Qualitatively different analysis of commonly available golden Chinese mushrooms(Cordyceps militaris) products

การวิ เคราะ ห์ความแตกต่าง เ ชิง

คุณภาพของ เ ห็ดถั่ ง เ ช่ า สีทอง

(Cordyceps mi l i ta r is ) ใน

ท้องตลาดPresenter: Urairat Mungmonsin

Master’s degree of biomedical scienceGraduate School, Khon Kaen University

Advisor: Assoc. Prof. Sakda Daduang

1

Outline

Introduction Objective Method & Result

Discussion Conclusion Acknowledgement

2

Introduction

Cordyceps militaris (C. militaris) is parasitized on the larva

of some species of insects and the dead caterpillar. It is a

well-known traditional Chinese medicine which distribute

over the plateau grass with an altitude of 3,500–5,000m in

Tibet, Qinghai, and Yunnan provinces.

(Cunningham et al.,1951; Meena et al., 2010)

3

Introduction

The important metabolites and its applications

▪ Adenosine: immunomodulatory

▪ Exo-polysaccharides: anti-virus, anti-tumor

▪ Peptides: peptides anti-tumor, immuno-potentiation activities and anti-fungus

▪ Cordycepin: abroad spectrum of bioactivity

This Photo by Unknown Author is licensed under CC BY-NC-ND

(Cui, 2015)

4

Introduction

Cordycepin: The chemical biomarker of C. militaris

This Photo by Unknown Author is licensed under CC BY-NC-ND

Cordycepin is a nucleoside analogue that

exhibits a broad spectrum of biological

activities including

o antitumor

o antileukemia

o antiviral activities

o immunoregulative effect

o antibacterial

o antifungal

(Ahn et al., 2000; Zhou et al., 2002)

5

The price and market demand

❖ The price of dried C. militaris approximately 4-70 THB/g DW

This Photo by Unknown Author is licensed under CC BY-SA

In current years, the utilization of natural medicinal products has expanded at the expansion rate of 10% yearly, and Western Europe nations have started to unwind the confinements on Chinese herbal drugs.

Source : Credence Research

6

(*g DW = grams of dried weight)

The products from different company are different price and may contain the various organic compound PROBLEM

7

Cordyceps militaris. Retrieved. 9 March 2020, from https://www.alibaba.com/showroom/cordyceps-militaris-fruiting-bodys.html

Objective

To analyze the quality of differences of

Cordyceps mil i taris products from the

market

8

Scope of work

9

Analyze the organic structure by ATR-FTIR

Cordyceps Products

Determination of total cordycepin content by HPLC

If products contaminate with

chemical or metal the IR spectra will be different

The total cordycepin content indicate the quality of products

*ATR-FTIR = Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

Methods & Results

10

1. To analyze the characteristics component by ATR-FTIR

IR spectra showed an individual characteristics of each samples at wavenumber 650-4000 cm-1

The samples were dried at 65oC until constantly weight and ground

11

Principle component analysis (PCA); considering at PC-1 (81%)

Fingerprint region

12

Peak position observe on IR spectra were analyzed by PCA

Prediction value(red line)

Wavenumbers (cm-1) Base group and vibration mode Functional group

2904 vas(C-H) CH3, CH2

2848 vs(C-H) CH3, CH2

1708 v(C=O) Carboxylic acid

1631-1655 v(C=C) alkene

~1015-1147 v(C-OH) alcohol

~993 b(CH) C=CH2 alkene

696 b(C-H) aromatic C-H aromatic

2. Determination of total cordycepin content by HPLC method

13

0.1 g of Sample

Added 1 mL of 50% v/v MeOH. Mixed 10 min

and soaked at room temp. overnight

Filtrated supernatant through 0.45 µm and

analyze the cordycepin content by HPLC under

optimal conditions

Pipetted supernatant and centrifuge

at 10,000 x g for 10 min.

Control

SPIKED SAMPLES

Adenosine

COR

14

The sample was spiked with standard cordycepin and adenosine

The concentration of total cordycepin content was calculated by

Total cordycepin (mg/g DW) = concentration (mg /mL ) x volume mL x dilution factor

dry weight (g DW )

Sample 1 Sample 2Control

The retention time of cordycepin was 19 min

Sample 3 Sample 4

COR

COR COR

CORCOR

15

2. Determination of total cordycepin content by HPLC method

Sample 3 and 4 showed the higher

cordycepin content when compare with control at significant 95%

Sample 1 and 2 showed the lower cordycepin

content when compare with control at significant 95%

b

a a

d

c

0.00

1.00

2.00

3.00

4.00

5.00

Control Sample1 Sample2 Sample3 Sample4

Tota

l cor

dyce

pin

per d

ried

weigh

t(m

g/g

DW)

Product

16

*Different lowercase letters indicate significant difference at the 5% level.

Conclusion

17

IR spectra of all samples had an individual characteristics at fingerprint region (wavenumber 1430-910 cm-1) which can use for identify the organic structural.

