黄芩采收规律及其数学模型的研究
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摘要
中药材质量好坏直接决定中药饮片和中成药的药效和临床疗效。如何确定中药材的适宜采收期以获取优质药材,一直是人们关注的焦点。黄芩(ScutellariabaicalensisGeorgi.)是唇形科植物,其干燥根为我国常用中药材。本文以黄芩作为研究对象,分别对北京延庆、山东莒县和吉林长春三个产地的黄芩,按不同物候期进行采样,分析其外观和内在品质的动态变化规律,从而确定黄芩的最佳采收期。同时本文还对黄芩各物候期的田间土壤和相关气候因子进行调查,并采用通径分析的方法,筛选出对黄芩产量和质量的主导影响因子。在此基础上,进一步采用matlab软件中Lars算法,建立了基于环境效应的黄芩根3种有效成分总量预测模型,以期为田间生产确定黄芩最佳采收期提供指导,并为根类中药材采收研究提供新的思路和方法。主要研究结果如下:
1黄芩根产量变化规律的研究
不同生长年限的黄芩,其根重和根直径在果实成熟期之前均增加缓慢;进入果实成熟期,两者均大幅增加,并在枯黄期达到最大值。不同生长年限的黄芩相比,在其生长发育的第一年和第二年,根重呈急剧上升趋势,到第三年增长速率显著减慢;而且随着生长年限的增加,黄芩根皮颜色加深,纵皱纹明显,有易剥落的栓皮,并开始出现枯心。上述三产地黄芩根重和根直径的变化趋势基本一致。
2黄芩化学成分变化规律的研究
本文采用高效液相色谱仪(HPLC)和超高效液相色谱仪(UPLC)分别建立了黄芩不同部位的指纹图谱和含量测定方法。
研究发现黄芩根中主要化学成分与茎、叶和花果存在明显差异,根中成分主要是黄芩苷、汉黄芩苷、黄芩素、汉黄芩素和千层纸素等,其它部位中主要成分则为野黄芩苷和黄芩苷。不同物候期的黄芩根指纹图谱相似度均在0.9以上,说明在不同物候期黄芩根中化学成分种类很相似,但各化学成分含量存在不同程度的差异。进一步比较分析其指纹图谱中9个共有峰所代表成分含量的变化,发现黄芩苷、汉黄芩苷等苷类成分以及一些极性较大的成分在展叶期或果实成熟期含量最大;黄芩素、汉黄芩素和千层纸素等苷元类成分含量多在花期最高。黄芩茎、叶和花果指纹图谱在不同物候期变化较大,化学成分在种类和含量上均发生改变,变化程度以花最显著,茎次之,叶最小。然而在相同物候期黄芩茎、叶和花果中化学成分比较相似,仅在含量上有差异。
结果显示在展叶期,不同产地黄芩根中黄芩苷含量均达到峰值,且北京延庆在果实成熟期还出现了第二个峰值;3种有效成分总量均在枯黄期达到最大值,其中吉林长春增加幅度较小。其中,3种有效成分总量计算公式为黄芩根重×(黄芩苷含量+黄芩素含量+汉黄芩素含量)。在展叶期不同产地黄芩茎和叶中野黄芩苷含量较高,而黄芩苷含量变化没有规律。不同产地花果中野黄芩苷和黄芩苷一般在显蕾期和始花期含量较高。文献报道野黄芩苷具有降血脂、抗氧化等作用,以上研究结果显示黄芩茎叶二者含量较高,因此将黄芩茎叶入药,可以有效地利用药用资源。
3黄芩生物量和营养元素的分配规律的研究
研究发现在不同物候期北京延庆黄芩不同器官生物量及其所含氮钙、镁、磷和钾等矿质元素分配情况存在显著变化。在展叶期和枯黄期黄芩根生物量最大,该时期正好是根中黄芩苷含量和3种有效成分总量出现峰值的时期;在展叶期或果实成熟期根中上述元素比例最大,这与根中黄芩苷含量达到峰值的时间较一致。
4黄芩关键物候期的气候特点及其适宜采收期确定
植物物候现象是过去和现在各种环境因素的综合反映,研究发现随着全球气候的变暖,植物春季物候期提前,秋季物候期推迟,生长季延长。因此本文采用物候期来划分中药材采收期较过去的月份或季节较为科学。研究发现展叶期和枯黄期是黄芩根中次生代谢产物形成和积累的关键时期,根中黄芩苷含量的变化与展叶期有较强的相关性,根中黄芩苷和3种有效成分总量的变化与枯黄期有较强的相关性。因此对上述产地气候数据进行分析,总结出黄芩展叶期的气候特点:日最高气温＞20℃的时间(天)占展叶期比例在90%以上。枯黄期的气候特点:平均气温、日最高气温、日最低气温和有效积温(＞10℃)的变化基本呈下降趋势;平均气温＞15℃和日最高气温＞25℃的时间(天)占枯黄期比例在20%以下。
中药材采收期的确定应综合考虑药材产量和化学成分含量,因此本文选择黄芩根中3种有效成分总量作为确定黄芩采收期的评价指标。研究发现在黄芩生长第三年的枯黄期,根中3种有效成分总量最高,但考虑到在其第二年的枯黄期时,根中3种有效成分总量已达到第三年枯黄期总量的80%左右,并且根部在第三年开始出现枯心,故选择黄芩的最佳采收期是第二年的枯黄期。
5影响黄芩3种有效成分总量的气候和土壤因子的筛?
