Transcript ХРОМАТОГРАММЫ

THE RESULTS OF BIOCHEMICAL RESEARCH AND
DEVELOPMENT OF NITROGEN-CONTAINING
COMPOUNDS OF NATURAL ORIGIN:
methodology of exploitation of biological properties as
universal natural regulators of metabolism and drugs
Leonid Nefyodov
Doktor of Medical Science, Professor,
Head of the Research Laboratory of
biochemistry of bioactive substances,
Professor of Biochemistry Department,
Yanka Kupala State University of Grodno
www.nil.grsu.by
1
BIOCHEMISTRY OF AMINO ACIDS AND
DEVELOPMENT OF METHODS OF THEIR
PRACTICAL USE AS UNIVERSAL ENDOGENOUS
REGULATORS OF METABOLISM, MEDICAL AND
PROPHYLACTIC AGENTS AND COMPONENTS OF
HUMAN NUTRITION
Grodno "amino acid" scientific school represented by 20
scientists, including 3 scientists of the second generation;
successfully were defended 12 candidate theses and 3
doctoral theses, one more is being prepared by a postgraduate.
2
ACCREDITATION OF THE RL “BIOCHEMISTRY OF
BIOACTIVE SUBSTANCES” ON ITS CONFORMANCE TO
THE REQUIREMENTS OF ISO/IEC 17025
Material and technical base of the RL was
created using directed funds of the Council
of Ministers and the Ministry of Education of
the Republic of Belarus, international
contracts (Austria, United Kingdom) and the
Central Fund of the University.
Due to this the laboratory acquired the
opportunities to develop and implement
modern methods of biochemical research,
perform researches in accordance with the
international standards of GLP at a
competitive level.
The Laboratory is a part of Grodno Regional
Clinical Hospital, and is integrated into its
Department of Clinical and Laboratory
Diagnosis, what contributes to the efficiency
of basic and applied research.
3
DEVELOPMENT OF DEMANDED, NEW AND ADAPTATION OF
KNOWN HIGHLY ANALYTICAL METHODS OF DETECTION OF
DIFFERENT NATURAL COMPOUNDS’ IN BIOLOGICAL
MATERIAL
Years of experience and qualification of the staff
enable to set and achieve goals not only with the
help of well-known and standard laboratory
methods but also by the
development or
adaptation of new methods, which often meet
unexpected and changing requirements of
customers (for example - combination of HPLC
with modern physical-chemical methods of
NMR,
mass
spectrometry,
differential
centrifugation, spectrofluorometry), what allows
to obtain evidential results, comparing them in
independent researches.
4
4
protopine
allokryptopine
chelidonine
coptisine
6
0
5
chelerythrine
stylopine
berberine
2
sanguinarine
One of the best used in practice,
evidence,
high-sensitive,
universal
and,
simultaneously,
specific
methods is high-efficient
liquid
chromatography
(HPLC).
10
15
20
25
min
5
0
10
20
30
40
50
60
70
mAU
DAD1 E, Sig=285,16 Ref=450,100 (D:\DATA\UKR00008.D)
8
0
5
77.109
74.735 - Orn
63.245
60.709 - Ctn
55.782 - Val
57.063 - Met
32.291 - Tau
25.318 - Arg
66.887 - Ile
48.929 - EA
67.417
