Application of phenylpropionic acid compounds in detecting monosaccharide composition of traditional Chinese medicine polysaccharides

Abstract

The application discloses application of a phenylpropionic acid compound in detection of monosaccharide composition of traditional Chinese medicine polysaccharide. The phenylpropionic acid compound has a structure as shown in formula (I). The application provides a new application of the phenylpropionic acid compound, and preliminary realization of quality control research on traditional Chinese medicine preknowing polysaccharide. Compared with a method for detecting monosaccharide composition of traditional Chinese medicine polysaccharide by using 1-phenyl-3-methyl-5-pyrazolone as a pre-column derivatization reagent, the application has the following advantages: 1. Derivatization of aldehyde sugar and ketose can be simultaneously realized; 2. The reaction temperature is more moderate, and the safety of the reaction is greatly improved; 3. The detection wavelength of HPLC-UV is improved from 245 nm to 274 nm, interference is reduced, and sensitivity is improved; and 4. The pre-column derivatization reagent is low in cost and easy to obtain.

Description

Technical Field

[0001] The present invention belongs to the technical field of pre-column derivatization reagents, and particularly relates to the application of phenylpropionic acid compounds in detecting the monosaccharide composition of traditional Chinese medicine polysaccharides. Background Art

[0002] Quality control is one of the problems faced by the application and development of traditional Chinese medicine. Akebia fruit is the dried nearly mature fruit of Akebia quinata (Thunb.) Decne., Akebia trifoliata (Thunb.) Koidz. or Akebia trifoliata (Thunb.) Koidz. var. australis (Diels) Rehd. of the family Lardizabalaceae, and is included in the first volume of the Chinese Pharmacopoeia 2005 Edition; it is bitter in taste and cold in nature, and has the effects of soothing the liver and regulating qi, promoting blood circulation to relieve pain, diuresis, and killing insects; it is used for epigastric and hypochondriac pain, amenorrhea and dysmenorrhea, dysuria, and snake and insect bites. In the 2005 Edition of the Chinese Pharmacopoeia, the identification and content determination items of Akebia fruit medicinal materials are vacant, making it difficult to guarantee the quality of the medicinal materials. It is urgent to establish a suitable quality control standard to ensure the safety of clinical medication.

[0003] Polysaccharide is the main chemical component in Akebia fruit and most traditional Chinese medicines. Since polysaccharides lack chromogenic groups and have complex structures, many existing techniques cannot detect them. Currently, pre-column derivatization reagents are used to carry out derivatization reactions with the monosaccharides after polysaccharide hydrolysis, and then separated by HPLC, so as to perform relatively good qualitative and quantitative analysis on the monosaccharides, indirectly realizing the quality control research of traditional Chinese medicine polysaccharides.

[0004] PMP is one of the most commonly used pre-column derivatization reagents at present and is widely used in the analysis of traditional Chinese medicine because it is simple to obtain and improves the detection sensitivity. Due to the low ionization efficiency of monosaccharides, the HPLC-MS / MS method is limited by sensitivity loss. Therefore, monosaccharide derivatives are essential for obtaining highly sensitive detection. In 1989, Honda [1] et al. first used PMP to label reducing sugars in an alkaline environment and achieved the separation of sugar chain derivatives by reversed-phase high-performance liquid chromatography (RP-HPLC). On this basis, PMP, as a derivatization reagent for saccharides, has been continuously promoted and applied. Many research groups have achieved the PMP derivatization of polysaccharide hydrolysis products and carried out HPLC-UV analysis. Fan et al. [2] established an HPLC-MS / MS method to apply 1-phenyl-3-methyl-5-pyrazolone (PMP) in the quality control of 3 kinds of osmanthus polysaccharides. The osmanthus polysaccharides were hydrolyzed into monosaccharides, and the PMP-derived monosaccharides were detected and analyzed by collecting the pairs of six characteristic fragment ions. The 6 kinds of monosaccharides detected were glucose, mannose, ribose, xylose, galactose, and fucose. Xu et al. [3]Using PMP as a derivatization reagent and Dendrobium officinale as a model medicinal material, a rapid, qualitative, and quantitative method for simultaneously characterizing sugar-based medicinal materials using high-performance liquid chromatography (HPGPC) was constructed. Liu [4] Multiple techniques were employed to perform fingerprint analysis on PMP-derived *Nematocystis jirovecii* polysaccharides. High-performance gel permeation chromatography (HPLC) and pre-column derivatization HPLC analysis showed high similarity among 18 batches of *Nematocystis jirovecii* polysaccharides. A schematic diagram of the PMP derivatization reaction principle for monosaccharides is shown below:

