Sargassum fusiforme symbolic active component group sequential content detection method

Abstract

The invention provides a method for detecting the sequential content of a sargassum fusiforme symbolic active component group, and relates to the field of analysis and detection. According to the detection method provided by the invention, an enzymolysis method and a solvent extraction method are combined, sequential analysis of four sargassum fusiforme symbolic active components, namely fucoidin, alginic acid, alginate oligosaccharide and brown algae polyphenol in one-time extraction is realized through design of an extraction process, target one-by-one extraction analysis is not needed, the experimental steps are simplified, and the detection efficiency is improved. The analysis time is shortened; and the analysis flux is improved. Meanwhile, the method improves the extraction and dissolution environment of each to-be-detected substance and avoids mutual interference between analysis of the to-be-detected substances, so that the content detection accuracy is improved.

Description

[0001] The present invention claims the priority of the Chinese patent application filed with the China Patent Office on August 15, 2024, with application number 202411121578.2 and invention name “A method for detecting the sequential content of a group of characteristic active components of Sargassum fusiformis”, the entire contents of which are incorporated into the application by reference. Technical Field

[0002] The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting the sequential content of a group of characteristic active components of Sargassum fusiformis. Background Art

[0003] Sargassum fusiforme, a plant of the Sargassum family, is a traditional Chinese medicine of marine origin. It is mainly used to eliminate phlegm, soften and disperse lumps, and promote diuresis and reduce swelling. Analysis of the chemical composition of Sargassum fusiforme shows that in addition to being rich in nutrients such as protein, amino acids, vitamins, minerals and trace elements, Sargassum fusiforme also contains many chemical components and active substances that are not found in terrestrial plants, including fucoidan, alginic acid and brown algae polyphenols. Fucoidan has attracted much research attention. Its unique active group, sulfate, gives it activities such as lowering blood sugar, lowering blood lipids, anti-coagulation and antioxidant.

[0004] Studies have shown that fucoidan from Sargassum fusiformis can reduce fasting blood sugar and blood lipid levels in type 2 diabetes model mice, improve their glucose tolerance and alleviate pathological changes in the heart and liver (Food & Function, 2021, 12 (8), 3572-3585). The fucoidan from Sargassum fusiformis obtained by three different extraction methods differed in chemical composition and spatial conformation. The antioxidant results showed that water-extracted fucoidan had the strongest antioxidant activity (International Journal of Biological Macromolecules, 2020, 155, 1386-1392). Alginate is the most abundant polysaccharide in Sargassum fusiformis. It is composed of mannuronic acid and guluronic acid. Based on its good biocompatibility and gel properties, it has been widely used in the field of biomedical materials. The researchers assembled melanin and alginate for cardiac mechanical support and regulation of the myocardial infarction microenvironment, improving the cardiac function of rats with myocardial infarction model (Advanced Science, 2021, 8 (20), 2100505).

[0005] The product of alginic acid degradation, brown algae oligosaccharides, showed more significant biological activity. Studies have reported that brown algae oligosaccharides improve the damage to reproductive function caused by the anticancer drug busulfan at the single-cell level (Applied Microbiology and Biotechnology, 2020, 104 (8), 3541-3554). In addition, brown algae oligosaccharides improve lipid metabolism and inflammation levels by regulating intestinal flora (Theranostics, 2020, 10 (7), 3308-3324). Brown algae polyphenols are important secondary metabolites of Sargassum fusiformis, composed of polymers of the basic unit phloroglucinol in different polymerization modes. It has strong antioxidant activity and has become a research hotspot in the fields of food, medicine, cosmetics, etc. In recent years, researchers have also discovered its lipid-lowering, anti-tumor, and antibacterial activities. Therefore, it is crucial to establish a method for determining the content of the characteristic active components in Sargassum fusiformis. This study helps to ensure the reliability and stability of drugs and the accuracy of quality evaluation. Due to the different physical and chemical properties of active components, different extraction methods are currently commonly used to extract the corresponding analytes, followed by corresponding tests.

