Determination of nucleosides, nucleobases and amino acids

in natural and cultured Cordyceps using HPLC-DAD-ELSD

By

Tan Miao

Master of Science

2012

Institute of Chinese Medical Sciences

University of Macau

Determination of nucleosides, nucleobases and amino acids

in natural and cultured Cordyceps using HPLC-DAD-ELSD

by

Tan Miao

A thesis submitted in partial fulfillment of the requirements

for the degree of

Master of Science

Institute of Chinese Medical Sciences

University of Macau

2012

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University of Macau, I agree that the Library and the Institute of Chinese Medical Sciences shall make

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碩士學位論文

HPLC-DAD-ELSD同時測定天然與人工蟲草中

核苷、堿基和氨基酸含量

研究生姓名： 譚 渺

導 師： 李紹平教授

專 業： 中 藥 學

日 期： 2012.7

澳門大學中華醫藥研究院

ACKNOWLEDGMENTS

When I came to graduate school to pursue a master’s degree, I was still unsure about what

my final career path would be. When I met Prof. Li Shao-Ping, things began to fall into place.

Prof. Li Shao-Ping is an exceptional advisor. He has taught me a great deal about the research

process and I am strongly inspired by his stringent realistic attitude, dedication and assiduous

work style and innovation spirit. I could not do the work well without his steady support.

Especially, sincere thanks to Director of Institute of Chinese Medical Sciences (ICMS), Prof.

Wang Yi-Tao, for his kindness and providing me the precious opportunity to accumulate

speculative knowledge and improve experimental skills which is significantly important for my

whole life.

Then, I would like to speak my very gratitude to Dr. Zhao Jing for her constant attention

and practical counseling to my experiments which help me avoid many tortuous paths, but also,

thanks to her meticulous care about my life.

I also owe my sincere thanks to Dr. Zhang Qing-wen, Prof. Simon Lee, Dr. Zheng Ying, Dr.

Yan Ru, Dr. Maggie Hoi, Ms. Hattie U and Ms. Chloe Lao for the support during the learning.

This research work was supported by all members of ICMS, University of Macau. Special

thanks to Leon Lai and Siokio Kuong for technical supports.

Thanks to Cheong Kit Leong and Li Jing for their guidance on experiment and thesis

writing. Thanks to Feng Kun, Qian Zheng-Ming, Chen Xiao-Jia, Huang Wei-Hua, Li De-Qiang,

Meng Lan-Zhen, Lv Guang-Ping, Wang Lan-Ying, Chen Yi-Wen for their help on my study.

Thanks to Aoli Mei-Jun, Li Hao-Ye, Chu Jun and Wang Sheng-Peng for sharing me with

the learning experience and the great happiness during my study life.

Thanks to my family for offering me great support all the time.

Abstract

Determination of nucleosides, nucleobases and amino acids in natural

and cultured Cordyceps using HPLC-DAD-ELSD

by

Tan Miao

Thesis Supervisor: Professor Li Shao-Ping

Major: Chinese Medicinal Sciences

Cordyceps sinensis, an entomogenous fungus also known as Dongcong Xiacao (winter

worm, summer grass) in Chinese, is one of the most valued medicinal herbs in traditional

Chinese medicine (TCM). With the appearances of cultured or alternative species Cordyceps, it

became an important problem for authentication and quality control of Cordyceps. Nucleosides

are believed to be one of the most important constitutes in Cordyceps for their pharmacological

activities. And amino acids are considered to be the key regulators of nutrient metabolism in cells.

And they may also provide tonic activities and act as the nutrition monitoring indexes in both

food and health care products, meanwhile, amino acids usually contribute the unique flavors to

fungus. Therefore, detecting of nucleosides and amino acids is required.

The samples were prepared through a heating reflux extraction. And in order to obtain more

information about the differences between micromolecule (nucleosides and amino acids) and

macromolecule (nucleotide, protein and etc.), this experiment also determined the compounds

hydrolyzed from macromolecule using the microwave-assisted hydrolysis. The chromatographic

separations were performed using an Agilent Zorbax SB-C18 column (5 μm, 4.6 mm×250 mm)

with an Agilent Zorbax SB-C18 guard column (5 μm, 4.6 mm×12.5 mm) at a constant

temperature of 30 ºC. The standards and samples were injected with 10 μL and separated using a

gradient mobile phase consisting of 4‰ TFA in water (A) and acetonitrile (B). At a flow rate of

0.6 mL/min, the gradient condition is : 0-15 min, 0% B; 15-20 min, 5% B; 20-25 min, 5% B;

25-35 min, 7% B; 35-40 min,7% B; 40-45 min, 7-40% B; 45-55 min, 40% B; 55-60 min, 40-0%

B; while recondition the column with 0% B for 15 min. The column was washed every five runs.

