O C p S N V a b B c d e a A R A A K A A D P P I c s f c “ a m t 0 ( Revista Brasileira de Farmacognosia 29 (2019) 191–197 ww w.elsev ier .com/ locate /b jp riginal Article hemical composition and biological properties of Ipomoea rocumbens aadia Batiga a, Marilia Valli b, Maria L. Zeraik c, Karina Fraige b, Gabriel M. Leme b, ayla S. Pitangui d, Ana Marisa F. Almeida d, Sylvie Michel a, Maria Claudia M. Young e, anderlan S. Bolzani b,∗ Laboratory of Pharmacognosy, University of Paris Descartes UMR CNRS (Centre national de la recherche scientifique), Faculty of Pharmacy, Paris, France Departamento de Química Orgânica, Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais, Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, razil Departamento de Química, Universidade Estadual de Londrina, Londrina, PR, Brazil Departamento de Análises Clínicas, Laboratório de Micologia Clínica, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara, SP, Brazil Instituto de Botânica, Secretaria do Meio Ambiente do Estado de São Paulo, Água Funda, São Paulo, SP, Brazil r t i c l e i n f o rticle history: eceived 20 April 2018 ccepted 17 August 2018 vailable online 14 November 2018 eywords: ntioxidant ntifungal ereplication henolics eroxyl radical a b s t r a c t Natural products have been the most valuable source of chemical compounds in the discovery of novel medicines. Secondary metabolites from terrestrial and marine organisms have found considerable use in the treatment of numerous diseases and have been considered lead molecules both in their natural form and as templates for medicinal chemistry. Brazil has an exceptionally rich biodiversity, and a valuable source of secondary metabolites that can be useful for the development of bioproducts. Ipomoea species, Convolvulaceae, are mostly found in tropical and sub-tropical regions, including South America and many are used for nutritional and medicinal purposes. Ipomoea procumbens Mart. & Choisy is endemic from South America, and this is the first study reported on the chemical composition and biological activities of this species. The present work reports the tentatively identification of natural products present in the extracts using a high performance liquid chromatography–high resolution mass spectrometry method. Additionally, the antioxidant and antifungal biological activities of the leaves, roots and steams extracts and fractions of this species were evaluated. While for the antioxidant activity the hydromethanol frac- tions (leaves, stem and roots) were more active, the methanol fractions of leaves and stem provided better results for the antifungal assay. © 2018 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Farmacognosia. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ ntroduction The Ipomoea genus belongs to the Convolvulaceae family, which ontains between 500 and 600 species mostly found in tropical and ub-tropical regions, like South America. This genus has been used or nutritional, medicinal, ritual and agricultural purposes. Con- erning the nutritional purpose, it is relevant to refer to Ipomoea as batata” also known as sweet potato, which is commonly cultivated nd consumed worldwide (Austin and Huamán, 1996). Some Ipomoea species are used to treat diseases, and the ost common use is as a purgative to treat constipation, using he roots (Pereda-Miranda and Bah, 2003). Additionally, they ∗ Corresponding author. E-mail: bolzaniv@iq.unesp.br (V.S. Bolzani). https://doi.org/10.1016/j.bjp.2018.08.010 102-695X/© 2018 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira http://creativecommons.org/licenses/by-nc-nd/4.0/). 