Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 5, Suppl 3, 2013

Research Article

ISOLATION AND STRUCTURAL ELUCIDATION OF FLAVONOIDS FROM AQUATIC FERN AZOLLA

MICROPHYLLA AND EVALUATION OF FREE RADICAL SCAVENGING ACTIVITY

K. SELVARAJ, RANJANA CHOWDHURY*, CHIRANJIB BHATTACHARJEE

Department of Chemical Engineering, Jadavpur University, Jadavpur, Kolkata 700032, India. Email: ranjana.juchem@gmail.com
Received: 26 Jun 2013, Revised and Accepted: 06 Aug 2013
ABSTRACT
Objective: The present study was designed for isolation of bioactive polyphenolic compounds from methanol extract of Azolla microphylla and their
subsequent characterization.
Methods: The flavonoid compounds were isolated and characterized by using thin layer chromatography (TLC), purified by preparative thin layer
chromatography (PTLC) and were identified using High performance chromatography (HPLC). Their structures and chemical bonds were analyzed
using Ultraviolet-Visible spectrophotomery (UV spec), Fourier Transform-Infra Red spectroscopy (FTIR) and Nuclear magnetic resonance NMR (13C
and 1H) techniques.
Results: Two flavonoids were identified as rutin and quercetin. The isolated compounds showed a potent antioxidant radical scavenging activity, as
assessed by non-physiological assays like DPPH (2, 2-diphenyl-1-picrylhydrazyl), ABTS (2, 2’-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid)
diammonium salt) and FRAP (Ferric reducing antioxidant power).
Conclusion: For the first time rutin and quercetin have been isolated successfully from the macrophyte aquatic fern Azolla microphylla under the
present study. The isolation of the above characterized flavonoids would be useful to prepare plant-based pharmaceutical preparation to treat
various complications linked with human diseases.
Keywords: Azolla microphylla, DPPH, ABTS, FRAP, Rutin, Quercetin.

