Analysis of Monosaccharide Composition of Polysaccharide Compounds in Wild Water chestnut Shell by Gas Chromatography-Mass Spectrometry
2019-09-14 06:05:43
Abstract Four kinds of polysaccharide compounds were extracted from wild water chestnut shell by ethanol fractionation method for the first time, using gas chromatography . By mass spectrometry , it was determined that the water chestnut polysaccharide is composed of arabinose, rhamnose, xylose, mannose, galactose, glucose, lactose and melibiose, respectively, among which glucose, galactose, mannose and xylose are dominant.
Keywords polysaccharide, gas chromatography-mass spectrometry, monosaccharide
1 Introduction Since the 1960s, people have gradually discovered that polysaccharides have complex and diverse biological activities and functions. For example, polysaccharides have immunomodulatory functions as broad-spectrum immune promoters, and can be used as drugs to treat rheumatism, chronic viral hepatitis, cancer, etc. It has anti-infection, anti-radiation, anti-coagulation, blood sugar lowering, blood fat reduction, and promotes the biosynthesis of nucleic acids and proteins. Ling, aliased water chestnut, water chestnut, wind ling, is the seed of the genus Trapa bicomis Osbeck. According to the literature, its shell, stalk, fruit, stem and petiole can be used as medicine. , anti-cancer and Other effects; can also be used to treat gastric ulcer, dysentery, esophageal cancer, cervical cancer and breast cancer.
In this study, the polysaccharide was extracted and extracted from wild water chestnut shell for the first time, and the monosaccharide composition of polysaccharide was studied in depth, which provided a scientific basis for further development and utilization of wild water chestnut resources.
2 Experimental part
2.1 Samples, reagents and instruments After the commercially available water chestnuts are removed, the shells are dried and pulverized for use. Monosaccharide and disaccharide standards (galactose, arabinose, xylose, rhamnose, mannose, glucose, lactose and melibiose) were purchased from Chem Service with a purity greater than 98%. The chemical reagents used were analytically pure except that methanol and ethanol were chromatographically pure. UV-2501 PC UV spectrometer (Shimadzu, Japan); Nicolet Magna 560 Fourier transform infrared spectrometer (Nikoli, USA); Varian Unity Plus 600 NMR spectrometer (Varian, USA); HP 6890 and HP 5973 Gas Chromatography-Mass Spectrometer (Agilent, USA).
2.2 Experimental methods (1) Extraction and purification of polysaccharide compounds Weigh 800g of washed and dried rhombohedral shells, add 1:1 ethanol-ether mixture, reflux at 65 °C for 1~3h in water bath; Add water, extract from 4O ~ 100 ° C water bath for 4 ~ 6 h, replace the water for 2 to 3 times, and combine the dark red polysaccharide extract, filter and concentrate. 3/7 volume of ethanol was added to the extract, the precipitate was separated by centrifugation, and lyophilized to obtain crude polysaccharide F1. 1.0 volume of ethanol was added to the supernatant, the precipitate was separated by centrifugation, and lyophilized to obtain crude polysaccharide F2. According to this method, 1.5 and 4.0 volumes of ethanol were successively added to the supernatant to obtain crude polysaccharides F3 and F4, respectively. . If 4 times by volume of absolute ethanol is directly added to the original polysaccharide extract, a crude mixed polysaccharide F5 is obtained. The protein in the crude polysaccharide was removed by the Sevag method, and the pigment was removed by the H2O2 method. After dialysis for 72 hours, it was concentrated and lyophilized to obtain a pure polysaccharide;
(2) Hydrolysis of polysaccharides 10 mg of polysaccharide sample was added to 2 mL of 3 mol/L trifluoroacetic acid in an ampoule. After sealing, it was hydrolyzed at 120 ° C for 6 h. The trifluoroacetic acid was blown dry with nitrogen, methanol was added and dried, and hydrolyzed. The product was added with 0.6 mL of 0.05 mol/L NaOH solution, and then added with 5 mg of NaBH4. The reaction was carried out for 8-10 h at room temperature, and a little acetic acid was added to decompose excess NaBH4 until no bubbles were formed.
Evaporating the reaction solution, washing the reaction product with acidic methanol, evaporating the methanol solution, repeating 3-4 times to remove the borate, adding methanol to the dried solution, and removing the water in an oven at 105 ° C;
(3) acetylation derivatization method 3 mg of monosaccharide obtained by hydrolysis, adding 0.5 mL of pyridine, 1 mL of acetic anhydride, heating for 2 h, cooling, adding 2 mL of toluene, drying with N2, adding chloroform, washing with distilled water, drying with N2 to obtain brown Yellow product. The acetylated monosaccharide alcohol was diluted with CHC13 and subjected to gas chromatography-mass spectrometry; (4) trimethylsilylation derivatization method was carried out by hydrolyzing a sample of monosaccharide 6 mg, adding pyridine 0.6 mL, hexamethyltetrasilamide 0.2 mL mL, trimethylchlorosilane 0.2 mL, shaken, left to stand, and the supernatant was taken for gas chromatography-mass spectrometry.
