1 Article

2 Exploring New Antioxidant Compounds from

3 Nymphaea alba wild-grown in Danube Delta
4 Biosphere
5 Mihaela Cudalbeanu 1, Ioana Otilia Ghinea 1, Bianca Furdui 1, Durand Dah-Nouvlessounon 2,
6 Robert Raclea 3, Teodor Costache 4, Iulia Elena Cucolea 4, Florentina Urlan 4 and Rodica Mihaela
7 Dinica 1,*
8 1 Faculty of Sciences and Environment, Department of Chemistry Physical and Environment, “Dunarea de
9 Jos” University of Galati, 111 Domneasca Street, 800201, Galati, Romania
10 2 Faculty of Sciences and Techniques, Department of Biochemistry and Cell Biology, Laboratory of Biology

11 and Molecular Typing in Microbiology, University of Abomey-Calavi, 05BP1604 Cotonou, Benin

12 3 Imperial College London, Faculty of Natural Sciences, Department of Chemistry, SW7 2AZ, London, UK

13 4 Research Center for Instrumental Analysis SCIENT, 1E Petre Ispirescu Street, 077167, Tancabesti, Ilfov,

14 Romania
15 * Correspondence: rodica.dinica@ugal.ro

16 Academic Editor: name

17 Received: date; Accepted: date; Published: date

18 Abstract: Nymphaea alba is an aquatic flowering plant from the Nymphaeaceae family used for
19 hundreds of years in traditional herbal medicine. The plant is characterized by different
20 phytochemicals, depending on the geographical location. Here in we have carried out, for the first
21 time, the separation and HPLC-MS/MS identification of some antioxidant compounds, such as
22 polyphenols and flavonoids from N. alba extracts from Danube Delta Biosphere, and we investigated
23 their possible antiradical properties. An ultrasonic green method has been exhaustively used for the
24 extraction of the antioxidant compounds from the different anatomic parts of N. alba (fruit, flower,
25 leaf, stem and root). The extracts obtained using the ultrasound irradiation, showed a large
26 polyphenol (19.42 mg EqGA/100mg extract) and flavonoid (0.97 mg EqQ/100mg extract) content.
27 The fruit and flower extracts showed the highest antioxidant activity index (AAI). Among the 27
28 phytochemical compounds identified in all N. alba extracts, rutin and coumaric acid were found as
29 the major components. FT-IR spectroscopy of N. alba extracts revealed the presence of active
30 functional groups of polyphenols. This research contributes to the study of Nymphaeaceae family,
31 being the first exhaustive phytochemical study of N. alba from a wild population in Romania.

32 Keywords: Nymphaea alba extracts; antioxidant compounds; Danube Delta Biosphere

33

34 1. Introduction
35 Nymphaea alba species (N. alba), also known as the European white water lily, belongs to the
36 Nymphaea genus and the Nymphaeaceae family [1, 2]. Species from the Nymphaeaceae family, such as N.
37 alba, have important medicinal properties, being used in the treatment of diabetes, inflammations,
38 liver disease, urinary tract infection, and they also have tonic and aphrodisiac properties [3]. Previous
39 studies have reported the antioxidant, anti-inflammatory and hepatoprotective effects of the N. alba
40 species. These effects can be attributed to some phytochemical components from the polyphenol
41 class, such as ellagic acid, gallic acid and their methyl and ethyl esters, in addition to flavonoids such
42 as quercetin or kaempferol [2, 4]. In Romania, the natural habitats of Nymphaea alba are found in the
43 Danube Delta landscape which shelters a broad large of water macrophyte communities.
44 Polyphenols and flavonoids are the largest group of phytochemical compounds and are
45 omnipresent secondary metabolites amongst plants. From ancient times, people have used plants for

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 Molecules 2018, 23, x FOR PEER REVIEW 2 of 14

