CN110305084A

CN110305084A - Nitrogen-containing organic acid compound in purslane and extraction and separation method and application thereof

Info

Publication number: CN110305084A
Application number: CN201910640052.8A
Authority: CN
Inventors: 英锡相; 英哲铭; 刘锡龙; 张文洁
Original assignee: Liaoning University of Traditional Chinese Medicine
Current assignee: Liaoning University of Traditional Chinese Medicine
Priority date: 2019-07-16
Filing date: 2019-07-16
Publication date: 2019-10-08
Anticipated expiration: 2039-07-16
Also published as: CN110305084B

Abstract

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to a novel compound extracted, separated and identified from a purslane medicinal material, an extraction and separation method and application thereof, and specifically relates to an extraction and separation method and application of a nitrogen-containing organic acid compound in purslane. The new compound is prepared by sequentially adopting ethanol extraction, silica gel column chromatography, polyamide column chromatography, macroporous resin column chromatography, ODS medium-pressure column and Sephadex LH-20 purification and liquid phase separation, and the structure adopts UHPLC-ESI-TOF-MS,¹H‑NMR、¹³The method of C-NMR and two-dimensional nuclear magnetic spectrum analysis determines a new nitrogenous organic acid compound. The compound has potential anti-inflammatory effectAnd neuroprotective effect, etc., and the new compound and its salt or derivative may be used as the material for synthesizing other compound and developing new medicine and pharmacological activity research.

Description

A kind of nitrogenous organic acid compound and its extraction separation method and purposes in purslane

Technical field

The present invention relates to traditional Chinese medicine extraction, separation field, more particularly to extracting and developing and identifies from purslane medicinal material Noval chemical compound and its extraction separation method a kind of nitrogenous organic acid compound extraction separation method and its are answered specially in purslane With.

Background technique

Purslane (Portulaca oleracea L.) also known as long life dish, horse three-coloured amaranth are portulacaceous plant.Purslane Drought-enduring waterlogging and fast light shade tolerant, widely distributed, resourceful, the wild plant as dual-purpose of drug and food is concerned, and 2015 editions The dry aerial parts that purslane is recorded in the Pharmacopoeia of the People's Republic of China are used as medicine, and have clearing heat and detoxicating, cooling blood and hemostasis, stop dysentery And other effects, for toxic-heat and blood stasis, carbuncle swells furunculosis, eczema, erysipelas, snakebite and bugbite, hematochezia, hemorrhoid blood, metrostaxis etc..

Purslane modern pharmacology research shows it with anti-inflammatory analgesic, anti-bacteria and anti-virus, lowering blood pressure and blood fat, antioxygen Change, anticancer, relaxation skeletal muscle and smooth muscle adjust the effects of immune function.Research shows that the numerous chemical components of purslane are it The pharmacological action of multiplicity provides material base, and purslane main chemical compositions include flavonoids, cumarin, terpene, steroid, life Alkaloids, amino acid, various pigments and minerals class etc..Wherein alkaloid is a kind of main chemical component, mesh in purslane Preceding reported composition of alkaloids have norepinephrine, dopamine, a small amount of DOPA, adenosine, uracil, adenine, N, N- Dicyclohexylurea (DCU), allantoin, N- be trans--asafoetide acyl group tyrasamine；There are also Cyclic dipeptides alkaloid and amide alkaloids: purslane acyl Amine A-S.

Most of chemical component isolated from purslane at present is known, and structure novel is lower, therefore, right The exploitation and separation of noval chemical compound urgently need in purslane.

Summary of the invention

In view of the above-mentioned problems, the present invention provides a kind of nitrogenous organic acid compound extraction separation method, specially from dent The noval chemical compound extracted in amaranth provides simultaneously it has been investigated that novel compound of present invention has the function of anti-inflammatory, neuroprotection A kind of extraction separation method easy, quick, environmentally friendly, with high purity for noval chemical compound of the present invention.

To achieve the above object, the present invention provides following technical scheme.

