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2022 Volume 1
Article Contents
Abstract
Introduction
Materials and methods
Plant materials
Chemicals and reagents
Preparation of marselan juices and wines
Total polyphenols and flavonoid content determination
Abts free-radical scavenging assay
Frap free-radical scavenging assay
Hs-gc-ims analysis
Statistical data analysis
Results
Changes in total polyphenol and flavonoid contents and antioxidant activity during fermentation
Analysis of the change in volatile organic compounds (vocs) during fermentation by gs-ims
Fingerprint analysis of vocs at different fermentation stages of marselan wine
Multivariate statistical analysis
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
Rights and permissions
References
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ARTICLE   Open Access    

Comparative analysis of chemical profiles of Radix Astragali between ultrafine granular powder and sliced traditional material

  • 1.

    National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, USA

  • 2.

    Natural Products Utilization Research Unit, Agricultural Research Service, United States Department of Agriculture, University, Mississippi 38677, USA

  • 3.

    Key Laboratory of Technologies and Applications of Ultrafine Granular Powder of Herbal Medicine, State Administration of Traditional Chinese Medicine, Zhongshan Zhongzhi Pharmaceutical Group Co., Ltd. Zhongshan 528437, P. R. China

  • 4.

    Hunan Provincial Key Laboratory of Dong Medicine, Hunan University of Medicine, Huaihua 41800, P. R. China

More Information
  • Radix Astragali, one of the most popular herbs in traditional Chinese medicine (TCM), is used to strengthen the immune system, protect liver function, fight bacteria and viruses, and treat diabetes, heart failure and seasonal allergies. In recent years, a new form of Radix Astragali material processed by cell wall disrupting technology, namely ultrafine granular powder (UGP) has been introduced into the market. In order to determine chemical consistency and homogeneity of the UGP material prepared from sliced traditional materials (TM) of Radix Astragali, multiple batches of the UGP and TM samples derived from Astragalus membranaceus var. mongholicus were analyzed by UHPLC/DAD-MS using isoflavones and triterpenoid glycosides as marker compounds. The results demonstrated that the chemical profiles of UGP was identical or similar to that of TM, but UGP was highly homogeneous in terms of marker compound contents as assessed, e.g., by the relative standard deviation values of the nine marker compounds in the range of 8.55%−43.80% for TM2 compared against 1.70%−8.38% for UGP2. Macromolecular component preparation and 1H NMR analyses indicated that TM4 and its corresponding UGP4 produced similar polysaccharides, but the later had approximately two-fold dissolution rate of the polysaccharides when compared to the former (yield 7.22 ± 0.35% vs 3.39 ± 0.20%). This study confirms that UGP of Radix Astragali is chemically consistent and homogenous, supporting its use as a promising material in TCM prescriptions.

  • Introduction

    Starting in the early 2000s, China has experienced rapid growth as an emerging wine market. It has now established itself as the world's second-largest grape-growing country in terms of vineyard surface area. Furthermore, China has also secured its position as the sixth-biggest wine producer globally and the fifth-most significant wine consumer in terms of volume[1]. The Ningxia Hui autonomous region, known for its reputation as the highest quality wine-producing area in China, is considered one of the country's most promising wine regions. The region's arid or semiarid climate, combined with ample sunlight and warmth, thanks to the Yellow River, provides ideal conditions for grape cultivation. Wineries in the Ningxia Hui autonomous region are renowned as the foremost representatives of elite Chinese wineries. All wines produced in this region originate from grapes grown in their vineyards, adhering to strict quality requirements, and have gained a well-deserved international reputation for excellence. Notably, in 2011, Helan Mountain's East Foothill in the Ningxia Hui Autonomous Region received protected geographic indication status in China. Subsequently, in 2012, it became the first provincial wine region in China to be accepted as an official observer by the International Organisation of Vine and Wine (OIV)[2]. The wine produced in the Helan Mountain East Region of Ningxia, China, is one of the first Agricultural and Food Geographical Indications. Starting in 2020, this wine will be protected in the European Union[3].

    Marselan, a hybrid variety of Cabernet Sauvignon and Grenache was introduced to China in 2001 by the French National Institute for Agricultural Research (INRA). Over the last 15 years, Marselan has spread widely across China, in contrast to its lesser cultivation in France. The wines produced from Marselan grapes possess a strong and elegant structure, making them highly suitable for the preferences of Chinese consumers. As a result, many wineries in the Ningxia Hui Autonomous Region have made Marselan wines their main product[4]. Wine is a complex beverage that is influenced by various natural and anthropogenic factors throughout the wine-making process. These factors include soil, climate, agrochemicals, and human intervention. While there is an abundance of research available on wine production, limited research has been conducted specifically on local wines in the Eastern Foot of Helan Mountain. This research gap is of significant importance for the management and quality improvement of Chinese local wines.

    Ion mobility spectrometry (IMS) is a rapid analytical technique used to detect trace gases and characterize chemical ionic substances. It achieves this through the gas-phase separation of ionized molecules under an electric field at ambient pressure. In recent years, IMS has gained increasing popularity in the field of food-omics due to its numerous advantages. These advantages include ultra-high analytical speed, simplicity, easy operation, time efficiency, relatively low cost, and the absence of sample preparation steps. As a result, IMS is now being applied more frequently in various areas of food analysis, such as food composition and nutrition, food authentication, detection of food adulteration, food process control, and chemical food safety[5,6]. The orthogonal hyphenation of gas chromatography (GC) and IMS has greatly improved the resolution of complex food matrices when using GC-IMS, particularly in the analysis of wines[7].

    The objective of this study was to investigate the changes in the physicochemical properties of Marselan wine during the winemaking process, with a focus on the total phenolic and flavonoids content, antioxidant activity, and volatile profile using the GC-IMS method. The findings of this research are anticipated to make a valuable contribution to the theoretical framework for evaluating the authenticity and characterizing Ningxia Marselan wine. Moreover, it is expected that these results will aid in the formulation of regulations and legislation pertaining to Ningxia Marselan wine in China.

    Materials and methods

    Plant materials

    All the grapes used to produce Marselan wines, grow in the Xiban vineyard (106.31463° E and 38.509541° N) situated in Helan Mountain's East Foothill of Ningxia Hui Autonomous Region in China.

    Chemicals and reagents

    Folin-Ciocalteau reagent, (±)-6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,20-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ), anhydrous methanol, sodium nitrite, and sodium carbonate anhydrous were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Reference standards of (+)-catechin, gallic acid, and the internal standard (IS) 4-methyl-2-pentanol were supplied by Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). The purity of the above references was higher than 98%. Ultrapure water (18.2 MΩ cm) was prepared by a Milli-Q system (Millipore, Bedford, MA, USA).

    Preparation of Marselan juices and wines

    Stage 1−Juice processing: Grapes at the fully mature stage are harvested and crushed, and potassium metabisulfite (5 mg/L of SO2) was evenly spread during the crushing process. The obtained must is transferred into stainless steel tanks. Stage 2−Alcoholic fermentation: Propagated Saccharomyces cerevisiae ES488 (Enartis, Italy) are added to the fresh must, and alcoholic fermentation takes place, after the process is finished, it is kept in the tanks for 7 d for traditional maceration to improve color properties and phenolics content. Stage 3−Malolactic fermentation: When the pomace is fully concentrated at the bottom of the tanks, the wine is transferred to another tank for separation from these residues. Oenococcus oeni VP41 (Lallemand Inc., France) is inoculated and malic acid begins to convert into lactic acid. Stage 4−Wine stabilization: After malolactic fermentation, potassium metabisulfite is re-added (35 mg/L of SO2), and then transferred to oak barrels for stabilization, this process usually takes 6-24 months. A total of four batches of samples during the production process of Marselan wine were collected in this study.

    Total polyphenols and flavonoid content determination

    Total polyphenols were determined on 0.5 mL diluted wine sample using the Folin-Ciocalteu method[8], using gallic acid as a reference compound, and expressed as milligrams of gallic acid equivalents per liter of wine. The total flavonoid content was measured on 0.05 mL of wine sample by a colorimetric method previously described[9]. Results are calculated from the calibration curve obtained with catechin, as milligrams of catechin equivalents per liter of wine.

    ABTS free-radical scavenging assay

    The antioxidative activity was determined using the ABTS·+ assay[10]. Briefly, the ABTS·+ radical was prepared from a mixture of 88 μL of potassium persulfate (140 mmol/L) with 5 mL of the ABTS·+ solution (7 mmol/L). The reaction was kept at room temperature under the absence of light for 16 h. Sixty μL samples were mixed with 3 mL of ABTS·+ solution with measured absorption of 0.700 ± 0.200 at 734 nm. After 6 min reaction, the absorbance of samples were measured with a spectrophotometer at 734 nm. Each sample was tested in triplicate. The data were expressed as mmol Trolox equivalent of antioxidative capacity per liter of the wine sample (mmol TE/L). Calibration curves, in the range 64.16−1,020.20 μmol TE/L, showed good linearity (R2 ≥ 0.99).

