CN110776579A - Process for optimizing sulfuric acid esterification of radix scrophulariae polysaccharide by using response surface method - Google Patents
Process for optimizing sulfuric acid esterification of radix scrophulariae polysaccharide by using response surface method Download PDFInfo
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Abstract
The invention relates to a process for optimizing sulfuric acid esterification of figwort polysaccharide by using a response surface method, which comprises the following steps of ⑴ sample preparation, wherein the dried roots of figwort are prepared into figwort polysaccharide powder, the reaction extraction is carried out according to different reaction temperatures, reaction times and esterification reagent proportions to obtain figwort polysaccharide sulfate powder corresponding to the esterification reagents with different proportions, ⑵ sample substitution degree is determined by using a barium chloride-gelatin turbidimetry method, and ⑶ sample ABTS
+Determination of clearance rate, ⑷ experimental Design and statistical analysis, ① single-factor test, ② response surface method optimization Design, namely establishing a multiple quadratic regression equation by taking reaction temperature, reaction time and esterification reagent proportion as investigation factors according to a single-factor test result, ⑸ experimental result analysis and optimization, namely performing drawing analysis by using Design Expert 8.0.6 software according to the multiple quadratic regression equation to obtain a response surface and a contour map of the regression equationThe rate is high.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a process for optimizing sulfuric acid esterification of figwort polysaccharides by using a response surface method.
Background
Radix scrophulariae
ScrophulariaeRadix) Is Scrophulariaceae (
Scrophulariaceae) Plant Figwort root (
Scrophularia ningpoensisHemsl
.) Dried root of (4). It is produced mainly in Zhejiang, Hubei, Jiangsu and Jiangxi provinces, among which Xuan Shen produced in Zhejiang is the best choice, and is known as one of eight Zhejiang flavors. It is sweet, bitter, salty and slightly cold in flavor, has the effects of clearing heat and cooling blood, purging fire and removing toxicity, nourishing yin and lowering fire, and is mainly used for heat entering nutrient blood, warm toxicity and macula, fever impairing yin, crimson tongue and polydipsia, body fluid impairment and constipation. At present, the main chemical components of the medicine are cyclohexene ether terpenoids, phenylpropanoid glycosides, phenylethanoid glycosides, sterols, flavonoids, polysaccharides and other chemical components, and the medicine has pharmacological effects of easing pain, resisting fatigue, resisting inflammation, inhibiting bacteria, protecting cardiac muscle, resisting blood coagulation, resisting cerebral ischemia injury, enhancing immunity and the like, and figwort is clinically used for treating yin deficiency and blood stasis type coronary heart disease angina pectoris.
The Chinese medicinal polysaccharide is used as a natural macromolecular compound, and has the characteristics of wide extraction source and basically no cytotoxicity. At present, the research on traditional Chinese medicine polysaccharide in China is very wide, such as ginseng polysaccharide, astragalus polysaccharide, Chinese date polysaccharide, ganoderma lucidum polysaccharide and the like; the research direction mainly focuses on the parts of extraction process optimization, separation and purification process research, component structure analysis, in-vivo and in-vitro antioxidant activity, pharmacological and immunological activity and the like. A large number of reports show that the Chinese medicinal polysaccharide has good effects of resisting tumors, inhibiting bacteria and viruses, resisting oxidation and aging, regulating immunity, reducing blood sugar and the like.
With the intensive research on the activity of polysaccharide components, modification of polysaccharide structure becomes one of the important methods for improving the bioactivity or bioavailability of polysaccharide. The common polysaccharide structure modification methods at present include carboxymethylation, acetylation, sulfation, selenization, alkylation and other modification methods. The polysaccharide derivative after structural modification shows high activity in many aspects.
Sulfated polysaccharides, i.e., derivatives of polysaccharides modified by sulfation, include naturally occurring polysaccharide sulfate esters and artificially synthesized polysaccharide sulfate ester derivatives. The natural polysaccharide sulfate, such as chondroitin sulfate, heparin and the like, discovered at present have very good pharmacological activity. The sulfated structure of the polysaccharide is modified, so that the functions of the polysaccharide such as oxidation resistance, tumor resistance, immunoregulation, antibiosis, antivirus and the like can be improved. At present, the Wblform method is often adopted to sulfate the pyran-type polysaccharide, and the Nagasawa method is commonly used for the bark-type polysaccharide. Common methods for sulfating polysaccharides include chlorosulfonic acid-pyridine method, sulfur trioxide-pyridine method, concentrated sulfuric acid method, chlorosulfonic acid-DMF method, and the like. Although the sulfur trioxide-pyridine method and the concentrated sulfuric acid method are simple to operate, the substitution degree of the derivative is low and the product recovery rate is low; the chlorosulfonic acid-pyridine method has the advantages of complex operation, long reaction period, high product substitution degree and convenient recovery.
For example, Wang et al have demonstrated that Lycium barbarum polysaccharide sulfate can significantly promote Cell proliferation, while increasing HI antibody titer and enhancing LBPS immune activity (Wang J M, Hu Y L, Wang D Y, et al. surface modified cancer of lysine barbararum polysaccharide [ J ]. Cell Immunol, 2010, 263 (2): 219-23.). Zhao et al found that sulfated-modified Tremella polysaccharides significantly enhanced The in vitro proliferative capacity of splenic lymphocytes (Zhao X N, Hu Y L, Wang D Y. The compatibility of immune-enhancing activity of sulfated polysaccharides from Tremella and dCondononpsis pilosula [ J ]. Carbohydr Polym, 2013, 98 (1): 438-443.).
However, there are few reports on the figwort polysaccharide, and no report is found on the study on the structural modification and the activity of the derivative product.
At present, researches on radix scrophulariae mainly focus on the research on iridoid glycoside components and the activities thereof, but few reports are made on extraction and separation of radix scrophulariae polysaccharide and the in vitro antioxidant activity of the radix scrophulariae polysaccharide, and researches on the in vivo pharmacological activity of the radix scrophulariae polysaccharide are rare.
Disclosure of Invention
The invention aims to solve the technical problem of providing a process for optimizing the sulfation of the figwort polysaccharide by utilizing a response surface method, which is simple to operate and high in extraction rate.