69.218 - Leu
62.833
50.342 - dAVA
44.584 - aABA
42.659 - Tyr
38.047 - GABA
33.122
29.324 - Ala
24.385 - Ctr
64.950 - Trp
LU
71.829
64.341
61.627
58.922
53.961
47.090
30.458
26.577
20
21.095 - Thr
35
17.280 - His
19.066 - MHis3
40
18.578 - Gly
15.628 - Gln
45
20.496
10
13.677 14.084
9.834 - Asn
6.345 - Glu
3.601 - Asp 3.170
25
11.757
15
2.479
30
7.291
4.097
HPLC of alkaloids Chelidonium majus L., free
amino acids, their derivatives, precursors and
metabolites
FLD1 A, Ex=231, Em=445, TT (AA CID\A A 000020.D)
FLD1 B, Ex=231, Em=313, TT (A A CID\A A 000020.D)
0
80
min
Methods of HPLC /mass-spectrometry, NMR and
flow cytometry
Inten.(x10,000)
582.35
1.75
1.50
1.25
1.00
817.65
0.75
534.00
0.50
392.75
564.90
0.25
719.00
616.75
877.15
652.95
746.00
892.80
829.75
0.00
400
500
600
700
800
m/z
6
Largely thanks to this, agreements with the
next organizations were implemented:
- Agreement with the Center for collective use of devices,
Federation of European Biochemical Communities (FEBS
SARS, London, UK) on free technical assistance;
- Agreement with Anticancer Institute in Vienna (Austria) on
the early diagnosis of malignant disease on the basis of
identification of specific compounds, which appear in the
range of the second and third lines of the anticancer
defense;
- Agreements with Institutions of the Ministry of Health of
the Republic of Belarus on new domestic medicines’ test, in
particular – on piracetam, produced by Grodno (Skidel)
factory.
7
- Agreement with Grodno executive committee to
implement the tasks of Regional Scientific and Technical
Programme (RSTP) and develop new products of
functional food on the basis of domestic herbal;
- Agreements with Belarusian Republican Fund for
Fundamental Research (BRFFR) and the Ministry of
Education to study mechanisms of therapeutic action of
natural nitrogen-containing compounds;
- Agreements with enterprises of meat and dairy industry
in the region to provide an advisory service in the
questions of safety and quality of food;
- There was made an independent examination of RSTP
and SSTP projects related to research and development
in the field of biochemistry of biologically active
substances.
8
STUDY OF BIOLOGICAL ACTIVITY AND EFECT
MECHANISMS, INCLUDING ANTICANCER EFFECT OF
NATURAL NITROGEN-CONTAINING COMPOUNDS
AND THEIR DERIVATIVES
Priority methodology of the RL based on the
exploitation of regulatory action of natural or
close to natural (endogenous) concentrations of
nitrogen-containing natural compounds in the
first place - amino acids and related substances,
which allows to fulfill biochemical and clinical
sections of the fourteenth tasks of VI section of
SSTP "Drugs: highly purified amino acids" and
to develop medicines manufactured by Grodno
(Skidel) factory (pills "taurine", "lejcine"
capsules "tavamin", "tryptophan").
9
10