[0006] Fucoidan with good water solubility is mostly extracted and prepared by acid extraction and water extraction. However, since alginate is slightly soluble in water, when hot water extraction of Sargassum fusiformis is carried out and the phenol-sulfuric acid method is used for detection, the fucoidan measurement value will be too high, thus causing errors.

[0007] Some studies have extracted and separated Sargassum fusiformis fucoidan by defatting, calcium coagulation, alcohol precipitation, and ion exchange chromatography. This method effectively removes alginate, but the steps are complicated and the process is cumbersome (Food Science, 2022, 43(12): 260-266).

[0008] For poorly water-soluble alginic acid, some studies have used alginic acid extraction by alginic acid extraction, and added ethanol to precipitate the alginic acid. The precipitate was collected, dried, and weighed to calculate the content. However, the calcium coagulation precipitation method is very likely to cause losses in the precipitation, filtration and other operation steps, causing large errors (Bioscience Biotechnology Research Communications, 2019, 12 (4): 894-903).

[0009] Some studies have used 95% ethanol reflux method to extract brown algae polyphenols (Journal of Chromatography A, 2016, 1428: 115-125), but due to the difference in polarity of phloroglucinol in different polymerization methods, it is impossible to achieve the most complete dissolution in the same solvent system at the same time. In addition, a corresponding extraction method must be formulated for each active component, which is cumbersome, time-consuming and labor-intensive, limiting the analysis throughput. Summary of the invention

[0010] In response to the above problems, the present invention provides a method for sequential content detection of the characteristic active component groups of Sargassum fusiformis, which takes into account the convenience and speed of the extraction process of the analytes and the accuracy and reliability of the detection process, avoids interference between the analytes, and realizes content detection with a coherent step sequence.

[0011] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0012] In one aspect, the present invention provides a method for sequentially detecting the content of a group of characteristic active components of Sargassum fusiformis, comprising the following steps:

[0013] S1: The Sargassum fusiformis sample is crushed, mixed with water, the pH is adjusted, and a composite enzyme is added for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0014] S2: Separate the enzymatic hydrolysate and take part of the supernatant to obtain enzymatic hydrolysate 1;

[0015] S3: taking the enzymatic hydrolysate 1, collecting the filtrate after ultrafiltration centrifugation to obtain the ultrafiltration enzymatic hydrolysate 1;

[0016] S4: Take the algae residue after enzymatic hydrolysis of S1 and the remaining supernatant of S2, extract them with alcohol 1-3 times, centrifuge, combine the supernatants, and obtain enzymatic hydrolyzate 2;

[0017] S5: using phenol-sulfuric acid method, m-hydroxybiphenyl method and Folin-phenol method to detect the content of the analyte in enzymatic hydrolysate 1, using phenol-sulfuric acid method to detect the content of the analyte in ultrafiltered enzymatic hydrolysate 1, and using Folin-phenol method to detect the content of the analyte in enzymatic hydrolysate 2;

[0018] S6: Calculate the content W of the characteristic active components fucoidan, alginic acid, brown algae oligosaccharides and brown algae polyphenols.

[0019] Preferably, in S1, the mass volume ratio of the Sargassum fusiformis sample to water is 1:15-25 g / mL; further preferably, in S1, the mass volume ratio of the Sargassum fusiformis sample to water is 1:18-22 g / mL.

[0020] Preferably, in S1, the mixing is stirring; the stirring time is 20-40 minutes;

[0021] Preferably, in S1, the pH is 5-6.5, and more preferably, the pH is 5.5-6.

[0022] Preferably, in S1, the mass of the complex enzyme is 16%-30% of the mass of the Sargassum fusiformis sample; further preferably, in S1, the mass of the complex enzyme is 20%-24% of the mass of the Sargassum fusiformis sample.

[0023] Preferably, in S1, the complex enzyme is selected from alginate complex enzyme and / or neutral protease; further preferably, in S1, the complex enzyme is alginate complex enzyme and neutral protease.

[0024] Preferably, the alginate complex enzyme comprises alginate lyase, pectinase and cellulase.

