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Journal of Research in Medical and Dental Science | Vol. 10 | Issue 4 | April 2022
Genistein: A new Isoflavone from Iraqi Chrozophora tinctoria
(Euphorbiaceae): Its Extraction, Isolation and Structure Elucidation
Abdul-aziz A Doghan*, Thukaa Z Abdul-Jalil
Department of Pharmacognosy and Medicinal Plants, College of Pharmacy, University of Baghdad, Baghdad, Iraq
ABSTRACT
In the current study, genistein proof of existence as a novel metabolite in the Iraqi Chrozophora tinctoria
(Euphorbiaceae) was the mainstay issue. Extraction of genistein was performed from the dried aerial parts of the
Iraqi Chrozophora tinctoria by using soxhelt apparatus with 85% Methanol followed by fractionation with petroleum
ether and ethyl acetate fraction. Preparative Reverse-phase high performance liquid chromatography HPLC analysis
of the ethyl acetate fraction for the aerial parts of the plant yielded eight phytoconstituents one of them (peak NO.2)
gave identical match with genistein standard, depending on which the isolation and quantitative determination
were done. A fruitful product of amorphous powder was yielded with a concentration 5.7 μg/g, the structure of
this compound was elucidated unambiguously by melting point, analytical RP-HPLC, Ultra Violet UV spectroscopic,
Fourier Transform Infrared Spectroscopy FTIR spectroscopic and High-performance compact mass spectrometer
(CMS) techniques revealing identical similarity to genistein.
Key words: Chrozophora tinctoria, Genistein, RP-HPLC, UV spectroscopic and High-performance compact mass
spectrometer (CMS)
HOW TO CITE THIS ARTICLE:
(Euphorbiaceae): Its Extraction, Isolation and Structure Elucidation, J Res Med Dent Sci, 2022, 10 (4):40-47.
Corresponding author: Abdul-aziz A Doghan
e-mail
: ali.mario28@yahoo.com
Received: 07-Mar-2022, Manuscript No. JRMDS-22-52361;
Editor assigned: 09-Mar-2022, Pre QC No. JRMDS-22-52361 (PQ);
Reviewed: 23-Mar-2022, QC No. JRMDS-22-52361;
Revised: 28-Mar-2022, Manuscript No. JRMDS-22-52361 (R);
Published: 04-April-2022
INTRODUCTION
Chrozophora tinctoria (Figure 1) is a medicinal plant
that belongs to the Euphorbiaceae family and contains
approximately nine species, the majority of which are
been found in the Mediterranean, tropical Africa, and
Southwest Asia [1]. The plant was traditionally used to
[2]. In some studies, Chrozophora was used as an
antioxidant [1], antimicrobial [2,3], anticancer [4], and
wound healing in diabetics [5]. The Chrozophora genus
is primarily composed of diterpenoids, alkaloids,
coumarins, chromones, xanthones, sterols, phenolic
acacetin [6,8-10].
Journal of Research in Medical and Dental Science
2022, Volume 10, Issue 4, Page No: 40-47
Copyright CC BY-NC 4.0
eISSN No. 2347-2367: pISSN No. 2347-2545
Figure 1: Photo of Iraqi Chrozophora tinctoria.
Abdul-aziz A Doghan, et al. J Res Med Dent Sci, 2022, 10 (4):40-47
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Journal of Research in Medical and Dental Science | Vol. 10 | Issue 4 | April 2022
with estrogen-like properties (phytoestrogen) [11,12].
. Genistein
Genistein (Figure 2) is a typical phytoestrogenic
. It is
treatment of many types of cancer. It has tumor-related
inhibits skin carcinogenesis and cutaneous aging in mice
and photo-damage in humans [16]. It is also used to treat
postmenopausal syndrome [17], osteoporosis disease
humans [19].
Aim of study
The hypothesis of the study concerned about other type
Chrozophora tinctoria aerial
future applications, leading the way by extraction,
isolation and structure elucidation of the genistein from
Chrozophora tinctoria.
