Rapid identification of growth years and profiling of bioactive ingredients in Astragalus membranaceus var. mongholicus (Huangqi) roots from Hunyuan, Shanxi

Abstract

Background: The content of medicinal bioactive constituents in huangqi is affected by plant age. In this study, we devised a quick and convenient method for determining the age of huangqi, which was cultivated in Hunyuan County (Shanxi Province).

Methods: 1, 2, 3, 4, 5, 8, 10 growth years huangqi had 38 samples, all samples were collected separately. The growth rings in these samples were observed after making paraffin section and freehand-section. The relationship between growth rings and its growth years was analyzed by SPSS 19.0 software. Histochemical localization of total flavones and saponins in huangqi was determined by color reactions. The concentration of four flavonoids and two saponins in the roots of huangqi of different ages and different organizational structure (normal roots and rotten heart roots) were determined by HPLC-DAD and HPLC-ELSD. The results were analyzed by SPSS 19.0 software.

Results: All huangqi samples had clear growth rings, and the statistical result about growth rings (X) and growth years (Y) showed significant correlation (r = 1, P = 0.000). The calibration curves of these six ingredients showed good linearity respectively, with significant correlation. All relative standard deviations (RSDs) of precision, recovery, repeatability, and stability experiments were less than 2%. Roots of 5-year-old plants contained the highest concentrations of total flavonoids and saponins. Saponin concentrations increased toward the center of the roots, whereas the four flavonoids showed an opposite trend in tissue distribution.

Conclusion: The growth year of huangqi (Hunyuan County, Shanxi Province) could be determined soon and conveniently by naked eyes after staining phloroglucinol-HCl solution on freehand section. The content of saponins and flavonoids in rotten heart root and the surrounding normal tissues were affected by the formation and the extent of rotten heart.

Keywords: Concentration; Freehand section; Growth rings; Histochemical localization; Rotten heart.

Fig. 1

Pictures of the root anatomy of Huangqi from Hunyuan County, Shanxi Province. a Secondary structure of the root. b Macroscopic examination. c Rotting microstructure in the upper portion of the root. d Middle portion of the root. e Lower portion of the root. f Cross-section of rotten heart root. g, h Early development of the rotten heart at low and high magnifications. i Microscopic cross-sectional examination at the junction of healthy and rotten tissue. The yellow arrows represent growth rings

Fig. 2

The growth rings in huangqi from Hunyuan County, Shanxi Province. a Growth rings in 1-year-old roots; b growth rings in 2-year-old roots; c growth rings in 3-year-old roots; d growth rings in 4-year-old roots; e growth rings in 5-year-old roots

Fig. 3

Influence of growth years on the concentrations of flavonoids and saponins in Huangqi from Hunyuan County, Shanxi Province. Sample analysis was conducted by HPLC using internal standards for quantification. Data are expressed as the mean ± standard deviations from three samples, each of which was determined in triplicate

Fig. 4

Quantitative localization of the main flavonoids and saponins in huangqi. (a1) Segment stained with 5% sodium hydroxide solution compared with the negative control segment (a2). (a3) Segment stained with 5% vanillin-sulfuric acid solution or unstained compared with the negative control segment (a4). b The concentration of four flavonoids and two saponins in different tissues. Data are expressed as the mean ± standard deviations from three samples, each of which was determined in triplicate. The slices were divided into seven areas, from the outside to inside, as follows: Pd, periderm; SPh1 and SPh2, secondary phloem zones 1–2; SX1, SX2, SX3, and SX4, secondary xylem zones 1–4; Ho, rotten heart

Fig. 5

Influence of rotten heart on the total concentrations of flavonoids and saponins in 8-year-old huangqi. 1 calycosin-7-glucoside, 2 ononin, 3 calycosin, 4 formononetin, 5 astragaloside IV, 6 astragaloside II. Sample analysis was conducted by HPLC using internal standards for quantification. Data are expressed as the mean ± standard deviations from three samples, each of which was determined in triplicate