全 文 ：食用菌学报 2011.18(2):49～ 52
收稿日期:2011-05-12 原稿;2011-06-03 修改稿
基金项目:上海市科技兴农重点攻关项目[ 编号:沪农科攻字(2009)第 2-2 号] 的部分研究内容
作者简介:王淑蕾(1988-), 女 ,中国药科大学中药专业在读硕士 , 主要从事天然产物研究。
＊本文通讯作者　E-mail:t angqingjiu@saas.sh.cn
文章编号:1005-9873(2011)02-0049-04
HPLC法测定香菇中香菇嘌呤含量
王淑蕾1 , 2 , 梁敬钰2 , 唐庆九1＊ , 刘艳芳1 , 周　帅1 , 杨　焱1 , 张劲松1
(1农业部南方食用菌资源利用重点实验室 ,国家食用菌工程技术研究中心 ,上海市农业遗传育种重点开放实验室,
上海市农业科学院食用菌研究所 , 上海 204303;2中国药科大学中药学院 , 江苏南京 210009)
摘　要:建立 1 种运用高效液相色谱(HPLC)测定香菇(Lentinula edodes)中香菇嘌呤含量的方法。优化后的
色谱条件为:Ultimate AQ-C18柱(5 μm , 4.6 mm×250 mm), 流动相为甲醇-磷酸盐缓冲液(7∶93 , pH 4.67)等
度洗脱 ,流速 1 mL/min , 柱温 30 ℃,检测波长 259 nm ,进样量 10 μL。研究结果发现该方法准确 、灵敏 、重现
性好 ,适用于香菇嘌呤的定量分析。
关键词:香菇;香菇嘌呤;HPLC
　　香菇(Lentinula edodes)是世界第二大食用
菌[ 1] ,具有独特香味 ,也是我国传统食品之一 。
研究发现 ,香菇中含有一种降血脂有效成分香
菇嘌呤(eritadenine),其降血脂作用比常用降血
脂药安妥明强 10倍 ,且口服有效[ 2] 。香菇嘌呤
还具有降低血浆中高半胱氨酸浓度的作用 ,从
而可以防治高半胱氨酸浓度过高引发的血栓和
血管疾病[ 3 , 4] , 同时香菇嘌呤还具有保肝[ 5]
作用 。
目前 ,有研究者用紫外分光光度法 、气质联
用法 、纸电泳法等对香菇嘌呤进行定性 、定量分
析。其中紫外分光光度法前处理(供试品溶液制
备)十分复杂 ,且干扰成分不易去除[ 6] ;气质联用
法须对样品预先进行衍生化 ,误差较大[ 7] ;而纸
电泳分析方法操作不方便 、重现性较差[ 8] 。本文
运用 HPLC 测定香菇中香菇嘌呤含量 ,并进行方
法学考察以寻找适合香菇中活性成分的定量分
析方法。
1　材料与方法
1.1 材料 、试剂和仪器
1.1.1 供试样品
　　香菇(L .edodes)子实体(商品名金钱菇)购
于上海百信食药用菌科贸有限公司 。
1.1.2 试剂
　　甲醇(色谱纯)为美国 Dikma 公司产品 ,磷酸
二氢钾 、磷酸 、无水乙醇等均为国产分析纯。
1.1.3 主要仪器
　　Waters 600 系列高效液相色谱仪 、Wate rs
717自动进样器 、Waters 2996光敏二极管阵列检
测器均为美国 Waters 公司产品 , KQ-600B 型超
声清洗器为昆山市超声仪器有限公司产品。
1.2 方法
1.2.1 香菇嘌呤标准品制备
　　参考 CHIBA TA I 等方法[ 9]运用阳离子和阴
离子交换树脂的分离纯化方法得到香菇嘌呤粗
品 ,并通过反复重结晶得到无色针状结晶 ,并进
行核磁共振验证 ,纯度大于 98%(另文报道)。
1.2.2 色谱条件优化
　　色谱柱:Ultimate AQ-C18柱(5 μm ,4.6 mm×
250 mm);流动相:甲醇-磷酸盐缓冲液(KH2 PO4
10 mmol/ L ,85%H3 PO4调节 pH);检测器:UV 检
