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Isolation and Identification of some Primary Metabolites, Micro-and Macroelements of Aesculus hippocastanum L. Seeds

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The qualitative composition and quantitative content of amino acids were studied by PC and HPLC methods. 15 free amino acids were identified in the endosperm and skin of A. hippocastanum. 16 bound amino acids were identified in the endosperm; 17 bound amino acids were identified in the skin. The GC/MS method shows that A. hippocastanum skin and endosperm contain 2 free sugars and 5 after hydrolysis. We can also distinguish specific sugars for seed endosperm which are not present in the skin of the seed: Rha, Fuc, and Sucrose. Specific sugars in the skin of the seed are Xyl and Man. The endosperm of seeds of Aesculus hippocastanum accumulates starch, WSPS, and lipophilic compounds. PS and HC are concentrated in the skin. The qualitative composition and quantitative content of 19 macro and microelements in skin and endosperm of seeds of A. hippocastanum was studied by atomic emission spectrophotometry. The skin of seeds of A. hippocastanum accumulates macro and microelements. The seeds do not accumulate toxic metals, and this enables their use as medicinal plant material. The results show big differences between endosperm and skin in their contents of primary metabolites and elements. Those differences depend on functions that the skin and endosperm are playing in plants, and they influence how medicines and food supplements might be created.
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International Journal of Pharmacognosy and Phytochemical Research 2017; 9(1); 108-113
ISSN: 0975-4873
Research Article
*Author for Correspondence: Zead.helmi@outlook.com
Isolation and Identification of some Primary Metabolites, Micro- and
Macroelements of Aesculus hippocastanum L. Seeds
Karpiuk U V1, Abudayeh Z H M2*, Kyslychenko V S3, Yemelianova O І1
1Department of Pharmacognosy and Botany, Bogomolets National Medical University, Kyiv, Ukraine.
.University, 11622 Amman, JordanFaculty of Pharmacy, Isra
2
3Department of Chemistry of Natural Compounds, National University of Pharmacy, Kharkiv, Ukraine.
Received: 16th Nov, 16; Revised: 23rd Nov, 16; Accepted: 27th Dec,16; Available Online: 15th January, 2017
ABSTRACT
The qualitative composition and quantitative content of amino acids were studied by PC and HPLC methods. 15 free
amino acids were identified in the endosperm and skin of A. hippocastanum. 16 bound amino acids were identified in the
endosperm; 17 bound amino acids were identified in the skin. The GC/MS method shows that A. hippocastanum skin and
endosperm contain 2 free sugars and 5 after hydrolysis. We can also distinguish specific sugars for seed endosperm
which are not present in the skin of the seed: Rha, Fuc, and Sucrose. Specific sugars in the skin of the seed are Xyl and
Man. The endosperm of seeds of Aesculus hippocastanum accumulates starch, WSPS, and lipophilic compounds. PS and
HC are concentrated in the skin. The qualitative composition and quantitative content of 19 macro and microelements in
skin and endosperm of seeds of A. hippocastanum was studied by atomic emission spectrophotometry. The skin of seeds
of A. hippocastanum accumulates macro and microelements. The seeds do not accumulate toxic metals, and this enables
their use as medicinal plant material. The results show big differences between endosperm and skin in their contents of
primary metabolites and elements. Those differences depend on functions that the skin and endosperm are playing in
plants, and they influence how medicines and food supplements might be created.
Keywords: Aesculus hippocastanum L. seeds, amino acids, monosaccharides, polysaccharides, lipophilic compounds,
micro- and macroelements.
INTRODUCTION
Plant cell produces two types of metabolites: primary
metabolites and secondary metabolites. Primary
metabolites are involved directly in growth and
metabolism. Secondary metabolites considered as end
products of primary metabolism that are not involved in
metabolic activity and typically act as chemical defense
or to attract pollinators. Their absence does not cause
adverse effects on the growth of the plant itself.
Primary metabolites comprise many different types of
organic compounds, including, but not limited to,
carbohydrates, lipids, proteins, and nucleic acids. They
are found universally in the plant kingdom because they
are the components or products of fundamental metabolic
pathways or cycles such as glycolysis, the Krebs cycle,
and the Calvin cycle1-4.