The nucleosides and nucleobases were monitored at 254 nm, and the ELSD was used for

detection of amino acids. Under this chromatographic condition, seven nucleosides and

nucleobases (uracil, hypoxanthine, uridine, adenine, inosine, adenosine, cordycepin), and eight

amino acids (alanine, histidine, valine, tyrosine, isoleucine, leucine, phenylalanine, tryptophane)

were baseline separated. And the method revealed good linear relationships, high repeatabilities

and recoveries.

The results suggested that the contents of nearly all the investigated compounds would

increase after hydrolysis. Among the cultured ones, only the contents of cordycepin and

adenosine decreased with hydrolysis. And for the natural group, except adenine and uridine were

almost disappeared, the others all showed a smaller growth with hydrolysis. On the other side,

the data of amino acids suggested that there were a larger increase in cultured Cordyceps after

hydrolysis. According the analysis of SPSS software, some of the same species could be

classified into one group through the data of nucleosides and nucleobases, and with only the

hydrolysis data, the samples could be generally divided into natural and cultured ones. This

suggested that there may be some significant difference in macromolecule between natural and

cultured Cordyceps.

To sum up, seven nucleosides and nucleobases (uracil, hypoxanthine, uridine, adenine,

inosine, adenosine, cordycepin), and eight amino acids (alanine, histidine, valine, tyrosine,

isoleucine, leucine, phenylalanine, tryptophane) have been selected as markers for analysis. And

a rapid qualitative and quantitative analysis HPLC method was established for natural and

cultured Cordyceps with good selectivity of detection and high repeatability.

Key words: Cordyceps; HPLC-DAD-ELSD; Nucleosides; Amino acids; Acid hydrolysis;

Microwave-assisted;

摘 要

HPLC-DAD-ELSD同時測定天然與人工蟲草中

核苷、堿基和氨基酸含量

碩士生：譚 渺

導 師：李紹平 教授

專 業：中藥學

冬蟲夏草是我國的名貴中藥，傳統中醫藥理論認為其有補腎益肺，止血化痰的功效，

用於治療腎虛精虧，陽痿遺精，腰膝酸痛，久咳虛喘，勞嗽咯血等症。現代藥理研究表明，

冬蟲夏草具有增強人體免疫功能和抗腫瘤等活性。隨著蟲草天然替代品的發掘及人工發酵

培養技術的發展，加之市場上的一些混亂現象，蟲草的質量顯得越來越重要。核苷類成份

是蟲草非常重要的活性成分之一，同時，蟲草作為藥食兩用的藥材，氨基酸被認為是其活

性成分及營養評價指標，此外它還是真菌類獨特風味的形成因素之一。故本實驗選擇了以

核苷類及氨基酸類物質為蟲草的質控指標。另一方面，本實驗還測定了水解后核苷及氨基

酸的含量，用以從一定程度上反應蟲草中核苷酸，蛋白質等大分子物質的情況。

本實驗採用平行迴流提取，微波輔助酸水解的方法，通過HPLC-DAD-ELSD對14種天

然及人工蟲草樣品分析，完成了對7種核苷（尿嘧啶，次黃嘌呤，尿苷，腺嘌呤，肌苷，腺

苷，蟲草素）和8種氨基酸（丙氨酸，組氨酸，纈氨酸，絡氨酸，異亮氨酸，亮氨酸，苯丙

氨酸，色氨酸）水解前後的含量測定與比較。方法如下：色譜柱Agilent Zorbax SB-C18 column

(5 μm, 4.6 mm×250 mm) ；預柱Agilent Zorbax SB-C18 guard column (5 μm, 4.6 mm×12.5 mm)；

柱溫30 ºC；進樣量10 μL。流動相：4‰ TFA(A)–乙腈(B)；流速：0.6 mL/min；梯度洗脫：

0-15 min，0% B；15-20 min，5% B；20-25 min，5% B；25-35 min，7% B；35-40 min，7%

B；40-45 min，7-40% B；45-55 min，40% B；55-60 min，40-0% B；回到初始流動相平衡

15 min。檢測波長；254 nm；ELSD參數：溫度為50 ºC，Gain值為8。本方法線性關係良好，

加樣回收率與重複性基本在要求內 。

結果表明，天然蟲草水解前後的核苷與氨基酸含量普遍低於人工蟲草。核苷類成份水

解后，人工蟲草中除蟲草素與腺苷的含量下降，其他皆呈上升趨勢。天然蟲草中大多數核

苷類成份也有相同趨勢但增幅較之減小很多，同時，部份成份如腺嘌呤與尿苷，幾乎完全

消失；氨基酸類成分中，水解后，人工蟲草的增幅遠大於天然蟲草。最後，通過SPSS分析

可得，利用蟲草遊離的核苷類成份數據能較好的區分其物種關係。水解后，則可將天然與

人工蟲草兩個大類大致區分出來。此結果可能預示著人工蟲草在核苷類的大分子上與天然

者有著較大差異。

此方法可以用於比較天然與人工蟲草在小分子（核苷類及氨基酸）和大分子（核苷酸

及蛋白質等）上的差異。結果表明，本方法穩定可靠，耗時較短，為蟲草的質量控制做出

了一定的參考。

關鍵字：蟲草；核苷；氨基酸；HPLC-DAD-ELSD；微波輔助酸水解

TABLE OF CONTENTS

Chapter 1 Review of Cordyceps.................................................................................................................. 1

