Qualitative and Quantitative Analysis of Ethanolic Extract and Phenolic

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Qualitative and quantitative analysis of ethanolic extract and phenolic fraction of Jatropha aethiopica (Euphorbiaceae) leaves and their hypoglycemic potential. Daylin Gamiotea-Turro, Nathalia Aparecida de Paula Camaforte, Alexander Barbaro Valerino-Diaz, Yarelis Ortiz Nunez, Daniel Rinaldo, Anne Ligia Dokkedal, José Roberto Bosqueiro, and Lourdes Campaner dos Santos J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05648 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Journal of Agricultural and Food Chemistry

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Qualitative and quantitative analysis of ethanolic extract and

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phenolic fraction of Jatropha aethiopica (Euphorbiaceae) leaves and

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their hypoglycemic potential.

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Daylin Gamiotea-Turro†,‡, Nathalia A.P. Camaforte§, Alexander B. Valerino-Diaz†, Yarelis Ortiz

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Nuñez‡, Daniel Rinaldo£, Anne L. Dokkedal§, José R. Bosqueiro§, and Lourdes Campaner dos

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Santos† *

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†

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Degni, 55 Bairro: Quitandinha, 14800-060 - Araraquara, SP, Brazil.

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‡

UNESP -São Paulo State University, Chemistry Institute – Araraquara. Rua Prof. Francisco

Institute of Fundamental Research in Tropical Agriculture “Alejandro de Humboldt” (INIFAT).

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Calles 1 y 2, No. 17200, Santiago de las Vegas, C.P. 17200, Havana, Cuba.

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§

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Bauru. Av. Eng. Luiz Edmundo C. Coube 14-01, Bairro: Núcleo Habitacional Presidente Geisel,

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CEP 17033-360, Bauru, SP, Brazil.

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£

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C. Coube 14-01, Bairro: Núcleo Habitacional Presidente Geisel, CEP 17033-360, Bauru, SP,

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Brazil.

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*Corresponding author, Tel: +55 16 3301-9657; Fax: +55 16 3322-2308. E-mail:

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[email protected]

UNESP - São Paulo State University, Department of Biological Sciences, Faculty of Sciences –

UNESP - São Paulo State University, Department of Chemistry –Bauru. Av. Eng. Luiz Edmundo

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ACS Paragon Plus Environment

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ABSTRACT

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While Jatropha aethiopica, popularly known in Cuba as “mata diabetes”, is used in salads and as

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a dietary supplement, its chemical composition and antidiabetic properties yet remains unclear. In

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this work, we evaluate the qualitative and quantitative composition of ethanolic extract (EE)

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and phenolic fraction (PF) of Jatropha aethiopica leaves and their hypoglycemic and

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hypolipidemic activity. Chemical fractionation of the ethanolic extract yielded nine

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compounds, which included protocatechuic acid (1), chlorogenic acid (2), caffeic acid (3),

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quercetin

3-O--L-rhamnopyranosyl-(12)-[-L-rhamnopyranolsyl-(16)]-β-D-

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galactopyranoside

(4),

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rhamnopyranolsyl-(16)]-β-D-galactopyranoside

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rhamnopyranosyl-(12)-[-L-rhamnopyranolsyl-(16)]-β-D-glucopyranoside

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(7), kaempferol 3-O--L-rhamnopyranosyl-(16)-β-D-glucopyranoside (8) and quercetin

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(9). The compounds (1, 4-7) were quantified by high performance liquid chromatography

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photodiode array detection (HPLC-PDA) in both the ethanolic extract (62.65  0.15 mg/g)

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and phenolic fraction (61.72  0.23 mg/g). The results obtained show that both ethanolic

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extract and phenolic fraction contributed towards the improvement of glucose tolerance,

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which in turn led to a decline in the glucose levels. Remarkably, the ethanolic extract

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presented a relatively higher promising effect compared to metformin.

a

new

kaempferol

3-O--L-rhamnopyranosyl-(14)-[-L(5),

kaempferol

3-O--L(6),

rutin

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Keywords: Jatropha aethiopica; medicinal plant; hypoglycemic; hypolipidemic; flavonol

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glycosides.

