 12mg radix pseudostellariae homogeneous polysaccharide to be dissolved in 10mL culture solution, concentration 1200mg/L is dilute with culture solution
It is interpreted into required 100,200,400,500,600 mg/L of concentration.
The preparation of gliclazide solution: it weighs gliclazide 32.34mg and dimethyl alum (DMSO) 10mL dissolution is added, i.e.,
It is 1 × 10-2The gliclazide of mol/L；Its 100 μ L is taken to be dissolved in the RPMI 1640 culture medium of 9.9mL to get 1 × 10-4mol/
L gliclazide solution；Its 10mL is taken to be dissolved in 1640 culture medium of RPMI of 9mL to get 1 × 10-5Mol/L gliclazide is molten
Liquid.4 DEG C of refrigerators of the solution prepared are protected from light storage.
Sugar-free KRBH buffer is prepared: being weighed each component one by one, is successively dissolved in 800 ml sterile waters, magnetic agitation
Hydrotropy, adjustment liquid pH value are 7.35, supply total volume to 1L, filtration sterilization, 4 DEG C of refrigerator packing preservations.
3. the influence of different time, various concentration radix pseudostellariae homogeneous polysaccharide to Ins-1 cell insulin secretion
The cell of logarithmic growth phase counts after digestion, adjustment cell density to 1 × 105A/mL is inoculated in 24 orifice plates.
Cell is adherent after 24 h, intervenes by following grouping administration.
1) blank group: RPMI-1640 culture solution is given.
2) PF40 Pseudostellaria Polysaccharide effect group: being prepared with RPMI-1640 culture solution, concentration 0,100,200,300,400,
500、600 mg/L。
3) positive drug gliclazide group: concentration 10-5 mol/L。
After polysaccharide and cell are incubated for jointly, in 12h, for 24 hours, 48h, 72h inhale and abandon supernatant, washed 3 times with the PBS of 37 DEG C of preheatings.
The KRBH buffer containing 3.3 mM glucose that preheating is added balances 1 h, abandons supernatant.It is added containing 16.7 mM glucose
37 DEG C of KRBH buffer 2 h of culture, take 4 DEG C of 1000rpm of supernatant to be centrifuged 5 min, and -80 DEG C of supernatant preservations are used for radioimmunoassay
Analytic approach detects insulin, is shown in Table 2.
4. result
2 different time of table, influence of the various concentration radix pseudostellariae homogeneous polysaccharide to Ins-1 cells secrete insulin
5. conclusion
INS-1 cell is derived from initially radiation-induced tumour, later by 2 mercapto ethanol (2-
Mercaptoethanol, 2-ME) anti-neoplastic is generated in culture solution.These cells shows go out many important spies of beta Cell of islet
Property, including relatively high insulin content and to the respond of physiological concentration glucose stimulation, therefore it is widely used in grinding
Study carefully insulin secretion mechanism.
Compared with the control group, it is remarkably improved after various concentration radix pseudostellariae homogeneous polysaccharide handles 12,24 and 48 h
The amount of insulin secretion of Ins-1 cell, and there is dosage and time dependence.400mg/L Pseudostellaria Polysaccharide, intervene 48 h after it is right
The stimulation of Ins-1 cell is most strong, and the insulin of secretion is most.With the extension of incubation time, insulin release is reduced, until
There is decompensation in the release of 72 h, Ins-1 cell insulin, but promotes the effect of Ins-1 cells secrete insulin not significant.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (2)
1. a kind of radix pseudostellariae homogeneous polysaccharide for promoting Islet Cells Insulin secretion, it is characterised in that: its chemical structural formula is as follows:
;
The preparation method of the radix pseudostellariae homogeneous polysaccharide the following steps are included: take the anti-T2DM active component polysaccharide PF40 of radix pseudostellariae,
It is dissolved in H2In O, make 25 ~ 50mg/mL of solution concentration；After chromatographic column loading, H is successively used2O、0.1mol/L~1.0mol/L
NaCl solution gradient elution, then isolated and purified repeatedly according to above-mentioned steps, obtain radix pseudostellariae homogeneous polysaccharide.
2. radix pseudostellariae homogeneous polysaccharide described in claim 1 is preparing the application in antidiabetic medicine.
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| CN113527531A (en) * | 2021-08-18 | 2021-10-22 | 安徽皖南烟叶有限责任公司 | Radix pseudostellariae polysaccharide and preparation method thereof |
| CN114280182A (en) * | 2021-12-23 | 2022-04-05 | 福建省中医药科学院 | HPLC-FLD detection method for homogeneous polysaccharides of radix pseudostellariae |
| CN114805622B (en) * | 2022-04-12 | 2023-04-21 | 福建中医药大学附属第二人民医院（福建省第二人民医院） | Radix pseudostellariae uniform polysaccharide capable of promoting healing of diabetic foot ulcers and preparation method thereof |
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