2.3 GC- MS analysis conditions HP-5 quartz capillary column (Agilent, USA), (50m × 0.22mm i.d., 0.25μm); EI source, electron energy 70 eV, molecular weight scanning range 33-550, Inlet temperature 260 ° C, interface temperature 270 ° C, carrier gas helium flow rate 1.2 mL / min, split ratio 20:1, program temperature: 100 ° C → 1O ° C / min 150 ° C → 15 ° C / min 260 ° C (40 min ).
3 Results and discussion
3.1 Identification of polysaccharides in the extract of the water chestnut shell Molisch experiment: The sulfuric acid and polysaccharide solution are divided into two layers, and a purple ring appears between the layers. The extract does contain sugar. FT-IR spectrum of pure polysaccharide F5 pure product. The characteristic peaks of polysaccharides can be seen in many places in the spectrum, such as: 3435/cm, the absorption peaks of 2924/cm are O-H, C-H stretching vibration peaks, 1193/cm, and the absorption peak at 1037/cm is C-O, C-O-C in polysaccharides
The stretching vibration characteristic of the stretching vibration and C-OH, and the absorption peak at 1000-700/cm are characteristic of the formation of α- and β-pyran monosaccharide.
H NMR spectrum of the pure polysaccharide F5 pure product. The response signal peaks of multiple saccharide protons can be seen between δ3.5-δ5.5 in the spectrum, where δ 4.605, δ4.7 and δ4.922 3 peaks are proton signals on β-pyran ring, and δ5. 238, δ5.348, δ5.446,
δ 5.465 and δ5.515 may be derived from protons on the α-pyran ring, and thus it may be seen that this polysaccharide may be composed of a plurality of monosaccharides.
3.2 Determination of Monosaccharide Composition in Polygonum Polysaccharide Samples The acetylated derivatives of the F1-F4 hydrolysate were analyzed by gas chromatography-mass spectrometry. The monosaccharide composition and molars were determined by comparison with standard chromatograms and mass spectrometry fragment analysis. ratio. The data show that the four polysaccharides are composed of several monosaccharides (such as arabinose, rhamnose, xylose, mannose, galactose, glucose) and disaccharides (lactose, melibiose) in different molar ratios, among which polysaccharides The monosaccharide composition of F2 to F4 is the same except that the molar ratio is slightly different; while F1 is composed of 5 monosaccharides and 2 disaccharides. Glucose, galactose and mannose account for a large proportion of the four polysaccharides and are the main components of the polysaccharide.
The GC-MS of the methylsilylated derivative of the mixed polysaccharide sample F5 was subjected to fragmentation analysis and searched using a standard mass spectrometer NIST library, and l1 peaks were confirmed as methylsilylated derivatives of monosaccharides. Due to the formation of alpha and beta pyran and furan isomers during derivatization, the same monosaccharide can form several derivatives. Therefore, only five monosaccharides were detected, including two pentoses (arabinose and xylose) and three hexoses (mannose, galactose and glucose). It can also be seen from the peak area percentage data that glucose, galactose, xylose and mannose account for a large proportion in the mixed polysaccharide, and are the main components of the water chestnut polysaccharide, which is basically consistent with the results of the acetylation analysis.
References
1 Bleton J, Mejanelle P, Sansoulet J, Goursaud S, Tchapla A. Journal of Chromatography, 1996, 720: 27 ~ 49
2 Cardoso SM, Silva AMS, Coimbra MA. Carbohydrate Research. 2002.337: 917~924
3 Doco T, O'Neill MA, Pellerin P. Carbohydrate Polymers, 2001, 46: 249 ~ 259
4 Fu DT, 0'Neill RA. Ana1. Biochem. , 1995, 227: 377 ~ 384
5 Li Yongchun (æŽæ°¸æ˜¥), Wang Shuqi (çŽ‹æ ‘æ§), Liu Ruchen (刘æ±ç›), xu Deliang (Xu Deliang), Zhang Dingchen (Zhang Dingchen).