46 infectious diseases, and scientific research has proved their therapeutic effects over time [5]. Recent
47 research supports the role of these types of secondary metabolites in the prevention of degenerative
48 diseases, especially cancer, cardiovascular and neurodegenerative diseases [ 6 ]. Polyphenols and
49 flavonoids are strong antioxidants that complete and add to the functionalities of vitamins and
50 antioxidant enzymes with the purpose of defense against oxidative stress caused by the excessive
51 presence of reactive oxygen species (ROS) [7, 8]. Some compounds, such as gallic acid, have a strong
52 antioxidant activity, while others, such as monophenols, are weak antioxidants [ 9 ]. In vacuoles,
53 flavonoids are found either in a free state or bound to carbohydrates (glucose, galactose, rhamnose,
54 mannose, etc.) and they tend to be soluble in water or organic solvents [10, 11]. Natural antioxidants
55 from plants play an important role in maintaining general health [12] and they have garnered interest
56 amongst consumers and scientific communities alike, since epidemiologic studies have shown that
57 frequent consumption of natural antioxidants is associated with lowered risk of cardiovascular
58 disease, chronic or degenerative, such as arthrosclerosis, cardiac and cerebral ischemia,
59 carcinogenesis, neurogenerative diseases, gestational diabetes, rheumatic disease, DNA deterioration
60 and skin aging [9, 11], also including infections of the respiratory, urinary and gastro-intestinal tract
61 and the biliary system [13].
62 This study is the first detailed chemical investigation that has been exhaustively done over all
63 the anatomic parts of N. alba the from Danube Delta Biosphere Reserve and includes ultrasound
64 irradiation as a green method of extraction, the identification and characterization of the
65 phytochemicals through chromatographic techniques, such as LC-MS/MS and ICP OES technique. A
66 correlation between flavonoid and polyphenol content with the antioxidant activity of the methanolic
67 extracts of N. alba was also illustrated. To the best of our knowledge this is the first phytochemical
68 study of N. alba from a wild population in Romania.

69 2. Results and Discussion

70 2.1. Total Polyphenol Content

71 The total polyphenol content was determined spectrophotometrically using gallic acid as the
72 calibration standard for the N. alba fruit, flower, leaf, stem and root extracts obtained by ultrasonic
73 extraction. The total polyphenol content showed a large variance, ranging from 7.95 to 19.42 mg
74 EqGA/100mg of extract. The largest polyphenol quantities were found in the leaf extract (19.42 mg
75 EqGA/100mg of extract) and flower extract (16.90 mg EqGA/100mg of extract) (Figure 1a). Literature
76 data about another Nymphaea species [14, 15, 2] confirms the total polyphenol content values for our
77 N. alba extracts.

(b)
(a)
78 Figure 1. Total concentration of polyphenols (a) and flavonoids (b) determined from the N. alba
79 methanolic fruit, flower, leaf, stem and root extracts.
 Molecules 2018, 23, x FOR PEER REVIEW 3 of 14

80 2.2. Total Flavonoid Content

81 Following the LC-MS/MS analyses conducted on the N. alba extracts, the presence of flavonoids
82 such as quercetin, catechin, epicatechin, naringenin and naringin was confirmed. The total flavonoid
83 content was spectrophotometrically quantified using quercetin as a calibration standard. The total
84 flavonoid content of the N. alba fruit, flower, leaf, stem and root extracts obtained by ultrasonic
85 extraction was determined. The values ranged from 0.09 to 0.97 mg EqQ/100mg of extract. The largest
86 flavonoid amount is present in the leaf extract with a value of 0.97 mgEqQ/100mg of extract, and in
87 the flower extract with a value of 0.85 mg EqQ/100mg of extract (Figure 1b).

88 2.3. Condensed Tannins Content

89 Condensed tannins are of interest because of their antioxidant activity and other potentially
90 beneficial effects for human health. Nevertheless, their biological activity depends on the structure
91 and their concentration [16, 17]. The total condensed tannins content was determined for the first time,
92 for the different anatomic parts of N. alba from Danube Delta Biosphere Reserve
93 spectrophotometrically using catechin as the calibration standard. The total content varied between
94 0.065 and 2.874 mg EqC/g. Condensed tannins are present in the methanolic fruit, leaf, stem and root
95 extracts data which is proven also by LC-MS/MS chromatograms. The largest content of condensed
96 tannins is present in the methanolic N. alba stem extract, with a value of 2.874 mg EqC/g (Table 1).

97 Table 1. Identification of the total condensed tannin content in different extracts of N. alba and
98 ±standard deviation (mg C/g).

Part of plant
Total Condensed
mg EqC/g or negative (-)
Tannin Content
Fruit Flower Leaf Stem Root

Methanolic
0.464 ± 0.011 - 0.065 ± 0.010 2.874 ± 0.125 1.452 ± 0.042
extracts

99 - unidentified

100 2.4. Antioxidant Activity

101 Natural antioxidants protect cells by the elimination of very reactive free radicals and ROS from
102 biological systems [11]. The antioxidant activity has been measured for the methanolic N. alba extracts
103 obtained by ultrasonic extraction. The IC50 value represents the summed concentration of compounds
104 that are necessary in order to reduce the initial DPPH concentration by 50%. IC 50 values for the
105 methanolic extracts have been determined using a graphical representation of the percentage of
106 inhibition versus concentration. The biggest antioxidant activity was found in the methanolic flower
107 extract. The IC50 values for the methanolic N. alba extracts are as follows: fruit - 17 µg/mL, flower - 17
108 µg/mL, leaf - 20 µg/mL, stem - 25 µg/mL and root-19 µg/mL (Figure 2a). The DPPH inhibition
109 percentages for all of the N. alba extracts after 50 minutes of incubation prove that the antioxidant
110 activity of these compounds is stable over time (Figure 2b).
 Molecules 2018, 23, x FOR PEER REVIEW 4 of 14

