CN104829737A - Crude raspberry leaf polysaccharide, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of traditional Chinese medicines, and in particular relates to a raspberry leaf crude polysaccharide, a preparation method and application thereof. The present invention extracts raspberry leaf crude polysaccharide from raspberry leaves through steps such as hot water reflux extraction, decolorization treatment and alcohol precipitation, and the crude polysaccharide can significantly inhibit the release of NO from RAW264.7 macrophages induced by LPS in vitro. Moreover, it can significantly inhibit the expression of TNF-α, iNOS and IL-6mRNA in RAW264.7 macrophages induced by LPS. In addition, the present invention further purifies the crude polysaccharide of raspberry leaf by ion exchange column chromatography to obtain three components, among which the component with the highest content is the refined polysaccharide of raspberry leaf, which is composed of five monosaccharides Heteropolysaccharide, wherein the molar ratio of sugar residues is: rhamnose:arabinose:xylose:glucose:galactose=2.47:4.75:4.12:1:2.48.

Description

A kind of raspberry leaf crude polysaccharide and its preparation method and application

technical field

The invention belongs to the field of traditional Chinese medicines, and in particular relates to a raspberry leaf crude polysaccharide, a preparation method and application thereof.

Background technique

One of the most promising current anti-inflammatory alternatives to classical antibiotic therapy is the use of immunomodulators that enhance host defense responses. A variety of immunomodulators are known, including mammalian proteins, such as: γ-interferon, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, and substances isolated and purified from microorganisms. For immune bacterial polysaccharides and synthetic compounds, their adverse reactions and side effects have attracted widespread attention. Most polysaccharides derived from higher plants are non-adverse substances and will not cause major side effects on the body. Therefore, polysaccharides isolated from plants have attracted great attention in biomedicine. The mechanism of action of these active substances is also constantly developing, and the immune mechanism induced by polysaccharides on non-specificity has received more attention. Studies have found that the immune activation mechanism of plant polysaccharides' anti-tumor, bactericidal and other therapeutic effects is accomplished through the regulation of the complement system and the stimulation of macrophages. Studies have found that plant-derived polysaccharides can exhibit various beneficial pharmacological effects by regulating the immune function of macrophages. Plant polysaccharides are the most ideal immune drug candidates for anti-inflammation, anti-tumor and promotion of wound healing.

Raspberry (Rubus chingii Hu), that is, palm leaf raspberry, is a plant of the genus Rubus of the family Rosaceae (Rosaceae), mainly produced in Zhejiang and Fujian, and also distributed in Anhui, Jiangxi, Jiangsu and other provinces, because it is mainly distributed in East China Also known as East China raspberry. Raspberry is included in the 2010 edition of the Pharmacopoeia of the People's Republic of China. Its immature and dried fruit is used as a medicinal part. It has the effects of nourishing the kidney, consolidating essence, and shrinking urine. It is used for enuresis due to kidney deficiency, frequent urination, impotence, premature ejaculation, and seminal emission. slippery essence etc. There are many studies on polysaccharides from raspberry fruit, but not on polysaccharides from raspberry leaves.

Contents of the invention

In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a method for preparing raspberry leaf crude polysaccharide.

Another object of the present invention is to provide the raspberry leaf crude polysaccharide prepared by the above preparation method.

Another object of the present invention is to provide the application of the above-mentioned raspberry leaf crude polysaccharide.

The object of the present invention is achieved through the following solutions:

A preparation method of raspberry leaf crude polysaccharide, comprising the steps of:

(1) Crush raspberry (Rubus chingii Hu) leaves and sieve to obtain raspberry leaf powder; reflux hot water at 75°C to 95°C to extract palm leaf raspberry leaf powder, then centrifuge the extract, and take the supernatant liquid, Filtrate 2 to 4 times, and concentrate the filtrate under reduced pressure to obtain a polysaccharide solution;

(2) Mix the polysaccharide solution with the pretreated macroporous adsorption resin, decolorize in a water bath at 40°C-60°C for 2-4 hours, then filter out the polysaccharide solution, and repeatedly wash out the polysaccharide adsorbed in the resin with water, and decolorize The polysaccharide solutions are combined and concentrated under reduced pressure to obtain concentrated sugar solution;

(3) adding 3 to 5 times the volume of ethanol to the concentrated sugar solution for alcohol precipitation, then centrifuging, discarding the supernatant, collecting the precipitate and drying it to obtain raspberry leaf crude polysaccharide (L-Ps);

The water-material ratio of the hot water reflux extraction described in step (1) is (10:1)～(30:1);

The time for hot water reflux extraction described in step (1) is 2 to 4 hours, and the number of hot water reflux extraction is 2 to 4 times;