A kind of nitrogenous organic acid compound, molecular formula C in purslane₁₁H₁₃NO₆, it is named as (2- (furan-2-yl)- 2-oxoethyl) glutamic acid, molecular formula are chemical structural formula are as follows:

A kind of nitrogenous organic acid compound (2- (furan-2-yl) -2-oxoethyl) glutamic acid in purslane Extraction separation method, specific steps are as follows:

Step 1 takes the dry medicinal material of purslane, is extracted using ethyl alcohol, and alcohol extract filtration, merging filtrate directly heats concentration, It cools to room temperature, it is spare to obtain medical fluid；

Step 2, upper silicagel column after being evaporated concentrate in step 1, are eluted with ethyl acetate, ethyl acetate are recovered under reduced pressure extremely Medicinal extract obtains ethyl acetate extract；

Step 3 separates ethyl acetate extract in step 2 through polyamide column, using alcohol-water gradient elution, cold water Part upper macroporous resin column after being evaporated, successively uses alcohol-water gradient elution, hot water section is evaporated, spare；

Step 4, by gains in step 3 pretreated ODS column (Octadecylsilyl, octadecylsilane key again Close silica filler) chromatography obtains several elution positions, detected through thin-layer chromatography with methanol-water gradient elution, it shows The elution position of colour developing is concentrated to dryness, it is spare to obtain concentrate by color；

Step 5, by step 4 gained the pretreated Sephadex LH-20 of concentrate (hydroxypropyl sephadex), It with methanol isocratic elution, is detected through thin-layer chromatography, develops the color, the elution position of colour developing is concentrated to dryness respectively, is obtained dense Contracting object is spare.

Step 6 carries out HPLC (efficient liquid phase) separation preparation to gained concentrate in step 5, with acetonitrile and 0.1% formic acid Volume ratio is 5:95 as mobile phase, and preparation finally obtains a kind of nitrogenous organic acid compound in purslane of the present invention.

The preprocessing process of the ODS is that methanol impregnated 24 hours, upper prop, is washed till in instillation water with methanol without muddiness, It is balanced each other again with initial flow.

Beneficial effects of the present invention compared with prior art.

A kind of separation of nitrogenous organic acid compound and pharmacology activity research be not existing in heretofore described purslane Paper periodical is reported；The present invention provides in the purslane a kind of nitrogenous organic acid compound and a kind of new for the present invention The extraction separation method of compound, using ethyl alcohol extraction, silica gel column chromatography, polyamide column chromatography, macroporous resin column chromatography, ODS Middle compression leg, Sephadex LH-20 and HPLC are isolated and purified and are prepared, and are successfully extracted and are isolated new compound, this method Operating procedure is only six steps, and operating method is easy and quick, extracts separation process and mainly uses ethyl alcohol extraction and ethyl acetate extraction Take, process environmental protection is and higher through the isolated compound purity of this method, be all larger than 90%, furthermore research has shown that The above compound has anti-inflammatory, neuroprotection, therefore noval chemical compound of the present invention and its salt and derivative can be used as other The raw material of compound synthesis primer and new drug development and pharmacology activity research also can be used for preparing anti-inflammatory, neuroprotection and make Drug.

Detailed description of the invention

Fig. 1 is the high-resolution matter of noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) glutamic acid Spectrogram.

Fig. 2 is noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) glutamic acid¹H-NMR spectrum Figure.

Fig. 3 is noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) glutamic acid¹³C-NMR spectrum Figure.

Fig. 4 is the nuclear magnetic resonance of noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) glutamic acid Carbon composes (DEPT) spectrogram.

The nuclear-magnetism that Fig. 5 is noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) glutamic acid is total Vibration¹H-¹HCOSY spectrogram.

Fig. 6 is the nuclear magnetic resonance of noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) glutamic acid HMBC spectrogram.

Fig. 7 is the nuclear magnetic resonance of noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) glutamic acid HSQC spectrogram.

Fig. 8 is the nuclear magnetic resonance of noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) glutamic acid ROESY spectrogram.

Specific embodiment

The present invention provides a kind of nitrogenous organic acid compound in purslane, molecular formula C₁₁H₁₃NO₆, chemical structural formula are as follows:

The noval chemical compound is named as (2- (furan-2-yl) -2-oxoethyl) glutamic acid, table according to structure 1 is the nuclear magnetic data of the noval chemical compound:¹H-NMR with¹³C-NMR is in DMSO.