通径分析和相关分析结果表明日最高气温、日最低气温、日较差、日照时数和＞10有效积温是影响黄芩根中3种有效成分总量的重要气候因子;有效磷和土壤水分是影响根中3种有效成分总量的重要土壤因子。
6基于环境效应的黄芩根3种有效成分总量预测模型的建立与验证
采用山东莒县和吉林长春的田间试验数据建立模型,以黄芩根中3种有效成分总量为因变量,把通径分析和相关分析筛选出的重要气候和土壤因子作为自变量,采用matlab软件中Lars算法建立基于环境效应的黄芩根3种有效成分总量预测模型,如下Y=-0.24429×X_2-1.7513×X_4+4.7647×X_7-5.066×X_9+1.929×X_2×X_4-0.93945×X_7~2
Y-黄芩根中3种有效成分总量、X_2-日最高气温、X_4-日较差、X_7-日照时数、X_9-＞10有效积温
利用北京延庆的田间试验数据对模型进行验证,预测的回归估计标准误差(RMSE)为24%,结果显示模拟值与观测值之间具有较好的一致性。本模型预测效果较好,可为黄芩采收期的确定提供帮助。但本模型的研究尚处于摸索阶段,有待于以后进一步地完善。
The quality of Chinese medicinal materials determined the efficacy and clinical effect.How to seize suitable collecting time and gain top-quality medicinal material has been the focus of attention.Scutellaria baicalensis is the dried root of Baical Skullcap belonging to labiate,and it is a well-known traditional Chinese medicine.The phenophase of Scutellaria baicalensis was investigated in the fields of Yanqing,Beijing, Juxian,Shandong province,and Changchun,Jilin province,the ruel of appearance character and internal quality was studied,and thus the optimum harvest time was determined.Meanwhile climatic factors and soil factors in the experimental field of Scutellaria baicalensis were investigated,and some of them were selected as the main impact factors.In order to provid guidance for determination of the best harvest and to provid new thinking and new methods for the research of the harvest time of root drugs, an environment model for predicting the three effective components in Scutellaria roots was set up by the lars method in matlab software on this basis.Main research resaults were as followings:
1 Research on the rule of root yields of Scutellaria baicalensis
Root weight and root diameter of Scutellaria baicalensis with different growth years increased slowly before fruit maturation period,rose rapidly after it,and reached maximum in leaf yellow period.Root weight rose markedly from the first year to the second year,and didn't slow down until the third year.It was found that the color of root bark deepened,the surface of root had obvious wrinkles and easily deciduous corks,and some roots were found with dead hearts with the increasing of cultivated years.
2 Research on the rule of chemical constituents in Scutellaria baicalensis
The analysis of content and fingerprint were developed by HPLC and UPLC method,respectively.