Plant primary metabolites are widely used in different
areas: food, pharmacy, medicines, and cosmetics.
Proteins and amino acids are primary components of
living organisms. Their presence in plants increases the
food value of these plants or of protein-based compounds
and extracts that could be isolated from them. Plant lipids
are used as auxiliary and active substances in many drugs.
Carbohydrates can serve main components of drugs and
food supplements, as stipulated by their pharmacological
action. Carbohydrates from plants are increasingly being
considered as ecofriendly alternatives to the use of
synthetic additives in many other products, including
plastics, detergents, pharmaceutical tablets, gels, and
others4,5.
Nowadays, interest in the analysis of chemical
composition of medicinal herbs is growing, owing to the
continuing developments in nutrition and in biochemical
surveys and mineral prospecting. Additionally, studies of
plant raw materials not only characterize the active
components but also seek scientific evidence of their
therapeutic properties. Macro, micro, and trace elements
are known to play a vital role in biological functions in
plants and in human metabolic reactions. Moreover, trace
elements play an important role in the formation of
bioactive chemical constituents in medicinal herbs and
accordingly are responsible for their medicinal and toxic
properties6,7.
Horse chestnut Aesculus hippocastanum L. of
Hippocastanaceae family is a well-known plant, widely
used in medicine and pharmacy. The study of its primary
metabolites, as well as the macro- and microelements,
will help to develop new medicines and food
supplements.
MATERIALS AND METHODS
Plant material
Karpiuk et al. / Isolation and Identification…
IJPPR, Volume 9, Issue 1: January 2017 Page 109
Horse chestnut seeds were collected in Kyiv region
(Ukraine) in September 2015. The average temperature at
harvest time was 14 °C. The raw material was dried in a
well-ventilated, shaded place. The dried seeds were
divided into two parts skin and endosperm which
were milled to powder. Dried material samples were kept
in a dry and dark place in multilayer paper bags at room
temperature.
Determination of amino acids
The presence of amino acids in the plant raw material was
confirmed by paper chromatography (PC) of the aqueous
extract obtained from the roots, using Filtrak FN 7
chromatography paper, n-BuOHHOAcH2O (1:4:2), and
three-fold chromatography. Amino acids were detected
using ninhydrin solution (0.1%) followed by heating in a
drying cabinet up to 96 °C until spots of amino acids
appeared8.
Free protein-forming amino acids in the plant raw
material were determined quantitatively after extraction
of free amino acids from the plant raw material, and
bound amino acids were determined after acid hydrolysis
of the preparations, followed by HPLC analysis of the
hydrolysates using pre-column derivatization by 9-
fluorenylmethoxycarbonyl chloride (FMOC) and o-
phthalaldehyde (OPA) and a fluorescence detector. Each
analysis used five determinations.
Samples of plant raw material were prepared and
analyzed as follows:
a) Free amino acids: 100 mg of powdered preparation
was placed into a vial, treated with 2 ml of 1 N aqueous
HCl solution, and held at 50 °C for 3 h in an ultrasonic
bath.
b) Total amino acids: 100 mg of preparation was placed
into a vial, treated with 2 ml of 6 N aqueous HCl solution,
and placed into a thermostatic chamber at 110 °C for 24
h. Then, 0.5 ml of the centrifuged extract/hydrolysate was
evaporated in a rotary vacuum evaporator, rinsed three
times with distilled H2O to remove HCl, re-suspended in
0.5 ml of distilled H2O, and filtered through a 0.2 μm
regenerated cellulose membrane. Amino acids were
identified by comparing their retention times with a
mixture of amino acid standards (Agilent 5061-3334).
The contents of bound amino acids were determined by
subtracting the contents of free amino acids from their
total contents.
Chromatographic separation was performed on an Agilent
1200 liquid chromatograph (Agilent Technologies, USA)
using a Zorbax AAA column (150 mm x 4.6 mm, 3 μm)
and mobile phase A (Na2HPO4, 40 mm, pH 7.8) and B
(AcCNMeOH H2O, 45:45:10, v/v/v) in gradient mode
at constant flow rate 1.5 ml/min. The column was
thermostatted at 40 °C. The pre-column derivatization
was carried out in automated programmed mode using
FMOC (Agilent 5061-3337) and OPA (Agilent 5061-
3335). Derivatized amino acids were detected using a
fluorescence detector8.
