WORLD SCIENCE AND TECHNOLOGY MODERNIZATION OF TRADITIONAL CHINESE MEDICINE AND MATERIA MEDICA Volume 11, Issue 3, June 2009 Online English edition of the Chinese language journal Cite this article as: Mode Tradit Chin Med Mater Med, 2009, 11(3): 375–381 RESEARCH REVIEW The Correlation between the Flavor and Quality of Radix Astragali: The Extraction and Characterization of Lipoxygenase in Radix Astragali Xie Daosheng1, 2, Wu Bin3*, Sun Haifeng1, 2, Guo Xiaoqing2, Zhang Lizeng2, Qin Xuemei1, 2* 1. 2. 3. College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China; Modern Research Centre for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China; Shanxi Medicine Group Co. Ltd., Taiyuan 030012, China Abstract: Lipoxygenase (LOX) is a key biotic factor producing the bean flavor in Radix Astragali. LOX was studied through spectro-photometry and Linolic acid. Extraction samples and detection methods were developed to investigate a range of biotic and abiotic factors, including the temperature, metal ion, chelate and age, and to evaluate the LOX activity of Radix Astragali. The results showed that LOX exists in the Radix Astragali grown in the Hunyuan County of Shanxi Province, China. Radix Astragali was ground into fine powder using liquid nitrogen, followed by being soaked for 1 h in the borate buffer. LOX crude extracts were obtained through centrifugation at 4000 r/min for 10 min at 4 °C. LOX crude extracts and the substrate solution were mixed at a ratio ranging from 1:10 to 1:3 when the LOX activity was detected at a wavelength of 236 nm. The results also suggested that the enzyme could be stable for 15 min at the temperature from 0 °C to 50 °C, but the enzymes rapidly became inactivated after 2 min at 70°C. The enzymes were activated by Fe3+, Fe2+ and Ca2+, but inhibited by Zn2+, Mg2+ and tartaric acid. The Radix Astragali plants of 5 years old had the highest LOX activity, suggesting that LOX activity was associated with the growth age. The findings were useful for further study of the mechanism of producing the bean flavor in Radix Astragali and for searching for authentic growing sites. Key Words: Radix Astragali; Bean flavor; Lipoxygenase; extraction; detection; biotic and abiotic factor 1 Introduction Radix Astragali (RA), known as Huangqi in China, is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or A. membranaceus (Fisch.) Bge, with bean flavor [1]. The plant is one of the most widely used Chinese herbs prescribed in many Chinese formulas to reinforce “Qi”, the vital energy. Today, RA is cultivated mainly in northern China provinces, such as Shanxi, Neimenggu and Gansu and northeastern provinces, such as Heilongjiang. The traditional Chinese medicine pays particular attention to “Shape, Color, Energy and Taste” of medicinal materials. The traditional application experience considers the “heavy bean flavor” as an excellent quality of RA. Thus, the strong bean flavor is used as a traditional evaluating indicator for RA. However, this traditional identification method is not characterized by modern technology. The bean flavor is a common characteristic of legume. It was indicated by Andre et al. study [2] that the bean flavor was caused by enzymatic reactions from polyunsaturated fatty acids (PUFAs). The key enzyme was Lipoxygenase (LOX, EC1.13.11.12). LOX is a ubiquitous plant enzyme that catalyzes the hydroperoxidation of PUFAs containing cis, cis-1, 4 pentadiene moiety such as linoleic acid and linolenic acid. PUFA hydroperoxides derived from the LOX activity are considered to be flavor precursors. Indeed, PUFA hydroperoxides are subsequently converted into flavor compounds, such as alcohol, ketone and aldehyde, by the hydroperoxide lyase (HPL) which produces the bean flavor [3]. Hunyuan County in Shanxi Province of China is regarded as the authentic growing region of RA, where the best quality of RA is grown with a significantly heavy bean flavor. However, there haven’t been any reported studies on the relationship between LOXs and the bean flavor of RA. In this study, wild RA samples with the heavy bean flavor were collected from Hunyuan County. The first purpose of this study was to