[0005]

[0006] PMP can derivatize aldoses, but not ketoses, making it impossible to detect ketose monosaccharide components in polysaccharides. Therefore, the development of pre-column derivatization reagents for ketose derivatization is urgently needed. Summary of the Invention

[0007] In view of the shortcomings of the prior art, this invention provides an application of phenylpropionic acid compounds in the detection of monosaccharide composition of polysaccharides in traditional Chinese medicine. This compound can simultaneously derivatize aldoses and ketoses, and its reaction conditions are milder, its sensitivity is higher, and its cost is lower than that of PMP reactions.

[0008] In a first aspect, the present invention provides the application of phenylpropionic acid compounds in the detection of the monosaccharide composition of polysaccharides in traditional Chinese medicine, the phenylpropionic acid compounds having the structure shown in formula (I);

[0009]

[0010] R is one of hydrogen, methyl, methoxy, nitro, hydroxy, or cyano, and all of these compounds are commercially available.

[0011] According to a specific embodiment of the present invention, R is a nitro group. In this case, phenylpropionic acid compounds are used as monosaccharide derivatizing reagents, and the monosaccharide derivative yield is the highest.

[0012] According to a specific embodiment of the present invention, the traditional Chinese medicine polysaccharide is a polysaccharide derived from *Evodia rutaecarpa*.

[0013] According to specific embodiments of the present invention, monosaccharides include aldoses and ketoses. Aldoses refer to sugars containing an aldehyde group, among which glyceraldehyde is the simplest aldose in terms of molecular structure. Other aldoses include glucose, maltose, ribose, and deoxyribose. Ketosaccharides refer to sugars containing a ketone group. The simplest ketose is dihydroxyacetone. Other ketoses include fructose, erythritolose, and xylulose.

[0014] According to a specific embodiment of the present invention, the detection method includes: hydrolyzing the polysaccharide of traditional Chinese medicine, centrifuging the hydrolysate to obtain the supernatant, concentrating and drying it to obtain a dried monosaccharide; reacting the dried monosaccharide with the compound shown in formula (Ⅰ) in the presence of anhydrous aprotic solvent, triphenylphosphine, and azodicarboxylic acid ester to obtain a monosaccharide derivatization reaction product; and separating and detecting the monosaccharide derivatization reaction product by high performance liquid chromatography.

[0015] The above monosaccharide derivatization reaction mechanism involves the Mitsunobu reaction, where a carboxylic acid, represented by phenylpropionic acid compounds, and two auxiliary reagents, triphenylphosphine and azodicarboxylic acid esters, convert an alcohol, represented by a monosaccharide, into an ester through configuration inversion. Anhydrous aprotic solvents such as tetrahydrofuran, toluene, dichloromethane, benzene, N,N-dimethylformamide, diethyl ether, acetonitrile, tertiary methyl ether, N,N-dimethylaniline, and 1,4-dioxane are preferred, with anhydrous 1,4-dioxane being preferred. Azodicarboxylic acid esters such as diisopropylazo-1,2-dicarboxylic acid ester or diethyl azodicarboxylate are preferred, with diisopropylazo-1,2-dicarboxylic acid ester being preferred.

[0016] According to a specific embodiment of the present invention, the molar ratio of the dried monosaccharide to the compound shown in formula (I) is 1-3:1-3.

[0017] According to a specific embodiment of the present invention, the molar ratio of the dried monosaccharide to the compound shown in formula (Ⅰ) is 1:1, at which point the yield of the monosaccharide derivative is the highest.

[0018] According to a specific embodiment of the present invention, the reaction temperature is 30–50°C and the reaction time is 8–12 h.