Determination of Sugars
Qualitative composition and quantitative content of
sugars in plant material were determined by GC/MS
based on the extraction of free sugars from plant material
and full acid hydrolysis of herbal preparations to
determine the total monosaccharide composition,
followed by obtaining acetates of their aldonitrile
derivatives and their analysis. Each analysis used five
determinations.
Sample preparation and analysis of plant raw materials:
a) free monosaccharides: 0.5 g of plant material was
placed in a vial and 5 ml of 80% ethanol was added.
Extraction of free monosaccharides was performed in an
ultrasonic bath at 80 °C for 4 h. Then, 2 ml of the extract
was collected, evaporated to dryness, and re-suspended
with 2 ml of an aqueous solution of the internal standard
(2.5 mg per sample);
b) monosaccharide composition after hydrolysis of plant
raw material: 5 ml of 2 M trifluoroacetic acid was added
to 0.5 g of raw material; hydrolysis held at 110 °C for 6 h.
Then, 2 ml of hydrolyzate was collected, evaporated, and
washed with water to remove trifluoroacetic acid. The
hydrolysate was then re-suspended with 2 ml of an
aqueous solution of the internal standard (2.5 mg per
sample).
Chromatographic separation was performed on the
chromatograph Agilent 6890N/5973inert (Agilent
technologies, USA) using capillary column HP-5ms
(3mm×0.25mm×0.25μm, Agilent technologies, USA).
Evaporator temperature was 250 °C, the interface
temperature 280 °C. Separation was carried out in the
programming mode of the temperature: initial
temperature of 160 °C was maintained for 8 min, then
raised with a gradient of 5 °C/min to 240 °C. The final
temperature was held for 6 min. A sample of 1 μl was
injected in a split flow mode 1:50. Detection was in the
SCAN mode in a range 38-400 m/z. The flow rate of
carrier gas through the column was 1.2 ml/min.
Identification was carried by retention time of
monosaccharide standards and by the library of mass
spectra NIST 02. Quantitative analysis was carried out by
adding a solution of internal standard to the test sample.
The following mixture of standard samples were used:
monosaccharides: Rib, Rha, Ara, Xyl, Fuc, Man, Glu,
Gal, Fru; disaccharides: Sucrose. For the internal standard
solution Sorbitol was used9.
Determination of polysaccharides and lipophilic
compounds
Lipophilic compounds
The content of lipophilic compound was determined
using the Soxhlet method. Plant raw material first was
defatted by exhaustive extraction with chloroform in the
Soxhlet apparatus, and the lipophilic extract (LE) was
received10. Each analysis used five determinations.
Fractioning polysaccharides
Isolation, purification and analysis of polysaccharides
was performed by polysaccharide fractionation method.
Plant raw material was dried and sequentially extracted
with different solvents after obtaining the LE: 82%
ethanol for alcohol soluble substances; purified water for
water soluble polysaccharides (WSPS); a mix of 0.5%
oxalic acid solution and 0.5% ammonium oxalate solution
for pectin substances (PS); 7% sodium hydroxide solution
for hemicellulose A (HC A) and hemicellulose B (HC B).
Karpiuk et al. / Isolation and Identification…
IJPPR, Volume 9, Issue 1: January 2017 Page 110
Each analysis used five determinations.
100 g of remaining raw material after extraction with
82% ethanol was mixed with purified water, at a ratio of
plant raw material to extract 1:20. Extraction was
performed twice at a constant temperature 30-35 °C for
3.5 hr with constant stirring. Extracts were combined and
evaporated on a rotary heater to minimum volume under
vacuum. WSPS were precipitated by fivefold volume of
96% alcohol. The precipitate was filtered and washed
successively by hot 96% ethanol and acetone, dried in a
drying oven to constant weight, and weighed. Plant
material was dried after extraction.
The raw material after extraction of WSPS was used for
obtaining PS. Extraction was done with hot mix of 0.5%
oxalic acid solution and 0.5% ammonium oxalate solution
in a ratio 1:1. Extraction was performed twice at a
constant temperature 30-35 °C for 2 hours with constant
stirring, with a ratio of plant raw material to extract 1:20.