The nuclear magnetic data of the noval chemical compound of the present invention of table 1

Number	δC	Type	δH,mult(J in Hz)
				1	174.92	C
2	59.93	CH	4.17(m)
				3	22.68	CH₂	2.00(m)
			2.30(m)
				4	28.82	CH	2.30(m)
5	174.92	C
				1′		N
2′	47.36	CH₂	4.30(d,18)
							4.87(d,17.88)
3′	182.95	C
				1″		O
2″	150.26	C
				3″	118.89	CH	7.56(d,3.54)
4″	112.56	CH	6.74(dd,1.56；3.48)
				5″	148.05	CH	8.03(d,1.02)

(2- (furan-2-yl) -2-oxoethyl) glutamic acid: purple oily is soluble in methanol, is slightly soluble in chlorine It is imitative.For point sample after silica gel thin-layer plate, spray bismuth potassium iodide test solution spot shows crocus, and prompting the compound is alkaloid component. UHPLC-ESI (+)-TOF-MS provides [M-H₂O+H]⁺, m/z:238.0710, molecular weight 255.0743.In conjunction with¹H-NMR,¹³C- NMR and DEPT data, thus it is speculated that the possible molecular formula of the compound is C₁₁H₁₃NO₆, degree of unsaturation 6.¹³C-NMR spectrum and DEPT Spectrum 11 carbon signals of display, respectively 3 CH₂(δ: 22.68；28.82；47.36), 4 CH (a fatty carbon, δ: 59.93； Three alkene carbon, δ: 112.56；118.89；148.05), 4 carbonyl carbons (δ: 150.26；174.92；182.95, wherein 174.92 being overlap peak).

¹H-NMR spectrum 7 hydrogen signals of display, 3 methylene signals, respectively δ 2.00 (1H, m), δ 2.30 (3H, m), δ 4.30 (1H, d, J=18Hz), δ 4.87 (1H, d, J=17.88Hz)；4 methine signals, respectively δ 4.17 (1H, m), δ 6.74 (1H, dd, J₁=1.56Hz, J₂=3.48Hz), δ 7.56 (1H, d, J=3.54Hz), δ 8.03 (1H, d, J=1.02Hz).

According to¹H-NMR,¹³Relevant peaks in C-NMR, HSQC and HMBC spectrum, H-4 " (δ 6.74,1H, dd, J₁=1.56Hz, J₂=3.48Hz) it is coupled with C-3 " (δ 118.89), C-5 " (δ 148.05), H-3 " (δ 7.56,1H, d, J=3.54Hz) and C- 4 " (δ 112.56), C-5 " are coupled, H-5 " (δ 8.03,1H, d, J=1.02Hz) and C-3 ", C-4 " are coupled, and illustrate C-3 ", C-4 " with C-5 " is the alkene carbon being connected.According to relevant peaks, H-4 " in HMBC spectrum, H-5 " respectively with C-2 " (δ 150.26) phase coupling It closes, illustrates that C-3 " with C-2 " is connected.C-2 " with C-5 " is in low field, prompts to be connected with O, illustrates that there are 1 furan nucleus.C-3′ (δ 182.95) is prompted there are carbonyl, and C-2 ' (δ 47.36) is in low field, and according to relevant peaks in HMBC spectrum, H-2 ' (δ 4.30, 1H, d, J=18Hz；δ 4.87,1H, d, J=17.88Hz) it is coupled with C-3 ', illustrate that C-2 ' is connected with C-3 '.It is composed according to HMBC Upper relevant peaks, H-5 " are coupled with C-3 ', are coupled according to relevant peaks, H-3 " in ROESY spectrum with H-2 ', illustrate C-2 " and C-3 ' It is connected.According to relevant peaks in HMBC spectrum, H-3 (δ 2.00,1H, m；δ 2.30,3H, m) and C-1 (δ 174.92), C-5 (δ 174.92) It is coupled, H-2 (δ 4.17,1H, m) and C-4 (δ 28.82) is coupled, and C-1, C-5 are located at low field, prompts to be connected with hydroxyl, explanation There are a glutaric acid structures.C-2 (δ 59.93) is located at low field, and H-2 (δ 4.17,1H, m) is the methine letter for connecting N atom Number, while being coupled according to relevant peaks, H-2 ' in HMBC spectrum with C-1, illustrate that C-2, C-2 ' and N atom are connected.According to the above letter Breath, it may be determined that this noval chemical compound is above structure.

