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Antidiabetic activity of Musa x paradisiaca extracts in streptozotocin-induced diabetic rats and chemical characterization by HPLC-DAD-MS
Journal Pre-proof Antidiabetic activity of Musa x paradisiaca extracts in streptozotocin-induced diabetic rats and chemical characterization by HPLC-DAD-MS R.O. Vilhena, I.D. Figueiredo, A.M. Baviera, D.B. Silva, B.M. Marson, J.A. Oliveira, R.G. Peccinini, I.K. Borges, R. Pontarolo PII:
S0378-8741(19)32117-8
DOI:
https://doi.org/10.1016/j.jep.2020.112666
Reference:
JEP 112666
To appear in:
Journal of Ethnopharmacology
Received Date: 27 May 2019 Revised Date:
9 January 2020
Accepted Date: 9 February 2020
Please cite this article as: Vilhena, R.O., Figueiredo, I.D., Baviera, A.M., Silva, D.B., Marson, B.M., Oliveira, J.A., Peccinini, R.G., Borges, I.K., Pontarolo, R., Antidiabetic activity of Musa x paradisiaca extracts in streptozotocin-induced diabetic rats and chemical characterization by HPLC-DAD-MS, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2020.112666. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
1 1
Graphical abstract
2 3 4
Antidiabetic activity of Musa x paradisiaca extracts in streptozotocin-
5
induced diabetic rats and chemical characterization by HPLC-DAD-MS
6 7
Vilhena, R. O.1; Figueiredo, I. D.2; Baviera, A. M.2; Silva, D. B.3; Marson, B. M.1;
8
Oliveira, J. A2; Peccinini, R. G.4; Borges, I. K.5; Pontarolo, R.1*
9 10
1
Departamento de Farmácia, Universidade Federal do Paraná, Curitiba, PR, Brazil
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2
Departamento de Análises Clínicas, Universidade Estadual Paulista Júlio de Mesquita
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Filho, Araraquara, SP, Brazil
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3
14
Farmacêuticas, Alimentos e Nutrição, Universidade Federal do Mato Grosso do Sul,
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Campo Grande, MS, Brazil
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4
17
Paulista Júlio de Mesquita Filho, Araraquara, SP, Brazil
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5
19
PR, Brazil
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Laboratório de Produtos Naturais e Espectrometria de Massas, Faculdade de Ciências
Departamento de Princípios Ativos Naturais e Toxicologia, Universidade Estadual
Departamento de Ciências Patológicas, Universidade Estadual de Londrina, Londrina,
2 21
*Corresponding author
22
Address: Department of Pharmacy, Federal University of Paraná, 632 Lothário
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Meissner Avenue, 80210-170, Curitiba – PR, Brazil
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E-mail: [email protected] (RP)
25 26
Declarations of interest: none
27 28 29
Authors e-mail:
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Vilhena, R. O.: [email protected]
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Figueiredo, I. D.: [email protected]
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Baviera, A. M.: [email protected]
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Silva, D. B.: [email protected]
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Marson, B. M.: [email protected]
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Oliveira, J. A: [email protected]
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Peccinini, R. G.: [email protected]
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Borges, I. K.: [email protected]
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Pontarolo, R.: [email protected]
39 40 41 42 43 44 45 46
3 47
ABSTRACT
48
Ethnopharmacological relevance
49
The Musa x paradisiaca L. inflorescence, known as banana blossom or banana heart, is
50
used in traditional medicine for the treatment of diabetes mellitus.
51
Aim of the study
52
The aim of the study was to investigate the antidiabetic activity of aqueous extracts and
53
fractions prepared from the bracts and flowers of Musa x paradisiaca in streptozotocin
54
(STZ)-induced diabetic rats and to chemically characterize the extracts.
55
Materials and Methods
56
Standard aqueous extracts of the flowers, bracts, and their fractions were prepared and
57
their chemical composition was determined tentatively by high-performance liquid
58
chromatography coupled to diode-array detection and mass spectrometry (HPLC-DAD-
59
MS). Changes in fasting glycemia and oral glucose tolerance were evaluated in STZ-
60
induced diabetic rats (n = 8) treated with aqueous extracts of Musa x paradisiaca (200
61
mg/kg) for 20 days.
62
Results
63
Chemical analyses detected 21 compounds and 17 metabolites were identified, among
64
which were glycosylated and acetylated phenylpropanoids of p-coumaric acid and caffeic
65
acid, as well as a glycosylated flavonol and anthocyanins. Following 15 days of treatment,
66
the bract aqueous extracts and the methanolic fraction of the flower had significant
67
effects on the glycemic profile after glucose load in diabetic rats as compared with the
68
untreated diabetic group.
69
Conclusions
70
The results of the present study show the antidiabetic potential of extracts of the flowers
71
and bracts of M. x paradisiaca.
4 72 73
Keywords: banana, hydroxycinnamic acid derivatives, HPLC-DAD-MS, anthocyanins,
74
diabetes mellitus
75 76
Abbreviations
77
HPLC-DAD-MS, high-performance liquid chromatography coupled to diode-array
78
detection and mass spectrometry; AFE, aqueous flower extract; MFF, methanolic
79
flower fraction; ABE, aqueous bract extract; MBF, methanolic bract fraction; AqE,
80
aqueous extract; MeF, methanolic; N, normal group; D, untreated diabetic group;
81
DAFE, diabetic group treated with aqueous flower extract; DMFF, diabetic group
82
treated with methanolic flower fraction; DABE, diabetic group treated with aqueous
83
bract extract; DMBF, diabetic group treated with methanolic bract fraction; Dp-CA,
84
diabetic group treated with p-coumaric acid; DINS, diabetic group treated with insulin;
85
b.w., body weight.
86
5 87
1. Introduction
88
Musa x paradisiaca L., commonly known as banana, is among the most important fruit
89
crops in the world (Jawla et al., 2012). In 2015, global banana production reached a
90
record growth rate of approximately 118 million tons (FAO, 2018). As a result of this
91
large production, it is estimated that more than 100 million tons of waste are generated
92
annually, which is mainly leaves, pseudostems, and inflorescences (IBGE, 2013).
93
Although inflorescences are economically regarded as waste, the use of these parts in
94
traditional medicine is common practice in many cultures. For example, inflorescence is
95
used for the treatment of diabetes through consumption of cooked inflorescence or in
96
the form of a decoction (Kumar et al., 2012). Additionally, a recent review showed the
97
in vitro and in vivo antidiabetic potential of Musa ssp. flowers (Vilhena et al., 2015).
98
Other studies have shown that the flowers exhibit antihyperglycemic properties (Borah
99
and Das, 2017; Jawla et al., 2012; Nisha and Mini, 2013) and the capacity to inhibit
100
carbohydrate digestion (Abdurrazak et al., 2015; Arun et al., 2017; Marikkar et al.,
101
2016) and improve glucose uptake (Arun et al., 2017; Bhaskar et al., 2011). However,
102
no studies have investigated the in vivo antidiabetic activity of bracts.
103
In this context, studies contributing to the verification of the ethnopharmacological use
104
of the inflorescence of banana, including the bracts, are of extreme importance for the
105
development and strengthening of complementary therapies for the treatment of
106
diabetes mellitus. Therefore, the aim of the present study was to investigate the
107
antidiabetic effects of aqueous extracts of the bracts and flowers of Musa x paradisiaca
108
in streptozotocin (STZ)-induced diabetic rats followed by chemical characterization.
109 110
2. Materials and Methods
111
2.1. Chemicals and Reagents
6 112
Streptozotocin and p-coumaric acid standards (≥ 98%) were purchased from
113
Sigma−Aldrich (St. Louis, MO, USA). Insulin was obtained from Lilly (Humulin® NPH
114
100 UI/mL, Indianapolis, USA). Isoflurane was purchased from Cristália (Isofurine®,
115
Itapira, SP, Brazil). Amberlite XAD-2 was obtained from Merck KGaA (Darmstadt,
116
Germany). Acetonitrile and methanol (HPLC grade) were obtained from Tedia
117
(Fairfield, CA, USA). Formic acid (98−100%; LC-MS grade) was purchased from
118
Sigma−Aldrich (St. Louis, MO, USA). Hydrochloric acid (36−38.0%) was purchased
119
from Mallinckrodit (Edo. de Mexico, Mexico). Ultrapure water was obtained using a
120
Milli-®purification system from Millipore (Milford, MA, USA).
