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Abstract Sulfuric acid-phenol and sulfuric acid-anthrone methods were used to detect polysaccharide content in shoots of Aralia elata （Miq.） Seem.， and the conversion factor to glucose was measured with refined polysaccharides. Comprehensive evaluation was carried out by linear relationship， precision， reproducibility， stability and recovery rate. The results showed that the linear relationship between glucose concentration and absorbance was good when glucose concentration was 0-40 μg/ml， and the average recovery rate was equal to or higher than 97.00% with good reproducibility （RSD<1.60%， n=5）. It revealed that the two methods were accurate and reliable， and suitable for the determination of polysaccharide content in the shoots of A. elata. Polysaccharide content detected by sulfuric acid-phenol and sulfuric acid-anthrone methods was 19.31% and 20.40% respectively.
Key words Shoots of Aralia elata （Miq.） Seem.； Polysaccharides； Sulfuric acid-phenol method； Sulfuric acid-anthrone method
Aralia elata （Miq.） Seem. is a perennial deciduous tree of Aralia Linn. of the Araliaceae. It is commonly known as northeast Chinese toon[1]. As a traditional medicinal and food plant with high medicinal and edible value， it is can be used to tonify Qi， tonify spleen， dispel wind and eliminate dampness， promote blood circulation and stop pain. Modern studies have shown that A. elata is mainly used to cure neurasthenia， constipation， stomach cramps， diabetes， rheumatoid arthritis， hepatitis and other diseases[2]. However， most studies on A. elata are focused on the content of chemical constituents such as total saponins and total flavonoids and their pharmacological activities in its root bark and stem bark， while there are few studies on the determination of polysaccharide content in the shoots of A. elata[3]. Polysaccharides in A. elata are a type of neutral heteropolysaccharides with the molecular weight of about 8 000， and can be mainly hydrolyzed into monosaccharides such as arabinose， glucose and galactose[4]. The pharmacological activities of polysaccharides in suppressing tumors， immune regulation， lowering the concentration of glucose in the blood， reducing blood fat， destroying or inhibiting the growth and reproduction of viruses， and resisting radiation have attracted the attention of the medical community at home and abroad. At present， there is no systematic report on the determination of polysaccharides in the shoots of A. elata[5]. In this study， polysaccharide content in shoots of A. elata was detected using sulfuric acid-phenol and sulfuric acid-anthrone methods respectively， and the differences between the two method were compared， which provide scientific reference for the determination of polysaccharide content in the shoots of A. elata and is of great significance for the effective control of intrinsic quality of shoots of A. elata. Materials and Methods
Materials and reagents
The shoots of A. elata were collected from Dandong City， Liaoning Province and identified by a Doctor of Pharmacognosy Professor Lian Lian of Liaoning Institute of Science and Technology.
The main reagents included anhydrous glucose （Sinopharm Chemical Reagent Co.， Ltd.）， phenol （Sinopharm Chemical Reagent Co.， Ltd.）， concentrated sulfuric acid （Sinopharm Chemical Reagent Co.， Ltd.）， anthrone （Sinopharm Chemical Reagent Co.， Ltd.）， diethyl ether （AR/Tianjin Fuyu Fine Chemical Co.， Ltd.）， ethyl acetate （AR/Tianjin Fuyu Fine Chemicals Co.， Ltd.）， anhydrous ethanol （AR/Tianjin Fuyu Fine Chemicals Co.， Ltd.）， and distilled water.
Instruments and equipment
The main instruments included a 7230 spectrophotometer （Hewlett-Packard Shanghai Analytical Instrument Co.， Ltd.）， an electronic analytical balance （Shanghai Jingtian Electronic Instrument Co.， Ltd.）， a FD-1A-50 refrigerant dryer （Beijing Boyikang Laboratory Equipment Co.， Ltd.）， and a water bath （Jiangsu Jintan Jincheng Guosheng Experimental Instrument Factory）， a rotary evaporator （Shanghai Yarong Biochemical Instrument Factory）， and a circulating water vacuum pump （Guoyi Yuhua Instrument Company）.