The centrifugation speed described in step (1) is 3000～5000rpm/min; the centrifugation time is 8～12min;

The macroporous adsorption resin described in step (2) is preferably D354FD resin;

The pretreatment method of the macroporous adsorption resin described in the step (2) is: the macroporous adsorption resin is first soaked in water for 12 to 24 hours, and then the mass fraction is 5% hydrochloric acid solution and the mass fraction is 5% hydrogen The sodium oxide solutions were soaked for 0.5-3 hours respectively, filtered, washed with water until neutral, and the pretreated macroporous adsorption resin was obtained;

The temperature of the concentrated under reduced pressure described in step (1) and (2) is 40～60 ℃;

The volume fraction of ethanol described in step (3) is 70%～100%;

The temperature of alcohol precipitation described in step (3) is 0～4 ℃, the time of alcohol precipitation is 8～24h;

The centrifugation speed described in step (3) is 3000～5000rpm/min; the centrifugation time is 12～15min;

The temperature of drying described in step (3) is 40～60 ℃;

A raspberry leaf crude polysaccharide (L-Ps), prepared by the above preparation method;

A raspberry leaf essence polysaccharide (L-Ps-1), obtained by further purifying the above-mentioned raspberry leaf crude polysaccharide;

The raspberry leaf essence polysaccharide (L-Ps-1) is a homogeneous component, and its molecular weight (Mn) is 12686Da;

The raspberry leaf extract polysaccharide (L-Ps-1) is a heteropolysaccharide comprising five monosaccharides, wherein the molar ratio of sugar residues is: rhamnose: arabinose: xylose: glucose: galactose=2.47 :4.75:4.12:1:2.48;

Described raspberry leaf essence polysaccharide (L-Ps-1) is prepared through the following steps:

(1) Transfer the pretreated DEAE-52 resin to a Z-type chromatography column after rotary evaporation at 40-60 ° C to remove air bubbles, and pump distilled water with a constant-flow pump to make the packing evenly packed into the column. After equilibrium, the flow rate is 5 ~ 10s/drop;

(2) Prepare the polysaccharide solution from the raspberry leaf crude polysaccharide, transfer it to the chromatographic column, and wash it successively with distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.3mol/L NaCl, and 0.5mol/L NaCl solution The flow rate is 5-10 s/drop, and each fraction is collected separately, and the eluent is tracked and detected by the phenol-sulfuric acid method, and the absorbance at 490nm is measured, and the elution curve is drawn, and the eluents in the same absorption peak are combined; and then concentrated by rotary evaporation , vacuum freeze-drying, three components were obtained after DEAE-52 ion exchange column chromatography, and the component with the highest content was raspberry leaf essence polysaccharide (L-Ps-1);

The method for the pretreatment of the DEAE-52 resin described in step (1) is: soak the DEAE-52 resin in water at 4-30° C. for 12-24 hours; Soak in L of NaOH solution for 0.5-2 hours, filter, and wash with water until neutral to obtain the pretreated DEAE-52 resin;

The temperature of the rotary evaporation and concentration described in the step (2) is 40～60° C.;

Application of the raspberry leaf crude polysaccharide or raspberry leaf essence polysaccharide in the preparation of anti-inflammatory drugs;

The raspberry leaf crude polysaccharide or raspberry leaf essence polysaccharide can be used as a novel anti-inflammatory drug and applied to the treatment of inflammation.

An anti-inflammatory drug containing the above-mentioned raspberry leaf crude polysaccharide or raspberry leaf essence polysaccharide;

Principle of the present invention: the plant-derived polysaccharide can exhibit various beneficial pharmacological effects by regulating the immune function of macrophages. The present invention discovers and confirms that the plant polysaccharide L-Ps has significant anti-inflammatory activity, and the dosage of the plant polysaccharide L-Ps is low, and the toxic and side effects are also low at the same dosage.

Compared with the prior art, the present invention has the following advantages and effects:

(1) The present invention found for the first time that raspberry leaf crude polysaccharide could significantly inhibit LPS-induced NO release from RAW264.7 macrophages in vitro.

(2) The present invention found for the first time that raspberry leaf crude polysaccharide could significantly inhibit LPS-induced expression of TNF-α, iNOS and IL-6 mRNA in RAW264.7 macrophages in vitro.

(3) The dosage of raspberry leaf crude polysaccharide is relatively low, and the toxicity is low within the range of effective dosage.

Description of drawings

Fig. 1 is the ultraviolet scanning spectrogram of the raspberry leaf crude polysaccharide prepared in Example 1.

