Download Amino acid composition of the aerial part of G. pratense L., G

Amino acid composition of the aerial part of G. pratense L., G.
sylvaticum L., G. palustre L.
© Razaryonova+ Ksenia Nikolaevna, Zaharova Anna Mikhailovna, Protasova Inna
Dmitrievna and Zhokhova* Elena Vladimirovna
Department of Pharmacognosy.
Saint-Petersburgs State Chemical pharmaceutical academy.
Prof. Popov str., 14. Saint-Petersburg 197376. Russia.
T. +7 (812) 234-43-62. E-mail: [email protected]
Analytical Laboratory Analyt Ltd St. Petersburg
Bolshoi pr. Vasilyevsky island, 31. Saint-Petersburg 199004. Russia.
Тел.: +7 (812) 325-55-02. E-mail: [email protected]
____________________________________
*Leader of the thematic; +Corresponding
Key words: amino acids, HPLC, Geranium pratense, Geranium sylvaticum, Geranium
palustre, phenylisothiocyanate
Abstract
Amino acid composition of the aerial part of G. pratense, G. sylvaticum, G. palustre was evaluated
by reversed-phase HPLC method with UV-detection. The method includes derivatization of amino acids
using phenylisothiocyanate solution in isopropanol.
Introduction
The genus Geranium L. comprises 400 species in temperate areas and tropical mountains
throughout most of the world [1]. There are occurring 70 species for this genus in Russia and
CIS (The Commonwealth of Independent States) [11]. Geranium pratense L. and Geranium
sylvaticum L. are perennial herbs widespread in Russia from European Russia to the Russian Far
East. Geranium palustre L. is a common species in the North-West Russia [4].
These species of Geranium have been used in folk medicine as astringent, anti-haemoragic,
anti-inflammatory and antimicrobial agent in treatment of digestive system dysfunctions
(diarrhea, gastritis, colitis, enterocolitis), inflammatory oral cavity diseases, haemorrhages, gout,
rheumatism [9].
The chemical composition of the aerial part of these species includes phenols (tannins,
flavonoids, phenolic acids), carbonhydrates, vitamines, organic acids [9].
Current problems of Pharmacy include searching for new remedies of plant origin. The
original chemical composition, wide spectrum of biological activities, wide distribution indicates that
G. pratense, G. sylvaticum, G. palustre can be potential sources of new medicines.
Studies have proved that amino acids can directly or indirectly influence the physiological
activities of the plant. Amino acids are active themselves and can act to improve synthesis,
bioavailability and enhance activity of the secondary metabolites [2].
20 amino acids were identified and measured in the aerial part of G. pratense harvested in
Bashkortostan [7]. There are no literature data on amino acid composition of G. sylvaticum and
G. palustre.
The aim of present investigation is quantification and quantitation of amino acids of the
aerial part of G. pratense, G. sylvaticum, G. palustre.
Typically, high performance liquid chromatography (HPLC) is used for the analysis of
amino acids [3]. Amino acids lack chromophores and do not give a UV-Vis response, hence
amino acids require derivatization prior to HPLC analysis performed with UV-Vis detectors. A
method to determine free amino acids by pre-column derivatization with phenylisothiocyanate
(PITC) is used in our investigations. The advantages of this derivatizing agent are: 1) the amino
acids are converted into the corresponding phenylthiohydantoin (PTH) derivatives, which are
stable; 2) the reaction of PITC with amino terminus or termini proceeds nearly quantitavely; 3) a
short-term analysis.
Experiment
Materials obtained from plants growing in Leningrad region at the stage of fruit development in
july-august of 2011. The aerial part of G. pratense and G. palustre was harvested in Pudost’ of
Gatchinskiy district. The raw material of G. sylvaticum was collected beside Lembolovo of
Vsevolozhskiy district. The aerial parts of G. pratense, G. sylvaticum, G. palustre included dried leaves,
collected with petiole 3 cm, and dried flowering tops 20-25 cm long [8]. Collected aerial organs of G.
pratense, G. sylvaticum, G. palustre were dried first in open air and then in sheds. Dried aerial parts were
cut and sifted to pieces 2 cm in diameter.