1.1 Introduction ....................................................................................................................................... 1

1.1.1 The natural resources of Cordyceps .................................................................................................. 2

1.1.2 Chemical constitutes and their pharmacological activities in Cordyceps ...................................... 3

1.1.3 Quality control of C. sinensis ............................................................................................................ 7

1.2. The application of Microwave ........................................................................................................ 8

1.2.1 The differences between water bath calefaction and microwave .................................................. 8

1.2.2 The selection of hydrolysis reagent ................................................................................................... 9

1.2.3 The principle and characteristics of microwave ............................................................................. 9

1.2.4 Other application of microwave in research ................................................................................. 10

Chapter 2 Simultaneous determination of nucleoside, nucleobases and amino acids in natural and

cultured Cordyceps using HPLC-DAD-ELSD ........................................................................................ 11

2.1. Introduction .................................................................................................................................... 11

2.2. Experiment materials .................................................................................................................... 11

2.2.1 Chemicals and medicinal materials ................................................................................................ 11

2.2.2 Instrumentals .................................................................................................................................... 13

2.3 HPLC analysis ................................................................................................................................. 14

2.3.1 Sample preparation ......................................................................................................................... 14

2.3.2 Standard preparation ...................................................................................................................... 14

2.3.3 HPLC condition ............................................................................................................................... 14

2.4 Validation of the methods............................................................................................................... 17

2.4.1 Calibration curves ............................................................................................................................ 17

2.4.2 LOD and LOQ .................................................................................................................................. 17

2.4.3 Intra-day and inter-day accuracy ................................................................................................... 20

2.4.4 Recovery............................................................................................................................................ 20

2.4.5 Repeatability ..................................................................................................................................... 20

2.5. Optimization of methods ............................................................................................................... 22

2.5.1 Optimization of extraction condition ............................................................................................. 22

2.5.2 Optimization of microwave-assisted acid hydrolysis .................................................................... 24

2.5.3 Optimization of Chromatographic condition ................................................................................ 26

2.5.4 Other Optimizations ........................................................................................................................ 29

2.6 Results and discussion .................................................................................................................... 29

2.6.1 Qualitative and quantitative of each sample ................................................................................. 29

2.6.2 Data analysis ..................................................................................................................................... 32

2.6.3 Discussion.......................................................................................................................................... 34

2.7 Conclusion ........................................................................................................................................... 36

References .................................................................................................................................................. 38

LIST OF TABLES

Table 1 Example of species Cordyceps and their geographical distribution .......................................... 3

Table 2 The differences between water bath calefaction and microwave .............................................. 8

Table 3 The characteristics of candidate reagent for hydrolysis ............................................................. 9

Table 4 Sample list of natural and cultured Cordyceps of different sources ........................................ 12

Table 5 Liner regression data, LOD and LOQ of the investigated compounds .................................. 17

Table 6 Intra-day precision of 15 standards ........................................................................................... 20

Table 7 Inter-day precision of 15 standards ........................................................................................... 21

Table 8 Recovery and repeatability of 15 standards .............................................................................. 22

Table 9 The effect of gain-value on ratio of signal to noise .................................................................... 28

Table 10 The effect of temperature on ratio of signal to noise .............................................................. 29

Table 11 Contents (mg/g ) of 15 investigated compounds in 14 samples of natural and cultured

Cordyceps (without hydrolyze) ......................................................................................................... 30

Table 12 Contents (mg/g ) of 15 investigated compounds in 14 samples of natural and cultured

Cordyceps (with hydrolyze) .............................................................................................................. 31

LIST OF FIGURES

Figure 1 The chemical structures of some nucleosides isolated from Cordyceps ................................... 4

Figure 2 The chemical structures of D-mannitol, sterols and amino acids isolated from Cordyceps ... 5

Figure 3 Chemical structures of 15 investigated compounds ................................................................ 12

Figure 4 HPLC-DAD-ELSD profile of 15 standard ............................................................................... 16

Figure 5 Calibration curves of 15 standards .......................................................................................... 20

Figure 6 Optimization of extraction condition ....................................................................................... 24

Figure 7 Optimization of the acid concentration.................................................................................... 25

Figure 8 Optimization of microwave treating time ................................................................................ 26

Figure 9 Optimization of pH value of mobile phase .............................................................................. 27

Figure 10 Dendrogram resulting from data of nucleosides and nucleobases without hydrolysis ...... 33

Figure 11 Dendrogram resulting from data of nucleosides and nucleobases with hydrolysis ............ 34