explore whether or not there are LOX activities in RA. The second purpose was to discover a desirable extraction process and detection conditions. Finally, the biotic and abiotic factors were studied in order to Received date: 7 April 2009 * Corresponding author. Tel: +86-351-7011202; E-mail: qinxm@sxu.edu.cn Foundation item: Supported by the Ministry of Science and Technology of the People's Republic of China (No.2006BA106A15-7) and the Science and Technology Key Project of Shanxi Province(No.052018). Copyright © 2009, World Science and Technology Press. Published by Elsevier BV. All rights reserved. Xie Daosheng et al. / Mode Tradit Chin Med Mater Med, 2009, 11(3), 375-381 understand their effects on LOX activities in RA. The findings would be useful for further study of the mechanism of forming the bean flavor in RA, the correlation between the quality and the bean flavor of RA, and RA’s special genuineness. 2 Materials and instruments 2.1 Plant Materials All roots of wild and half-wild varieties were obtained from Hunyuan County in Shanxi Province, China. The wild variety was provided by the Wansheng Company in Hunyuan, Shanxi for the extraction of LOX, determination of the LOX activity, and the study of biotic and abiotic factors’ effects on the LOX activity. The half-wild plants with growth ages ranging from two to seven years were collected from the cultivating farms run by the Lizhu Company in Hunyuan for studying the effect of growth ages on the LOX activity. All of the samples were authenticated by one of the authors, Prof. Qin Xuemei. All samples were immediately frozen in liquid nitrogen prior to be stored at -80°C. 2.2 Chemicals and instruments Linoleic acid with the concentration greater than 97% was purchased from the Alfa Aesar Company in the United States. The other chemicals were of analytical grade except Tween-20 at a chemical grade was used. Deionized distilled water was used in all experiments. Spectrophotometer 7501 UV-VIS was purchased from the Wuxi Instrument Factory in Wuxi City of Jiangsu Province, China. 3 Methods 3.1 Extraction of LOX from RA The determination condition was similar to what has been described by Gardner et al. [4] with some modifications. 5 g RA was ground into fine powder using liquid nitrogen, followed by being soaked in 40 mL 0.05 mol·L-1 borate buffer solution (pH9.0) in an ice bath for 2 h. LOX crude extracts were obtained through centrifugation at 4000 r·min-1 and 4°C for 10 min. The supernatant was collected in a volumetric flask to detect the LOX activities using the method to be described in section 3.2. 3.2 Determination of LOX activity To assess the LOX activity, linoleic acid was used as the substrate. Its stock solution was prepared in the borate buffer (pH 9.0, 0.05 mol·L-1) at a concentration level of 0.5 mg·mL-1 according to the procedure described by Gardner et al. [4] with some modifications. The substrate medium was composed of 0.25 mL of Tween-20 and 0.27 mL of linoleic acid. 10 mL borate buffer solution (pH9.0, 0.05 mol·L-1) was processed at 25°C by blowing O2 into the substrate medium while the stirred solution was irradiated under light by adding several drops of the NaOH solution (1 mol·L-1). Then the mixture solution was diluted to a final volume of 500 mL with the borate buffer (0.05 mol·L-1) at pH 9.0. The prepared substrate was stored in sealed tubes at 4°C under a steam of nitrogen. The LOX activity was spectrophotometrically assayed at 25°C using a 7501 UV-VIS spectrophotometer. The reaction mixture consisted of 0.2 mL of enzymatic extract, 0.8 mL of the linoleic acid stock solution, 5 mL of absolute ethanol and 5 mL of distilled water. Eight-tenth mL of the substrate solution was constant at 20°C for 5 min. Then 0.2 mL of the enzymatic extract was added to the reaction mixture and incubated at 25°C for 4 min. The reaction was stopped by adding 5 mL of absolute ethanol, followed by 5 mL of distilled water to dilute the solution. LOX specific activities were monitored by increasing the absorbance at 236 nm. All LOX assays were performed in duplicates and run in tandem with blanks, containing all components of the assay except that absolute ethanol was added into the mixture before the enzymatic extract was added. 