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INTRODUCTION

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Euphorbiaceae, the spurge family, is a large family of flowering plants with 300 genera

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and around 7.500 species. A number of plants of the spurge family are of considerable

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economic importance.1

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In medicine, some Euphorbiaceae species have proven to be effective as anti-diabetic

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and/or hypoglycemic agents.24 Among the Euphorbiaceae family, the Jatropha genus is

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known to be used in traditional medicine for the treatment of diabetes mellitus (DM).57

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Jatropha aethiopica Mül-Arg (Euphorbiaceae) was introduced in Cuba from an unspecified

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origin and at an unknown date.8 The tree has many branches and produces a milky sap. In

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spite of the abundant flowering, which characterizes the species, very few fruits are borne

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by the tree.8

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Jatropha aethiopica leaves are used in Cuba both as medicinal plant and as food in

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salads. In several Cuban towns, this specie is known as “mata diabetes”.9 People ensure that

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consumption of the tea made from fresh leaves of J. aethiopica is capable not only

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controlling diabetes but also curing it.9

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Diabetes mellitus (DM) is a metabolic disease characterized by chronic

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hyperglycemia caused by defects in insulin action and/or secretion which affects protein, fat

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and carbohydrate metabolism.10 Type 1 is characterized by the absolute deficiency of insulin

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production and represents 5-10% of all diabetes cases; it results from an autoimmune

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destruction of pancreatic β cells. In type 2, which represents 90-95% of diabetes cases,

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insulin resistance and progressive β cell failure (decrease of β cell mass, glucose sensitivity

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and secretory capacity) are characteristics features and several drugs to increase insulin

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sensitivity are used in clinic.11 Polyphagia, polydipsia, polyuria and weight loss are the major


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characteristics of DM installation. Diabetes mellitus regarded by the World Health

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Organization (WHO) as one of the four main non-communicable diseases (NCDs).12 It has

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been estimated that 52% of premature deaths are due to NCDs.

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Diabetes increases 2-3 folds the risk of heart attacks and strokes.13 Its treatment options

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include the sole application of exogenous insulin or combining it with allopathic drugs such as

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biguanides (metformin), sulfonylureas (glibenclamide) and alpha-glucosidase inhibitors

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(acarbose and miglitol), which act by decreasing fasting blood glucose through many pathways.

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However, the prolonged use of these drugs is likely to produce adverse side effects and may also

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lead to a decline in their efficacy. 13

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While J. aethiopica leaves are used as a source of nourishment and in folk medicine,

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no empirical evidence has, to date, proven their hypoglycemic properties9. Furthermore, the

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chemical composition of J. aethiopica has not yet been fully defined. The aim of this study

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was to investigate the hypoglycemic effects of J. aethiopica leaves and quantify the main

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metabolites of ethanolic extract and phenolic fraction of these leaves in a model of

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streptozotocin-induced diabetes.

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MATERIALS AND METHODS

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Chemicals. Methanol and trifluoroacetic acid (TFA), HPLC grade, were purchased

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from Tedia Company (Fairfield, OH, USA). The water used in the experiments was purified

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using a Milli-Q system (Millipore, Billerica, MA, USA). All solutions prepared for HPLC

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were filtered through a 0.22-µm GHP filter (Waters, Milford, MA, USA) before use.

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Plant material.

J. aethiopica Müll-Arg. leaves were obtained from some adult

specimens in Havana, Cuba from August to September 2014. The specimens were


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authenticated by Victor Fuentes-Fiallo (PhD), full researcher from the “Dr. Juan T. Roig”

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Experimental Station of Medicinal Plants in Cuba. A voucher specimen (1165) was deposited

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at the Herbarium of the Institute of Fundamental Research in Tropical Agriculture (INIFAT)

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in Havana, Cuba.