(a) (b)

111 Figure 2. DPPH inhibition percentage of the methanolic N. alba fruit, flower, leaf, stem and root
112 extracts after 20 minutes of incubation (a), and after 50 minutes of incubation (b).

113 The large-scale use of the DPPH method and the lack of standardization of the results make it
114 difficult to compare the antioxidant resistances of different plant extracts and pure compounds. For
115 this reason, an antioxidant activity index (AAI) has been determined for the DDPH method [18]. For
116 plant extracts or pure compounds, data such as the percentage of DPPH inhibition of IC 50 values
117 modify as a function of the final concentration of used DPPH.

118
119 Figure 3. Antioxidant activity index (AAI) of the methanolic N. alba fruit, flower, leaf, stem and root
120 extracts.

121 Antioxidant activity index (AAI) value indicates the success of a compound as antioxidant. All
122 the tested extracts showed a strong antioxidant activity (AAI>2). No significant differences were
123 observed between the fruit and flower extracts which showed the higher AAI value than leaf and
124 root extracts, which were similar to each other. The steam extract showed the lowest AAI value. Fruit
125 and flower extracts exhibited strong antioxidant activity due to the presence of flavonoids as apigenin
126 or luteolin, who had hydroxyl groups on the A ring, that could be involved in increasing the
127 antioxidant potential of the extracts.

128 2.5. HPLC-MS/MS Analysis of Polyphenolic Compounds

129 A new and sensitive method that combines high-performance liquid chromatography and
130 ionization mass spectrometry for the separation, identification and quantification of polyphenols,
131 flavonoids and tannins in the methanolic N. alba extracts has been adapted and optimized. Caffeic
132 acid, chlorogenic acid, coumaric acid, catechin, epicatechin, naringin, naringenin, vanillic acid,
133 quercetin and rutin were used as reference standards. 17 MRM experiments from literature were also
 Molecules 2018, 23, x FOR PEER REVIEW 5 of 14

134 used: HHDP-hexoside, corilagin, tannic acid, gallic acid, ferulic acid, ellagic acid, ellagic acid-
135 pentoside, quinic acid, kaempferol, castalin, orientin, apigenin, luteolin, brevifolin, geraniin, ellagic
136 acid, rhamnosyl and cinnamic acid derivatives.
137 The qualitative LC-MS/MS analysis of the methanolic N. alba extracts obtained by ultrasonation
138 has shown a wide range of polyphenolic compounds. Using the reference standards and comparing
139 the obtained mass spectra with literature data has led to the identification of different fragments of
140 polyphenolic compounds split into multiple compound subgroups: phenolic acids, flavonoids,
141 tannins and other non-flavonoid polyphenols. In total, 27 phytochemical compounds were identified
142 in the methanolic N. alba extracts, and their retention time (TR), chemical formula, parent ion and the
143 fragment negative ion are presented in Table 2.

144 Table 2. Peak assignments and identification of the phytochemical compounds in the N. alba extracts
145 by HPLC-MS/MS in the negative ion mode.

[M-H]−b
TR*a m/z
Compounds Formula References
(min) Parent
Fragment ion
ion
HHDP-hexosidec 6.15 481.06 301.14, 463.2 19, 15, 2

C15H14O6x
Catechin (+) 39.67 289 245, 205, 179, 261 19, 15, 20
H2O
Chlorogenic acid 18.05 C16H18O9 355 163 21