    FRAP free-radical scavenging assay

    The FRAP assay was conducted according to a previous study[11]. The FRAP reagent was freshly prepared and mixed with 10 mM/L TPTZ solution prepared in 20 mM/L FeCl3·6H2O solution, 40 mM/L HCl, and 300 mM/L acetate buffer (pH 3.6) (1:1:10; v:v:v). Ten ml of diluted sample was mixed with 1.8 ml of FRAP reagent and incubated at 37 °C for 30 min. The absorbance was determined at 593 nm and the results were reported as mM Fe (II) equivalent per liter of the wine sample. The samples were analyzed and calculated by a calibration curve of ferrous sulphate (0.15−2.00 mM/mL) for quantification.

    HS-GC-IMS analysis

    The volatile compounds were analyzed on a GC-IMS instrument (FlavourSpec, GAS, Dortmund, Germany) equipped with an autosampler (Hanon Auto SPE 100, Shandong, China) for headspace analysis. One mL of each wine was sampled in 20 mL headspace vials (CNW Technologies, Germany) with 20 μL of 4-methyl-2-pentanol (20 mg/L) ppm as internal standard, incubated at 60 °C and continuously shaken at 500 rpm for 10 min. One hundred μL of headspace sample was automatically loaded into the injector in splitless mode through a syringe heated to 65 °C. The analytes were separated on a MxtWAX capillary column (30 m × 0.53 mm, 1.0 μm) from Restek (Bellefonte, Pennsylvania, USA) at a constant temperature of 60 °C and then ionized in the IMS instrument (FlavourSpec®, Gesellschaft für Analytische Sensorsysteme mbH, Dortmund, Germany) at 45 °C. High purity nitrogen gas (99.999%) was used as the carrier gas at 150 mL/min, and drift gas at 2 ml/min for 0−2.0 min, then increased to 100 mL/min from 2.0 to 20 min, and kept at 100 mL/min for 10 min. Ketones C4−C9 (Sigma Aldrich, St. Louis, MO, USA) were used as an external standard to determine the retention index (RI) of volatile compounds. Analyte identification was performed using a Laboratory Analytical Viewer (LAV) 2.2.1 (GAS, Dortmund, Germany) by comparing RI and the drift time of the standard in the GC-IMS Library.

    Statistical data analysis

    All samples were prepared in duplicate and tested at least six times, and the results were expressed as mean ± standard error (n = 4) and the level of statistical significance (p < 0.05) was analyzed by using Tukey's range test using SPSS 18.0 software (SPSS Inc., IL, USA). The principal component analysis (PCA) was performed using the LAV software in-built 'Dynamic PCA' plug-in to model patterns of aroma volatiles. Orthogonal partial least-square discriminant analysis (OPLS-DA) in SIMCA-P 14.1 software (Umetrics, Umeă, Sweden) was used to analyze the different volatile organic compounds in the different fermentation stages.

    Results

    Changes in total polyphenol and flavonoid contents and antioxidant activity during fermentation

    The results of the changes in the antioxidant activity of Marselan wines during the entire brewing process are listed in Table 1. It can be seen that the contents of flavonoids and polyphenols showed an increasing trend during the brewing process of Marselan wine, which range from 315.71−1,498 mg CE/L and 1,083.93−3,370.92 mg GAE/L, respectively. It was observed that the content increased rapidly in the alcoholic fermentation stage, but slowly in the subsequent fermentation stage. This indicated that the formation of flavonoid and phenolic substances in wine mainly concentrated in the alcoholic fermentation stage, which is consistent with previous reports. This is mainly because during the alcoholic fermentation of grapes, impregnation occurred to extract these compounds[12]. The antioxidant activities of Marselan wine samples at different fermentation stages were detected by FRAP and ABTS methods[11]. The results showed that the ferric reduction capacity and ABST·+ free radical scavenging capacity of the fermented Marselan wines were 2.4 and 1.5 times higher than the sample from the juice processing stage, respectively, indicating that the fermented Marselan wine had higher antioxidant activity. A large number of previous studies have suggested that there is a close correlation between antioxidant activity and the content of polyphenols and flavonoids[13−15]. Previous studies have reported that Marselan wine has the highest total phenol and anthocyanin content compared to the wine of Tannat, Cabernet Sauvignon, Merlot, Cabernet Franc, and Syrah[13]. Polyphenols and flavonoids play an important role in improving human immunity. Therefore, Marselan wines are popular because of their high phenolic and flavonoid content and high antioxidant capacity.