Dictionary of Chinese Herbalist Doctor, Fascicule of Chinese Traditional Medicine (Chinese Medicine Dictionary). 1st ed. (*
Edition), Beijing (Beijing) People Sanitation Press. (People's Health Publishing House) 1980: 313
6 Osbom HMI, 1. z~hey F, Moshy L, Read D. Chromatogr. A, 1999, 831: 267 ~ 276
7 Zhang Lina (å¼ ä¿å¨œ), Ding Qiong (ä¸ç¼), Zhang Pingyi (å¼ å¹³ä¹‰), Feng Hanqiao (冯汉桥). Chem. Chinese
Universities (Chemical Journal of Higher Education), 1997, 6: 990 ~ 993
Keywords polysaccharide, gas chromatography-mass spectrometry, monosaccharide
1 Introduction Since the 1960s, people have gradually discovered that polysaccharides have complex and diverse biological activities and functions. For example, polysaccharides have immunomodulatory functions as broad-spectrum immune promoters, and can be used as drugs to treat rheumatism, chronic viral hepatitis, cancer, etc. It has anti-infection, anti-radiation, anti-coagulation, blood sugar lowering, blood fat reduction, and promotes the biosynthesis of nucleic acids and proteins. Ling, aliased water chestnut, water chestnut, wind ling, is the seed of the genus Trapa bicomis Osbeck. According to the literature, its shell, stalk, fruit, stem and petiole can be used as medicine. , anti-cancer and Other effects; can also be used to treat gastric ulcer, dysentery, esophageal cancer, cervical cancer and breast cancer.
In this study, the polysaccharide was extracted and extracted from wild water chestnut shell for the first time, and the monosaccharide composition of polysaccharide was studied in depth, which provided a scientific basis for further development and utilization of wild water chestnut resources.
2 Experimental part
2.1 Samples, reagents and instruments After the commercially available water chestnuts are removed, the shells are dried and pulverized for use. Monosaccharide and disaccharide standards (galactose, arabinose, xylose, rhamnose, mannose, glucose, lactose and melibiose) were purchased from Chem Service with a purity greater than 98%. The chemical reagents used were analytically pure except that methanol and ethanol were chromatographically pure. UV-2501 PC UV spectrometer (Shimadzu, Japan); Nicolet Magna 560 Fourier transform infrared spectrometer (Nikoli, USA); Varian Unity Plus 600 NMR spectrometer (Varian, USA); HP 6890 and HP 5973 Gas Chromatography-Mass Spectrometer (Agilent, USA).
2.2 Experimental methods (1) Extraction and purification of polysaccharide compounds Weigh 800g of washed and dried rhombohedral shells, add 1:1 ethanol-ether mixture, reflux at 65 °C for 1~3h in water bath; Add water, extract from 4O ~ 100 ° C water bath for 4 ~ 6 h, replace the water for 2 to 3 times, and combine the dark red polysaccharide extract, filter and concentrate. 3/7 volume of ethanol was added to the extract, the precipitate was separated by centrifugation, and lyophilized to obtain crude polysaccharide F1. 1.0 volume of ethanol was added to the supernatant, the precipitate was separated by centrifugation, and lyophilized to obtain crude polysaccharide F2. According to this method, 1.5 and 4.0 volumes of ethanol were successively added to the supernatant to obtain crude polysaccharides F3 and F4, respectively. . If 4 times by volume of absolute ethanol is directly added to the original polysaccharide extract, a crude mixed polysaccharide F5 is obtained. The protein in the crude polysaccharide was removed by the Sevag method, and the pigment was removed by the H2O2 method. After dialysis for 72 hours, it was concentrated and lyophilized to obtain a pure polysaccharide;
(2) Hydrolysis of polysaccharides 10 mg of polysaccharide sample was added to 2 mL of 3 mol/L trifluoroacetic acid in an ampoule. After sealing, it was hydrolyzed at 120 ° C for 6 h. The trifluoroacetic acid was blown dry with nitrogen, methanol was added and dried, and hydrolyzed. The product was added with 0.6 mL of 0.05 mol/L NaOH solution, and then added with 5 mg of NaBH4. The reaction was carried out for 8-10 h at room temperature, and a little acetic acid was added to decompose excess NaBH4 until no bubbles were formed.
Evaporating the reaction solution, washing the reaction product with acidic methanol, evaporating the methanol solution, repeating 3-4 times to remove the borate, adding methanol to the dried solution, and removing the water in an oven at 105 ° C;
(3) acetylation derivatization method 3 mg of monosaccharide obtained by hydrolysis, adding 0.5 mL of pyridine, 1 mL of acetic anhydride, heating for 2 h, cooling, adding 2 mL of toluene, drying with N2, adding chloroform, washing with distilled water, drying with N2 to obtain brown Yellow product. The acetylated monosaccharide alcohol was diluted with CHC13 and subjected to gas chromatography-mass spectrometry; (4) trimethylsilylation derivatization method was carried out by hydrolyzing a sample of monosaccharide 6 mg, adding pyridine 0.6 mL, hexamethyltetrasilamide 0.2 mL mL, trimethylchlorosilane 0.2 mL, shaken, left to stand, and the supernatant was taken for gas chromatography-mass spectrometry.