[0025] Preferably, the mass ratio of alginate lyase, pectinase and cellulase is 2-4: 1-2: 2-4. Preferably, the mass ratio of alginate complex enzyme and neutral protease is 1: 0.8-1.2.

[0026] Preferably, the alginate lyase has an activity of 50,000-100,000 U / g, the pectinase has an activity of 30,000-80,000 U / g, the cellulase has an activity of 80,000-150,000 U / g, and the neutral protease has an activity of 80,000-150,000 U / g.

[0027] Preferably, in S1, the temperature of the enzymatic hydrolysis is 40-50°C, and the time is 4-7h; further preferably, in S1, the temperature of the enzymatic hydrolysis is 43-47°C, and the time is 4.5-5.5h.

[0028] Preferably, in S1, the enzymatic hydrolysis is carried out under heating in a water bath.

[0029] Preferably, in S2, the volume of the supernatant is 30%-50% of the volume of the enzymatic hydrolysate in S1; further preferably, in S2, the volume of the supernatant is 35%-45% of the volume of the enzymatic hydrolysate in S1.

[0030] Preferably, in S3, the ultrafiltration molecular weight cutoff is 2.5-3.5 kDa, and further preferably, in S3, the ultrafiltration molecular weight cutoff is 3 kDa.

[0031] Preferably, in S3, the ultrafiltration speed is 10000-15000 rpm, and the time is 15-25 min; further preferably, in S3, the ultrafiltration speed is 10000-13000 rpm, and the time is 15-20 min.

[0032] Preferably, in S4, the alcohol extracted is anhydrous ethanol;

[0033] Preferably, in S4, the temperature of the alcohol extraction is 70-85°C, and the time is 1-3h; further preferably, in S4, the temperature of the alcohol extraction is 70-80°C, and the time is 1-2h.

[0034] Preferably, in S4, the centrifugal speed is 3500-4200 rpm, and the time is 8-12 min; further preferably, in S4, the centrifugal speed is 3500-4000 rpm, and the time is 8-10 min.

[0035] Preferably, in S5, the concentration value obtained by detecting the analyte in the enzymatic hydrolyzate 1 by the phenol-sulfuric acid method, the m-hydroxybiphenyl method, or the folin phenol method is C 1A , C 1B , C 1C The concentration value of the analyte in ultrafiltration enzymatic hydrolysate 1 detected by phenol-sulfuric acid method is C u1A The concentration value of the analyte in enzymatic solution 2 detected by the Folin phenol method is C 2C .

[0036] Preferably, in S6, the calculation formula of the content W of each characteristic active component is as follows:

[0037] W 岩藻多糖 =(C 1A -C 1B )*V1 / m 样品 ;

[0038] W 海藻酸 =C 1B *V1 / m 样品 ;

[0039] W 褐藻寡糖 =C u1A *V1 / m 样品 ;

[0040] W 褐藻多酚 =(0.4*C 1C *V1+C 2C *V2) / m 样品 ;

[0041] in,

[0042] V1 is the volume of the enzymatic solution described in S1;

[0043] V2 is the volume of the enzymatic hydrolysis solution 2 described in S4.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1) The m-hydroxybiphenyl method can be used to detect uronic acid, while the neutral sugars fucose and glucose have no significant effect on the color development of the m-hydroxybiphenyl method. Based on the characteristic that the monosaccharide composition of A. fusogenum is mainly fucose and glucose, the present invention replaces the traditional single analyte extraction and impurity removal analysis by the idea of ​​co-extraction and analysis of multiple analytes to detect the content of fucoidan and alginate, avoiding mutual interference between the two, while promoting the full extraction of the analytes and improving the accuracy of detection.

[0046] 2) Based on the characteristics of brown algae polyphenols being diverse in structure and easy to degrade, the present invention adopts a mild extraction method to extract brown algae polyphenols in two solvents of different polarity. Compared with the extraction using a single solvent, the dissolution environment of brown algae polyphenols is optimized and the accuracy of detection is improved.