[0019] According to a specific embodiment of the present invention, the reaction temperature is 40°C and the reaction time is 10 hours, at which point the yield of the monosaccharide derivative is the highest.

[0020] According to a specific embodiment of the present invention, the chromatographic conditions for separation and detection by high performance liquid chromatography include: an Agilent XB-C8 column (4.6×250mm, 5μL); a set temperature of 25℃; a detection wavelength of 274nm; an injection of 5μL at a flow rate of 1.2mL / min; mobile phase B being methanol and mobile phase A being pure water; and gradient elution.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention provides a novel application of phenylpropionic acid compounds, enabling preliminary research on the quality control of polysaccharides from the traditional Chinese medicine *Physalis alkekengi*. Compared with the traditional method using 1-phenyl-3-methyl-5-pyrazolone as a pre-column derivatization reagent to detect the monosaccharide composition of *Physalis alkekengi* polysaccharides, this invention has the following advantages: 1. It can simultaneously derivatize aldoses and ketoses; 2. The reaction temperature is 30–50℃, which is milder than the reaction temperature of 1-phenyl-3-methyl-5-pyrazolone (generally 70℃), greatly improving reaction safety; 3. Most compounds have UV absorption around 245 nm and below, where the absorption of analytes and impurities is detected, easily leading to mutual interference. This invention increases the HPLC-UV detection wavelength from 245 nm to 274 nm, reducing interference and improving sensitivity (e.g., ...). Figure 3 (as shown); 4. Pre-column derivatization reagents are low in cost and readily available. Attached Figure Description

[0023] Figure 1 This is the monosaccharide derivatization reaction formula for Example 1;

[0024] Figure 2 The graphs are standard solution curves for glucose and galacturonic acid; where A: standard solution curve for glucose; B: standard solution curve for galacturonic acid.

[0025] Figure 3 Comparison of UV absorption spectra of PMP and NPA;

[0026] Figure 4 High-performance liquid chromatograms of 10 batches of polysaccharides from different origins;

[0027] Figure 5 HPLC fingerprints of 10 batches of samples from different origins. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Screening of Monosaccharide Derivatization Reagents and Derivatization Conditions

[0030] Powdered D-(+)-mannose (2 mmol, 360 mg) was added to 40 mL of anhydrous 1,4-dioxane in a three-necked flask. The mixture was sonicated for 15 min until suspended. Then, 3-phenylpropionic acid (0.66 mmol, 100 mg) and triphenylphosphine (4 mmol, 1060 mg) were added. The mixture was evacuated and purged with nitrogen. Diisopropylazo-1,2-dicarboxylic acid ester (4 mmol, 800 μL) was slowly added dropwise to the mixture under ice bath conditions. Finally, the reaction temperature was raised to 40 °C and the reaction was carried out for 10 h. The resulting product was a pale yellow solid with a yield of approximately 20%. NMR analysis of the prepared compound confirmed its identity as the target compound. 1 H NMR (600MHz, MeOD) δ7.24(dt,J=21.1,7.5Hz,4H),7.18(t,J=6.6Hz,1H),4.92(s,1H),3.93-3.66(m,6H),2.95(t,J=7.7Hz,2H),2.71(t,J=7.7Hz,2H). 13 C NMR (151MHz, MeOD) δ172.7,142.0,141.5,129.5,127.1,95.2,76.77,72.0,70.9,67.7,62.3,36.9,31.8.

[0031] The reaction formula is as follows Figure 1 As shown.

[0032] Other monosaccharide derivatives were synthesized using the same method, resulting in six monosaccharide derivatives with different 4-position substituents for the R group: -H, -CH3, -CN, -OCH3, -NO2, and -OH. The yields of the bound derivatives were determined by UV spectroscopy and were 20%, 17%, 11%, 42%, 55%, and 27%, respectively. The highest yield was observed when the R group was -NO2. The optimal pre-column derivatization reagent was determined to be 3-(4-nitrophenyl)propionic acid, which was named NPA. The derivatization conditions were then optimized. Optimization was performed using the RSM response surface methodology, with reaction time (A), temperature (B), and molar ratio (C) as independent variables, and the yield of the monosaccharide derivative as the response value. The optimized parameters were: time (8 h, 10 h, 12 h): temperature (30 °C, 40 °C, 50 °C); pre-column derivatization reagent: mannose (3:1, 1:1, 1:3). The optimal derivatization conditions were determined to be: temperature: 40 °C; time: 10 h; molar ratio: 1:1. The response surface data analysis is shown in Table 1.