Corilagin 18.17 C27H22O18 633.07 301.14, 589.22 2

Vanillic acid 15.35 C8H8O4 167 123, 125, 152 19

Caffeic acid 20.37 C9H8O4 181 163 21

Tannic acid 24.61 C76H52O46 183.5 123.7 20

Gallic acid 16.50 C7H6O5 169 125 19, 15, 20

Epicatechin (-) 39.67 C15H14O6 289 245, 205, 179, 261 19, 15

p - Coumaric acid 23.85 C9H8O3 163 145, 119, 103, 89, 127 22

Naringenin 32.46 C15H12O5 271 151, 177, 165, 107, 125 19

Naringin 34.30 C27H32O14 579 151, 119, 271 23

Rutin 26.16 C27H30O16 609.2 301 21

Quercetin 31.94 C15H10O7 301 151 21

Kaempferol 36.60 C15H10O6 285.19 241.16, 217.25 15

Quinic acid 6.16 C7H12O6 191.06 127.07, 173.12 15

Ellagic acid 27.31 C14H6O8 301.15 257.22, 229.10 19, 15

Castalin 16.93 C27H20O18 631.06 301.29, 299.12 2

Orientin 49.74 C21H20O11 447.02 403.16, 233.03 2

Apigenin 47.80 C15H10O5 269.03 223.01, 179.07 21, 2

Luteolin 36.92 C15H10O6 285.04 257.16, 241.13 21, 2

Brevifolin 19.53 C10H12O4 247.02 203.11, 175.13 15, 2

Ferulic acid 38.09 C10H10O4 193 177, 149, 145, 117, 89 22

291.15, 405.17, 301.18,

Ellagic acid-pentoside 32.07 - 433 19, 2
275.23, 247, 229
C41H28O27 933.10, 613.20, 301.13,
Geraniin 17.29 951.07 2
631.15
359.17 403.2 385.11
Ellagic acid rhamnosyl 27.80 - 447.02 315.25 2

301.13 275.18
Cinnamic acid -
38.65 329.09 197.10 239.1 169.07 2
derivative
146 aTR – Retention time

147 b [M-H] − - Negative Ionization Mass/Mass spectrometry

148 c HHDP - Hexahydroxydiphenic acid

 Molecules 2018, 23, x FOR PEER REVIEW 6 of 14

149 The methanolic N. alba flower extract was separated by HPLC-MS/MS in the [M-H] − mode.
150 Overall, 25 different polyphenolic compounds (caffeic acid, coumaric acid, chlorogenic acid,
151 naringin, naringenin, vanillic acid, quercetin, rutin, HHDP-hexoside, corilagin, tannic acid, gallic
152 acid, ferulic acid, ellagic acid, ellagic acid-pentoside, quinic acid, kaempferol, castalin, orientin,
153 apigenin, luteolin, brevifolin, geraniin, cinnamic acid derivative and ellagic acid rhamnosyl) were
154 separated and identified in the methanolic N. alba flower extract (Figure 4). In the N. alba leaf extract
155 (Figure 5), 24 polyphenolic compounds were separated and identified. Compared to the flower
156 extract, this extract contained the catechin (+) and epicatechin (-) isomers, which eluted together at a
157 TR of 39.67, with a [M-H] − parent ion at 289 m/z and a fragment ion at 245 m/z, whereas kaempferol,
158 apigenin and ferulic acid were not identified. The stem extract contained 24 compounds that were
159 identified and separated: caffeic acid, coumaric acid, catechin, epicatechin, naringin, naringenin,
160 vanillic acid, corilagin, tannic acid, gallic acid, ferulic acid, ellagic acid, quinic acid, kaempferol,
161 castalin, orientin, apigenin, luteolin, brevifolin, geraniin and ellagic rhamnosyl acid, quercetin, rutin,
162 HHDP-hexoside, For the methanolic N. alba root extract (Figure 6), 21 out of the 27 polyphenolic
163 compounds were identified, the missing ones being quercetin, chlorogenic acid, kaempferol, luteolin,
164 ellagic pentoside acid and the cinnamic acid derivative.

165

166 Figure 4. The LC-MS/MS chromatographic separation of the methanolic N. alba flower extract (H -
167 HHDP-hexoside, Qu – quinic acid, C – corilagin, V – vanillic acid, Cas – castalin, Ga – gallic acid, G –
168 geraniin, Ca – caffeic acid, p-C – coumaric acid, T – tannic acid, R – rutin, El – ellagic acid, Elr – ellagic
169 rhamnosyl acid, Elp – ellagic pentoside acid, Cn – cinnamic acid derivative, Na – naringenin, N –
170 naringin, F – ferulic acid, Cl – chlorogenic acid, Q – quercetin, A – apigenin, L – luteolin, B –
171 brevifolin, K – kaempferol, O – orientin).
 Molecules 2018, 23, x FOR PEER REVIEW 7 of 14

172

173 Figure 5. The LC-MS/MS chromatographic separation of the methanolic N. alba leaf extract (H -
174 HHDP-hexoside, Qu – quinic acid, C – corilagin, V – vanillic acid, Cas – castalin, Ga – gallic acid, G –
175 geraniin, Ca – caffeic acid, p-C – coumaric acid, T – tannic acid, R – rutin, El – ellagic acid, Elr – ellagic
176 rhamnosyl acid, Elp – ellagic pentoside acid, Cn – cinnamic acid derivative, Na – naringenin, N –
177 naringin, Ch – catechin, Ep – epicatechin, Cl – chlorogenic acid, Q – quercetin, L – luteolin, B –
178 brevifolin, O – orientin).