Methods
Extraction and refinement of polysaccharides in the shoots of A. elata
The shoots of A. elata were dried with the FD-1A-50 refrigerant dryer， smashed and sieved with a 60-mesh sieve. Afterwards， 30.0 g of the powder was soaked for 24 h in 500 ml of 75% alcohol， and then the mixture was refluxed for 1 h in a water bath. After suction filtration， the filter cake was washed twice with 75% ethanol， and the ethanol was removed under reduced pressure. After it was dried， 1 000 ml of distilled water was added to it， and then polysaccharides were extracted for 1 h in a water bath at 95 ℃. After suction filtration was repeated twice， all filtrate was merged and concentrated to 50 ml under reduced pressure. Protein was removed from it by using Sevag method， and extraction lasted for 25 min and was repeated 15 times. The supernatant was dialyzed with distilled water for 24 h， to which alcohol was added until its volume fraction was up to 80%. It stayed overnight and centrifuged. The precipitate was washed with ethyl ether， ethyl acetate， and anhydrous ethanol respectively， and was dried under reduced pressure to obtain polysaccharides[6].
Drawing of glucose standard curves
Preparation of standard solution Firstly， 50 mg of glucose standard that was dried at 105 ℃ until its weight was constant in advance was placed in a small dry beaker， to which an appropriate amount of distilled water was added. The solution was stirred with a glass rod to make it dissolved and transferred to a volumetric flask （50 ml in volume）， and distilled water was added to it until its volume was up to 50 ml. Afterwards， 10 ml of the above solution was placed in another volumetric flask （100 ml in volume）， and distilled water was added to it until its volume was up to 100 ml. Finally， 100 μg/ml standard solution was obtained.
Preparation of sulfuric acid-anthrone solution
At first， 100 ml of concentrated sulphuric acid was added to 0.21 g of anthrone， and then the mixture was placed in a brown bottle. Afterwards， it was shaken well to obtain 2.1 mg/ml sulfuric acid-anthrone solution and put in a refrigerator （it was prepared when it was needed）.
Sulfuric acid-phenol method
Firstly， 0， 0.1， 0.2， 0.3， 0.4， 0.5， 0.6， 0.7 and 0.8 ml of the glucose standard solution was taken with a pipette and put in colorimetric tubes （10 ml in volume） respectively. Afterwards， distilled water was added to each of the colorimetric tubes until the volume was up to 2.0 ml. After 1.0 ml of 5% phenol solution was added to each of the colorimetric tubes， the mixed liquor was shaken well. Then 5.0 ml of concentrated sulphuric acid was added to each of the colorimetric tubes quickly， and the mixed liquor was shaken well again. Developing lasted for 20 min at room temperature. After absorbance was detected at 490 nm， a standard curve was drawn， and a regression equation was obtained[7-8].
Sulfuric acid-anthrone method
At first， 0， 0.1， 0.2， 0.3， 0.4， 0.5， 0.6， 0.7 and 0.8 ml of the glucose standard solution was taken with a pipette and put in colorimetric tubes （10 ml in volume） respectively. Then distilled water was added to each of the colorimetric tubes until the volume was up to 2.0 ml， and the mixed liquor was shaken well. Afterwards， it was put in an ice-water bath， to which 5.0 ml of the sulfuric acid-anthrone solution was added. After the mixed liquor was heated for 7 min in a boiling water bath， it cooled down at room temperature. After absorbance was detected at 625 nm， a standard curve was drawn， and a regression equation was obtained[9].