121 122
2.2. Plant material
123
Musa x paradisiaca inflorescence was collected after approximately 60 days of fruit
124
development in Morretes, PR, Brazil (geographical coordinates 25º29'45,59 "S and
125
48º48'40,25 "W, 10.93 m) and was identified by a botanist from the herbarium of
126
Museu Botânico Municipal de Curitiba (MBM – 348145). The inflorescence was
127
separated into flowers and bracts and dried in a drying cabinet for seven days at 30ºC.
128
Following drying, the flowers and bracts were ground into separate powders using a
129
cutting mill and were subsequently sieved (3 mm). The dried powder was stored in air-
130
tight containers until analysis.
131 132
2.3. Extraction procedure
133
The dried flower and bract powders were extracted twice with water (1:10, w/v) via an
134
ultrasound-assisted extraction process. The equipment was operated at a frequency of 40
135
kHz, a power of 100 W, a temperature of 25ºC, and a sonication time of 30 min. The
136
liquid was filtered, and the total volume was lyophilized at -80°C and 100 mTorr for 48
7 137
h (Virtis Advantage Plus, SP Scientific, Warminster, England). The obtained flower
138
(AFE) and bract (ABE) extracts were fractionated in amberlite resin and solubilized in
139
water (8 g/100 mL), followed by adjustment of the pH to 2.0 using HCl. The extracts
140
were mixed with 12 g amberlite XAD-2 resin (9 nm pore and 20−60 mesh particles) for
141
1 h using a magnetic stirrer and then packed into a glass column (45 x 3.5 cm).
142
Amberlite impregnated with crude extract was washed with 1 L water. The fraction
143
adsorbed on the column was eluted with 370 mL methanol and dried under reduced
144
pressure in a Centrivap Sample Concentrator (Labconco, Kansas City, USA) at 40°C.
145
The methanolic fractions of flower (MFF) and bract (MBF), in addition to AFE and
146
ABE, were stored in at -40°C in air-tight and light-protected containers until analysis.
147
The extraction yields obtained were 15,2% (ABE), 18,1% (AFE), 3,2 % (MBF), and
148
6,7% (MFF).
149 150
2.4. HPLC-DAD-MS analysis
151
Extracts and methanolic fractions (AFE, ABE, MFF, and MBF; 1 mg/mL) were
152
analyzed using a high-performance liquid chromatography (Prominence UFLC,
153
Shimadzu, Kyoto, Japan) coupled to a diode-array detector (190–400 nm) and a mass
154
spectrometer with an electrospray ionization source (ESI) and the quadrupole-time-of-
155
flight (QTOF) (MicrOTOF-Q III, Bruker Daltonics, Billerica, USA) analyzers operating
156
in negative and positive ion modes. Nitrogen was used as gas of nebulization (4 Bar),
157
dry (9.1 L/min at 200 ° C) and collision. The capillary voltage was set at 3,500 V, and
158
the scan range was m/z 100-1300. The analysis was performed on a C-18 column
159
(Kinetex, 150 mm × 2.1 mm id, 2.6 µm), with an oven temperature of 40°C. The mobile
160
phase was deionized water (A) and acetonitrile (B), both containing 0.1% formic acid
161
(v/v), under the following gradient profile: 0–2 min 3% B, 2–25 min 3–25% B, and 25–
8 162
40 min 25–80% B. The flow rate was 0.3 mL/min and the injection volume was 1 µL.
163
The extracts were prepared at 1 mg/mL using acetonitrile and water (6:4, v/v) and
164
filtered on a 0.22 µm × 3.0 mm PTFE membrane (Millex®, Millipore).
165 166
2.5. Antidiabetic activity of M. x paradisiaca extracts
167
2.5.1. Animals
168
Male Wistar rats (Rattus norvegicus) weighing 140–160 g (6 weeks old) were
169
maintained under environmentally controlled conditions, with a temperature of 23±1°C,
170
a humidity of 55 ± 5%, and a 12 h light/dark cycle. Rats received water and normal lab
171
chow diet (Presence, Paulínia, São Paulo, Brazil) ad libitum. The experiments were
172
conducted during the light phase following approval of the experimental protocol by the
173
Committee for Ethics in Animal Experimentation of the School of Pharmaceutical
174
Sciences, UNESP, Araraquara (protocol number CEUA/FCF/CAr: 31/2017).
175 176
2.5.2. Induction of the experimental diabetes mellitus model
177
Following four days of acclimation, experimental DM was induced by a single
178
intravenous injection of STZ (40 mg/kg b.w.) dissolved in 0.01 M citrate buffer (pH
179
4.5) in 12 h-fasted rats. Normal rats received only citrate buffer. All animals were
180
anesthetized with isoflurane for this procedure. Three days after STZ administration,
181
rats with post-prandial glycemia values ≥ 350 g/dL were used in the experiments
182
(Furman, 2015). Plasma glucose levels were determined by the glucose oxidase method
183
(Trinder, 1969) using a commercial kit (Biotécnica, Varginha, MG, Brazil).
184 185
2.5.3. Experimental design
9 186
Diabetic animals were stratified into seven different experimental groups (8 rats/group)
187
using matched glycemia and body weight values: diabetic rats treated with water (D); 4
188
UI insulin (DINS); 200 mg/kg b.w. AFE (DAFE); 200 mg/kg b.w. ABE (DABE); 200
189
mg/kg b.w. MFF (DMFF); 200 mg/kg b.w. MBF (DMBF); and 20 mg/kg b.w. p-
190
coumaric acid (Dp-CA). Additionally, a group of normal rats (n = 8) treated orally with
191
vehicle (water) were used as a control. The treatments, except for DINS, were
192
performed by gavage twice a day, with a half-dose (0.5 mL) at 08:00 h and 17:00 h, for
193
a total of 20 days. Extract and methanolic fraction doses were solubilized in water,
194
whereas p-coumaric acid was suspended in carboxymethyl cellulose (1%, w/v) due to
195
its poor water solubility. DINS rats received subcutaneous injections of insulin twice
196
daily (2 UI/rat per injection).
197 198
2.5.4. Oral glucose tolerance test (OGTT)
199
Following 15 days of treatment, an OGTT was performed in 12 h-fasted rats. A glucose
200
solution (2 g/kg b.w.) was administered orally, and blood samples were collected from
201
the tip of the tail before (t = 0) and 15, 30, 60, 90, and 120 min after glucose loading.
202
Results are expressed as mg/dL, and the area under the curve (AUC, g/dL/120 min) was
203
calculated (GraphPad Prism® 6.0 Software, GraphPad, La Jolla, USA).
204 205
2.5.6. Statistical analysis
206
Differences between groups were analyzed with one-way ANOVA followed by a
207
Student−Newman−Keuls test (p < 0.05) (GraphPad Prism® 6.0 Software, GraphPad, La
208
Jolla, USA).
209 210
3. Results and Discussion
10 211 212
3.1. Identification of the constituents by HPLC-DAD-MS
213
The constituents of M. x paradisiaca extracts and fractions were identified based on
214
UV, MS, and MS/MS data as compared with data described in the literature (Abdallah
215
et al., 1994; Abid et al., 2017; Shirota et al., 1997; Zhang et al., 2015). The
216
chromatograms of AFE, ABE, MFF, and MBF are shown in Fig. 1. Seventeen
217
compounds were identified from the extracts (Table 1).
218
219 220
Fig. 1. Total ion chromatograms (negative ion mode) of aqueous bract extract (A);
221
methanolic bract fraction (B); aqueous flower extract (C); and methanolic flower
222
fraction (D) of M. x paradisiaca. The fractions were obtained by clean-up procedures
223
using Amberlite XAD2. The identification of chromatographic peaks is described in
224
Table 1 and all the chromatograms are in the same intensity range.