Fig. 2 is the infrared spectrogram of the raspberry leaf crude polysaccharide prepared in Example 1.

Fig. 3 is the ion-exchange column chromatography elution curve chart of embodiment 5.

Fig. 4 is the GPC graph of the raspberry leaf essence polysaccharide prepared in Example 5.

Fig. 5 is a graph showing the effect of raspberry leaf crude polysaccharide on NO release from RAW264.7 macrophages induced by LPS.

Figure 6 is a graph showing the effect of raspberry leaf crude polysaccharide on the expression of TNF-αmRNA in RAW264.7 macrophages induced by LPS, where, ##: compared with the control group, P<0.01.

Figure 7 is a graph showing the effect of raspberry leaf crude polysaccharides on the expression of iNOS mRNA in RAW264.7 macrophages induced by LPS, where ##: compared with the control group, P<0.01.

Fig. 8 is a graph showing the effect of raspberry leaf crude polysaccharide on the expression of IL-6 mRNA in RAW264.7 macrophages induced by LPS, wherein, ##: compared with the control group, P<0.01.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

In the examples, mouse macrophage RAW 264.7 cells were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; raspberry leaves were collected from a farm in Zhejiang.

Embodiment 1 prepares raspberry leaf crude polysaccharide (L-Ps) from palm leaf raspberry leaf

(1) Raspberry palmate leaves are crushed and passed through a 20-mesh sieve, weighed 100g, and heated under reflux with distilled water for extraction. The number of times is 3 times; then the extracts are combined, centrifuged at 4000rpm/min for 10min, the supernatant liquid is taken, filtered through filter paper for 3 times, and the filtrate is concentrated to 200mL under reduced pressure at 40°C with a rotary evaporator to obtain a polysaccharide solution;

(2) D354FD resin was first soaked in distilled water for 12 hours, then soaked in 5% hydrochloric acid solution and 5% sodium hydroxide solution for 0.5 hours respectively, filtered through 200 mesh gauze, and washed with distilled water until neutral. Obtain pretreated D354FD resin;

(3) Mix the polysaccharide solution prepared in step (1) with 200 mL of pretreated D354FD resin, place in a constant temperature water bath at 50°C for 3 hours for decolorization, stir at intervals, and then filter out the polysaccharide solution with a 200-mesh filter cloth , and repeatedly washed out the polysaccharide adsorbed in the resin with water, combined the decolorized polysaccharide solution, and concentrated it to 200mL under reduced pressure at 40°C with a rotary evaporator to obtain a concentrated sugar solution;

(4) Add 4 times the volume of ethanol with a volume fraction of 95% to the concentrated sugar solution prepared in step (3), magnetically stir while adding, alcohol precipitation at 4°C for 8h, and then centrifuge at 4000rpm/min After 15 minutes, the supernatant was discarded, and the precipitate was taken out and dried at 45° C. to obtain raspberry leaf crude polysaccharide (L-Ps).

Embodiment 2 prepares raspberry leaf crude polysaccharide (L-Ps) from the palm leaf raspberry leaf

(1) Raspberry palmate leaves are crushed and passed through a 20-mesh sieve, weighed 100g, and then heated and refluxed with distilled water for extraction, wherein the ratio of water to material is 20:1, the reflux extraction temperature is 85°C, the reflux extraction time is 3 hours, and the number of reflux extractions 2 times; then combine the extracts, centrifuge at 3000rpm/min for 8min, take the supernatant liquid, filter it twice with filter paper, and concentrate the filtrate to 200mL under reduced pressure at 50°C with a rotary evaporator to obtain a polysaccharide solution;

(2) D354FD resin was first soaked in distilled water for 24h, then soaked in 5% hydrochloric acid solution and 5% sodium hydroxide solution for 2h respectively, filtered through 200 mesh gauze, washed with distilled water until neutral, and obtained Pretreated D354FD resin;

(3) Mix the polysaccharide solution prepared in step (1) with 200 mL of pretreated D354FD resin, place in a constant temperature water bath at 40°C for 2 hours for decolorization, stir at intervals, and then filter out the polysaccharide solution with a 200-mesh filter cloth , and repeatedly washed out the polysaccharide adsorbed in the resin with water, combined the decolorized polysaccharide solution, and concentrated it to 200mL under reduced pressure at 50°C with a rotary evaporator to obtain a concentrated sugar solution;

(4) Add 3 times the volume of ethanol with a volume fraction of 95% to the concentrated sugar solution prepared in step (3), magnetically stir while adding, alcohol precipitation at 0°C for 12h, and then centrifuge at 3000rpm/min After separation for 12 minutes, the supernatant was discarded, and the precipitate was taken out and dried at 50°C to obtain raspberry leaf crude polysaccharide (L-Ps).