The research resaults indicated that main ingredients in roots were distinct obviously from those in stems,leaf,flowers and fruits,major constituents in roots included baicalin, wogonoside,baicalein,wogonin and oroxylin,etc,those in other locations of Scutellaria baicalensis were scutellarin and baicalin,etc.The resualts of fingerprint similarity evaluation showed that chemical constituents in Scutellaria roots collected in differet phenophases had good consistency in number,but made a difference only in content. Based on the comparasion of the content change of 9 common peaks representing elements,it was found that the contents of baicalin,wogonoside and some components with high polarity reached peaks in leaf expansion period or fruit maturation period,while the maximum content of baicalein,wogonin and oroxylin was found in blooming period. The resaults also showed that main ingredients in stems,leafs,flowers and fruits changed greatly in different phenophases,which varied in number and in content.Chemical constituents in flowers and fruits changed in the highest degree,those in stems took second place,those in leafs varied lowest.However,samples collected from the three areas only changed in content in the same phenophase.
The content peak of baicalin in Scutellaria roots collected from the three places above_mentioned appeared in leaf expansion period,but in Yanqing,Beijing the content arrived at the second one in fruit maturation period.The total contents of the three effective components all reached the maximum in leaf yellow period,and in Changchun,Jilin province they increased less than in the other two places.The content of scutellarin in Scutellaria stems and leaves was higher in leaf expansion period than in the other phenophases,but that of baicalin varied differently,and in the period of squaring and early flowering the contents of scutellarin and baicalin in flowers and fruits were higher than in the other phenophases.It was reported that scutellarin had the function of antioxidation and reducing blood lipid.Because the contents of scutellarin in Scutellaria stems and leaves were in a high level,they can be used as medicines on effectively utilizing resources.
3 Distribution laws of biomass and nutrient elements in different locations of Scutellaria baicalensis
The proportion of biomass of different locations of Scutellaria baicalensis and that of N,Ca,Mg,P and K content were computed.The distribution law showed that in leaf expansion period and in leaf yellow period the proportion of biomass of root reached the highest,and in leaf expansion period or in fruit maturation period that of N,Ca,Mg,P and K was the biggest.The distribution laws showed that the phenophases when the biggest percentage of biomass of roots and that of nutrient element content in roots were consistent with the time when the contents of baicalin and the three effective components in roots were in the highest level.
The harvest time of Chinese medicinal materials was determined on the basis of contents of chemical components and yields.So this paper selected the three effective components in Scutellaria roots as evaluation index,and found that the total content in three-year-old roots were higher than in one-year-old or two-year-old roots.Considering that the proportion of the total content in two-year-old roots was about 80%of that in three-year-old roots,and some three-year-old roots were found with dead hearts,So Scutellaria baicalensis should be harvested in leaf yellow period in two-year-old.
4 Climatic characteristic of the key phenophase of Scutellaria baicalensis and determination of the best haverst time
Plant phenology phenomenon was synthetical reflection of the environmental factor of past and present.The plant phenophase had an earlier onset of spring,a delay of autumn, and a lengthening of growing season with the global warming.So the division of the harvest time by phenomenon was more scientific than by month or season.It showed that leaf expansion period and leaf yellow period were the key stages for the growth and the development and secondary metabolism.There was a strong correlation betweent the contents of baicalin and the three effective components and phenophases.Based on sorting and analyzing the daily climate data,the climatic characteristics of leaf expansion period and leaf yellow period were as followed,respectively.In leaf expansion period,the days appearing daily maximum temperature above 20℃were more than 90%of the days of leaf expansion period.In yellow period,average air temperature,daily maximum temperature,daily minimum temperature and effective accumulated temperature above 10℃showed a downward trend;the days appearing average air temperature above 15℃and daily maximum temperature above 25℃were less than 20%of the days of leaf yellow period.
5 Selection of climatic factors and soil factors related to the three effective components in Scutellaria roots
Path analysis and correlative analysis revealed that cliamatic factors such as daily maximum temperature,daily minimum temperature,daily range of temperature,sunshine hours and effective accumulated temperature above 10℃were important for the total content of three effective components in Scutellaria roots,soil factors including available P and soil moisture were important for it.
6 Development and validation of an environment model for predicting the total content of the three effective components in Scutellaria roots
Based on the experiments conducted in Shandong province,and Changchun,Jilin province,the total content of the three effective components in Scutellaria roots independent variable and climatic factors and soil factors dependent variable were made respectively,and an environment model for predicting it was established by the lars method in matlab software.The model was as followings.