4.0/). also present therapeutic effects or biological activities such as antimicrobial, analgesic, spasmolitic, spasmogenic, hypotensive, psychotomimetic and anticancer (Meira et al., 2012). The bioac- tive compounds found in the plants of this genus are ergoline alkaloids, indolizidine alkaloids, nortropane alkaloids, phenolic compounds, coumarins, norisoprenoids, diterpenes, isocoumarins, benzenoids flavonoids, anthocyanins, glycolipids, lignans and triterpenes (Meira et al., 2012). Several studies have shown that flavonoids and phenolic compounds contribute significantly to the antioxidant and anti-inflammatory activities of many plants used in traditional medicine (Zeraik et al., 2011; Fraige et al., 2017). The interest in polyphenols, particularly flavonoids, is due to their great abundance in our diet and their role in the prevention of various diseases associated with oxidative stress (Arwa et al., 2015). An interesting class of compounds named glycolipids, such as batatins, batatinosides and orizabins can be found in several species de Farmacognosia. This is an open access article under the CC BY-NC-ND license https://doi.org/10.1016/j.bjp.2018.08.010 www.elsevier.com/locate/bjp http://crossmark.crossref.org/dialog/?doi=10.1016/j.bjp.2018.08.010&domain=pdf https://orcid.org/0000-0001-8760-9676 https://orcid.org/0000-0003-1106-183X https://orcid.org/0000-0002-0559-8502 https://orcid.org/0000-0002-8298-6568 https://orcid.org/0000-0002-7082-4553 https://orcid.org/0000-0003-3248-5180 https://orcid.org/0000-0002-2115-8988 https://orcid.org/0000-0002-3993-4041 https://orcid.org/0000-0002-2860-6659 https://orcid.org/0000-0001-7019-5825 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ mailto:bolzaniv@iq.unesp.br https://doi.org/10.1016/j.bjp.2018.08.010 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ 1 de Far o R h a B w l l C w g t a w w a N ( d a s 2 f e i p a a fl a l a t M a a s c t h a t t b t c e a M P s r D v i T p 92 S. Batiga et al. / Revista Brasileira f Ipomoea from Mexico (Hernandez-Carlos et al., 1999; Rosas- amírez and Pereda-Miranda, 2013). Ipomoea batatas is reported to ave chlorogenic acids (Clifford et al., 2003), mono-caffeoylquinic cids and dicaffeoylquinic acids namely isochlorogenic acids A, and C (Ishiguro et al., 2007). Isochlorogenic acids A, B and C ere also isolated from I. pes-caprae (Teramachi et al., 2005). The eaves, stem and roots of I. batatas cultivated in China were ana- yzed by LC–MS in the search for chlorogenic acids (Zheng and lifford, 2008). These compounds were not detected in the root, hereas caffeoylquinic acids were the main subgroup of chloro- enic acids detected in the stem and the only subgroup detected in he leaves. Three feruloylquinic acids, 3,5- and 4,5-dicaffeoylquinic cid, and small amounts of four caffeoyl-feruloylquinic acids ere also detected in the stem. Five caffeoylquinic acids ere isolated from I. batatas using AB-8 macroresin absorption nd semipreparative HPLC–DAD and identified by ESI/MS and MR as 5-caffeoylquinic acid, 6-O-caffeoyl-�-d-fructofuranosyl- 2-1)-�-d-glucopyranoside, trans-4,5-dicaffeoylquinic acid, 3,5- icaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. The scavenging ctivity of the compounds was evaluated by DPPH• assay and howed IC50 values ranging from 7.6 to 12.4 �g mL−1 (Zhao et al., 014). Rehman et al. (2011) evaluated the antioxidant activity of dif- erent extracts of I. hederacea by four methods and reported that an thyl acetate fraction showed an IC50 of 60.28 �g mL−1 of DPPH• nhibition, as well as the highest content of total phenolic com- ounds. The aqueous extract of leaves of I. fistulosa exhibited high ntioxidant activity (IC50 = 59.94 �g mL−1) in DPPH assay, that was ssociated with the presence of higher amount of phenolic and avonoid compounds in the extract (Phulera et al., 2014). From Ipomoea asarifolia were isolated acylated anthocyanins nd ergoline alkaloids (chanoclavine I, ergine, ergobalansinine and ysergic acid �-hydroxyethylamide) (Jenett-Siems et al., 1994). I. sarifolia is toxic to goats, sheep and cattle and causes depression, remors of the head and hypermetria (Carvalho et al., 2014). Ipomoea procumbens species was described by the authors Von artius and Choisy, (Muséum National d’Histoire Naturelle, Paris), nd it is found in South America (Flora do Brasil, 2017). Despite wide research on scientific resources, no additional information uch as chemical studies or biological activities about this plant ould be found further than the taxonomy studies. The reported herapeutic effects are achieved by the use of the plant extract or erbal preparations. Therefore, the