Chemical composition, antioxidant activity, neuroprotective and anti-inflammatory effects of cipó-pucá (Cissus sicyoides L.) extracts obtained from supercritical extraction

Accepted Manuscript Title: Chemical composition, antioxidant activity, neuroprotective and anti-inflammatory effects of cip´o-puc´a (Cissus sicyoides L.) extracts obtained from supercritical extraction ´ Authors: Marielba de los Angeles Rodr´ıguez Salazar, Jonabeto Vasconcelos Costa, Glides Rafael Olivo Urbina, Vˆania Maria Borges Cunha, Marcilene Paiva da Silva, Priscila do Nascimento Bezerra, Wandson Braamcamp de Souza Pinheiro, Walace Gomes-Leal, Alessandra Santos Lopes, Raul Nunes de Carvalho Junior PII: DOI: Reference:

S0896-8446(17)30784-2 https://doi.org/10.1016/j.supflu.2018.03.022 SUPFLU 4238

To appear in:

J. of Supercritical Fluids

Received date: Revised date: Accepted date:

26-10-2017 25-3-2018 25-3-2018

´ Please cite this article as: Marielba de los Angeles Rodr´ıguez Salazar, Jonabeto Vasconcelos Costa, Glides Rafael Olivo Urbina, Vˆania Maria Borges Cunha, Marcilene Paiva da Silva, Priscila do Nascimento Bezerra, Wandson Braamcamp de Souza Pinheiro, Walace Gomes-Leal, Alessandra Santos Lopes, Raul Nunes de Carvalho, Chemical composition, antioxidant activity, neuroprotective and anti-inflammatory effects of cip´o-puc´a (Cissus sicyoides L.) extracts obtained from supercritical extraction, The Journal of Supercritical Fluids https://doi.org/10.1016/j.supflu.2018.03.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Chemical

composition,

antioxidant

activity,

neuroprotective

and

anti-

inflammatory effects of cipó-pucá (Cissus sicyoides L.) extracts obtained from supercritical extraction

Marielba de los Ángeles Rodríguez Salazara, Jonabeto Vasconcelos Costab, Glides

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Rafael Olivo Urbinaa, Vânia Maria Borges Cunhaa, Marcilene Paiva da Silvaa, Priscila do Nascimento Bezerraa, Wandson Braamcamp de Souza Pinheiroc, Walace Gomes-

LABEX/FEA (Extraction Laboratory/Faculty of Food Engineering), ITEC (Institute of

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a

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Lealb, Alessandra Santos Lopesd, Raul Nunes de Carvalho Juniora,

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Technology), UFPA (Federal University of Para), Rua Augusto Corrêa S/N, Guamá,

LNNE/ICB (Laboratory of Neuroprotection and Experimental Neurorregeneration/

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b

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66075-900 Belém, PA, Brazil

Institute of Biological Sciences), UFPA (Federal University of Para), Rua Augusto

c

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Corrêa S/N, Guamá,66075-900 Belém, PA, Brazil Central Extraction Laboratory, ICEN (Institute of Exact and Natural Sciences), UFPA

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Brazil

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(Federal University of Para), Rua Augusto Corrêa S/N, Guamá,66075-900 Belém, PA,

d

LABIOTEC/FEA (Laboratory of Biotechnological Processes/Faculty of Food

Engineering), ITEC (Institute of Technology), UFPA (Federal University of Para), Rua

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Augusto Corrêa S/N, Guamá,66075-900 Belém, PA, Brazil

*

Corresponding author.

E-mail address: [email protected] (M.A.R. Salazar), [email protected] e [email protected] (R.N. de Carvalho Junior).

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Graphical abstract

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Highlights

 Box–Behnken design allowed prediction of the optimal conditions in SFE.

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 Pressure and percentage of cosolvent were the main factors that influenced SFE.

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 HPTLC of extracts showed the presence of terpenes and phenolic compounds.

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 The extract obtained with SC–CO2 + EtOH showed high antioxidant activity.

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Abstract

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 Supercritical extract presents neuroprotective and anti-inflammatory effects.

Cissus sicyoides L. is a plant used in the treatment of stroke and abscesses in popular Brazilian medicine. The aim of this work was to determine the best operational

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condition of the supercritical fluid extraction (SFE) of C. sicyoides with CO2 and CO2 + EtOH using the Box–Behnken design based on global yield, phenolic compounds, and flavonoids and antioxidant activity, compared to conventional extraction (Soxhlet), and to evaluate the extract’s neuroprotective effect. The optimal process condition corresponded to 400 bar, 40 °C, and 10% EtOH. Pressure and the cosolvent percentage

were the factors that influenced SFE. Extracts with increased antioxidant activity were obtained in both extractions (SFE and Soxhlet). HPTLC analysis of the extracts showed the presence of terpenes, phenolic compounds, and flavonoids. The preliminary in vivo assay showed that the extract obtained by SFE has a neuroprotective and antiinflammatory effect in a model of focal cerebral ischemia.