3.3 Optimization of the LOX extraction condition Based on the method described above in section 3.1, the effects of different material ratios were discussed. The material ratios (w:v) of 1:5, 1:7.5 and 1:10 were used to extract enzymes. According to the optimum material ratio, the effects of extracting time of 1 h, 2 h, 3 h, 4 h, 6 h and 8 h were also discussed. 3.4 Optimization of the enzymatic reaction condition The optimum LOX extraction condition described in section 3.3 was used to obtain enzymes. One-tenth mL of the enzyme extract was added into 0.5 to 3 mL substrate solution to select the suitable amounts of the substrate solution. Based on the optimum substrate solution, the effect of the enzyme amount was also discussed. 3.5 Enzymatic properties of LOX The secondary metabolites were usually the main pharmacodynamics compositions of traditional Chinese medicines. The growing environment, especially environmental stress, is an important factor which alters the secondary metabolites formation and its accumulation in plants. The published reports by Guo et al. [5, 6] have shown that the growth of A. lancea in Mt. Mao is faced with nutrient and high temperature stress. The study carried by Huang et al. [7] has shown that the authentic medicinal materials formation is affected by stress. The special bean flavor of RA from Hunyuan may be influenced by the local growth environment related to some abiotic factors such as the temperature and soil element content. It may also be correlated with some biotic factors such as growth ages. This experiment has studied the effects on the LOX activity from factors such as temperature, growth age and selected additives, including metal ions and complex agents. 3.5.1 Thermo-stability The thermo-stability of LOX activities was determined by the pre-incubating enzymatic extract in a wide range of temperatures from 0°C to 70°C for 15 min. The residual LOX activities were measured using the standard assays. Xie Daosheng et al. / Mode Tradit Chin Med Mater Med, 2009, 11(3), 375-381 Based on the stability data of LOX at different temperatures, the stability of LOX activities at 50°C and 70°C was dynamically monitored. Each reaction mixture was removed at a specific time, immediately cooled in an ice-water bath to be assayed for LOX activities. 3.5.2 Metal ions RA contains various trace elements, especially high concentration levels of Zn2+, Mg2+, Fe3+/Fe2+ and Ca2+, depending on the growing regions. A series of metal ion concentrations (Table 1) were designed to discuss their effects on the LOX activity according to the reported contents of metal ions in RA [8]. 25 µL metal ions was added to 0.5 mL LOX extract and incubated at 25°C for 15 min. The LOX activity was assayed. 3.5.3 Complex agents The effects of complex agents, including EDTA, citric acid and tartaric acid, on the LOX activity were studied by varying their concentrations (Table 1) in the reaction mixtures based on the method described in section 3.5.2. 3.5.4 Growth ages The half-wild samples were used to extract and determinate the LOX activities based on the optimum methods described in section 3.4. 4 Results and discussion 4.1 The LOX activity detected in RA The result showed that the maximum absorption of the enzymatic reaction happened at 236 nm (Figure 1). The LOX activity existed in the RA grown in Hunyuan. Compared with the maximum absorption of the soybean LOX reaction at 234 nm [4], the RA LOX was distinct in catalytic character from the former, which might lead to the different product structures. 4.2 Optimization of the LOX extraction condition 4.2.1 The material ratio Figure 2 showed that the same LOX extract of RA using different material ratios had different increases in A236, and reached the maximum increase of A236 when the w:v ratio was 1:7.5. Based on these experimental data, the material ratio 1:7.5 was used throughout this study for further extraction. 