The present invention also provides the extraction separation method of nitrogenous organic acid compound a kind of in above-mentioned purslane, specific steps For.

Step 1: weighing the dry medicinal material 150kg of purslane, extracted using 50% alcohol reflux, 50% ethanol consumption is medicinal material 8~16 times, twice, ethyl alcohol is recovered under reduced pressure in each 2h to refluxing extraction, cools to room temperature, it is spare to obtain medical fluid.

Step 2: it is separated after gained medical fluid part in step 1 is evaporated through silica gel column chromatography, with ethyl acetate isocratic elution, Wherein silica gel is 100-200 mesh, and 40 DEG C or less are recovered under reduced pressure ethyl acetate to medicinal extract, obtain ethyl acetate extract.

Step 3: ethyl acetate extract in step 2 is separated through polyamide column, using cold water, hot water, 70% ethyl alcohol, 100% ethanol gradient elution separates after being evaporated at 90-100 DEG C of cold water part through macroporous resin column chromatography, successively using cold water, Hot water, 70% ethyl alcohol, 100% ethanol gradient elution, it is spare by 90-100 DEG C of hot water section down toward dry.

Step 4: by gains in step 3, pretreated ODS medium pressure column chromatography is separated again, wherein filler particle size be 40~ 70 μm, with methanol-water (30/70,50/50,70/30,100/0, v/v) gradient elution, (pressurization, makes flow velocity 1mL/min, temperature For room temperature), 13 positions (i.e. gradient elution obtains 13 bottles, every bottle of 500mL) is obtained, is detected through thin-layer chromatography, is developed the color, it will 3~6 positions of colour developing retain, and 50 DEG C or less are concentrated to dryness, spare.The preprocessing process of the ODS is that methanol impregnated For 24 hours, upper prop is washed till to instill in water with methanol and be balanced each other without muddiness, then with initial flow.

Step 5: the pretreated Sephadex LH-20 column in gained colour developing position in step 4 is chromatographed, it is isocratic with methanol Elution, obtains 45 positions (i.e. gradient elution obtains 45 bottles, every bottle of 25mL), is detected through thin-layer chromatography, develops the color, will develop the color 4~15 positions merge, 50 DEG C or less are concentrated to dryness, spare.The preprocessing process of the Sephadex LH-20 gel It was impregnated for 24 hours for methanol, upper prop, and was washed till to instill in water with methanol and be balanced each other without muddiness, then with initial flow.

Step 6: gained colour developing position in step 5 is separated through HPLC and is prepared, with acetonitrile and 0.1% formic acid volume ratio for 5: 95 are used as mobile phase, Detection wavelength 210,280nm, and noval chemical compound of the present invention is prepared in separation, and it is equal that normalization method measures purity It is 90~99%.

A kind of anti-inflammatory effect of nitrogenous organic acid compound in purslane of the present invention.

1, main material.

1.1, it drug and reagent: tests new alkaloids compound used and is prepared by the above method, purity is 90~99%, essence It is close to weigh, solution needed for being diluted to following each dosage groups with DMSO.(U.S. Hyclone is public for DMEM high glucose medium, fetal calf serum Department)；Penicillin, streptomysin (Hangzhou Chinese holly company)；LPS (Sigma Co., USA)；IL-6,TNF-α,PGE₂ELISA examination Agent box (Cayman company, the U.S.)；Cell pyrolysis liquid, Griess reagent (green skies Bioisystech Co., Ltd).

1.2 cell strains: RAW264.7 macrophage (U.S.'s ATCC cell bank).

1.3 groupings: being divided into control group, LPS group and experimental group, and each one group.

2 experimental methods.

The fetal calf serum of l0%, l% antibiotic (100U/mL penicillin is added in 2.1 cell culture, DMEM high glucose medium With 100 μ g/mL streptomysins), it is placed in 37.5%, CO₂It is cultivated in incubator.