11
225
Table 1. Compounds identified from the aqueous extracts and methanolic fractions of the bracts and flowers of M. x paradisiaca by
226
HPLC-DAD-MS
1 2
RT (min) 1.1 1.2
3
9.6
di-O-hexosyl coumaric acid
4
10.7
UI di-O-acetyl di-O-hexosyl coumaric acid O-hexosyl-deoxihexosyl quercetin UI
Peak
Compound Quinic acid Hexose
5
17.8
6
18.1
7
18.4
8
19.6
9
20.0
10
21.5
11
21.9
12
22.9
13
24.2
14
25.1
15
25.3
16
26.1
Nonanedioic acid O-deoxyhexosyl-hexosyl cyanidin tri-O-acetyl di-O-hexosyl coumaric acid tri-O-acetyl di-O-hexosyl coumaric acid tri-O-acetyl di-O-hexosyl coumaric acid tetra-O-acetyl di-O-hexosyl caffeic acid tetra-O-acetyl di-O-hexosyl coumaric acid O-deoxyhexosyl-hexosyl Oacetyl cyanidin UI
17
26.4
tetra-O-acetyl di-O-hexosyl
UV (nm) 299, 312 285 299, 313 260, 352 299, 325 274, 520 299, 313 299, 313 299, 313 299, 325 299, 313 265, 517 276, 313 299,
Bract
Flower
MF
Ident. levela
MS (+ / -) (m/z)
MS/MS (m/z)
C7H12O6 C6H12O6 C21H28O13
1 4 3
191.0568 (-) 179.0566 (-) 487.1488 (-)
163
C12H13NO3 C25H32O15
4 3
218.0832 (-) 571.1695 (-)
216, 188, 162 487, 341, 307, 163, 145
C27H30O16
3
609.1482 (-)
300, 271, 255, 179
X
C17H16O10
4
379.0697 (-)
185
X
C9H16O4 C27H31O15+
4 3
187.0989 (-) 595.1673 (+)
287
C27H34O16
3
613.1803 (-)
C27H34O16
3
C27H34O16
3
C29H36O18
3
C29H36O17
3
C29H33O16+
3
C31H34O17 C29H36O17
MeF
AqE
MeF
X
X X X
X
X
X X
X X
X X
X X
X X
383, 341, 307, 163, 145
X
X
X
613.1803 (-)
383, 341, 307, 163, 145
X
X
X
613.1794 (-)
341, 323, 163, 145
X
X
X
383, 179, 163
X
425, 383, 341, 163, 145
X
637.1770 (+)
329
X
X
4
679.1861 (+)
391, 373
X
X
3
655.1906 (-)
383, 341, 163, 145
X
671.1856 (-) 655.1872 (-)
AqE X X
X
X X
X X
X
X
X
12
coumaric acid 312 3 655.1911 (-) 383, 341, 323, 163, 145 X tetra-O-acetyl di-O-hexosyl 299, C29H36O17 coumaric acid 313 27.5 tetra-O-acetyl di-O-hexosyl O299, C30H38O18 3 685.2023 (-) 193, 175, 160 X X 19 methyl caffeic acid 325 30.0 penta-O-acetyl di-O-hexosyl 299, C31H38O18 3 697.2022 (-) 425, 383, 163, 145 X X 20 coumaric acid 313 30.9 UI C18H34O5 4 329.2351 (-) 229, 211, 183, 171 21 Note: +: positive ion mode; -: negative ion mode; UI: unidentified; MF: molecular formula: RT: retention time; UV: ultraviolet; AqE aqueous extract; MeF: methanolic; X: presence of identified compound. aIdentification level of compounds using Metabolomics Standards as reported by Schymanski et al., 2014. All MFs were determined from the accurate mass considering a mass error and mSigma lower than 8 ppm and 30, respectively. 18
227 228 229 230
26.9
13
231
Chromatographic peaks 3, 5, 10−12, 14, 17−18, and 20 revealed absorption bands in the
232
ultraviolet spectra, with wavelengths near 299 and 310 nm, which is compatible with
233
the coumaric acid moiety (Zhang et al., 2015). All these compounds revealed fragment
234
ions at m/z 163, which is in accordance with a neutral loss of coumaric acid molecule. It
235
is yielded from the loss of two hexoses (324 u - compound 3) or two hexoses together
236
with acetyl groups, such as two (408 u - compound 5), three (450 u - compounds
237
10−12), four (508 u-compounds 14, 17−18), or five (534 u-compound 20) acetyl groups.
238
Compound 5 exhibited an ion at m/z 571.1695 [M-H]-, indicating a molecular formula
239
of C25H32O15, and also showed a fragment ion at m/z 487 produced from the loss of two
240
acetyl groups (42 + 42 = 84 u), confirming the acetyl substituents. Thus, it was possible
241
to identify the compounds as di-O-hexosyl coumaric acid (3), di-O-acetyl di-O-hexosyl
242
coumaric acid (5), tri-O-acetyl di-O-hexosyl coumaric acid), tetra-O-acetyl di-O-
243
hexosyl coumaric acid (14, 17-18), and penta-O-acetyl di-O-hexosyl coumaric acid
244
(20). These spectral data are compatible with those described in the literature (Zhang et
245
al., 2015).
246
Peaks 13 and 19 exhibited two absorption bands at wavelengths of 299 and 325 nm in
247
the UV spectra, suggesting the presence of a caffeic acid unit. These compounds
248
revealed fragment ions at m/z 179 and 193, confirming the presence of caffeic and O-
249
methyl caffeic acids, respectively. From comparison with previously published data,
250
substances 13 and 19 were identified as tetra-O-acetyl di-O-hexosyl caffeic acid (13)
251
(Abdallah et al., 1994) and tetra-O-acetyl di-O-hexosyl O-methyl caffeic acid (19)
252
(Shirota et al., 1997).
253
A glycosylated flavonol, O-hexosyl-deoxyhexosyl quercetin (6), and quinic acid (1),
254
hexose (2), and nonanedioic acid (8) were also identified in the samples. In addition,
14
255
peaks 9 and 15 showed UV spectra typical of anthocyanins (λmax ≈ 270 and 515 nm) and
256
protonated ions (m/z 595.1673 and 637.1770), indicating a molecular formula of
257
C27H31O15+ and C29H33O16+, respectively. They showed losses of 308 u, suggesting O-
258
deoxyhexosyl-hexosyl substituents, and yielded fragment ions m/z 287 (C15H11O6+) and
259
329 (C17H13O7+), which confirmed the aglycones cyanidin and O-acetyl cyanidin (Abid
260
et al., 2017). Thus, compounds 9 and 15 were identified as O-deoxyhexosyl-hexosyl
261
cyanidin and O-deoxyhexosyl-hexosyl O-acetyl cyanidin.
262 263
3.1. Antidiabetic activity of M. x paradisiaca extracts
264
3.1.1. Glucose tolerance following treatment of diabetic rats with M. x paradisiaca
265
extracts
266
STZ administration destroys β-cells, leading to the development of metabolic
267
disturbances due to severe insulin deficiency in a profile similar to the changes observed
268
in type 1 diabetes mellitus (Lenzen, 2008). Therefore, an abnormality in the glycemic
269
profile of diabetic animals is expected. On day 15 of treatment, the OGTT was
270
performed to evaluate the effects of the aqueous extracts, methanolic fractions, and p-
271
coumaric acid on fasting glycemia and glucose tolerance in diabetic rats. The OGTT
272
results for the control and diabetic-treated groups are shown in Fig. 2.
15
273 274
Fig. 2. Oral glucose tolerance in normal and STZ-induced diabetic rats treated with the
275
extracts and fractions of M. x paradisiaca. (A) Glycemia levels before (t = 0) and after
276
glucose load in rats treated for 20 days; (B) area under the curve (AUC) of the oral
277
glucose tolerance test; and (C) fasting glycemia of the control and treated groups. The
278
values are expressed as the mean ± SD, n = 8 per group. Differences between groups
279
were analyzed with one-way ANOVA followed by a Student−Newman−Keuls test.
280
Asterisks denote the significant differences from the D group (*p < 0.05, **p < 0.01);
281
the pound signs denote the significant differences from the N group (#p < 0.05).
282
Note: N, normal group; D, untreated diabetic group; DAFE, diabetic group treated with aqueous flower
283
extract (200 mg/kg/day); DMFF, diabetic group treated with methanolic flower fraction (200 mg/kg/day);
284
DABE, diabetic group treated with aqueous bract extract (200 mg/kg/day); DMBF, diabetic group treated
285
with methanolic bract fraction (200 mg/kg/day); Dp-CA, diabetic group treated with p-coumaric acid (20
286
mg/kg/day); and DINS, diabetic group treated with insulin (4 UI/rat/day).
287 288
For the D group, increased values of fasting glycemia, in addition to glucose intolerance
289
after oral load, were observed, indicating impairments in the control of carbohydrate
16
290
metabolism in STZ-induced diabetic rats, likely due to an insulin-deficient state. On the
291
other hand, in comparison with the D group, all groups of diabetic rats treated with
292
extracts or fractions of M. x paradisiaca had decreases in fasting glycemia, not
293
significantly different from the DINS group (Fig. 2C).