In order to solve the problems, the process for optimizing the sulfation of the figwort polysaccharide by using the response surface method comprises the following steps:
⑴ sample preparation:
drying dried roots of radix scrophulariae at 50 ℃ for 24h, then crushing to 10 meshes, and mixing the powder according to the weight ratio of 200 g: soaking 6L of the above materials in deionized water for 1 hr, decocting for 2 hr, filtering the decoction, decocting the residue once again, and mixing filtrates; concentrating the filtrate under reduced pressure to 350-450 mL to obtain a concentrated solution, adding absolute ethyl alcohol with the volume 4 times that of the concentrated solution, uniformly mixing, standing overnight, and removing a supernatant to obtain a precipitate; dissolving the precipitate with 100mL of deionized water, removing pigment through a macroporous resin column DM-130, concentrating the eluate under reduced pressure, and removing protein by Sevage method to obtain radix scrophulariae polysaccharide extract with pigment and protein removed; concentrating the figwort polysaccharide extracting solution without pigment and protein, and freeze-drying to obtain figwort polysaccharide powder;
adding 20 mL of anhydrous formamide into 0.50 g of the figwort polysaccharide powder to dissolve the figwort polysaccharide powder to obtain a figwort polysaccharide formamide solution of 25 mg/mL; slowly adding the solution into esterification reagents with different proportions, heating in water baths at different temperatures of 60-100 ℃ under full stirring, and reacting for different time of 1-5 h to obtain reaction liquid; adjusting the pH of the reaction solution to be neutral by using a NaOH solution with the mass concentration of 40%, filling the reaction solution into a dialysis bag with the molecular interception amount of 1000, and dialyzing for 3d under normal flow water; concentrating the dialyzed solution under reduced pressure, and freeze-drying to obtain radix scrophulariae polysaccharide sulfate powder corresponding to esterification reagents with different proportions; the esterification reagent is prepared by adding 4.0-6.0 mL of pyridine pre-frozen for 2 hours at 4 ℃ into a three-neck flask placed in an ice-water bath; dropwise adding 6.0-9.0 mL of chlorosulfonic acid into a three-necked flask, reacting with pyridine under vigorous stirring, and dropwise adding for 30-40 min; when no dense smoke is generated, the reaction is finished to obtain a light yellow esterification reagent;
⑵ sample substitution was determined by barium chloride-gelatin turbidimetry:
① Standard Curve preparation:
accurately weighing 100.0 mg K
2SO
4Using 1 mol/L hydrochloric acid to adjust the volume to 100mL to obtain 1.0 mg/mL K
2SO
4A standard solution; respectively taking 20 μ L, 60 μ L, 100 μ L, 140 μ L, 180 μ L and 200 μ L with mass concentration of 1.0 mg/mLK
2SO
4Putting the standard solution in a test tube, and complementing 200 mu L of hydrochloric acid with 1 mol/L; adding 3.8mL of trichloroacetic acid with the mass concentration of 3% and 0.5% of BaCl in sequence
21mL of gelatin solution; taking 1 mol/L hydrochloric acid as a blank, measuring at a wavelength of 360nm within 5-15 min after sample adding, and recording the absorbance as A
1(ii) a Replacing BaCl with gelatin solution with mass concentration of 0.25%
2Gelatin solution, determination of the recorded absorbance A
2(ii) a With K
2SO
4The standard solution mg/mL is the abscissa, and the difference of absorbance values A
1-A
2Drawing a standard curve for a vertical coordinate;
② measurement of degree of substitution of sample:
precisely weighing 10mg sulfated radix scrophulariae polysaccharide in a test tube, adding 5mL of 1 mol/L hydrochloric acid, hydrolyzing in 90 deg.C water bath for 5 hr, supplementing hydrochloric acid to 5mL after hydrolysis to obtain 2 mg/mL sulfated radix scrophulariae polysaccharide hydrolysate, placing 200 μ L hydrolysate in the test tube, and finding out K according to the standard curve, wherein the rest operation is the same as that of the standard curve prepared in step ①
2SO
4Concentration;
⑶ sample ABTS
+Determination of clearance rate:
a) ABTS
+preparing a solution: weighing 10mg ABTS in a test tube, adding 2.6 mL of 0.66 mg/mL potassium persulfate solution, mixing thoroughly, and reacting for 16 h at room temperature in the dark to obtain ABTS
+(ii) a After the reaction is finished, diluting the mixture by absolute ethyl alcohol, and measuring the mixture at 734 nm until the absorbance is 0.7 +/-0.02;
b) determination of sample clearance: dissolving 2.5 mg of sulfated figwort polysaccharide powder in 5mL of distilled water to obtain 0.5mg/mL of sample solution; precisely transferring 1mL of sample solution into a test tube, and adding diluted ABTS
+3mL of solution is reacted for 1h at room temperature in a dark place; after the reaction, the absorbance A was measured at a wavelength of 734 nm
1Replacing ABTS with absolute ethyl alcohol
+Solution, absorbance measured by the same method as A
x(ii) a Replacing sample liquid with distilled water as blank, measuring by the same method, and recording absorbance as A
0(ii) a (ii) a Calculating ABTS
+Clearance (%);
ABTS
+clearance (%) = (a)
1-A
x）/ A
0×100%；
⑷ Experimental design and statistical analysis:
① Single-factor test:
sequentially changing the reaction temperature, the reaction time and the proportion of the esterification reagent to carry out a single-factor test, calculating the percentage content S% of sulfur and the substitution degree DS of the sulfate radical in the sulfated figwort polysaccharide obtained by measuring by a barium chloride-gelatin turbidimetry according to the following formula, and repeating the treatment for 3 times each time:
S%=0.092 Cs×100%；
wherein 162 is relative molecular weight of monosaccharide, 32 is sulfur atom mass fraction, and 102 is relative molecular weight increase value of monosaccharide after hydroxyl in monosaccharide group is substituted by sulfate group;
② response surface method optimization design:
according to the result of the single-factor test, the reaction temperature, the reaction time and the ratio of the esterification reagent are taken as investigation factors, and the substitution degree and ABTS are taken as
+The clearance is used as a survey index, Design Expert 8.0.6 software is used for carrying out experimental Design according to the Box-Behnken Design principle, the reaction temperature A, the reaction time B and the esterification reagent proportion C are used as independent variables, the substitution degree DS is used as a response value y and ABTS
+And (3) establishing a multivariate quadratic regression equation with the clearance rate as a response value y':
y=1.21+8.925×10
-3A+0.047B+7.925×10
-3C+0.033AB-0.039AC+0.016BC-
0.14A
2-0.061B
2-0.099C
2；
y´=（83.34+1.83A+1.81B+3.41C-0.9AB-1.85AC+3.23BC-9.18A
2-3.58B
2
-10.46C
2）×100%；
⑸ analysis and optimization of experimental results:
and (4) performing drawing analysis by using Design Expert 8.0.6 software according to the multiple quadratic regression equation to obtain a response surface of the regression equation and a contour map thereof.
The 0.25% gelatin solution in the step ① is obtained by dissolving 1.250 g of gelatin in 100 ℃ water bath by heating and fixing the volume to 500 mL.
0.5% BaCl in said step ①
2Gelatin solution means that 1.250 g of BaCl
2The volume of the solution is adjusted to 250mL by 0.25% gelatin solution.
The 3% trichloroacetic acid solution in the step ① is obtained by dissolving 15.0 g trichloroacetic acid in water to a constant volume of 500 mL.
Compared with the prior art, the invention has the following advantages:
1. compared with an orthogonal method, the method takes reaction temperature, reaction time and esterification reagent ratio as investigation factors, a three-factor three-level model response surface analysis method is designed by adopting a Box-Behnken principle through Design-Expert 8.0.6 software, an optimization result can be obtained by using 3 change factors, 3 levels and a small amount of experiment groups (only 17 groups of experiments), and the optimal substitution degree and ABTS are obtained
+Clearance rate, increased degree of substitution and increased ABTS
+The clearance rate has practical significance for industrial production; and the resulting optimum extraction conditions are not set values but are within the range of the set conditions.
2. The optimal reaction conditions obtained by analyzing the quadratic regression model are that the reaction temperature is 81 ℃, the reaction time is 200 min, and the ratio of esterification reagents is 2:1, and the substitution degree of the figwort polysaccharide reaches 1.2175, ABTS
+The clearance rate reaches 82.21%. At the same time, the degree of substitution and ABTS
+The basic coincidence of the optimal clearing capacity points shows that the figwort polysaccharide sulfate ABTS
+The clearance ability has close relation with the degree of substitution, and ABTS is increased along with the improvement of the degree of substitution
+The clearance rate also shows an upward trend.