Detection of content of crude polysaccharides in the samples
Determination of conversion factor Firstly， 5.0 mg of refined polysaccharides extracted from the shoots of A. elata was put in a volumetric flask （25 ml in volume）， and then distilled water was added to the volumetric flask until its volume was up to 25 ml. After being shaken well， the solution was as the stock solution of polysaccharides. After 0.2 ml of the stock solution was taken， chromogenic reaction was conducted according to the above two methods， and absorbance was detected. Glucose concentration in the stock solution of refined polysaccharides extracted from the shoots of A. elata was calculated based on the standard curve， and conversion factor （f） was calculated according to the formula （1） as follows：
f =W/（C×D）（1）
where W is the weight of refined polysaccharides extracted from the shoots of A. elata （mg）； C is glucose concentration in the stock solution of polysaccharides extracted from the shoots of A. elata （μg/ml）； D is the dilution ratio of refined polysaccharides extracted from the shoots of A. elata[6，9].
Preparation of the test solution
At first， 1.0 g of powder of shoots of A. elata was soaked overnight in 150 ml of 75% alcohol， and then the mixture was refluxed for 1 h in a water bath. After suction filtration， the filter cake was washed twice with 75% ethanol， and the ethanol was removed under reduced pressure. After it was dried， 90 ml of distilled water was added to it， and then polysaccharides were extracted for 1 h at 95 ℃. After suction filtration was repeated twice， all filtrate was merged and concentrated under reduced pressure. The filtrate was placed in a volumetric flask （100 ml in volume）， to which distilled water was added until the volume was up to 100 ml. It was shaken well and as the test stock solution.
Determination of polysaccharide content in the shoots of A. elata
Firstly， 0.2 ml of the above test stock solution was taken， to which a certain amount of distilled water was added until the volume was up to 2 ml. Afterwards， absorbance （A） was detected using the above two methods mentioned in "Drawing of glucose standard curves"， and glucose concentration （C） in the test solution was calculated based on the regression equation. Finally， polysaccharide content （B） in the shoots of A. elata was calculated according to the formula （2） as follows：
B=（C×D×f）/1 000m×100%（2）
where C is glucose concentration in the samples （μg/ml）； D is the dilution ratio of refined polysaccharides extracted from the shoots of A. elata； f is conversion factor； m is the mass of shoots of A. elata （mg）. Precision test
At first， a certain amount of glucose control solution was taken， and chromogenic reaction was conducted according to the above two methods， and absorbance was detected five times to study the precision of the instrument.
Stability test
Firstly， 0.2 ml of stock solution of polysaccharides extracted from the shoots of A. elata was put in a colorimetric tube， to which distilled water was added until the volume was up to 2.0 ml. Meanwhile， 2.0 ml of distilled water was as the control group， and chromogenic reaction was conducted according to the above two methods. Finally， absorbance was detected every 30 min and lasted for 2 h to study its stability.
Reproducibility test
At first， five samples of powder of shoots of A. elata was taken to make five test solutions at the same time according to the methods mentioned above. Afterwards， 0.2 ml of each of the test solutions was taken， and chromogenic reaction was conducted according to the above two methods. Finally， absorbance was detected to study its reproducibility.
Recovery rate test
The test solution of polysaccharides extracted from the shoots of A. elata was taken precisely and put in a colorimetric tube， to which glucose standard solution was added. Afterwards， chromogenic reaction was conducted according to the above two methods. Finally， absorbance was detected to calculate recovery rate.
Results and Analysis
Standard curves and regression equations
Sulfuric acid-phenol method
A standard curve was drawn using glucose concentration as the abscissa and absorbance as the ordinate （Fig.1）. The regression equation was y=0.013 4x+0.023 5， R2=0.999 3. There was a good linear relationship between absorbance and glucose concentration （0-40 μg/ml）.
Sulfuric acid-anthrone method
A standard curve was drawn using glucose concentration as the abscissa and absorbance as the ordinate （Fig.2）. The regression equation was y=0.014 6x+0.029 7， R2=0.999 6. There was also a good linear relationship between absorbance and glucose concentration （0-40 μg/ml）.
Results of conversion factor
The values of conversion factor measured by sulfuric acid-phenol and sulfuric acid-anthrone methods were 1.16 and 1.74 respectively.