294
Observing the post glucose load profiles (Fig. 2A), there was a reduction in glucose
295
intolerance in the groups of diabetic rats that received aqueous extracts of flower and
296
bract, methanolic flower fraction, and insulin (DAFE, DABE, DMFF, and DINS
297
groups). Moreover, the glycemia levels in the DABE and DMFF groups returned to
298
values close to those in the DINS group at the last evaluation time after oral glucose
299
overload (120 min).
300
Comparison of the AUCs of the different groups (Fig. 2B) shows that MFF, ABE, and
301
insulin treatment in diabetic rats promoted statistically significant alterations in the post
302
glucose load profile in relation to the D group, indicating that these treatments improved
303
the glucose tolerance in animals with DM. Considering that the effects in the DABE and
304
DMFF groups were not statistically different from each other, ABE presents an
305
advantage over MFF due to the ease of obtaining an aqueous extract. Although the
306
glycemic profile in the DAFE group was not significantly different as compared with
307
the D group, it is important to note that the profile up to 60 min following the glucose
308
load is very similar to that in the DINS, DMFF, and DABE groups, indicating the
309
potential of this extract.
310
Considering the relationship between the results of the antidiabetic properties of the M.
311
x paradisiaca preparations and the chemical composition of the bract extracts, it can be
312
inferred that the antidiabetic potential of this part of the inflorescence could be
313
attributed to a possible synergistic action of the total components of the extract, since
17
314
ABE promoted improvements in the post glucose load profile and fasting glycemia to a
315
greater extent than MBF. Although the procedure to obtain MBF was used to get rid of
316
the most of the sugars, some of the potent bioactive metabolites might also be eluted
317
with water from the amberlite XAD-2 column, which could explain the lower activity.
318
The effect of rutin, a flavonol glycoside, on glycemia levels has been evaluated in in
319
vitro and in vivo studies. The antihyperglycemic activity of rutin in animal models of
320
acute and chronic hyperglycemia induced by alloxan and improvements in the glycemic
321
profile of normoglycemic mice submitted to OGTT are examples of the antidiabetic
322
potential attributed to doses of 30 mg/kg of this compound (Calzada et al., 2017). In
323
addition, rutin, quercetin, and isoquercetin have shown in vitro inhibitory activity
324
against α-glycosidase (Jo et al., 2009; Sohretoglu et al., 2018). Therefore, the presence
325
of flavonol glycosides in the bract extracts may have contributed to the observed results.
326
Anthocyanins, compounds present in the bracts of Musa spp. (Kitdamrongsont, 2018),
327
appear to play a role in the prevention or reversal of pathogenic processes related to
328
type 2 DM by inhibiting body weight gain, preventing the production of free radicals
329
and lipid peroxidation, regulating the inflammatory response, reducing the glucose and
330
lipids levels in the blood, and improving insulin resistance (Guo and Ling, 2015). Thus,
331
these compounds may also be related to the effects obtained by treatment with bract
332
extracts.
333
Additionally, the presence of glycosylated and acetylated phenylpropanoid derivatives
334
could also be related to the antidiabetic activity of the extracts. Studies have shown that
335
acetylated derivates of p-coumaric acid glycosides have platelet antiaggregant activity
336
(Yoshikawa et al., 2002) and moderate aldose reductase inhibitory activity (Fujimoto et
337
al., 2014). For compounds belonging to this group, α-glycosidase inhibitory activity,
18
338
such as lapatoside D (Fan et al., 2010), and β-glycosidase inhibitory activity, such as
339
vanicoside A and B isolated from the root of Polygonum sachalinense were also
340
observed (Kawai et al., 2006). In these studies, in addition to the glycosylated and
341
acetylated compounds, p-coumaric and ferulic acids were also evaluated; however, they
342
did not show any activity. Thus, the authors suggested that the complete structure of the
343
compounds is related to the action, not just hydroxycinnamic acids. Additionally, poorly
344
bonded compounds, such as ester bonding, have been reported to be hydrolyzed in the
345
gut prior to absorption (Lafay et al., 2007). Thus, the present study also evaluated the
346
activity of p-coumaric acid alone for the purpose of comparing the phenylpropanoyl
347
portion of the compounds identified in the extracts. Similarly, p-coumaric acid
348
administered alone showed no activity in diabetic animals in the present study (Fig. 2).
349 350
4. Conclusion
351
The results of the present study show the antidiabetic potential of the flower and bract
352
extracts and fractions of M. x paradisiaca. To the best of our knowledge, this is the first
353
time that the antidiabetic effect of bracts has been evaluated in STZ-induced diabetic
354
rats. All diabetic rats treated with the extracts and fractions of M. x paradisiaca had
355
decreased fasting glycemia as compared with the untreated diabetic group. ABE
356
treatment was highlighted as presenting improvements in the post glucose load profile
357
in diabetic rats as compared with the untreated group. These findings provide novel
358
evidence for traditional antidiabetic use of M. x paradisiaca inflorescence and potential
359
for the application of these extracts in the development of novel antidiabetic medicines.
360 361
Conflicts of interest
19
362
The authors have no conflicts of interest.
363 364
Acknowledgments
365
The authors acknowledge the Brazilian agencies CAPES and CNPq for fellowships and
366
the biochemistry and clinical enzymology laboratory of UNESP-Araraquara for help
367
with in vivo experiments. The authors would also like to thank FUNDECT (Fundação
368
de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do estado de Mato
369
Grosso do Sul).
370 371
Author’s contributions
372
ROV contributed to the collection and identification of plant samples, running of the
373
herbarium, performing laboratory work, analysis of the data, and drafting of the paper.
374
IDF contributed to the design of in vivo experiments, performing laboratory work, and
375
analysis of the data. AMB designed the in vivo experiments, analyzed the data, critically
376
read the manuscript, and contributed to reagents/materials. DBS performed the LC-
377
DAD-MS analyses and identified the potential bioactive compounds, contributed to
378
reagents/materials, and critically read the manuscript. BMM contributed to the
379
collection and identification of plant samples, running of the herbarium, performing
380
laboratory work, and analysis of the data. JO contributed to in vivo experiments,
381
performing laboratory work, and analysis of the data. RGP contributed to the laboratory
382
support
383
reagents/materials. IKB contributed to the critical reading of the manuscript. RP
384
designed the study, supervised the laboratory work, contributed to reagents/materials,
of
in
vivo
experiments,
critical
reading
of
the
manuscript,
and
20
385
and critically read the manuscript. All authors read the final manuscript and approved
386
the submission.
387 388
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Furman, B.L., 2015. Streptozotocin-induced diabetic models in mice and rats. Curr. Protoc. Pharmacol. 70, 5.47.1−5.47.20. Guo, H. and W. Ling, 2015. The update of anthocyanins on obesity and type 2 diabetes: experimental evidence and clinical perspectives. Rev. Endocr. Metab. Disord. 16, 1−13. IBGE, 2013. Levantamento Sistemático da Produção Agrícola. Instituto Brasileiro de Geografia e Estatística. 1−88. Jawla, S., Kumar, Y., and Khan, M. S. Y., 2012. Antimicrobial and antihyperglycemic activities of Musa paradisiaca flowers. Asian Pac. J. Trop. Biomed. 2, S914−S918. Jo, S. H., Ka, E. H., Lee, H. S., Apostolidis, E., Jang, H. D., Kwon, and Y. I., 2009. Comparison of antioxidant potential and rat intestinal alpha-glucosidases inhibitory activities of quercetin, rutin, and isoquercetin. Int. J. Appl. Res. Nat. Prod. 2, 9. Kawai, Y., Kumagai, H., Kurihara, H., Yamazaki, K., Sawano, R., and Inoue, N., 2006. β-Glucosidase inhibitory activities of phenylpropanoid glycosides, vanicoside A and B from Polygonum sachalinense rhizome. Fitoterapia. 77, 56−459. Kitdamrongsont, K., Pothavorn, P., Swangpol, S., Wongniam, S., Atawongsa, K., Svasti, J., and Somana, J., 2008. Anthocyanin composition of wild bananas in Thailand. J. Agric. Food Chem. 56, 10853−10857. Kumar, K. P. S., Bhowmik, D., Duraivel, S., and Umadevi, M., 2012. Traditional and medicinal uses of banana. J. Pharmacogn. Phytochem. 1, 51−63. Lafay, S., Morand, C., Manach, C., Besson, C., and Scalbert, A., 2007. Absorption and metabolism of caffeic acid and chlorogenic acid in the small intestine of rats. Br. J. Nutr. 96(1), 39-46. Lenzen, S., 2008. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia. 51, 216−26. Marikkar, J. M. N., Tan, S. J., Salleh, A., Azrina, A., and Shukri, M. A. M., 2016. Evaluation of banana (Musa sp.) flowers of selected varieties for their antioxidative and anti-hyperglycemic potentials. Int. Food Res. J. 23, 1988−1995. Nisha, P. and S. Mini, 2013. Flavanoid rich ethyl acetate fraction of Musa paradisiaca inflorescence down-regulates the streptozotocin induced oxidative stress, hyperglycaemia and mRNA levels of selected inflammatory genes in rats. J. Funct. Foods. 5, 1838−1847.