2.2 MTT colorimetric method for determining cell viabilities, above-mentioned three groups respectively logarithmic growth phase RAW264.7 macrophage connect For kind in 96 well culture plates, cell density is 1 × 10⁴A/mL, every 100 μ L of hole, 37 DEG C of temperature, 5%CO₂Under the conditions of overnight incubation Afterwards, noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) glutamic acid of various concentration is added in experimental group (1-20 μM) is separately added into the LPS of final concentration of 1 μ g/mL to LPS group and experimental group after being incubated for 1h, separately sets zeroing group and (contain DMSO The culture solution of solvent), every group sets 3 multiple holes, investigates the influence being added after drug to cell.After above-mentioned group of cells culture for 24 hours, The addition 20 μ L of 5mg/mL MTT in each hole cell, 37 DEG C of temperature, 5%CO₂Under the conditions of continue be incubated for 4h after, terminate culture, inhale Liquid in hole is abandoned, 100 μ L dimethyl sulfoxides (DMSO) are added in every hole, vibrate 10min, make to crystallize sufficiently dissolution, enzyme mark into the cell Each hole light absorption value is measured at instrument 570nm wavelength.

2.3 measure the content of NO using Ge Lisi (Griess) method, investigate the mouse that noval chemical compound of the present invention induces LPS The inhibiting effect of the NO yield of macrophage RAW264.7.Containing 10% tire ox blood after mouse macrophage RAW264.7 passage It is cultivated in the sugared cell culture medium DMEM of clear height, the noval chemical compound of the present invention (2- (furan-2- of various concentration is added in experimental group Yl) -2-oxoethyl) glutamic acid (1-20 μM), at 37 DEG C, 5%CO₂Under the conditions of be incubated for 1h after use LPS (final concentration Inflammatory reaction is induced for 1 μ g/mL), collects supernatant afterwards for 24 hours, every group of processing repeats 3 holes.Griess method measures cell supernatant The content of middle NO, the influence according to various concentration noval chemical compound of the present invention to the LPS RAW264.7 cell release NO induced, to Reflect that NO is horizontal.

2.4 ELISA methods measure inflammatory factor IL-6, TNF-α and inflammatory mediator PGE₂: by logarithmic growth phase RAW264.7 Macrophage is inoculated in 24 well culture plates, and cell density is 1 × 10⁵A/mL, every hole 1mL, 37 DEG C of temperature, 5%CO₂Under the conditions of New alkaloids compound (2- (furan-2-yl) -2-oxoethyl) glutamic of the present invention is added in overnight incubation, experimental group Acid (1-20 μM) after cultivating 1h, is added LPS (final concentration of 1 μ g/mL) in every hole, is incubated for altogether for 24 hours, every group of processing repeats 3 Hole.ELISA method measures IL-6, TNF- of purslane source new alkaloids compound treated RAW264.7 macrophages secrete α and PGE₂Content.

3 experimental results.

The experimental results showed that special compound of the present invention on the proliferation of the LPS macrophage RAW264.7 induced without influence, It is safe and non-toxic；And excess inflammatory cytokine IL-6, TNF- produced by the macrophage RAW264.7 of LPS induction can be effectively suppressed α and inflammatory mediator NO, PGE₂, and be in concentration dependant.

Comparative survival rate of cells experimental result is as shown in table 2.

Influence of 2 present invention of table to RAW264.7 macrophage relative survival rate.

Note:^*P < 0.05 compared with the control group (high concentration group has significant difference),

3 are shown in Table using the content experimental result of Ge Lisi (Griess) method measurement NO.

Influence (mean ± standard deviation, n=3) of 3 present invention of table to the RAW264.7 cell release NO of LPS induction

Note: P < 0.05 * compared with the control group,^#P < 0.05 is compared with LPS group.

ELISA method measures inflammatory factor IL-6, TNF-α and inflammatory mediator PGE₂The results are shown in Table 4.

IL-6, TNF-α and PGE of 4 present invention of table to the RAW264.7 cell secretion of LPS induction₂Influence (the mean of content ± standard deviation, n=3)

Note:^*P < 0.05 compared with the control group,^#P < 0.05 is compared with LPS group.

A kind of neuroprotection of nitrogenous organic acid compound in purslane of the present invention.

1 main material.