Embodiment 3 prepares raspberry leaf crude polysaccharide (L-Ps) from the palm leaf raspberry leaf

(1) Raspberry palmate leaves were crushed and passed through a 20-mesh sieve, weighed 100g, and heated under reflux with distilled water for extraction. The number of times is 4 times, then the extracts are combined, centrifuged at 5000rmp/min for 12min, the supernatant liquid is taken, filtered 4 times with filter paper, and the filtrate is concentrated to 200mL under reduced pressure at 60°C with a rotary evaporator to obtain a polysaccharide solution;

(2) D354FD resin was first soaked in distilled water for 18h, then soaked in 5% hydrochloric acid solution and 5% sodium hydroxide solution for 3h respectively, filtered through 200 mesh gauze, washed with distilled water until neutral, and obtained Pretreated D354FD resin;

(3) Mix the polysaccharide solution prepared in (1) with 200 mL of pretreated D354FD resin, place in a constant temperature water bath at 60°C for 4 hours for decolorization, stir at intervals, and then filter out the polysaccharide solution with a 200-mesh filter cloth , and repeatedly washed out the polysaccharide adsorbed in the resin with water, combined the decolorized polysaccharide solution, and concentrated it to 200 mL under reduced pressure with a rotary evaporator at 60 ° C to obtain a concentrated sugar solution;

(4) Add 5 times the volume of ethanol with a volume fraction of 95% to the concentrated sugar solution prepared in step (3), magnetically stir while adding, alcohol precipitation at 4°C for 24h, and then centrifuge at 5000rpm/min After 14 minutes, the supernatant was discarded, and the precipitate was taken out and dried at 60° C. to obtain raspberry leaf crude polysaccharide (L-Ps).

Embodiment 4 ultraviolet scanning collection of illustrative plates and infrared spectrum scanning

(1) Take 4 mg of raspberry leaf crude polysaccharide (L-Ps) prepared in Examples 1 to 3 respectively, and prepare it into a 1 mg/mL polysaccharide solution, and measure its absorbance at 190 to 500 nm with distilled water as a contrast.

(2) Take 2 mg of raspberry leaf crude polysaccharide (L-Ps) prepared in Examples 1-3, mix it with KBr and grind it into a uniform thin layer, and perform infrared scanning in the wavenumber range of 4000-400 cm ⁻¹ .

Result analysis:

(1) Polysaccharide UV spectrum analysis results: the raspberry leaf crude polysaccharides prepared in Example 1 all have strong absorption at 200-280nm, indicating that the sample may contain unsaturated carbonyl and carboxyl groups, and there is a strong absorption at 266nm. Absorption peaks, indicating that the sample contains conjugated nucleic acids or proteins (Figure 1). The detection result of embodiment 2 and embodiment 3 is the same as embodiment 1.

(2) Polysaccharide infrared spectrum analysis results: the raspberry leaf crude polysaccharides prepared in Example 1 all have a strong absorption peak at a wave number of 3345 cm ^-1 , which is the OH stretching vibration peak between sugar molecules or intramolecules, and at a wave number of 2933 cm ^-1 The absorption peak at the place is the CH vibration absorption peak of methine or methyl group, and the absorption peak at the wave number 1430～1200cm ^-1 is the CO stretching vibration peak of carboxyl group. The characteristic peaks of these three groups of sugars can be judged as in Example 1 The raspberry leaf crude polysaccharide prepared in is a polysaccharide. The absorption peak at wavenumber 1000～1200cm ^-1 is the vibration peak of uronic acid, and the relatively weak absorption peak at wavenumber 1617cm ^-1 is the characteristic infrared absorption peak of protein (Figure 2). These results further illustrate that the raspberry leaf crude polysaccharide prepared in Example 1 contains polysaccharide, protein and uronic acid. The detection result of embodiment 2 and embodiment 3 is the same as embodiment 1.

The preparation of embodiment 5 raspberry leaf essence polysaccharide (L-Ps-1)

(1) DEAE-52 pretreatment: Take 70g of DEAE-52, soak in distilled water at 4°C for 24 hours, then pour out the upper layer of water carefully to remove impurities, then soak in 0.5mol/L hydrochloric acid solution for 0.5h, and remove the upper layer of acid Carefully pour out the lye, filter it to dryness, and then wash it with distilled water to neutrality, then soak it in 0.5mol/L NaOH solution for 0.5h, carefully pour out the upper layer of lye, filter it to dryness, and then wash it with distilled water until it is neutral. spare;