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Shirota, O., Sekita, S., and Satake, M., 1997. Two phenylpropanoid glycosides from Sparganium stoloniferum. Phytochem. 44, 695−698. Sohretoglu, D., Sari, S., Soral, M., Barut, B., Ozel, A., and Liptaj, T., 2018. Potential of Potentilla inclinata and its polyphenolic compounds in alpha-glucosidase inhibition: kinetics and interaction mechanism merged with docking simulations. Int. J. Biol. Macromol. 108, 81−87. Trinder, P., 1969. Determination of blood glucose using 4-amino phenazone as oxygen acceptor. J. Clin. Pathol. 22, 246−246. Vilhena, R. O., Fachi, M. M., Marson , B. M., Dias, B. L., Pontes, F. L. D., Tonin, F. S., and Pontarolo, R., 2018. Antidiabetic potential of Musa spp. inflorescence: a systematic review. J. Pharm. Pharmacol. 70, 1583−1595. Yoshikawa, M., Murakami, T., Ishiwada, T., Morikawa, T., Kagawa, M., Higashi, Y., and Matsuda, H., 2002. New flavonol oligoglycosides and polyacylated sucroses with inhibitory effects on aldose reductase and platelet aggregation from the flowers of Prunus mume. J. Nat. Prod. 65, 1151−1155. Zhang, X., Lin, Z., Fang, J., Liu, M., Niu, Y., Chen, S., and Wang, H., 2015. An on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-total antioxidant capacity detection system applying two antioxidant methods for activity evaluation of the edible flowers from Prunus mume. J. Chromatogr. A. 1414, 88−102.
S0378-8741(19)32117-8
DOI:
https://doi.org/10.1016/j.jep.2020.112666
Reference:
JEP 112666
To appear in:
Journal of Ethnopharmacology
Received Date: 27 May 2019 Revised Date:
9 January 2020
Accepted Date: 9 February 2020
Please cite this article as: Vilhena, R.O., Figueiredo, I.D., Baviera, A.M., Silva, D.B., Marson, B.M., Oliveira, J.A., Peccinini, R.G., Borges, I.K., Pontarolo, R., Antidiabetic activity of Musa x paradisiaca extracts in streptozotocin-induced diabetic rats and chemical characterization by HPLC-DAD-MS, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2020.112666. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
1 1
Graphical abstract
2 3 4
Antidiabetic activity of Musa x paradisiaca extracts in streptozotocin-
5
induced diabetic rats and chemical characterization by HPLC-DAD-MS
6 7
Vilhena, R. O.1; Figueiredo, I. D.2; Baviera, A. M.2; Silva, D. B.3; Marson, B. M.1;
8
Oliveira, J. A2; Peccinini, R. G.4; Borges, I. K.5; Pontarolo, R.1*
9 10
1
Departamento de Farmácia, Universidade Federal do Paraná, Curitiba, PR, Brazil
11
2
Departamento de Análises Clínicas, Universidade Estadual Paulista Júlio de Mesquita
12
Filho, Araraquara, SP, Brazil
13
3
14
Farmacêuticas, Alimentos e Nutrição, Universidade Federal do Mato Grosso do Sul,
15
Campo Grande, MS, Brazil
16
4
17
Paulista Júlio de Mesquita Filho, Araraquara, SP, Brazil
18
5
19
PR, Brazil
20
Laboratório de Produtos Naturais e Espectrometria de Massas, Faculdade de Ciências
Departamento de Princípios Ativos Naturais e Toxicologia, Universidade Estadual
Departamento de Ciências Patológicas, Universidade Estadual de Londrina, Londrina,
2 21
*Corresponding author
22
Address: Department of Pharmacy, Federal University of Paraná, 632 Lothário
23
Meissner Avenue, 80210-170, Curitiba – PR, Brazil
24
E-mail: [email protected] (RP)
25 26
Declarations of interest: none
27 28 29
Authors e-mail:
30
Vilhena, R. O.: [email protected]
31
Figueiredo, I. D.: [email protected]
32
Baviera, A. M.: [email protected]
33
Silva, D. B.: [email protected]
34
Marson, B. M.: [email protected]
35
Oliveira, J. A: [email protected]
36
Peccinini, R. G.: [email protected]
37
Borges, I. K.: [email protected]
38
Pontarolo, R.: [email protected]
39 40 41 42 43 44 45 46
3 47
ABSTRACT
48
Ethnopharmacological relevance
49
The Musa x paradisiaca L. inflorescence, known as banana blossom or banana heart, is
50
used in traditional medicine for the treatment of diabetes mellitus.
51
Aim of the study
52
The aim of the study was to investigate the antidiabetic activity of aqueous extracts and
53
fractions prepared from the bracts and flowers of Musa x paradisiaca in streptozotocin
54
(STZ)-induced diabetic rats and to chemically characterize the extracts.
55
Materials and Methods
56
Standard aqueous extracts of the flowers, bracts, and their fractions were prepared and
57
their chemical composition was determined tentatively by high-performance liquid
58
chromatography coupled to diode-array detection and mass spectrometry (HPLC-DAD-
59
MS). Changes in fasting glycemia and oral glucose tolerance were evaluated in STZ-
60
induced diabetic rats (n = 8) treated with aqueous extracts of Musa x paradisiaca (200
61
mg/kg) for 20 days.
62
Results
63
Chemical analyses detected 21 compounds and 17 metabolites were identified, among
64
which were glycosylated and acetylated phenylpropanoids of p-coumaric acid and caffeic
65
acid, as well as a glycosylated flavonol and anthocyanins. Following 15 days of treatment,
66
the bract aqueous extracts and the methanolic fraction of the flower had significant
67
effects on the glycemic profile after glucose load in diabetic rats as compared with the
68
untreated diabetic group.
69
Conclusions
70
The results of the present study show the antidiabetic potential of extracts of the flowers
71
and bracts of M. x paradisiaca.
4 72 73
Keywords: banana, hydroxycinnamic acid derivatives, HPLC-DAD-MS, anthocyanins,
74
diabetes mellitus
75 76
Abbreviations
77
HPLC-DAD-MS, high-performance liquid chromatography coupled to diode-array
78
detection and mass spectrometry; AFE, aqueous flower extract; MFF, methanolic
79
flower fraction; ABE, aqueous bract extract; MBF, methanolic bract fraction; AqE,
80
aqueous extract; MeF, methanolic; N, normal group; D, untreated diabetic group;
81
DAFE, diabetic group treated with aqueous flower extract; DMFF, diabetic group
82
treated with methanolic flower fraction; DABE, diabetic group treated with aqueous
83
bract extract; DMBF, diabetic group treated with methanolic bract fraction; Dp-CA,
84
diabetic group treated with p-coumaric acid; DINS, diabetic group treated with insulin;
85
b.w., body weight.
86
5 87
1. Introduction
88
Musa x paradisiaca L., commonly known as banana, is among the most important fruit
89
crops in the world (Jawla et al., 2012). In 2015, global banana production reached a
90
record growth rate of approximately 118 million tons (FAO, 2018). As a result of this
91
large production, it is estimated that more than 100 million tons of waste are generated
92
annually, which is mainly leaves, pseudostems, and inflorescences (IBGE, 2013).
93
Although inflorescences are economically regarded as waste, the use of these parts in
94
traditional medicine is common practice in many cultures. For example, inflorescence is
95
used for the treatment of diabetes through consumption of cooked inflorescence or in
96
the form of a decoction (Kumar et al., 2012). Additionally, a recent review showed the
97
in vitro and in vivo antidiabetic potential of Musa ssp. flowers (Vilhena et al., 2015).