[0047] 3) The present invention realizes the sequential analysis of the four characteristic active components of Sargassum fusiformis, namely fucoidan, alginic acid, brown algae oligosaccharide and brown algae polyphenols, in one extraction. Through process design, the extraction method that is conducive to the dissolution of the analyte is optimized, so that the content determination results are more accurate and reliable. Compared with the target-based content determination, the extraction and impurity removal processes are simplified, the analysis time is shortened by more than 60% and the analysis throughput is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a flow chart for the sequential content detection of the characteristic active component groups of Sargassum fusiformis.

[0049] Figure 2 This is the monosaccharide composition diagram of Sargassum fusiformis determined by PMP pre-column derivatization HPLC.

[0050] Figure 3 This is a single factor investigation diagram of enzymatic hydrolysis time for the extraction of Sargassum fusiformis enzyme.

[0051] Figure 4 This is a single factor investigation diagram of enzymatic hydrolysis temperature for the extraction of Sargassum fusiformis enzyme.

[0052] Figure 5 The present invention is a flow chart for detecting the content of characteristic active component groups of Sargassum fusiformis using the existing technical methods. DETAILED DESCRIPTION

[0053] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further explained below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without making creative work all belong to the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited. The technology and scientific terms used in the embodiments have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0054] Some materials and equipment of the present invention are as follows:

[0055] (1) Test materials

[0056] Sargassum fusiforme is produced in Dongtou District, Wenzhou City, Zhejiang Province.

[0057] Sodium tetraborate, concentrated sulfuric acid, sodium hydroxide, and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0058] Alginate complex enzyme (including alginate lyase, pectinase, and cellulase) was purchased from Ningxia Xiasheng Industrial Group Co., Ltd.

[0059] m-Hydroxybiphenyl and neutral protease were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0060] Sodium alginate reference substance was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0061] Gallic acid reference substance was purchased from Thermo Fisher Scientific Holdings Co., Ltd.

[0062] Fucose was purchased from Bio Basic.

[0063] Ultrafiltration tubes (molecular weight cutoff 3 kDa) were purchased from Sigma.

[0064] Folin phenol reagent was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0065] (2) Instruments and Equipment

[0066] Eppendorf MiniSpin centrifuge.

[0067] Eppendorf Centrifuge 5804R centrifuge.

[0068] Agilent Cary 60 UV-Vis spectrophotometer.

[0069] Example 1

[0070] (1) Sample preparation

[0071] S1: Weigh 0.50g of Sargassum fusiformis powder, add 10mL of deionized water, stir for 30min, adjust the pH to 5.5-6.0, add 12% of alginate complex enzyme and 12% of neutral protease based on the mass of Sargassum fusiformis powder, and perform enzymolysis at 45°C for 5h.

[0072] S2: After the enzymatic hydrolysis is completed, centrifuge at 4000 rpm for 15 min and take 4 mL of the supernatant to obtain enzymatic hydrolyzate 1.

[0073] S3: Take the enzymatic hydrolysate 1, and ultrafilter and centrifuge it at 13000 rpm for 20 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, and then collect the filtrate to obtain the ultrafiltered enzymatic hydrolysate 1.

[0074] S4: Add anhydrous ethanol to the algae residue after enzymatic hydrolysis of S1 and the remaining supernatant of S2 to make the volume fraction of ethanol in the solution reach 40%. After mixing, extract at 80°C for 2h, centrifuge at 4000rpm for 10min, collect the supernatant, repeat this process once, combine the supernatant of the extract, and obtain enzymatic hydrolyzate 2.

[0075] (2) Phenol-sulfuric acid method

[0076] Accurately pipette 1.0, 1.4, 1.8, 2.2, 2.6, and 3.0 mL of 0.5 g / L fucose standard solution into a 10 mL volumetric flask to make up to volume to obtain reference solutions of different concentrations for later use.

[0077] Take 1mL of reference solution of different concentrations in a stoppered test tube, add 1mL of 5% phenol solution, put in an ice bath for 5min, slowly add 5mL of concentrated sulfuric acid, and mix well. Heat the test tube in a boiling water bath for 30min, cool to room temperature, measure the absorbance at a wavelength of 490nm, adjust to zero with concentrated sulfuric acid, and draw a fucose standard curve.