Dried and ground aerial parts were extracted by 40% ethanol at boiling water bath. The extraction
method was maceration with 3 steps: every step of maceration continued for 1 h with solvent to solid ratio
10:1. Combined filtered extracts were evaporated to dryness. The dry extract was ground to powder.
We used paper chromatography to separate and identify amino acids. For this separation a solution,
containing n-butanol, acetic acid and water (4:1:2) was a mobile phase. About 0.5 g of dry powdered
extract, obtained from geraniums as described earlier, was dissolved in 2 ml of 40% ethanol. 10µL of
solution was applied at the bottom of a rectangular piece of filter paper. The spots of amino acids were
visualized by spraying the paper with 2% acetonic solution of ninhydrin [10].
Amino acid composition of the aerial part of G. pratense, G. sylvaticum, G. palustre was analyzed
by HPLC.
All analysis was performed using Shimadzu LC-20 Prominence HPLC system equipped with LC20AD pump, the SPD-20A UV-Vis detector. Data analysis was carried out by Shimadzu’s LCsolution
software.
The separation was carried out on a SUPELCO DiscoveryTM LC-18 column (250 mm × 4.6 mm
i.d., 5 μm) under the following chromatographic conditions: sample injection volume, 20 μL; flow rate,
1.2 mL/min; mobile phase: sodium acetate 0.06 M adjusted to pH=5.5 (eluent A), solution of isopropanol
in acetonitrile 1% (v/v) (eluent B), sodium acetate 0.06 M adjusted to pH= 4.05 (eluent C) [6].
We used following amino acid standards: Asparagine (ASN), Glutamine (GLN), Hydroxyproline
(H-PRO), Serine (SER), Glycine (GLY), Histidine (HIS), Arginine (ARG), Threonine (THR), Alanine
(ALA), Proline (PRO), Tyrosine (TYR), Valine (VAL), Lysine (LYS), Leusine (LEU), Isoleusine (ILE),
Phenylalanine (PHE), Methionine (MET), Cystine (CYS), Cysteine (CYS-CYS), Tryptophan (TRP).
The calibration curves were obtained by the external standard method on three levels of
concentration of standard compounds. Chromatograms of derivatized amino acid standards are given in
Figure 1.
Figure 1. HPLC chromatograms of mixture of amino acids standards derivatized by PITC; Shimadzu LC-20
Prominence HPLC system, SPD-20A UV-Vis detector, SUPELCO DiscoveryTM LC-18 column, wavelength 254 nm
For HPLC-analysis of amino acids (with the exception of Tryptophan and Cystine +Cysteine)
about 0.1g of accurately weight powdered dry extract of G. pratense, G. sylvaticum, G. palustre was
dissolved in 5 mL of 40% ethanol, solution was set in ultrasonic bath for 10 min (Solution A).
0.1-0.2 mL of Solution A was hot air dried at 60°C. 0.1 mL of 0.15 M sodium hydroxidum were
added to dry Solution A (solution B). Then Solution B was mixed with 0.35 mL of PITC solution in
isopropanol and 0.05 mL of distilled water. The composition stood for 20 min. After that it was dried at
65°C. Dry mixture was dissolved in 1 mL of distilled water, and this solution passed through filter and
injected into the «entrance» of the column.
Measuring of Tryptophan included no PITC-derivatization. Described earlier Solution A of
Tryptophan was directly injected into the column (wavelength 278 nm).
For identification and quantitaion of Cystine +Cysteine Solution A was oxidized by peroxyformic
acid. To obtain peroxyformic acid 1 part of hydroxidi peroxidum was mixed with 9 parts of formic acid.
Convertion of Cystine +Cysteine to cysteic acid was followed by PITC-derivatization of cysteic acid and
HPLC-analysis.
Results and discussion
The paper chromatography experiment indicated that the aerial part of G. pratense, G.
sylvaticum, G. palustre contained free amino acids. When the solvent front came near the top of
the filter paper the paper was removed from the beaker and allowed to dry. After spraying the
paper with 2% acetonic solution of ninhydrin paper showed a number of blue-violet (in the case
of Proline the spot was yellow) spots each one corresponding to an amino acids.