179

180 Figure 6. The LC-MS/MS chromatographic separation of the methanolic N. alba root extract (H -
181 HHDP-hexoside, Qu – quinic acid, C – corilagin, V – vanillic acid, Cas – castalin, Ga – gallic acid, G –
182 geraniin, Ca – caffeic acid, p-C – coumaric acid, T – tannic acid, R – rutin, El – ellagic acid, Elr – ellagic
183 rhamnosyl acid, Na – naringenin, N – naringin, F – ferulic acid, Ch – catechin, Ep – epicatechin, A –
184 apigenin, B – brevifolin, O – orientin).

185 Table 3 contains a comparative presentation of the identified compounds in the methanolic
186 extracts from the N. alba flower, leaf, stem and root. The largest number of polyphenolic compounds
 Molecules 2018, 23, x FOR PEER REVIEW 8 of 14

187 was found in the flower extract, while the root extract contained the lowest number of polyphenolic
188 compounds.

189 Table 3. Identification of the phytochemical compounds in different N. alba extracts.

Part of plant
Polyphenols Positive (+) or negative (-)
Flower Leaf Stem Root
HHDP-hexoside* + + + +
Catechin (+) - + + +
Chlorogenic acid + + - -
Corilagin + + + +
Vanillic acid + + + +
Caffeic acid + + + +
Tannic acid + + + +
Gallic acid + + + +
Epicatechin (-) - + + +
p - Coumaric acid + + + +
Naringenin + + + +
Naringin + + + +
Rutin + + + +
Quercetin + + + -
Kaempferol + - + -
Quinic acid + + + +
Ellagic acid + + + +
Castalin + + + +
Orientin + + + +
Apigenin + - + +
Luteolin + + + -
Brevifolin + + + +
Ferulic acid + - + +
Ellagic acid-
+ + - -
pentoside
Geraniin + + + +
Ellagic acid
+ + + +
rhamnosyl
Cinnamic acid
+ + - -
derivative
190 - unidentified

191 + identified

192 2.6. Macro- and Microelement Contents

193 The bioavailability of macro- and microelements depends on their nature and their association
194 with the components in the soil. Plants easily assimilate macro- and microelements through the roots.
195 Other sources for these elements are rainfall, atmospheric dust, fertilizers and chemicals used to
196 protect plants that can be absorbed through the leaf blades. Plants used for therapeutic purposes must
197 be collected from contamination-free areas. Regardless, as it can be observed in literature, the raw
198 plant medical materials significantly differ with regards to their macro- and microelement content.
199 This content represents one of the criteria that have to be met in order for raw plant material to be
200 used for medical purposes [24].
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201 The positive effects of plant-based medicines were attributed to the presence of polyphenols and
202 flavonoids and can be correlated with their antioxidant activity, but they are also influenced by other
203 organic and inorganic substances (macro- and microelements) [25] Usually, research in this field only
204 focuses on the organic components, but it is well-known that some diseases require both macro- and
205 micronutrients to be treated and their effect depends on the dose they are administered at . Relatively
206 high quantities of macronutrients, such as calcium, phosphorus, sodium, potassium and magnesium,
207 are necessary for a regular human diet [25].
208 The content of macro- and microelements from the N. alba samples is shown in Table 4. Large
209 quantities of potassium (4931.32 – 10724.93 mg/kg), magnesium (1954.15 – 4084.08 mg/kg),
210 phosphorus (1762.59 – 5181.95 mg/kg) and sodium (4692.36 – 36712.48 mg/kg) were found in all 4
211 samples. While the root sample contained the largest quantities of magnesium (4692.36 – 36712.48
212 mg/kg) and calcium (8621.94 mg/kg), the flower sample had the highest content of potassium
213 (10724.93 mg/kg) and phosphorus (5181.95 mg/kg), and the stem sample contained the largest
214 amount of sodium (36712.48 mg/kg).
215 Micronutrients such as iron, zinc, manganese, copper, molybdenum, cobalt and chromium are
216 present in the human body in very small quantities, but, unlike the other nutritive substances, they
217 are essential to our survival [25]. The cobalt and molybdenum concentrations were under the detection
218 limit in all of the N. alba samples. The chromium content varies from <1.0 to 14.56 mg/kg. The
219 concentration of other microelements (Al, As, B, Ba, Cd, Hg, Li, Pb, Se, Sn, Ni) was determined as
220 well, and most concentrations are under the detection limit. The lead content varied from <0.5 to 2.81
221 mg/kg, while all samples had a mercury and cadmium content that was under 0.5 mg/kg and 1.0
222 mg/kg, respectively. The lithium content was very small, varying between <1.0 to 6.46 mg/kg.