    Table 1.  GC-IMS integration parameters of volatile compounds in Marselan wine at different fermentation stages.
    No. Compounds Formula RI* Rt
    [sec]**    		 Dt
    [RIPrel]***    		 Identification
    approach    		 Concentration (μg/mL) (n = 4)
    Stage 1 Stage 2 Stage 3 Stage 4
    Aldehydes
    5 Furfural C5H4O2 1513.1 941.943 1.08702 RI, DT, IS 89.10 ± 4.05c 69.98 ± 3.22c 352.16 ± 39.06b 706.30 ± 58.22a
    6 Furfural dimer C5H4O2 1516.6 948.77 1.33299 RI, DT, IS 22.08 ± 0.69b 18.68 ± 2.59c 23.73 ± 2.69b 53.39 ± 9.42a
    12 (E)-2-hexenal C6H10O 1223.1 426.758 1.18076 RI, DT, IS 158.17 ± 7.26a 47.57 ± 2.51b 39.00 ± 2.06c 43.52 ± 4.63bc
    17 (E)-2-pentenal C5H8O 1129.2 333.392 1.1074 RI, DT, IS 23.00 ± 4.56a 16.42 ± 1.69c 18.82 ± 0.27b 18.81 ± 0.55b
    19 Heptanal C7H14O 1194.2 390.299 1.33002 RI, DT, IS 17.28 ± 2.25a 10.22 ± 0.59c 14.50 ± 8.84b 9.11 ± 1.06c
    22 Hexanal C6H12O 1094.6 304.324 1.25538 RI, DT, IS 803.11 ± 7.47c 1631.34 ± 19.63a 1511.11 ± 26.91b 1526.53 ± 8.12b
    23 Hexanal dimer C6H12O 1093.9 303.915 1.56442 RI, DT, IS 588.85 ± 7.96a 93.75 ± 4.67b 92.93 ± 3.13b 95.49 ± 2.50b
    29 3-Methylbutanal C5H10O 914.1 226.776 1.40351 RI, DT, IS 227.86 ± 6.39a 33.32 ± 2.59b 22.36 ± 1.18c 21.94 ± 1.73c
    33 Dimethyl sulfide C2H6S 797.1 193.431 0.95905 RI, DT, IS 120.07 ± 4.40c 87.a02 ± 3.82d 246.81 ± 5.62b 257.18 ± 3.04a
    49 2-Methylpropanal C4H8O 828.3 202.324 1.28294 RI, DT, IS 150.49 ± 7.13a 27.08 ± 1.48b 19.36 ± 1.10c 19.69 ± 0.92c
    Ketones
    45 3-Hydroxy-2-butanone C4H8O2 1293.5 515.501 1.20934 RI, DT, IS 33.20 ± 3.83c 97.93 ± 8.72b 163.20 ± 21.62a 143.51 ± 21.48a
    46 Acetone C3H6O 836.4 204.638 1.11191 RI, DT, IS 185.75 ± 8.16c 320.43 ± 12.32b 430.74 ± 3.98a 446.58 ± 10.41a
    Organic acid
    3 Acetic acid C2H4O2 1527.2 969.252 1.05013 RI, DT, IS 674.66 ± 46.30d 3602.39 ± 30.87c 4536.02 ± 138.86a 4092.30 ± 40.33b
    4 Acetic acid dimer C2H4O2 1527.2 969.252 1.15554 RI, DT, IS 45.25 ± 3.89c 312.16 ± 19.39b 625.79 ± 78.12a 538.35 ± 56.38a
    Alcohols
    8 1-Hexanol C6H14O 1365.1 653.825 1.32772 RI, DT, IS 1647.65 ± 28.94a 886.33 ± 32.96b 740.73 ± 44.25c 730.80 ± 21.58c
    9 1-Hexanol dimer C6H14O 1365.8 655.191 1.64044 RI, DT, IS 378.42 ± 20.44a 332.65 ± 25.76a 215.78 ± 21.04b 200.14 ± 28.34b
    13 3-Methyl-1-butanol C5H12O 1213.3 414.364 1.24294 RI, DT, IS 691.86 ± 9.95c 870.41 ± 22.63b 912.80 ± 23.94a 939.49 ± 12.44a
    14 3-Methyl-1-butanol dimer C5H12O 1213.3 414.364 1.49166 RI, DT, IS 439.90 ± 29.40c 8572.27 ± 60.56b 9083.14 ± 193.19a 9152.25 ± 137.80a
    15 1-Butanol C4H10O 1147.2 348.949 1.18073 RI, DT, IS 157.33 ± 9.44b 198.92 ± 3.92a 152.78 ± 10.85b 156.02 ± 9.80b
    16 1-Butanol dimer C4H10O 1146.8 348.54 1.38109 RI, DT, IS 24.14 ± 2.15c 274.75 ± 12.60a 183.02 ± 17.72b 176.80 ± 19.80b
    24 1-Propanol C3H8O 1040.9 274.803 1.11042 RI, DT, IS 173.73 ± 4.75a 55.84 ± 2.16c 80.80 ± 4.99b 83.57 ± 2.34b
    25 1-Propanol dimer C3H8O 1040.4 274.554 1.24784 RI, DT, IS 58.20 ± 1.30b 541.37 ± 11.94a 541.33 ± 15.57a 538.84 ± 9.74a
    28 Ethanol C2H6O 930.6 231.504 1.11901 RI, DT, IS 5337.84 ± 84.16c 11324.05 ± 66.18a 9910.20 ± 100.76b 9936.10 ± 101.24b
    34 Methanol CH4O 903.6 223.79 0.98374 RI, DT, IS 662.08 ± 13.87a 76.94 ± 2.15b 61.92 ± 1.96c 62.89 ± 0.81c
    37 2-Methyl-1-propanol C4H10O 1098.5 306.889 1.35839 RI, DT, IS 306.91 ± 4.09c 3478.35 ± 25.95a 3308.79 ± 61.75b 3313.85 ± 60.88b
    48 1-Pentanol C5H12O 1257.6 470.317 1.25222 RI, DT, IS 26.13 ± 2.52c 116.50 ± 3.71ab 112.37 ± 6.26b 124.17 ± 7.04a
    Esters
    1 Methyl salicylate C8H8O3 1859.6 1616.201 1.20489 RI, DT, IS 615.00 ± 66.68a 485.08 ± 31.30b 470.14 ± 23.02b 429.12 ± 33.74b
    7 Butyl hexanoate C10H20O2 1403.0 727.561 1.47354 RI, DT, IS 95.83 ± 17.04a 62.87 ± 3.62a 92.59 ± 11.88b 82.13 ± 3.61c
    10 Hexyl acetate C8H16O2 1298.6 524.366 1.40405 RI, DT, IS 44.72 ± 8.21a 33.18 ± 2.17d 41.50 ± 4.38c 40.89 ± 4.33b
    11 Propyl hexanoate C9H18O2 1280.9 499.577 1.39274 RI, DT, IS 34.65 ± 3.90d 70.43 ± 5.95a 43.97 ± 4.39b 40.12 ± 4.05c
    18 Ethyl hexanoate C8H16O2 1237.4 444.749 1.80014 RI, DT, IS 55.55 ± 5.62c 1606.16 ± 25.63a 787.24 ± 16.95b 788.91 ± 28.50b
    20 Isoamyl acetate C7H14O2 1127.8 332.164 1.30514 RI, DT, IS 164.22 ± 1.00d 243.69 ± 8.37c 343.51 ± 13.98b 365.46 ± 1.60a
    21 Isoamyl acetate dimer C7H14O2 1126.8 331.345 1.75038 RI, DT, IS 53.61 ± 4.79d 4072.20 ± 11.94a 2416.70 ± 49.84b 2360.46 ± 43.29c
    26 Isobutyl acetate C6H12O2 1020.5 263.605 1.23281 RI, DT, IS 101.65 ± 1.81a 15.52 ± 0.67c 44.87 ± 3.21b 45.96 ± 1.41b
    27 Isobutyl acetate dimer C6H12O2 1019.6 263.107 1.61607 RI, DT, IS 34.60 ± 1.05d 540.84 ± 5.64a 265.54 ± 8.31c 287.06 ± 3.66b
    30 Ethyl acetate dimer C4H8O2 885.2 218.564 1.33587 RI, DT, IS 1020.75 ± 6.86d 5432.71 ± 6.55a 5052.99 ± 9.65b 5084.47 ± 7.30c
    31 Ethyl acetate C4H8O2 878.3 216.574 1.09754 RI, DT, IS 215.65 ± 3.58a 38.29 ± 2.37c 71.59 ± 2.99b 69.32 ± 2.85b
    32 Ethyl formate C3H6O2 838.1 205.127 1.19738 RI, DT, IS 175.48 ± 3.79d 1603.20 ± 13.72a 1472.10 ± 5.95c 1509.08 ± 13.26b
    35 Ethyl octanoate C10H20O2 1467.0 852.127 1.47312 RI, DT, IS 198.86 ± 36.71b 1853.06 ± 17.60a 1555.51 ± 24.21a 1478.05 ± 33.63a
    36 Ethyl octanoate dimer C10H20O2 1467.0 852.127 2.03169 RI, DT, IS 135.50 ± 13.02d 503.63 ± 15.86a 342.89 ± 11.62b 297.28 ± 14.40c
    38 Ethyl butanoate C6H12O2 1042.1 275.479 1.5664 RI, DT, IS 21.29 ± 2.68c 1384.67 ± 8.97a 1236.52 ± 20.21b 1228.09 ± 5.09b
    39 Ethyl 3-methylbutanoate C7H14O2 1066.3 288.754 1.26081 RI, DT, IS 9.70 ± 1.85d 200.29 ± 4.21a 146.87 ± 8.70b 127.13 ± 12.54c
    40 Propyl acetate C5H10O2 984.7 246.908 1.48651 RI, DT, IS 4.57 ± 1.07c 128.63 ± 4.28a 87.75 ± 3.26b 88.49 ± 1.99b
    41 Ethyl propanoate C5H10O2 962.1 240.47 1.46051 RI, DT, IS 10.11 ± 0.34d 107.08 ± 3.50a 149.60 ± 5.39c 167.15 ± 12.90b
    42 Ethyl isobutyrate C6H12O2 971.7 243.229 1.56687 RI, DT, IS 18.29 ± 2.61d 55.22 ± 1.07c 98.81 ± 4.67b 104.71 ± 4.73a
    43 Ethyl lactate C5H10O3 1352.2 628.782 1.14736 RI, DT, IS 31.81 ± 2.91c 158.03 ± 2.80b 548.14 ± 74.21a 527.01 ± 39.06a
    44 Ethyl lactate dimer C5H10O3 1351.9 628.056 1.53618 RI, DT, IS 44.55 ± 2.03c 47.56 ± 4.02c 412.23 ± 50.96a 185.87 ± 31.25b
    47 Ethyl heptanoate C9H18O2 1339.7 604.482 1.40822 RI, DT, IS 39.55 ± 6.37a 38.52 ± 2.47a 28.44 ± 1.52c 30.77 ± 2.79b
    Unknown
    1 RI, DT, IS 15.53 ± 0.18 35.69 ± 0.80 12.70 ± 0.80 10.57 ± 0.86
    2 RI, DT, IS 36.71 ± 1.51 120.41 ± 3.44 198.12 ± 6.01 201.19 ± 3.70
    3 RI, DT, IS 44.35 ± 0.88 514.12 ± 4.28 224.78 ± 6.56 228.32 ± 4.62
    4 RI, DT, IS 857.64 ± 8.63 33.22 ± 1.99 35.05 ± 5.99 35.17 ± 3.97
    * Represents the retention index calculated using n-ketones C4−C9 as external standard on MAX-WAX column. ** Represents the retention time in the capillary GC column. *** Represents the migration time in the drift tube.
     | Show Table
    DownLoad: CSV

    Analysis of the change in volatile organic compounds (VOCs) during fermentation by GS-IMS

    This study adopted the GC-IMS method to test the volatile organic compounds (VOCs) in the samples from the different fermentation stages of Marselan wine. Figure 1 shows the gas phase ion migration spectrum obtained, in which the ordinate represents the retention time of the gas chromatographic peaks and the abscissa represents the ion migration time (normalized)[16]. The entire spectrum represents the aroma fingerprints of Marselan wine at different fermentation stages, with each signal point on the right of the relative reactant ion peak (RIP) representing a volatile organic compound detected from the sample[17]. Here, the sample in stage 1 (juice processing) was used as a reference and the characteristic peaks in the spectrum of samples in other fermentation stages were compared and analyzed after deducting the reference. The colors of the same component with the same concentration cancel each other to form a white background. In the topographic map of other fermentation stages, darker indicates higher concentration compared to the white background. In the 2D spectra of different fermentation stages, the position and number of peaks indicated that peak intensities are basically the same, and there is no obvious difference. However, it is known that fermentation is an extremely complex chemical process, and the content and types of volatile organic compounds change with the extension of fermentation time, so other detection and characterization methods are needed to make the distinction.