2.3 GC- MS analysis conditions HP-5 quartz capillary column (Agilent, USA), (50m × 0.22mm i.d., 0.25μm); EI source, electron energy 70 eV, molecular weight scanning range 33-550, Inlet temperature 260 ° C, interface temperature 270 ° C, carrier gas helium flow rate 1.2 mL / min, split ratio 20:1, program temperature: 100 ° C → 1O ° C / min 150 ° C → 15 ° C / min 260 ° C (40 min ).
3 Results and discussion
3.1 Identification of polysaccharides in the extract of the water chestnut shell Molisch experiment: The sulfuric acid and polysaccharide solution are divided into two layers, and a purple ring appears between the layers. The extract does contain sugar. FT-IR spectrum of pure polysaccharide F5 pure product. The characteristic peaks of polysaccharides can be seen in many places in the spectrum, such as: 3435/cm, the absorption peaks of 2924/cm are O-H, C-H stretching vibration peaks, 1193/cm, and the absorption peak at 1037/cm is C-O, C-O-C in polysaccharides
The stretching vibration characteristic of the stretching vibration and C-OH, and the absorption peak at 1000-700/cm are characteristic of the formation of α- and β-pyran monosaccharide.
H NMR spectrum of the pure polysaccharide F5 pure product. The response signal peaks of multiple saccharide protons can be seen between δ3.5-δ5.5 in the spectrum, where δ 4.605, δ4.7 and δ4.922 3 peaks are proton signals on β-pyran ring, and δ5. 238, δ5.348, δ5.446,
δ 5.465 and δ5.515 may be derived from protons on the α-pyran ring, and thus it may be seen that this polysaccharide may be composed of a plurality of monosaccharides.
3.2 Determination of Monosaccharide Composition in Polygonum Polysaccharide Samples The acetylated derivatives of the F1-F4 hydrolysate were analyzed by gas chromatography-mass spectrometry. The monosaccharide composition and molars were determined by comparison with standard chromatograms and mass spectrometry fragment analysis. ratio. The data show that the four polysaccharides are composed of several monosaccharides (such as arabinose, rhamnose, xylose, mannose, galactose, glucose) and disaccharides (lactose, melibiose) in different molar ratios, among which polysaccharides The monosaccharide composition of F2 to F4 is the same except that the molar ratio is slightly different; while F1 is composed of 5 monosaccharides and 2 disaccharides. Glucose, galactose and mannose account for a large proportion of the four polysaccharides and are the main components of the polysaccharide.
The GC-MS of the methylsilylated derivative of the mixed polysaccharide sample F5 was subjected to fragmentation analysis and searched using a standard mass spectrometer NIST library, and l1 peaks were confirmed as methylsilylated derivatives of monosaccharides. Due to the formation of alpha and beta pyran and furan isomers during derivatization, the same monosaccharide can form several derivatives. Therefore, only five monosaccharides were detected, including two pentoses (arabinose and xylose) and three hexoses (mannose, galactose and glucose). It can also be seen from the peak area percentage data that glucose, galactose, xylose and mannose account for a large proportion in the mixed polysaccharide, and are the main components of the water chestnut polysaccharide, which is basically consistent with the results of the acetylation analysis.
References
1 Bleton J, Mejanelle P, Sansoulet J, Goursaud S, Tchapla A. Journal of Chromatography, 1996, 720: 27 ~ 49
2 Cardoso SM, Silva AMS, Coimbra MA. Carbohydrate Research. 2002.337: 917~924
3 Doco T, O'Neill MA, Pellerin P. Carbohydrate Polymers, 2001, 46: 249 ~ 259
4 Fu DT, 0'Neill RA. Ana1. Biochem. , 1995, 227: 377 ~ 384
5 Li Yongchun (æŽæ°¸æ˜¥), Wang Shuqi (çŽ‹æ ‘æ§), Liu Ruchen (刘æ±ç›), xu Deliang (Xu Deliang), Zhang Dingchen (Zhang Dingchen).
Dictionary of Chinese Herbalist Doctor, Fascicule of Chinese Traditional Medicine (Chinese Medicine Dictionary). 1st ed. (*
Edition), Beijing (Beijing) People Sanitation Press. (People's Health Publishing House) 1980: 313
6 Osbom HMI, 1. z~hey F, Moshy L, Read D. Chromatogr. A, 1999, 831: 267 ~ 276
7 Zhang Lina (å¼ ä¿å¨œ), Ding Qiong (ä¸ç¼), Zhang Pingyi (å¼ å¹³ä¹‰), Feng Hanqiao (冯汉桥). Chem. Chinese
Universities (Chemical Journal of Higher Education), 1997, 6: 990 ~ 993
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