INTRODUCTION ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) were

obtained from Sigma–Aldrich, MO, USA. Folin-Ciocalteu’s reagent and
Azolla microphylla is an aquatic fern that freely floats on the surface of gallic acid were obtained from Himedia laboratories Pvt. Ltd. Mumbai,
the water body. It is a pteridophyte plantae belonging to the India. Other chemicals and solvents such as silica gel (GF254), IR grade
Salvinacea family [1]. It has been traditionally used as a green manure potassium bromide, methanol, petroleum ether, chloroform, ethyl
for wetland paddy fields for the fixation of atmospheric- nitrogen (N2) acetate and formic acid were purchased from Merck, India Ltd. Azolla
with the help of Azolla-anabaena - a cyanobacterium [2]. They can microphylla, an aquatic fern, was purchased from Vivekananda
accumulate elements like P and K and minerals from the environment. Kendra-NARDEP (Natural Resources Development Project),
These essential elements become available to the soil on the Vivekanandapuram, Kanyakumari, Tamil Nadu, India.
decomposition of Azolla microphylla [3, 4]. This fern also serves as one
of the main components in the food for the omnivorous– Sample Preparation
phytoplanktonophagous tilapia (Oreochromis niloticus) [5, 6]. One or
more Azolla species are found worldwide in wetlands [7]. The The whole plant, Azolla microphylla were washed thoroughly with
phytochemical investigation on the Azolla microphylla shows that tap water followed by rinsing with double distilled water and shade
tannins, phenols, sugar, anthroquinone glycosides and steroids are drying for 7days. The fine powder (≈60 mesh size) was obtained
from dried plant by using kitchen mixer grinder (Bajaj electronics
present [8]. Azolla microphylla is also rich in protein, vitamin and
Ltd, India). The plant powder was sterilized at 121˚C for 15 min. The
minerals and is used as food supplements for cattle, pigs, ducks and
plant powder was stored under desiccator for further studies.
chickens resulting in increased milk production, enhancement of
weight of cattle, pigs, ducks and broiler chickens and for production of Instruments
eggs with layered yolk, as compared to conventional ones[9,10]. More
than 4000 chemically unique flavonoids have been isolated and Nuclear Magnetic Resonance (1H and 13C) spectra were recorded in
identified in plant extracts obtained through solvent extraction either CD3OD or CDCl3 on a Bruker NMR Avance with TCI cyroprobe
processes [11, 12]. More recently, a few flavonoids have also been operating at 600MHz. UV absorption was carried out by Varian, Cary
isolated from microorganisms as their secondary metabolites [13]. 50 UV-Visible double beam spectrophotometer. High performance
Flavonoids are of great importance for the bioactivities, related to liquid chromatography (HPLC) was performed on Model LC-8,
their antioxidant activities and many enzymatic reactions, resulting in Shimadzu, Japan and FT-IR spectrum was generated using Perkin
a decrease of platelet activation and aggregation, against Elmer, Spectrum 100 instrument.
cardiovascular diseases, cancer chemoprevention and anti- Extraction of flavonoids
inflammatory activity [14-20].Although from phytochemical analysis
of Azolla microphylla, it is evident that this fern is rich in flavonoids, no Solvent extraction of dried powder (40g) of Azolla microphylla was
work has so far been reported on the isolation of flavonoids from done using 2L of 80% methanol (50mL/g of sample) in a soxhlet
them. The present study focuses on the isolation and free radical extractor for 24h. The extract was concentrated by evaporation (40-
scavenger activities of flavonoids from methanolic extract of Azolla 50°C) in a rotary vacuum evaporator. The concentrated methonolic
microphylla. Two flavonoids namely, rutin and quercetin have been extract (10mL) was suspended in 50mL of distilled water and was
isolated from Azolla microphylla. Their structures have been further extracted successively with petroleum ether, chloroform,
elucidated through UV, FTIR, 13C NMR, and 1 H NMR. and ethyl acetate. For each solvent, extraction procedure was
repeated thrice for complete extraction. The petroleum ether extract
MATERIALS AND METHODS (Fraction-I) was discarded because it contained fatty substances.
The cyanidine test was performed for the chloroform (Fraction-II)
Chemicals and Plant material and ethyl acetate (Fraction-III) extracts for further selection. The
Rutin, Quercetin, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 4, 6- ethyl acetate extract (Fraction-III) was analyzed for flavonoids using
tripyridyl-s-triazine (TPTZ) and 2, 2’-Azinobis (3- chromatographic separation.
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Isolation of flavonoids by thin layer chromatography (TLC) ABTS scavenging assay

