Cordyceps militaris) products
Transcript of Cordyceps militaris) products
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
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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)
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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)
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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)
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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
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(*g DW = grams of dried weight)
The products from different company are different price and may contain the various organic compound PROBLEM
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Cordyceps militaris. Retrieved. 9 March 2020, from https://www.alibaba.com/showroom/cordyceps-militaris-fruiting-bodys.html
Scope of work
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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
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
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Principle component analysis (PCA); considering at PC-1 (81%)
Fingerprint region
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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
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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
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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
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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
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*Different lowercase letters indicate significant difference at the 5% level.
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
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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
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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.
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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.
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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.
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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)