    Figure 1.  2D-topographic plots of volatile organic compounds in Marselan wine at different fermentation stages.
    DownLoad: Full-Size Img PowerPoint

    Fingerprint analysis of VOCs at different fermentation stages of Marselan wine

    To visually display the dynamic changes of various substances in the fermentation process of Marselan wine, peaks with obvious differences were extracted to form the characteristic fingerprints for comparison (Fig. 2). Each row represents all signal peaks selected from samples at the same stage, and each column means the signal peaks of the same volatile compound in samples from different fermentation stages. Figure 2 shows the volatile organic compounds (VOCs) information for each sample and the differences between samples, where the numbers represent the undetermined substances in the migration spectrum library. The changes of volatile substances in the process of Marselan winemaking is observed by the fingerprint. As shown in Fig. 2 and Table 2, a total of 40 volatile chemical components were detected by qualitative analysis according to their retention time and ion migration time in the HS-GC-IMS spectrum, including 17 esters, eight alcohols, eight aldehydes, two ketones, one organic acid, and four unanalyzed flavor substances. The 12 volatile organic compounds presented dimer due to ionization of the protonated neutral components before entering the drift tube[18]. As can be seen from Table 2, the VOCs in the winemaking process of Marselan wine are mainly composed of esters, alcohols, and aldehydes, which play an important role in the construction of aroma characteristics.

    Figure 2.  Fingerprints of volatile organic compounds in Marselan wine at different fermentation stages.
    DownLoad: Full-Size Img PowerPoint
    Table 2.  Antioxidant activity, total polyphenols, and flavonoids content of Marselan wine at different fermentation stages.
    Winemaking stage TFC (mg CE/L) TPC (mg GAE/L) FRAP (mM FeSO4/mL) ABTs (mM Trolox/L)
    Stage 1 315.71 ± 0.00d 1,083.93 ± 7.79d 34.82c 38.92 ± 2.12c
    Stage 2 1,490.00 ± 7.51c 3,225.51 ± 53.27c 77.32b 52.17 ± 0.95b
    Stage 3 1,510.00 ± 8.88a 3,307.143 ± 41.76b 77.56b 53.04 ± 0.76b
    Stage 4 1,498.57 ± 6.34b 3,370.92 ± 38.29a 85.07a 57.46 ± 2.55a
    Means in the same column with different letters are significantly different (p < 0.05).
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    Esters are produced by the reaction of acids and alcohols in wine, mainly due to the activity of yeast during fermentation[19], and are the main components of fruit juices and wines that produce fruit flavors[20,21]. In this study, it was found that they were the largest detected volatile compound group in Marselan wine samples, which is consistent with previous reports[22]. It can be observed from Table 2 that the contents of most esters increased gradually with the extension of fermentation time, and they mainly began to accumulate in large quantities during the stage of alcohol fermentation. The contents of ethyl hexanoate (fruity), isoamyl acetate (banana, pear), ethyl octanoate (fruity, pineapple, apple, brandy), ethyl acetate (fruity), ethyl formate (spicy, pineapple), and ethyl butanoate (sweet, pineapple, banana, apple) significantly increased at the stage of alcoholic fermentation and maintained a high level in the subsequent fermentation stage (accounting for 86% of the total detected esters). These esters can endow a typical fruity aroma of Marselan wine, and played a positive role in the aroma profiles of Marselan wine. Among them, the content of ethyl acetate is the highest, which is 5,153.79 μg/mL in the final fermentation stage, accounting for 33.6% of the total ester. However, the content of ethyl acetate was relatively high before fermentation, which may be from the metabolic activity of autochthonous microorganisms present in the raw materials. Isobutyl acetate, ethyl 3-methyl butanoate, propyl acetate, ethyl propanoate, ethyl isobutyrate, and ethyl lactate were identified and quantified in all fermentation samples. The total contents of these esters in stage 1 and 4 were 255.28 and 1,533.38 μg/mL, respectively, indicating that they may also have a potential effect on the aroma quality of Marselan wine. The results indicate that esters are an important factor in the formation of flavor during the brewing process of Marselan wine.

    Alcohols were the second important aromatic compound in Marselan wine, which were mainly synthesized by glucose and amino acid decomposition during alcoholic fermentation[23,24]. According to Table 2, eight alcohols including methanol, ethanol, propanol, butanol, hexanol, amyl alcohol, 3-methyl-1-butanol, and 2-methyl-1-propanol were detected in the four brewing stages of Marselan wine. The contents of ethanol (slightly sweet), 3-methyl-1-butanol (apple, brandy, spicy), and 2-methyl-1-propanol (whiskey) increased gradually during the fermentation process. The sum of these alcohols account for 91%−92% of the total alcohol content, which is the highest content of three alcohols in Marselan wine, and may be contributing to the aromatic and clean-tasting wines. On the contrary, the contents of 1-hexanol and methanol decreased gradually in the process of fermentation. Notably, the content of these rapidly decreased at the stage of alcoholic fermentation, from 2,026.07 to 1,218.98 μg/mL and 662.08 to 76.94 μg/mL, respectively, which may be ascribed to volatiles changed from alcohols to esters throughout fermentation. The reduction of the concentration of some alcohols also alleviates the strong odor during wine fermentation, which plays an important role in the improvement of aroma characteristics.

    Acids are mainly produced by yeast and lactic acid bacteria metabolism at the fermentation stage and are considered to be an important part of the aroma of wine[22]. Only one type of acid (acetic acid) was detected in this experiment, which was less than previously reported, which may be related to different brewing processes. Acetic acid content is an important factor in the balance of aroma and taste of wine. Low contents of volatile acids can provide a mild acidic smell in wine, which is widely considered to be ideal for producing high-quality wines. However, levels above 700 μg/mL can produce a pungent odor and weaken the wine's distinctive flavor[25]. The content of acetic acid increased first and then decreased during the whole fermentation process. The content of acetic acid increased rapidly in the second stage, from 719.91 to 3,914.55 μg/mL reached a peak in the third stage (5,161.81 μg/mL), and decreased to 4,630.65 μg/mL in the last stage of fermentation. Excessive acetic acid in Marselan wine may have a negative impact on its aroma quality.

    It was also found that the composition and content of aldehydes produced mainly through the catabolism of amino acids or decarboxylation of ketoacid were constantly changing during the fermentation of Marselan wines. Eight aldehydes, including furfural, hexanal, heptanal, 2-methylpropanal, 3-methylbutanal, dimethyl sulfide, (E)-2-hexenal, and (E)-2-pentenal were identified in all stage samples. Among them, furfural (caramel bread flavor) and hexanal (grass flavor) are the main aldehydes in Marselan wine, and the content increases slightly with the winemaking process. While other aldehydes such as (E)-2-hexenal (green and fruity), 3-methylbutanol (fresh and malt), and 2-methylpropanal (fresh and malt) were decomposed during brewing, reducing the total content from 536.52 to 85.15 μg/mL, which might potently affect the final flavor of the wine. Only two ketones, acetone, and 3-hydroxy-2-butanone, were detected in the wine samples, and their contents had no significant difference in the fermentation process, which might not affect the flavor of the wine.

    Multivariate statistical analysis

    To more intuitively analyze the differences of volatile organic compounds in different brewing stages of Marselan wine samples, principal component analysis was performed[26−28]. As presented in Fig. 3, the points corresponding to one sample group were clustered closely on the score plot, while samples at different fermentation stages were well separated in the plot. PC1 (79%) and PC2 (18%) together explain 97% of the total variance between Marselan wine samples, indicating significant changes in volatile compounds during the brewing process. As can be seen from the results in Fig. 3, samples of stages 1, 2, and 3 can be distinguished directly by PCA, suggesting that there are significant differences in aroma components in these three fermentation stages. Nevertheless, the separation of stage 3 and stage 4 samples is not very obvious and both presented in the same quadrant, which means that their volatile characteristics were highly similar, indicating that the volatile components of Marselan wine are formed in stage 3 during fermentation (Fig. S1). The above results prove that the unique aroma fingerprints of the samples from the distinct brewing stages of Marselan wine were successfully constructed using the HS-GC-IMS method.

    Figure 3.  PCA based on the signal intensity obtained with different fermentation stages of Marselan wine.
    DownLoad: Full-Size Img PowerPoint

    Based on the results of the PCA, OPLS-DA was used to eliminate the influence of uncontrollable variables on the data through permutation test, and to quantify the differences between samples caused by characteristic flavors[28]. Figure 4 revealed that the point of flavor substances were colored according to their density and the samples obtained at different fermentation stages of wine have obvious regional characteristics and good spatial distribution. In addition, the reliability of the OPLS-DA model was verified by the permutation method of 'Y-scrambling'' validation. In this method, the values of the Y variable were randomly arranged 200 times to re-establish and analyze the OPLS-DA model. In general, the values of R2 (y) and Q2 were analyzed to assess the predictability and applicability of the model. The results of the reconstructed model illustrate that the slopes of R2 and Q2 regression lines were both greater than 0, and the intercept of the Q2 regression line was −0.535 which is less than 0 (Fig. 5). These results indicate that the OPLS-DA model is reliable and there is no fitting phenomenon, and this model can be used to distinguish the four brewing stages of Marselan wine.