The glass plates (100×200mm) coated with silica gel G (0.2-0.3mm) ABTS˙* radical scavenging activity was measured according to the
paste were dried naturally (atmospheric). Subsequently they were reference method [21] with some modifications. The ABTS˙* was
activated at 100 °C for 30 minutes and were cooled at room generated by the reaction between 7mM ABTS (2, 2’-Azinobis (3-
temperature (≈ 25oC). The extract was separated by TLC with the ethylbenzothiazoline-6-sulphonic acid) diammonium salt) solution
following mobile phases: ethyl acetate: n-butanol: water (50:30:10 and 2.45mM potassium persulphate solution incubated in the dark
v/v), ethyl acetate: acetic acid: water (60:30:10) and benzene: acetic at room temperature for 16h. Before use, the absorbance at 734nm
acid: water (60:35: 5 v/v). The plates were developed using was adjusted to 0.700(±0.0020) by dilution with ethanol. To 3mL of
ammonia fumes and visualized under UV lamp. The Rf values of the ABTS solution, 0.1mL of aqueous solution of each isolated
separated bands were calculated. compound (1-100µg/mL) was mixed vigorously. The reaction
mixture was incubated for 6min and the absorbance was
Purification of flavonoids by preparative thin layer determined at 734nm by a UV-vis spectrophotometer. A standard
chromatography (PTLC) curve was obtained by using rutin in 80% ethanol. The % ABTS
The extract was reduced to 5mL and loaded in preparative TLC which was scavenged (%ABTSSC) was calculated using the formula;
plates coated with silica gel GF254 (0.4-0.5mm). The chromatogram % ABTSSC = (A0 - A1) × 100/A0
was developed with benzene: acetic acid: water (60:35:5 v/v) and
examined under UV lamp. The fluorescing spots were scraped out Where A0= absorbance of the control; A1 = absorbance of the sample.
from the 100 plates and extracted with ethanol. The diluted mixture
was then centrifuged at 10000 rpm for 10minutes at 4˚C. The Ferric reducing antioxidant potential (FRAP) assay
supernatant was collected for further experimental (HPLC, FT-IR, The ferric reducing antioxidant power activity was measured
NMR) analysis. The eluted compounds were co-chromatographed according to the reference method [22]. The FRAP reagent was
along with standard flavonoids. prepared using 300mM acetate buffer (3.1g Sodium acetate, and
High Performance Liquid Chromatography analysis 16mL Acetic acid) at pH 3.6, 10mM TPTZ (2,4,6-tripyridyl -s-
triazine) solution in 40mM hydrochloric acid solution, and 20mM
The isolated compounds (in ethanol) were filtered through FeCl3.6H2O solution in distilled water. The acetate buffer (25mL) and
membrane filter (Millipore, USA) and were injected (10µL) through TPTZ (2.5mL) were mixed together with FeCl3.6H2O (2.5mL). The
the BDS Hypersil RP-C18 column (Thermo, 5µm, 120Å, 250mm × temperature of the solution was adjusted to 37˚C before it was used.
4.6mm) at column temperature 25˚C. The mobile phase composed of The different concentrations (10-100µg/mL) of the isolated
methanol, water and formic acid (70:30:1 vol. %) was eluted at a compounds (1mL each) were allowed to react with the FRAP
flow rate of 1mL/min and the effluent was monitored at 280nm by solution (3mL) for 30min under dark conditions. The absorbance
UV detector. The peaks were detected and compared with the was measured at 593nm and the reducing power was expressed as
standards. percentage ferric reducing activity of the compounds.
Spectral analysis Statistical analysis
The isolated compounds 1 and 2 were dissolved in methanol and All experiments were repeated at least three times. Results are
their maximum UV absorption ranges were recorded using the UV- reported as mean ± standard deviation.
Vis double-beam spectrophotometer. The compounds 1 and 2 were
dissolved in CDCl3 and 1H and 13C NMR spectra using NMR RESULTS AND DISCUSSIONS
spectroscopy with TCI Cyroprobe were recorded. Tetramethylsilane Physical and Chemical Characteristics of isolated compounds
(TMS) was used as an internal standard. The chemical shift values
were reported in ppm (δ) unit and the coupling constants (J) are in The methanol extract of A. microphylla on phytochemical screening
Hz. FTIR spectra of the compounds 1 and 2 were measured using IR showed the presence of large number of compounds. The
grade potassium bromide (KBr). The compounds 1 and 2 were methonolic extract was fractionated with petroleum ether,
separately mixed with 200mg KBr to obtain round disc with the help chloroform and ethyl acetate. Cyanidine test indicated that the
of hydraulic press. Round disc was later subjected to FTIR in the maximum quantities of phenolic compounds were present in the
range of 4000-400cm-1 at a resolution of 4cm-1. ethyl acetate extract (Fraction-III). Thus the Fraction-III was only
subjected to further analysis to characterize the flavonoids. TLC was
In vitro radical scavenging assays done with Fraction-III and the Rf values (compound-1:0.31;
DPPH scavenging assay compound-2: 0.53) of two TLC spots almost matched with two
therapeutically valuable flavonoids, namely rutin and quercetin
The 2, 2-diphenyl-1-picrylhydrazil (DPPH˙*) free radical scavenging (Table.1). The results of preparative TLC further established that the
activity of the isolated compounds (1 and 2) was determined spots of compound-1 and compound-2 coincided with those of rutin
according to previous method [19]. To 0.1mL of aqueous solution and quercetin respectively. The Chromatographic analysis using
(10-50µg/mL) of each isolated compound, 3mL of ethanolic solution HPLC indicated that compound-1 and compound-2 had the same
of DPPH (0.1µM) was added. The mixture was shaken vigorously retention time (Rt: 2.8 and 3.4min) as the standard flavonoids,
and allowed to stand for 30minutes in the dark, and the absorbance namely rutin and quercetin (Fig 1A-2B) respectively. The structural
was measured at 517nm against a blank. The capability to scavenge identification of each compound was carried out by UV, FTIR, 1H
the free radical DPPH (%DPPHSC) was calculated using the formula; NMR and 13C NMR. The elucidation of the structure is in consonance
with the previous report [23]. Significant free radical scavenging
%DPPHSC = (A0 - A1) × 100/A0
activities of non-physiological assay of crude methanol, chloroform
Where A0= absorbance of the control; A1 = absorbance of the sample. and ethyl acetate extracts were performed as shown in table. 2.
[