[0078] Take 1mL of appropriately diluted enzymatic hydrolysate 1 and ultrafiltered enzymatic hydrolysate 1 into a stoppered test tube, add 1mL of 5% phenol solution, ice bath for 5min, slowly add 5mL of concentrated sulfuric acid, and mix well. Heat the test tube in a boiling water bath for 30min, cool to room temperature, measure the absorbance at a wavelength of 490nm, and adjust to zero with concentrated sulfuric acid. Calculate C according to the fucose standard curve 1A and C u1A .

[0079] (3) m-Hydroxybiphenyl method

[0080] Accurately pipette 0.30mL, 0.40mL, 0.50mL, 0.60mL, 0.70mL, and 0.80mL of 0.2g / L sodium alginate standard solution into a stoppered test tube, and then add water to a total volume of 1.00mL, and mix well. After the test tube is ice-bathed for 5 minutes, slowly add 6mL of 0.0125g / L sodium tetraborate sulfuric acid solution and mix well. Heat the test tube in a boiling water bath for 30 minutes and cool to room temperature. Add 100μL of 1.5g / L m-hydroxybiphenyl solution as a color developer to each test tube and mix well. After standing at room temperature for 10 minutes, measure the absorbance at a wavelength of 520nm, adjust to zero with sodium tetraborate sulfuric acid solution, and draw a sodium alginate standard curve.

[0081] Take 1mL of appropriately diluted enzymatic hydrolyzate 1 in a stoppered test tube, place in an ice bath for 5 minutes, slowly add 6mL of 0.0125g / L sodium tetraborate sulfuric acid solution, and mix well. Heat the test tube in a boiling water bath for 30 minutes, and cool to room temperature. Add 100μL of 1.5g / L m-hydroxybiphenyl solution as a color developer to each test tube and mix well. After standing at room temperature for 10 minutes, measure the absorbance at a wavelength of 520nm and adjust to zero with sodium tetraborate sulfuric acid solution. Calculate C according to the sodium alginate standard curve 1B .

[0082] (4) Folin phenol method

[0083] Accurately pipette 1.0mL, 1.5mL, 2.0mL, 2.5mL, and 3.0mL of 50μg / mL gallic acid standard solution into a 25mL volumetric flask, add 3mL of Folin phenol reagent, shake well, let stand for 30s, then add 6mL of 12% Na2CO3 solution, make up to volume with deionized water, place at 25℃ in the dark for 2h, measure the absorbance at 760nm, adjust to zero with deionized water, and draw a gallic acid standard curve.

[0084] Take 1mL of appropriately diluted enzymatic hydrolysate 1 and enzymatic hydrolysate 2 in a 25mL volumetric flask, add 3mL of Folin phenol reagent, shake well and let stand for 30s, then add 6mL of 12% Na2CO3 solution, make up to volume with deionized water, place at 25℃ in the dark for 2h, measure the absorbance at 760nm, and adjust to zero with deionized water. According to the gallic acid standard curve, C 1C and C 2C .

[0085] (5) Calculation of the content of characteristic active components

[0086] The calculation formula of the content W of each characteristic active component is as follows:

[0087] W 岩藻多糖 =(C 1A -C 1B )*V1 / m样品 ;

[0088] W 海藻酸 =C 1B *V1 / m 样品 ;

[0089] W 褐藻寡糖 =C u1A *V1 / m 样品 ;

[0090] W 褐藻多酚 =(0.4*C 1C *V1+C 2C *V2) / m 样品 ;

[0091] in,

[0092] V1 is the volume of the enzymatic solution described in S1;

[0093] V2 is the volume of the enzymatic hydrolysis solution 2 described in S4.

[0094] Embodiment 2-4

[0095] Compared with Example 1, only the analysis samples were changed to Sargassum from different origins, Example 2 was Sargassum from Wenling, Taizhou, Zhejiang, Example 3 was Sargassum from Rongcheng, Weihai, Shandong, and Example 4 was Sargassum from Xiapu, Ningde, Fujian, and the rest of the steps were the same. All samples were collected from April to May when Sargassum was commercially mature, and the content of the characteristic active component group was determined by a sequential content detection method.

