Effects of Different Decolorants on Retention Rate of Total Triterpenes in Fruit and Rattan Stems of Schisandra chinensis（Turcz.）Baill

Junxia LlU，Yinping JlN，Fengming DOU，Yushuai WANG，Yingping WANG*

1.Jilin Agricultural Science And Technology University，Jilin 132101，China；

2.Institute of Special Animal and Plant Sciences of CAAS，Changchun 130112，China

Effects of Different Decolorants on Retention Rate of Total Triterpenes in Fruit and Rattan Stems of Schisandra chinensis（Turcz.）Baill

Junxia LlU1，2，Yinping JlN2，Fengming DOU1，Yushuai WANG2，Yingping WANG1*

1.Jilin Agricultural Science And Technology University，Jilin 132101，China；

2.Institute of Special Animal and Plant Sciences of CAAS，Changchun 130112，China

This study was to investigate the decoloration effects of activated clay，activated carbon，diatomite，Ca group bentonite，kaolin，activated aluminum oxide，magnesium oxide and attapulgite.Ultraviolet-visible spectroscopywas adopted to detect the absorbances of the extracts of Schisandra chinensis fruit and rattan stems，the contents of total triterpenoids in S.chinensis fruit and rattan stems were determined，and the decoloration rate and the retention rate of total triterpenoids of S.chinensis fruit and rattan stems were calculated.The results indicated that，attapulgite exhibited the best decoloration effect on the extract of S.chinensis fruit，with a decoloration rate up to 60.47%，activated carbon exhibited a better decoloration effect on the extract of S.chinensis rattan stems，with a decoloration rate up to 69.24%，and they had relatively higher retention rates of total triterpenoids.In the eight decolorants，attapulgite showed the best decoloration effect on the extract of S.chinensis fruit，and activated carbon exhibited the best decoloration effect on the extract of S.chinensisrattan stems.

Schisandra chinensis（Turcz.）Baill；Total triterpenoids；Decoloration；Attapulgite；Activated carbon；Retention rate

S chisandra chinensis（Turcz.）Baill contains the main components which are lignans，triterpenoids，volatile oils and polysaccharides，in which lignans are the main active components.S.chinensis has the pharmacological activities of protecting liver，inhibiting nervus centralis，and resisting oxidation，aging and tumor.In recent years，researchers have found a variety of nortriterpenoid compounds including nortriterpenoids，dinortriterpenoids， trinortriterpenoids，pentanortriterpenoids and octanortriterpenoids with novel structures and high oxidation degree subjected to skeleton rearrangement［1］.Currently，the technique for extracting effective components from S.chinensis mainly is ethanol extraction method，the extract obtained by which has low purity and high pigment content and is dull black orbrown.Therefore，decoloration is very important in the production process of plant extracts，otherwise the appearances and quality of products could be affected seriously.It is necessary to adopt physical-chemical methods to remove pigments.The methods for removing pigments mainly include adsorption method，chemical methodandmacroporousresin adsorption method.Diatomite is composed of homogeneous amorphous opal having unique pore structure with large porosity，strong adsorbability，and especially large adsorption capacity to liquid，and can serve as a filtering agent and decolorant［2］.Attapulgite clay is a kind of white powder that taking attapulgite as raw material，as well as a kind ofhydrous magnesium sili-cate mineral with a layer-chain transitional type structure，belonging to sepiolite family，having specialfibrous structure with larger specific surface area and strong adsorbability［3］.The raw ore can obtain greatly-improved adsorptive property after isolation，purification and modification，resulting in higher decolorizing ability by adsorption.Ca group bentonite is a kind of private soil widely produced in nature，and contains montmorillonite as its essential mineral which is a typical phyllosilicate of 2∶1 type having tremendous specific surface area，specific surface energy and higher cation exchange capacity and adsorbability capable of removing various harmful components（metal ions，organic matters）［4］.Kaolin is a dioctahedral phyllosilicate of 1∶1 type for which a larger specific surface area results in an larger exchange capacity，and can serve as a adsorbing material［5］.Magnesium oxide（light-burned magnesia）has stronger buffer performance（pH not higher than 9）and higher activity［6］. Activated clay is a modified bentonite product obtained from bentonite by acidation modification with inorganic acids which can improve the whiteness，specific surface area and acid strength of bentonite，having the functions of adsorption and decoloration and catalysis［7-8］.Activated carbon as one of the most ancient and most important industrial adsorbent has highlydeveloped pore structure and huge internal specific surface area，the surface of which contains （or can be added with）a variety of functional groups，and it thus has stable catalytic performance，can be used at different temperature and pH values and can be regenerated［9］.Activated aluminum oxide （r-Al2O3）is a porous and highly dispersive solid material with the characteristics of large specific surface area，good adsorption performance，acidic surface and good thermal stability，and can serve as the catalyst and catalyst support for a variety of chemical reactions［10］.In this study，the effects of above eight adsorption materials as decolorants on the retention rates of total triterpenoids in S.chinensis fruit and rattan stems were investigated，in order to select the suitable adsorbents for decoloration of extracts of S.chinensis fruit and rattan stems from them.

