CN1148193C - Application of oligose treating stress brain injury - Google Patents

Abstract

The present invention relates to an application of oligose in preparing medicine for treating stress brain injury, wherein the oligose is disclosed in a formula I.

Description

The purposes of oligosaccharide in the anti-stress brain injury

The present invention relates to the purposes of oligosaccharide in the anti-stress brain injury derived from the Chinese medicine Radix Morindae Officinalis.

Chinese medicine Radix Morindae Officinalis (morinda officinalis How) has kidney invigorating and YANG supporting, bone and muscle strengthening, rheumatism effect.Chinese patent application No.94103414.3 has disclosed the medicine that a kind of active component is the anti-psychotic disorder of Radix Morindae Officinalis.Chinese patent application No.94103415.1 has disclosed formula (I) oligosaccharide that obtains from Radix Morindae Officinalis extraction and as antipsychotic drug, wherein n=1-8.

But formula I oligosaccharide yet there are no report in the activity aspect the anti-stress brain injury.

Goal of the invention:

The objective of the invention is to seek the new purposes of formula I oligosaccharide.

The invention summary

The now unexpected after deliberation discovery of the inventor, the oligosaccharide of formula I has good anti-stress brain injury effect.

Therefore, first aspect present invention relates to formula I oligosaccharide and is used for the treatment of purposes in the treating stress brain injury medicine in preparation.

What further aspect of the present invention related to is the method for treatment treating stress brain injury, and it comprises the treating stress brain injury patient who the formula I oligosaccharide of above-mentioned definition is needed treatment.

Describe in detail:

The oligosaccharide that the present invention relates to formula I is used for the treatment of the treating stress brain injury medicine in preparation

In purposes, n=1-8 wherein.

The invention still further relates to the method for treatment treating stress brain injury, it comprises the oligosaccharide with formula I

Need the treating stress brain injury patient of treatment, n=1-8.

According to the present invention, the formula I oligosaccharide of effect of the present invention obtains by extraction plant Radix Morindae Officinalis.Solvent for use is organic solvent, water-containing organic solvent or water in the extraction.Organic solvent is alcohols such as methanol, ethanol, propanol or butanols etc., halo alkanes such as dichloromethane or chloroform etc., esters such as methyl acetate, ethyl acetate, propyl acetate etc., or ethers such as petroleum ether or ether etc.

According to the present invention, formula I oligosaccharide can use separately or with composition forms in being used for the treatment for the treatment of stress brain injury.Administering mode can be oral or parenteral route.Form of administration can be peroral dosage form such as tablet or capsule etc., parenterai administration dosage form such as aseptic injection, drip liquid etc.

According to the present invention, preferred formula I oligosaccharide is the formula I oligosaccharide of n=4.

The present invention will be described further by the following examples and biological activity test, but this and do not mean that any limitation of the invention.

Embodiment 1

The preparation of the formula I oligosaccharide of n=4

Get Radix Morindae Officinalis 1KG and pulverized 50 mesh sieves, sieved the thing Petroleum ether extraction.Each with 5 liters of petroleum ether, extract three times.Merge, reduction vaporization is done, and obtains ligroin extraction 100g; The medicinal residues ethanol extraction.Each 8 liters of ethanol of using are carried three times, merge, and reduction vaporization is done, and obtains ethanol extraction 160g.Extract reuse water dissolution filters, and filtered solution distributes extraction in n-butyl alcohol and water, obtain dissolving in the extract 120g and the water-soluble part 300g of n-butyl alcohol.Water-soluble part 100g is separated with the carbon column chromatography, and developing solvent is the ethanol of 10%-70%.Obtain formula I oligosaccharide mixture, heavy 26g (purity almost 100%).Get its part and carry out high pressure liquid chromatography (post: Spherisorb C ₁₈(4.5 * 250mm), detect: RI Waters DifferentialRefractometer 410 (US), pump: Waters ^TM600, eluting: 5% methanol-water, flow velocity: 1.0ml/ minute), the retention time that records formula I oligosaccharide is:

The n retention time (minute)

1 2.39

2 3.05

3 3.43

4 4.27

5 4.53

6 6.34

7 7.48

8 8.47

The formula I oligosaccharide of solid n=4, i.e. (2 → 1) fruit furyl glycosyl sucrose six aggressiveness simultaneously obtain being white in color.

〔α〕 _D-18.2(C＝1.0，H ₂O)。

IRλ _max ^cm-1：3411(br，S，OH)，2931，2895，1420(w)，1032(s)。

Anion FAB-MS m/z:989[M-H] ^-

Elementary analysis: measured value, C, 39.28, H, 6.89 (C ₃₆H ₆₂O ₃₁6H ₂O)

, value of calculation, C, 39.35, H, 6.79.

