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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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

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