Table1: Thin layer Chromatographic characteristics of isolated compounds

Solvent systems Isolated compounds (Rf) UV NH3 fumes AlCl3 and FeCl3
Compound 1 Compound 2
Ethyl acetate: n-butanol: water (50:30:10 v/v) 0.29 0.45 Violet Yellow Dark yellowish
brown
Ethyl acetate: acetic acid: water (60:30:10) 0.52 0.84 Violet Yellow Dark yellowish
brown
Benzene: acetic acid: water (60:35: 5 v/v) 0.31 0.53 Violet Yellow Dark yellowish
brown

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Fig. 1A: HPLC chromatogram of Isolated Rutin

Fig. 1B: HPLC chromatogram of Standard Rutin

Fig. 2A: HPLC chromatogram of Isolated Quercetin

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Fig. 2B: HPLC chromatogram of Standard Quercetin

Table 2: Antioxidant activity of various extracts of Azolla microphylla

Sample DPPHsc (%) ABTSsc (%) % Ferric reducing power (%)
Methanol extract 88.42 50.3 55
Chloroform 86.69 48.2 52
Ethyl acetate 92.61 55.4 56

Compound-1 Compound-2
Light yellow powder (ethanol); m. p. 190 ˚C; λmax. 300nm; mol. Pale yellow powder (ethanol); m. p. 300 ˚C; λmax. 360nm; mol.
formula: C27 H30 O16; IR (KBr) Vmax cm-1: 3408, 3321 (O-H stretching), formula: C 15 H10 O7; IR (KBr) V max cm-1:3428, 3369 (O-H
2924,2843( CH2-stretching), 2714 (C-H bonding), 1462( C=O stretching), 2986 (CH 2-stretching), 2872 (CH-stretching),
groups) and 1383(C-OH vibrations) (shown in Figure 3A); 1H-NMR 1457(C=O), 1362 (C-OH vibrations) (shown in figure 3B); 1H-
(600MHz in CH3OD, δ ppm) 3.33-3.64 (m,12H of sugar moieties), NMR (600MHz in CH 3OD, δ ppm) 6.18(1H, d, J=2Hz, H-6),
3.81(d, J=1.15Hz, 1H-Rham), 1.10(3H, d, J=6Hz, CH3-Rham), 4.52(4H, 6.38(1H, d, J=2Hz, H-8), 6.88(1H, d, J=8Hz, H-5’), 7.63(1H, d,
d, J=7.8Hz, H-1 Glu), 5.11 (1H, d, J=2Hz H-6), 6.19 (1H, d, J=2Hz, H-8), J=7.5Hz, H-6’), 7.72(1H, d, J=2Hz, H-2’); 13C-NMR(600MHz in
6.38(1H, d, J=8Hz, H-5’), 7.66 (1H, m, H-2’, H-6’); 13C –NMR (600MHz CH3OD, δ ppm): shown in Table 3. It was characterized as 2-(3,
in CH3OD, δ ppm): shown in Table 3. It was characterized as 3, 3’, 4’, 4-Dihydroxyphenyl)-3, 5, 7-trihydroxy-4H-1-benzopyran-4-one
5, 7-pentahydroxy flavones-3-rutinoside (rutin) [24]. (quercetin) [25].