    Figure 4.  Scores plot of OPLS-DA model of volatile components in Marselan wine at different fermentation stages.
    DownLoad: Full-Size Img PowerPoint
    Figure 5.  Permutation test of OPLS-DA model of volatile components in Marselan wine at different fermentation stages (n = 200).
    DownLoad: Full-Size Img PowerPoint

    VIP is the weight value of OPLS-DA model variables, which was used to measure the influence intensity and explanatory ability of accumulation difference of each component on classification and discrimination of each group of samples. In previous studies, VIP > 1 is usually used as a screening criterion for differential volatile substances[28−30]. In this study, a total of 22 volatile substances had VIP values above 1, indicating that these volatiles could function as indicators of Marselan wine maturity during fermentation (see Fig. 6). These volatile compounds included furfural, ethyl lactate, heptanal, dimethyl sulfide, 1-propanol, ethyl isobutyrate, propyl acetate, isobutyl acetate, ethanol, ethyl hexanoate, acetic acid, methanol, ethyl formate, ethyl 3-methylbutanoate, ethyl acetate, hexanal, isoamyl acetate, 2-methylpropanal, 2-methyl-1-propanol, and three unknown compounds.

    Figure 6.  VIP plot of OPLS-DA model of volatile components in Marselan wine at different fermentation stages.
    DownLoad: Full-Size Img PowerPoint

    Conclusions

    This study focuses on the change of volatile flavor compounds and antioxidant activity in Marselan wine during different brewing stages. A total of 40 volatile aroma compounds were identified and collected at different stages of Marselan winemaking. The contents of volatile aroma substances varied greatly at different stages, among which alcohols and esters were the main odors in the fermentation stage. The proportion of furfural was small, but it has a big influence on the wine flavor, which can be used as one of the standards to measure wine flavor. Flavonoids and phenols were not only factors of flavor formation, but also important factors to improve the antioxidant capacity of Marselan wine. In this study, the aroma of Marselan wines in different fermentation stages was analyzed, and its unique aroma fingerprint was established, which can provide accurate and scientific judgment for the control of the fermentation process endpoint, and has certain guiding significance for improving the quality of Marselan wines (Table S1). In addition, this work will provide a new approach for the production management of Ningxia's special wine as well as the development of the native Chinese wine industry.

  • The authors confirm contribution to the paper as follows: study conception and design: Gong X, Fang L; data collection: Fang L, Li Y; analysis and interpretation of results: Qi N, Chen T; draft manuscript preparation: Fang L. All authors reviewed the results and approved the final version of the manuscript.

  • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    • Acknowledgments

    This work were supported by the project of Hainan Province Science and Technology Special Fund (ZDYF2023XDNY031) and the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences in China (Grant No. 1630122022003).

  • The authors declare that they have no conflict of interest.

  • Supplemental Fig. S1 Typical UHPLC-MS-UV chromatograms of six standard compounds. Standard compounds: calycosin (1), calycosin-7-O-β -D-glucose (2), formononetin (4), ononin (5), astragaloside IV (7),and  astragaloside II (8).
    Supplemental Fig. S2 ESIMS spectra of two minor compounds calycosin 7-O-β -D-(6''-acetyl)-glucoside (P1) and 6''-acetylononin (P2) in UGP1 and TM1. A: ESI (+) and B: ESI (-) of P1; C: ESI (+) and D: ESI (-) of P2.
    Supplemental Fig. S3 Peak areas (UHPLC/DAD-MS UV254 nm) of isoflavonoids (1-6) extracted by solvent systems of 0%, 50%, 80%, and 100% methanol in water. Marker compounds: calycosin (1), calycosin-7-O-β-D-glucose (2), calycosin-7-O-β-D-glucose-6''-O-malonate (3), formononetin (4), ononin (5) and formononetin-7-O-β-D-glucoside-6''-O-malonate (6).
    Supplemental Fig. S4 Contents (mg/g) of marker compounds (1-6 and 8-10) in UGP2 and TM2 from the same source of Astragali Radix (n=10). Marker compounds: calycosin (1), calycosin-7-O-β-D-glucose (2), calycosin-7-O-β-D-glucose-6''-O-malonate (3), formononetin (4), ononin (5), formononetin-7-O-β-D-glucoside-6''-O-malonate (6), astragaloside II (8), astragaloside I (9), and acetylastragaloside Ⅰ (10).
    Supplemental Fig. S5 Contents (mg/g dry material) of marker compounds (1-6, 8-10) in UGP3 and TM3 materials (n=10). Marker compounds: calycosin (1), calycosin-7-O-β-D-glucose (2), calycosin-7-O-β-D-glucose-6''-O-malonate (3), formononetin (4), ononin (5), formononetin-7-O-β-D-glucoside-6''-O-malonate (6), astragaloside II (8), astragaloside I (9), and acetylastragaloside Ⅰ (10).
    Supplemental Table S1 Peak areas (UHPLC/DAD-MS UV 254 nm) of isoflavonoids (1-6)  a extracted by different solvent systems of methanol/water (0%, 50%, 80%, and 100% of methanol in water).
    Supplemental Table S2 Regression data for the marker compounds in UHPLC/DAD-MS.
    Supplemental Table S3 Intra-day and inter-day precisions by analyzing UGP2.
    Supplemental Table S4 Recovery test for determination of six standard compounds in Radix Astragali (by using sample of UGP2, n = 3).
    Supplemental Table S5 Contents (mg/g dry material) of marker compounds (1-6 and 8-10) a in UGP2 and TM2 from the same source of Radix Astragali (n=10).
    Supplemental Table S6 Contents (mg/g dried material) of marker compounds (1-6 and 8-10) a in UGP3 and TM3 materials (n=10).
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  • Cite this article

    Cao L, Wang M, Zhao J, Peng LH, Cheng JL, et al. 2022. Comparative analysis of chemical profiles of Radix Astragali between ultrafine granular powder and sliced traditional material. Medicinal Plant Biology 1:4 doi: 10.48130/MPB-2022-0004
    Cao L, Wang M, Zhao J, Peng LH, Cheng JL, et al. 2022. Comparative analysis of chemical profiles of Radix Astragali between ultrafine granular powder and sliced traditional material. Medicinal Plant Biology 1:4 doi: 10.48130/MPB-2022-0004
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Comparative analysis of chemical profiles of Radix Astragali between ultrafine granular powder and sliced traditional material

  • 1.

    National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, USA

  • 2.

    Natural Products Utilization Research Unit, Agricultural Research Service, United States Department of Agriculture, University, Mississippi 38677, USA

  • 3.

    Key Laboratory of Technologies and Applications of Ultrafine Granular Powder of Herbal Medicine, State Administration of Traditional Chinese Medicine, Zhongshan Zhongzhi Pharmaceutical Group Co., Ltd. Zhongshan 528437, P. R. China

  • 4.

    Hunan Provincial Key Laboratory of Dong Medicine, Hunan University of Medicine, Huaihua 41800, P. R. China

  • Received: 05 May 2022
  • Accepted: 05 September 2022
  • Published online: 30 September 2022
Medicinal Plant Biology  1,  Article number: 4  (2022)  |  Cite this article

Abstract: Radix Astragali, one of the most popular herbs in traditional Chinese medicine (TCM), is used to strengthen the immune system, protect liver function, fight bacteria and viruses, and treat diabetes, heart failure and seasonal allergies. In recent years, a new form of Radix Astragali material processed by cell wall disrupting technology, namely ultrafine granular powder (UGP) has been introduced into the market. In order to determine chemical consistency and homogeneity of the UGP material prepared from sliced traditional materials (TM) of Radix Astragali, multiple batches of the UGP and TM samples derived from Astragalus membranaceus var. mongholicus were analyzed by UHPLC/DAD-MS using isoflavones and triterpenoid glycosides as marker compounds. The results demonstrated that the chemical profiles of UGP was identical or similar to that of TM, but UGP was highly homogeneous in terms of marker compound contents as assessed, e.g., by the relative standard deviation values of the nine marker compounds in the range of 8.55%−43.80% for TM2 compared against 1.70%−8.38% for UGP2. Macromolecular component preparation and 1H NMR analyses indicated that TM4 and its corresponding UGP4 produced similar polysaccharides, but the later had approximately two-fold dissolution rate of the polysaccharides when compared to the former (yield 7.22 ± 0.35% vs 3.39 ± 0.20%). This study confirms that UGP of Radix Astragali is chemically consistent and homogenous, supporting its use as a promising material in TCM prescriptions.

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    INTRODUCTION

    • Radix Astragali prepared from dried roots of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. is one of the most popular herbs in traditional Chinese medicine (TCM). It is used to strengthen the immune system, protect liver function, fight bacteria and viruses, and treat diabetes, heart failure and seasonal allergies[1, 2]. Isoflavones and triterpenoid glycosides are major small-molecule active compounds present in Radix Astragali[2] (Fig. 1). For example, ononin (5) and astragaloside IV (7) exhibited anti-gastric ulcer effects[3]; and formononetin (4) inhibited the proliferation and metastasis of ovarian cancer cells[4]. Polysaccharides are another major chemical component of Radix Astragali[5], which have demonstrated antioxidant activities[6] and therapeutic potential for glomerulonephritis[7] and cognitive dysfunction[8].