L. Nefyodov, M. Velichlo, A. Karavay, and others. Medication containing
derivative of L-glutamine, which possesses the anti-cancer activity /Inventor’s
application 19990937 d/d 15.10. 1999

L. Nefyodov , M. Velichko, A. Karavay, I. Lednyova, E. Doroshenko, D. Maslakov
New counter-drugs agent based on L-triptophane / Inventor’s application
19991021 d/d 17.11. 1999.

V. Soldatov, L. Nefyodov, N. Kurbat Medicament “taurine” / Registration
certificate МЗ РБ N 08-1-6/856.

V. Soldatov, L. Nefyodov, N. Kurbat Medicament “leucine” / Registration
certificate МЗ РБ N 12-1-6.

L. Nefyodov, E. Doroshenko, V. Smirnov Medication with hepatoprotective,
antioxidant and antiradiation effect “tavamine“ / Inventor’s application 19991021
d/d 17.09.2001. МПК А 61 К 31/195

L. Nefyodov, M. Velichko, A. Karavay, I. Lednyova, T. Valyuk Medication
containing derivative of L-glutamine, which possesses the anti-cancer activity /
Official bulletin of the Center for Intellectual Property of Belarus - 2004.-N1.p.103. Patent BY N 5882 CI.

Zh. Motylevich, L. Nefyodov, E. Sudnikovich, T. Rudyak, S. Zabrodskaya
Medication which is a magnesium salt hydrate of - toluydine acid with and has
an anti-cancer effect / Official bulletin of the Center for Intellectual Property of
Belarus - 2005.-N.- Patent BY N 5882 CI.
11
We were the first to demonstrate that endogenous levels of free
amino acids’ of men’s fluids and tissues are the most
important integral indicators and regulators of metabolism.
This enables to prove the use of individual amino acids or their
combinations for guided correction of metabolism with
specific human diseases and significantly expand the area of
practical application of these compounds.
12
Under Regulator effect of amino acids is
understood their effect on the biochemical
processes and vital functions, which is
manifested in natural (endogenous) or close to
natural concentrations of these compounds.
A regulatory effect after introduction of amino
acids can be achieved by using either individual
amino acids, or a combination of their small sets.
The structure of these compositions in specific
ratios may include almost any amino acids, their
structural analogs or derivatives with a known
mechanism of action.
13
The trend we develop is notable for the fund
of amino acids in biological fluids and
tissues is assessed as a single information
unit, and while analyzing it, it is possible to
estimate the segments which disrupt the
metabolic balance (changes in the metabolic
flows ratio and their balance shifts).
Thus, the studied characteristics represent a
kind of "chemical projection" of the genome
that is being undertaken through the
proteome.
14
Thus, field of our studies and developments
includes new, postgenome technologies
which are on the list of EU scientific
priorities, but not so developed in the
Republic of Belarus
These research solve not only fundamental
tasks but also enable us to solve key
problems of biotechnology, medicine and
pharmacology, in this connection most
countries devote great funds to perform such
researches.
15
The results of such researches enable to
distinguish "normal" metabolism from a
“modified“ one and since the year 2002 has
being successfully applied in USA, Britain,
Japan and EU countries in the development
of new medicines and methods of molecular
diagnostics. They accelerate the development
of new medicines and increase the chances
for their safe and effective application.
16
It is believed that research in this area will
have more significant impact on medicine
than the project “Genome Project”, as they
reflect more accurately physiological and
biochemical processes in the body, what
enables to use them not only for early
diagnosis of human diseases, but also to
assess the aptitude to them.
17
Performance of tasks of RSTP "Sustainable
development: science, innovation and technology
in Grodno region," grants of BRFFR and the
Ministry of Education, scientific and methodical
support of clinical trials of new home made
drugs
The methodology of the regulatory effect of amino acids
and their derivatives was used by us for:
 realization of the project on the development of new
domestic sorts of functional food based on vegetable
supplement “Lofantus anisatis” together with JSC
“Oshmyany meat-packing factory” and farm “Gury”.
 development of alternative, applied and used as
evidence methods for assessing nutritional quality and
safety of functional food, and addition of vegetarian
supplements with unique chemistry and similarity in
the basic qualitative and quantitative characteristics
with the amino acid Fund of human plasma;
18
Percentage of free amino acids in
anise giant hyssop
Cys
1,5%
Hcy
0,2%
CysGly
0,2%
GSH
1,7%
CA
0,8%
Asp
7,6%
Pro
17,1%
Glu
8,4%
Lys
0,3%
Orn
1,2%
HPro
0,0%
Leu
2,4%
Asn
20,0%
Ile
2,3%
Phe
1,6%
Trp
0,5%
CysCys
0,8%
Ctn
0,5%
Met
0,0%
Ser
1,5%
Val
2,2%
EA
1,3%
aABA
0,0%
Tyr
2,2%
GABA
5,9%
bABA
0,0%
Ala
3,7%bAla
Arg
Tau
0,5%
2,5%
0,2%
1MHis
0,0%
PEA
Thr
0,2%
Ctr
7,1%
0,1%
Gly
1,0%
His
2,2%
aAAA
0,4%
Gln
1,3%
3MHis
0,2%
19
Profiles of free amino acids and their derivatives
anise giant hyssop and human plasma
40
35
25
20
15
10
5
His
Lys
Orn
EA
bAla
Phe
Tyr
Leu
Ile
Cys
Val
Ala
Gly
Pro
Gln
Glu
Ser
Thr
Asp
Tau
0
CA
%, от сум.
30
плазма крови человека
Лофант анисовый
20
Content of free amino acids and their derivatives in food production
before and after addition of anise giant hyssop’s extract
800
700
600
400
300
200
100
0
CA
Asp
Glu
Asn
Ser
aAAA
Gln
His
Gly
3-MHis
PEA
Thr
1-MHis
Ctr
Arg
bAla
Ala
Tau
bABA
GABA
Tyr
aABA
EA
Val
Met
Ctn
Trp
Phe
Ile
Leu
HPro
Orn
Lys
Pro
Cys
Hcy
CysGly
GSH
С, мг/кг
500
пищевая продукция с лофантом
пищевая продукция без лофанта
21