(2) Transfer the pretreated DEAE-52 to a 2.6×30cm Z-type chromatography column after rotary evaporation at 48°C to remove air bubbles, and then pump distilled water with a constant flow pump to make the packing uniform. 7s/drop after equilibration;

(3) Weigh 100 mg of the raspberry leaf crude polysaccharide (L-Ps) prepared in Example 1, prepare it into a polysaccharide solution, drain it into a chromatography column with a glass rod, and use distilled water, 0.05mol/L NaCl, 0.1mol /LNaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution elution, automatically collect each fraction, each tube collects about 5mL, track and detect the eluate by phenol-sulfuric acid method, measure the absorbance at 490nm, and draw the elution curve, the eluents in the same absorption peak were combined; then the eluent was concentrated by rotary evaporation at 40°C, and vacuum freeze-dried. Among them, L-Ps was subjected to DEAE-52 ion exchange column chromatography to obtain three components, the highest content The component (48.0%) is raspberry leaf essence polysaccharide (L-Ps-1), wherein, Fig. 3 is the elution curve of ion exchange column chromatography in this embodiment.

The preparation of embodiment 6 raspberry leaf essence polysaccharide (L-Ps-1)

(1) DEAE-52 pretreatment: take 70g of DEAE-52, soak in distilled water at 20°C for 12h, then pour out the upper layer of water carefully to remove impurities, then soak in 0.5mol/L hydrochloric acid solution for 1h, and remove the upper layer of acid solution Pour it out carefully, filter it until it is dry, and elute it with distilled water until it is neutral, then soak it in 0.5mol/L NaOH solution for 1 hour, pour out the lye in the upper layer carefully, filter it until it is dry, wash it with distilled water until it is neutral, and set it aside;

(2) Transfer the pretreated DEAE-52 to a 2.6×30cm Z-type chromatography column after rotary evaporation at 48°C to remove air bubbles, and then pump distilled water with a constant flow pump to make the packing uniform. 7s/drop after equilibration;

(3) Weigh 100 mg of the raspberry leaf crude polysaccharide (L-Ps) prepared in Example 1, prepare it into a polysaccharide solution, drain it into a chromatography column with a glass rod, and use distilled water, 0.05mol/L NaCl, 0.1mol /LNaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution elution, automatically collect each fraction, each tube collects about 5mL, track and detect the eluate by phenol-sulfuric acid method, measure the absorbance at 490nm, and draw the elution curve, the eluents in the same absorption peak were combined; then the eluent was concentrated by rotary evaporation at 50°C, and vacuum freeze-dried. Among them, L-Ps was subjected to DEAE-52 ion exchange column chromatography to obtain three components, the highest content The component (48.0%) is raspberry leaf essence polysaccharide (L-Ps-1), wherein, the elution result of the ion exchange column chromatography in this example is the same as that in Example 5.

The preparation of embodiment 7 raspberry leaf essence polysaccharide (L-Ps-1)

(1) DEAE-52 pretreatment: Take 70g of DEAE-52, soak in distilled water at 25°C for 20h, then pour out the upper layer of water carefully to remove impurities, then soak in 0.5mol/L hydrochloric acid solution for 0.8h, and remove the upper layer of acid Carefully pour out the lye, filter it to dryness, and then wash it with distilled water to neutrality, then soak it in 0.5mol/L NaOH solution for 0.8h, carefully pour out the upper layer of lye, filter it to dryness, and then wash it with distilled water until it is neutral. spare;

(2) Transfer the pretreated DEAE-52 to a 2.6×30cm Z-type chromatography column after rotary evaporation at 48°C to remove air bubbles, and then pump distilled water with a constant flow pump to make the packing uniform. 7s/drop after equilibration;

(3) Weigh 100 mg of the raspberry leaf crude polysaccharide (L-Ps) prepared in Example 1, prepare it into a polysaccharide solution, drain it into a chromatography column with a glass rod, and use distilled water, 0.05mol/L NaCl, 0.1mol /LNaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution elution, automatically collect each fraction, each tube collects about 5mL, track and detect the eluate by phenol-sulfuric acid method, measure the absorbance at 490nm, and draw the elution The eluents in the same absorption peak of the curve were combined; then the eluents were concentrated by rotary evaporation at 60°C, and vacuum freeze-dried.

Among them, L-Ps obtained three components after DEAE-52 ion exchange column chromatography, and the component (48.0%) with the highest content was raspberry leaf essence polysaccharide (L-Ps-1), wherein, the present embodiment The ion exchange column chromatography elution result is the same as embodiment 5.

