1. LC-DAD-MS (ESI+) ANALYSIS OF AMARANTHUS RETROFLEXUS-
ANTIOXIDANT, ANTI-INFLAMMATORY AND PROTECTIVE EFFECT AGAINST LONG TERM DIABETIC COMPLICATIONS.
Savvanaki Vasilikia, Chatzopoulou Mariab, Kefalas Panosc, Kokkalou Evgeniosa,*
aDepartment of Pharmacognosy-Pharmacology, Faculty of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Aristotle university of Thessaloniki, 54124 Thessaloniki, Greece
cMediterranean Agronomic Institute of Chania, 73100 Chania, Crete, Greece
*Corresponding author. Tel.:2310997634, fax: 2310997662, E-mail address: kokkalou@pharm.auth.gr
Introduction
For many centuries, the leaves and seeds of Amaranthus species such as A. retroflexus have been
sources of food for native people from North and South America to Asia, Africa and Europa.
Amaranth seeds contain high-quality protein with amino acid composition close to ideal protein. The
main properties of Amaranthus products are presented below:
•Amaranth flour represents a suitable foodstuff for patients with gluten free diet, as amaranth affects
positively plasma lipid profile.
•Amaranth oil is applicable as an effective natural antioxidant supplement especially due to the high
content of unsaturated fatty acids and unique presence of squalene.
•Tea with the leaves of amaranth for prophylactic and therapeutic purposes was developed in Russia.
•Amaranth was applied as means of cleaning the stomach, a diuretic, to cure intestinal colic, cough,
headaches and tumors.
•Antioxidant activities of the amaranth seed extract positively correlated with the presence of total
polyphenols. [1]
•Amaranth leaves in Greece are used as salad.
Purpose
The current study aims to evaluate LC-DAD-MS (ESI+) from the phytochemical analysis of
Amaranthus Retroflexus, and the antioxidant, anti-inflammatory and protective effects of it against
long term diabetic complications.
Materials and methods
The inflorescence of the plant Amaranthus Retroflexus were collected from Velestino/Volos in May
2011. All plant material (290g) after defatting were exhaustively extracted with methanol. The dry
remaining of the methanolic extract (22.8g) was dissolved in hot water, filtrated and partitioned with
the following solvents: diethyl ether, ethyl acetate and butanol respectively. The dry remainings were
1.58g for diethyl ether, 730mg for ethyl acetate and 3.4g for butanol.
Instruments
For the antioxidant activity all the spectrophotometric data were acquired using UV-VIS, Hitachi U-2000 in a 0-mm quartz
cuvette and for the anti-inflammatory activity we used UV-VIS, Perkin Elmer Lambda 20 in a 10-mm quartz cuvette.
LC-DAD-MS (ESI +) analysis of Amaranthus Retroflexus
Mass spectra were obtained via liquid chromatographic introduction into a Finnigan AQA Thermoquest, equipped with a
Finnigan Surveyor Modular HPLC system. The chromatographic separation of the compounds was achieved using a Synergi
hydro-RP C18 80Å (150×2mm; 4u)(Phenomenex) column at a flow rate of 0.3ml/min. Mobile phases A and B were aqueous
2.5% Acetic acid and Methanol, respectively. Gradient elution was conducted as follows: 0–2min for 0% B with a linear
gradient, followed by 2–52min of 100%B, followed by isocratic step for 8 min solvent B, then 60 to 65min 0%B. The MS
system consisted of a Surveyor autosampler AS3000, pump P4000, UV-Vis diode array detector UV6000LP (DAD), and MS
detector finnigan AQA Thermoquest equipped with an ESI source and run by Xcalibur® 1.2.
Probe at 4KV and 400ºC. Kinetic energies at 10 and 50 eV. Mass range: 121-787 amu UV-Vis at 278 and 340 nm
Plus Full range UV-Vis at 230-650 nm.
Anti-inflammatory activity
The anti-inflammatory activity of the above extracts were defined through measuring the
ability of inhibition of the enzyme lipoxygenase (LOX) [2]. The diethyl ether extract showed
the highest activity, but the action of the other extracts wasn’t high.
Protective effect against long term diabetic complications
Aldose reductase and the polyol pathway of glucose metabolism have been implicated on
the onset and progression of long-term diabetic complications. Inhibitory activity on
partially purified rat lens ALR2 was measured spectrophotometrically by determining
NADPH consumption at 340 nm, with DL-glyceraldehyde used as substrate. The samples
were tested at the concentration of 50 mg/ml and were dissolved in 10% aqueous DMSO.