identification of the biologically ctive compounds using modern analytical techniques without ime consuming isolation is an excellent strategy to understand he plant biological properties and detect compounds that could e further investigated for medicine properties. The main objec- ive of the present work was to tentatively identify the chemical ompounds present in the leaves, roots and stems of I. procumbens xtracts by HPLC–HRMS method and to evaluate the antioxidant nd antifungal of the extracts fractions. aterials and methods lant material Ipomoea procumbens Mart. & Choisy, Convolvulaceae, leaves, tem and roots were collected at Itirapina Ecological Station, Iti- apina, SP, Brazil on March 2002. The species was identified by r. Inês Cordeiro from the Institute of Botany of São Paulo and a oucher specimen (MRSilva 402) was deposited in the State Herbar- um ‘Maria Eneyda P. Kaufmann Fidalgo (SP)’, São Paulo, Brazil. he plant material was dried at 40 ◦C in a circulating air oven and ulverized by a mechanical grinder. macognosia 29 (2019) 191–197 Extraction and solid phase extraction (SPE) microfractionation Analysis of the extracts The dried, powdered leaves (5.4 mg), stem (5.5 mg), and roots (5.2 mg) of I. procumbens were extracted by sonication with 1 mL MeOH/H2O (9:1), for 30 min, at room temperature. The extracts were subjected to a clean-up procedure in SPE C18 car- tridges (Chromabond C18 ec, 500 mg, 3 mL cartridge) initially activated with 5 mL MeOH and then conditioned with 5 mL MeOH/water (9:1). The cartridge was loaded with 1 mL sample extract (5 mg mL−1) that was eluted with 3 mL MeOH/water (9:1). The solvents were removed by evaporation under reduced pressure at 40 ◦C using a Buchi Rotavapor ® R-114 to yield dry extracts. These extracts were resolubilized, filtered and analyzed by HPLC–UV/PDA (SPD-M20A model, Shimadzu Lab Solutions, Kyoto, Japan) and eval- uated for antioxidant and antifungal activity. The water user was purified by a Milli-Q system (Millipore, Bedford, MA, USA). Microfractionation The microfractionation of the extracts of I. procumbens (50 mg each, in 1 mL H2O/MeOH 8:2,) was performed with SPE C18 car- tridges activated as previously described and conditioned with the initial solution (H2O/MeOH 8:2, v/v), then the elution was per- formed using a mixture of H2O/MeOH, 5 mL, in the following ratios: 8:2, 1:1, 0:1. The separation yielded 3 fractions of each different part of the plant, a total of 9 fractions (SPE1–9). These samples were analyzed by HPLC–UV/PDA under the same conditions used for the extract analysis and the dereplication using HPLC–HRMS was per- formed as described further. Each fraction was evaluated for the antioxidant and antifungal assay. HPLC–UV/PDA and HPLC–HRMS analysis Chromatographic analyses were carried out on a LC-10AD liquid chromatography system equipped with a binary pump, autosam- pler a photodiode array detector SPD-M20A model (Shimadzu Lab Solutions, Kyoto, Japan). The samples were filtered with a NORM- JECT ® syringe and a Simplepure ® NY (0.22 �m) filter, and then injected automatically (20 �L) with a flow rate of 1 mL min−1. The separation was performed using a Phenomenex Luna ® C18 column (250 mm × 4.6 mm i.d.; 5.0 �m, Torrance, CA, USA). The solvent sys- tem was a mixture of H2O (A) and MeOH (B), both with 0.1% formic acid in a linear gradient mode from 5 to 100% B in 50 min. The chro- matogram was monitored simultaneously at 254, 280 and 366 nm, and the UV spectra of individual peaks were recorded in the range of 200–400 nm. HPLC–HRMS data were obtained in a MicrOTOF II (Bruker, Biller- ica, MA, USA) series system equipped with an electrospray interface (ESI), an auto sampler and a high-pressure mixing pump. The column and the chromatographic conditions were the same as those used for the HPLC–UV/PDA analysis. The ESI-MS conditions consisted of capillary voltage set at 3500 V, dry heater tempera- ture 220 ◦C, and nitrogen as the sheath gas flow. The analyses were performed in positive and negative ion modes. For data comparison and tentative identification of the com- pounds, a database containing 108 