3. The barium chloride-gelatin turbidimetry method adopted by the invention is examined in methodology, and the correlation coefficient R =0.9995 of a standard curve of the method indicates that the method is good in linear regression (see figure 1).
⑴ stability examination:
precisely transferring 200 μ L sulfated polysaccharide hydrolysate, measuring at 0min, 3min, 6 min, 9 min, 12 min, 15min, 20min, 25 min and 30 min after sample application, and calculating substitution degree and RSD (see Table 1).
Table 1 stability test results
Through stability investigation, when the degree of substitution of a sample is measured by a barium chloride-gelatin turbidimetry method, the absorbance is gradually increased within 0-3 min, and the time is the time for the barium chloride to react with sulfate radicals to generate barium sulfate precipitate; is stable within 3-15 min, and is the best time for determination; after 15min, the turbidity of the solution decreased due to the natural sedimentation of barium sulfate, and the absorbance decreased as well. Therefore, when the barium chloride-gelatin turbidimetry is used for measurement, the measurement is finished within 3-15 min after the sample is added, so that the phenomenon that the measurement result is inaccurate due to natural sedimentation of barium sulfate precipitate is avoided, and the error is generated on the experimental result.
⑵ precision investigation:
precisely transferring 200 mu L of sulfated polysaccharide hydrolysate into six test tubes with plugs. The measurement was carried out according to the standard curve measurement method, and the degree of substitution and RSD of six parallel groups were calculated, and the precision of the parallel measurement was examined by this method. The results are shown in Table 2.
TABLE 2 results of precision investigation
According to the precision investigation result, the barium chloride-gelatin turbidimetry has good precision, the difference among experiments of each parallel group is small, and the measurement result is credible.
⑶ repeatability test:
taking a sulfated radix scrophulariae polysaccharide sample, repeatedly performing six groups of measurement according to the sample substitution degree measurement method, calculating the substitution degree of each measurement and calculating RSD. The results are shown in Table 3.
TABLE 3 results of repeated investigation
The result difference of the barium chloride-gelatin turbidimetry in repeated determination is small and the experimental data is reliable through repeated investigation experiments.
⑷ sample recovery test:
precisely transferring 1.0 mg/mL K respectively
2SO
4Adding 100 μ L of standard solution into six-support plug test tube, adding sulfated radix scrophulariae polysaccharide hydrolysate with potassium sulfate equivalent of 0.1 mg, and supplementing with 1 mol/L hydrochloric acid to 200 μ L. And (4) measuring according to the same method as the standard curve measuring method, calculating the equivalent weight of each group of potassium sulfate according to the measuring result, and calculating the RSD. The results are shown in Table 4.
TABLE 4 investigation results of sample recovery
From the investigation result of the sample recovery rate, the average sample recovery rate of the barium chloride-gelatin turbidimetry is in the range of 95-105%, the average sample recovery rate is 99.3%, and the RSD is 2.0%, which shows that the method has no significant difference in sensitivity to the sample and the barium chloride and is reliable.
4. The invention discovers that the sugar chain configuration and the charge distribution are changed due to the key input of the sulfate group through comparison. Adjacent sulfate groups repel each other due to steric hindrance, and sugar chain distortion modification is caused; simultaneously, due to the fact that a large number of electron-donating groups are input, sugar chain charge distribution is changed, and compared with figwort polysaccharide, the figwort polysaccharide sulfate is ABTS
+The cleaning capability is obviously improved, and IC
50From 1.36 mg/mL before esterification to 0.230 mg/mL after esterification (as shown in FIG. 13).
The infrared spectrogram analysis also proves that the hydroxyl vibration peak of the figwort polysaccharide sulfate is obviously weakened compared with the spectrogram of the figwort polysaccharide, and characteristic absorption peaks of S = O and C-O-S are newly appeared, thereby verifying the success of the sulfation modification of the figwort polysaccharide.
In the infrared spectrum 14 of the figwort polysaccharide, the value can be seen at 3378 cm
-1There is a clear absorption peak for VOH; 1053 cm
-1The absorption peak of (a) is a stretching vibration absorption peak of a C-O bond in a hydroxyl group; 2930 cm
-1The sharper peak is a-C-H stretching vibration absorption peak; 1411 cm
-1The position is a-C-H bond bending vibration absorption peak; 1609 cm
-1The strong and stable absorption peak is-C = O vibration absorption peak [42,43]。
Comparing the infrared spectrogram 14 and 15 of the figwort polysaccharide sulfate shows that the figwort polysaccharide sulfate well keeps the characteristic absorption peaks of the parent body, and certain characteristic peaks are correspondingly changed due to the key-in of the sulfate group. Firstly, 3354-3363 cm
-1The large and slow peak is the-OH shock absorption peak on the polysaccharide chain, and the comparison shows that the-OH absorption peak of the esterified polysaccharide is smaller and narrower than that of the figwort polysaccharide because a part of-OH is replaced by sulfation. Second, V
C=OThe absorption peak shifts to high frequencies, indicating that some of the intramolecular hydrogen bonds are broken. The spectrum of the esterified polysaccharide is 1230 cm
-1Characteristic absorption peak by C-O-S and 837 cm appearing nearby
-1The characteristic absorption peaks of S = O appearing nearby are all due to the introduction of a sulfate group. Therefore, the sulfuric acid groups are successfully input into the sulfuric acid esterification derivatization of the figwort polysaccharides, and the sulfuric acid groups can be fully embodied in the infrared spectrum.
5. The obtained sulfated figwort polysaccharide and figwort polysaccharide have strong anti-inflammatory effect, and the anti-inflammatory mechanism of the sulfated figwort polysaccharide is related to the inhibition of the phosphorylation of NF-kappa Bp 65. And the figwort polysaccharide has better anti-inflammatory effect after being modified by sulfation. Therefore, the figwort polysaccharide can effectively improve the pharmacological activity after being modified by persulfuric acid esterification, and has wider application prospect.
[ PROPHYLENE ACHIATUM POLYSACCHARIDE AND SULPHATED PROPHYLENE ACHIATUM POLYSACCHARIDE SOURCE ]
The dried radix scrophulariae slices purchased from regular medicinal materials are identified as Scrophulariaceae by Wangjian auxiliary professor of traditional Chinese medicine of pharmaceutical institute of Oenongmedical institute
ScrophulariaceaeScrophularia genus
ScrophulariaPlant radix scrophulariae
ScrophularianingpoensisHemslSlicing root, oven drying → pulverizing → sieving with 50 mesh sieve→ degreasing with acetone → hot water reflux extraction → macroporous resin decolorant → sevag reagent decolorant → freeze drying → radix scrophulariae polysaccharide → pyridine chlorosulfonate method to obtain sulfated radix scrophulariae polysaccharide.
[ animal origin ]
All the mice are purchased from stockbreeding limited of great-grained city of Qingdao Daren. The specification of the mice is 40 male SPF grade ICR mice (18-22 g). The experimental animal is fed with conventional feed in room at 24 + -2 deg.C, and is fed with free drinking water for one week.