Implementation of grants of BRFFR and the Ministry of
Education of the Republic of Belarus, which result not
only in development and optimization of highly
selective HPLC, NMR, mass and flow cytometric
methods of nitrogen-containing biologically active
substances’ determination (free α-amino acids and
their metabolites, as well as the alkaloids of celandine),
which allow to identify them in one biological sample,
but in vitro a specific selective binding of these
compounds with components of plasma and formed
blood elements of cancer patients were determined .
High sensitivity (91.7%), specificity (83.3%) and
efficiency (88.9%) of the proposed method of malignant
tumors’ diagnosis enable us to develop a new testsystem for diagnosis and monitoring the treatment of
cancer patients.
22
Plane projection of two main components and
groups’ clustering according to the levels of
specific compounds in blood plasma of healthy
people and patients with malignant or benign
tumors
23
Improving the quality of education and
upbringing, work with talented student youth,
and training of higher qualified personnel
We pay particular attention to education
and training of talented students:
search and find students with fresh
thinking, ability to analyze and solve
tasks with the help of not just wellknown but original ways. In reality, as
shows the experience, according to the
normal
distribution
law,
the
percentage of researchers among
population doesn’t exceed 5%. When
invited
to
the
Laboratory
undergraduates work together with
the lab’s personnel in the normal
working conditions, perform their
daily work, get practice and learn. Few
of them remain till the 5th year.
Diligence, active research work and
guidance bring results. In such a way,
the Laboratory prepared Anton
Glazov, who became an Associate
Professor (new representative of
"amino acid" school), a JRS of the
Laboratory, a lecturer at the Faculty of
Biology and Ecology and a talented
biochemist. The others are coming
soon.
24
Today the Laboratory:




- Submitted all the documentation for new RSTP tasks 2010;
- Completed the preparation of two SSTP projects for 2011 2015. ("Metabolism" and "Development of new effective
domestic drugs and blood substitutes with directional
effect");
- Accumulated experience in development and production
of new domestic products of functional food, delivery of
consultative help to companies of meat and dairy industry
in questions concerning quality assurance and food safety,
food staples, premixes and feed additives, as production of
the indicated enterprises is marketing in both the Republic
of Belarus, and in Russia and EU countries;
- Negotiates for participation in establishment of a crossborder inter-agency center for promotion of food quality
assurance and safety in accordance with EU regulations.
25
The publications of Laboratory:
1. Nefyodov L.I. Taurine, Grodno, 1999, 140 p.
2. Uglyanitsa K.N., Nefyodov L.I., Bzosko W. Ukrain® (biological activity & medical
applications), Grodno, 2000, 315 p.
3. Ledneva I.O., Velichko M.G., Nefyodov L.I. Effects of deryvativs of L- glutamin and Lphenilalanin in the tumorbraiding, Grodno, GSU, 2003, 108 p.
4. Amino Acids And Their Derivatives//Proc of Internat. Symp/Ed. V. Soldatov, L.
Nefydov, 1996, Grodno, 125 p
5. Nefyodov L.I., Uglynitsa K.N., Godysh Yu. et al. Biochemical mechanisms of realization
of Ukrain antitumor activity // European Journal of Cancer, 1997, V. 33, Supp.5, S27.
6. Nefyodov L.I., Mechanisms of regulatory effects and strategy of using amino acids and
their derivatives as effective agents for metabolic therapy and medicinal preparations Proc
of Internat. Symp. Biological activity and transport of drugs. /Ed. L. Nefydov, Grodno,
1999, 189p.
7. Nefyodov L.I., Smirnov V.Y. Brzosko W. et al. Mechanisms of the anticancer effect of
Ukrain in the treatment of cancer of the urinary bladder // Drugs under Exptl. Clin. Res.,
2000,
V.XXVI,
N
5/6,
p.
334
-344
8. Nefyodov L.I., Planowa regulacja równowagi metabolizmu aminokwasami i strategia
ich zastosowania w jakości nowych efektywnych preparatów leczniczych // XXXVII
Zjazd Polskiego towarzystwa biochemicznego. Torun, 10-14 IX 2001. s.327
9. Biologically active compounds in control of metabolic homeostasis //Proc of Internat.
Symp /Ed. L. Nefyodov, 2000, Grodno, 325 p
10. VI Ordinary General Assembly Society of Biochemistry of Belarus /Ed. L. Nefyodov,
2000,
Grodno,
225
p
11. Amino Acids And Their Derivatives // Proc Internat. Symp. /Ed. L. Nefyodov, 2001
Grodno, 124 p.
12. Nefyodov LI., Doroshenko E.M., Smirnov V. et al. Metabolic control and treatment of
malignant growth: tumor development, patterns of amino acid imbalance and Ukrain //
Int. J. Immunotherapy, 2003,V.XIX (2-4), p.109-114.
26
13. Nefyodov LI., Doroshenko E.M., Smirnov V. Yu. at al. Metabolic control
and treatment of malignant growth: tumor development, patterns of amino
acid imbalance and Ukrain/ FEBS Journal", 2005, V 272, N1 (Suppl), p.558,
// Abst. 30th FEBS Congress & 9th IUBMB Conference 'The Protein World
Proteins and Peptides: Structure, Function & organization", 3-7 July 2005,
V1-067P Budapest
14. Nefyodov L., Nowicky J., Glazev A., Karavay A. Anticancer drug
Ukrain®: mechanisms of anticancer effects and perspectives of its
application in the cancer diagnostics // Abstracts of the 41st Meeting of the
Polish Biochemical Society, September 12-15, 2006, Bialystok, Poland. –
2006. – P. 92.
15. Glazev A, L. Nefyodov Changes in amino acid patterns of blood plasma
in tumor patients with anticancer drug NSC-631570: possible approaches to
cancer diagnostics// Biochemistry (Moscow). Suppl. series B. Biomedical
chemistry, 2008, V. 2, № 3, p. 318–324.
16. Glazev, A. Nefyodov L., Nowicky W.HPLC–method for detection of
alkaloids and its derivatives in anticancer drug Ukrain® for its using in the
cancer detection, effective treatment and investigating the mechanisms of
carcinogenesis // Abstracts of the 41st Meeting of the Polish Biochemical
Society, September 12-15, 2006, Bialystok, Poland. – 2006. – P. 88.
17. W. Nowicky, L. Nefyodov, A. Glazev, [et al] The effect of NSC 631570 on
amino acid profile as a possible assay for early cancer diagnostics // Int.
Sypm. Clinical Biomarкer Summit, March 29-31, 2006, Coronado,
California, USA. – 2006. – P. 25.
27
24.
L. Nefyodov, E. Doroshenko, V. Smirnov Medication with
hepatoprotective, antioxidant and antiradiation effect “tavamine“ /
Inventor’s application 19991021 d/d 17.09.2001. МПК А 61 К 31/195
25. L. Nefyodov, M. Velichko, A. Karavay, I. Lednyova, T. Valyuk
Medication containing derivative of L-glutamine, which possesses the anticancer activity / Official bulletin of the Center for Intellectual Property of
Belarus
2004.-N1.p.103.
Patent
BY
N
5882
CI.
26. Zh. Motylevich, L. Nefyodov, E. Sudnikovich, T. Rudyak, S.
Zabrodskaya Medication which is a magnesium salt hydrate of - toluydine
acid with and has an anti-cancer effect / Official bulletin of the Center for
Intellectual Property of Belarus - 2005.-N.- Patent BY N 5882 CI.