测器检测;检测波长:259 nm;流速:1 mL/min;柱
温:40 ℃;进样量:10μL。对甲醇-磷酸盐缓冲液的
比例(甲醇所占比例分别为 3%、4%、5%、6%、
7%、10%、15%和 20%)及 pH(4.45 、4.67和 5.12)
等条件进行优化 ,确定色谱分析条件。
1.2.3 样品前处理方法优化
1.2.3.1 乙醇提取溶剂浓度筛选
称取 6份香菇子实体粉 1 g ,分别加入 5%、
10%、15%、20%、25%、30%的乙醇溶液 100 mL ,
超声提取 30 min ,放冷至室温 ,用溶剂补足减失
食　用　菌　学　报 第 18 卷
的重量 ,过滤去除菌粉沉淀 ,续滤液经 0.45 μm
膜过滤 ,进行 HPLC测定 ,分析提取量。以加入
蒸馏水 100 mL , 100 ℃热回流提取 1 h 为对照 ,
每个处理重复 3次。
1.2.3.2 料液比的筛选
称取 6 份香菇子实体粉 1 g ,分别加入筛选
浓度乙醇溶液 50 、100 、150 、200 、250 和 300 mL ,
超声提取 1 h ,后续操作同 1.2.3.1 。
1.2.3.3 提取时间的优化
称取 6份香菇子实体粉 1 g ,按照 1.2.3.2和
1.2.3.1试验筛选的料液比和浓度加入乙醇溶剂
后 ,超声提取 10 、15 、20 、30 、60 、120和 180 min ,
后续操作同 1.2.3.1。
1.2.4 方法学考察
1.2.4.1 标准曲线的绘制
称取烘至恒重的香菇嘌呤标准品 10.0 mg ,
置于 10 mL容量瓶中 ,加超纯水定容至刻度作储
备液 。将储备液稀释至不同浓度(10 、15 、20 、25 、
30 、35和 40μg/mL),进样分析香菇嘌呤 ,根据峰
面积和香菇嘌呤含量绘制标准曲线 。
1.2.4.2 精密度实验
吸取 20 μg/mL 的标准品溶液 10 μL ,连续
进样 5次 ,计算 RSD ,考察精密度。
1.2.4.3 重复性实验
取同一批样品 ,按优化后的方法平行制备供
试液 5份 ,分别进样 ,测定峰面积。
1.2.4.4 稳定性实验
吸取 20 μg/mL 的标准品溶液 10 μL ,分别
于配制后的 0 、5 、10 、15 和 20 h 时进样 ,测定峰
面积 。
1.2.4.5 加样回收率实验
取香菇样品粉末 6 份 ,每份 0.1 g ,按优化的
样品前处理方法平行制备供试液 , 分别加入
1 mg/mL标准品溶液 0.2 mL ,并按优化色谱条
件进行测定分析 ,计算回收率 。
1.2.5 香菇样品中香菇嘌呤含量的测定
　　取香菇样品粉末 5 份 ,每份 0.1 g ,按优化的
样品前处理方法平行制备供试液 ,并按优化色谱
条件进行测定分析。
2　结果与分析
2.1 色谱条件优化结果
　　利用 Ultimate AQ-C18分析柱 ,在柱温30 ℃、流
速为 1 mL/min 、流动相甲醇-磷酸盐缓冲液条件
下 ,样品中香菇嘌呤的峰形及分离度较好 ,随着甲
醇比例的增加 ,香菇嘌呤的保留时间提前 ,当甲醇
比例大于 10%时 ,香菇嘌呤的峰形和分离度不能
达到分析的要求。综合考虑保留时间和分离效果 ,
选取甲醇∶磷酸盐缓冲液=7∶93来进行等度洗脱。
同时发现缓冲液 pH 对香菇嘌呤的峰形和保留时
间也有较大影响 ,pH 4.67时香菇嘌呤同其他成分
可以达到最佳的基线分离且峰形好。
图 1　标准品(A)及样品(B)的 HPLC图谱
Fig.1　RP-HPLC chromatograms of eritadenine standard(A)and test samples(B)
50