Results of precision test
According to Table 1， after absorbance was detected five times， the values of relative standard deviation （RSD） detected by sulfuric acid-phenol and sulfuric acid-anthrone methods were 1.39% and 0.75% respectively， showing that the precision of the instrument was good. Results of polysaccharide content in the shoots of A. elata
In the same series of shoots of A. elata， polysaccharide content detected by sulfuric acid-phenol and sulfuric acid-anthrone methods was 19.31% and 20.40% respectively.
Discussion
The methods used for the determination of polysaccharide content mainly include colorimetric method and titration method at the present， in which the content of monosaccharide obtained through hydrolysis is used to reflect polysaccharide content， and the content of total sugar is detected in fact[10]. Sulfuric acid-phenol and sulfuric acid-anthrone methods are commonly used colorimetric methods， and their developing principles are basically the same. That is， polysaccharides are hydrolyzed into monosaccharides under the action of concentrated sulfuric acid， and furfural derivatives were formed after rapid dehydration； they can react with phenol to form an orange-yellow compound or condense with anthrone to form a blue-green compound， which has a maximum absorption peak at wavelengths of 490 and 625 nm respectively[11].
Conclusions
In this study， polysaccharide content in the shoots of A. elata was detected using sulfuric acid-phenol and sulfuric acid-anthrone methods respectively， and the methodological evaluation was performed by the linear relationship， precision， stability， reproducibility， and recovery rate tests. Moreover， the conversion factor of glucose was corrected by the detection of the refined polysaccharides extracted from the shoots of A. elata to reduce measured deviation when glucose was used as the standard control. The results showed that the methods of detecting polysaccharide content in the shoots of A. elata had good accuracy and precision， and the average recovery rate was higher than 97%. The average content of polysaccharides in the shoots of A. elata detected by sulfuric acid-phenol and sulfuric acid-anthrone methods was 19.31% and 20.40% respectively. That is， the value detected by sulfuric acid-anthrone method was higher， because sulfuric acid-anthrone method can determine almost all of the carbohydrates， and the total amount of all soluble carbohydrates was measured by this method. The results provide scientific basis for the scientific evaluation of quality of shoots of A. elata.
References
[1] Editorial Board of Chinese Academy of Sciences. Chinese flora[M]. Beijing： Science Press， 1998： 165-166.
[2] ZHANG ZQ. Research status of Aralia elata （Miq.） Seem. at home and abroad[J]. Heilongjiang Medicine Journal， 2008， 21（1）：46-20. [3] REN FZ， LUAN XH， QU HH. Research progress on chemical composition and pharmacological activities of Aralia plants[J]. Guang Zhou Chemical Industry and Technology， 2000，28（4）：124-130.
[4] AYIGULI AHMT， WANG Y， YANG Xj， et al. Study on extraction method and assay of polysaccharides of Lycium barbarum L. from two different source[J]. Xinjiang Agricultural Sciences，2007，25（5）：724-728.
[5] GUO XL， ZHU SC， ZHAI XF， et al. Comparison of methods in determination of polysaccharide in Ganoderma lucidum[J]. Chinese Archives of Traditional Chinese Medicine，2010，28（9）：2000-2002.
[6] OUYANG Z， LI YH， SHU SL， et al. Determination of polysaccharide in leaves of Morus alba L.[J]. Food Science， 2003，24（11）：118-120.
[7] XU GY， YAN J， GUO XJ， et al. The betterment and apply of phenol-sulphate acid method[J]. Food Science， 2006，26（8）：342-346.
[8] HOU HH， WANG YF， ZHAO B， et al. Ultrasonic extraction and determination of polysaccharides from Aralia chinensis L. roots[J]. Chinese Journal of Bioprocess Engineering，2015，13（2）：19-23.
[9] FENG Y， HAN N， XU DS. Study on the determination of polysaccharides from Ophiopogon japonicus[J]. Chinese Traditional Patent Medicine，2006，28（5）：705-707.