223 Table 4. Concentration of different elements in the dry residue of N. alba after ultrasonic extraction
224 and ±standard deviation (mg/kg).

Part of plant
Elements mg/kg (dry weight)
Flower Leaf Stem Root
Al 646.69±15.2 1075.76±57.8 224.96±12.1 7151.95±57.9
As <1.0 <1.0 <0.5 <2.5
B 36.28±1.3 32.18±1.1 23.06±1.2 <2.5
Ba <2.5 13.47±0.8 5.72±1.0 55.11±2.2
Ca 3817.11±69.80 8103.09±58.89 7293.97±38.07 8621.94±113.28
Cd <0.1 <0.1 <0.1 <0.1
Co <1.0 <1.0 <1.0 2.042±0.1
Cr <1.0 <2.5 <1.0 14.56±0.5
Cu 6.28±0.5 2.99±0.3 <1.0 10.23±1.1
Fe 149.19±6.6 817.05±4.0 24.56±1.8 539.628±20.5
Hg <0.5 <0.5 <0.5 <0.5
K 10724.93±45.2 4931.32±17.8 6876.60±19.9 7453.42±18.8
Li <1.0 <1.0 <1.0 6.46±0.5
Mg 2857.22±82.7 2057.35±68.5 1954.15±24.9 4084.08±36.3
Mn 69.17±1.7 508.36±14.6 345.79±6.3 379.02±6.9
Mo <1.0 <1.0 <1.0 <1.0
Na 9745.81±55.9 16908.65±580.2 36712.48±589.1 4692.36±67.7
Ni <1.0 <2.5 <1.0 7.55±0.5
P 5181.95±57.2 2051.31±34.3 1762.59±14.7 3467.95±23.8
Pb <0.5 <0.5 <0.5 2.81±0.3
Zn 64.09±2.4 16.07±1.8 12.73±1.0 44.79±2.9
Se <1.0 <0.5 <0.5 <0.5
Sn <2.5 <2.5 <2.5 <2.5
 Molecules 2018, 23, x FOR PEER REVIEW 10 of 14

225 3. Materials and Methods

226 3.1. Plant Material

227 The collection of plant samples is an important step in their analysis. The N. alba samples were
228 collected in June 2017 from the Somava-Parcheș Lagoon Complex situated in the Danube Delta
229 Biosphere Reserve. Specimens were deposited at the Botanical Garden of Galati, Romania.

230 3.2. Extraction

231 In order to extract the bioactive compounds, dry samples of N. alba fruit, flowers, leaves, stems
232 and roots were used. The basic procedures, such as preliminary washing with distilled water and
233 ultrapure water, drying the plant product at room temperature (7-12 days) until a constant weight
234 and grinding to granulometry lower than 2 mm prior to extraction, to obtain a homogenous sample
235 were followed. The main solvent used for the extraction of bioactive compounds was methanol. Since
236 the target compounds can be both polar and non-polar and also thermally unstable, the extraction
237 methods must be adapted for this case. The extraction procedure used was ultrasonation as a green
238 extraction method.

239 3.3. Microplate Determination of Total Polyphenol Content

240 The total polyphenol content was determined by applying the Folin-Ciocalteu method [17] to a
241 96-well plate analysis. 25 µL of Folin-Ciocalteu reagent was added to 10 µL of extract. After 5 minutes
242 of incubation, 25 µL of a 20% aqueous sodium carbonate solution and ultrapure water was added
243 until the final volume reached 200 µL. Blanks were also prepared for each sample by replacing the
244 Folin-Ciocalteu reagent with ultrapure water. A freshly prepared gallic acid solution was used as a
245 standard reference and the results are given in equivalents of gallic acid per 1 g of sample. After 30
246 minutes, the absorbance values of the samples at 760 nm were recorded using a multiwell plate reader
247 (Tecan Pro 200).

248 3.4. Microplate Determination of Total Flavonoid Content

249 The total flavonoid content was quantified by a 96-well plate analysis using aluminum chloride.
250 100 µL of an aqueous 2% aluminum chloride solution was added to 100 µL of sample. After 15
251 minutes of incubation, the sample absorbance values at 415 nm were read using the Tecan Pro 200
252 multiwell plate reader. Quercetin was used as a reference standard and the results are given in
253 equivalents of quercetin per 1 g of sample [26].