第 2 期 王淑蕾 , 等:H PLC 法测定香菇中香菇嘌呤含量
2.2 样品前处理优化方法
　　由于乙醇具有较好的穿透细胞壁作用 ,同
时香菇嘌呤具有水溶性的特点 ,考察不同浓度
乙醇超声提取和蒸馏水加热回流的提取效率 ,
结果发现 5%～ 20%乙醇超声提取的效率优于
蒸馏水加热回流 ,由于超声提取方式简便易操
作 , 且使用 5%乙醇提取时提取液所含杂质最
少 ,所以确定最佳提取方式为 5%乙醇超声
提取 。
图 2显示不同超声时间对香菇嘌呤提取量
的影响 , 15 min 超声提取可以达到较好提取效
果 ,而 30 min超声提取时香菇嘌呤提取量的差异
最小 ,较稳定 ,因此选用 30 min 为适宜超声提取
时间 。由图 3结果可知 ,随着提取溶剂体积的增
加 ,香菇嘌呤的提取率也相应增加。当提取溶剂
增加到 100 mL 时 ,提取所得香菇嘌呤含量与其
他组无显著性差异 ,而此时料液比较为适中 ,因
此料液比选为 1∶100。
图 2　不同提取时间的香菇嘌呤提取量
Fig.2　Effect of extraction time on eritadenine yields
图 3　不同料液比的香菇嘌呤提取量
Fig.3　Effect of sample-solvent ratio on eritadenine yields
2.3 方法学考察
2.3.1 标准曲线
　　以标准品的进样浓度(μg/mL)为横坐标 ,色
谱峰的面积为纵坐标 ,得回归方程:y =40 200x -
15 600 , r2 =0.999 87。线性范围为 5 ～ 40μg/mL 。
2.3.2 精密度 、重复性和稳定性实验
　　精密度 、重复性和稳定性实验中香菇嘌呤含
量的 RSD 分别为0.40%、0.08%和 0.46%,说明
运用建立的 HPLC分析方法测定香菇嘌呤含量 ,
精密度 、重复性和稳定性均良好。
2.3.3 加样回收率
　　加样回收率实验结果表明 ,加样回收率在
95.31%～ 97.85%之间 ,平均回收率为 96.42%,
RSD=0.86%。
2.4 样品中香菇嘌呤含量
　　采用优化的前处理方法及色谱条件平行测
定 5 份香菇样品 , 其香菇嘌呤的平均含量为
1.993 6 mg/g , RSD=0.08%。
3　讨论
　　卫生部发布的《2002年中国居民营养与健康
现状调查报告》中就宣布:全国血脂异常人数估
计达 1.6亿 ,其中约有 9 000万人患有高血脂症
(俗称高血脂)。目前高血脂症是导致心脑血管
疾病的元凶 ,发病率高 ,已成为威胁中老年人生
命的主要祸首 。早在 60 年代 ,日本就发现香菇
具有降血脂作用 ,并得到降血脂活性成分香菇嘌
呤。近年来 ,对其降血脂的作用机制也进行了大
量深入研究 ,发现香菇嘌呤通过调控脂肪酸代
谢[ 10 , 11] 及改 变磷脂 酰胆碱 分子谱 图的组
成[ 10 , 12-14]来降低血液胆固醇的含量。香菇是日常
食品之一 ,毒副作用小 ,香菇嘌呤含量较高 ,因此
开发降血脂的香菇嘌呤保健品或药品具有很好
的应用前景。
香菇嘌呤是由腺嘌呤上接一个丁酸组成的 ,
因此极性较大 ,运用传统的核苷分析条件即水与
甲醇梯度洗脱[ 15] ,分离度和峰形都不好。笔者采
用单核苷酸的色谱条件 ,使香菇嘌呤与其它的核
苷类成分得到了很好的分离。本文所建立的运
用 HPLC测定香菇嘌呤的分析方法 ,操作简单 、
重复性好 、准确度高 ,应用此方法对样品进行测
定的结果显示数据稳定 ,误差小 ,适用于香菇嘌
呤的定量分析 。
参考文献
[ 1] 张树庭 ,陈明杰.香菇产业的过去现在和未来[ J] .食
用菌 , 2003 , 25(1):2-4.