The extracts were separated from the raw material,
combined, concentrated, and precipitated by fivefold
volume of 96% alcohol. Formed PS precipitate was
filtered, washed successively with hot 96% ethanol and
acetone, dried in a drying oven to constant weight, and
weighed. Plant material was dried after extraction.
Hemicelluloses (HC) were obtained from the raw
material that remained after PS was removed. Extraction
was performed twice by 10% sodium hydroxide solution
in the ratio of raw material to extract 1:5, at room
temperature for 12 h. Alkaline extract was filtered, and
the filtrate was acidified with glacial acetic acid to
precipitation. The precipitate was filtered, dried to
constant weight, and weighed. In this way, HC A was
isolated. Twice the volume of 96% ethanol was added to
the filtrate; in this case it formed a precipitate which was
filtered, washed successively with hot 96% ethanol and
acetone, dried in a drying oven to constant weight, and
weighed. Thus, were obtained the fractions of HC B11,12.
Isolation of starch
Starch was extracted from horse chestnuts, using alkaline
steeping method as described by Sun et al. (2014) and
Perez & Lares (2004), with slight modification. The
endosperm was cut into pieces (2 cm2), steeped in 0.25 %
NaOH solution (w/v) in the ratio of 1:3 and stored at 4o C
for 24 h. The steeped endosperm, along with alkali, was
ground in a laboratory grinder and filtered through a
100 mesh sieve and allowed to settle, then given 23
washings with distilled water. The slurry was again
filtered through a 300 mesh sieve and the liquid was
centrifuged at 3000 rpm for 15 min. The aqueous phase
obtained upon centrifuging was discarded. The white
starch layer was re-suspended in distilled water and
centrifuged 23 times. The starch was then collected and
dried in a hot air oven at 40 °C. Saponins, the bitter
compounds present in horse chestnut, were removed by
continuous washings of the starch13. The analysis used
five determinations.
Determination of micro- and macroelements
To study the qualitative composition and quantitative
content of macro and microelements in the skin and
endosperm of horse chestnut seed, atomic absorption
spectrophotometry with atomization in an air-acetylene
flame was used. Analysis used five determinations.
Standard stock solutions with a concentration (1000 mg
L-1) of the individual metal element were used to prepare
the requested concentrations by dilution using a 1% (v/v)
nitric acid solution. The diluted standard solutions were
used to build the calibration curves. Metal element
standards were purchased from Sigma-Aldrich (St Louis,
MO, USA). An analytical reagents grade of concentrated
nitric acid (70%) and hydrogen peroxide (30%) were also
purchased from Sigma- Aldrich (St Louis, MO, USA). In
all the laboratory work, the glass and plastic containers
were cleaned by soaking in 10% v/v HNO3 for at least 24
h and rinsing with distilled water prior to use. All
chemicals used were of analytical grade. Ultrapure
deionized water, obtained from a Milli-Q water
purification system (Millipore, Bedford, MA, USA), was
used for preparing the solutions and for all dilutions.
An AAS instrument (Perkin Elmer AAnalyst 700 model
AAS) with deuterium background corrector was used for
the determination of Fe, Si, P, Al, Mn, Mg, Pb, Ni, Mo,
Ca, Cu, Zn, Na, K, Sr, Co, Cd, As, and Hg. Pb, Cd, and
Ni were determined by HGA graphite furnace using high
purity argon, while other measurements were carried out
in an air/acetylene flame. The operating parameters for
working elements were set according to the
recommendations of the manufacturer.
Procedure for drying ash
One gram of sample was transferred into a porcelain
crucible. The muffle furnace temperature was gradually
increased from room temperature to 450 ̊C in 1 h. The
sample was re-dried for 1 h in the oven, cooled, and
reweighed. The steps were repeated at 1 h drying
intervals until the differences in the variations in the
released water were less than 0.05%. The obtained
sample was ashed for about 8 h until a gray or white ash
residue was obtained. The residue was dissolved in 5 ml
of HNO3 (25% v/v) and, if necessary, the mixture was
heated slowly to dissolve the residue. Then the mixture
was heated up using an electric hot plate at 150 °C until
evaporated to near dryness. The residue was filtered
through Whatman filter paper and transferred into a
volumetric flask and filled to 25 ml with 3% HNO3. The
control experiment was also prepared in the same way.