[0033] Table 1. Analysis of variance of the experimental results of monosaccharide-derived BBD

[0034]

[0035] Example 2: Construction of NPA pre-column derivatization-HPLC method for detecting the monosaccharide composition of predictive polysaccharides

[0036] 1. Raw material collection: Ten batches of dried yarrow seeds were collected from different parts of the country. Batch 1 to 4 were collected from Enshi and Yichang, Hubei (2 batches each), batch 5 and 6 were collected from Zhengzhou, Henan, batch 7 was collected from Fenghuang County, Hunan, and batches 8 to 10 were collected from Yuexi, Anhui (2 batches) and Jinzhai, Anhui.

[0037] 2. Extraction of polysaccharides from *Evodia rutaecarpa*: Weigh 150g of each batch of dried *Evodia rutaecarpa*. First, boil in 800mL of distilled water for 1 hour, then collect the decoction. Add another 800mL of distilled water and boil for approximately 45 minutes, collecting the decoction again. Concentrate the decoction, then add ethanol (prepared from anhydrous ethanol to 80% ethanol) to the polysaccharide solution at a volume ratio of 1:4. Stir for 1 hour, allow to stand, centrifuge, discard the supernatant, dry (to remove any possible ethanol), dissolve in water, centrifuge again, and retain the solution. Finally, deproteinize using the Sevage method to ensure sample purity, then freeze-dry and weigh. The polysaccharide yields of 10 batches of *Evodia rutaecarpa* ranged from 2.1% to 6.5%.

[0038] 3. Determination of total sugar content and uronic acid content of polysaccharides: A standard curve was obtained by plotting glucose concentration (C) on the x-axis and absorbance value (A) on the y-axis: Y = 12.47x + 0.5153, with a correlation coefficient R0. 2 =0.9973. A good linear relationship was observed between glucose concentration and absorbance value within the range of 0–0.05 mg / mL. The total sugar content of the 10 batches of *Gynostemma pentaphyllum* polysaccharides ranged from 65% to 85%. Among them, the fifth batch from Zhengzhou, Henan Province, had a relatively high total sugar content, accounting for 85% of the total sugar content, while the fourth batch from Yichang, Hubei Province, had a relatively low total sugar content, accounting for 65% of the total sugar content. The standard curve for galacturonic acid obtained using the m-hydroxybiphenyl method was Y = 0.8304x + 0.2783, with a correlation coefficient R... 2 =0.9992, proving that the galacturonic acid concentration has a good linear relationship with absorbance in the range of 0.05-0.4 mg / mL. By measuring the absorbance values ​​at 525 nm of 10 different batches of samples, the uronic acid content in the total sugar of *Gnaphalium affine* ranged from 32% to 51%. The uronic acid content in the 7th batch of *Gnaphalium affine* polysaccharide from Fenghuang County, Hunan Province, was higher than that from other places, while the 4th batch from Yichang, Hubei Province, had the lowest uronic acid content in the total sugar. The standard curve of total sugar and uronic acid content of *Gnaphalium affine* polysaccharide is shown below. Figure 2 As shown:

[0039] 4. Hydrolysis of Predicted Polysaccharide: Weigh 200 mg of each batch of Predicted Polysaccharide into a 10 mL sealed container, add 3 mL of water to dissolve, add 2 mL of TFA (2 mmol / L), and hydrolyze at 110 °C for 5 h to obtain a liquid. Cool to room temperature. Centrifuge at 4000 r / min for 5 min to obtain the supernatant liquid, concentrate under vacuum at 70 °C, evaporate to dryness, and weigh.