98
Other studies have shown that the flowers exhibit antihyperglycemic properties (Borah
99
and Das, 2017; Jawla et al., 2012; Nisha and Mini, 2013) and the capacity to inhibit
100
carbohydrate digestion (Abdurrazak et al., 2015; Arun et al., 2017; Marikkar et al.,
101
2016) and improve glucose uptake (Arun et al., 2017; Bhaskar et al., 2011). However,
102
no studies have investigated the in vivo antidiabetic activity of bracts.
103
In this context, studies contributing to the verification of the ethnopharmacological use
104
of the inflorescence of banana, including the bracts, are of extreme importance for the
105
development and strengthening of complementary therapies for the treatment of
106
diabetes mellitus. Therefore, the aim of the present study was to investigate the
107
antidiabetic effects of aqueous extracts of the bracts and flowers of Musa x paradisiaca
108
in streptozotocin (STZ)-induced diabetic rats followed by chemical characterization.
109 110
2. Materials and Methods
111
2.1. Chemicals and Reagents
6 112
Streptozotocin and p-coumaric acid standards (≥ 98%) were purchased from
113
Sigma−Aldrich (St. Louis, MO, USA). Insulin was obtained from Lilly (Humulin® NPH
114
100 UI/mL, Indianapolis, USA). Isoflurane was purchased from Cristália (Isofurine®,
115
Itapira, SP, Brazil). Amberlite XAD-2 was obtained from Merck KGaA (Darmstadt,
116
Germany). Acetonitrile and methanol (HPLC grade) were obtained from Tedia
117
(Fairfield, CA, USA). Formic acid (98−100%; LC-MS grade) was purchased from
118
Sigma−Aldrich (St. Louis, MO, USA). Hydrochloric acid (36−38.0%) was purchased
119
from Mallinckrodit (Edo. de Mexico, Mexico). Ultrapure water was obtained using a
120
Milli-®purification system from Millipore (Milford, MA, USA).
121 122
2.2. Plant material
123
Musa x paradisiaca inflorescence was collected after approximately 60 days of fruit
124
development in Morretes, PR, Brazil (geographical coordinates 25º29'45,59 "S and
125
48º48'40,25 "W, 10.93 m) and was identified by a botanist from the herbarium of
126
Museu Botânico Municipal de Curitiba (MBM – 348145). The inflorescence was
127
separated into flowers and bracts and dried in a drying cabinet for seven days at 30ºC.
128
Following drying, the flowers and bracts were ground into separate powders using a
129
cutting mill and were subsequently sieved (3 mm). The dried powder was stored in air-
130
tight containers until analysis.
131 132
2.3. Extraction procedure
133
The dried flower and bract powders were extracted twice with water (1:10, w/v) via an
134
ultrasound-assisted extraction process. The equipment was operated at a frequency of 40
135
kHz, a power of 100 W, a temperature of 25ºC, and a sonication time of 30 min. The
136
liquid was filtered, and the total volume was lyophilized at -80°C and 100 mTorr for 48
7 137
h (Virtis Advantage Plus, SP Scientific, Warminster, England). The obtained flower
138
(AFE) and bract (ABE) extracts were fractionated in amberlite resin and solubilized in
139
water (8 g/100 mL), followed by adjustment of the pH to 2.0 using HCl. The extracts
140
were mixed with 12 g amberlite XAD-2 resin (9 nm pore and 20−60 mesh particles) for
141
1 h using a magnetic stirrer and then packed into a glass column (45 x 3.5 cm).
142
Amberlite impregnated with crude extract was washed with 1 L water. The fraction
143
adsorbed on the column was eluted with 370 mL methanol and dried under reduced
144
pressure in a Centrivap Sample Concentrator (Labconco, Kansas City, USA) at 40°C.
145
The methanolic fractions of flower (MFF) and bract (MBF), in addition to AFE and
146
ABE, were stored in at -40°C in air-tight and light-protected containers until analysis.
147
The extraction yields obtained were 15,2% (ABE), 18,1% (AFE), 3,2 % (MBF), and
148
6,7% (MFF).
149 150
2.4. HPLC-DAD-MS analysis
151
Extracts and methanolic fractions (AFE, ABE, MFF, and MBF; 1 mg/mL) were
152
analyzed using a high-performance liquid chromatography (Prominence UFLC,
153
Shimadzu, Kyoto, Japan) coupled to a diode-array detector (190–400 nm) and a mass
154
spectrometer with an electrospray ionization source (ESI) and the quadrupole-time-of-
155
flight (QTOF) (MicrOTOF-Q III, Bruker Daltonics, Billerica, USA) analyzers operating
156
in negative and positive ion modes. Nitrogen was used as gas of nebulization (4 Bar),
157
dry (9.1 L/min at 200 ° C) and collision. The capillary voltage was set at 3,500 V, and
158
the scan range was m/z 100-1300. The analysis was performed on a C-18 column
159
(Kinetex, 150 mm × 2.1 mm id, 2.6 µm), with an oven temperature of 40°C. The mobile
160
phase was deionized water (A) and acetonitrile (B), both containing 0.1% formic acid
161
(v/v), under the following gradient profile: 0–2 min 3% B, 2–25 min 3–25% B, and 25–
8 162
40 min 25–80% B. The flow rate was 0.3 mL/min and the injection volume was 1 µL.
163
The extracts were prepared at 1 mg/mL using acetonitrile and water (6:4, v/v) and
164
filtered on a 0.22 µm × 3.0 mm PTFE membrane (Millex®, Millipore).
165 166
2.5. Antidiabetic activity of M. x paradisiaca extracts
167
2.5.1. Animals
168
Male Wistar rats (Rattus norvegicus) weighing 140–160 g (6 weeks old) were
169
maintained under environmentally controlled conditions, with a temperature of 23±1°C,
170
a humidity of 55 ± 5%, and a 12 h light/dark cycle. Rats received water and normal lab
171
chow diet (Presence, Paulínia, São Paulo, Brazil) ad libitum. The experiments were
172
conducted during the light phase following approval of the experimental protocol by the
173
Committee for Ethics in Animal Experimentation of the School of Pharmaceutical
174
Sciences, UNESP, Araraquara (protocol number CEUA/FCF/CAr: 31/2017).
175 176
2.5.2. Induction of the experimental diabetes mellitus model
177
Following four days of acclimation, experimental DM was induced by a single
178
intravenous injection of STZ (40 mg/kg b.w.) dissolved in 0.01 M citrate buffer (pH
179
4.5) in 12 h-fasted rats. Normal rats received only citrate buffer. All animals were
180
anesthetized with isoflurane for this procedure. Three days after STZ administration,
181
rats with post-prandial glycemia values ≥ 350 g/dL were used in the experiments
182
(Furman, 2015). Plasma glucose levels were determined by the glucose oxidase method
183
(Trinder, 1969) using a commercial kit (Biotécnica, Varginha, MG, Brazil).
184 185
2.5.3. Experimental design
9 186
Diabetic animals were stratified into seven different experimental groups (8 rats/group)
187
using matched glycemia and body weight values: diabetic rats treated with water (D); 4
188
UI insulin (DINS); 200 mg/kg b.w. AFE (DAFE); 200 mg/kg b.w. ABE (DABE); 200
189
mg/kg b.w. MFF (DMFF); 200 mg/kg b.w. MBF (DMBF); and 20 mg/kg b.w. p-
190
coumaric acid (Dp-CA). Additionally, a group of normal rats (n = 8) treated orally with
191
vehicle (water) were used as a control. The treatments, except for DINS, were
192
performed by gavage twice a day, with a half-dose (0.5 mL) at 08:00 h and 17:00 h, for
193
a total of 20 days. Extract and methanolic fraction doses were solubilized in water,
194
whereas p-coumaric acid was suspended in carboxymethyl cellulose (1%, w/v) due to
195
its poor water solubility. DINS rats received subcutaneous injections of insulin twice
196
daily (2 UI/rat per injection).
197 198
2.5.4. Oral glucose tolerance test (OGTT)
199
Following 15 days of treatment, an OGTT was performed in 12 h-fasted rats. A glucose
200
solution (2 g/kg b.w.) was administered orally, and blood samples were collected from
201
the tip of the tail before (t = 0) and 15, 30, 60, 90, and 120 min after glucose loading.
202
Results are expressed as mg/dL, and the area under the curve (AUC, g/dL/120 min) was
203
calculated (GraphPad Prism® 6.0 Software, GraphPad, La Jolla, USA).
