Studies On The Quality Control Of Asparagus Officinalis L. By Modern Analysis Techniques

Asparagus officinalis L. is one of the ten most famous vegetables of the world. It is delicious and eutrophy, and is good for health. Recent studies have found that Asparagus officinalis L. can be used to cure cancer, lower blood-lipid, enhance immunity, anti-aging, anti-fatigue and protect liver. So far as we know, there are only a few papers about the chemical composition of Asparagus officinalis L. These papers are about the separation and purification of total flavonoids and total saponins. So, it is difficult to control the quality of Asparagus officinalis L. fully and efficiently. This study detected the nutritional ingredients in Asparagus officinalis L. and then identified the purified compounds in Asparagus officinalis L. Next, total flavonoids of Asparagus officinalis L. was prepared and analyzed. The preparation method of the five flavonoid glycosides from Asparagus officinalis L. was optimized using two dimensional prep-HPLC and the total flavonoids of Asparagus officinalis L. was analyzed with comprehensive two dimensional HPLC to study the interactions between the components in Asparagus officinalis L. and the liposome membrane. Finally, the antioxidant activities of total flavonoids and five flavonoid glycosides from Asparagus officinalis L. were studied. The material basis for the efficiency of Asparagus officinalis L. and the structure-function relationship for the antioxidant activities of flavonoids were revealed and validated.1. Quality control of the nutritional ingredients in Asparagus officinalis L.1.1 Amino acids react with ninhydrin to generate a kind of hepatic complex at pH 5.5 and the shade of the color varies directly with the contents of the amino acids. The complex has the strongest absorption at the wavelength of 570 nm. So, the contents of the amino acids can be determined with this principle. This paper fully studied the technology of this method using L-Glu as reference substance. The equation of the linear regression was A=0.0073C-0.1133 (r=0.9998) and the linear range was 30.30-151.50μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of amino acids in ten batches of Asparagus officinalis L. were 17.43-22.43‰.1.2. Coomassie brilliant blue G-250 is red at free state. It changes into blue when combines with proteins in dilute acid and the wavelength with the strongest absorption changes into 595 nm instead of 465 nm. The absorbance of the conjugate at 595 nm varies directly with the contents of proteins. This paper fully studied the technology of this method using BSA as reference substance. The equation of the linear regression was A=0.0077C+0.0524 (r=0.9996) and the linear range was 19.80-99.00μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of proteins in ten batches of Asparagus officinalis L. were 1.08-1.69‰.1.3. The total polysaccharides in dry Asparagus officinalis L. was determined by coloration with sulphuric acid and anthrone. This paper fully studied the technology of this method using glucose as reference substance. The equation of the linear regression was A=0.0081C+0.0907 (r=0.9990) and the linear range was 20.00-100.00μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of total polysaccharides in ten batches of dry Asparagus officinalis L. were 1.16-1.64 %.1.4 The fingerprint of hydrolytic amino acids in Asparagus officinalis L. was established on a common C18 column using AQC as the derivatization reagent. Sixteen peaks were defined as common peaks and 11 of them were identified. Eleven amino acids in ten batches of Asparagus officinalis L. were determined. The results were as follows: histidine 2.97-5.56‰, glycine 0.39-0.53‰, aspartic acid 1.64-3.63‰, glutamic acid 0.89-1.55‰, threonine 0.32-0.46‰, alanine 0.32-0.57‰, cystine 0.47-1.01‰, tyrosine 0.23-0.28‰, valine 0.36-0.65‰, leucine 0.37-0.60‰, phenylalanine 0.35-0.44‰.2. Studies on the chemical constituents from Asparagus officinalis L.2.1 Separation and purification of two purines from Asparagus officinalis L. The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins. The fraction of 20% ethanol was collected and decolored with polydextran gel. The purified sample was separated with prep-HPLC and two compounds were prepared. They were identified as 9-(2-methylthio-3-sulfo-4-hydroxy)-furan-6-aminopurine and adenosine by 1HNMR and 13CNMR.2.2 Separation and purification of five flavonoid glycosides from Asparagus officinalis L.The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins. The fraction of 40% ethanol was collected and decolored with polydextran gel. The purified sample was separated with prep-HPLC and five compounds were prepared. They were identified as quercetin-3-O-glucosyl- rutinoside, rutin, isorhamnetin-3-O-glucosyl- rutinoside, nicotiflorin and narcissin by HPLC-DAD-MS.2.3 Separation and purification of an organic acid from Asparagus officinalis L.The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins. The fraction of 60% ethanol was collected and purified with polydextran gel. The compound was identified as cinnamic acid by 1HNMR and 13CNMR.3. Preparation of purified flavonoids extract of Asparagus officinalis L. and quantitative analysis3.1. The dry Asparagus officinalis L. was extracted with 60% ethanol and the extraction was processed with macroporous adsorptive resins (60×4cm). The extract was adsorbed by the resins for 1 h, and then eluted with 1500 mL of water, 2000 mL of 20% ethanol and 2000 mL of 40% ethanol. The fraction of 40% ethanol was collected and evaporated to dryness by rotary evaporation at 50℃under reduced pressure. The extract was dissolved in 30% methanol and then processed with polydextran gel (100×3cm). It was eluted with 300 mL of water, 200 mL of 50% methanol and 1000 mL of methanol. The fractions containing flavonoids were combined and evaporated to dryness by