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General Apparatus. The analytical HPLC system used was a JASCO HPLC (Jasco,

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Tokyo, Japan), equipped with a PU-2089S Plus pump, an MD-2018 Plus Photodiode Array

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Detector (PDA), an AS-2055 Plus auto sampler, and a column oven (CO-2065 plus). The

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ChromNav (Workstation JASCO-ChromNav v.1.18.03) software was used for controlling the

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analytical system and for carrying out the data collection and processing as well as quantifying

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the isolated compounds. For compounds isolation, a preparative HPLC JASCO equipped with

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a PU-2086 Plus pump, an MD-2010 Plus Photodiode Array Detector (PDA) and manual

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injection were used.

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The MPLC system employed was that of a Buchi®, equipped with a C-615 pump.

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1D and 2D NMR spectra were recorded on a Bruker Advance III HD 600

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spectrometer (14.1 Tesla) using an inverse detection 5-mm (1H, 13C, 15N) cryoprobe and a z

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gradient, as well as automated tuning and matching (ATM) in (CD3)2SO-d6 (99.95%, Sigma-

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Aldrich) as solvent purchased from Sigma-Aldrich TM, chemical shifts were referenced to

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tetramethylsilane (TMS).

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HRESIMS data were detected in the negative ion mode on a Bruker Maxis Impact mass spectrometer with ESI-QqTOF-MS configuration.

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Preparation of the extract. J. aethiopica leaves were first shade dried and were then

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placed in an oven set at 400C. They were subsequently ground and stored at room temperature.


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The plant extract was prepared by percolation from J. aethiopica leaves (800.0 g) at room

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temperature using ethanol. The solvent was evaporated to dryness under low pressure,

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yielding 80.4 g of ethanolic extract crude (EE) (10%). The ethanolic extract (40.0 g) was

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redissolved in water/ethanol (1:1, v/v) while a liquid-liquid partition was carried out with n-

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hexane, ethyl acetate and n-butanol (thrice with each solvent, respectively). The yield from

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the extraction were n-hexane (2.4 g, 6 %,) ethyl acetate (10.0 g, 25%), butanolic (8.0 g, 20%)

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and aqueous (12.0 g, 30%).

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Qualitative and quantitative determination of polyphenols in the ethanolic extract

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and phenolic fraction from J. aethiopica leaves. The ethyl acetate fraction (3.0 g) obtained

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was subjected to size exclusion chromatography using a Sephadex LH-20 column (85 x 2.5

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cm; H x d.i.) with peristaltic pump and automatic collector, using methanol as eluent. One

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hundred and eighty-six eluents (5.0 mL each) were taken and combined into eighteen major

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fractions (F1 – F18) based on thin layer chromatography (TLC) evaluation. Fraction F17 yielded

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a pure compound 9 (6.0 mg Rt = 20.07 min). Fraction F9 (190.0 mg) was fractionated by

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HPLC-PDA preparative liquid chromatography using a Hypersil Gold (Thermo) (250 x 30

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mm, 5 m) reversed-phase column protected by a Hypersil Gold Thermo guard column

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(Thermo) (25 x 3 mm, 5 m) aiming at isolating the compounds. The elution system used for

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the HPLC-PDA assay was a binary gradient elution system with solvent A (0.1% TFA in

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H2O) and solvent B (0.1% TFA in methanol) eluted at an initial linear gradient of 5:13 % (B)

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in 10 min, which was changed to 13:65% (B) in 20 min under flow rate of 13 mL min-1. The

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sample injection volume was 400 L. The signal was monitored at 254 nm. Five compounds

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were isolated of the Fraction F9, which included the following: 1 (5.0 mg, Rt = 9.50 min.), 2


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(6.0 mg, Rt = 12.34 min.), 3 (4.0 mg, Rt = 13.00 min.), 7 (59.0 mg, Rt = 16.58 min.) and 8 (59

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mg, Rt = 18.60 min.).