[ Experimental methods ]
⑴ grouping administration and material selection of mice
① grouping of mice:
40 ICR mice were randomly divided into 4 groups (10/group): (1) normal control group (gavage and intraperitoneal injection of the same dose of normal saline); (2) model group (lavage of normal saline, intraperitoneal injection of LPS 1 mg/kg); (3) a positive control group (aspirin for intragastric administration 10 mg/kg, LPS for intraperitoneal injection 1 mg/kg); (4) sulfated radix scrophulariae polysaccharide dose group (200 mg/kg, i.e. 1 mg/kg of LPS for intraperitoneal injection).
② dosing and modeling:
according to the experimental grouping, different groups of mice are respectively administered with corresponding doses of sulfated figwort polysaccharide and aspirin through gastric gavage for 3 days, and normal control groups and model groups are administered with corresponding doses of normal saline to ensure the experimental consistency. LPS was injected intraperitoneally 30 min after the last gavage.
③ mouse selection:
the body weight of the mice was accurately weighed 90min after LPS injection. Placing the venous blood of the mobile mouse at room temperature for 2h, and centrifuging to obtain serum for later use; mouse liver tissue was taken and accurately weighed and recorded and stored in a-80 ℃ freezer.
⑵ ELISA method for detecting protein expression of IL-6 and TNF- α in serum:
the mouse serum stored in the refrigerator at-20 ℃ in step ③ was allowed to stand at room temperature.
I determination of IL-6 content in serum:
adding sample, incubating and developing according to the specification of an IL-6 ELISA kit of Invitrogen company, detecting the absorbance of each sample at 450 nm by using a multifunctional microplate reader after developing, and calculating the protein content of IL-6 in serum according to the specification of the kit.
Ii determination of TNF- α content in serum:
adding sample, incubating and developing according to the specification of a TNF- α ELISA kit of Peprotech company, detecting the absorbance of each sample at 450 nm by using a multifunctional microplate reader after developing, and calculating the protein content of TNF- α in serum according to the specification of the kit.
⑶ Real time RT-PCR method for detecting mRNA expression of IL-6 and TNF- α in liver:
① Total RNA extraction, content determination and cDNA Synthesis in liver tissue
I, fully cracking liver tissues, operating according to a UNIQ-10 Column Trizol kit, extracting total RNA in the tissues, collecting the RNA and calculating the concentration of the total RNA.
Ii, reverse transcription of the extracted total RNA into cDNA using a reverse transcription kit. Adding random primer 5 μ l and sample 5 μ l, mixing on ice, extending at 50 deg.C for 15min, standing at 80 deg.C for 5min to denature at high temperature, and performing real-time fluorescence quantitative polymerase chain reaction with the primer.
② fluorescence labeling and relative quantification calculation of Real-time RT-PCR
Fluorescent dye SYBR Green I is added into the PCR reaction solution, and the dye is combined with DNA to emit fluorescence, so that quantitative detection is realized. Then, solubility curve analysis is carried out to ensure that the target gene can be specifically amplified. Finally, an ABI 7500 real-timePCR instrument is used for amplification to obtain C
TValue and adopt 2
-ΔΔC TThe method carries out data statistics.
⑷ Western blot method for detecting the protein expression of NF-kappa Bp65 and phospho-NF-kappa Bp65 in mouse liver tissues:
taking mouse liver tissues stored in a refrigerator at ③ -80 ℃, adding RIPA lysate with corresponding volume according to m: V =1:9, ultrasonically crushing the lysed tissues, extracting and separating proteins, determining the protein concentration by adopting a BCA method, specifically referring to BCA kit specification of Solebao company, uniformly mixing the obtained proteins with SDS-PAGE, boiling for 3-5 min, fully deforming the proteins, performing polyacrylamide gel electrophoresis, adding the quantified proteins into SDS-PAGE gel adding holes (40 mu g per hole), setting the voltage to be 100V, setting the time to be 90-120 min for electrophoresis, installing a wet rotation instrument, transferring the proteins onto a PVDF membrane by using the wet rotation method (switching on an external power supply for 300mA for 30-90min), washing with PBTS, sealing the PVDF membrane chamber temperature for 2h by using PBTS containing 5% skimmed milk powder, diluting primary antibody (NF-kappa Bp65, Phospb-Bp-65, performing constant current scanning on a constant current-constant current scanning bed containing 5% skimmed milk powder for 2h, and performing slow light emission scanning on a constant current scanning bed containing PBTS 19-34% of a constant current-stabilized bed containing peroxidase, and a constant current scanning on a constant current scanning bed (20-34) containing PBTS) containing 5% skimmed milk powder, and performing slow light emission on a constant current scanning bed, and a constant current scanning bed for 2h, and a constant time, and a constant current scanning on a constant speed scanning bed for 2h, and a constant speed scanning bed for detecting mouse by using a constant speed scanning bed (20-stabilized bed) for 20-20 th, and a constant current scanning bed.
⑸ statistical processing analysis:
the experimental data among the groups are analyzed by adopting one-factor variance analysis, the data analysis results are expressed by Mean +/-standard error (Mean +/-SE), the variance analysis is adopted for the comparison among the groups, the t test is adopted for the comparison between the two groups,
Pa value of < 0.05 indicates statistical significance.
[ results and discussion ]
⑴ effects on LPS-induced protein expression of the inflammatory factors IL-6 and TNF- α:
① effect on LPS-induced protein expression of the inflammatory factor IL-6:
the results of the experiment are shown in FIG. 16: the expression of IL-6 protein in the serum of mice in the model group was significantly increased compared with that in the normal control group (
P< 0.01). After aspirin and sulfated figwort polysaccharide are given for protection, the expression of IL-6 protein in each group of blood serum is obviously reduced compared with that in a model group (
P<0.01), sayThe figwort polysaccharide sulfating Ming can effectively relieve acute inflammatory injury of mice and has good anti-inflammatory effect. And the expression of IL-6 protein in the serum of the sulfated radix scrophulariae polysaccharide group is similar to that of the aspirin group, which shows that the anti-inflammatory effect of the sulfated radix scrophulariae polysaccharide is similar to that of the aspirin.
② effect on LPS-induced protein expression of the inflammatory factor TNF- α:
the results of the experiment are shown in FIG. 17: the expression of IL-6 protein in the serum of mice in the model group was significantly increased compared with that in the normal control group (
P< 0.01) the expression of TNF- α protein in serum was reduced after aspirin protection compared to the model group
P<0.01), after the sulfated figwort polysaccharide is given for protection, the expression of TNF- α protein in serum of each group is obviously reduced compared with that of a model group (
P<0.001), and the expression of TNF- α protein in the sulfated radix scrophulariae polysaccharide group is obviously reduced compared with that of the aspirin group, which shows that the sulfated radix scrophulariae polysaccharide can effectively relieve acute inflammatory injury of mice and has good anti-inflammatory effect, and the sulfated radix scrophulariae polysaccharide has more obvious anti-inflammatory effect compared with that of aspirin.