Y=-0.24429×X_2-1.7513×X_4+4.7647×X_7-5.066×X_9+1.929×X_2×X_4-0.93945×X_7~2
Y- the total content of the three effective components、X_2- daily maximum temperature、X_4- daily range of temperature、X_7- sunshine hours、X_9- effective accumulated temperature above 10℃
The model was tested by the different data in Yanqing,Beijing,the RMSE values was less than 30%.The results indicated a better fit between the simulated and observed values of the total content of the three effective components.The prediction effect of the model was good,so it can provide help for the determination of the best harvest time.Nevertheless,this model study was still at the beginning stage at present,and it should be perfected further.
1黄芩根产量变化规律的研究
不同生长年限的黄芩,其根重和根直径在果实成熟期之前均增加缓慢;进入果实成熟期,两者均大幅增加,并在枯黄期达到最大值。不同生长年限的黄芩相比,在其生长发育的第一年和第二年,根重呈急剧上升趋势,到第三年增长速率显著减慢;而且随着生长年限的增加,黄芩根皮颜色加深,纵皱纹明显,有易剥落的栓皮,并开始出现枯心。上述三产地黄芩根重和根直径的变化趋势基本一致。
2黄芩化学成分变化规律的研究
本文采用高效液相色谱仪(HPLC)和超高效液相色谱仪(UPLC)分别建立了黄芩不同部位的指纹图谱和含量测定方法。
研究发现黄芩根中主要化学成分与茎、叶和花果存在明显差异,根中成分主要是黄芩苷、汉黄芩苷、黄芩素、汉黄芩素和千层纸素等,其它部位中主要成分则为野黄芩苷和黄芩苷。不同物候期的黄芩根指纹图谱相似度均在0.9以上,说明在不同物候期黄芩根中化学成分种类很相似,但各化学成分含量存在不同程度的差异。进一步比较分析其指纹图谱中9个共有峰所代表成分含量的变化,发现黄芩苷、汉黄芩苷等苷类成分以及一些极性较大的成分在展叶期或果实成熟期含量最大;黄芩素、汉黄芩素和千层纸素等苷元类成分含量多在花期最高。黄芩茎、叶和花果指纹图谱在不同物候期变化较大,化学成分在种类和含量上均发生改变,变化程度以花最显著,茎次之,叶最小。然而在相同物候期黄芩茎、叶和花果中化学成分比较相似,仅在含量上有差异。
结果显示在展叶期,不同产地黄芩根中黄芩苷含量均达到峰值,且北京延庆在果实成熟期还出现了第二个峰值;3种有效成分总量均在枯黄期达到最大值,其中吉林长春增加幅度较小。其中,3种有效成分总量计算公式为黄芩根重×(黄芩苷含量+黄芩素含量+汉黄芩素含量)。在展叶期不同产地黄芩茎和叶中野黄芩苷含量较高,而黄芩苷含量变化没有规律。不同产地花果中野黄芩苷和黄芩苷一般在显蕾期和始花期含量较高。文献报道野黄芩苷具有降血脂、抗氧化等作用,以上研究结果显示黄芩茎叶二者含量较高,因此将黄芩茎叶入药,可以有效地利用药用资源。
3黄芩生物量和营养元素的分配规律的研究
研究发现在不同物候期北京延庆黄芩不同器官生物量及其所含氮钙、镁、磷和钾等矿质元素分配情况存在显著变化。在展叶期和枯黄期黄芩根生物量最大,该时期正好是根中黄芩苷含量和3种有效成分总量出现峰值的时期;在展叶期或果实成熟期根中上述元素比例最大,这与根中黄芩苷含量达到峰值的时间较一致。
4黄芩关键物候期的气候特点及其适宜采收期确定
植物物候现象是过去和现在各种环境因素的综合反映,研究发现随着全球气候的变暖,植物春季物候期提前,秋季物候期推迟,生长季延长。因此本文采用物候期来划分中药材采收期较过去的月份或季节较为科学。研究发现展叶期和枯黄期是黄芩根中次生代谢产物形成和积累的关键时期,根中黄芩苷含量的变化与展叶期有较强的相关性,根中黄芩苷和3种有效成分总量的变化与枯黄期有较强的相关性。因此对上述产地气候数据进行分析,总结出黄芩展叶期的气候特点:日最高气温＞20℃的时间(天)占展叶期比例在90%以上。枯黄期的气候特点:平均气温、日最高气温、日最低气温和有效积温(＞10℃)的变化基本呈下降趋势;平均气温＞15℃和日最高气温＞25℃的时间(天)占枯黄期比例在20%以下。
中药材采收期的确定应综合考虑药材产量和化学成分含量,因此本文选择黄芩根中3种有效成分总量作为确定黄芩采收期的评价指标。研究发现在黄芩生长第三年的枯黄期,根中3种有效成分总量最高,但考虑到在其第二年的枯黄期时,根中3种有效成分总量已达到第三年枯黄期总量的80%左右,并且根部在第三年开始出现枯心,故选择黄芩的最佳采收期是第二年的枯黄期。
5影响黄芩3种有效成分总量的气候和土壤因子的筛?