[10] HUANG RS. A summary of determination methods of polysaccharide content in Chinese herbal medicine[J]. China Pharmacist，2005，8（1）：68-70.
[11] DUAN JF， WANG YX， LI XY， et al. Diversity of polysaccharide content in five species of zizphus jujube in Alar area and their in vitro antioxidative activities[J]. Xinjiang Agricultural Sciences， 2014，51（5）：831-838.
Key words Shoots of Aralia elata （Miq.） Seem.； Polysaccharides； Sulfuric acid-phenol method； Sulfuric acid-anthrone method
Aralia elata （Miq.） Seem. is a perennial deciduous tree of Aralia Linn. of the Araliaceae. It is commonly known as northeast Chinese toon[1]. As a traditional medicinal and food plant with high medicinal and edible value， it is can be used to tonify Qi， tonify spleen， dispel wind and eliminate dampness， promote blood circulation and stop pain. Modern studies have shown that A. elata is mainly used to cure neurasthenia， constipation， stomach cramps， diabetes， rheumatoid arthritis， hepatitis and other diseases[2]. However， most studies on A. elata are focused on the content of chemical constituents such as total saponins and total flavonoids and their pharmacological activities in its root bark and stem bark， while there are few studies on the determination of polysaccharide content in the shoots of A. elata[3]. Polysaccharides in A. elata are a type of neutral heteropolysaccharides with the molecular weight of about 8 000， and can be mainly hydrolyzed into monosaccharides such as arabinose， glucose and galactose[4]. The pharmacological activities of polysaccharides in suppressing tumors， immune regulation， lowering the concentration of glucose in the blood， reducing blood fat， destroying or inhibiting the growth and reproduction of viruses， and resisting radiation have attracted the attention of the medical community at home and abroad. At present， there is no systematic report on the determination of polysaccharides in the shoots of A. elata[5]. In this study， polysaccharide content in shoots of A. elata was detected using sulfuric acid-phenol and sulfuric acid-anthrone methods respectively， and the differences between the two method were compared， which provide scientific reference for the determination of polysaccharide content in the shoots of A. elata and is of great significance for the effective control of intrinsic quality of shoots of A. elata. Materials and Methods
Materials and reagents
The shoots of A. elata were collected from Dandong City， Liaoning Province and identified by a Doctor of Pharmacognosy Professor Lian Lian of Liaoning Institute of Science and Technology.
The main reagents included anhydrous glucose （Sinopharm Chemical Reagent Co.， Ltd.）， phenol （Sinopharm Chemical Reagent Co.， Ltd.）， concentrated sulfuric acid （Sinopharm Chemical Reagent Co.， Ltd.）， anthrone （Sinopharm Chemical Reagent Co.， Ltd.）， diethyl ether （AR/Tianjin Fuyu Fine Chemical Co.， Ltd.）， ethyl acetate （AR/Tianjin Fuyu Fine Chemicals Co.， Ltd.）， anhydrous ethanol （AR/Tianjin Fuyu Fine Chemicals Co.， Ltd.）， and distilled water.
Instruments and equipment
The main instruments included a 7230 spectrophotometer （Hewlett-Packard Shanghai Analytical Instrument Co.， Ltd.）， an electronic analytical balance （Shanghai Jingtian Electronic Instrument Co.， Ltd.）， a FD-1A-50 refrigerant dryer （Beijing Boyikang Laboratory Equipment Co.， Ltd.）， and a water bath （Jiangsu Jintan Jincheng Guosheng Experimental Instrument Factory）， a rotary evaporator （Shanghai Yarong Biochemical Instrument Factory）， and a circulating water vacuum pump （Guoyi Yuhua Instrument Company）.