⑵ effects on LPS-induced mRNA expression of IL-6 and TNF- α in liver tissue:
① Effect on LPS-induced mRNA expression of IL-6 in liver tissue:
the experimental results are shown in fig. 18: significantly increased expression of IL-6mRNA in liver tissue of the model group compared with that of the normal control group: (
P< 0.01). After aspirin and sulfated radix scrophulariae polysaccharide are given for protection, the expression of IL-6mRNA in liver tissues of aspirin group is obviously reduced compared with that of model group (the expression level of the mRNA in liver tissues of aspirin group is lower than that in liver tissues of aspirin group by the method of (1)
P<0.01), the expression of IL-6mRNA in the sulfated figwort polysaccharide liver tissue is more obviously reduced than that of the model group (
P<0.001), which shows that the sulfated figwort polysaccharide can effectively relieve acute inflammatory injury of mice and has good anti-inflammatory effect.
② effect on LPS-induced mRNA expression of TNF- α in liver tissue:
the results of the experiment are shown in FIG. 19: compared with the normal control group,significantly elevated expression of TNF- α mRNA in liver tissue of the model group: (
P< 0.01) after aspirin and sulfated scrophularia polysaccharide are given for protection, the expression of liver TNF- α mRNA in aspirin group is reduced compared with that in model group
P<0.01), the expression of TNF- α mRNA in the sulfated figwort polysaccharide liver tissue is more obviously reduced than that in the model group, and the expression of TNF- α mRNA in the sulfated figwort polysaccharide liver tissue is lower than that in the aspirin group (1)
P<0.001), which shows that the sulfated figwort polysaccharide can effectively relieve acute inflammatory injury of mice and has good anti-inflammatory effect.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a standard curve of the barium chloride-gelatin turbidimetry of the present invention.
FIG. 2 shows the effect of reaction temperature on the degree of substitution according to the invention.
FIG. 3 is a graph showing the effect of reaction time on the degree of substitution according to the present invention.
FIG. 4 is a graph showing the effect of the ratio of esterifying reagents on the degree of substitution according to the present invention.
FIG. 5 is a model diagnostic map of the present invention. Wherein: the left graph is a residual probability normal distribution graph, and the right graph is a corresponding graph of the residual and an equation predicted value.
FIG. 6 shows the interaction of the reaction temperature and reaction time on the degree of substitution according to the present invention.
FIG. 7 is a graph showing the interaction of reaction temperature and reagent ratio on the degree of substitution according to the present invention.
FIG. 8 is a graph showing the interactive effect of reagent ratio and reaction time on the degree of substitution according to the present invention.
FIG. 9 shows ABTS of the present invention
+Clearance response surface model diagnostic map. Wherein: the left graph is a residual probability normal distribution graph, and the right graph is a corresponding graph of the residual and an equation predicted value.
FIG. 10 shows ABTS of the present invention
+Clearance temperature-time interaction model.
FIG. 11 shows ABTS of the present invention
+Clearance temperature-reagent ratio interaction model.
FIG. 12 ABTS of the present invention
+Clearance reagent ratio-time interaction model.
FIG. 13 shows polysaccharide sulfate and figwort polysaccharide ABTS of the present invention
+The cleaning ability is compared.
FIG. 14 shows an infrared spectrum of figwort polysaccharide (potassium bromide as background film, 400-4000 cm)
-1Scanning at wavenumbers).
FIG. 15 is an infrared spectrum of sulfated radix scrophulariae polysaccharide (potassium bromide as background tablet at 400-4000 cm)
-1Scanning at wavenumbers).
FIG. 16 shows the effect of sulfated figwort polysaccharides of the present invention on LPS-induced mouse liver IL-6 protein expression (
##It is shown that compared to the blank control group,
P<0.01；
**presentation compared to LPS group
P<0.01）。
FIG. 17 shows the effect of sulfated figwort polysaccharides of the present invention on LPS-induced mouse liver TNF- α protein expression (
##It is shown that compared to the blank control group,
P<0.01；
*presentation compared to LPS group
P<0.05；
**Presentation compared to LPS group
P<0.01）。
FIG. 18 shows the effect of sulfated figwort polysaccharides of the present invention on LPS-induced mouse liver IL-6mRNA expression (
##It is shown that compared to the blank control group,
P<0.01；
**presentation compared to LPS group
P<0.01）。
FIG. 19 shows the effect of sulfated figwort polysaccharides of the present invention on LPS-induced mouse liver TNF- α mRNA expression (
##It is shown that compared to the blank control group,
P<0.01；
**presentation compared to LPS group
P<0.01）。
Detailed Description
A process for optimizing sulfuric acid esterification of figwort polysaccharide by using a response surface method comprises the following steps:
⑴ sample preparation:
drying dried roots of radix scrophulariae at 50 ℃ for 24h, then crushing to 10 meshes, and mixing the powder according to the weight ratio of 200 g: soaking 6L of the above materials in deionized water for 1 hr, decocting for 2 hr, filtering the decoction, decocting the residue once again, and mixing filtrates; concentrating the filtrate to 350-450 mL by a rotary evaporator under reduced pressure to obtain a concentrated solution, adding absolute ethyl alcohol with the volume 4 times that of the concentrated solution, uniformly mixing, standing overnight, and removing a supernatant to obtain a precipitate; dissolving the precipitate with 100mL of deionized water, removing pigment with macroporous resin column DM-130, concentrating the eluate under reduced pressure with rotary evaporator, and removing protein with Sevage method to obtain radix scrophulariae polysaccharide extract with pigment and protein removed; and (3) carrying out reduced pressure concentration on the figwort polysaccharide extract without pigments and proteins by using a rotary evaporator, and carrying out freeze drying by using a vacuum freeze dryer to obtain figwort polysaccharide powder.
20 mL of anhydrous formamide is added into 0.50 g of figwort polysaccharide powder for dissolving to obtain figwort polysaccharide formamide solution of 25 mg/mL; slowly adding the solution into esterification reagents with different proportions, heating in water baths at different temperatures of 60-100 ℃ under full stirring, and reacting for different time of 1-5 h to obtain reaction liquid; adjusting the pH of the reaction solution to be neutral by using a NaOH solution with the mass concentration of 40%, filling the reaction solution into a dialysis bag with the molecular interception amount of 1000, and dialyzing for 3d under normal flow water; and (3) concentrating the dialyzed solution under reduced pressure by using a rotary evaporator, and freeze-drying by using a vacuum freeze-drying machine to obtain the figwort polysaccharide sulfate powder corresponding to the esterification reagents in different proportions.
The esterification reagent is prepared by adding 4.0-6.0 mL of pyridine pre-frozen for 2 hours at 4 ℃ into a three-neck flask placed in an ice-water bath; dropwise adding 6.0-9.0 mL of chlorosulfonic acid into a three-necked flask, reacting with pyridine under vigorous stirring, and dropwise adding for 30-40 min; and when no dense smoke is generated, finishing the reaction to obtain the light yellow esterification reagent.
⑵ sample substitution was determined by barium chloride-gelatin turbidimetry:
① Standard Curve preparation:
accurately weighing 100.0 mg K
2SO
4Using 1 mol/L hydrochloric acid to adjust the volume to 100mL to obtain 1.0 mg/mL K
2SO
4A standard solution; respectively taking 20 μ L, 60 μ L, 100 μ L, 140 μ L, 180 μ L and 200 μ L of K with mass concentration of 1.0 mg/mL
2SO
4Putting the standard solution in a test tube, and complementing 200 mu L of hydrochloric acid with 1 mol/L; adding 3.8mL of trichloroacetic acid with the mass concentration of 3% and 0.5% of BaCl in sequence
2-gelatin solution1mL of the solution; taking 1 mol/L hydrochloric acid as a blank, measuring at a wavelength of 360nm within 5-15 min after sample adding, and recording the absorbance as A
1(ii) a Replacing BaCl with gelatin solution with mass concentration of 0.25%
2Gelatin solution, determination of the recorded absorbance A
2(ii) a With K
2SO
4The standard solution mg/mL is the abscissa, and the difference of absorbance values A
1-A
2The ordinate is a standard curve.