Molecular weight, purity identification and monosaccharide composition analysis of embodiment 8L-Ps-1

(1) The molecular weight and purity of the raspberry leaf essence polysaccharide (L-Ps-1) prepared in Examples 5-7 were respectively measured by high performance liquid chromatography. Chromatographic conditions: TSK G-5000PXL column (7.8×300mm) and TSK G-3000PXL column (7.8×300mm) in series, mobile phase is 0.02mol/L KH ₂ PO ₄ buffer solution, flow rate is 0.6mL/min, 2414 differential Detector, column temperature 35°C.

(2) Weigh 10 mg of the raspberry leaf extract polysaccharide (L-Ps-1) prepared in Examples 5 to 7 and place it in a 5 mL ampere tube, add 4 mL of 2 mol/L trifluoroacetic acid solution, seal it with an alcohol blowtorch, 110 ℃ hydrolysis for 6 hours, then the hydrolyzed sample was concentrated and evaporated to dryness under reduced pressure at 48°C, and an appropriate volume of methanol was added, then concentrated and evaporated to dryness under reduced pressure at 48°C, and this was repeated 3 times until the sample was neutral. Take 10mg of standard glucose, fucose, rhamnose, xylose, mannose, arabinose, galactose and each hydrolyzate in a 10mL screw tube, add 10mg of hydroxylamine hydrochloride and 1mL of pyridine, and place in a water bath at 90°C React at low temperature for 30 minutes, shake once every 5 minutes during the reaction, add 1 mL of acetic anhydride after natural cooling to room temperature, and continue to react for 30 minutes in a water bath at 90°C, shake once every 5 minutes during the reaction, take it out, and cool it to room temperature naturally. Finally, volatile sugar nitrile acetate derivatives are generated, processed through a membrane (0.22 μm), and then directly analyzed by GC. According to the retention time comparison between samples and standards, the monosaccharide composition of polysaccharides in various samples is obtained, and according to The area ratio of each peak was used to calculate the weight percentage of various monosaccharides. Chromatographic conditions: Agilent6890N gas chromatograph; DB-1701 capillary column (30m×0.25m, 0.25μm); vaporization chamber temperature 250°C, detector temperature 300°C, column temperature 180°C; temperature program: 180 (2min) at 2°C Raise the temperature to 220°C at a speed of 5°C/min, keep it for 1 minute, then raise the temperature to 250°C at a speed of 5°C/min, and keep it for 2 minutes. Detector: FID detector; carrier gas flow rate: 1mL/min; purge: 4.0mL/min.

Result analysis:

(1) The GPC figure of the L-Ps-1 prepared in Example 5 is a single symmetrical peak, showing that L-Ps-1 is a homogeneous polysaccharide, its number-average molecular mass (Mn) is 12686Da, and its weight-average molecular mass (Mw) It is 16073Da, and the peak molecular mass (Mp) is 17415Da (Figure 4). Embodiment 6 and 7 result are the same as embodiment 5.

(2) According to the area normalization method, the ratio of the peak area is equal to the ratio of the contained substance mass, and according to the ratio of the quality of each substance to its molar mass, it is the amount of substance of each substance, which is prepared in Example 5. The ratio of the amount of each monosaccharide in the raspberry leaf essence polysaccharide (L-Ps-1) is: rhamnose: arabinose: xylose: glucose: galactose=2.47:4.75:4.12:1:2.48. It can be seen that the content of arabinose and xylose in L-Ps-1 is relatively high, which are 32.05% and 27.80%, respectively, while the content of glucose is the lowest, only accounting for 6.74%. Embodiment 6 and 7 result are the same as embodiment 5.

Example 9 Griess method to detect the effect of L-Ps on the release of NO from RAW264.7 cells

Routine culture cells, take RAW264.7 cells in the logarithmic growth phase, pipette the cells into a single cell suspension, count with a hemocytometer under a microscope, centrifuge at 1000rpm/min for 5min to remove the supernatant, resuspend the medium and adjust the cell concentration , inoculated in a 24-well culture plate at a cell density of 200,000/well, cultured in a 5% CO ₂ incubator at 37°C, adhered to the wall for 24 hours, discarded the supernatant, and administered according to the following grouping requirements, with 3 cells in each group. Multiple wells: Control group, lipopolysaccharide (LPS, final concentration 1 μg/mL) group, LPS (final concentration 1 μg/mL)+dexamethasone (DXM, final concentration 50 μg/mL) group, LPS (final concentration 1 μg/mL)+L-Ps (the raspberry leaf crude polysaccharide prepared in Example 1, the final concentration is 2, 20, 200, 400 μg/mL) group, and the same volume of medium was added to the control group. After administration, place them in a 37°C, 5% CO ₂ incubator and continue to cultivate for 24h. Take 100 μL of the cell supernatant from each well and add it to another new 96-well plate. Add 50 μL of Griess reagent A to each well, place it in a 37°C incubator for 10 minutes, add 50 μL of Griess reagent B to each well, place it in a 37°C incubator and react for 10 minutes, and immediately place the plate on a multi-labeled microplate detector. The absorbance value of each well was detected at a wavelength of 550 nm.