204 205
2.5.6. Statistical analysis
206
Differences between groups were analyzed with one-way ANOVA followed by a
207
Student−Newman−Keuls test (p < 0.05) (GraphPad Prism® 6.0 Software, GraphPad, La
208
Jolla, USA).
209 210
3. Results and Discussion
10 211 212
3.1. Identification of the constituents by HPLC-DAD-MS
213
The constituents of M. x paradisiaca extracts and fractions were identified based on
214
UV, MS, and MS/MS data as compared with data described in the literature (Abdallah
215
et al., 1994; Abid et al., 2017; Shirota et al., 1997; Zhang et al., 2015). The
216
chromatograms of AFE, ABE, MFF, and MBF are shown in Fig. 1. Seventeen
217
compounds were identified from the extracts (Table 1).
218
219 220
Fig. 1. Total ion chromatograms (negative ion mode) of aqueous bract extract (A);
221
methanolic bract fraction (B); aqueous flower extract (C); and methanolic flower
222
fraction (D) of M. x paradisiaca. The fractions were obtained by clean-up procedures
223
using Amberlite XAD2. The identification of chromatographic peaks is described in
224
Table 1 and all the chromatograms are in the same intensity range.
11
225
Table 1. Compounds identified from the aqueous extracts and methanolic fractions of the bracts and flowers of M. x paradisiaca by
226
HPLC-DAD-MS
1 2
RT (min) 1.1 1.2
3
9.6
di-O-hexosyl coumaric acid
4
10.7
UI di-O-acetyl di-O-hexosyl coumaric acid O-hexosyl-deoxihexosyl quercetin UI
Peak
Compound Quinic acid Hexose
5
17.8
6
18.1
7
18.4
8
19.6
9
20.0
10
21.5
11
21.9
12
22.9
13
24.2
14
25.1
15
25.3
16
26.1
Nonanedioic acid O-deoxyhexosyl-hexosyl cyanidin tri-O-acetyl di-O-hexosyl coumaric acid tri-O-acetyl di-O-hexosyl coumaric acid tri-O-acetyl di-O-hexosyl coumaric acid tetra-O-acetyl di-O-hexosyl caffeic acid tetra-O-acetyl di-O-hexosyl coumaric acid O-deoxyhexosyl-hexosyl Oacetyl cyanidin UI
17
26.4
tetra-O-acetyl di-O-hexosyl
UV (nm) 299, 312 285 299, 313 260, 352 299, 325 274, 520 299, 313 299, 313 299, 313 299, 325 299, 313 265, 517 276, 313 299,
Bract
Flower
MF
Ident. levela
MS (+ / -) (m/z)
MS/MS (m/z)
C7H12O6 C6H12O6 C21H28O13
1 4 3
191.0568 (-) 179.0566 (-) 487.1488 (-)
163
C12H13NO3 C25H32O15
4 3
218.0832 (-) 571.1695 (-)
216, 188, 162 487, 341, 307, 163, 145
C27H30O16
3
609.1482 (-)
300, 271, 255, 179
X
C17H16O10
4
379.0697 (-)
185
X
C9H16O4 C27H31O15+
4 3
187.0989 (-) 595.1673 (+)
287
C27H34O16
3
613.1803 (-)
C27H34O16
3
C27H34O16
3
C29H36O18
3
C29H36O17
3
C29H33O16+
3
C31H34O17 C29H36O17
MeF
AqE
MeF
X
X X X
X
X
X X
X X
X X
X X
X X
383, 341, 307, 163, 145
X
X
X
613.1803 (-)
383, 341, 307, 163, 145
X
X
X
613.1794 (-)
341, 323, 163, 145
X
X
X
383, 179, 163
X
425, 383, 341, 163, 145
X
637.1770 (+)
329
X
X
4
679.1861 (+)
391, 373
X
X
3
655.1906 (-)
383, 341, 163, 145
X
671.1856 (-) 655.1872 (-)
AqE X X
X
X X
X X
X
X
X
12
coumaric acid 312 3 655.1911 (-) 383, 341, 323, 163, 145 X tetra-O-acetyl di-O-hexosyl 299, C29H36O17 coumaric acid 313 27.5 tetra-O-acetyl di-O-hexosyl O299, C30H38O18 3 685.2023 (-) 193, 175, 160 X X 19 methyl caffeic acid 325 30.0 penta-O-acetyl di-O-hexosyl 299, C31H38O18 3 697.2022 (-) 425, 383, 163, 145 X X 20 coumaric acid 313 30.9 UI C18H34O5 4 329.2351 (-) 229, 211, 183, 171 21 Note: +: positive ion mode; -: negative ion mode; UI: unidentified; MF: molecular formula: RT: retention time; UV: ultraviolet; AqE aqueous extract; MeF: methanolic; X: presence of identified compound. aIdentification level of compounds using Metabolomics Standards as reported by Schymanski et al., 2014. All MFs were determined from the accurate mass considering a mass error and mSigma lower than 8 ppm and 30, respectively. 18
227 228 229 230
26.9
13
231
Chromatographic peaks 3, 5, 10−12, 14, 17−18, and 20 revealed absorption bands in the
232
ultraviolet spectra, with wavelengths near 299 and 310 nm, which is compatible with
233
the coumaric acid moiety (Zhang et al., 2015). All these compounds revealed fragment
234
ions at m/z 163, which is in accordance with a neutral loss of coumaric acid molecule. It
235
is yielded from the loss of two hexoses (324 u - compound 3) or two hexoses together
236
with acetyl groups, such as two (408 u - compound 5), three (450 u - compounds
237
10−12), four (508 u-compounds 14, 17−18), or five (534 u-compound 20) acetyl groups.
238
Compound 5 exhibited an ion at m/z 571.1695 [M-H]-, indicating a molecular formula
239
of C25H32O15, and also showed a fragment ion at m/z 487 produced from the loss of two
240
acetyl groups (42 + 42 = 84 u), confirming the acetyl substituents. Thus, it was possible
241
to identify the compounds as di-O-hexosyl coumaric acid (3), di-O-acetyl di-O-hexosyl
242
coumaric acid (5), tri-O-acetyl di-O-hexosyl coumaric acid), tetra-O-acetyl di-O-
243
hexosyl coumaric acid (14, 17-18), and penta-O-acetyl di-O-hexosyl coumaric acid
244
(20). These spectral data are compatible with those described in the literature (Zhang et
245
al., 2015).
246
Peaks 13 and 19 exhibited two absorption bands at wavelengths of 299 and 325 nm in
247
the UV spectra, suggesting the presence of a caffeic acid unit. These compounds
248
revealed fragment ions at m/z 179 and 193, confirming the presence of caffeic and O-
249
methyl caffeic acids, respectively. From comparison with previously published data,
250
substances 13 and 19 were identified as tetra-O-acetyl di-O-hexosyl caffeic acid (13)
251
(Abdallah et al., 1994) and tetra-O-acetyl di-O-hexosyl O-methyl caffeic acid (19)
252
(Shirota et al., 1997).
253
A glycosylated flavonol, O-hexosyl-deoxyhexosyl quercetin (6), and quinic acid (1),
254
hexose (2), and nonanedioic acid (8) were also identified in the samples. In addition,
14
255
peaks 9 and 15 showed UV spectra typical of anthocyanins (λmax ≈ 270 and 515 nm) and
256
protonated ions (m/z 595.1673 and 637.1770), indicating a molecular formula of
257
C27H31O15+ and C29H33O16+, respectively. They showed losses of 308 u, suggesting O-
258
deoxyhexosyl-hexosyl substituents, and yielded fragment ions m/z 287 (C15H11O6+) and
259
329 (C17H13O7+), which confirmed the aglycones cyanidin and O-acetyl cyanidin (Abid
260
et al., 2017). Thus, compounds 9 and 15 were identified as O-deoxyhexosyl-hexosyl
261
cyanidin and O-deoxyhexosyl-hexosyl O-acetyl cyanidin.
262 263
3.1. Antidiabetic activity of M. x paradisiaca extracts
264
3.1.1. Glucose tolerance following treatment of diabetic rats with M. x paradisiaca
265
extracts
266
STZ administration destroys β-cells, leading to the development of metabolic
267
disturbances due to severe insulin deficiency in a profile similar to the changes observed
268
in type 1 diabetes mellitus (Lenzen, 2008). Therefore, an abnormality in the glycemic
269
profile of diabetic animals is expected. On day 15 of treatment, the OGTT was
270
performed to evaluate the effects of the aqueous extracts, methanolic fractions, and p-
271
coumaric acid on fasting glycemia and glucose tolerance in diabetic rats. The OGTT
272
results for the control and diabetic-treated groups are shown in Fig. 2.