Materials and Methods

lnstruments

Following instruments were used: Guohua HY-2 multi-speed multi-purpose oscillator （Changzhou Guohua Electric Appliances Co.，Ltd）；PTT-A+ 100 electronic scale （Fuzhou Huazhi Scientific Instrument Co.，Ltd.）；SFTDL-5A table-top low-speed centrifuge（Shanghai Fitjar Analytic Instrument Co.，Ltd.）；HH-6 type thermostatic water bath （Changzhou Aohua Instrument Co.，Ltd.）；PS-80AJeken digital ultrasonic cleaner（Dongguan Jeken UltrasonicwaveCo.，Ltd.）；FW100 high-speed universal pulverizer（Tianjin TaisiteInstrument Co.，Ltd.）；TU-1810 ultraviolet-visible spectrophotometer（Beijing Purkinje GeneralInstrument Co.，Ltd.）；and Electro-thermostatic blast oven（Shanghai Yiheng Scientific Instruments Co.，Ltd.）.

Reagents

Oleanolicacid waspurchased from National Institute for the Control of Pharmaceutical and Biological Products. Absolute ethyl alcohol，vanillicaldehyde，glacial acetic acid，perchloric acid and concentrated sulfuric acid were analytically pure.Other reagents were as follows:food-grade activated clay（200 mesh）（Huangshan Taikeactive Bleaching Earth，Ltd.）；analytically-pure activated carbon（particle）（Tianjin Tianda Scavenging MaterialFine ChemicalFactory）；chemically-pure diatomite （Tianjin Sheng Ao Chemical Reagent Co.，Ltd.）；chemically-pure Ca group bentonite（200 mesh）（Aladdin）；chemically-pure kaolin （Sinopharm Chemical Reagent Co.，Ltd.）；chemically-pure activiated aluminum oxide（Sinopharm Chemical Reagent Co.，Ltd.）；analytically-pure magnesium oxide （light weight）（Sinopharm Chemical Reagent Co.，Ltd.）；and attapulgite （200 mesh）（Wuxi Ouwei Light Industry Science and Technology Co.，Ltd.）.

Materials

Fruit and rattan stems of S.chinensis were purchased from Tonghua Town of Jilin Province，and identified by associate professor Xu Shiquan from Medicinal Plant Research Department，Institute of Special Animal and Plant Sciences，CAAS，as dry fruit and 4-year-old rattan stems of S.chinensis，respectively.