Analytical procedure
AAS is a widely used technique for determining a large
number of metals. In AAS, an aqueous sample containing
the metal analyte of interest is aspirated into an air-
acetylene flame, causing evaporation of the solvent as
well as vaporization of the free metal atoms. Fe, Si, P, Al,
Mn, Mg, Pb, Ni, Mo, Ca, Cu, Zn, Na, K, Sr, Co, Cd, As,
and Hg in samples was analyzed using AAS equipped
with flame and graphite furnace. A graphite furnace was
used for the determination of trace and ultra-trace
concentrations (Pb, Ni, Mo, Co, Cd, As, Hg). The
operational conditions used to operate AAS instrument
were as recommended by the manufacturer. Data were
rounded off properly based on the value of standard
deviation from measurement conducted in triplicate6,14.
Karpiuk et al. / Isolation and Identification…
IJPPR, Volume 9, Issue 1: January 2017 Page 111
RESULTS AND DISCUSSION
Amino acids
PC identified these free amino acids in the endosperm
and skin of A. hippocastanum: Ser, Val, Glu, Arg, and
Ala (violet); His, Tyr, and Phe (gray-violet); Asp (blue-
violet); and Pro (yellow)15.
HPLC identified 15 free amino acids in the endosperm of
A. hippocastanum and 15 in the skin (Table 1).
Free Glu was present in endosperm in the greatest amount
(1.62 μg/mg). The amounts of the other amino acids were
less. Free amino acids in endosperm of A. hippocastanum
included seven essential amino acids (His, Thr, Val, Phe,
Ile, Leu, and Lys) in addition to Arg, which is considered
conditionally essential because it is extremely necessary
for young people8,16. Asp and Met were not found.
Free amino acids in A. hippocastanum skin were detected
in trace amounts. Among them were eight essential amino
acids His, Thr, Val, Met, Phe, Ile, Lys, and Arg. Leu was
not found.
Sixteen bound amino acids were identified in A.
hippocastanum endosperm. The quantitative contents of
all amino acids showed a tendency to increase
significantly after hydrolysis. The contents of bound Glu
(11.26±0.99 μg/mg), Asp (6.56±0.28μg/mg), and Cys
(8.24±0.58 μg/mg) were the greatest. However, Asp was
found only in bound form. The content of bound Glu
increased compared with the other acids from 1.62±0.07
to 11.26±0.99 μg/mg; the content of bound Cys increased
from 0.51±0.06 to 8.24±0.58 μg/mg.
Seventeen bound amino acids were identified in the skin
of A. hippocastanum. The quantitative contents of all
amino acids also showed a tendency to increase
Table 1: Quantitative content of identified amino acids in endosperm and skin of seeds from A. hippocastanum L.,
μg/mg.
п/п
Aminoa
cid
Retentio
n time
Free
Bound
Total
skin
endosperm
skin
endosperm
skin
1.
Asp
2.464
0.002288±0
.00025
6.56±0.28
2.98±0.05
6.56±0.05
2.98±0.58
2.
Glu
4.808
0.002683±0
.0002
11.26±0.99
1.61±0.58
12.88±2.83
1.61±0.44
3.
Ser
7.332
0.001513±0
.00038
2.61±0.06
1.91±0.44
2.94±0.34
1.92±0.39
4.
His٭
8.254
0.00256±0.
00028
0.87±0.07
2.54±0.29
0.98±0.05
2.54±0.68
5.
Gly
8.554
0.001262±0
.00031
2.61±0.17
1.02±0.77
2.86±0.38
1.02±0.49
6.
Thr٭
8.731
0.001463±0
.0003
2.09±0.19
2.54±0.05
2.38±0.22
2.54±0.21
7.
Arg٭
9.352
0.003815±0
.00019
3.27±0.05
0.86±0.05
4.04±0.75
0.86±0.28
8.