254 3.5. Microplate Determination of Total Condensed Tannins Content

255 For the determination of the total condensed tannins content, a 96-well plate adaptation of Zou’s
256 method was used [ 27 ]. 150 µL of a 4% methanolic vanillin solution and 75 µL of a concentrated
257 aqueous solution of HCl were added to 10 µL of sample. After 15 minutes, the absorbance values at
258 500 nm were read against a blank solution which replaced the extract sample with 10 µL of ultrapure
259 water. Aqueous solutions of (±) catechin with concentrations ranging from 10 to 100 µg/mL were
260 used as standards, and the results are reported in milligrams of catechin per 1 g of sample (mg EqC/g).

261 3.6. Microplate Determination of Antioxidant Activity using DPPH

262 The antioxidant activity has been reported in various manners, such as the percentage of utilized
263 reagent and the percentage of oxidation inhibition. Quercetin was used as a reference standard for
264 an easier way of measuring antioxidant activity [18]. The DPPH free radical has a maximum
265 absorbtion at 517 nm, which gives a purple colour. The colour shifts from purple to yellow resulting
266 in a reduced form of DPPH. Antioxidant compounds can be water - soluble, insoluble or bound to
267 the cell walls. As such, the efficiency of extraction is an important factor in quantifying a plant’s
268 antioxidant activity [9]. In a 96-well plate, 100 µL samples of a 100 µg/mL DPPH solution were added
 Molecules 2018, 23, x FOR PEER REVIEW 11 of 14

269 to 100 µL methanolic extract samples with a 200 µg/mL concentration. The resulting solutions were
270 then homogenized and then left in the dark at room temperature for 20, 35 and 50 minutes. The
271 absorbance values at 517 nm were recorded after each time period. The blank sample used was a 1:1
272 mixture of methanol and DPPH solution. A 40 µg/mL solution of quercetin was used as a reference
273 standard. The DPPH inhibition percentage, which relates to the antioxidant activity of quercetin and
274 the extracts, was calculated using the following formula: % inhibition = (𝐴𝑏𝑙𝑎𝑛𝑘 – 𝐴𝑠𝑎𝑚𝑝𝑙𝑒)/
275 𝐴𝑏𝑙𝑎𝑛𝑘 ∗ 100 [26]. The concentration of sample required to scavenge 50% of DPPH (IC50) were
276 graphically determined by plotting the percentage of inhibition against concentration of the inhibitor.
277 The antioxidant activity index (AAI) was calculated according to the following formula: 𝐴𝐴𝐼 =
278 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝐷𝑃𝑃𝐻 (µ𝑔/𝑚𝐿)/𝐼𝐶50 (µ𝑔/𝑚𝐿).Thus, the AAI value is calculated taking into
279 consideration both the mass of DPPH and the mass of the sample tested in the reaction, resulting in
280 a constant for each compound, independent of the DPPH concentration used in the sample [18].

281 3.7. HPLC-ESI-MS/MS Analysis of Polyphenolic Compounds

282 The HPLC-MS/MS analysis was carried out using a Q Trap 3200 Triple Quadrupole Mass
283 spectrometer from Perkin Elmer. In the chromatographic analysis, the Thermo C18 (150 x 4.6 mm,
284 particle size of 5 μm) column was used with an injection volume of 25 μL. The solvents used were
285 (A) formic acid (1%) and (B) methanol. The gradient elution was from 5 to 100% B at 30 °C and the
286 elution flow was set at 500 μL/min. Modifying the mobile phase’s pH value caused a significant
287 change in the resolution of our compounds, especially the phenolic acids. Formic acid is volatile and
288 is thus compatible with the LC-MS system. For the mass spectra, the ionization source temperature
289 was 500 °C and the spectra were recorded in the negative ion mode between 50 and 650 m/z using
290 argon as a collision gas. The pressure of the gas flux to the nebulizer was set at 1000 psi.

291 3.8. Determination of Macro- and Microelement Contents

292 Due to the importance of minerals and oligoelements present in medicinal plants [24], the
293 quantities of macro- and microelements from the N. alba extracts were experimentally determined.
294 The amount of the following 23 elements was determined in 3 parallel measurements. For Al, B, Ba,
295 Ca, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Zn and Sn, the Inductively Coupled Plasma Optical
296 Emission Spectrometer, Optima 8300DV (ICP-OES) from Perkin Elmer was used. For Hg, the Atomic
297 Absorption Spectrophotometer PinAAcle 900T FIAS FLAME from Perkin Elmer was used, and
298 because As, Cd, Pb and Se showed quantities smaller than the detection limit, the Atomic Absorption
299 Spectrophotometer PinAAcle 900Z equipped with a longitudinally heated graphite oven (AAS) from
300 Perkin Elmer was used instead. The general detection limit was equal to the stock values from the
301 Perkin Elmer machines. The standard solutions were prepared from the Merck standards (ICP, FIAS
302 and AAS) and are found in the same matrix as in the samples. Sampling: 1 g from each N. alba sample
303 with 8 mL of p.a. HNO3 and 2 mL concentrated HCl were introduced in the quartz flasks of the Anton
304 Paar oven. After mineralization, ultrapure water was added to the samples until a volume of 25 mL.