¹H-NMR(D ₂O)δ：5.42(1H，d，J＝3.9Hz，1-H)，4.27，4.20，4.21，4.22，4.17，4.08，4.06，4.03，3.52(1H，dd，J＝10.5，3.9Hz；2-H)，3.45(1H，t，J＝9.5Hz；4-H)。

Biological activity test:

The formula I oligosaccharide of embodiment 2, n=4 (being called for short compd A from now on) is to the adjusting of corticosterone and testosterone levels in the chronic stress rat serum

1. method and result

The Wistar male rat, body weight 160 ± 20g is divided into 4 groups at random, 8 every group, stress control animals every day shock by electricity, forced swimming, vibration at a high speed, fixing, fasting pessimal stimulations such as (water), time-histories 28 days makes animal be in the chronic stress state.Then at formula I oligosaccharide that stress back 30 minutes lumbar injection (ip) n=4, normal group is not then accepted any stress stimulation to the administration group, and broken end was got blood and separation of serum in the 15th day, utilized corticosterone, testosterone test kit to measure.The result adds up and sees Table 1 with one factor analysis of variance.

Table 1

Group	Dosage (μ g/kg)	Corticosterone (ng/ml)	Testosterone (ng/dL)
				Normal control stress control compound A	- - 50 500	55.96±41.22 89.99±21.86 55.81±7.12 *** 19.11±9.90 ***	646.91±235.43 ***88.12±60.27 475.35±236.23 **564.91±428.60 *

^{* *}P＜0.001, ^*P＜0.01, ^*P＜0.05vs stress contrast.

2. discuss:

One of key feature of chronic stress is exactly that the corticosterone level significantly raises in the blood, testosterone levels reduces.Can see that by table 1 two dosage groups of compd A can reverse this result, expression can the antagonism chronic stress.

Embodiment 3, compd A are to the protective effect of the PC12 cell of corticosterone damage

1. method

(contain penicillin sodium 200kU/L, streptomycin 100mg/L is about 2 * 10 with cell dilution pH7.4) with the DMEM culture fluid that contains 5% hyclone and 5% horse serum ⁸/ L is inoculated in 96 well culture plates of using poly-D-lysine (0.1g/L) to handle in advance, and every hole 100 μ L put into CO ₂37 ℃ of incubators (production of U.S. Napco company), 5%CO ₂Cultivated under the condition 3～4 days, treat promptly to can be used for experiment after cell covers with at the bottom of the hole, reference literature (Liu Jinggen etc., Acta Pharmaceutica Sinica 1998,33 (3): 165) method, improve a little, Cell sap is removed in suction, change and contain respective concentration medicine and 0.2mmol/L corticosterone (corticosterone, serum-free DMEM Cort) (matched group does not contain any medicine), 37 ℃ of 5% CO ₂Cultivated 48 hours, culture fluid is removed in suction, wash 2 times with D-Hanks liquid, every hole adds the serum-free DMEM that contains 0.5g/L MTT and cultivates to inhale after 4 hours and remove culture fluid, every hole adds 10% sodium lauryl sulphate, 100 μ L, treat that blue particle dissolves (about 12～16 hours) fully, utilize porous scanning spectrophotometer (model: MCC/340) measure absorbance (A) value of specimen, be used for the quantitative response viable count and calculate the suppression ratio of medicine the PC12 cell injury of corticosterone damage at 570nm.

Table 2

Medicine (mg/ml)	Absorbance (A)
		Normal control corticosterone corticosterone+compd A 0.0625 0.125 0.5 2.0	1.97±0.12 *1.09±0.78 1.71±0.29 *1.94±0.06 ***1.73±0.10 *1.59±0.20

^{* *}P＜0.001vs corticosterone damage contrast.

2. result

The PC12 cell is after the 0.2mmol/L corticosterone is handled 48 hours, to MTT picked-up ability drop, the A value significantly is lower than the normal control group, showing that cell sustains damage or the part cell is in heaven dies, but when compd A existed, the PC12 cell obviously strengthened MTT picked-up ability, shows that the PC12 cell injury obviously weakens, the living cells showed increased has the concentration dependence.This result shows the damage of the cranial nerve cell that compd A can cause anti-stress.

Claims (4)

1. formula I oligosaccharide is used for the treatment of purposes in the treating stress brain injury medicine in preparation,

N=1-8 wherein.

2. according to the purposes of claim 1, wherein said medicine uses with tablet or capsule form.

3. according to the purposes of claim 1, wherein said medicine uses with aseptic injection or drip liquid form.

4. according to the purposes of claim 1, its Chinese style I oligosaccharide is the formula I oligosaccharide of n=4.