Methods
Extraction and refinement of polysaccharides in the shoots of A. elata
The shoots of A. elata were dried with the FD-1A-50 refrigerant dryer， smashed and sieved with a 60-mesh sieve. Afterwards， 30.0 g of the powder was soaked for 24 h in 500 ml of 75% alcohol， and then the mixture was refluxed for 1 h in a water bath. After suction filtration， the filter cake was washed twice with 75% ethanol， and the ethanol was removed under reduced pressure. After it was dried， 1 000 ml of distilled water was added to it， and then polysaccharides were extracted for 1 h in a water bath at 95 ℃. After suction filtration was repeated twice， all filtrate was merged and concentrated to 50 ml under reduced pressure. Protein was removed from it by using Sevag method， and extraction lasted for 25 min and was repeated 15 times. The supernatant was dialyzed with distilled water for 24 h， to which alcohol was added until its volume fraction was up to 80%. It stayed overnight and centrifuged. The precipitate was washed with ethyl ether， ethyl acetate， and anhydrous ethanol respectively， and was dried under reduced pressure to obtain polysaccharides[6].
Drawing of glucose standard curves
Preparation of standard solution Firstly， 50 mg of glucose standard that was dried at 105 ℃ until its weight was constant in advance was placed in a small dry beaker， to which an appropriate amount of distilled water was added. The solution was stirred with a glass rod to make it dissolved and transferred to a volumetric flask （50 ml in volume）， and distilled water was added to it until its volume was up to 50 ml. Afterwards， 10 ml of the above solution was placed in another volumetric flask （100 ml in volume）， and distilled water was added to it until its volume was up to 100 ml. Finally， 100 μg/ml standard solution was obtained.
Preparation of sulfuric acid-anthrone solution
At first， 100 ml of concentrated sulphuric acid was added to 0.21 g of anthrone， and then the mixture was placed in a brown bottle. Afterwards， it was shaken well to obtain 2.1 mg/ml sulfuric acid-anthrone solution and put in a refrigerator （it was prepared when it was needed）.
Sulfuric acid-phenol method
Firstly， 0， 0.1， 0.2， 0.3， 0.4， 0.5， 0.6， 0.7 and 0.8 ml of the glucose standard solution was taken with a pipette and put in colorimetric tubes （10 ml in volume） respectively. Afterwards， distilled water was added to each of the colorimetric tubes until the volume was up to 2.0 ml. After 1.0 ml of 5% phenol solution was added to each of the colorimetric tubes， the mixed liquor was shaken well. Then 5.0 ml of concentrated sulphuric acid was added to each of the colorimetric tubes quickly， and the mixed liquor was shaken well again. Developing lasted for 20 min at room temperature. After absorbance was detected at 490 nm， a standard curve was drawn， and a regression equation was obtained[7-8].
Sulfuric acid-anthrone method
At first， 0， 0.1， 0.2， 0.3， 0.4， 0.5， 0.6， 0.7 and 0.8 ml of the glucose standard solution was taken with a pipette and put in colorimetric tubes （10 ml in volume） respectively. Then distilled water was added to each of the colorimetric tubes until the volume was up to 2.0 ml， and the mixed liquor was shaken well. Afterwards， it was put in an ice-water bath， to which 5.0 ml of the sulfuric acid-anthrone solution was added. After the mixed liquor was heated for 7 min in a boiling water bath， it cooled down at room temperature. After absorbance was detected at 625 nm， a standard curve was drawn， and a regression equation was obtained[9].
Detection of content of crude polysaccharides in the samples
Determination of conversion factor Firstly， 5.0 mg of refined polysaccharides extracted from the shoots of A. elata was put in a volumetric flask （25 ml in volume）， and then distilled water was added to the volumetric flask until its volume was up to 25 ml. After being shaken well， the solution was as the stock solution of polysaccharides. After 0.2 ml of the stock solution was taken， chromogenic reaction was conducted according to the above two methods， and absorbance was detected. Glucose concentration in the stock solution of refined polysaccharides extracted from the shoots of A. elata was calculated based on the standard curve， and conversion factor （f） was calculated according to the formula （1） as follows：
f =W/（C×D）（1）
where W is the weight of refined polysaccharides extracted from the shoots of A. elata （mg）； C is glucose concentration in the stock solution of polysaccharides extracted from the shoots of A. elata （μg/ml）； D is the dilution ratio of refined polysaccharides extracted from the shoots of A. elata[6，9].