Ala
9.941
0.001287±0
.00029
2.39±0.05
1.09±0.29
2.98±0.55
1.09±0.54
9.
Tyr
11.033
0.001344±0
.00072
0.68±0.03
0.29±0.06
0.82±0.48
0.29±0.05
10.
Cys
12.162
0.004722±0
.00056
8.24±0.58
3.44±0.55
8.85±0.61
3.44±0.05
11.
Val٭
12.980
0.001014±0
.00037
2.15±0.08
0.99±0.01
2.47±0.51
0.99±0.04
12.
Met٭
13.507
0.000468±0
.00032
-
0.05±0.03
-
0.05±0.03
13.
Phe٭
14.320
0.010514±0
.00068
2.28±0.15
0.96±0.21
2.65±0.82
0.96±0.05
14.
Ile٭
14.506
0.00087±0.
00029
2.34±0.85
1.13±0.21
2.52±0.59
1.13±0.21
15.
Leu٭
15.109
-
3.54±0.52
0.56±0.08
3.83±0.12
0.56±0.04
16.
Lys٭
15.369
0.000866±0
.00077
2.64±0.47
1.95±0.09
2.91±0.23
1.95±0.45
17.
Pro
18.864
0.000918±0
.00032
2.3±0.29
0.96±0.49
2.50±0.28
0.96±0.09
The sum of essential
amino acids
0.02157
19,16
11.56
21.78
11.58
The sum of nonessential
amino acids
0.016017
36.76
13.29
40.39
13.31
The sum of amino acids
0.037587
55.92
24.85
62.17
24.89
Note: ٭ - essential amino acid.
Karpiuk et al. / Isolation and Identification…
IJPPR, Volume 9, Issue 1: January 2017 Page 112
Table 3: Content of polysaccharides and lipophilic
compounds in A. hippocastanum seeds.
Fraction
Yield, %
skin
endosperm
LE
3.3±0.66
18.80±1.06
WSPS
0.56±0.04
3.23±0.36
PS
15.0±1.33
1.5±0.39
HC A
6.7±0.83
4.2±1.01
HC B
3.8±0.72
1.7±0.33
Starch
-
54.5±2.75
significantly after hydrolysis, but compared with contents
in the endosperm they were found in lower amounts. The
concentrations of bound Asp (2.98±0.05 μg/mg), His
(2.54±0.29 μg/mg), Thr (2.54±0.05 μg/mg), and Cys
(3.44±0.55 μg/mg) were the greatest. Several amino acids
that were found in trace quantities in the free state
occurred in greater quantities in the bound form. These
were Ile (1.13±0.21 g/mg), Lys (1.95±0.09 μg/mg), and
Pro (0.96±0.49 μg/mg). The quantitative content of one
amino acid that was not identified in the free state but
was found in the bound form was 0.56 μg/mg (Leu).
Bound amino acids from A. hippocastanum seed skin
included nine essential amino acids (His, Thr, Arg, Val,
Met, Phe, Ile, Leu, and Lys).
Sugars
The results of identification of monosaccharides by
GC/MS are in Table 2. Free sugars in the endosperm of
Aesculus hippocastanum seeds consist of the
monosaccharide Glu and the disaccharide Sucrose.
Sucrose is present in a higher amount. Free sugars in the
skin consist of Glu and Ara.
Rha, Ara, Fuc, Glu, and Gal were found in a composition
of monosaccharides in the endosperm of A.
hippocastanum seeds by GC/MS method after hydrolysis.
The content of Glu increased after hydrolysis. Rha, Ara,
Fuc, and Gal were found only after hydrolysis (Table 2).
Ara, Xyl, Man, Glu, and Gal were identified as a part of
the sum of sugars from skin of A. hippocastanum seeds.
Also, the amount of Glu and Ara increased significantly
after hydrolysis, compared to its amount as a free sugar.
Xyl, Man, and Gal were found only after hydrolysis.
We can distinguish specific sugars for the endosperm of
seeds, which are not present in the skin of seeds: Rha,
Fuc, and Sucrose. The specific sugars in the skin of seeds
are Xyl and Man. The amount of Glu is higher in the
endosperm than in the skin, both in free form and after
hydrolysis. The amount of Ara and Gal in skins exceeds
its content in endosperms, both in free form and after
hydrolysis.