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The ethanolic extract (EE, 1.5 g) of the J. aethiopica leaves was dissolved in 5.0 mL of

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MeOH and 4.5 g of C18 were added with subsequent rotaevaporation of the solvent (repeated 13

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times). The semi-preparative fractionation of the EE was performed with the aid of a medium-

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pressure liquid chromatography (MPLC) system, equipped with a reverse phase column C18 (150

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x 40 mm, 5µm). The pellet with extract (EE) was placed in the matched column with reverse phase.

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The mobile phase used consisted of water (eluent A) and methanol (eluent B), in step gradient

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mode of increasing polarity 13 to 100% B with a flow of 7.0 mL. min.-1, yielding 10.0 g of

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phenolic fraction (PF).

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The Phenolic fraction (PF) (160.0 mg) was purified by HPLC-PDA using gradient of

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5:13% methanol in 10 min. followed by the application of gradient of 13:65% methanol in 20

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min. The flow rate applied was 13 mL min-1. The PF sample injection volume was 400 L

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and the signal was monitored at 254 nm, yielding compounds 4 (10.0 mg, Rt = 14.70 min.)

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and 5 + 6 (6.0 mg, Rt = 15.30 min.).

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The isolated compounds (1, 4, 5+6 and 7) were quantified by HPLC-PDA using an external

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calibration standard.14,15

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The curves were constructed using protocatechuic acid (>97 % purity), quercetin (95% purity)

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and kaempferol (90% purity) standards (Sigma). A stock solution of 1000 𝜇g/mL of the

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standards was prepared, and serial dilutions of 250.0 – 3.90 and 500.0 - 7.8 𝜇g/mL were made,

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respectively. Each concentration level was analyzed in triplicate and measurements were

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performed at 254 nm. The mean areas of the chromatographic peaks obtained were

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interpolated as a function of concentration using linear regression and were used to generate


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the calibration curves. The correlation coefficient (𝑟2), linear coefficients (a) and angle (b)

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were obtained from the calibration curves.

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The accuracy of the HPLC method was estimated from the isolated rutin (7) recovery tests.

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The recovery tests were performed by adding known concentrations (low, medium, and high)

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of the isolated rutin (7) (15, 60, and 250 𝜇g/mL). The intra and interday repeatability were

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carried out so as to determine the accuracy of the developed method (in sextuplicate).

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Precision was expressed as a relative standard deviation (RSD) of the results.

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Animals. Male Swiss mice (aged 60 days, weighing 40.0 g) were obtained from

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Central Animal House in Botucatu (SP, Brazil) of Universidade Estadual Paulista “Julio de

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Mesquita Filho” (UNESP). The animals were kept under standard environmental conditions:

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22±2ºC, 12/12h dark/light cycle. They were fed with industrialized food (Labina®, Purina,

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Brazil) and water ad libitum. The local Ethics Committee (CEP-FC) approved the procedures,

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wich followed all the recommendations for ethical usage of animals stated by the Brazilian

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College of Animal Experimentation -COBEA (www.cobea.org.br).

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Induction of experimental diabetes. The diabetes induction was performed using a

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single injection of 150 mg/kg b.w. of streptozotocin (STZ, Sigma-Aldrich®, St. Louis, MO,

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USA) in mice, which had been subjected to fasting for 12-14 h. The STZ was dissolved in

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citrate buffer (pH 4.5) and immediately injected intraperitoneally in the mice. The animals

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were subjected to fasting for 3 h after induction where they received a glucose solution (10%)

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for the subsequent 24h to protect them against hypoglycemia. On the 7th day after the STZ-

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injection, the animals with glycaemia above 250 mg/dL were included in the study.16

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Treatment with J. aethiopica crude extract and glycaemia measurement. The

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animals were randomly divided into five groups (n=8/group), which comprised the following:


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CTLSAL – non-diabetic mice treated with saline; CTLEXT – non-diabetic mice treated with

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J. aethiopica extract at 500 mg/kg b.w.; STZSAL – diabetic mice treated with saline;

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STZMET- diabetic mice treated with metformin at 300 mg/kg b.w.; STZEXT – diabetic mice

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treated with J. aethiopica extract at 500 mg/kg b.w. Saline, extract and metformin were

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administered orally by gavage once a day for 14 consecutive days. Fasting glycaemia was

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measured weekly using a glucometer (One touch, Johnson & Johnson).