1.1 drugs and reagent: testing noval chemical compound used and prepared by the above method, and purity is 90~99%, and precision weighs, Solution needed for being diluted to following each dosage groups with DMSO.DMEM high glucose medium, fetal calf serum (Hyclone company, the U.S.)；It is green Mycin, streptomysin (Hangzhou Chinese holly company), phosphate buffer (PBS), (Wuhan doctor's moral Co., Ltd), ROS detection examination Agent box (the green skies Reagent Company in Haimen)

1.2 cell strains: human neuroblastoma cells' strain (SH-SY5Y, IMR-32) (Chinese Academy of Sciences's Shanghai cell

1.3 groupings: it is divided into control group, H₂O₂Damage model group and experimental group.

2 experimental methods.

10% fetal calf serum, l% antibiotic (100U/mL penicillin is added in 2.1 cell culture, DMEM high glucose medium With 100 μ g/mL streptomysins), it is placed in 37 DEG C, 5%CO₂It is cultivated in incubator.

2.2 MTT colorimetric method for determining cell viabilities, above-mentioned three groups of difference logarithmic growth phase SH-SY5Y cell and IMR-32 For cell inoculation in 96 well culture plates, cell density is 1 × 10⁴A/mL, every 100 μ L of hole, 37 DEG C of temperature, 5%CO₂Under the conditions of train After supporting overnight, the noval chemical compound of the present invention (2- (furan-2-yl) -2-oxoethyl) of various concentration is added in experimental group To H after glutamicacid (1-20 μM), incubation 1h₂O₂Group and experimental group are separately added into the H of final concentration of 800 μM/L₂O₂, separately If zeroing group (culture solution of the solvent containing DMSO), every group sets 3 multiple holes, investigates the influence being added after drug to cell.It is above-mentioned each After group cell culture for 24 hours, the addition 20 μ L of 5mg/mL MTT in each hole cell, 37 DEG C of temperature, 5%CO₂Under the conditions of continue to be incubated for After 4h, culture is terminated, inhales and abandons liquid in hole, 100 μ L dimethyl sulfoxides (DMSO) are added in every hole, vibrate 10min, make to tie into the cell It is brilliant sufficiently to dissolve, each hole light absorption value (A) value of measurement at microplate reader 450nm wavelength, calculating cell survival rate, cell survival rate= (AH₂O₂Damage-A blank)/(A control-A blank).

2.3 DCFH-DA methods detect SH-SY5Y cell and the intracellular ROS of IMR-32, after group of cells gives respective substance It is incubated for for 24 hours, incubation terminates preceding 30min, and each hole is added DCFH-DA, makes final concentration of 10 μm of ol/L, continue to be incubated in 37 DEG C 30min collects cell, and PBS is washed 2 times, and the cell suspension of same concentrations is made in group of cells by cell count.Take 100 μ L cells Suspension fluorescence intensity, excitation wavelength 485nm, launch wavelength 538nm.With control group fluorescence intensity for 100%, remaining each group Compared with control group fluorescence intensity, ROS variation intracellular is calculated.

2.4 INT chromogenic reaction methods measure the burst size of LDH, remove above-mentioned control group, H₂O₂Damage model group and experimental group Outside, it separately sets up blank control group (blank control group not inoculating cell), respective substance culture is added for 24 hours in group of cells, takes each hole For 120 μ L of supernatant into 96 new orifice plates, the LDH detection working solution for adding 60 μ L to prepare is protected from light incubation at room temperature 30min, at 490nm A value is measured with multi-function microplate reader, calculates the LDH burst size percentage relative to control tube.LDH release rate=(A administration-A Blank)/(A control-A blank)

3 experimental results.

Comparative survival rate of cells experimental result is as shown in table 5.

Influence of 5 present invention of table to human neuroblastoma cells' strain SH-SY5Y and IMR-32 comparative survival rate of cells

Note:^*P < 0.05 and H₂O₂Damage model group compares.

SH-SY5Y cell and the intracellular ROS amount testing result of IMR-32 are as shown in table 6.

Influence of 6 present invention of table to human neuroblastoma cells' strain intracellular ROS amount of SH-SY5Y and IMR-32

Note:^*P < 0.05 compared with the control group,^#P < 0.05 and H₂O₂Damage model group compares

The results are shown in Table 7 for the influence of SH-SY5Y cell and the intracellular LDH release of IMR-32.

Influence of 7 present invention of table to the intracellular LDH release of human neuroblastoma cells' strain SH-SY5Y and IMR-32

Note:^*P < 0.05 compared with the control group,^#P < 0.05 and H₂O₂Damage model group compares.