L-Ps can inhibit LPS-induced NO release from RAW264.7 macrophages in a gradient-dependent manner (Figure 5).

Example 10 Effect of L-Ps on LPS-induced RAW264.7 cell TNF-α, iNOS and IL-6mRNA expression

Cells were routinely cultured. Cells in several stages of growth were taken and inoculated in a 6-well plate at a rate of 1 million cells/well. After incubation for 24 hours, the supernatant was discarded and administered according to the following grouping requirements. Each group had 3 replicate wells. Control group, lipopolysaccharide (LPS, final concentration of 1 μg/mL) group, LPS (final concentration of 1 μg/mL)+dexamethasone (DXM, final concentration of 50 μg/mL) group, LPS (final concentration of 1 μg/mL )+L-Ps (the raspberry leaf crude polysaccharide prepared in Example 1, the final concentration is 5, 25, 50, 100 μg/mL), and the same volume of medium was added to the control group. After administration, place in a 37°C, 5% CO ₂ incubator and continue to cultivate for 12h. After 12 hours of action, the cell supernatant was discarded and washed twice with PBS. Add 1 mL of Trizol to each well, let it stand for 5 minutes, blow it with a pipette until the liquid has no viscous, absorb the cell lysate and transfer it to an EP tube, add 0.2 mL of chloroform to each tube, cover the EP tube cap, shake it up and down for 10 seconds, and let it stand at room temperature 5min, centrifuge at 12,000rpm at 4°C for 15min, transfer the upper aqueous phase to another EP tube after centrifugation, add 0.5mL isopropanol, let stand at room temperature for 10min, centrifuge at 12,000rpm at 4°C for 10min, carefully aspirate and discard the upper layer Clear, wash twice with 1 mL of cold 75% ethanol, centrifuge at 7,500 rpm for 5 min at 4 °C, discard the supernatant carefully, dry with air for about 15 min, add 0-50 μL RNase-free pure water, heat at 60 °C for 10 min Dissolve the precipitate. Determine the purity and concentration of mRNA. And use RevertAid First Strand cnthesis Kit (Thermo Company) reverse transcription kit, 20 μ L reaction system, carry out reverse transcription to RNA. Using DyNAmo Flash SYRB Green qPCR Kit (Thermo Company) kit, ABI real-time fluorescent quantitative PCR instrument (Rrism 7500, Applied Biosystems, FosterCity, CA, USA) to amplify mRNA, after amplification, use RelativeQuantification ( ddCt) Study method for automatic analysis to obtain the relative expression of the target gene. The relative gene mRNA primer sequence is TNF-α (forward, 5'-GGG GAT TAT GGC TCA GGG TC-3', reverse, 5'-CGA GGC TCC AGT GAA TTC GG-3'), IL-6 (forward, 5 '-GTA CTCCAG AAG ACC AGA GG-3',reverse,5'-TGC TGG TGA CAA CCA CGG CC-3'),iNOS(forward,5'-CGG CAA ACA TGA CTT CAG GC-3',reverse,5 '-GCA CATCAA AGC GGC CAT AG-3') and GAPDH (forward, 5'-CAC TCA CGG CAA ATTCAA CGG CAC-3', reverse, 5'-GAC TCC ACG ACA TAC TCA GCA-3').

Compared with the control group, the secretion of TNF-α, iNOS and IL-6 in the LPS group was significantly increased (P<0.01), indicating that the inflammatory model of LPS-induced NO release from RAW264.7 macrophages was established. L-Ps can significantly inhibit the secretion of TNF-α, iNOS and IL-6 (P<0.05), and present a gradient dependence (Fig. 6-8).

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. a preparation method for leaf of Palmleaf Raspberry Crude polysaccharides, is characterized in that comprising following steps:

(1) by raspberry leaf grinding and sieving, leaf of Palmleaf Raspberry powder is obtained; 75 DEG C ~ 95 DEG C hot water returns extract Rubus chingii leaf powder, then that extracting solution is centrifugal, get supernatant liquid, filter 2 ~ 4 times, by filtrate reduced in volume, obtain polysaccharide soln;

(2) mix by polysaccharide soln with through pretreated macroporous adsorbent resin, decolour in 40 DEG C ~ 60 DEG C water-bath 2 ~ 4h, then filter out polysaccharide soln, and the polysaccharide adsorbed in resin is repeatedly washed out with water, merged by polysaccharide soln after decolouring, concentrating under reduced pressure, obtains concentrated liquid glucose;

(3) ethanol adding 3 ~ 5 times of volumes in concentrated liquid glucose carries out alcohol precipitation, then centrifugal, abandoning supernatant, and collecting precipitation thing is also dry, obtains leaf of Palmleaf Raspberry Crude polysaccharides.