[ 2] ROKUJO T , KIKUCHI H , TENSHO A , et al.
Lenty sine: a new hypolipidemic agent from a
mushroom [ J] .Life Science s , 1970 , 9:379-385.
[ 3] SEKIYA A , FUKADA S , MORITA T , et al.
51
食　用　菌　学　报 第 18 卷
Suppression of methionine-induced hyperhomo-
cy steinemia by die tary eritadenine in rats[ J] .Bio sci
Biotechno l Biochem , 2006 , 70(8):1987-1991.
[ 4] FUKADA S , SETOUE M , MORITA T , et al.
Dietar y eritadenine suppresse s guanidinoacetic acid-
induced hyperhomocy steinemia in ra ts[ J] .J Nutr ,
2006 , 136:2797-2802.
[ 5] TUCHWEBER B , SA LAS M.Prevention of CeCl3-
induced hepa to tox icity by hypo lipidemic compounds
[ J] .Arch Toxico l , 1978 , 41:223-232.
[ 6 ] SAITO M , YASUMOTO T , KANEDA T.
Quantita tive analy ses of e ritadenine in Shiitake
mushro om and o ther edible fung i[ J] .J Jap Soc Food
Nutr , 1975 , 28:503-513.
[ 7] VIT NYI G , LELIK L , BIH TSI-KARSAI , et
al.Detection o f eritadenine in ex tracts f rom Shiitake
mushro om by gas chroma to g raphy/ mass
spectr ometry [ J] .Rapid Commun M ass Spec trom ,
1998 , 12:120-122.
[ 8] 孙培龙 , 吴学谦 , 季培军 , 等.香菇及其他食用菌中香
菇嘌呤含量的检测[ J] .食品工业科技 , 2000 , 21(5):
70-72.
[ 9] CHIBATA I , OKUMURA K , TAKEYAMA S , et
al.Lentinacin:a new hypocho lestero lemic substance
in Lentinus edodes[ J] .Specialia , 1969 , 15(12):
1237-1238.
[ 10] SHIMADA Y , MORITA T , SUGIYAMA K.Eri-
tadenine-induced alternations o f plasma lipopr otein
　 lipid concentrations and phospha- tidy lcholine
molecular species pr ofile in rats fed chole ster ol-f ree
and choleste rol-enriched diets[ J] .Bio sci Bio technol
Biochem , 2003 , 67(5):996-1006.
[ 11] S UGIYAM A K , YAM AKAWA A , S AEKI S.
Cor rela tion of suppressed lino leic acid metabolism
with the hypocho lestero lemic action of e ritadenine in
ra ts[ J] .Lipids , 1997 , 32:859-866.
[ 12] S UGIYAM A K , YAM AKAWA A. Dietary
eritadenine-induced alte ration of mo lecular species
composition of pho spholipids in r ats[ J] .Lipids ,
1996 , 31:399-404.
[ 13] SUGIYAMA K , YAMAKAWA A , KAWAGISH I
H , et al .Die tary eritadenine modifies plasma
pho sphatidylcho line mo lecular species pro file in rats
fed diffe rent types of fat[ J] .J Nut r , 1997 , 127:593-
599.
[ 14] SUGIYAMA K , KUM AZAWA A , ZHOU H , et
al .Die tary methionine leve l affects linoleic acid
metabolism thr ough pho sphatidylethano lamine N-
methylation in r ats[ J] .Lipids , 1998 , 33:235-242.
[ 15] ENMAN J , ROVA U , BERGLUND K.
Quantification of the bioac tive compound eritadenine
in selected str ains of Shiitake mushro om (Lentinus
edodes)[ J] . J Ag ric Food Chem , 2007 , 55:
1177-1180.