[0096] Table 1. Contents of characteristic active components of Sargassum fusiformis from different origins

[0097] sample Fucoidan(%) Alginic acid (%) Brown algae oligosaccharide (%) Brown algae polyphenols (%) Example 1 8.24 32.21 27.82 0.86 Example 2 6.57 32.65 29.53 0.76 Example 3 8.57 34.93 28.93 0.70 Example 4 10.97 34.24 32.86 0.75

[0098] Embodiment 5-9

[0099] Compared with Example 1, the analysis sample was changed to another batch of Sargassum fusiformis produced in Dongtou, Wenzhou, Zhejiang, and the enzymatic hydrolysis times were 2h, 3h, 4h, 5h, and 6h, respectively, and the remaining steps were the same.

[0100] Table 2. Enzymatic hydrolysis time investigation

[0101]

[0102] Under the condition of keeping other enzymatic hydrolysis parameters unchanged, the enzymatic hydrolysis time was changed to investigate the extraction amount of alginate and brown algae polyphenols ( Figure 3 The results showed that the extraction amount of both alginate and brown algae polyphenols reached the highest level when the enzymatic hydrolysis time was 5 h.

[0103] Examples 10-14

[0104] Compared with Example 1, the analysis sample was changed to another batch of Sargassum fusiformis produced in Dongtou, Wenzhou, Zhejiang, the enzymatic hydrolysis temperature was 35°C, 40°C, 45°C, 50°C, and 55°C, and the remaining steps were the same.

[0105] Table 3. Enzymatic hydrolysis temperature investigation

[0106]

[0107]

[0108] The enzymatic hydrolysis temperature was changed while other enzymatic hydrolysis parameters were kept constant, and the extraction amount of alginate and brown algae polyphenols in the two enzymatic hydrolysates was further investigated ( Figure 4 ). The results showed that the extraction amount of alginate reached the highest at 40°C, which was not significantly different from the extraction amount at 45°C. The optimal extraction temperature of brown algae polyphenols in hydrolysate 1 was 50°C, and the optimal extraction temperature in hydrolysate 2 was 40°C. In addition, there was little difference between the two extraction amounts at 45°C, and the total amount of brown algae polyphenols in hydrolysate 1 and hydrolysate 2 was the highest at 45°C. Taking all factors into consideration, 45°C was selected as the hydrolysis temperature parameter for subsequent experiments.

[0109] Examples 15-16

[0110] Compared with Example 1, the only difference is that Example 15 replaces the original 12% alginate complex enzyme and 12% neutral protease with 24% alginate complex enzyme, and Example 16 replaces the original 12% alginate complex enzyme and 12% neutral protease with 24% neutral protease, and the other steps are the same.

[0111] Table 4. Contents of characteristic active components of Sargassum fusiformis under different enzyme compositions

[0112] sample Fucoidan(%) Alginic acid (%) Brown algae oligosaccharide (%) Brown algae polyphenols (%) Embodiment 15 10.77 31.66 27.98 0.90 Example 16 2.55 14.47 7.08 0.61

[0113] Comparing the contents of the characteristic active component groups of Sargassum fusiformis in Example 1 and Example 15, it can be seen that the use of neutral protease to replace a part of the alginate complex enzyme during the enzymatic hydrolysis process will not have a significant effect on the full extraction and detection of the active components. Therefore, the addition of neutral protease can reduce the amount of alginate complex enzyme, thereby saving costs. Comparing Example 1 and Example 16, it can be seen that only adding neutral protease cannot completely extract the active component group, and the full extraction of each active component of Sargassum fusiformis needs to rely on the synergistic effect of multiple enzymes.

[0114] Test Example 1

[0115] According to the detection method of Example 1, the linear regression equation, linear range, stability within 0-12 hours, repeatability and accuracy were investigated.