4.2.2 Extracting time Figure 3 summarized the LOX activities of RA at different extracting time points from 1 h to 8 h. It showed that the LOX activity was relatively stable within 1 h to 2 h, slightly increased at 3 h, and decreased to the 1 h level at 4 h, followed by continuous decreases. The extracting time of 1 h was selected in consideration of the extracting efficiency and LOX activity. 4.3 Determination of the enzymatic reaction condition One-tenth mL of the enzyme extract was incubated with different volumes of substrate stock solutions, and the LOX activities were assayed (Figure 4a). A positive linear correlation was found between the LOX activities and the 0.5 to 1.5 mL substrate solutions (Figure 4b). The enzyme activity showed slightly increase within 1.5 to 4.0 mL of substrate solutions. Therefore, the optimal volume of substrate solution was determined to be 0.75 mL, in agreement with the documented report [9]. Figure 5a showed the results of different volumes of enzyme extracts in the reaction system. A positive linear correlation was found to exist within 0.05--0.3 mL of enzyme extracts (Figure 5b). Subsequently, the increase of LOX activities became slow, and the value of A236 became greater than 1. As a result, 0.1--0.2 mL of the enzyme extract was selected. 4.4 Effects of biotic and abiotic factors on the LOX activity 4.4.1 Temperature The thermo-stability profiles of LOX showed that LOX had certain levels of stability at elevated temperatures (Figure 6a). LOX showed the best thermo-stability at 4°C. However, an abrupt decline of thermo-stability occurred when the temperature was above 60°C. The incubation at 70°C completely inactivated the enzyme activity. With the incubation time extension at 50°C and 70°C, the activities of LOX first increased, followed by decreases (Figure 6b). A greater increase of the residual LOX activities was found within the first 1 min, which might be caused by heat stress [10]. The residual LOX activities were 102% and 47% of their initial values after 6 min incubation at 50°C and 70°C, respectively. Afterwards, LOX activities were decreased to 93% and 8% at 50°C and 70°C for 15 min. In all trials, the LOX activity was relatively stable when the temperature was between 0°C to 50°C. 4.4.2 Effect of metal ions on the LOX activity LOX is an important class of non-heme iron enzymes including Fe(ⅱ)-OH2 and Fe(ⅲ)-OH-1 that catalyze the hydroperoxidation of UFAs with Fe(ⅲ)-OH-1 as the active site base [11]. In conclusion, Fe3+ and Fe2+ are key factors of the enzymatic reaction of LOX. The effects of Fe3+ and Fe2+ were studied in terms of their efficiency as the chemical additives in the maintenance of LOX activities. Figure 7(a) and 7(b) summarized the LOX activities of the enzymatic extract at different concentration levels (Table 1). The results showed that the additions of Fe3+ and Fe2+ activated the LOX activities of the enzymatic extract, and that the increase in LOX activities was associated with the concentration level of the additive. The low Mg2+ concentrations at dose 1, 2 and 4 inactivated the LOX activities. The addition of Mg2+ at dose 2 led to an 8% decrease in LOX activities. However, the results also showed that the addition of Mg2+ at dose 3 activated the LOX activities, and the addition of Mg2+ at dose 5 resulted in a 100% recovery of LOX activities (Figure 7c). Tian et al. [12] reported that the addition of a low concentration of Mg2+ into the enzyme extract of soybeans Xie Daosheng et al. / Mode Tradit Chin Med Mater Med, 2009, 11(3), 375-381 resulted in an increase in the residual specific activities, and the same conclusion was drawn at a high concentration of Mg2+. This is in agreement with the reported results. The experimental findings (Figure 7d) indicated that in contrast to the other studied additives, the presence of Ca2+ (Table 1) in the enzymatic assay resulted in an increase in LOX activities. On the other hand, the addition of Ca2+ at dose 1 and dose 3 resulted in 110% and 102% increases in LOX activities, respectively. These findings suggested that the increase in LOX activities was associated with the addition of Ca2+. Figure 7(e) showed that the LOX activity could be activated by a low concentration of Zn2+ at dose 1 and dose 2. By contrast, a high dose of Zn2+ inactivated the LOX activity, and LOX showed the lowest activity at dose 3. 