Polysaccharides
Polysaccharide contents are listed in a Table 3. Skin and
endosperm have different contents of polysaccharides and
lipophilic compounds. The yields of LE and WSPS in
endosperm vastly exceeds those in skin, but the content of
PS, HC A, and HC B is higher in skin. Starch, WSPS, and
fats are an energy source and are needed for complex
physiological and biochemical processes during seeds
stratification and germination. Accordingly, the higher
content of these compounds is more important in the
endosperm than in the skin of seeds.
Micro- and macroelements
Our research identified 19 macro- and microelements
(Table 4). Elemental composition showed the following
pattern in the content of macroelements in the skin: K>
Ca> Mg, Si > P > Na. Microelements accumulated in this
way: Fe> Al> Zn> Sr> Mn> Cu> Ni> Mo. In the
endosperm metals were found in very small amounts;
they showed this pattern in their contents: macroelements
Ca>P>Mg>Si>K>Na; microelements Cu>Fe, Al> Ni,
Mo>Mn, Zn, Sr.
Heavy metals were found in very small quantities: Co
<0.03 mg/100g; Pb <0.03 mg/100g; Cd <0.01 mg/100g;
As <0.01 mg/100g; Hg <0.01 mg/100g. The seeds do not
accumulate toxic metals, and this enables their use as
medicinal plant material. This absence of toxic metals is
also important because of anthropogenic factors,
pollution, and quality control methods for raw materials.
CONCLUSIONS
The results showed big differences between primary
metabolites and content of elements in endosperm and
skin of A. hippocastanum. It depends on the functions
that skin and endosperm have in the plant. The qualitative
composition and quantitative content of amino acids were
studied by PC and HPLC method. 15 free amino acids
were identified in the endosperm and skin of A.
hippocastanum. 16 bound amino acids were identified in
A. hippocastanum endosperm. 17 bound amino acids
were identified in A. hippocastanum skin. The GC/MS
Table 2: Free monosaccharides and sum of monosaccharides in endosperm and skin of seeds from Aesculus
hippocastanum L., mg/kg.
Standard
Retention time
Endosperm
Skin
Free
Sum
Free
Sum
Rha
8.0442
-
0.66±0.04
-
-
Ara
7.976
-
3.53±0.08
0.09±0.02
24.97±0.93
Xyl
8.627
-
-
-
4.12±0.29
Fuc
9.005
-
0.65±0.02
-
-
Man
14.334
-
-
-
6.63±0.77
Glu
14.628
1.84±0.06
158.35±1.57
0.06±0.02
21.64±0.85
Gal
15.163
-
8.03±0.09
-
20.29±1.06
Sorbitol
18.07
i/s
i/s
i/s
i/s
Sucrose
34.041
31.94±0.78
-
-
-
Total
33.78
171.22
0.15
77.65
Karpiuk et al. / Isolation and Identification…
IJPPR, Volume 9, Issue 1: January 2017 Page 113
Table 4: Content of minerals in endosperm and skin of
A. hippocastanum seeds.
Content
mg/100g
Sample
Skin
Endosperm
Fe
5.0±0.21
0.03±0.002
Si
64±1.08
3.0±0.27
P
24±0.97
15±0.98
Al
4.0±0.72
0.03±0.002
Mn
0.48±0.03
<0.01
Mg
64±2.2
8,7±0,74
Ni
0.016±0.004
<0.03
Mo
<0.03
<0.03
Ca
255±3.25
15±1.12
Cu
0.16±0.03
0.058±0.0019
Zn
1.6±0.3
<0.01
Na
16±1.6
<0.1
K
720±3.8
<1.0
Sr
0.8±0.07
<0.01
Pb <0.03; Co <0.03; Cd <0.01; As <0.01; Hg <0.01.
method showed that A. hippocastanum skin and
endosperm contain 2 free sugars and 5 after hydrolysis.
We can also distinguish specific sugars in the endosperm
which are not present in the skin: Rha, Fuc, and Sucrose.