15
273 274
Fig. 2. Oral glucose tolerance in normal and STZ-induced diabetic rats treated with the
275
extracts and fractions of M. x paradisiaca. (A) Glycemia levels before (t = 0) and after
276
glucose load in rats treated for 20 days; (B) area under the curve (AUC) of the oral
277
glucose tolerance test; and (C) fasting glycemia of the control and treated groups. The
278
values are expressed as the mean ± SD, n = 8 per group. Differences between groups
279
were analyzed with one-way ANOVA followed by a Student−Newman−Keuls test.
280
Asterisks denote the significant differences from the D group (*p < 0.05, **p < 0.01);
281
the pound signs denote the significant differences from the N group (#p < 0.05).
282
Note: N, normal group; D, untreated diabetic group; DAFE, diabetic group treated with aqueous flower
283
extract (200 mg/kg/day); DMFF, diabetic group treated with methanolic flower fraction (200 mg/kg/day);
284
DABE, diabetic group treated with aqueous bract extract (200 mg/kg/day); DMBF, diabetic group treated
285
with methanolic bract fraction (200 mg/kg/day); Dp-CA, diabetic group treated with p-coumaric acid (20
286
mg/kg/day); and DINS, diabetic group treated with insulin (4 UI/rat/day).
287 288
For the D group, increased values of fasting glycemia, in addition to glucose intolerance
289
after oral load, were observed, indicating impairments in the control of carbohydrate
16
290
metabolism in STZ-induced diabetic rats, likely due to an insulin-deficient state. On the
291
other hand, in comparison with the D group, all groups of diabetic rats treated with
292
extracts or fractions of M. x paradisiaca had decreases in fasting glycemia, not
293
significantly different from the DINS group (Fig. 2C).
294
Observing the post glucose load profiles (Fig. 2A), there was a reduction in glucose
295
intolerance in the groups of diabetic rats that received aqueous extracts of flower and
296
bract, methanolic flower fraction, and insulin (DAFE, DABE, DMFF, and DINS
297
groups). Moreover, the glycemia levels in the DABE and DMFF groups returned to
298
values close to those in the DINS group at the last evaluation time after oral glucose
299
overload (120 min).
300
Comparison of the AUCs of the different groups (Fig. 2B) shows that MFF, ABE, and
301
insulin treatment in diabetic rats promoted statistically significant alterations in the post
302
glucose load profile in relation to the D group, indicating that these treatments improved
303
the glucose tolerance in animals with DM. Considering that the effects in the DABE and
304
DMFF groups were not statistically different from each other, ABE presents an
305
advantage over MFF due to the ease of obtaining an aqueous extract. Although the
306
glycemic profile in the DAFE group was not significantly different as compared with
307
the D group, it is important to note that the profile up to 60 min following the glucose
308
load is very similar to that in the DINS, DMFF, and DABE groups, indicating the
309
potential of this extract.
310
Considering the relationship between the results of the antidiabetic properties of the M.
311
x paradisiaca preparations and the chemical composition of the bract extracts, it can be
312
inferred that the antidiabetic potential of this part of the inflorescence could be
313
attributed to a possible synergistic action of the total components of the extract, since
17
314
ABE promoted improvements in the post glucose load profile and fasting glycemia to a
315
greater extent than MBF. Although the procedure to obtain MBF was used to get rid of
316
the most of the sugars, some of the potent bioactive metabolites might also be eluted
317
with water from the amberlite XAD-2 column, which could explain the lower activity.
318
The effect of rutin, a flavonol glycoside, on glycemia levels has been evaluated in in
319
vitro and in vivo studies. The antihyperglycemic activity of rutin in animal models of
320
acute and chronic hyperglycemia induced by alloxan and improvements in the glycemic
321
profile of normoglycemic mice submitted to OGTT are examples of the antidiabetic
322
potential attributed to doses of 30 mg/kg of this compound (Calzada et al., 2017). In
323
addition, rutin, quercetin, and isoquercetin have shown in vitro inhibitory activity
324
against α-glycosidase (Jo et al., 2009; Sohretoglu et al., 2018). Therefore, the presence
325
of flavonol glycosides in the bract extracts may have contributed to the observed results.
326
Anthocyanins, compounds present in the bracts of Musa spp. (Kitdamrongsont, 2018),
327
appear to play a role in the prevention or reversal of pathogenic processes related to
328
type 2 DM by inhibiting body weight gain, preventing the production of free radicals
329
and lipid peroxidation, regulating the inflammatory response, reducing the glucose and
330
lipids levels in the blood, and improving insulin resistance (Guo and Ling, 2015). Thus,
331
these compounds may also be related to the effects obtained by treatment with bract
332
extracts.
333
Additionally, the presence of glycosylated and acetylated phenylpropanoid derivatives
334
could also be related to the antidiabetic activity of the extracts. Studies have shown that
335
acetylated derivates of p-coumaric acid glycosides have platelet antiaggregant activity
336
(Yoshikawa et al., 2002) and moderate aldose reductase inhibitory activity (Fujimoto et
337
al., 2014). For compounds belonging to this group, α-glycosidase inhibitory activity,
18
338
such as lapatoside D (Fan et al., 2010), and β-glycosidase inhibitory activity, such as
339
vanicoside A and B isolated from the root of Polygonum sachalinense were also
340
observed (Kawai et al., 2006). In these studies, in addition to the glycosylated and
341
acetylated compounds, p-coumaric and ferulic acids were also evaluated; however, they
342
did not show any activity. Thus, the authors suggested that the complete structure of the
343
compounds is related to the action, not just hydroxycinnamic acids. Additionally, poorly
344
bonded compounds, such as ester bonding, have been reported to be hydrolyzed in the
345
gut prior to absorption (Lafay et al., 2007). Thus, the present study also evaluated the
346
activity of p-coumaric acid alone for the purpose of comparing the phenylpropanoyl
347
portion of the compounds identified in the extracts. Similarly, p-coumaric acid
348
administered alone showed no activity in diabetic animals in the present study (Fig. 2).
349 350
4. Conclusion
351
The results of the present study show the antidiabetic potential of the flower and bract
352
extracts and fractions of M. x paradisiaca. To the best of our knowledge, this is the first
353
time that the antidiabetic effect of bracts has been evaluated in STZ-induced diabetic
354
rats. All diabetic rats treated with the extracts and fractions of M. x paradisiaca had
355
decreased fasting glycemia as compared with the untreated diabetic group. ABE
356
treatment was highlighted as presenting improvements in the post glucose load profile
357
in diabetic rats as compared with the untreated group. These findings provide novel
358
evidence for traditional antidiabetic use of M. x paradisiaca inflorescence and potential
359
for the application of these extracts in the development of novel antidiabetic medicines.
360 361
Conflicts of interest
19
362
The authors have no conflicts of interest.
363 364
Acknowledgments
365
The authors acknowledge the Brazilian agencies CAPES and CNPq for fellowships and
366
the biochemistry and clinical enzymology laboratory of UNESP-Araraquara for help
367
with in vivo experiments. The authors would also like to thank FUNDECT (Fundação
368
de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do estado de Mato
369
Grosso do Sul).
370 371
Author’s contributions
372
ROV contributed to the collection and identification of plant samples, running of the
373
herbarium, performing laboratory work, analysis of the data, and drafting of the paper.
374
IDF contributed to the design of in vivo experiments, performing laboratory work, and
375
analysis of the data. AMB designed the in vivo experiments, analyzed the data, critically
376
read the manuscript, and contributed to reagents/materials. DBS performed the LC-
377
DAD-MS analyses and identified the potential bioactive compounds, contributed to
378
reagents/materials, and critically read the manuscript. BMM contributed to the
379
collection and identification of plant samples, running of the herbarium, performing
380
laboratory work, and analysis of the data. JO contributed to in vivo experiments,
381
performing laboratory work, and analysis of the data. RGP contributed to the laboratory
382
support
383
reagents/materials. IKB contributed to the critical reading of the manuscript. RP
384
designed the study, supervised the laboratory work, contributed to reagents/materials,
of
in
vivo
experiments,
critical
reading
of
the
manuscript,
and
20
385
and critically read the manuscript. All authors read the final manuscript and approved
386
the submission.
387 388
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