Fig. 3A: FTIR spectra of rutin

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Fig. 3B: FTIR spectra of Quercetin

Table 3: 13C-NMR data for isolated rutin and quercetin in comparison to 13C-NMR of rutin and quercetin obtained by the references [24, 25].
Position Compound 1 Reference rutin Compound 2 Reference quercetin
2 158.5 158.5 148.8 147.9
3 135.7 135.6 137.3 137.2
4 179.4 179.4 177.4 177.3
5 163.0 162.5 162.5 162.5
6 100.0 99.9 99.3 99.3
7 166.0 166.0 165.6 165.7
8 95.0 94.8 94.5 94.4
9 159.4 159.3 158.3 158.2
10 105.7 105.6 104.6 104.4
1’ 123.7 123.1 124.2 124.1
2’ 117.8 117.6 116.0 116.0
3’ 145.9 145.8 146.3 146.2
4’ 149.9 149.7 148.1 148.7
5’ 116.1 116.1 116.3 116.2
6’ 123.2 123.5 121.7 121.6
1’’ 104.8 104.7
2’’ 75.8 75.7
3’’ 77.2 77.2
4’’ 71.5 71.4
5’’ 78.2 78.1
6’’ 68.6 68.6
1’’’ 102.5 102.4
2’’’ 72.3 72.0
3’’’ 72.2 72.2
4’’’ 74.0 73.9
5’’’ 69.8 69.7
6’’’ 18.0 17.9

Anti-free radical activity of quercetin and rutin scavenging activity as a function of concentration of rutin and
quercetin in the range of 92.6% at 35µg/ml and 91.5% at
The antioxidant activity of isolated rutin and quercetin was measured by 30µg/ml concentration, whereas ABTS radical scavenging value
different non- physiological methods. Mainly DPPH, ABTS radical of rutin and quercetin was 93.5% in 35µg/ml and 90.8% in
scavenging and ferric (FRAP) reducing assays have been made. The 35µg/ml respectively. From the analysis of the figures, it is
antioxidant parameter is influenced by various factors such as oxidation evident that free radical scavenging activities increase with
substrate, oxidation mechanism and reaction medium [26]. The DPPH increase of concentration of both rutin and quercetin up to a
radical can be reduced by the en-1, 2-diol and dien-1, 4-diole moieties in certain level and beyond this concentration saturation is
antioxidants [27]. From the scavenging activity of the stable DPPH, ABTS observed. For scavenging activity against DPPH, the
cation radical and ferric reducing power assay of presently isolated rutin concentrations of rutin and quercetin corresponding to
and quercetin it was observed that the flavonoids and their glycosides saturation value are 35µg/ml and 30µg/ml respectively. For
exhibited potent antioxidant activity. scavenging activity against ABTS, the concentrations of rutin and
Fig.4 and 5 respectively show the potential free radical quercetin corresponding to saturation value are 35µg/ml and
scavenging activity against DPPH and ABTS free radicals 35µg/ml respectively.

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Fig. 4: Scavenging of DPPH radical potential of rutin and quercetin in DPPH assay

Fig. 5: Scavenging of ABTS radical potential of rutin and quercetin in ABTS assay

It has been reported that ferric reducing power is associated with quercetin corresponding to saturation of ferric reducing power are
antioxidant activity and may serve as a significant reflection of the 40µg/ml for 85% and 40µg/ml for 80% respectively. The reducing
antioxidant activity [28]. The ferric reductive potential of the properties are generally associated with the presence of
presently isolated rutin and quercetin were also dose dependent reductones, which have been shown to exert antioxidant action by
as shown in Fig. 6. The ferric reducing power of rutin and breaking the free radical chain by donating a hydrogen atom. Thus,
quercetin correlate similarly as the antioxidant activity with the bioactive flavonoids isolated under the present investigation
concentration of flavonoids. The concentrations of rutin and are potent antioxidants.