Specific sugars in the skin are Xyl and Man. Endosperms
accumulate starch, WSPS, and lipophilic compounds. PS
and HC are concentrated in skin. The qualitative
composition and quantitative content of 19 macro and
microelements in skin and endosperm of seeds of A.
hippocastanum were studied by atomic emission
spectrophotometry. The skins accumulate macro- and
microelements. Seeds do not accumulate toxic metals,
and this enables their use as medicinal plant material. The
contents of primary metabolites provide opportunities for
creating medicine and food supplements.
REFERENCES
1. Heldt H-W, Piechulla B. Plant Biochemistry. 4-th
Edition, Elsevier or Academic Press, Amsterdam,
Boston, Heidelberg, London, New York, Oxford,
Paris, San Diego, San Francisco, Singapore, Sydney,
Tokyo, 2011, 622.
2. Siddiqui M W, Bansal V, Prasad K. Plant Secondary
Metabolites. Volume Two: Stimulation, Extraction,
and Utilization. ALPEL PUB (PQ), 2016, 375.
3. Irchhaiya R, Kumar A, Yadav A, Gupta N, Kumar S,
Gupta N, Kumar S, Yadav V, Prakash A, Gurjar H.
Metabolites in plants and its classification. World
journal of pharmacy and pharmaceutical sciences
2015; 4(1):287-305.
4. Evans W. C. Trease and Evans' Pharmacognosy. 16-
th Edition, Elsevier Health Sciences, 2009, 616.
5. Sasikala A, Linga Rao M, Savithramma N.
Quantification of primary and secondary metabolites
from leaves and stem bark of Cochlospermum
religiosum (L.) alston. International research journal
of pharmacy 2013; 4(8):228-231.
6. Karpiuk U V, Al Azzam K M, Abudayeh Z H M,
Kislichenko V, Naddaf A, Cholak I, Yemelianova O.
Qualitative and quantitative content determination of
macro-minor elements in Bryonia alba L. roots using
flame atomic absorption spectroscopy technique.
Advanced pharmaceutical bulletin 2016; 6(2):285-
291.
7. Guardia M, Garrigues S. Handbook of mineral
elements in food. John Wiley & Sons, 2015, 792.
8. Karpyuk U V, Kislichenko V S, Gur’eva I G. HPLC
Determination of free and bound amino acids in
Bryonia alba. Chemistry of natural compounds 2015;
51(2):399-400.
9. Karpyuk U V, Kislichenko V S, Gur’eva I G.
Carbohydrate composition of Bryonia alba.
Chemistry of natural compounds 2016; 52(4):672-
673.
10. Cukanovic J, Ninic-Todorovic J, Ognjanov V,
Mladenović E, Ljubojević M, Kurjakov A.
Biochemical composition of the horse chestnut seed
(Aesculus hippocastanum L.). Archives of biological
sciences 2011; 63(2):345-351.
11. Deng J, Shi Z-J, Li X-Z, Liu H-M. Soluble
polysaccharides isolation and characterization from
rabbiteye blueberry (Vaccinium ashei) fruits.
BioResources 2013; 8(1):405-419.
12. Li J-J, Bi H-t, Yan J H, Sun F, Fan Sh-sh, Cao G,
Zhou Y-f, Chen X-G, Comparative analysis of
polysaccharides from two ecological types of Leymus
chinensis. Chemical research in chinese universities
2012; 28(4):677-681.
13. Rafiq S I, Jan K, Singh S, Saxena D C.
Physicochemical, pasting, rheological, thermal and
morphological properties of horse chestnut starch.
Journal of food science and technology 2015;
52(9):56515660.
14. Karpiuk U V. Mineral composition of corn silk of
different sorts and hybrids of Zea mays. Collection of
scientific works of staff members of P.L. Shupyk
NMAPE 2011; 20(3):483-487.
15. Harborne J B. Phytochemical Methods. A guide to
modern techniques of plant analysis. 3rd Edition,
Chapman and Hall, London, Weinheim, New York,
Tokyo, Melbourne, Madras, 1998, 302.
16. Radovick S, Margaret H, MacGillivray. Pediatric
Endocrinology: A Practical Clinical Guide, Second
Edition Contemporary Endocrinology, 2d Ed.,
Springer Science & Business Media, 2013, 624.
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