[0116] According to the guidelines for analytical method validation in the 2020 edition of the Chinese Pharmacopoeia, the sequential content detection method of the characteristic active component group of Sargassum fusiformis was methodologically validated, including the linear regression equation, linear range, stability within 0-12 hours, repeatability and accuracy. The results are shown in Tables 5-6 below:

[0117] Table 5. Results of linearity, stability and repeatability of the method

[0118]

[0119]

[0120] Table 6. Results of sample recovery determination of the method

[0121] Object to be tested Added amount (mg) RSD Sample recovery rate Fucoidan 49.6 0.98％ 95.22％ alginic acid 22.1 1.91％ 96.33％ Brown algae oligosaccharide 34.1 1.84％ 95.89％ Brown algae polyphenols 3.91 0.95％ 97.70％

[0122] It can be seen from the correlation coefficient values ​​in Table 5 that the four characteristic active components of Sargassum fusiformis show a good linear relationship within the linear range, meeting the quantitative requirements. Referring to the table "Relationship between the content of the components to be determined in the sample and the acceptable range of precision" in the "Chinese Pharmacopoeia 2020 Edition", it can be seen that the test object has good stability within 12 hours, and the detection method has good repeatability and accuracy (Table 6). Therefore, the sequential content detection method of the characteristic active component group of Sargassum fusiformis established by the present invention is convenient, fast, accurate and reliable.

[0123] Comparative Example 1

[0124] Compared with Example 1, the only difference is that the content of the characteristic active component group in Sargassum fusiformis is determined by adopting the traditional idea of ​​extracting and removing impurities from a single analyte and then analyzing it.

[0125] The content determination process is as follows: Figure 5 It can be seen that the specific steps are as follows:

[0126] (1) Determination of alginate content

[0127] Weigh 5.00g of Sargassum fusiformis, add 100mL of deionized water, and soak at room temperature for 4h. After soaking, wash the Sargassum fusiformis and cut it into pieces. Then add 10% Na2CO3 solution and digest it at 60℃ for 3h and leave it at room temperature overnight. After digestion, collect the filtrate by suction, adjust the pH value to 6-7 and add 10% CaCl2 solution. Collect the precipitate by suction, add 15% NaCl solution and ultrasonicate for 1h for ion exchange. Finally, add 95% ethanol and leave it overnight, collect the precipitate by suction, weigh the solid mass after drying, and convert it to the alginate content.

[0128] (2) Determination of fucoidan content

[0129] Weigh 1.00 g of Sargassum fusiformis powder, add 10 mL of deionized water, heat and reflux for extraction for 1 h, cool and centrifuge to collect the supernatant. Add 10% CaCl2 solution to the supernatant to remove alginic acid, centrifuge again to collect the supernatant and determine the fucoidan content according to the phenol-sulfuric acid method in Example 1.

[0130] (3) Determination of brown algae polyphenol content

[0131] 1.00 g of Sargassum fusiformis powder was weighed, and 10 mL of 10% ethanol solution was added, heated under reflux for extraction for 1 h. After cooling, the supernatant was collected by centrifugation and the brown algae polyphenol content was determined according to the Folin phenol method in Example 1.

[0132] Table 7. Content of characteristic active components of Sargassum fusiformis detected by prior art methods

[0133] Object to be tested Produced in Dongtou, Wenzhou, Zhejiang (%) Fucoidan 5.12 alginic acid 22.12 Brown algae polyphenols 0.73

[0134] Comparing Table 1 with Table 7, it can be seen that for the same analysis sample, the values ​​of fucoidan, alginic acid, and brown algae polyphenol detected by the sequential content detection method are all higher. This suggests that the extraction process used in the method of the present invention may be more conducive to the dissolution of the active component group of Sargassum fusiformis, thereby improving the accuracy of the detection. Figure 1 and Figure 5 It can be seen that the analysis steps of measuring the content of the characteristic active component groups of Sargassum fusiformis one by one using the existing technical methods are cumbersome, time-consuming and labor-intensive, while the method of the present invention simplifies the experimental steps and shortens the analysis time by more than 60%, which is convenient and fast.