4.4.3 Effect of complex agents on RA LOX Figure 7(f) illustrateed the LOX activity profile in the presence of three different complex agents. The result demonstrated that the LOX activity was relatively stable in a broader range of additive concentrations, such as at dose 1, 2 and 5. Dose 3 inactivated the LOX activity. Dose 4 with EDTA activated the LOX activity. Dose 4 with citric acid and tartaric acid inactivated the LOX activity. Overall, the results suggested that tartaric acid was the appropriate additive to inactivate the LOX activity in RA. 4.4.4 Effect of growth ages on the LOX activity Figure 8 demonstrated the data of LOX activities at different growth ages. The LOX activity was at the highest level in the plants of 5 years old, at lower and relatively stable levels in the plants of 2 to 4 years old and 6 to 7 years old, respectively. These datas suggested that the LOX activity was related to growth ages. The traditional harvest time of RA in Hunyuan is when the plants are 5 years old, which is consistent with our results. extraction and determination of the LOX activity. The result suggested that many biotic and abiotic factors affected the LOX activity of RA. The thermo-stability study indicated that the LOX activity was relatively stable at 0 °C to 50 °C, and was increased quickly under heat stress. It was activated by Fe3+, Fe2+ and Ca2+, but inhibited by Zn2+, Mg2+ and tartaric acid. Dose 3 and dose 4 of complex agents obviously affected the LOX activity of RA. In addition, the LOX activity of RA was related to growth ages. The LOX activity is at the highest level in the plants of 5 years old, which may be used as evidence for the traditional harvest time of RA in Hunyuan. In Hunyuan, the average temperature in July is 21.6 °C, and the annual average temperature is 6.2 °C. The iron resource is abundant in this area. Therefore, it is speculated that a lower temperature and rich metal ions could promote the LOX activity expression and lead to the special bean flavor in RA. These findings will be useful for further study on the mechanism of producing the bean flavor in RA, the correlation between the RA quality and the bean flavor, and RA’s special genuineness. They would also help to interpret the traditional Chinese medication experience with modern technology. 5 Conclusions [3] Eskin NA, Grossman S, Pinsky A. Biochemistry of lipoxygenase Several documents and historical data have indicated that Hunyuan County in Shanxi Province, China is the authentic growing area of RA. An earlier record of such evaluation appeared in “Ben Cao Meng Quan” as “The Best Quality of Mianqi is Grown in Qinzhou of China”. An Illustrated Book of Plants written by Wu Qijun of Qing Dynasty stated that “Huangqi has been widely distributed in China. Those grown in Shanxi and Neimenggu have the best quality”. Recent studies also indicated that Shanxi of China produces RA with the best quality. RA grown in Hunyuan possesses a significantly heavy bean flavor which was recorded in “Shanxi Zhong Cao Yao”. These records are consistent with the traditional application experience that considers the heavy bean flavor as an excellent quality of RA. Overall, RA grown in Hunyuan is a typical representation as far as the germ plasma resource and genuineness are concerned. In studying the LOX activity existing in RA grown in Hunyuan, this work first established the methods of Acknowledgements This work was supported by the Ministry of Science and Technology of the People's Republic of China (No.2006BA106A15-7) and the Science and Technology Key Project of Shanxi Province (No.052018). References [1] Ch P (2005), Vol I[S]. 