From the samples tested: diethyl ether (1), ethyl acetate (2), butanol (3), aqueous (4), the
second exhibited the highest inhibitory activity. [3]
Extract Retention
time
Structures Basic
fragm
ents
Diethyl ether 17.90
Caffeoyl-ferulic or isoferulic acid derivative
195,
180
19.98
Ferrulic or isoferrulic acid/ dihydrocoumaroyl ester derivative
195,
180,
177,
149
Ethyl acetate 23.72
Coumaric-caffeoyl ether 7-o quercetin
465,
303,
180,
147
24.77
Coumaroyl-caffeic ester derivative
180,
147
26.30
Coumaric 3-0-kaempferol- 7-O- caffeic
595,
449,
287,
179
Butanol 20.43
Coumaroyl 3-O-quercetin/ 7-O feruloyl or isoferuloyl derivative
611,
303,
287,
181,
165,
147
23.42
Coumaric-caffeoyl ether-7-O-quercetin
633,
465,
303,
147
OH
OH
HO
O
*
HO
OH
O
-H2O
OO
OH O
OH
OH
OH
*
*
HO
O
O
HO
HO
OH
O
-H20
O
O
OH
OH
HO
OHO
O
OH
O
OH
OH
O
OHO
H3CO
HO
O
-2H
O
OH
H3CO
HO
HO
HO
O
OH
-H2O
Evaluation of antiradical efficiency
The antioxidant activity of four extracts [diethylether (1), ethyl acetate (2), butanol (3),
aqueous (4) ] of Amaranthus retroflexus inflorences was evaluated by the 2,2-diphenyl-1-
picrylhydrazyl (DPPH.) free radical assay. The exact initial DPPH. concentration ( C DPPH.) in
the reaction medium was calculated from the calibration curve with the equation, A(515nm)
= 0.0174 CDPPH. (μg/ml) + 0.0194, R2= 0.9996, as determined by linear regression. For each
antioxidant concentration tested the precentage of DPPH. remaining at the steady state was
calculated as follows : %DPPH.rem = [DPPH.]T / [DPPH.]T=0, where T is the time necessary to
reach the steady state. These values were plotted vs. moles antioxidant/mole DPPH. to
obtain the amount of antioxidant necessary to decrease the initial DPPH. concentration by
50% (EC50). The conscentration of antioxidant needed to decrease the initial DPPH (EC50)
by 50% is a parameter widely used to measure the antioxidant activity. [4]
In the present study, we found out that extracts of the Amaranthus Retroflexus exhibit
important antioxidant, anti-inflammatory and protective effect against long term diabetic
complications. The strongest results are related to the inhibitory activity on partially
purified rat lens ALR2. Three out of 4 extracts, except the aqueous, present remarkable
inhibitory properties. Therefore, the phytochemical study focuses on the extracts with the
strongest results. The phytochemical analysis and the identification of the full structures
of LC-DAD-MS analysis of the plant Amaranthus retroflexus is still in process.
The results showed that all four extracts were efficient and effective in inhibiting the free
radical DPPH., and the scavenging activity of them was decreased in the following order
3>1>2>4.
Refrences:
[1] J.Kalinova, E. Dadakova (2009) Rutin and Total Quercetin Content in Amaranth (Amaranthus spp.) Plant Foods Hum Nutr 64:68–74
[2]D. Hadjipavlou-Litina, G.E. Magoulas, S.E. Bariamis, D. Drainas, K. Avgoustakis, D. Papaioannou, Does conjugation of antioxidants improve their antioxidative/anti-inflammatory potential?, Bioorg. Med. Chem. 18 (2010) 8204-8217
[3] P.Alexiou, K. Pegklidou, M. Chatzopoulou, I. Nicolaou, V.J.Demopoulos* (2009) Aldose Reductase Enzyme and its Implication to Major Health Problems of the 21st Century Current Medicinal Chemistry, 2009, 16, 734-752
[4]I. Parejo, C. Codina, C. Petrakis, P. Kefalas, Journal of Pharmacological and Toxicological Methods , 44, (2000), 507-512
OH
OH
HO
O
*
HO
OH
O
-H2O
OO
OH O
OH
OH
OH
*
* Conclusion
OO
OH O
O
OH
OH
O
HO
O
OH
OH
-H20
-H20
0
0.5
1
1.5
2
2.5
diethylether (1) ethyl-acetate(2) butanol (3) aqueous (4)
Antiradical efficiency
0
10
20
30
40
50
60
diethyl ether (1) ethyl acetate (2) butanol (3) aqueous (4)
(%) Inhibition of soybean lipoxygenase (LOX)
0
10
20
30
40
50
60
70
80
90
diethylether (1) ethyl acetate (2) butanol (3) aqueous (4)
% Inhibition of ALR2