Wherein: the 0.25% gelatin solution is obtained by dissolving 1.250 g gelatin in 100 deg.C water bath under heating, and diluting to 500 mL.
0.5% BaCl
2Gelatin solution means that 1.250 g of BaCl
2The solution is prepared in situ after being prepared into 250mL by 0.25 percent gelatin solution.
The 3% trichloroacetic acid solution is obtained by dissolving 15.0 g trichloroacetic acid in water to a constant volume of 500mL, and storing in dark place.
② measurement of degree of substitution of sample:
precisely weighing 10mg sulfated radix scrophulariae polysaccharide in a test tube, adding 5mL of 1 mol/L hydrochloric acid, hydrolyzing in 90 deg.C water bath for 5 hr, supplementing hydrochloric acid to 5mL after hydrolysis to obtain 2 mg/mL sulfated radix scrophulariae polysaccharide hydrolysate, placing 200 μ L hydrolysate in the test tube, and making standard curve according to the standard curve in step ①, wherein K is found out according to the standard curve
2SO
4And (4) concentration.
⑶ sample ABTS
+Determination of clearance rate:
a) ABTS
+preparing a solution: weighing 10mg ABTS in a test tube, adding 2.6 mL of 0.66 mg/mL potassium persulfate solution, mixing thoroughly, and reacting for 16 h at room temperature in the dark to obtain ABTS
+(ii) a After the reaction, the mixture was diluted with absolute ethanol and measured at 734 nm until the absorbance became 0.7. + -. 0.02.
b) Determination of sample clearance: dissolving 2.5 mg of sulfated figwort polysaccharide powder in 5mL of distilled water to obtain 0.5mg/mL of sample solution; precisely transferring 1mL of sample solution into a test tube, and adding diluted ABTS
+3mL of solution is reacted for 1h at room temperature in a dark place; after the reaction, the absorbance A was measured at a wavelength of 734 nm
1Replacing ABTS with absolute ethyl alcohol
+Solution ofThe absorbance of the assay is recorded as A
x(ii) a Replacing sample liquid with distilled water as blank, measuring by the same method, and recording absorbance as A
0(ii) a Calculating ABTS
+Clearance (%).
ABTS
+Clearance (%) = (a)
1-A
x）/ A
0×100%。
⑷ Experimental design and statistical analysis:
① Single-factor test:
sequentially changing the reaction temperature, the reaction time and the proportion of the esterification reagent to carry out a single-factor test, measuring the sulfate radical concentration Cs of the sample in the obtained sulfated radix scrophulariae polysaccharide by using a barium chloride-gelatin turbidimetry, calculating the percentage content S% of sulfur and the substitution degree DS according to the following formula, and repeating the treatment for 3 times each time:
S%=0.092 Cs×100%；
wherein 162 is the relative molecular weight of monosaccharide, 32 is the sulfur atom mass fraction, and 102 is the relative molecular weight increase of monosaccharide having hydroxyl group substituted by sulfate group
② response surface method optimization design:
according to the result of the single-factor test, the reaction temperature, the reaction time and the ratio of the esterification reagent are taken as investigation factors, and the substitution degree and ABTS are taken as
+The clearance is used as a survey index, Design Expert 8.0.6 software is used for carrying out experimental Design according to the Box-Behnken Design principle (the survey factors and the level are shown in table 5), the reaction temperature A, the reaction time B and the esterification reagent ratio C are used as independent variables, the substitution degree DS is used as a response value y, ABTS
+The clearance rate is a response value y' (the experimental scheme and the result are shown in the table 6), and a multivariate quadratic regression equation is established:
y=1.21+8.925×10
-3A+0.047B+7.925×10
-3C+0.033AB-0.039AC+0.016BC-
0.14A
2-0.061B
2-0.099C
2。
y´=（83.34+1.83A+1.81B+3.41C-0.9AB-1.85AC+3.23BC-9.18A
2-3.58B
2
-10.46C
2）×100%。
from the regression model analysis table (table 7), it is found that the P value of the entire model is less than 0.001, and extremely significant. F =2.3 and P =0.2188 in the differential terms, indicating that the model differential terms are not significant. As shown in fig. 5, the left graph shows the normal distribution of the residual probability, and the residuals are substantially distributed near the straight line and are more concentrated at the center of the straight line, which indicates that the residual distribution conforms to the normal distribution curve. The right graph shows the corresponding values of the residual error and the equation predicted value, points in the graph are dispersed, and therefore the model is well fitted to the experimental result, the model fitting is consistent with the actual situation, and the model reliability is high.
The quadratic polynomial regression equation shows that the sum of coefficients of each factor term is-0.22615, which indicates that the image of the equation has a downward opening and has a maximum value point, so that the optimal substitution reaction condition can be optimized. The coefficients of the first order term of the equation can be found: of the three factors affecting the degree of substitution, the reaction time > reaction temperature > ratio of esterification reagents.
As can be seen from the data in Table 8, the ABTS
+The clearance response surface model has the overall P =0.0065, which shows that the model achieves a very significant level; the values of the differential terms F =3.6 and P =0.1238, which indicates that the model differential terms are not significant, and it can be found from the diagnostic model diagram (left diagram) in fig. 9 that the residual normal probability distribution diagram is centered on a straight line, is dense in the middle and sparse on two sides, and conforms to the normal distribution rule; the residual error is dispersed with the corresponding value of the equation predicted value (right graph), which shows that the experimental data is well fitted and is consistent with the actual situation, and the model is reliable.
Among the three influencing factors, the influence of the reagent ratio on the clearance rate is more obvious; the influence of the reaction temperature and the reaction time on the clearance is not significant. The impact of the two-factor interaction model on clearance is not significant, possibly due to the impact on ABTS
+The reason for the cleaning ability is more, and the mechanism is more complicated. In addition, the temperature quadratic term sum of the quadratic terms of all the factors reaches a very significant level, and the reaction time quadratic term is not significant.
The sum of the coefficients of the terms of each factor of the quadratic polynomial regression equation is-15.69, which shows that the image of the equation has a downward opening and has a maximum value point, so that the reaction condition with the optimal clearance rate can be optimized. The coefficients of the first order term of the equation can be found: of the three factors affecting clearance, the ratio of esterification reagents > reaction time > reaction temperature.
TABLE 5 response surface consideration factors and levels
Table 6 response surface analysis protocol and results
TABLE 7 regression model analysis Table
Note: indicates very significant levels.
TABLE 8 ABTS
+Return model analysis table of clearance response surface
Note: indicates significant levels; indicates that there are very significant levels.
⑸ analysis and optimization of experimental results:
and (4) performing drawing analysis by using Design Expert 8.0.6 software according to the multiple quadratic regression equation to obtain a response surface of the regression equation and a contour map thereof.