rotary evaporation at 50℃under reduced pressure. That was the purified flavonoids extract of Asparagus officinalis L..3.2. The contents of total flavonoids in the purified flavonoids extract of Asparagus officinalis L. were determined with ultraviolet spectrophotometry. This paper fully studied the technology of this method using rutin as reference substance. The equation of the linear regression was A=0.0024C-0.0174 (r=0.9992) and the linear range was 62.00-310.00μg/mL. The sample was stable for at least 1.5 h after color reaction. The results of the precision, reproducibility and recovery were within the criterion. The contents of total flavonoids in three batches of samples were >50 %.3.3. The contents of quercetin-3-O-glucosyl-rutinoside, rutin, isorhamnetin-3-O-glucosyl- rutinoside, nicotiflorin and narcissin in the purified flavonoids extract of Asparagus officinalis L. were determined with HPLC. Diamonsil○R C18 column (200×4.6mm,5μm, Dikma Technologies) was used for separation. The mobile phase was acetonitrile-methanol-0.1% HAc (8﹕16﹕76). The flue rate was 1.0 mL/min. The column temperature was set at 30℃and the detection was set at 260 nm. The injection volume was 20μL. This paper fully studied the technology of this method and all the results were within the criteria. The contents of quercetin-3-O-glucosyl-rutinoside, rutin, isorhamnetin-3-O-glucosyl-rutinoside, nicotiflorin and narcissin in three batches of samples were 11.26-12.62%, 22.64-24.78%, 4.37-5.09%, 4.13-4.89% and 3.22-3.63%, respectively.4. Preparation of five flavonoid glycosides from Asparagus officinalis L. by two-dimensional methods and the separation of the flavonoids using comprehensive two dimensional HPLC4.1. Five flavonoid glycosides (quercetin-3-O-glucosyl- rutinoside, rutin, isorhamnetin-3-O-glucosyl- rutinoside, nicotiflorin and narcissin) were purified from Asparagus officinalis L. by two-dimensional prep-HPLC with column switch technology. A six-port two-position switching valve instead of a sample loop was used in this system. This approach assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension were not restricted. Compared with traditional prep-HPLC, this method enhanced the production rate greatly and saved much organic solvent.4.2. Five flavonoid glycosides (quercetin-3-O-glucosyl-rutinoside, rutin, isorhamnetin-3-O-glucosyl-rutinoside, nicotiflorin and narcissin) were purified from Asparagus officinalis L. by HSCCC-PHPLC. In this method, HSCCC was used as a pretreatment process. Two fractions containing the target compounds were obtained by HSCCC. Then, they were further separated using PHPLC and the target compounds with high purity were finally obtained. This method was simple and high-performance without tedious pretreatment process using column chromatography.4.3. The purified flavonoids extract of Asparagus officinalis L. was analyzed by comprehensive two dimensional HPLC. In the first dimension, a liposome column was used with a flow rate of 0.1 mL/min. And in the second dimension, a Chromolith Performance RP-18e (100×4.6mm) column was used. The mobile phase consisted of A: acetonitrile, B: 10% acetonitrile. The gradient progress was as follows: 0-2min 3%A,2-4min 3%A-17% A,4-4.5min,17% A -3%A,4.5-5.0min,3%A, and the flow rate was 3.0 mL/min. The interactions between the components in Asparagus officinalis L. and liposome membrane could be judged according to the chromatographic behavior of these components on a liposome column. And the ADME of the drugs could also be concluded.5. Studies on the antioxidant activities of total flavonoids and five flavonoid glycosides from Asparagus officinalis L.The in vitro scavenging activities of total flavonoids of Asparagus officinalis L. and five flavonoid glycosides on superoxide radical, hydroxyl radical and DPPH, and their protective effects on H2O2-induced hemolysis of rat erythrocytes were investigated. The results suggested that total flavonoids and five flavonoid glycosides from Asparagus officinalis L. showed significant antioxidant activities in vitro. The material basis for the efficiency of Asparagus officinalis L. and the structure-function relationship for the antioxidant activities of flavonoids were revealed and validated. In a word, this study was carried out to control the quality of Asparagus officinalis L. The quantitation methods were developed to determine total free amino acids, total proteins and total polysaccharides in Asparagus officinalis L.. The fingerprint of hydrolytic amino acids in Asparagus officinalis L. was established and 11 amino acids were quantitated. To further understand the material basis for the efficiency of Asparagus officinalis L., the chemical constituents in Asparagus officinalis L. were studied. Two purines, five flavonoid glycosides and one organic acid were separated from Asparagus officinalis L. for the first time. The methods for preparation of total flavonoids of Asparagus officinalis L. and its quantitative analysis were developed. It was very meaningful for the further study of flavonoids in Asparagus officinalis L.. This paper also established the preparation method for five flavonoid glycosides from Asparagus officinalis L. with two-dimensional column switch technology or HSCCC-PHPLC method. The two methods were superior to the traditional ones for its higher efficiency, less reagent consumption et al.. The purified flavonoids extract of Asparagus officinalis L. was analyzed by comprehensive two dimensional HPLC. The interactions between the components in Asparagus officinalis L. and liposome membrane were studied. Finally, the antioxidant activities of total flavonoids and five flavonoid glycosides from Asparagus officinalis L. were studied. The material basis for the efficiency of Asparagus officinalis L. and the structure-function relationship for the antioxidant activities of flavonoids were revealed and validated.