      Figure 1. 

      Structures of 10 marker compounds of Radix Astragali.

      It is well known that chemical consistency can be an issue within individual medicinal plants used in TCM due to genetic and environmental variations[9−11], which has a significant impact on the quality of the TCM material from batch to batch. In recent years, a new form of TCM material, namely ultrafine granular powder (UGP), has been introduced into the market with the application of cell wall-disrupting micronization technology. Technically, crude TCM slices (or pieces) are crushed into ultrafine powder (particle size distribution, D90 < 45 μm) with airflow crushing technology, and then are prepared as granules without excipients[12]. UGP material generally maintains the same chemical profiles as the sliced traditional material (TM), as demonstrated in the UGP material of Salvia miltiorrhiza[12]. Additional studies have shown that the UGP materials of S. miltiorrhiza and Panax quinquefolius possess improved oral bioavailability and pharmacokinetic profiles for selected marker compounds compared to the corresponding TM materials[13, 14].

      With the growing use of the UGP material of Radix Astragali in China, an understanding of its chemical profile is critical in assessing its clinical utility. In the present study, the aforementioned two chemotypes of small-molecule compounds, isoflavones and triterpenoid glycosides, were selected as marker compounds to compare the chemical consistency and homogeneity between UGP and TM of Radix Astragali. In addition, polysaccharides, important biologically active constituents of Radix Astragali, were prepared from the two materials for comparison of their chemical profiles and dissolution rates.

    RESULTS

      Chemical consistency between UGP and TM materials of Radix Astragali

    • The bioactive compounds in TCM are the material basis for clinical application. In order to confirm that the UGP material of Radix Astragali retains the same or similar chemical profiles as the corresponding TM material that is commonly prescribed by TCM practitioners, a comparative study between the two materials was conducted using UHPLC/DAD-UV (see the method in the experimental section). The six isoflavones including calycosin (1), calycosin-7-O-β-D-glucose (2), calycosin-7-O-β-D-glucose-6''-O- malonate (3), formononetin (4), ononin (5), and formononetin-7-O-β-D-glucoside-6''- O-malonate (6) and four triterpenoid glycosides including astragaloside IV (7), astragaloside II (8), astragaloside I (9), and acetylastragaloside I (10) (Fig. 1) previously identified in Radix Astragali, were used as marker compounds to facilitate chromatographic comparison (refer to Supplemental Fig. S1 for typical UHPLC-MS-UV chromatograms of the six commercially available standards 1, 2, 4, 5, 7, and 8). The retention time and ESI-MS data (both positive and negative) of all the 10 compounds are summarized in Table 1.

      Table 1.  Retention time (tR) and characteristic MS data of 10 marker compounds.

      AnalytetR (min)MS (m/z)MW
      DAD 254 nmMSESI (+)ESI (–)
      17.187285 [M + H]+283 [M − H]−284
      25.219447 [M + H]+491 [M + HCOO]−446
      35.810533 [M + H]+475 [M − Malonyl + HCOO]−532
      48.791269 [M + H]+267 [M − H]−268
      56.411431 [M + H]+475 [M + HCOO]−430
      66.928517 [M + H]+267 [M – Malonyl – Glc -H]−516
      77.939807 [M + Na]+829 [M + HCOO]−784
      88.571849 [M + Na]+871 [M + HCOO]−826
      99.737891 [M + Na]+913 [M + HCOO]−868
      1010.166933 [M + Na]+955 [M + HCOO]−910

      For a direct qualitative analysis by UHPLC/DAD-UV, extraction of TM1 and its corresponding UGP1 was conducted in MeOH under sonication at room temperature for 30 min. It is evident that the UV254 nm chromatograms (Fig. 2a) and the negative and positive ESI-MS chromatograms (Fig. 2b & c) of UGP1 and TM1 are basically identical, indicating the same material basis for the two. A different extraction method involving sonication of the samples in 80% MeOH at room temperature for 30 min was used for UGP2 and TM2, a different batch of Radix Astragali prepared from the same source of the plant material. The results indicated that UGP2 and TM2 produced almost the same chromatographic fingerprints, except for two minor peaks marked as P1 and P2 in the UV- and MS detected chromatograms of UGP2 (Fig. 2d− f). Based on the MS data and UV absorption characteristics (Supplemental Fig. S2), it was speculated that P1 was calycosin 7-O-β-D-(6''-acetyl)-glucoside, while P2 was 6''-acetylononin. Actually, these two compounds were also detected to be present in trace amounts in the extracted ion chromatograms of TM2 (by extracting quasi-molecular ions for both positive and negative ESI-MS modes). They were likely enriched in UGP2 due to different compound dissolution rates in extraction or intermolecular acetyl transfer during the micronization process. Thus, it can be concluded that the UGP and TM materials of Radix Astragali derived from the same source have the same material basis, with slight differences for individual compounds between the two materials.

      Figure 2. 

      Comparison of UHPLC/DAD-MS UV254 nm, ESI negative, ESI positive chromatograms between ultrafine granular powder (UGP) and sliced traditional material (TM) of Radix Astragali. UV 254 nm chromatograms (a) UGP1 (top) and TM1 (down), (d) UGP2 (top) and TM (down); ESI negative chromatograms (b) UGP1 (top) and TM1 (down), (e) UGP2 (top) and TM2 (down); ESI positive chromatograms (c) UGP1 (top) and TM1 (down), (f) UGP2 (top) and TM2 (down).

      Chemical homogeneity between UGP and TM materials of Radix Astragali

    • It is readily assumed that the UGP material would be a chemically homogenized product because it is produced from large amounts of a particular TCM material, whether or not it is from the same or multiple batches of raw plant material. To provide convincing evidence for the homogeneity of the UGP material from Radix Astragali, a quantitative UHPLC/DAD-MS analytical approach was employed to assess the UGP materials in comparison with the TM materials for the contents of the selected marker compounds. The UHPLC/DAD-UV conditions were based on those used for the chemical consistency study.

      To achieve good quantitative analysis, the sample extraction method was optimized by using different solvents (0%, 50%, 80%, and 100% methanol in water), and 80% methanol was determined to possess the best extraction efficiency based on relative peak areas of the six isoflavones (Supplemental Fig. S3 and Supplemental Table S1). Therefore, 80% methanol was used to extract the TM and UGP samples. For quantification, four standard isoflavones (1, 2, 4, and 5) and two standard triterpenoid glycosides (7 and 8) were used to construct calibration curves. The standard curves of the isoflavones generated from the UV254 nm chromatograms possessed good correlation coefficients and a wide concentration range, while the triterpenoid glycosides produced good linearity from the negative ESI-MS chromatograms. Quantification of the two commercially unavailable isoflavones 3 and 6 was approximated using the calibration curves of the structurally close compounds 2 and 5, respectively, assuming they produced similar UV responses. Similarly, quantification of the commercially unavailable triterpenoid glycosides 9 and 10 was based on the calibration curve of compound 8. The equations of calibration curves of the ten marker compounds and the limit of quantification (LOQ) of the standard compounds are summarized in Supplemental Table S2. The analytical method was further validated by the accuracy and precision experiments (see experimental section and Supplemental Tables S3 and S4).

      Ten accessions of UGP2 and TM2 from the same source were assessed by paired sample t-test for the content of marker compounds 1−6 and 8−10 (Supplemental Fig. S4 and Supplemental Table S5) using statistic software SPSS (Statistical Package for the Social Sciences) (Table 2). Astragaloside IV (7) was shown to have a very low concentration in these samples (under the LOQ), and its quantification was not reliable, thereby not included. Relative standard deviation (RSD) values for the marker compounds were used to judge the homogeneity of the 10 accessions. The RSD values of the nine marker compounds in UGP2 were in the range of 1.70%−8.38%, in comparison with 8.55%−43.80% for these compounds in TM2. For example, compound 1 in UGP2 had a content of 0.059−0.065 mg/g with an RSD value of 3.05%, compared to 0.017−0.071 mg/g with an RSD value of 43.80% in TM2. Comparison of the content of three representative compounds 1, 4, and 8 is illustrated in Fig. 3a. These data indicated that UGP2 possessed greater homogeneity than TM2 in terms of the marker compound content in different accessions. It was also observed that the content of compounds 1 and 4 demonstrated significant differences (p < 0.01) in UGP2 and TM2, with UGP2 having greater concentrations, while the concentrations of their corresponding isoflavone glycosides (compounds 2, 3, 5, and 6) (p < 0.05) and triterpenoid glycosides (8−10) were slightly greater in TM2. This suggests that, to some degree, chemical conversion of glycosides to corresponding aglycones or dissolution rates for some compounds may have occurred in the UGP preparation process.

      Table 2.  Content of marker compounds 1−6 and 8−10 (mg/g dry material) in UGP2 and TM2 from the same source of Radix Astragali (n = 10).