compounds reported for Ipomoea genus was created from the calculated the m/z [M+H]+ and [M−H]− using Chem Draw Ultra 8.0 (CambridgeSoft, Cambridge, USA). Antioxidant DPPH• scavenging assay The DPPH• method reported by Brand-Williams et al. (1995), with some modifications (Zeraik et al., 2016) was used to eval- uate the antioxidant capacity of the I. procumbens extracts and S. Batiga et al. / Revista Brasileira de Farmacognosia 29 (2019) 191–197 193 F of leaves (A), stem (B) and roots (C) of Ipomoea procumbens. Column: Phenomenex Luna® C nd methanol (B), both acidified with 0.5% formic acid and eluted in gradient mode, from 5 f a w d s u T c t o c c A w e p s a p ( w t ( m i n a p s u m p b e A C d a M u m s t S Table 1 Codes of the fractions obtained after the microfractionation of the extracts of leaves, stem and roots of Ipomoea procumbens. Sample/H2O:MeOH ratio Fraction code Leaves (8:2) SPE1 Leaves (1:1) SPE2 Leaves (0:1) SPE3 Stem (8:2) SPE4 Stem (1:1) SPE5 Stem (0:1) SPE6 Root (8:2) SPE7 ig. 1. Representative HPLC–UV analysis at 254 nm of the hydro-methanol extract 18 column (250 mm × 4.6 mm i.d.; 5.0 �m). Mobile phase components: water (A) a % B to 100% B in 50 min. Flow-rate: 1.0 mL min−1. Injection volume: 20 �L. ractions. A range of concentrations (5–120 �g mL−1) of the extracts nd fractions, as well as the positive controls (rutin and gallic acid) ere incubated for 30 min with 100 �mol l−1 of a DPPH• (2,2- iphenyl-1-picrylhydrazyl) solution in methanol in the dark. The cavenger activity was evaluated spectrophotometrically at 517 nm sing a microplate reader (Synergy 2 Multi-Mode, BioTek, USA). he absorbance of the unreacted DPPH• radical was used as the ontrol. The scavenging activity was calculated using the equa- ion [(absorbance of control − absorbance of sample)/absorbance f control] × 100. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was pur- hased from Sigma-Aldrich Chemical Co. The experiments were arried out in triplicate. ntioxidant peroxyl radical scavenging assay The peroxyl radical (ROO•)-scavenging capacity of the extracts as evaluated using the pyranine based procedure (Campos t al., 2004) with some modifications. The fluorescent com- ound pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium alt, 5 �mol l−1) was incubated with 20 mmol l−1 AAPH (2,2′- zobis(2-methyl-propionamidine) hydrochloride)) in 10 mmol l−1 hosphate buffered saline (PBS, pH 7.4) at 37 ◦C in the absence control) or presence of the tested extracts and fractions in the ells of a microplate. The final reaction volume was 300 �L and he extracts and fractions were tested in a range of concentrations 5–120 �g mL−1). The fluorescence bleaching of the pyranine was easured at 485 nm (�exc) and 528 nm (�em) at 37 ◦C during 90 min n a Synergy2 Multi-Mode microplate reader (BioTek, EUA). Pyra- ine with AAPH (control 1) and without AAPH (control 2) were used s controls. Rutin and gallic acid were used as positive controls. The ercentage of peroxyl radical scavenged was expressed by [(area ample − area control 2)/(area control 1 − area control 2)] × 100, sing the values of the areas below the kinetic curves. 2,2′-azobis(2- ethyl-propionamidine) hydrochloride (AAPH) (purity 97%), was urchased from Sigma-Aldrich Chemical Co. All reagents used for uffer preparation and mobile phases were of analytical grade. The xperiments were carried out in triplicate. ntifungal susceptibility test Susceptibility tests of Candida krusei, Cryptococcus neoformans, andida parapsilosis, Cryptococcus gattii, Candida albicans and Can- ida tropicalis to the I. procumbens fractions, amphotericin B (AmB) nd fluconazole (FLZ) were performed according to the document -27-A3 (Clinical and Laboratory Standards Institute, 2008). Inoc- la were prepared in Roswell Park Memorial Institute (RPMI-1640) edium purchased from Sigma-Aldrich, with l-glutamine, without odium bicarbonate, supplemented with 2% glucose, and buffered o pH 7.0 using 0.165 M morpholinepropanesulfonic acid (MOPS; igma-Aldrich) to achieve a final concentration in microdilution Root (1:1) SPE8 Root (0:1) SPE9 plates of 2.5 × 103 colony-forming units (CFU)/mL. Work solutions of AmB and FLZ were tested in concentrations ranged from 64 to 0.0625 �g mL−1 and from 16 to 0.03 �g mL−1, respectively. For the I. procumbens fractions, stock solution was prepared in appropriate quantities of dimethyl sulfoxide (DMSO) (purity 99.9%, Sigma- Aldrich Chemical Co.) and work solutions in RPMI medium. The amount of DMSO used was previously tested and did not affect the fungal viability (data not shown). The range of concentration tested was from 0.48 to 250 �g mL−1. The plates were incubated at 37 ◦C under agitation of 150 rpm for up to 48 h. The readings were performed visually and confirmed using Alamar Blue ® (Sigma- Aldrich). All the assays were performed in triplicate and in three independent experiments. All reagents used for buffer preparation and mobile phases were of analytical grade. Results and discussion The phytochemical screening of the leaves, stem and roots of hydro-methanol extracts of I. procumbens were performed by HPLC–UV and are shown in Fig. 1. The comparison of the chromato- graphic profiles obtained for each part of the plant showed that the leaves extract presents a richer composition in secondary metabo- lites that absorbs around 250 nm and 360 nm, probably belonging to the class of polyphenols. The extracts of stem and roots are very similar, with differences concerning the intensity of the peaks. The extract of each part of the plant was microfractioned to yield three fractions each with different polarities (SPE1–9, Table 1), with the objective to make it easier to identify compounds present in the species by dereplication and to focus the antioxidant and antifungal activities. These fractions were analyzed by HPLC–UV under the same conditions used for the extracts and are shown in Fig. 2. In an attempt to identify the compounds present in I. procum- bens, we used the fast analytical technique dereplication, aimed at rapid on-line identification of known natural products contained in crude extracts or fractions. The database created for data com- parison includes eighteen alkaloids, 34 phenolic compounds, 45 194 S. Batiga et al. / Revista Brasileira de Farmacognosia 29 (2019) 191–197 F (SPE3 a g c n f M c m s 2 p d y a a o ig. 2. HPLC–UV analysis at 254 nm of the SPE fractions of leaves (SPE1–SPE3), stems re the same as described in Fig. 1. lycolipids, one lignan, seven triterpenes, among others. The appli- ation of LC coupled to MS in the analysis and characterization of atural products is a major breakthrough and it is frequently used or profiling extracts (Allard et al., 2017). Indeed, coupling LC with S is extremely powerful in terms of time, detection, quantifi- ation and identification of a wide range of natural product. This ethod provides excellent sensitivity and selectivity, important tructural information such as molecular weight (Wolfender et al., 010). The observed m/z obtained for the major chromatographic eaks were compared with the m/z from the compounds in the atabase created (error less than ± 10 ppm). I. procumbens anal- sis allowed the detection of two isomers of dihydroxycinnamic cid, caffeoyl-quinic acid derivatives, the coumarins scopoletin nd umbelliferone, and the alkaloids feruloyl tyramine and chan- clavine described in Tables 2 and 3 and Fig. 3. –SPE6) and roots (SPE7–SPE9) of Ipomoea procumbens. Chromatographic conditions Caffeoylquinic acids are phenolic compounds widely distributed in plants, and they are formed by esterification of quinic acid and caffeic acid, being chlorogenic acid the most known (Azuma et al., 2000). These compounds were also found in I. batatas, I. aquatica, I. pes-caprae and I. fistulosa (Meira et al., 2012; Zhao et al., 2014) and are associated with antioxidant and anti-inflammatory prop- erties, and the dimers are reported to possess higher antioxidant activity than the monomer (Iwai et al., 2004), but poor bioavail- ability. Pale et al. (2003) reported the presence of two isomers of dihydroxycinnamic acids (caffeic acid and 3,5-dihydroxycinnamic acid) in the flowers of I. asarifolia. Scopoletin and umbellifer- one were also found in I. batatas, I. cairica