2. the preparation method of leaf of Palmleaf Raspberry Crude polysaccharides according to claim 1, is characterized in that:

The ratio of water to material that hot water return described in step (1) extracts is (10:1) ~ (30:1);

The time that hot water return described in step (1) extracts is 2 ~ 4h, and the number of times that hot water return extracts is 2 ~ 4 times.

3. the preparation method of leaf of Palmleaf Raspberry Crude polysaccharides according to claim 1, is characterized in that:

Macroporous adsorbent resin described in step (2) is D354FD resin;

The pretreated method of the macroporous adsorbent resin described in step (2) is: be first soaked in water macroporous adsorbent resin 12 ~ 24h, then successively with massfraction be 5% hydrochloric acid soln and massfraction be 5% sodium hydroxide solution soak 0.5 ~ 3h separately, filter, be washed to neutrality, obtain through pretreated macroporous adsorbent resin.

4. the preparation method of leaf of Palmleaf Raspberry Crude polysaccharides according to claim 1, is characterized in that:

The temperature of step (1) and the concentrating under reduced pressure described in (2) is 40 ~ 60 DEG C;

The temperature of the alcohol precipitation described in step (3) is 0 ~ 4 DEG C, and the time of alcohol precipitation is 8 ~ 24h.

5. the preparation method of leaf of Palmleaf Raspberry Crude polysaccharides according to claim 1, is characterized in that:

Centrifugal rotating speed described in step (1) is 3000 ~ 5000rpm/min; The described centrifugal time is 8 ~ 12min;

Centrifugal rotating speed described in step (3) is 3000 ~ 5000rpm/min; The described centrifugal time is 12 ~ 15min.

6. a leaf of Palmleaf Raspberry Crude polysaccharides, is characterized in that being prepared by the preparation method described in any one of Claims 1 to 5.

7. a raspberry folic acid polysaccharide, is characterized in that obtaining by being further purified by leaf of Palmleaf Raspberry Crude polysaccharides according to claim 6;

Described raspberry folic acid polysaccharide is homogeneous components, and its molecular weight is 12686Da;

Described raspberry folic acid polysaccharide is the mixed polysaccharide comprising five kinds of monose, and wherein, saccharide residue mol ratio is: rhamnosyl: pectinose: wood sugar: glucose: semi-lactosi=2.47:4.75:4.12:1:2.48.

8. raspberry folic acid polysaccharide according to claim 7, is characterized in that:

Described raspberry folic acid polysaccharide prepares as follows:

(1) will proceed to after 40 ~ 60 DEG C of rotary evaporation bubble removings in Z-type chromatography column through pretreated DEAE-52 resin, even to make filler fill post with constant flow pump pumping distilled water, after balance, flow velocity 5 ~ 10s/ drips;

(2) leaf of Palmleaf Raspberry Crude polysaccharides is mixed with polysaccharide soln, proceed in chromatography column, use distilled water, 0.05mol/L NaCl, 0.1mol/L NaCl, 0.3mol/L NaCl, 0.5mol/L NaCl solution wash-out successively, flow velocity 5 ~ 10s/ drips, collect each stream part respectively, phend-sulphuric acid tracing detection elutriant, measure the absorbancy under 490nm, draw elution curve, the elutriant in same absorption peak merges; Then concentrated by rotary evaporation, vacuum lyophilization, obtains three components after DEAE-52 ion-exchange chromatography, and wherein, the component that content is the highest is raspberry folic acid polysaccharide.

9. raspberry folic acid polysaccharide according to claim 8, is characterized in that:

The pretreated method of DEAE-52 resin described in step (1) is: 4 ~ 30 DEG C, DEAE-52 resin water is soaked 12 ~ 24h; 0.5 ~ 2h is soaked again with the hydrochloric acid soln of 0.5mol/L; The NaOH solution of 0.5mol/L soaks 0.5 ~ 2h, filters, is washed to neutrality, obtains through pretreated DEAE-52 resin.

10. leaf of Palmleaf Raspberry Crude polysaccharides according to claim 6 or the raspberry folic acid polysaccharide described in any one of claim 7 ~ 9 are preparing the application in anti-inflammatory drug.