The G. uralensis seeds and seedlings were grown in Hangjingqi (Inner Mongolia, China). The plants were cultivated in April 2011 in a growth chamber in the Beijing University of Chinese Medicine (39º55′ N, 116º28′ E, at an altitude of 54.7 m). The plants were cultivated at an average temperature of 11.8ºC and an annual rainfall of 577 mm. The average evaporation capacity was 1,861 mm, while the comparative humidity was 62%.

The G. uralensis was grown in a polyvinyl chloride (PVC) tube, which was 5 cm above the ground. The tube was filled with sandy loam, containing organic matter (0.286%), alkali-hydrolyzable nitrogen (35.88 mg/kg), soil-available phosphorus (3.0 mg/kg), soil-available potassium (85.18 mg/kg) and CaCO₃ (2.71%). The pH value of the sandy loam was maintained at 7.87. The diameter and length of the tube were 30 and 80 cm, respectively, and the bottom of the tube was sealed with gauze. The seeding and transplantation of the G. uralensis were conducted on May 10 each year. Four G. uralensis plants were cultivated in one PVC tube. The relative water content in the tube was maintained between 60 and 70%. No fertilizer was given to the plants.

In order to evaluate the rate of respiration, the root was cleaned with distilled water and wrapped with wet gauze. The categorization of the root was performed as previously described (18). A zero-class root is also called an axial root and first-class roots mean the lateral roots growing on the axial root. Subsequently, the rate of respiration was calculated in accordance with the method from a previous study (19). To avoid wound respiration, Vaseline was smeared onto the section of the root system. The root was then placed in a Li-7000 CO₂/H₂O analyzer (Li-Cor, Lincoln, NE, USA), in which the concentration of CO₂ released by the root system was analyzed subsequent to constant airflow being obtained. In addition, the rate of respiration was calculated based on the CO₂ flux.

The root systems were scanned at a resolution of 3,000 dpi using an Epson Expression 10000XL scanner (Epson Co., Ltd., Long Beach, CA, USA). Root growth was measured by analyzing the scanned images using WinRHIZO version Pro2007d software (Regent Instruments Inc., Quebec Canada). All the experiments were performed at least six times.

The samples were oven-dried at 50ºC until a constant weight was achieved, prior to the roots being weighed and crushed. A 40-mesh screen was used as the threshold separating thick and fine roots. Subsequently, the active components of the G. uralensis were analyzed using a high-performance liquid chromatography (HPLC) assay with ultraviolet detection, based on a previous description (20). In brief, 5 ml G. uralensis (0.01 g) was added to 5 ml ethanol (70%), prior to ultrasound extraction being performed at 40ºC for 30 min (250 W). Following cooling at room temperature, 70% ethanol was added and the mixture was filtered using a Millipore filter with a diameter of 0.45 μm (Millipore, Billerica, MA, USA). A chromatographic column (Diamonsil^® C18, 250×4.6 mm, 5 μm; Agilent Technologies Inc., Santa Clara, CA, USA) was used for the HPLC analysis. A methyl cyanide-phosphoric acid mixture was used as the mobile phase. The wavelengths used for the analysis were as follows: λ₁=237 nm, λ₂=365 nm.

SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for the data analysis. P<0.05 was considered to indicate a statistically significant difference.

In this study, samples of annual and biennial G. uralensis, harvested in June and November, 2011, were used to determine the rate of respiration. A significant difference was observed in the volume rate of respiration between the annual and biennial G. uralensis (P<0.05, Figs. 1 and 2). In the annual and biennial G. uralensis, the maximum and minimum values for the rate of respiration were obtained in July (P<0.05) and November (P<0.05), respectively. Compared with the axial root, a marked increase in the rate of respiration was detected in the first-class root in the annual (0.688 versus 0.375 μmol CO₂cm⁻³sec⁻¹; P<0.05) and biennial (0.686 versus 0.411 μmol CO₂cm⁻³sec⁻¹; P<0.05) G. uralensis for months 6–11. These results were consistent with those obtained in a previous study by Ren et al(21).

The maximum rate of respiration was affected by the soil temperature, water content and nutritional intake. In the present study, higher rates of respiration were observed in July and August, which was likely to be correlated with the soil temperature and root metabolism.

Five active components, specifically, glycyrrhizic acid, glycyrrhizin, isoliquiritin, liquiritigenin and isoliquiritigenin, were analyzed in G. uralensis in our study.

Fig. 3 shows the fluctuations in the percentages of glycyrrhizin, liquiritigenin, glycyrrhizic acid, isoliquiritin and isoliquiritigenin present in the G. uralensis harvested in June and November, respectively. The data shown in the figure indicate that significant fluctuations occurred in the percentages of the five major components (P<0.05), depending on the season. The percentage of glycyrrhizic acid measured in the axial root in the present study was consistent with the value obtained in a previous study (3). Furthermore, the maximum percentage of glycyrrhizic acid in annual G. uralensis was apparent in September, which was also consistent with a previous investigation (22).

As shown in Table I, the levels of the five major components in the axial roots were higher than those of the first-class roots. In addition, the percentages of glycyrrhizin, isoliquiritin, liquiritigenin and isoliquiritigenin were higher in the biennial G. uralensis than in the annual G. uralensis.

Table II summarizes the correlation between root respiration and the percentages of the active components in G. uralensis. A previous study indicated that the rate of respiration in the root increased as the nitrogen concentration in the root increased (23). In general, the first-class root exhibited the maximum content of nitrogen and rate of respiration. In plants, ~90% of nitrogen has been shown to be present in the form of various proteins (24). The levels of glycyrrhizic acid and glycyrrhizin have been demonstrated to be negatively correlated with primary metabolites, including soluble protein, in the roots (25). Therefore, compared with the axial root, a higher nitrogen content and rate of respiration and a lower secondary metabolite content were apparent in the first-class root.