and I. digitate (Meira et al., 2012). Alkaloids such as chanoclavine, were also found in I. asarifolia, I. hederacea, I. muelleri, I. corymbose, I. tricolor and I. violacea. S. Batiga et al. / Revista Brasileira de Farmacognosia 29 (2019) 191–197 195 Table 2 Compounds tentatively identified by dereplication studies in Ipomoea procumbens using HPLC–HRMS in the negative ion mode. Retention time (min) Observed m/z [M−H]− Calculated [M−H]− Error (ppm) Compound Fraction analyzed 14.3 179.0359 179.0350 5.02 Dihydroxycinnamic acid SPE4 14.3 353.0888 353.0878 2.83 Caffeoyl-quinic acid SPE4 20.2 529.1389 529.1351 7.18 Di-caffeoyl-quinic acid methyl ester SPE8 22.4 677.153 677.1512 2.65 Tri-caffeoyl-quinic acid SPE8 Table 3 Compounds tentatively identified by dereplication studies in Ipomoea procumbens using HPLC–HRMS in the positive mode. Retention time (min) Observed m/z [M+H]+ Calculated [M+H]+ Error (ppm) Compound Fraction analyzed 15.8/16.3 163.0397 163.039 4.29 Umbelliferone SPE1/SPE4 18.2 193.0946 193.096 −7.25 Scopoletine SPE2 23.7 314.1386 314.1387 −0.31 Feruloyl tyramine SPE2 28.7/28.7 203.1055 203.1067 −5.90 Trimethyl-dihydro-hydroxy-naftalenone SPE3/SPE9 14.3 179.0337 179.0339 −1.11 Esculetine/methylisocoumarins SPE4 16.3 181.0498 181.0495 1.65 Dihydroxycinnamic acid SPE4 12.1 257.1645 257.1648 −1.16 Chanoclavine SPE8 F s in Ip w o a w m c f t r r g c e 1 m 2 a D f a w s t p ig. 3. Representative chemical structures of the tentatively identified compound ere not identified). In Brazil there are a few studies that report the composition f the genus Ipomoea. To the best of our knowledge, none of the uthors studying Ipomoea species from Brazil, including the present ork, reported the glycolipid class of compounds found in Ipo- oea species from Mexico. Apparently, there is a difference in the omposition of Ipomoea genus found in these two countries and urther studies could bring new information needed for confirma- ion. A difference in composition and biological activity was also eported when comparing the crude extract of I. pes-caprae, that eversibly inhibited the contractions induced by several spasmo- ens (Pongprayoon et al., 1989) and a similar study with the plant ollected in Brazil (Emendorfer et al., 2005). The antioxidant properties of the I. procumbens extracts were valuated by two distinct methods, namely DPPH• (2,2-diphenyl- -picrylhydrazyl) and peroxyl radical assays (ROO•), to reflect ultifunctional properties in physiological processes (Muller et al., 011). The DPPH• assay is based on the transfer of a hydrogen atom nd an electron (ET), between the antioxidant compound and the PPH radical, involving the change of color of the reaction medium rom purple to yellow, wherein DPPH• itself reacts as a radical and probe (Brand-Williams et al., 1995). This assay has been used idely to evaluate the radical scavenging activity of a variety of ubstances and plant extracts. The peroxyl radical scavenging capacity procedure represents a ypical hydrogen atom transfer based method, since it uses a com- etitive reaction scheme between antioxidants and a fluorescence omoea procumbens (the positions for substitutions are merely representative and probe (in this case pyranine), in which antioxidants and sub- strate compete for thermally generated peroxyl radicals generated by AAPH (2,2′-Azobis(2-methyl-propionamidine) hydrochloride) (Huang et al., 2005). The results of the in vitro assays showed that the extracts of I. procumbens were able to scavenge DPPH• and ROO• in a dose dependent manner and presented antioxidant capacity comparable to that of the positive controls rutin and gallic acid, as well as most fractions as indicated in Table 4. The leaves and steam fractions extracted with 1:1 H2O:MeOH (SPE2 and SPE5) presented the higher antioxidant capacity, with EC50 values of 1.24 ± 0.08 and 1.29 ± 0.10 �g mL−1, respectively, while the most polar were the less active fractions. These results compared to the chromatograms presented in Fig. 2 suggest that the compounds related to the peaks observed can be the responsible for the activity. Considering the roots, the most polar