Fig. 6: Ferric reducing power of rutin and quercetin in ferric reducing antioxidant power assay

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For bio-evaluation studies, rutin is used for the treatment of various 13. Leonard, E., Yan, Y., Fowler, Z.L., Li, Z., Lim C.G., Kok-Hong
conditions related to capillary bleeding and increased capillary Lim,K.H., Koffas, M.A.G. Strain Improvement of Recombinant
fragility and permeability [29]. The combination of flavonoids such Escherichia coli for Efficient Production of Plant Flavonoids.
as rutin and quercetin has been frequently used in the allergic Molecular Pharmaceutics. 2008., 5: 257-265.
conditions. The quercetin alone exerts cytotoxic effects against the 14. Yao, L. H., Jiang, Y. M., Shi, J., Tomás-Barberán, F. A., Datta, N.,
human cancer cell line [30]. Additionally, flavonoids are also widely Singanusong, R., Chen, S. S. Flavonoids in food and their health
used in food industry for the preservation of food to elongate the benefits. Plant Foods Hum. Nutr. 2004., 59: 113-122.
shelf life by preventing or delaying the oxidation process [31].The 15. Sadhu, S. K., Okuyama, E., Fujimoto, H., Ishibashi, M., Yesilada, E.
study suggests that the bioactive flavonoids, namely rutin and Prostaglandin inhibitory and antioxidant components of Cistus
quercetin may be extracted from Azolla microphylla in an laurifolius, a Turkish medicinal plant. J. Ethnopharmacol. 2006.,
inexpensive route and the plant may be a potential source for the 108:371-378.
isolation of these bioactive flavonoids. Further studies are, however, 16. Kong, L.D., Abliz, Z., Zhou, C.X., Li, L. J., Cheng, C. H. K., Tan, R. X.
required to investigate the hypoglycemic, anti-hepatotoxicity, Glycosides and xanthine oxidase inhibitors from Conyza
anticancer and anti-inflammatory activities of the flavonoids, when bonariensis. Phytochemistry. 2001., 58:645-651.
applied to living animals. 17. Sadik, C. D., Sies, H., Schewe, T. Inhibition of 15-lipoxyigenase
by flavonoids: structure-activity relations and mode of action.
CONCLUSION Biochem. Pharmacol. 2003., 65:773-781.
It may be mentioned that two bioactive flavonoids have been 18. Fayez, M.A., Cai, H., Tunstall, R., Steward, W. P., Gescher, A. J.
isolated successfully from the macrophyte aquatic fern Azolla Differential modulation of cyclooxygenase-mediated
microphylla under the present study. On the basis of spectral data, prostaglandin production by the putative cancer
the compounds were identified as rutin and quercetin. The in-vitro chemopreventive flavonoids tricin, apigenin and quercetin.
free radical scavenging assays of the compounds strongly indicate Cancer Chemother. Pharmacol. 2006., 58: 816-825.
their potent antioxidant activity. 19. Sampath, M and Vasathi, M. Isolation, structural elucidation of
Flavonoids from Polyalthia longifolia (SONN.) thawaits and
ACKNOWLEDGMENTS evaluation of antibacterial, antioxidant and anticancer
potential. International Journal of Pharmacy and
The authors are grateful to Prof. T. K. Pal, Bioequivalence study
Pharmaceutical Sciences. 2013., 5: 336-341.
center, Department of Pharmaceutical Technology, Jadavpur
20. Khatri, D.K., Juvekar, P and Juvekar, A.R. Phytochemical
University and Prof. E. Padmanaban, Indian Institute of Chemical
investigation and in vitro antioxidant activities indigofera
Biology, Kolkata for HPLC and NMR measurements respectively.
cordifolia seed extracts. International Journal of Pharmacy and
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