[本文编辑] 　于荣利
52
ACTA EDULIS FUNGI 2011.18(2):53 ～ 56
Received:May 12 , 2011;　Accepted:June 3 , 2011
Sponsored by the Sh anghai Municipal Government Foundation
＊Corresponding author.　E-mail:tangqingjiu@saas.sh.cn
Determinat ion of Eritadenine in Lentinula edodes
Fruit bodies Using HPLC
WANG Shule i
1 , 2 , LIANG Jingyu2 , TANG Qingjiu1＊ , LIU Yanfang1 ,
ZHOU Shuai
1 , YANG Yan1 , ZHANG Jingsong1
(1National Engineer ing Resear ch Center of Edible Fungi;Shangh ai Key Laboratory of Agr icultural Genetics
and Breeding;Institute of Edible Fungi , Shanghai Academy of Agr icultur al Sciences , Shanghai 201403 , China;
2 Traditional Chinese Medicine College , China Pha rmaceutical University , Nanjing , Jiangsu 210009 , China)
Abstract:A simple and sensitive H PLC-based method for the accur ate de te rmination of e ritadenine in
Lentinula edodes fr uit bodies is descr ibed.Optim ization of sample work-up procedures showed th at the
eff iciency of e ritadenine extrac tion was highest when powde red f ruit bodies were suspended in 5%(w/v)
ethanol and subjected to ultr asound for 30 min using a 1∶100 sample-solvent ra tio.Optimal separa tion of
er itadenine in the extr acts was achieved by loading 10μL samples on to an Ultimate AQ-C18 column (5 μm ,
4.6 mm×250 mm)and e luting with methanol-phosphate buf fer (7∶93 , pH 4.67)a t 30 ℃ using a f low ra te
of 1.0 mL/min.Eritadenine in eluted fr actions was monitor ed at 259 nm.
Key words:Lentinula edodes ;er itadenine;HPLC
　　Lent inula edodes r anks among the top
three mushroom species available on the
inter national market
[ 1] , mushroom frui t bodies
contain er itadenine , the cholesterol-lower ing
effects of which ar e repor ted to be ten times
str onger than the widely used lipid-lower ing
dr ug , clofibrate[ 2] .Er itadenine also reduces
homocysteine concentrat ions in plasma ,
thereby preventing high-level homocyste ine-
induced thrombosis and vascular dise ase
[ 3 , 4] ,
and exhibits hepatoprotect ive act ivi ty
[ 5] .
Curr ently , UV spectr ophotometry , GC-
MS or paper electrophoresis are used for the
qualitat ive and quant itative determina tion of
eritadenine , although each of these methods
has inherent def iciencies.Prepar at ion of the
test samples for UV spect rophotometr y is
complex and it is often diff icult to remove
inter fering components
[ 6] , the required pre-
der ivat izat ion of samples for GC-MS causes
large errors
[ 7] , and poor reproducibility is a
regular problem with the paper elect rophoresis
method
[ 8] .In this paper , we have used HPLC
to determine er itadenine levels in L .edodes
fruit bodies , and have examined various
features of the methodology (i.e.accur acy ,
reproducibility , er itadenine stabili ty and
recove ry rate) to establ ish if the method is
suitable for the r outine quant itative analysis of
this bioact ive component .
1　Materials and methods
1.1 Materials
　　L .edodes fruit bodies was purchased f rom
Shanghai Baixin Bio-Tech Co., Ltd.
1.2 Reagents
　　Methanol (HPLC gr ade)was from Dikma
Technologies Inc (CA , USA), while other
reagents were fr om Chinese sour ces and of
analyt ical grade.
1.3 Methods
1.3.1 Eri tadenine standar d
　　A sample of er itadenine , shown by nucle ar
magne tic resonance imaging to be 98% pur e ,
was fur ther pur ified by chromatogr aphy using
cation and anion exchange resins
[ 10]
followed by
ACTA EDULIS FUNGI Vol.18
repeated r ecr ystallizat ion to give colorless
needle-l ike crystals (details to be repor ted
elsewhere).