MATERIALS AND METHODS
Equipment and chemical
(Knauer Germany 10AV-LC)], Fourier Transform
Infrared Spectroscopy [FTIR (Schimadzu/Japan 1900)],
High-performance compact mass spectrometer [CMS
of analytical grade and obtained from Riedel-de Haen,
which are HPLC grade purchased from Sigma-Aldrich,
Germany. The standard genistein were purchased from
Chengdu Biopurify Phytochemicals, China (purity >97).
Collection of plant materials and authentication
The plant materials of Chrozophora tinctoria (aerial
Mohammed Amer taxonomist in biology department,
by electrical milled and then weighed.
Extraction of plant materials
Chrozophora
tinctoria was packed in a thimble of soxhlet apparatus
and extracted with 85% methanol until complete
exhaustion (750ml for 12hrs). The methanolic extract
brown residue designated as a crude fraction [6,20]. The
crude fraction was partitioned with Petroleum ether
and Ethyl acetate (3x200 ml) for each fraction. The
[7]. The optimized process for the extraction, isolation
powdered plant material (aerial parts) of Chrozophora
tinctoria is shown in following scheme Figure 3.
Preliminary phytochemical investigation
ethyl acetate fraction of Chrozophora tinctoria aerial
parts were carried out using the procedures as described
by Harborne et al. [21] and Lallianrawna et al [22].
Alkaline reagent test
Few milligrams of the crude extract and ethyl acetate
fraction were suspended in ethanol and few drops of 5%
HCl were added. The changes in colors were recorded.
Shinoda test
1ml from extracts (crude and ethyl acetate) of
Chrozophora tinctoria aerial parts treated with
magnesium turning and concentrated HCl after few
Reverse - phase high performance liquid
chromatography analysis (RP-HPLC)
making a comparison of their retention time and the
shape of the UV spectrum with calibration (linear least
with that for authenticated reference standards at
identical chromatographic conditions. In this study, the
ethyl acetate fraction for Chrozophora tinctoria aerial
Figure 2: Genistein structure.
Abdul-aziz A Doghan, et al. J Res Med Dent Sci, 2022, 10 (4):40-47
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Journal of Research in Medical and Dental Science | Vol. 10 | Issue 4 | April 2022
Preparations of standard and samples for analysis
0.04 mg in 1 ml of methanol HPLC grade. Dried sample (ethyl acetate
and subjecting it to ultrasonication at 60% duty cycles for 25 minutes
at 25°C followed by centrifugation at 7500 rpm for 15 minutes. The
sample was injected into HPLC system for analysis [20].
analysis
A:90% B:10%,5-20min A:60% B:40%,20-40 min A:
40% B:60%,40-50min A:10% B:90%,50-55min A:90%
B:10%).
Column: Knauer/Germany LC C18 (250 mm x 4.6 mm, 5
Sample: Ethyl acetate fraction for Chrozophora tinctoria
aerial parts
Standard: Genistein.
Flow rate: 0.7 ml/min.
.
Injection concentration: 1 mg/ml.
Chrozophora tinctoria aerial parts.
Abdul-aziz A Doghan, et al. J Res Med Dent Sci, 2022, 10 (4):40-47
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Journal of Research in Medical and Dental Science | Vol. 10 | Issue 4 | April 2022
RP-HPLC conditions for isolation and quantitative
analysis
A:90% B:10%,5-20min A:70% B:30%,20-40min A:50%
B:50%,40-50min A:30% B:70%,50-55min A:10%
B:90%).
Column: Nucleodur, Macherey –Angel/Germany LC C18
Sample: ethyl acetate fraction for Chrozophora tinctoria
aerial parts
Standard: Genistein.
Flow rate: 3 ml / min.
.
Injection concentration: 1 mg /ml.
Characterization and structure elucidation of the
isolated constituent (G)
9Measuring the melting point and compare it to that
of the standard.
9
same retention time with the standard
9Analyzing the UV/Vis spectra and IR spectra
9
molecular mass information.
RESULTS AND DISCUSSIONS
In spite of the importance of Chrozophora tinctoria as
genistein in this study is the main target for its different
and perhaps worldwide about the present of genistein
Chrozophora tinctoria. The aerial parts were
extracted by Soxhlet method, as this method is heat
to moderately polar phytochemicals that exists in the
Chrozophora tinctoria, hence; 85% methanol was used in
this study in an attempt to obtain as much extract yield
differences in polarities is used as a crucial separation
step in order to analyse interconnected chemicals in
extracts preceded by different polarity petroleum ether
yield of each fraction is illustrated in the Table 1.