Preparation of the test solution
At first， 1.0 g of powder of shoots of A. elata was soaked overnight in 150 ml of 75% alcohol， and then the mixture was refluxed for 1 h in a water bath. After suction filtration， the filter cake was washed twice with 75% ethanol， and the ethanol was removed under reduced pressure. After it was dried， 90 ml of distilled water was added to it， and then polysaccharides were extracted for 1 h at 95 ℃. After suction filtration was repeated twice， all filtrate was merged and concentrated under reduced pressure. The filtrate was placed in a volumetric flask （100 ml in volume）， to which distilled water was added until the volume was up to 100 ml. It was shaken well and as the test stock solution.
Determination of polysaccharide content in the shoots of A. elata
Firstly， 0.2 ml of the above test stock solution was taken， to which a certain amount of distilled water was added until the volume was up to 2 ml. Afterwards， absorbance （A） was detected using the above two methods mentioned in "Drawing of glucose standard curves"， and glucose concentration （C） in the test solution was calculated based on the regression equation. Finally， polysaccharide content （B） in the shoots of A. elata was calculated according to the formula （2） as follows：
B=（C×D×f）/1 000m×100%（2）
where C is glucose concentration in the samples （μg/ml）； D is the dilution ratio of refined polysaccharides extracted from the shoots of A. elata； f is conversion factor； m is the mass of shoots of A. elata （mg）. Precision test
At first， a certain amount of glucose control solution was taken， and chromogenic reaction was conducted according to the above two methods， and absorbance was detected five times to study the precision of the instrument.
Stability test
Firstly， 0.2 ml of stock solution of polysaccharides extracted from the shoots of A. elata was put in a colorimetric tube， to which distilled water was added until the volume was up to 2.0 ml. Meanwhile， 2.0 ml of distilled water was as the control group， and chromogenic reaction was conducted according to the above two methods. Finally， absorbance was detected every 30 min and lasted for 2 h to study its stability.
Reproducibility test
At first， five samples of powder of shoots of A. elata was taken to make five test solutions at the same time according to the methods mentioned above. Afterwards， 0.2 ml of each of the test solutions was taken， and chromogenic reaction was conducted according to the above two methods. Finally， absorbance was detected to study its reproducibility.
Recovery rate test
The test solution of polysaccharides extracted from the shoots of A. elata was taken precisely and put in a colorimetric tube， to which glucose standard solution was added. Afterwards， chromogenic reaction was conducted according to the above two methods. Finally， absorbance was detected to calculate recovery rate.
Results and Analysis
Standard curves and regression equations
Sulfuric acid-phenol method
A standard curve was drawn using glucose concentration as the abscissa and absorbance as the ordinate （Fig.1）. The regression equation was y=0.013 4x+0.023 5， R2=0.999 3. There was a good linear relationship between absorbance and glucose concentration （0-40 μg/ml）.
Sulfuric acid-anthrone method
A standard curve was drawn using glucose concentration as the abscissa and absorbance as the ordinate （Fig.2）. The regression equation was y=0.014 6x+0.029 7， R2=0.999 6. There was also a good linear relationship between absorbance and glucose concentration （0-40 μg/ml）.
Results of conversion factor
The values of conversion factor measured by sulfuric acid-phenol and sulfuric acid-anthrone methods were 1.16 and 1.74 respectively.
Results of precision test
According to Table 1， after absorbance was detected five times， the values of relative standard deviation （RSD） detected by sulfuric acid-phenol and sulfuric acid-anthrone methods were 1.39% and 0.75% respectively， showing that the precision of the instrument was good. Results of polysaccharide content in the shoots of A. elata
In the same series of shoots of A. elata， polysaccharide content detected by sulfuric acid-phenol and sulfuric acid-anthrone methods was 19.31% and 20.40% respectively.