1.3.2 HPLC
　　HPLC was carried out using a Waters 600
Series high-performance liquid chromatograph ,
attached to a Waters 717 autosampler and a
Waters 2996 photodiode array detector.The
chromatograph was fit ted with an Ultimate AQ-
C18 column(5 μm , 4.6 mm ×250 mm), and
the mobile phase was methanol-phosphate buffer
(10 mmol/L KH2 PO4 adjusted with 85%H3 PO4
to give different pH values). Operating
conditions were as follows:column temperature ,
40 ℃;inject ion volume , 10 μL;flow rate ,
1 mL/min;detect ion wavelength , 259 nm.The
shape of the eritadenine peak and the degree of
separation var ied according to the methanol-
phosphate buffer ratio.In order to opt imize the
HPLC conditions , the effects of using different
proportions(3%, 4%, 5%, 6%, 7%, 10%,
15% and 20%) of methanol in the mobile
phase , and different pH values(4.45 , 4.67 and
5.12), were evaluated.
1.3.3 Optimizat ion of sample pr e-t reatment
1.3.3.1 Concentr at ion of ext ract ion solvent
L .edodes fruit body powder (1 g)was
suspended in 100 mL aqueous ethanol (5%,
10%, 15%, 20%, 25%or 30%)and subjected
to ul tr asound (KQ-600B ultr asonic cle aner ,
Kunshan Ult rasonic I nst rument Co., Ltd ,
Kushan , China)for 30 min.After cool ing to
room temperature and addit ion of solvent to
compensate for losses dur ing extr action ,
suspensions were coarse-filte red to remove
residual solids.Filtr ates were then membrane
filtered (0.45 μm)and the extract ion yields
determined using HPLC. Controls were
pr epared by suspending 1 g fr uit body powde r
in 100 mL dist illed water and extract ing unde r
reflux at 100 ℃ for 1 h.Each tre atment was
carried out in tr ipl icate.
1.3.3.2 Sol id-liquid r atio
L .edodes fruit body powder (1 g)was
suspended in different volumes (50 , 100 , 150 ,
200 , 250 or 300 mL)of 5% aqueous ethanol
and extracted using ult rasound as above.
Subsequent work-up procedures wer e the same
as descr ibed in Sect ion 1.3.3.1.
1.3.3.3 Extract ion time
L .edodes fruit body powder (1 g)was
suspended in 100 mL of 5% aqueous ethanol
and extr acted using ul tr asound for 10 , 15 , 20 ,
30 , 60 , 120 or 180 min.Subsequent work-up
pr ocedures were the same as described in
Sect ion 1.3.3.1.
1.3.4 Analysis of methodology
1.3.4.1 Standar d Cur ve
A standar d cur ve relat ing peak area with
eritadenine concentr ation was pr epared using
dif ferent concentrat ions of eritadenine (10 ,
15 , 20 , 25 , 30 , 35 and 40μg/mL)dissolved in
ult ra-pure water.
1.3.4.2 Precision test
The pr ecision level of the methodology was
assessed fr om the RSD value calcula tedafter
five r epl icate de terminat ions of the peak ar ea
obtained following HPLC of a 10 μL sample of
a standard(20 μg/mL)eritadenine solut ion.
1.3.4.3 Repr oducibility
Repr oducibility was assessed fr om the RSD
value calculated fr om the peak are as obtained
following HPLC of five test solut ions prepared
from the same batch of fr uit bodies using the
opt imized pre-tre atment and HPLC conditions.
1.3.4.4 Stabil ity test
Eritadenine stability was assessed from the
RSD value calculated from peak ar eas obtained
following HPLC of five repl icate 10 μL samples
of a standard (20 μg/mL)er itadenine solution
assayed af ter 0 , 10 , 15 and 20 h.
1.3.4.5 Recovery r ate
Eritadenine r ecovery r ates were
de termined by adding 0.2 mL aliquots of a
standard er itadenine solut ion(1 mg/mL)to six
powder ed f rui t body samples pr ior to extraction
and analysis adopt ing the optimized pre-
tr eatment and HPLC procedures.
54
No.2 WANG Shulei , LIANG Jingyu , TANG Qingjiu , et al
1.3.5 Determination of eritadenine in L .
edodes fr uit bodies
　　Eri tadenine levels were determined in f ive
powdered L .edodes fruit body samples(0.1 g)
adopt ing the opt imized pre-trea tment and
HPLC procedures.