As shown in fig. 2, the substitution degree of figwort polysaccharide increases and decreases gradually with the gradual increase of the reaction temperature, and the substitution degree is highest at 80 ℃. The degree of substitution gradually increased as the reaction temperature increased from 60 ℃ to 80 ℃, probably because the increase in temperature was favorable for increasing the activity of a part of the hydroxyl groups; the substitution degree is gradually reduced along with the temperature rise at 80-100 ℃, and experiments show that the reaction liquid after the reaction is finished has a slight coking phenomenon at 90-100 ℃, and the fluidity is relatively reduced, so that the strong oxidizing property of chlorosulfonic acid at high temperature is supposed to damage certain structures in polysaccharide chains, so that the substitution reaction capability is reduced.
As shown in fig. 3, the substitution degree of figwort polysaccharide increases and then decreases with the increase of the reaction time, and reaches a maximum in 3 hours. The substitution degree is continuously increased in the process of increasing the reaction time from 1h to 3 h, probably because the substitution reaction is slow and needs a long time to complete the reaction. After 3 h, the substitution degree shows a slow reduction trend along with the continuous increase of the reaction time, which can be caused by the oxidation of residual chlorosulfonic acid due to the overlong reaction time, or the occurrence of side reaction due to the increase of heating time, or the breaking of bonds of originally associated sulfate groups.
As shown in FIG. 4, the substitution degree of the figwort polysaccharide is increased and then decreased with the increase of the ratio of the esterifying reagents, and the substitution degree is maximized when the volume ratio of chlorosulfonic acid to pyridine is 2: 1. The degree of substitution increases as the volume ratio increases from 1:1 to 2:1, and should be increased because increasing the volume ratio of chlorosulfonic acid leads to gradual completion of the esterification reaction of the esterification reagents. The substitution degree was decreased as the volume ratio of the two was increased from 2:1 to 3:1, and it was estimated that the amount of pyridine produced was decreased gradually to decrease the amount of the esterifying reagent. In the experimental process, the phenomenon that the color of the dialysate is obviously darkened firstly and then lightened after the dialysis of the esterification reagent proportion group is completed is also found, and the linkage of a sulfate group is presumed to be one of the reasons for deepening the color.
As can be seen from fig. 6, in the model of the interaction between the reaction time and the reaction temperature (right graph), the influence of the reaction temperature on the degree of substitution is more significant. As can be seen from the contour plot (left panel), the temperature-time interaction has a significant effect on the degree of substitution: when the reaction time is 2-3.4 h and the reaction temperature is 70-80 ℃, the substitution degree is continuously increased, and the substitution degree is continuously reduced in the process of 3.4-4 h and 80-90 ℃.
As can be seen from the temperature-reagent ratio interaction contour diagram (left panel) of FIG. 7, the degree of substitution of the esterified polysaccharide increases as the reaction temperature increases from 70 ℃ to 80 ℃ and the reagent ratio increases from 1.5:1 to 2:1, reaching a maximum at approximately 80 ℃ and 2: 1. The curvature of the change in the reaction temperature and the reagent ratio are approximately equivalent in the 3D model (right panel), which shows that both have a certain effect on the degree of substitution in the temperature-reagent ratio interaction.
In the reaction time-reagent ratio interaction contour plot (left plot) of FIG. 8, it can be seen that the degree of substitution peaks at 3.5h and a reagent ratio of 2: 1. It can be seen in the 3D model (right panel) that the curvature of the reaction time is larger than the curvature of the reagent ratio, indicating that the reaction time has a greater effect on the degree of substitution in the time-ratio interaction.
Temperature-time vs. ABTS from FIG. 10
+From the contour plot of the cross-influence of clearance, ABTS increased from 2h to 3 h as the reaction temperature increased from 70 ℃ to 90 ℃ and the reaction time increased
+The clearance rate is continuously increased and reaches the maximum clearance rate at about 80 ℃ for 3.4 h. It can be seen from the 3D model that the effect of reaction temperature on clearance is greater than the effect of reaction time in the temperature-time interaction.
From the temperature-reagent ratio interaction model diagram of FIG. 11, ABTS
+Clearance rate with reaction temperature from 70 ℃ to 80 ℃ and reagent ratio from 1.5:1 to 2:1
+The clearance rate is increasing. It can be found in the 3D model that the temperature-reagent ratio is to ABTS
+The effect of the ratio of reagents is greater than the effect of temperature in the clearance interaction.
Reaction time-reagent ratio vs ABTS from FIG. 12
+Interaction of clearance rates ABTS in contour plot
+The clearance rate is increased continuously along the process of increasing from 2h to 3.4 h and increasing the reagent ratio from 1.5:1 to 2: 1. It can be found in the 3D model that the curvature of the change in reagent ratio is greater than the curvature of the change in reaction time, indicating that in this interaction, the change in reagent ratio is to ABTS
+Clearance plays a more important role.
[ regression model results and verifications ]
The regression model was built as per table 9:
TABLE 9 regression model prediction conditions
Through modeling analysis, the optimal conditions of the figwort polysaccharide sulfation process are predicted to be 80.60 ℃, 202.8 min of reaction time and 2.07:1 of reagent ratio, the substitution degree can reach 1.2145 under the conditions, and the clearance can reach 84.01%.
In consideration of actual conditions, the reaction temperature of 81 ℃, the reaction time of 200 min and the ratio of esterification reagents of 2:1 are selected as optimal conditions, and verification is performed based on the conditions. The average degree of substitution of three groups of parallel verification reaches 1.2175 ABTS
+The clearance rate reaches 82.21% and basically accords with model prediction, which shows that the model is basically reliable, the obtained conditions have better reference value for process optimization, and the prediction result and the verification result are shown in a table 10.
TABLE 10 comparison of prediction and verification results
In the model, the basic coincidence of the optimal point of the substitution degree and the optimal point of the clearance rate explains the ABTS of the figwort polysaccharide sulfate
+The clearance ability is closely related to the degree of substitution, but due to ABTS
+The clearance capability has the influence of a more complex mechanism and a plurality of factors, so the clearance capability and the clearance capability are not in a perfect linear relation, but the ABTS can be inferred by a model as the degree of substitution rises
+The scavenging capacity is also significantly improved. Other factors affecting clearance remain to be investigated.
The experimental methods used in the above examples are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the above examples are commercially available unless otherwise specified.
The rotary evaporator used in the above examples was RV-211M of Shanghai-Hengscientific instruments, Inc.; the vacuum freeze-drying machine is SCIENTZ-18N of Ningbo New technology Biotech.
The radix scrophulariae is purchased from Bozhou of Anhui province, and is identified as Scrophulariaceae plant radix scrophulariae by professor Wangjian auxiliary professor of traditional Chinese medicine of economic Ningjian academy of medicine
Scrophularia ningpoensisHemsl
.) Dried root of (4).