305 3.9. FT-IR Spectra Analysis

306 The methanolic N. alba extracts from the fruit, flower, leaf, stem and root were analyzed using
307 the modernized Thermo Scientific Nicolet 6700 FT-IR Spectrometer. The samples were scanned in the
308 absorbtion range between 4000 and 600 cm-1. The analysis was repeated twice for the spectrum
309 confirmation.

310 3.10. Statistical Analysis

311 All data were expressed as means ± standard deviation of the mean of three independent assays
312 and analyzed through unpaired Student’s t-test. Correlation between the data was carried out using
313 the Microsoft Excel program.

314 5. Conclusions
 Molecules 2018, 23, x FOR PEER REVIEW 12 of 14

315 To the best of our knowledge, this is the first comparative study that presents an exhaustive
316 survey on the extraction, separation, identification and analysis of the bioactive compounds of all
317 anatomic parts of N. alba (fruit, flower, leaf, stem and root) from the Danube Delta Biosphere Reserve.
318 The evaluation of the bioactive compounds has been made using both qualitative and quantitative
319 methods of analysis. The HPLC-MS/MS analysis highlighted the presence of polyphenolic and
320 flavonoid compounds with antioxidant activities. The quantitative analysis included
321 spectrophotometric determinations in order to evaluate polyphenols, flavonoids, tannins, condensed
322 tannins and the antioxidant activity. The antioxidant activity of the methanolic N. alba extracts,
323 evaluated by using DPPH assay, is comparable with literature data gained through different
324 techniques. The focus of antioxidant studies in the Nymphaeaceae family has been mainly on leaves
325 and fruit and any data from romanian N. alba extracts has not been reported. As secondary
326 metabolites, polyphenols and flavonoids are strongly influenced by ecological stress, such as water
327 deficit. The total content of polyphenols and flavonoids is also influenced by the activity of specific
328 enzymes [10] in N. alba, which depends on the genetic material of the plant. We have proven that the
329 ultrasound mediated extracts of N. alba from Danube Delta Biosphere Reserve had a high total
330 polyphenol and flavonoid content.
331 Samples from aquatic plants usually contain many different types of bioactive compounds. We
332 have proven that the ultrasound mediated extracts of N. alba from Danube Delta Biosphere Reserve
333 had a high total polyphenol and flavonoid content. As secondary metabolites, polyphenols and
334 flavonoids are strongly influenced by ecological stress, such as water deficit [10]. The total content of
335 polyphenols and flavonoids is also influenced by the activity of specific enzymes in N. alba, which
336 depends on the genetic material of the plant.
337 The ICP OES method, applied for screening analysis of macro and micronutrients, revealed also
338 the presence of K, P, Na, Ca and Mg in all the parts of the plant.
339 Our results highlight the valuable contribution to the knowledge about the bioactive properties
340 of native N. alba species from Danube Delta Biosphere Reserve.
341 Further studies (unpublished results) are conducted in order to isolate, characterize and
342 demonstrate other biological properties of N. alba species from Danube Delta Biosphere Reserve.
343 Supplementary Materials: The following are available online at www.mdpi.com/link, Figure S1: title, Table S1:
344 title, Video S1: title.

345 Acknowledgments: The authors wish to thank the SCIENT, Research Center for Instrumental Analysis for
346 technical assistance.

347 Author Contributions: For research articles with several authors, a short paragraph specifying their individual
348 contributions must be provided. The following statements should be used “X.X. and Y.Y. conceived and
349 designed the experiments; X.X. performed the experiments; X.X. and Y.Y. analyzed the data; W.W. contributed
350 reagents/materials/analysis tools; Y.Y. wrote the paper.” Authorship must be limited to those who have
351 contributed substantially to the work reported.

352 Conflicts of Interest: The authors declare that they have no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

© 2018 by the authors. Submitted for possible open access publication under the
terms and conditions of the Creative Commons Attribution (CC BY) license
(http://creativecommons.org/licenses/by/4.0/).