2　Results and Analysis
2.1 Optimized HPLC conditions
　　An Ult imate AQ-C18 analytical column
opera ted at 30 ℃, and using a methanol-
phosph ate buffer mobile phase and a flow rate
of 1 mL/min , gave a well-defined er itadenine
peak and a good degree of separa tion.
Eritadenine retent ion times wer e shorter with
incr easing concentrat ions of methanol in the
mobile phase but peak characterist ics and the
degree of separ at ion made analysis more
dif ficult , especially when the proport ion of
methanol in the mobile phase > 10%.A
methanol∶phosphate buffer r at io of 7∶93 was
selected as opt imal based on re tent ion time and
degree of separ at ion. Peak fe atures and
retention times were also gr eatly affected by
the pH of the buffer , with pH 4.67 providing
the best basel ine separ at ion and a well-defined
eritadenine peak (see Fig.1 in the Chinese
version).
2.2 Optimized sample pre-treatment conditions
　　I n view of the higher cel l wall penet rat ing
pr opert ies of ethanol and the water-soluble
nature of eri tadenine , two extract ion methods
wer e compared in terms of er itadenine
extract ion efficiency:(1) extr act ion with
ult rasound using different concentrat ions of
ethanol as the solvent , and (2) refluxing at
100 ℃ using distilled water.Extract ion with
ult rasound using 5%-20% ethanol was more
efficient than refluxing with boiling water and
5% ethanol was selected on the basis of e asy
opera tion and lowest impur ity levels.Maximum
eritadenine yields wer e achieved after 15 min
ult rasound treatment (see Fig.2 in the Chinese
version)but , since the extr action efficiency at
30 min was more reproducible , the longer
ext ract ion time was adopted.The ra tio of fr uit
body sample to extract ion solvent had no
significant effect on eri tadenine yields over the
range tested (Fig.3 in Chinese version)and
the sol id-liquid r at io of 1∶100 was selected as
standard.
2.3 Methodology data
2.3.1 Standard cur ve
　　 The following regression equat ion was
derived from the experimental data:y =40 200 x -
15 600 , r2 =0.99987 , linear range 5-40 μg/mL ,
x :standard sample concentration (μg/mL),
y :peak area.
2.3.2 Pr ecision , repr oducibility and stabili ty
　　RSD values for er itadenine levels
de termined in precision , repr oducibility and
stabili ty tests were 0.40%, 0.08% and 0.46%,
respect ively , indicat ing that the HPLC method
gave accura te , reproducible and stable r esults.
2.3.3 Recovery rate
　 　 The rates of recover y ranged between
95.31% and 97.85%, with an average value of
96.42%(RSD =0.86%).
2.4 Content of eritadenine in L. edodes
fruit bodies
　　The average eritadenine content of L .
edodes fr uit bodies was 1.993 6 mg/g(RSD =
0.08%).
3　Discussion
　　Accor ding to the “ 2002 China National
Nutrit ion and Health Survey Report” issued by
the Ministr y of Health , there are
appr oximately 160 million dyslipidemic people
living in China , of which about 90 million are
suffer ing fr om hyperl ipidemia.Hyperl ipidemia
is a leading cause of car diovascular dise ase and
a major life-threatening f actor affect ing the
elder ly.The lipid-lower ing effect of L .edodes
fruit bodies was f irst recor ded in the 1960s and
the active lipid-lowering component was
subsequent ly identif ied as eri tadenine.More
recent studies on the mech anism underlying the
55
ACTA EDULIS FUNGI Vol.18
lipid-lower ing effects have shown that
eritadenine reduces blood cholesterol by
regulat ing fat ty acid me tabol ism
[ 10 , 11]
and
ch anging the molecular composi tion of
phosph atidylcholine
[ 10 , 12-14] . Eritadenine has
wide appl ication and exploita tion potential for
use in cholesterol-lowering nutriceut ical and
ph armaceutical pr oducts.