Claims (4)
1. A process for optimizing sulfuric acid esterification of figwort polysaccharide by using a response surface method comprises the following steps:
⑴ sample preparation:
drying dried roots of radix scrophulariae at 50 ℃ for 24h, then crushing to 10 meshes, and mixing the powder according to the weight ratio of 200 g: soaking 6L of the above materials in deionized water for 1 hr, decocting for 2 hr, filtering the decoction, decocting the residue once again, and mixing filtrates; concentrating the filtrate under reduced pressure to 350-450 mL to obtain a concentrated solution, adding absolute ethyl alcohol with the volume 4 times that of the concentrated solution, uniformly mixing, standing overnight, and removing a supernatant to obtain a precipitate; dissolving the precipitate with 100mL of deionized water, removing pigment through a macroporous resin column DM-130, concentrating the eluate under reduced pressure, and removing protein by Sevage method to obtain radix scrophulariae polysaccharide extract with pigment and protein removed; concentrating the figwort polysaccharide extracting solution without pigment and protein, and freeze-drying to obtain figwort polysaccharide powder;
adding 20 mL of anhydrous formamide into 0.50 g of the figwort polysaccharide powder to dissolve the figwort polysaccharide powder to obtain a figwort polysaccharide formamide solution of 25 mg/mL; slowly adding the solution into esterification reagents with different proportions, heating in water baths at different temperatures of 60-100 ℃ under full stirring, and reacting for different time of 1-5 h to obtain reaction liquid; adjusting the pH of the reaction solution to be neutral by using a NaOH solution with the mass concentration of 40%, filling the reaction solution into a dialysis bag with the molecular interception amount of 1000, and dialyzing for 3d under normal flow water; concentrating the dialyzed solution under reduced pressure, and freeze-drying to obtain radix scrophulariae polysaccharide sulfate powder corresponding to esterification reagents with different proportions; the esterification reagent is prepared by adding 4.0-6.0 mL of pyridine pre-frozen for 2 hours at 4 ℃ into a three-neck flask placed in an ice-water bath; dropwise adding 6.0-9.0 mL of chlorosulfonic acid into a three-necked flask, reacting with pyridine under vigorous stirring, and dropwise adding for 30-40 min; when no dense smoke is generated, the reaction is finished to obtain a light yellow esterification reagent;
⑵ sample substitution was determined by barium chloride-gelatin turbidimetry:
① Standard Curve preparation:
accurately weighing 100.0 mg K
2SO
4Using 1 mol/L hydrochloric acid to adjust the volume to 100mL to obtain 1.0 mg/mL K
2SO
4A standard solution; respectively taking 20 μ L, 60 μ L, 100 μ L, 140 μ L, 180 μ L and 200 μ L of K with mass concentration of 1.0 mg/mL
2SO
4Putting the standard solution in a test tube, and complementing 200 mu L of hydrochloric acid with 1 mol/L; adding 3.8mL of trichloroacetic acid with the mass concentration of 3% and 0.5% of BaCl in sequence
21mL of gelatin solution; taking 1 mol/L hydrochloric acid as a blank, measuring at a wavelength of 360nm within 5-15 min after sample adding, and recording the absorbance as A
1(ii) a Replacing BaCl with gelatin solution with mass concentration of 0.25%
2Gelatin solution, determination of the recorded absorbance A
2(ii) a With K
2SO
4The standard solution mg/mL is the abscissa, and the difference of absorbance values A
1-A
2Drawing a standard curve for a vertical coordinate;
② measurement of degree of substitution of sample:
precisely weighing 10mg sulfated radix scrophulariae polysaccharide in a test tube, adding 5mL of 1 mol/L hydrochloric acid, hydrolyzing in 90 deg.C water bath for 5 hr, supplementing hydrochloric acid to 5mL after hydrolysis to obtain 2 mg/mL sulfated radix scrophulariae polysaccharide hydrolysate, placing 200 μ L hydrolysate in the test tube, and finding out K according to the standard curve, wherein the rest operation is the same as that of the standard curve prepared in step ①
2SO
4Concentration;
⑶ sample ABTS
+Determination of clearance rate:
a) ABTS
+preparing a solution: weighing 10mg ABTS in a test tube, adding 2.6 mL of 0.66 mg/mL potassium persulfate solution, mixing thoroughly, and reacting for 16 h at room temperature in the dark to obtain ABTS
+(ii) a After the reaction is finished, diluting the mixture by absolute ethyl alcohol, and measuring the mixture at 734 nm until the absorbance is 0.7 +/-0.02;
b) determination of sample clearance: getDissolving 2.5 mg of sulfated figwort polysaccharide powder in 5mL of distilled water to obtain 0.5mg/mL of sample solution; precisely transferring 1mL of sample solution into a test tube, and adding diluted ABTS
+3mL of solution is reacted for 1h at room temperature in a dark place; after the reaction, the absorbance A was measured at a wavelength of 734 nm
1Replacing ABTS with absolute ethyl alcohol
+Solution, absorbance measured by the same method as A
x(ii) a Replacing sample liquid with distilled water as blank, measuring by the same method, and recording absorbance as A
0(ii) a (ii) a Calculating ABTS
+Clearance (%);
ABTS
+clearance (%) = (a)
1-A
x）/ A
0×100%；
⑷ Experimental design and statistical analysis:
① Single-factor test:
sequentially changing the reaction temperature, the reaction time and the proportion of the esterification reagent to carry out a single-factor test, calculating the percentage content S% of sulfur and the substitution degree DS of the sulfate radical in the sulfated figwort polysaccharide obtained by measuring by a barium chloride-gelatin turbidimetry according to the following formula, and repeating the treatment for 3 times each time:
S%=0.092 Cs×100%；
wherein 162 is relative molecular weight of monosaccharide, 32 is sulfur atom mass fraction, and 102 is relative molecular weight increase value of monosaccharide after hydroxyl in monosaccharide group is substituted by sulfate group;
② response surface method optimization design:
according to the result of the single-factor test, the reaction temperature, the reaction time and the ratio of the esterification reagent are taken as investigation factors, and the substitution degree and ABTS are taken as
+The clearance is used as a survey index, Design Expert 8.0.6 software is used for carrying out experimental Design according to the Box-Behnken Design principle, the reaction temperature A, the reaction time B and the esterification reagent proportion C are used as independent variables, the substitution degree DS is used as a response value y and ABTS
+And (3) establishing a multivariate quadratic regression equation with the clearance rate as a response value y':
y=1.21+8.925×10
-3A+0.047B+7.925×10
-3C+0.033AB-0.039AC+0.016BC-
0.14A
2-0.061B
2-0.099C
2；
y´=（83.34+1.83A+1.81B+3.41C-0.9AB-1.85AC+3.23BC-9.18A
2-3.58B
2
-10.46C
2）×100%；
⑸ analysis and optimization of experimental results:
and (4) performing drawing analysis by using Design Expert 8.0.6 software according to the multiple quadratic regression equation to obtain a response surface of the regression equation and a contour map thereof.
2. The process for optimizing sulfation of radix scrophulariae polysaccharide by using the response surface method as claimed in claim 1, wherein said 0.25% gelatin solution in step ① is prepared by dissolving 1.250 g gelatin in 100 deg.C water bath under heating to 500 mL.
3. The process for optimizing sulfation of scrophularia polysaccharide using the response surface method as claimed in claim 1, wherein 0.5% BaCl is added in said step ①
2Gelatin solution means that 1.250 g of BaCl
2The volume of the solution is adjusted to 250mL by 0.25% gelatin solution.
4. The process for optimizing sulfation of radix scrophulariae polysaccharide by using the response surface method as claimed in claim 1, wherein said 3% trichloroacetic acid solution in step ① is prepared by dissolving 15.0 g trichloroacetic acid in water to a volume of 500 mL.
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