通径分析和相关分析结果表明日最高气温、日最低气温、日较差、日照时数和＞10有效积温是影响黄芩根中3种有效成分总量的重要气候因子;有效磷和土壤水分是影响根中3种有效成分总量的重要土壤因子。
6基于环境效应的黄芩根3种有效成分总量预测模型的建立与验证
采用山东莒县和吉林长春的田间试验数据建立模型,以黄芩根中3种有效成分总量为因变量,把通径分析和相关分析筛选出的重要气候和土壤因子作为自变量,采用matlab软件中Lars算法建立基于环境效应的黄芩根3种有效成分总量预测模型,如下Y=-0.24429×X_2-1.7513×X_4+4.7647×X_7-5.066×X_9+1.929×X_2×X_4-0.93945×X_7~2
Y-黄芩根中3种有效成分总量、X_2-日最高气温、X_4-日较差、X_7-日照时数、X_9-＞10有效积温
利用北京延庆的田间试验数据对模型进行验证,预测的回归估计标准误差(RMSE)为24%,结果显示模拟值与观测值之间具有较好的一致性。本模型预测效果较好,可为黄芩采收期的确定提供帮助。但本模型的研究尚处于摸索阶段,有待于以后进一步地完善。
The quality of Chinese medicinal materials determined the efficacy and clinical effect.How to seize suitable collecting time and gain top-quality medicinal material has been the focus of attention.Scutellaria baicalensis is the dried root of Baical Skullcap belonging to labiate,and it is a well-known traditional Chinese medicine.The phenophase of Scutellaria baicalensis was investigated in the fields of Yanqing,Beijing, Juxian,Shandong province,and Changchun,Jilin province,the ruel of appearance character and internal quality was studied,and thus the optimum harvest time was determined.Meanwhile climatic factors and soil factors in the experimental field of Scutellaria baicalensis were investigated,and some of them were selected as the main impact factors.In order to provid guidance for determination of the best harvest and to provid new thinking and new methods for the research of the harvest time of root drugs, an environment model for predicting the three effective components in Scutellaria roots was set up by the lars method in matlab software on this basis.Main research resaults were as followings:
1 Research on the rule of root yields of Scutellaria baicalensis
Root weight and root diameter of Scutellaria baicalensis with different growth years increased slowly before fruit maturation period,rose rapidly after it,and reached maximum in leaf yellow period.Root weight rose markedly from the first year to the second year,and didn't slow down until the third year.It was found that the color of root bark deepened,the surface of root had obvious wrinkles and easily deciduous corks,and some roots were found with dead hearts with the increasing of cultivated years.
2 Research on the rule of chemical constituents in Scutellaria baicalensis
The analysis of content and fingerprint were developed by HPLC and UPLC method,respectively.
The research resaults indicated that main ingredients in roots were distinct obviously from those in stems,leaf,flowers and fruits,major constituents in roots included baicalin, wogonoside,baicalein,wogonin and oroxylin,etc,those in other locations of Scutellaria baicalensis were scutellarin and baicalin,etc.The resualts of fingerprint similarity evaluation showed that chemical constituents in Scutellaria roots collected in differet phenophases had good consistency in number,but made a difference only in content. Based on the comparasion of the content change of 9 common peaks representing elements,it was found that the contents of baicalin,wogonoside and some components with high polarity reached peaks in leaf expansion period or fruit maturation period,while the maximum content of baicalein,wogonin and oroxylin was found in blooming period. The resaults also showed that main ingredients in stems,leafs,flowers and fruits changed greatly in different phenophases,which varied in number and in content.Chemical constituents in flowers and fruits changed in the highest degree,those in stems took second place,those in leafs varied lowest.However,samples collected from the three areas only changed in content in the same phenophase.