Convent ional methods for nucleotide
analysis based on gr adient elut ion using wate r
and me thanol
[ 15]
are not part icularly suited for
separ at ing eritadenine due to the highly polar
nature of a molecular st ructure consist ing of
adenine and butyrate. We have now
demonst rated that er itadenine can be re adily
separ ated from other pur ine nucleoside
components adopting chroma tographic
conditions suitable f or separat ing single
nucleot ides.Our HPLC-based determination
method is str aight forward , highly accur ate and
reproducible , and is ther efor e sui table for the
quanti tat ive determinat ion of this bioact ive
component.
References
[ 1] ZHANG ST , CHEN MJ.Mushroom industry in the
past , present and futur e [ J] .Edible Fungi , 2003 , 25
(1):2-4.(In Chinese)
[ 2] ROKUJO T , KIKUCHI H , TENSHO A , et al .
Lentysine: a new hypolipidemic agent f rom a
mushroom [ J] .Lif e Sc iences , 1970 , 9:379-385.
[ 3] SEKIYA A , FUKADA S , MORITA T , et al .
Suppression of methionine-induced hype rhomo-
cysteinem ia by dietary e ritadenine in ra ts[ J] .Biosci
Biotechnol Biochem , 2006 , 70(8):1987-1991.
[ 4] FUKADA S , SETOUE M , MORITA T , et al .
Die tary er itadenine suppresses guanidinoacetic a cid-
induced hype rhomocyste inemia in r ats[ J] .J Nutr ,
2006 , 136:2797-2802.
[ 5] TUCHWEBER B , SALAS M.Prevention of CeCl3-
induced hepa totoxicity by hypolipidemic compounds
[ J] .Arch Toxicol , 1978 , 41:223-232.
[ 6] SAITO M , YASUMOTO T , KANEDA T.
Quantitative analyses of e ritadenine in Shiitake
mushroom and other edible fungi[ J] .J Jap Soc Food
Nutr , 1975 , 28:503-513.
[ 7] VIT NYI G , LELIK L , BIH TSI-KARSAI , et
al . De tection of e ritadenine in extr acts fr om
Shiitake mushroom by gas chromatography/mass
spectrometry[ J] .Rapid Commun Mass Spectrom ,
1998 , 12:120-122.
[ 8] SUN PL , WU XQ , JI PJ , e t al .The determination
of er itadenine in Lentinus edodes and other edible
fungi [ J] .Sc ience and Technology of the Food
Industry , 2000 , 21(5):70-72.(I n Chinese)
[ 9] CHIBATA I , OKUMURA K , TAKEYAMA S , et
al . Lentinacin: a new hypocholesterolemic
substance in Lentinus edodes [ J] .Specialia , 1969 ,
15(12):1237-1238.
[ 10 ] SHIMADA Y , MORITA T , SUGIYAMA K.
Er itadenine-induced alternations of plasma
lipoprotein lipid concentr ations and phospha-
tidylcholine molecular species profile in r ats fed
cholesterol-fr ee and cholesterol-enr iched diets[ J] .
Biosci Biotechnol Biochem , 2003 , 67(5):996-1006.
[ 11] SUGIYAMA K , YAMAKAWA A , SAEKI S.
Corr elation of suppressed linole ic acid me tabolism
with the hypocholesterolemic action of er itadenine
in r ats[ J] .Lipids , 1997 , 32:859-866.
[ 12] SUGIYAMA K , YAMAKAWA A. Die tary
er itadenine-induced altera tion of molecular species
composition of phospholipids in r ats[ J] .Lipids ,
1996 , 31:399-404.
[ 13] SUGIYAMA K , YAMAKAWA A , KAWAGISHI
H , e t al .Die tary er itadenine modif ies plasma
phosph atidylcholine molecular species prof ile in ra ts
f ed dif fer ent types of f at[ J] .J Nutr , 1997 , 127:
593-599.
[ 14] SUGIYAMA K , KUMAZAWA A , ZHOU H , et
al .Dietary methionine level af fects linoleic acid
me tabolism through phosphatidylethanolamine N-
methylation in r ats[ J] .Lipids , 1998 , 33:235-242.
[ 15] ENMAN J , ROVA U , BERGLUND K.
Quantification of the bioactive compound
er itadenine in selec ted stra ins of Shiitake mushroom
(Lentinus edodes)[ J] .J Agric Food Chem , 2007 ,
55:1177-1180.
56