fraction presented the higher antioxidant capacity, with EC50 of 1.48 ± 0.10 �g mL−1 (SPE7), followed by the fraction 1:1 H2O:MeOH (SPE8). Polyphenols are one of the most abundant groups of phyto- chemical compounds from plants, being phenolic compounds and flavonoids reported to present high antioxidant activity (Erkan et al., 2011; Kamiyama and Shibamoto, 2012). The content of total phenols, flavonoids, reducing power and antioxidant activity were compared for six I. batatas varieties commonly found in Malaysia. All varieties showed high radical scavenging activity by DPPH• 196 S. Batiga et al. / Revista Brasileira de Farmacognosia 29 (2019) 191–197 Table 4 Antioxidant capacity of Ipomoea procumbens extracts and fractions (SPE1–9) by DPPH• and ROO• scavenging methods. Sample Peroxyl radical scavenging EC50 (�g mL−1) DPPH• radical scavenging EC50 (�g mL−1) Leaves extract 4.14 ± 0.19 40.0 ± 0.50 Stem extract 5.32 ± 0.23 38.5 ± 0.41 Roots extract 3.34 ± 0.12 41.1 ± 0.41 SPE1 17.79 ± 0.27 >120.0 SPE2 1.24 ± 0.08 11.85 ± 0.35 SPE3 16.40 ± 0.25 83.10 ± 0.62 SPE4 17.27 ± 0.19 >120.0 SPE5 1.29 ± 0.10 13.8 ± 0.28 SPE6 4.70 ± 0.21 20.4 ± 0.32 SPE7 1.48 ± 0.10 16.0 ± 0.27 SPE8 3.13 ± 0.26 17.8 ± 0.33 SPE9 5.34 ± 0.29 19.1 ± 0.19 Rutin 1.36 ± 0.14 6.86 ± 0.13 Gallic acid 0.92 ± 0.07 2.01 ± 0.09 Table 5 Antifungal activity of SPE fractions of Ipomoea procumbens. Fraction code Minimum inhibitory concentration (MIC, �g mL−1) Candida krusei ATCC 6258 Cryptococcus neoformans 90012 Candida parapsilosis ATCC 22019 Cryptococcus gattii ATCC 56990 Candida albicans ATCC 90028 Candida tropicalis ATCC 750 SPE1 250 250 ≥250 250 ≥250 ≥250 SPE2 125 125 ≥250 250 ≥250 ≥250 SPE3 31.25 31.25 62.5 15.6 ≥250 ≥250 SPE4 ≥250 250 ≥250 125 ≥250 ≥250 SPE5 31.25 125 ≥250 125 ≥250 ≥250 SPE6 15.6 15.6 31.25 62.5 ≥250 ≥250 SPE7 ≥250 250 ≥250 ≥250 ≥250 ≥250 SPE8 250 125 ≥250 125 ≥250 ≥250 SPE9 ≥250 ≥250 ≥250 ≥250 ≥250 ≥250 FLZa 64 4 8 8 1 0.125–4 AmBb 0.25–2 0.25 1 0.125 1 0.5–2 a a fl i A p p d I I l e ( a a t a t w l h w C l t a a a a Fluconazole. b Amphotericin B. ssay, ranging from 372.4 �g mL−1 (IC50) to 597.61 �g mL−1 (IC50) nd these values were related to the high content of phenolics and avonoids also reported in the varieties (Hue et al., 2012). The evaluation of the antifungal properties of I. procumbens was mportant because of the medicinal plants use especially in South merica, contributing significantly to primary health care. Many lants are used in Brazil in the form of crude extracts or herbal reparations to treat common infections without any scientific evi- ence of efficacy (Holetz et al., 2002). The phytochemistry of the pomoea genus has been studied since 1950 and some species of pomoea showed fungicide activity in the traditional use, particu- arly I. batata (Meira et al., 2012). The antifungal properties were valuated in the present work according to the document M-27-A3 Clinical and Laboratory Standards Institute, 2008) and the results re available in Table 5. For this assay, it was considered that if the sample displayed minimum inhibitory concentration (MIC) less than 100 �g mL−1, he antimicrobial activity was good; from 100 to 500 �g mL−1 the ntimicrobial activity was moderate; from 500 to 1000 �g mL−1 he antimicrobial activity was weak; over 1000 �g mL−1 the sample as considered inactive (Holetz et al., 2002). Candida parapsilopsis is sensitive to the methanol fractions of the eaf and the stem only (SPE3 and SPE6). The fractions SPE1 and SPE2 ave a moderate activity on C. krusei, C. neoformans and C. gattii, hereas fraction SPE3 has a very good activity on all four C. krusei, . parapsilosis, C. neoformans and C. gattii. The activity is particu- arly excellent on C. gatti with a MIC of 15.6 �g mL−1. Concerning he stem, SPE4 fraction has a moderate activity on C. neoformans nd C. gatti but has a better activity on C. gatti. The SPE5 fraction lso has a moderate activity on C. neoformans and C. gatti, and the ctivity on C. krusei is very good. The methanol fraction (SPE6) has an excellent activity on C. krusei and C. neoformans, and very good activity on C. parapsilosis and C. gattii. The root