Preparation of S.chinensis samples

Preparation of extraction solutions of S.chinensis fruit and rattan stems The Fruit and rattan stems of S.chinensis contain a variety of active components，suchaslignansand triterpenoids，which are extracted by ethanol refluxing method generally. The specific technological process including the steps of pulverizing dried ripe fruit and rattan stems of S.chinensis，sieving with a 40 mesh sieve，and drying in an oven at 50℃to constant weight；and weighing 50.0 g of S.chinensis fruit and rattan stems，respectively，adding 95%ethanol solution at a ratio of material to liquid of 1 g∶10 ml to perform reflux extraction for 3 times，and filtering to obtain filtrates，which were diluted to 500 ml，respectively.

Pretreatment of decolorants The eight decolorants（100 g for each）was soaked with 95%ethanol solution and were precipitated naturally until the supernatants were clear，and the precipitates were dried in an oven at 50℃for later use.

Decoloration method The extract（20.0 ml）of each of the fruit and rattan stems of S.chinensis was added with 5%colorant，and oscillated for 2 h.After decoloration，the corresponding filtrate was centrifuged in a centrifuge at 4 500 r/min for 10 min to obtain a supernatant.The supernatant was diluted with 95%ethanol to 20.0 ml，and then determined to obtain decoloration rates，and contents of total triterpenoids，and each treatment was repeated for three times to give a mean value.

Results and Analysis

Determination of decoloration rate

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Determination of determination wavelength With distilled water as reference，the extracts of S.chinensis fruit and rattan stems free of decoloration were scanned under 400-800 nm，and it was determined that the maximum absorption wavelengths of the extracts of S.chinensis fruit and rattan stems were 535 and 660 nm，respectively，as shown in Fig.1.

Table 1 Results of standard recovery test（n=5）

Determination ofsample decoloration rate With distilled water as reference，decolorized extracts of S.chinensis fruit and rattan stems were determined at 535 and 660 nm to obtain absorbances，respectively，for calculating decoloration rates.The calculation formula of decoloration rate is shown as following:

Decoloration rate= （D1-D2）/D1× 100%.

In which:D1represents the absorbance before decoloration；and D2represents the absorbance after decoloration.

lnvestigation of Methodology

Standard curve plotting A certain amount（1.0 mg）oleanolic acid dried to constant weight was weighed accurately to a 10 ml volumetric flask，into which absolute ethylalcoholwas added to dissolve the oleanolic acid，and the obtained solution was then diluted to constant volume and shaken to obtain 0.1 mg/ml standard solution. Certain amounts（0.2，0.4，0.6，0.8，1.0 and1.2 ml）of the standard solutions were accurately sucked into test tubes，heated in boiling water baths to vaporize ethanol and added with 50 g/L vanillic aldehyde-glacialacetic acid（0.2 ml）and perchloric acid（0.8 ml）to obtain solutions which were subjected to heating in water baths at 60℃for 15 min followed by cooling，addition of glacial acetic acid （5.0 ml）and shaking.The final solutions were determined at547 nm to obtain absorbances （D547nm）.A standard curve was constructed with the concentrations of oleanolic acid as horizontal coordinates and absorbances （D547nm）as vertical coordinates.Regression was performed to obtain a following equation:y=0.006 8x+0.009 7，r2= 0.999 1，and it was shown that there was a good linear relation between D547nmand the concentration of oleanolic acid in the range of 0-120 μg/ml（Fig.2）.

Precision test The standard solution（0.5 ml）was accurately sucked into six test tubes，respectively，and other steps were the same as the preparation of the standard curve.The obtained solutions were determined at 547 nm to obtained absorbances，the RSD value of which was 0.83%，indicating that the precision of the instruments used by this experiment was good.

Stability test The No.4 sample for the precision test was determined at 10，20，30，60，90 and 120 min to obtain D547nm，and the standard solution was stable substantially within 30 min after development，indicating the two test solutions were stable within 2 h.

Repeatability test Sixpartsof samples were weighed，respectively，to prepare test solutions which were determined，showing a RSD value of 1.96% ，indicating the method had good repeatability.