Discussion
The methods used for the determination of polysaccharide content mainly include colorimetric method and titration method at the present， in which the content of monosaccharide obtained through hydrolysis is used to reflect polysaccharide content， and the content of total sugar is detected in fact[10]. Sulfuric acid-phenol and sulfuric acid-anthrone methods are commonly used colorimetric methods， and their developing principles are basically the same. That is， polysaccharides are hydrolyzed into monosaccharides under the action of concentrated sulfuric acid， and furfural derivatives were formed after rapid dehydration； they can react with phenol to form an orange-yellow compound or condense with anthrone to form a blue-green compound， which has a maximum absorption peak at wavelengths of 490 and 625 nm respectively[11].
Conclusions
In this study， polysaccharide content in the shoots of A. elata was detected using sulfuric acid-phenol and sulfuric acid-anthrone methods respectively， and the methodological evaluation was performed by the linear relationship， precision， stability， reproducibility， and recovery rate tests. Moreover， the conversion factor of glucose was corrected by the detection of the refined polysaccharides extracted from the shoots of A. elata to reduce measured deviation when glucose was used as the standard control. The results showed that the methods of detecting polysaccharide content in the shoots of A. elata had good accuracy and precision， and the average recovery rate was higher than 97%. The average content of polysaccharides in the shoots of A. elata detected by sulfuric acid-phenol and sulfuric acid-anthrone methods was 19.31% and 20.40% respectively. That is， the value detected by sulfuric acid-anthrone method was higher， because sulfuric acid-anthrone method can determine almost all of the carbohydrates， and the total amount of all soluble carbohydrates was measured by this method. The results provide scientific basis for the scientific evaluation of quality of shoots of A. elata.
References
[1] Editorial Board of Chinese Academy of Sciences. Chinese flora[M]. Beijing： Science Press， 1998： 165-166.
[2] ZHANG ZQ. Research status of Aralia elata （Miq.） Seem. at home and abroad[J]. Heilongjiang Medicine Journal， 2008， 21（1）：46-20. [3] REN FZ， LUAN XH， QU HH. Research progress on chemical composition and pharmacological activities of Aralia plants[J]. Guang Zhou Chemical Industry and Technology， 2000，28（4）：124-130.
[4] AYIGULI AHMT， WANG Y， YANG Xj， et al. Study on extraction method and assay of polysaccharides of Lycium barbarum L. from two different source[J]. Xinjiang Agricultural Sciences，2007，25（5）：724-728.
[5] GUO XL， ZHU SC， ZHAI XF， et al. Comparison of methods in determination of polysaccharide in Ganoderma lucidum[J]. Chinese Archives of Traditional Chinese Medicine，2010，28（9）：2000-2002.
[6] OUYANG Z， LI YH， SHU SL， et al. Determination of polysaccharide in leaves of Morus alba L.[J]. Food Science， 2003，24（11）：118-120.
[7] XU GY， YAN J， GUO XJ， et al. The betterment and apply of phenol-sulphate acid method[J]. Food Science， 2006，26（8）：342-346.
[8] HOU HH， WANG YF， ZHAO B， et al. Ultrasonic extraction and determination of polysaccharides from Aralia chinensis L. roots[J]. Chinese Journal of Bioprocess Engineering，2015，13（2）：19-23.
[9] FENG Y， HAN N， XU DS. Study on the determination of polysaccharides from Ophiopogon japonicus[J]. Chinese Traditional Patent Medicine，2006，28（5）：705-707.
[10] HUANG RS. A summary of determination methods of polysaccharide content in Chinese herbal medicine[J]. China Pharmacist，2005，8（1）：68-70.
[11] DUAN JF， WANG YX， LI XY， et al. Diversity of polysaccharide content in five species of zizphus jujube in Alar area and their in vitro antioxidative activities[J]. Xinjiang Agricultural Sciences， 2014，51（5）：831-838.