      CompoundDetectorUGP2 (mean ± SD mg/g)RSD (%)TM2 (mean ± SD mg/g)RSD (%)
      1UV 2540.062 ± 0.002**3.050.038 ± 0.016**43.80
      2UV 2540.190 ± 0.010*5.090.239 ± 0.040*17.94
      3UV 2540.768 ± 0.015*1.940.909 ± 0.185*20.31
      4UV 2540.078 ± 0.002**1.910.064 ± 0.006**8.55
      5UV 2540.123 ± 0.004*3.130.148 ± 0.020*13.65
      6UV 2540.388 ± 0.0071.700.403 ± 0.08218.96
      Total isoflavonoids1.609 ± 0.0291.831.827 ± 0.32918.00
      8ESI-0.091 ± 0.0077.680.096 ± 0.01718.30
      9ESI-0.283 ± 0.0217.580.292 ± 0.0289.58
      10ESI-0.113 ± 0.0098.380.115 ± 0.01512.82
      Total astragalosides0.487 ± 0.0193.820.503 ± 0.0469.11

      Figure 3. 

      Comparison of content (mg/g dry material) for marker compounds 1, 4, and 8 between ultrafine granular powder (UGP) and sliced traditional material (TM) of Radix Astragali. (a) UGP2 and TM2 accessions (n = 10); (b) UGP3 and TM3 accessions (n = 10).

      To expand the comparative scope for chemical homogeneity of Radix Astragali, a sliced traditional material (TM3) and a UGP material (UGP3) were purchased from a local TCM drug store. It is unknown whether TM3 and UGP3 were derived from the same or different batches of raw plant materials. Analysis of UGP3 and TM3 (each with 10 accessions, Supplemental Fig. S5 and Supplemental Table S6) indicated that the two materials had significant differences in terms of the contents of marker compounds 1−6 and 8−10 (Table 3). The RSD values for the nine marker compounds were in the range of 1.25%−10.90% in UGP3 versus 13.63%−37.74% in TM3 (Table 3), indicating UGP3 was much more chemically homogenized than TM3. Significant variances for the concentrations of compounds 1, 4, and 8 in the 10 accessions of TM3 are also evident in Fig. 3b.

      Table 3.  Content of marker compounds 1−6 and 8−10 (mg/g dry material) in UGP3 and TM3 from unknown batch sources of Radix Astragali (n = 10).

      CompoundDetectorUGP3 (mean ± SD mg/g)RSD (%)TM3 (mean ± SD mg/g)RSD (%)
      1UV 2540.162 ± 0.0042.210.179 ± 0.06737.74
      2UV 2540.212 ± 0.0083.820.188 ± 0.04423.23
      3UV 2540.656 ± 0.010**1.470.392 ± 0.126**32.14
      4UV 2540.139 ± 0.0021.400.136 ± 0.04029.31
      5UV 2540.124 ± 0.003**2.360.088 ± 0.012**13.63
      6UV 2540.312 ± 0.004**1.250.151 ± 0.029**18.88
      Total isoflavonoids1.603 ± 0.0271.671.133 ± 0.24121.29
      8ESI-0.196 ± 0.012**5.890.082 ± 0.024**29.20
      9ESI-0.498 ± 0.026**5.180.228 ± 0.047**20.71
      10ESI-0.182 ± 0.020**10.900.072 ± 0.023**31.66
      Total astragalosides0.876 ± 0.0262.960.382 ± 0.08822.90

      Chemical profiles and content of polysaccharides in UGP and TM of Radix Astragali

    • The polysaccharides present in Radix Astragali are considered to play an important role in the efficacy of this TCM. The preparation of Radix Astragali polysaccharides (RAPS) was achieved by extraction of TM4 and its corresponding UGP4 (prepared from TM4) with hot water, and then treated with the Sevag reagent to remove proteins, followed by ultrafiltration to remove small molecules and oligosaccharides. The resultant RAPS were subjected to 1H NMR analyses, which demonstrated that RAPS from both TM4 and UGP4 were very similar (Fig. 4), indicating chemical consistency of the two materials. It was also confirmed that these 1H NMR profiles were similar to those of APS-I and APS-II, two anti-tumor polysaccharides purified from Astragalus mongholicus[15]. The yields of RAPS resulted from TM4 and UGP4 were 3.39 ± 0.20% and 7.22 ± 0.35%, respectively. This indicated that the UGP materials had excellent dissolution rates for RAPS, and appear to be more efficient than TM in terms of releasing RAPS during the course of usage.

      Figure 4. 

      1H NMR spectra (500 MHz) of RAPS from (a) TM4 and (b) UGP4, recorded at a concentration of 10 mg/mL in D2O.

    DISCUSSION

    • Small-molecule compounds in A. membranaceus var. mongholicus and A. membranaceus were reported to have significant organ-, age,- and variety-specificity based on LC-MS and NMR analyses[9−11]. In cultivated Radix Astragali, isoflavonoids and astragalosides vary in concentrations in different organs of the plants harvested from different regions[16]. The Radix Astragali polysaccharides also demonstrate notable differences of carbohydrate composition in the plant materials from different geographic origins[17]. This implies the traditional slices of Radix Astragali differ from batch-to-batch and even within the same batch, as demonstrated in this study (refer to Fig. 3) in terms of the content of bioactive compounds, which significantly affects the material quality and potential clinical efficacy. Production of the Radix Astragali UGP material generally involves a large amount (kg to ton scale) of sliced material. Even though the sliced material is from different batches of the plant, the UGP production process will physically improve chemical homogeneity, with minor chemical conversions in the UGP production process which would not negatively impact the overall metabolite profile.

      It should be pointed out that UGP can be used like tea leaves in hot water for direct administration while TM is generally decocted in a mixture form in routine TCM practice. For any UGP or TM material, the content of bioactive compounds in the extracts depend on the extraction process. For example, decoction of a specific TCM prescription involves multiple TCM materials in boiling water, which is a complex extraction process leading to specific content and ratios of bioactive compounds required for pharmacological effects. The current study was performed under the same extraction conditions to compare their chemical consistency, homogeneity, and dissolution. It appears that our results are consistent with previous studies demonstrating a higher dissolution rate of ginsenosides from UGP of Panacis Quinquefolii Radix[18] and an improved bioavailability of flavonoid compounds present in UGP of Radix Astragali compared to respective TM material[19].

    CONCLUSIONS

    • This study confirmed that the UGP material of Radix Astragali was not only identical or similar to the traditional slices in terms of small-molecule metabolite profiles, but also chemically homogeneous over traditional slices, laying a scientific foundation to support the use of UGP in TCM. In addition, the UGP material demonstrated chemical profiles of the bioactive polysaccharides similar to those of TM, however, it possessed a greater dissolution rate compared to the traditional slices. The UGP material is an advancement in overcoming the batch-to-batch inconsistency of traditional slices, and has great potential to be used in TCM prescriptions.

    MATERIALS AND METHODS

      Material and chemicals

      Plant material

    • Three batches of sliced traditional material TM1 (code 20170802), TM2 (code 181101), and TM4 (code 20170601) of Radix Astragali prepared from Astragalus membranaceus var. mongholicus and their corresponding ultrafine granular powder UGP1 (code 20170908), UGP2 (code DP20190130), and UGP4 (code 20180706), as well as two batches of sliced traditional material TM3 (code 181005) and ultrafine granular powder UGP3 (code 20190105) purchased from a TCM drug store were supplied by Zhongzhi Pharmaceutical Co. Ltd. Each accession of TM and UGP were randomly selected and powdered with a grinder to a particle size of < 0.25 mm.

      Chemicals and solvents

    • Reference standards of calycosin (> 99%), calycosin-7-O-β-D-glucose (> 98%), formononetin (> 98%), ononin (> 98%), astragaloside II (> 99%), astragaloside IV (> 99%) were purchased from AvaChem Scientific (San Antonio, TX, USA). HPLC grade methanol and acetonitrile were purchased from Merck. Formic acid (HPLC grade) was from Sigma-Aldrich. Deuterated trimethylsilylpropanoic acid (TMSP, 2,2,3,3-D4, 98%) in D2O (D, 99.9%) (0.02% (W/V)) was purchased from Cambridge Isotope Laboratories. Inc. (50 Frontage Road, Andover, MA 01810, USA). Chloroform and n-butanol (ACS grade) were obtained from Merck. 10 kDa molecule weight cut-off (MWCO) Spin–X UF concentrators was manufactured in Corning, NY, USA.

      UHPLC/DAD-MS analysis

      UHPLC/DAD–MS conditions

    • Agilent UHPLC 1290 (Agilent Technologies, Santa Clara, CA, USA) consists of a dualistic solvent delivery system, an autosampler, and a column temperature controller. An Agilent Eclipe Plus C18 1.8 µm, 2.1 × 100 mm column was used for the separation. The mobile phase consisted of 0.05% formic acid (v/v) in water (A) and acetonitrile (B) with a flow rate of 0.25 mL/min. The gradient program was as follows: 0–1 min, 5% B; 1–15 min, 5%–95% B; 15–16 min, 95%–100% B. The DAD wavelength was set at 254 nm. The column temperature was set to 30 °C and the injection volume was 2 µL. The mass spectrometer was an Agilent 6120 quadrupole. The ESI source was set in both positive and negative modes for isoflavones and astragalosides. The parameters were as follows: capillary voltage 4.0 kV; gas temperature: 325 °C; gas flow 12 L/min; nebulizer 35 psi; vaporizer temperature: 225 °C; mass range (m/z): 100−1000.