Data of quantitative analyses are listed in Table 1.
Chromatograms of dry extracts obtained from the aerial part of G. pratense, G. sylvaticum,
G. palustre are given in Figures 2-4.
Table 1. The content of identified amino acids in the aerial part of G. pratense, G. sylvaticum, G. palustre
(mg/g on the weight of the absolutely dry herb material)
Amino acid
G. pratense
G. sylvaticum
G. palustre
Asparagine
1.17
0.36
0.05
Glutamine
0.93
0.58
0.23
Hydroxyproline
0.43
0.09
0.05
Serine
1.10
0.21
2.65
Glycine
0.16
0.17
0.22
Histidine
0.08
0.18
0.12
Arginine
1.60
1.32
1.84
Threonine
0.31
not identified
not identified
Alanine
0.11
1.21
0.32
Proline
0.87
4.00
1.40
Tyrosine
0.12
0.22
0.39
Valine
0.06
0.36
0.27
Isoleusine
0.16
0.48
4.15
Leusin
0.22
0.07
0.40
Phenylalanine
0.09
0.08
0.04
Lysine
0.30
0.16
0.04
Cystine + Cysteine
0.84
0.16
0.11
Methionine
0.19
3.48
1.15
Tryptophan
0.016
0.037
0.03
Total content
8.76
13.17
13.46
20 amino acids were identified by HPLC-analysis in the aerial part of G. pratense: the
quantities of Arginine, Serine, Asparagine were maximal (Tab.1, Fig.2). The amino acid
composition of G. sylvaticum included 19 substances with maximal content of Arginine, Proline,
Methionine, Alanine (Fig.3). Among 19 identified amino acids of the aerial part of G. palustre
the highest concentration was established for Arginine, Proline, Methionine, Serine, Isoleusine
(Fig.4).
Figure 2. HPLC chromatograms of dry extract obtained from G. pratense after derivatization by PITC;
Shimadzu LC-20 Prominence HPLC system, SPD-20A UV-Vis detector, SUPELCO DiscoveryTM LC-18
column, wavelength 254 nm
Figure 3. HPLC chromatograms of dry extract obtained from G. sylvaticum after derivatization by PITC;
Shimadzu LC-20 Prominence HPLC system, SPD-20A UV-Vis detector, SUPELCO DiscoveryTM LC-18
column, wavelength 254 nm
Figure 4. HPLC chromatograms of dry extract obtained from G. palustre after derivatization by PITC;
Shimadzu LC-20 Prominence HPLC system, SPD-20A UV-Vis detector, SUPELCO DiscoveryTM LC-18
column, wavelength 254 nm
10 amino acids detected in G. pratense herb are essential (Histidine, Arginine, Threonine,
Valine, Lysine, Leusine, Isoleusine, Phenylalanine, Methionine, Tryptophan). The same essential
amino acids with exception of Threonine were measured in the aerial part of G. sylvaticum, G.
palustre.
The total content of amino acids in the aerial part of G. pratense, G. sylvaticum, G.
palustre was respectively 8.76, 13.17, 13.46 mg/g.
The significant amounts of Methionine in G. sylvaticum and G. palustre, Isoleusine in G.
palustre are ought to be underlined. According to literature data therapeutic daily dose of G.
sylvaticum and G. palustre herb is 10 g by infusion [5]. This dosage of G. sylvaticum herb
correspondes 15% of recommrnded daily amounts of Methionine [12]. The said dose of G.
palustre herb correspondes 5% of recommended daily intake of Methionine and Isoleusine [12].
Results of our assay approve necessity of complete investigation of Geranium species as
sources of biologically active components.
Conclusions
1. Reversed-phase HPLC method with UV-detection of PTH derivatives of amino acids was
used for quantification and quantitation of amino acids in the aerial part of G. pratense, G.
sylvaticum, G. palustre.
2. 20 amino acids (10 of them are essential) were measured in G. pratense herb. It is
established that amino acid composition of the aerial part G. sylvaticum, G. palustre included 19
constituents with 9 essential.
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