The content peak of baicalin in Scutellaria roots collected from the three places above_mentioned appeared in leaf expansion period,but in Yanqing,Beijing the content arrived at the second one in fruit maturation period.The total contents of the three effective components all reached the maximum in leaf yellow period,and in Changchun,Jilin province they increased less than in the other two places.The content of scutellarin in Scutellaria stems and leaves was higher in leaf expansion period than in the other phenophases,but that of baicalin varied differently,and in the period of squaring and early flowering the contents of scutellarin and baicalin in flowers and fruits were higher than in the other phenophases.It was reported that scutellarin had the function of antioxidation and reducing blood lipid.Because the contents of scutellarin in Scutellaria stems and leaves were in a high level,they can be used as medicines on effectively utilizing resources.
3 Distribution laws of biomass and nutrient elements in different locations of Scutellaria baicalensis
The proportion of biomass of different locations of Scutellaria baicalensis and that of N,Ca,Mg,P and K content were computed.The distribution law showed that in leaf expansion period and in leaf yellow period the proportion of biomass of root reached the highest,and in leaf expansion period or in fruit maturation period that of N,Ca,Mg,P and K was the biggest.The distribution laws showed that the phenophases when the biggest percentage of biomass of roots and that of nutrient element content in roots were consistent with the time when the contents of baicalin and the three effective components in roots were in the highest level.
The harvest time of Chinese medicinal materials was determined on the basis of contents of chemical components and yields.So this paper selected the three effective components in Scutellaria roots as evaluation index,and found that the total content in three-year-old roots were higher than in one-year-old or two-year-old roots.Considering that the proportion of the total content in two-year-old roots was about 80%of that in three-year-old roots,and some three-year-old roots were found with dead hearts,So Scutellaria baicalensis should be harvested in leaf yellow period in two-year-old.
4 Climatic characteristic of the key phenophase of Scutellaria baicalensis and determination of the best haverst time
Plant phenology phenomenon was synthetical reflection of the environmental factor of past and present.The plant phenophase had an earlier onset of spring,a delay of autumn, and a lengthening of growing season with the global warming.So the division of the harvest time by phenomenon was more scientific than by month or season.It showed that leaf expansion period and leaf yellow period were the key stages for the growth and the development and secondary metabolism.There was a strong correlation betweent the contents of baicalin and the three effective components and phenophases.Based on sorting and analyzing the daily climate data,the climatic characteristics of leaf expansion period and leaf yellow period were as followed,respectively.In leaf expansion period,the days appearing daily maximum temperature above 20℃were more than 90%of the days of leaf expansion period.In yellow period,average air temperature,daily maximum temperature,daily minimum temperature and effective accumulated temperature above 10℃showed a downward trend;the days appearing average air temperature above 15℃and daily maximum temperature above 25℃were less than 20%of the days of leaf yellow period.
5 Selection of climatic factors and soil factors related to the three effective components in Scutellaria roots
Path analysis and correlative analysis revealed that cliamatic factors such as daily maximum temperature,daily minimum temperature,daily range of temperature,sunshine hours and effective accumulated temperature above 10℃were important for the total content of three effective components in Scutellaria roots,soil factors including available P and soil moisture were important for it.
6 Development and validation of an environment model for predicting the total content of the three effective components in Scutellaria roots
Based on the experiments conducted in Shandong province,and Changchun,Jilin province,the total content of the three effective components in Scutellaria roots independent variable and climatic factors and soil factors dependent variable were made respectively,and an environment model for predicting it was established by the lars method in matlab software.The model was as followings.
Y=-0.24429×X_2-1.7513×X_4+4.7647×X_7-5.066×X_9+1.929×X_2×X_4-0.93945×X_7~2
Y- the total content of the three effective components、X_2- daily maximum temperature、X_4- daily range of temperature、X_7- sunshine hours、X_9- effective accumulated temperature above 10℃
The model was tested by the different data in Yanqing,Beijing,the RMSE values was less than 30%.The results indicated a better fit between the simulated and observed values of the total content of the three effective components.The prediction effect of the model was good,so it can provide help for the determination of the best harvest time.Nevertheless,this model study was still at the beginning stage at present,and it should be perfected further.
引文
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