Principle component analysis results showed sample 3 and 4 had similarly vibration of organic structure when compare with control (PC-1).

Chromatogram of all samples showed the single peak position of cordycepin at retention time at 19 min. The concentration of cordycepin content of sample 3 and 4 were higher than control while sample 1 and 2 were lower than control.

Conclusion

18

19

Conclusion (Cont.)IR spectra showed that the samples had

an individual characteristics of IR

spectra because it contained different chemical components.

The different fingerprints showed

different chemical constitutes which

can be determined for any chemical or heavy metal contaminations.

Chromatogram from HPLC analysis

showed the amount of total cordycepin content in sample.

ATR-FTIR and HPLC techniques have

proved the practicability of the method, which

can be applied to the quality estimation of

other Cordyceps militaris products.

โครงการวจิยัน้ีไดร้บัการสนบัสนุนจาก (1) โครงการสง่เสรมิผลงานวจิยัแสงซนิโครตรอน ส าหรบันกัวจิยัใหมท่ีไ่มเ่คยใชเ้ทคโนโลยแีสงซนิโครตรอน

มหาวทิยาลยัขอนแก่น ประจ าปีงบประมาณ 2561 (2) โครงการพฒันานกัวจิยัและงานวจิยัเพือ่อุตสาหกรรม (พวอ.) ระดบัปรญิญาโท ส านกังานกองทุน

สนบัสนุนการวจิยั (สกว.) ประจ าปีงบประมาณ 2561

Acknowledgement

20

References

Cui JD. Biotechnological production and applications of Cordyceps militaris, a valued traditional

Chinese medicine. Critical reviews in biotechnology 2015; 35(4): 475-84.

Cunningham KG, Hutchinson SA, Manson W, Spring FS. 508. Cordycepin, a metabolic product

from cultures of Cordyceps militaris (Linn.) link. Part I. Isolation and characterisation.

Journal of the Chemical Society 1951: 2299-300.

Kang C, Wen TC, Kang JC, Meng ZB, Li GR, Hyde KD. Optimization of large-scale culture

conditions for the production of cordycepin with Cordyceps militaris by liquid static

culture. The scientific world journal 2014; 2014.

Kodama EN, McCaffrey RP, Yusa K, Mitsuya H. Antileukemic activity and mechanism of action of

cordycepin against terminal deoxynucleotidyl transferase-positive (TdT+) leukemic cells.

Biochemical pharmacology 2000; 59(3): 273-81.

Tuli HS, Sharma AK, Sandhu SS, Kashyap D. Cordycepin: a bioactive metabolite with

therapeutic potential. Life sciences 2013; 93(23): 863-9.

Yang P, Song P, Sun SQ, Zhou Q, Feng S, Tao JX. Differentiation and quality estimation of

Cordyceps with infrared spectroscopy. Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy 2009; 74(4): 983-90.

Zhou X, Meyer CU, Schmidtke P, Zepp F. Effect of cordycepin on interleukin-10 production of

human peripheral blood mononuclear cells. European journal of pharmacology 2002;

453(2-3): 309-17.

21

Thank you

22

Sample 1

Sample 2

control

SPIKED SAMPLES

COR

Adenosine

Adenosine

COR

Adenosine

COR

23

SPIKED SAMPLES

Sample 3

Sample 4

COR

Adenosine

Adenosine

COR

24

25

Principle of ATR-FTIR technique

An ATR accessory operates by measuring the changes that occur

in an internally reflected IR beam when the beam comes into

contact with a sample. An IR beam is directed onto an optically

dense crystal with a high refractive index at a certain angle. This

internal reflectance creates an evanescent wave that extends

beyond the surface of the crystal into the sample held in contact

with the crystal.

In regions of the IR spectrum where the sample absorbs energy,

the evanescent wave will be attenuated. The attenuated beam

returns to the crystal, then exits the opposite end of the crystal and

is directed to the detector in the IR spectrometer. The detector

records the attenuated IR beam as an interferogram signal, which can then be used to generate an IR spectrum.

26

Principle of HPLC

High Performance Liquid Chromatography (HPLC) :

Principle, Types, Instrumentation and Applications

by Editorial Team on January 11, 2020 in Biochemistry

Chromatography is a technique to separate mixtures of

substances into their components on the basis of their

molecular structure and molecular composition.

This involves a stationary phase (a solid, or a liquid

supported on a solid) and a mobile phase (a liquid or a

gas). The mobile phase flows through the stationary phase

and carries the components of the mixture with it. Sample

components that display stronger interactions with the

stationary phase will move more slowly through the column than components with weaker interactions.

27

Dosage

Cordyceps has been used in human trials in the dosage range of 1,000-3,000mg

daily, either in one single dose or multiple doses with meals.

Maximum tolerance dose of cordycepin (3600 mg/kg) daily.

(Ma et al., 2015)

28

PCA