Standard recovery test Five parts of fruit and rattan stems of the same batch with known contents were accurately weighed，respectively，and were added with the same amounts of standard solutions，respectively，to prepare solutions.The recovery rates were calculated，showing RSD values of 2.75%and 2.20% ，respectively（Table 1）.

Decoloration rate and retention rates of total triterpenoids by various adsorbents

As shown in Fig.3，the decoloration effects of different decolorants on the extract of S.chinensis fruit were in the order of:attapulgite＞activated carbon＞activated aluminum oxide＞kaolin＞magnesium oxide＞diatomite＞Ca group bentonite＞activated clay（from good to bad），in which attapulgite exhibited the best decoloration effect on S.chinensis fruit，with a decoloration rate up to 60.47%，and had the highest retention rate of total triterpenoids.In fact，attapulgite not only adsorbs pigments in decoloration process，but also removes peroxides，saponins，trace metals，phosphorus，sulfide and oxidation products，and it ischeap as well［11］.Therefore，attapulgite was chosen as the decolorant for the extract of S.chinensis fruit.

As shown in Fig.4，the decoloration effects of different decolorants on the extract of S.chinensis rattan stems were in the order of:activated carbon＞diatomite＞attapulgite＞magnesium oxide＞kaolin＞activated aluminum oxide＞activated clay＞Ca group bentonite，in which activated carbon exhibited the best decoloration effect on S.chinensis rattan stems，with a decoloration rate up to 69.24%，and had a higher retention rate of total triterpenoids.

Conclusion

The results of this research indicated that，the main pigments in S.chinensis fruit were anthocyanin，etc.；the main pigment in S.chinensis rattan stems was chlorophyll；and attapulgite，activated carbon，activated aluminum oxide，kaolin，magnesium oxide，diatomite，Ca group bentonite and activated clay all had certain adsorption effects on the pigments including anthocyanin and chlorophyll.In addition，attapulgite showed the strongest adsorption effect on anthocyanin，activated carbon exhibited the strongest adsorption effect on chlorophyll，and they had relatively higher retention rates of total triterpenoids.

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Responsible editor：Yingzhi GUANG

Responsible proofreader：Xiaoyan WU

不同脱色剂对五味子果实、藤茎中总三萜保留率的影响

刘俊霞1，2，金银萍2，窦凤鸣1，王玉帅2，王英平1*
（1．吉林农业科技学院，吉林吉林132101；2．中国农业科学院特产研究所，吉林长春130112）

研究吸附活性白土、活性炭、硅藻土、钙质膨润土、高岭土、活性氧化铝、氧化镁、凹凸棒土的脱色效果，采用紫外－可见分光光度法检测五味子果实溶液、藤茎溶液吸光度，测定五味子果实、藤茎中总三萜含量，计算五味子果实、藤茎样品的脱色率、总三萜的保留率。结果表明，凹凸棒土对五味子果实提取液脱色效果最好，脱色率可达60.47%，活性炭对五味子藤茎提取液的脱色效果较好，脱色率达到69.24%，并且对总三萜的保留率相对较高。8种吸附脱色剂中，凹凸棒土对五味子果实溶液脱色效果最佳，活性炭对藤茎溶液的脱色效果最佳。

五味子；总三萜；脱色；凹凸棒；活性炭；保留率

吉林省重点科技攻关项目（20140204068YY，20140204062YY）；吉林省医药产业发展专项资金（YYZW201246）。

刘俊霞（1977-），女，新疆石河子人，博士研究生，主要从事中药新药开发与利用研究，E-mail：zyljx2007123@163.com。*通讯作者，博士生导师，研究员，主要从事药用植物资源研究，E-mail：yingpingw@126.com。

2015-07-24

Supported by Key Science and Technology Research and Development Program of Jilin Province （20140204068YY，20140204062YY）；Special Fund for Pharmaceutical Industry Development of Jilin Province（YYZW201246）.

*Corresponding author.E-mail:yingpingw@126.com

Received:July 24，2015 Accepted:November 23，2015

修回日期 2015-11-23