[0040] 5. Monosaccharide derivatization: Add the sample (0.66 mmol, 1.0 eq) and anhydrous 1,4-dioxane (40 mL) to a three-necked flask. After supersuspension for 15 min, add pre-column derivatization reagent NPA (129 mg, 0.66 mmol, 1.0 eq) and triphenylphosphine (1060 mg, 4 mmol, 6.0 eq). Evacuate the container and purge with nitrogen. Slowly add diisopropylazo-1,8-dicarboxylic acid ester (4 mmol, 800 μL) to the mixture under ice bath conditions. Finally, raise the reaction temperature to 40 °C and react for 10 h.

[0041] 6. High-Performance Liquid Chromatography (HPLC) Injection Analysis: HPLC conditions were as follows: Agilent XB-C8 column (4.6 × 250 mm, 5 μL); set temperature 25℃; detection wavelength 274 nm. 5 μL was injected at a flow rate of 1.2 mL / min. Mobile phase B was methanol, and mobile phase A was pure water. Gradient elution was used, with the following gradient: 0–7 min, 35% B; 9–18 min, 62% B; 20–25 min, 100% B; 26–30 min, 35% B. Figure 4 As can be seen, each component can be effectively separated with relatively good peak shapes. The retention times of glucose, fructose, mannose, and ribose derivatives are 10.03, 11.67, 15.24, and 21.63 min, respectively. Glucose, mannose, and ribose are aldoses, and fructose is a ketose. Under these chromatographic conditions, the four monosaccharides can be effectively separated, indicating that pre-column derivatization reagents can achieve qualitative and quantitative analysis of these four monosaccharides under these conditions. A monosaccharide fingerprint chromatogram based on pre-column derivatization-HPLC was established, and the similarity was examined. The similarity values ​​were all greater than 0.8, indicating good results, as shown in Tables 2-3. Figure 5 As shown.

[0042] Table 2. Quantitative analysis of monosaccharides from 10 batches of predictor seeds.

[0043]

[0044] Table 3. Similarity Examination of 10 Batches of Predictors

[0045]

[0046] References

[0047] [1]Lamari F N,Kuhn R,Karamanos N K.Derivatization of carbohydratesfor chromatographic,electrophoretic and mass spectrometric structure analysis[J].Journal of Chromatography B,2003,793(1):15-36.

[0048] [2]Fan B,Li T,Song X,et al.A rapid,accurate and sensitive method fordetermination of monosaccharides in different varieties of Osmanthus fragransLour by pre-column derivatization with HPLC-MS / MS[J].International Journal ofBiological Macromolecules,2019,125:221-231.

[0049] [3]Jun X,Songlin L,Ruiqi Y,et al.2014.A novel and rapid HPGPC-basedstrategy for quality control of saccharide-dominant herbal materials:Dendrobium officinale,a case study[J].Anal Bioanal Chem,406:6409-6417.

[0050] [4]Huan L,Xingqun G,Zichen W,et al.2018.Multiple fingerprint profileand chemometrics analysis of polysaccharides from Sarcandraglabra.International Journal of Biological Macromolecules,123:957-967.

[0051] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

The application of 1,3-(4-nitrophenyl)propionic acid in the detection of the monosaccharide composition of predictor polysaccharides, characterized in that, The monosaccharides include aldoses and ketoses; The detection method includes: Take dried ginseng, boil it with distilled water, and collect the decoction; concentrate it, add ethanol, stir, and let it stand; centrifuge, discard the supernatant, and dry it; dissolve it in water again, centrifuge, and keep the solution; remove the protein using the Sevage method, and then freeze-dry it to obtain crude ginseng polysaccharide; Take the crude polysaccharide of the seed, dissolve it in water, add TFA to hydrolyze it, and obtain a liquid; cool to room temperature, centrifuge, and obtain the supernatant liquid; concentrate under vacuum, evaporate to dryness, and obtain the dried monosaccharide product; Take the dried monosaccharide and anhydrous 1,4-dioxane, sonicate, add 3-(4-nitrophenyl)propionic acid and triphenylphosphine to obtain a mixture; evacuate, purge with nitrogen, slowly add diisopropylazo-1,8-dicarboxylic acid ester to the mixture under ice bath, finally raise the reaction temperature to 40 °C, react for 10 h to obtain the monosaccharide derivatization product; The monosaccharide derivatization reaction products were separated and detected by high performance liquid chromatography.

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