      Sample extraction

    • To 500.0 mg of each TM or UGP material, 10 mL of solvent was added. The suspension was sonicated at room temperature for 30 min. After removal of the supernatant, the residual material was extracted twice in the same manner. The combined supernatants were filtered by a 0.22 µm micro membrane prior to UHPLC-MS-UV analysis. First, the TM1 sample and its corresponding UGP1 sample were extracted with MeOH for chemical consistency study. Next, the extraction method was evaluated using different solvent systems (0%, 50%, 80%, and 100% methanol), leading to the identification of 80% methanol possessing the greatest extraction efficiency based on relative peak areas of six isoflavonoids (Supplemental Fig. S3), which is consistent with previous reports[20]. Thus, 80% methanol was used to extract TM2, UGP2, TM3, and UGP3.

      Preparation of calibration solutions

    • The stock solutions of calycosin (1) (3,200 µg/mL), calycosin-7-O-β-D-glucose (2) (3,000 µg/mL), formononetin (4) (1,500 µg/mL), ononin (5) (1,200 µg/mL), astragaloside IV (7) (3,400 µg/mL), and astragaloside II (8) (2,900 µg/mL) were individually prepared with methanol. All stock solutions were stored at 4 ℃. The stock solutions of the standards were further diluted with methanol to produce combined standard working solutions at a series of concentration of 0.11–533.33 µg/mL for 1; 0.10–500.00 µg/mL for 2; 0.05–250.00 µg/mL for 4; 0.04–200.00 µg/mL 5; 0.11–566.67 µg/mL for 7; 0.09–483.33 µg/mL for 8.

      Method validation

    • The intra-day and inter-day precision were determined by analyzing UGP2 sample during a day and on three different days, respectively. Accuracy of this method was verified by a recovery test. The standard solution was spiked to the UGP2 material to make two different final concentrations of 10.67 (5.33), 10.00 (5.00), 5.00 (2.50), 4.00 (2.00), 11.33 (5.67), 9.67 (4.83) µg/mL for calycosin (1), calycosin-7-O- β -D-glucose (2), formononetin (4), ononin (5), astragaloside IV (7), astragaloside II (8), respectively. The detailed data are shown in Supplemental Table S3 & S4.

      Statistical analysis

    • The statistical analysis was performed using the software SPSS (Statistical Package for the Social Sciences, IBM, NY, USA)

      Analysis of RAPS in UGP and TM materials

      RAPS preparation

    • Five hundred mg of TM4 or UGP4 material (n = 3) was extracted twice with 10 ml of water at 100 ℃ for 15 min. The combined filtrates were fixed to a final volume of 20 ml, which was treated with Sevag reagent (CHCl3: n-BuOH = 4:1)[7] (20 mL × 3) to remove proteins by centrifugation. The resulting water layer was subjected to ultrafiltration[21] using a 10 kDa Spin–X UF concentrator (20 mL Corning). Briefly, the water layer was added to the sample reservoir of the concentrator, and centrifuged at 3000 rpm. After the water layer was reduced to approximately 1.5 mL, the concentrator was refilled with 20 mL of deionized water and centrifugation was continued. This procedure was repeated twice. Finally, the residual solution in the sample reservoir was collected and lyophilized to afford the desired polysaccharides with yields of 3.39 ± 0.20% and 7.22 ± 0.35% from TM4 and from UGP4, respectively.

      1H NMR analysis

    • 1H NMR profiles were used to characterize the RAPS obtained from UGP and TM materials. The 1H NMR measurements were conducted on an Agilent DD2-500 NMR spectrometer equipped with a One NMR probe operating at 499.86 MHz for 1H, operating at a temperature of 300 K (27 °C). A scan number of 128 for each sample was used for data collection, with a relaxation delay of 5.0 s and a pulse width of 7.8 ms (90 degree). A 0.3 Hz line broadening was used for apodization and the data sets were zero-filled to 256 k points prior to processing. Chemical shifts (δ) expressed in ppm and referenced to TMSP proton signals at δH = 0.0 ppm.

      Acknowledgments

      • This research was supported by the USDA Agricultural Research Service Specific Cooperative Agreement No. 58‐6060‐6‐001 and Zhongshan Zhongzhi Pharmaceutical Co. Ltd. We thank Mr. Mohammad A. Daghestani for his sustenance of drying samples during the work.

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Supplementary information
      • Supplemental Fig. S1 Typical UHPLC-MS-UV chromatograms of six standard compounds. Standard compounds: calycosin (1), calycosin-7-O-β -D-glucose (2), formononetin (4), ononin (5), astragaloside IV (7),and  astragaloside II (8).
      • Supplemental Fig. S2 ESIMS spectra of two minor compounds calycosin 7-O-β -D-(6''-acetyl)-glucoside (P1) and 6''-acetylononin (P2) in UGP1 and TM1. A: ESI (+) and B: ESI (-) of P1; C: ESI (+) and D: ESI (-) of P2.
      • Supplemental Fig. S3 Peak areas (UHPLC/DAD-MS UV254 nm) of isoflavonoids (1-6) extracted by solvent systems of 0%, 50%, 80%, and 100% methanol in water. Marker compounds: calycosin (1), calycosin-7-O-β-D-glucose (2), calycosin-7-O-β-D-glucose-6''-O-malonate (3), formononetin (4), ononin (5) and formononetin-7-O-β-D-glucoside-6''-O-malonate (6).
      • Supplemental Fig. S4 Contents (mg/g) of marker compounds (1-6 and 8-10) in UGP2 and TM2 from the same source of Astragali Radix (n=10). Marker compounds: calycosin (1), calycosin-7-O-β-D-glucose (2), calycosin-7-O-β-D-glucose-6''-O-malonate (3), formononetin (4), ononin (5), formononetin-7-O-β-D-glucoside-6''-O-malonate (6), astragaloside II (8), astragaloside I (9), and acetylastragaloside Ⅰ (10).
      • Supplemental Fig. S5 Contents (mg/g dry material) of marker compounds (1-6, 8-10) in UGP3 and TM3 materials (n=10). Marker compounds: calycosin (1), calycosin-7-O-β-D-glucose (2), calycosin-7-O-β-D-glucose-6''-O-malonate (3), formononetin (4), ononin (5), formononetin-7-O-β-D-glucoside-6''-O-malonate (6), astragaloside II (8), astragaloside I (9), and acetylastragaloside Ⅰ (10).
      • Supplemental Table S1 Peak areas (UHPLC/DAD-MS UV 254 nm) of isoflavonoids (1-6)  a extracted by different solvent systems of methanol/water (0%, 50%, 80%, and 100% of methanol in water).
      • Supplemental Table S2 Regression data for the marker compounds in UHPLC/DAD-MS.
      • Supplemental Table S3 Intra-day and inter-day precisions by analyzing UGP2.
      • Supplemental Table S4 Recovery test for determination of six standard compounds in Radix Astragali (by using sample of UGP2, n = 3).
      • Supplemental Table S5 Contents (mg/g dry material) of marker compounds (1-6 and 8-10) a in UGP2 and TM2 from the same source of Radix Astragali (n=10).
      • Supplemental Table S6 Contents (mg/g dried material) of marker compounds (1-6 and 8-10) a in UGP3 and TM3 materials (n=10).
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      • Copyright: © 2022 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (4)  Table (3) References (21)
  • About this article
    Cite this article
    Cao L, Wang M, Zhao J, Peng LH, Cheng JL, et al. 2022. Comparative analysis of chemical profiles of Radix Astragali between ultrafine granular powder and sliced traditional material. Medicinal Plant Biology 1:4 doi: 10.48130/MPB-2022-0004
    Cao L, Wang M, Zhao J, Peng LH, Cheng JL, et al. 2022. Comparative analysis of chemical profiles of Radix Astragali between ultrafine granular powder and sliced traditional material. Medicinal Plant Biology 1:4 doi: 10.48130/MPB-2022-0004
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Catalog

  • Html

  • Introduction
  • Results

  • Chemical consistency between ugp and tm materials of radix astragali

  • Chemical homogeneity between ugp and tm materials of radix astragali

  • Chemical profiles and content of polysaccharides in ugp and tm of radix astragali

  • Discussion
  • Conclusions
  • Materials and methods

  • Material and chemicals

  • Plant material
  • Chemicals and solvents

  • Uhplc/dad-ms analysis

  • Uhplc/dad–ms conditions
  • Sample extraction
  • Preparation of calibration solutions
  • Method validation
  • Statistical analysis

  • Analysis of raps in ugp and tm materials

  • Raps preparation
  • 1h nmr analysis
  • Acknowledgments
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
  • About this article
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