fraction SPE7 has a moderate activity on C. neoformans, the SPE8 has a moderate action on C. krusei, but better on both Cryptococcus. There is no possibil- ity to conclude on the methanol root fraction (SPE9) results. The methanol fractions of leaves and stem provided the best results for the antifungal activity. On C. albicans and C. tropicalis, all the results cannot be qualified; indeed, the MIC (>250 �g mL−1) could not precisely be determined, so neither the activity. While for the antioxidant activity the hydromethanol 1:1 frac- tions were more active, the methanol fractions (stems and leaves) provided better results for the antifungal assay. Several species of Ipomoea (I. batatas, I. muricata and I. aquatica) have been explored extensively by various research groups for the search of compounds with good antifungal activity (Meira et al., 2012). In the present study, the methanol fractions of the leaf and the stem of I. procumbems, SPE3 and SPE6, showed a potent in vitro antifungal activity against Candida and Cryptococcus species and can be considered as promising antifungal compounds. In this sce- nario, more pharmacological studies will be necessary to evaluate these molecules as antifungal prototypes. The findings herein reported are a preliminary study of chem- ical composition and biological activities and additional chemical studies are necessary to support biological studies. Authors’ contributions SB (MSc student) contributed in running the laboratory work, extractions, acquisition and analysis of the data. MV supervised laboratory work, analyzed data and drafted the paper. MLZ and KF contributed to antioxidant biological studies and aided in the de Far d a t l V m a E P t t C a R n C A F g a F e f f R A A A A B C C C C E S. Batiga et al. / Revista Brasileira rafting of the paper. GML contributed to chromatographic analysis nd aided in the drafting of the paper. NSM and AMFA contributed o antifungal biological studies. MCMY contributed to plant col- ection, plant identification and herbarium confection. SM and SB designed the study and contributed to critical reading of the anuscript. All the authors have read the final manuscript and pproved the submission. thical disclosures rotection of human and animal subjects. The authors declare hat no experiments were performed on humans or animals for his study. onfidentiality of data. The authors declare that no patient data ppear in this article. ight to privacy and informed consent. The authors declare that o patient data appear in this article. onflicts of interest The authors declare no conflicts of interest. cknowledgments The authors gratefully acknowledge financial support from APESP (Fundaç ão de Amparo à Pesquisa do Estado de São Paulo, rants #2010/52327-5 and #2013/07600-3), CNPq and CAPES. The uthors thank the EU for funding this work under the scope of the P7-PEOPLE CHEMBIOFIGHT (Project ID: 269301). MV acknowl- dges scholarship #2010/17329-7 from FAPESP and #120/2017 rom Finatec. We would like to thank Rosângela Simão-Bianchini or providing I. procumbens pictures. eferences llard, P.-M., Genta-Jouve, G., Wolfender, J.-L., 2017. Deep metabolome annotation in natural products research: towards a virtuous cycle in metabolite identification. Curr. Opin. Chem. Biol. 36, 40–49. rwa, P.S., Zeraik, M.L., Ximenes, V.F., da Fonseca, L.M., Bolzani, V.S., Silva, D.H.S., 2015. 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http://refhub.elsevier.com/S0102-695X(18)30223-0/sbref0175 http://refhub.elsevier.com/S0102-695X(18)30223-0/sbref0175 Chemical composition and biological properties of Ipomoea procumbens Introduction Materials and methods Plant material Extraction and solid phase extraction (SPE) microfractionation Analysis of the extracts Microfractionation HPLC–UV/PDA and HPLC–HRMS analysis Antioxidant DPPH scavenging assay Antioxidant peroxyl radical scavenging assay Antifungal susceptibility test Results and discussion Authors’ contributions Ethical disclosures Protection of human and animal subjects Confidentiality of data Right to privacy and informed consent Conflicts of interest Acknowledgments References