In conclusion the present invention provides noval chemical compound and its extraction separation method, successively extracted using ethyl alcohol, silica gel column layer Analysis, polyamide column chromatography, macroporous resin column chromatography, ODS medium pressure column chromatography, Sephadex LH-20 column chromatography and HPLC separation system Standby, a kind of successful isolated noval chemical compound, this method is easy, quickly, environmental protection, and the compound isolated through this method Purity is higher, since gained compound chemical structure is unique, extracts from conventional Chinese medicine purslane, with anti-inflammatory, refreshing Through protective effect, therefore noval chemical compound of the present invention and its salt and derivative can be used as natural products exploitation new Chinese medicine, have Bright prospects.

Claims

1. a kind of nitrogenous organic acid compound (2- (furan-2-yl) -2-oxoethyl) glutamic acid in purslane, Molecular formula is C₁₁H₁₃NO₆, chemical structural formula are as follows:

。

2. a kind of nitrogenous organic acid compound (2- (furan-2-yl) -2- in purslane as described in claim 1 Oxoethyl) the extraction separation method of glutamic acid, which is characterized in that specific steps are as follows:

Step 1 takes the dry medicinal material of purslane, is extracted using ethyl alcohol, alcohol extract filtration, merging filtrate directly heats concentration, cools To room temperature, it is spare to obtain medical fluid；

Step 2, upper silicagel column after being evaporated concentrate in step 1, are eluted with ethyl acetate, and ethyl acetate is recovered under reduced pressure to leaching Cream obtains ethyl acetate extract；

Step 3 separates ethyl acetate extract in step 2 through polyamide column, using alcohol-water gradient elution, cold water part Upper macroporous resin column, successively uses alcohol-water gradient elution, hot water section is evaporated after being evaporated, spare；

Step 4, by gains in step 3 pretreated ODS column (Octadecylsilyl, octadecylsilane bonded silica again Glue filler) chromatography obtains several elution positions, detected through thin-layer chromatography with methanol-water gradient elution, develops the color, and it will The elution position of colour developing is concentrated to dryness, and it is spare to obtain concentrate；

Step 5, by gained concentrate pretreated Sephadex LH-20(hydroxypropyl sephadex in step 4), with first Alcohol isocratic elution, is detected through thin-layer chromatography, and the elution position of colour developing is concentrated to dryness respectively, obtains concentrate by colour developing It is spare；

Step 6 carries out HPLC (efficient liquid phase) separation preparation to gained concentrate in step 5, with acetonitrile and 0.1% formic acid volume Than being 5:95 as mobile phase, preparation finally obtains a kind of nitrogenous organic acid compound (2- in purslane of the present invention (furan-2-yl)-2-oxoethyl)glutamic acid。

3. extraction separation method as claimed in claim 2, which is characterized in that 50% alcohol reflux extracts every time in the step 1, Reflux 2 hours every time, amount of alcohol are 8~16 times of medicinal material.

4. extraction separation method as claimed in claim 2, which is characterized in that ODS the and Sephadex LH-20 gel Preprocessing process is that methanol impregnated 24 hours, upper prop, is washed till with methanol equal without muddiness, then with initial flow in instillation water Weighing apparatus.

5. extraction separation method as claimed in claim 2, which is characterized in that mobile phase elution program used in the step 2 For isocratic elution.

6. extraction separation method as claimed in claim 2, which is characterized in that in the step 3

Alcohol-water gradient elution used is cold water, hot water, 70% ethyl alcohol, 100% ethanol gradient elution.

7. extraction separation method as claimed in claim 2, which is characterized in that methanol-water gradient elution used in the step 4 The volume ratio of middle first alcohol and water is 30:70,50:50,70:30 and 100:0.

8. a kind of nitrogenous organic acid compound (2- (furan-2-yl) -2- in purslane as described in claim 1 Oxoethyl) glutamic acid is preparing the purposes in anti-inflammatory drug or health care product.

9. a kind of nitrogenous organic acid compound (2- (furan-2-yl) -2- in purslane as described in claim 1 Oxoethyl) glutamic acid is preparing the purposes in nerve protection medicine or health care product.