Preliminary phytochemical investigation
and ethyl acetate fraction. The results gained resembles
Reverse - phase high performance liquid
chromatography analysis (RP-HPLC)
Chrozophora tinctoria aerial parts ethyl
acetate fraction, in which both genistein standard and one
minutes) as shown in Figure 4.
the new detected compound in fraction of ethyl acetate
was used to determine the concentration as shown in
Table 2 and Figure 5.
elucidation of isolated G
The characterization and structural elucidation of the
amorphous powder isolated G constituent was done by
Melting point
useful information, which in its turn, helps to identify
the sample and its purity. The isolated G constituent had
a melting point at 300 C0- 302C0 (with decomposition)
compared with standard genistein which has a melting
point of 301C0-302C0 (with decomposition).
Analytical RP-HPLC
was applied on the isolated G constituent, in which the
times obtained at identical chromatographic conditions
of the isolated G constituent and the authentic standard
(genistein), as shown in Figure (6A), the HPLC
chromatograms were identical for both the isolated
compound G and genistein standard.
Table 1: Difference in weight yields of extract’s content of Iraqi
Chrozophora tinctoria aerial parts.
Fracons name Weight Yields
Crude methanolic extract 5.8 gm
Petroleum ether 670 mg
Ethyl acetate 220 mg
Abdul-aziz A Doghan, et al. J Res Med Dent Sci, 2022, 10 (4):40-47
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Journal of Research in Medical and Dental Science | Vol. 10 | Issue 4 | April 2022
Figure 5: A: preparative HPLC chromatogram of ethyl acetate fraction, B: calibration curves for the new isolated compound.
Table 2: The concentration of the isolated compound G from Iraqi Chrozophora tinctoria aerial parts ethyl acetate fraction.
Sample Peak area Concentraon in ethyl acetate fracon µg/ml Concentraon in plant µg/mg
Ethyl acetate 710.106 6.1779222 5.702697415
UV/Vis spectra
Determination of UV spectrum analysis was done by UV
computerized spectrophotometer (SHIMADUZU-1900/
range 200-1000 nm) using 100% methanol as standard
reference. The isolated G constituent showed identical
spectrum and same lambda max with genistein standard
at same condition, as shown in Figure (6B).
Fourier Transform Infrared Spectroscopy (FTIR)
model: SHIMADUZU1900/range 550-4000 nm was used
presence of substitution of benzene ring of isolated
Figure 4: RP-HPLC analysis chromatogram of ethyl acetate fraction with genistein standard where red curve NO. 1=ethyl acetate chromatogram
and blue curve number NO. 2= genistein standard chromatogram.
Abdul-aziz A Doghan, et al. J Res Med Dent Sci, 2022, 10 (4):40-47
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Journal of Research in Medical and Dental Science | Vol. 10 | Issue 4 | April 2022
G constituent has identical peaks at same region to
genistein standard as shown in Figure (6C).
High-performance compact mass spectrometer (CMS)
methanol and pumped through a capillary inside an
𝑚/
Figure 6: characterization and structural elucidation of isolated G compound. A: HPLC, B: UV, C: FT-IR and D: MS.
Abdul-aziz A Doghan, et al. J Res Med Dent Sci, 2022, 10 (4):40-47
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Journal of Research in Medical and Dental Science | Vol. 10 | Issue 4 | April 2022
𝑧=271.5 which has a moderate abundance with other
mass peaks (105,110,121,136,149,152,171,257) which
were identical with the peaks of genistein standard, as
hydroxyl groups, all of these lead to the conclusions
CONCLUSION
Diet is predicted to contribute to about 50% of cancers
world population, while incorporating food rich with
traditional medicinal systems around the world, had
Chrozophora tinctoria aerial parts
study and perhaps worldwide about the presence of
Chrozophora tinctoria. A simple and
easy method of extraction accompanied with rapid and
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