[0135] In summary, the method for sequential content detection of the characteristic active component groups of Sargassum fusiformis of the present invention is convenient and fast, has a short analysis time and high throughput, and can complete the analysis of four characteristic active components in one extraction. In addition, through methodological verification, it can be known that the method of the present invention is accurate and reliable, and meets the analysis requirements of Sargassum fusiformis samples.

[0136] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting the sequential content of a group of characteristic active components of Sargassum fusiformis, characterized in that: The following steps are involved: S1: The Sargassum fusiformis sample is crushed, mixed with water, the pH is adjusted, and a composite enzyme is added for enzymatic hydrolysis to obtain an enzymatic hydrolyzate; S2: Separate the enzymatic hydrolysate and take part of the supernatant to obtain enzymatic hydrolysate 1; S3: taking the enzymatic hydrolysate 1, collecting the filtrate after ultrafiltration centrifugation to obtain the ultrafiltration enzymatic hydrolysate 1; S4: Take the algae residue after enzymatic hydrolysis of S1 and the remaining supernatant of S2, extract them with alcohol 1-3 times, centrifuge, combine the supernatants, and obtain enzymatic hydrolyzate 2; S5: using phenol-sulfuric acid method, m-hydroxybiphenyl method and Folin-phenol method to detect the content of the analyte in enzymatic hydrolysate 1, using phenol-sulfuric acid method to detect the content of the analyte in ultrafiltered enzymatic hydrolysate 1, and using Folin-phenol method to detect the content of the analyte in enzymatic hydrolysate 2; S6: Calculate the content W of the characteristic active components fucoidan, alginic acid, brown algae oligosaccharides and brown algae polyphenols.

2. The detection method according to claim 1, characterized in that: In S1, the mass volume ratio of the Sargassum fusiformis sample to water is 1:15-25 g / mL; In S1, the pH is 5-6.5; In S1, the mass of the complex enzyme is 16%-30% of that of the Sargassum fusiformis sample; In S1, the enzymatic hydrolysis temperature is 40-50°C and the time is 4-7h.

3. The detection method according to claim 1, characterized in that: In S1, the complex enzyme is selected from alginate complex enzyme and / or neutral protease.

4. The detection method according to claim 3, characterized in that: The complex enzyme is alginate complex enzyme and neutral protease.

5. The detection method according to claim 4, characterized in that: The alginate complex enzyme comprises alginate lyase, pectinase and cellulase.

6. The detection method according to claim 1, characterized in that: In S2, the volume of the supernatant is 30%-50% of the volume of the enzymatic hydrolysate in S1.

7. The detection method according to claim 1, characterized in that: In S3, the ultrafiltration molecular weight cut-off is 2.5-3.5 kDa; In S3, the ultrafiltration speed is 10000-15000 rpm, and the time is 15-25 min.

8. The detection method according to claim 1, characterized in that: In S4, the alcohol extraction temperature is 70-85°C and the time is 1-3h; In S4, the centrifugal speed is 3500-4200 rpm and the time is 8-12 min.

9. The detection method according to claim 1, characterized in that: In S5, the concentration value obtained by detecting the analyte in the enzymatic solution 1 by the phenol-sulfuric acid method, the m-hydroxybiphenyl method, and the folin phenol method is C 1A , C 1B , C 1C The concentration of the substance to be tested in ultrafiltration enzymatic hydrolysate 1 was C u1A The concentration value of the analyte in enzymatic solution 2 detected by the Folin phenol method is C 2C .

10. The detection method according to claim 1, characterized in that: In S6, the calculation formula of the content W of each characteristic active component is as follows: W 岩藻多糖 =(C 1A -C 1B )*V1 / m 样品 ; W 海藻酸 =C 1B *V1 / m 样品 ; W 褐藻寡糖 =C u1A *V1 / m 样品 ; W 褐藻多酚 =(0.4*C 1C *V1+C 2C *V2) / m 样品 ; in, V1 is the volume of the enzymatic solution described in S1; V2 is the volume of the enzymatic hydrolysis solution 2 described in S4.

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