2005, 212. [2] Andre E, Hou KW. Sur la presence dcune oxydase des lipids ou lipoxydase dans la grain de so ja, glycine so ja life. Compte Rendu Acad Sci (paris) 1932; 194: 645–647. EC-1.13.11.12 in relation to food quality. Crit Rev Food Sci Nutr 1977; 1: 1-4. [4] Gardner HW. Isolation of a pure isomer of linoleic acid hydroperoxide. Lipids 1974; 4: 248–252. [5] Guo LP, Yan YN. Habitat characteristics for the growth of Atractylodes lancea based on GIS. China Journal of Chinese Materia Medica 2002; 4: 245-250. [6] Guo LP, Huang LQ, Shao AJ, et al. The status and changes of soil nutrients in rhizosphere of cultivated Atractylodes lancea. China Journal of Chinese Materia Medica 2005; 19: 1504-1507. [7] Huang LQ, Chen ML, Xiao PG. Biological basis of modern and model hypothesis of genuineness of Chinese traditional medicinal herbs. China Journal of Chinese Materia Medica 2004; 6: 494. [8] Shen XF, Zhang Y, Yang C, et al., Speciation analysis of trace elements in Radix Astragali by flame atomic adsorption spectrophotometry. Chinese Journal of Analytical Chemistry 2006; 3: 396-398. Xie Daosheng et al. / Mode Tradit Chin Med Mater Med, 2009, 11(3), 375-381 [9] Cai K, Fang Y, Xia YM. Extraction of soybean lipoxygenase functional characterization of second-coordination sphere and factors affecting its enzyme activity. Chemistry and mutants of soybean lipoxygenase-1. Biochemistry 2001; 25: Industry of Forest Products 2004; 2: 52-56. 7509-7517. [10] Melan MA, Enriquez A, Peterman TK. The LOX1 gene of Arabidopsis is temporally and spatially regulated [12] Tian QY,Yin GZ,Hua Y F. Influencing factors on activity of in soy lipoxygenase isozymes. Science and Technology of Food germinating seedlings. Plant Physiology 1994; 1: 385–393. Industry 2008; 1:156–159. [11] Tomchick DR,Phan P,Cymborowski M,et a1. Structural and Table 1 Concentrations of metal ions and complex agents in different groups Dose 0 Dose 1 Dose 2 Dose 3 Dose 4 Dose 5 (µmol·L-1) (µmol·L-1) (µmol·L-1) (µmol·L-1) (µmol·L-1) (µmol·L-1) Fe3+ 0 0.1 0.5 1 10 100 Fe2+ 0 0.005 0.02 0.1 0.5 2.5 Mg2+ 0 0.02 0.08 0.4 2 10 2.507 (0.104) Ca2+ 0 0.032 0.16 0.8 4 20 1807 (45.175) Zn2+ 0 0.0004 0.002 0.01 0.05 0.25 41 (0.631) 0 8 40 200 1000 5000 Volume of RA µg·g-1 (µmol·g-1) 305 (5.446) EDTA/ citric acid /tartaric acid nnm Fig. 1 UV spectra of samples. 冰水浸提时间考察 0.3 0.2 A236 A236 0.25 0.15 0.1 0.05 0 0 11:5 1:7.5 2 2 1/2 31:10 3 1/2 Material ratio (g·mL-1) Fig. 2 Determination of the optimal ratio of samples to extraction. 1/2 1 1/2 buffer 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 2 4 6 8 Extracting time (h ) 冰水浴浸提时间/h Fig. 3 Determination of the optimal extraction time. 10 Xie Daosheng et al. / Mode Tradit Chin Med Mater Med, 2009, 11(3), 375-381 底物加入量考察(前四点) 0.6 0.5 (a) 0.5 0.4 A236 OD236 A236 0.4 0.3 0.2 y = 0.1944x + 0.156 R2 = 0.9861 (b) 0.3 0.2 0.1 0.1 0 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 1 1.5 Volume of substrate (mL) 底物加入量/mL Fig. 4 Determination of the optimal volume of the substrate solution. Volume of substrate (mL) 0.5 酶液加入量考察 6 酶液加入量考察 2.5 (a) 5 (b) y = 7.5071x - 0.2004 2 R = 0.9998 2 AOD236 236 4 3 2 1.5 1 0.5 1 0 0 0 0.5 1 1.5 Volume of enzyme (mL) 酶液加入量/mL 2 2.5 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Volume of enzyme (mL) 酶液加入量/mL Fig. 5 The determination of the LOX volume. 脂合酶温度耐受性考察 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 70摄氏度 0.5 (a) 50摄氏度 (b) 0.4 A236 OD236 A23 6 OD236 AOD236 236 2 0.3 50°C 0.2 70°C 0.1 0 20 40 温度/摄氏度 60 Temperature (°C) 80 0 0 5 10 15 Time (min) 时间/min Fig.6 (a) Stability of Astragalus membranaceus LOX at (b) Stability of Astragalus membranaceus LOX at 50°C and different temperatures for 15 min. 70°C for different times. 20 Xie Daosheng et al. / Mode Tradit Chin Med Mater Med, 2009, 11(3), 375-381 不同浓度二价铁离子对酶活的影响 0.32 0.33 0.32 (a) 0.3 A236 OD236 A236 OD236 0.31 0.29 0.28 不同浓度镁离子对酶活的影响 0 1 2 3 4 0.27 5 0 6 0.28 0.32 0.275 0.31 0.27 0.265 A236 OD236 A236 OD236 0.3 0.29 0.28 0.27 (c) 0.26 不同浓度锌离子对酶活的影响 1 2 3 4 5 浓度编号(代表浓度由0至0.01mmoL/L) 0.25 0 6 (d) 0.29 0.27 0 6 不同浓度络合剂对酶活的影响 1 2 3 4 5 6 浓度编号(代表浓度由0至0.02mmoL/L) 柠檬酸 EDTA citric acid EDTA 酒石酸 tartaric acid 0.350.35 0.30.3 0.250.25 0.2 0.20.15 0.150.1 0.10.05 0.05 0 0 1 2 3 4 1 2 3 4 5 6 0 Dose group Dose group 浓度编号(代表浓度由0至250nmoL/L) 0 1 2 3 4 Fig. 7 The effects of metal ions and complex agents on RA LOX activities. 浓度编号 0.28 (e) OD236 0.27 0.26 0.25 0.24 0 (f) 5 6 5 0.6 0.5 A236 OD236 0.3 A236 OD236 0.29 不同浓度钙离子对酶活的影响 1 2 3 4 5 浓度编号(代表浓度由0至2500nmoL/L) 0.28 0.255 A236 (b) 0.31 0.4 0.3 0.2 0.1 0 0 2 4 6 8 growth ages (years) Fig. 8 The relationship between LOX activities and growing ages of Astragalus membranaceus. 6