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Oral glucose tolerance test (oGTT) following J. aethiopica treatment. All the groups

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of mice were subjected to fasting for 8-10h. Their glycaemia level was measured prior to the

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beginning of the fasting (time zero). Afterwards, the animals received an oral load of D-

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glucose (2.0 g/kg b.w.). Their blood glucose was measured at 15, 30, 60, 90 and 120 min after

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glucose administration. Blood samples were obtained from the tail tip under anesthesia

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(Tiopental® 60 mg/kg b.w.) and glucose levels were measured using an enzymatic kit

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(Dolles®, Goiás, Brazil).

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Glucose tolerance test (oGTT) for dose determination of the phenolic fraction.

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The animals were divided into eight groups (n=10/group): CTLSAL: non-diabetic mice

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treated with saline; STZSAL: diabetic mice treated with saline; PF50: diabetic mice treated

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with 50 mg/kg of the phenolic fraction; PF100: diabetic mice treated with 100 mg/kg of the

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phenolic fraction; and PF200: diabetic mice treated with 200 mg/kg of the phenolic fraction.

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All the groups fasted for 8-10h and received their respective treatment by gavage 30 min prior

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to the measurement of the glycaemia at time zero. Afterwards, the animals received an oral

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load of D-glucose (2.0 g/kg b.w.). Their blood glucose was measured at 30, 60 and 90 min

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following glucose administration. Blood samples were obtained from the tail tip of the animals


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under anesthesia (Tiopental® 60 mg/kg b.w.) and their glucose levels were measured using

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an enzymatic kit (Dolles®, Goiás, Brazil).

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Treatment with phenolic fraction and glycaemia measurement. The animals were

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randomly divided into five groups (n=8/group), which comprised the following: CTLSAL –

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non-diabetic mice treated with saline; STZSAL – diabetic mice treated with saline; STZMET-

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diabetic mice treated with metformin at 300 mg/kg b.w.; PF200 – diabetic mice treated with

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phenolic fraction at a dose of 200 mg/kg b.w. Saline fractions and metformin were

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administered orally by gavage once a day for seven consecutive days. Fasting glycaemia was

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measured weekly using a glucometer (One touch, Johnson & Johnson).

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Oral glucose tolerance test (oGTT) following treatment with the fraction. All the

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groups were subjected to fasting for 8-10h. Their glycaemia level was measured prior to the

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commencement of the fasting (at time zero). Thereafter, the animals received an oral load of

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D-glucose (2.0 g/kg b.w.). Their blood glucose was measured at 15, 30, 60 and 90 min after

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glucose administration. Blood samples were obtained from the tail tip of the animals under

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anesthesia (Tiopental® 60 mg/kg b.w.) and their glucose levels were measured using an

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enzymatic kit (Dolles®, Goiás, Brazil).

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Biochemical parameters. At the end of the treatment with crude extract or phenolic

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fraction, the animals were subjected to fasting for 8-10h. Their blood samples were collected

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and centrifuged at 1500 rpm for 10 min in order to obtain serum, which was stored at -80ºC.

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Urea, total proteins, total cholesterol (TC), HDL-cholesterol and triglycerides (TG) were

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measured by spectrophotometry with the aid of an A15 equipment from Bioclin® using

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Biosystems kits. The VLDL-cholesterol was measured according to the formula: VLDL =

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TG/5.


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Statistical Analysis. The results were expressed as means ± standard error of the means

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(S.E.M.). Statistical analysis was performed using Instat 3® software. To perform multiple

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comparisons, ANOVA was employed, which was then followed by Tukey’s post test. For

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comparison between two groups, Student’s t test was used. The significance level adopted

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was p

Qualitative and Quantitative Analysis of Ethanolic Extract and Phenolic

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