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Keywords: Cissus sicyoides L.; Supercritical fluid extraction; Design of Box–Behnken;

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HPTLC; Antioxidant activity; Neuroprotective effect.

1. Introduction

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Cissus sicyoides L. or Cissus verticillata L., which belongs to the Vitaceae family,

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is also known as cipó-pucá, vegetal insulin, or anil–trepador. It is considered a plant of

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the Neotropical region and is generally found in the Amazon region [1, 2]. According to

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studies on the chemical composition of C. sicyoides, it was discovered that contains

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bioactive compounds with increased antioxidant activity. Thus, the leaves and stems of the plant are characterized by the presence of carotenoids (α-carotene and β-carotene)

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[3] and phenolic compounds (flavonoids, stilbenes, tannins, and coumarins), as well as the presence of steroids and essential oils [4–8]. This species is traditionally used by

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Brazilian popular medicine to treat rheumatism, epilepsy, stroke, abscesses, arthritis, and diabetes [9–11]. It has been demonstrated that C. sicyoides has a potent anti-

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inflammatory activity in rats with induced edema, owing to the abundant presence of phenolic compounds since these compounds are mainly responsible for the pharmacological effects associated with the plant [5, 8, 12]. In addition to its antitumor activity, whereby it inhibits sarcoma–180 and Ehrlich's carcinoma [13], and antihypertensive activity for the treatment and/or prevention of dysfunctions, such as

hypertension, vasoconstriction of arteries, veins and capillaries [14], phenolic compounds act by preventing or retarding oxidative stress, thus acting as sequestrants of free radicals and reduce the appearance of different chronic diseases [15, 16]. Furthermore, they have a large capacity to inhibit inflammatory mediators, such as

role in the progression and development of inflammatory diseases [17].

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nuclear factor κB (NF–κB) and protein activator 1, thus identified to play an important

The World Health Organization (WHO) defines stroke as the sudden focal or

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global deficit of neurological functions lasting longer than 24 h or leading to death, and

whose only cause is of vascular origin [18, 19]. Because of the limitations of therapeutic effects, stroke is the third largest cause of mortality in the world, thereby constituting a

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significant public health problem [20, 21]. Currently, there are no available therapeutic

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approaches (with the exception of the recombinant tissue plasminogen activator

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thrombolysis) that reduce secondary neural damage after the onset of symptoms.

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However, few patients benefit from the use of this drug [22, 23]. The ineffectiveness of

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animal-tested drugs for acute neural disorders [24, 25] emphasizes the need to seek new and more effective anti-inflammatory and/or neuroprotective agents for diseases of the

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central nervous system.

Supercritical fluid extraction (SFE) has already been studied to obtain bioactive

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compounds from natural sources. Botelho et al. [26] demonstrated that the application of SFE technology is successful in obtaining extracts from copaiba leaves (Copaifera

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sp.) with neuroprotective effects against induced focal cerebral ischemia in rats. SFE is a technology particularly used for the extraction of bioactive compounds without the drawbacks associated with conventional extraction processes, such as the use of organic solvents, which induce toxicity and contaminate the extracts. Supercritical carbon dioxide (SC–CO2) is a solvent with increased applicability and is mainly used to extract

apolar compounds (lipids and carotenoids). However, polar compounds, such as phenolic compounds, have reduced solubility in this solvent. To circumvent this limitation, a cosolvent or polar modifier is usually used [27–30]. Thus, the aim of this work was to determine the best operational condition of the supercritical fluid extraction (CO2 and CO2 + EtOH) of the stem and leaves of cipó-pucá (C. sicyoides L.) by means

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of the Box–Behnken's experimental design on global yield, total phenolic compounds, and total flavonoids and antioxidant activity, compared to conventional extraction

focal cerebral ischemia in rats based on in vivo studies.

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2.1. Characterization of the raw material

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2. Material and methods

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(Soxhlet), and to evaluate the neuroprotective and anti-inflammatory effect in induced

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The leaves and stems of C. sicyoides were collected in the municipality of Salinópolis, at the Para state of Brazil, and the voucher specimen (Number 194.178)

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was deposited in the Herbarium of the Agroforestry Research Center of the Eastern Amazon (EMBRAPA). The samples were dried at 35 °C for 48 h in an oven with air

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circulation (Fabbe-Primar®, model 225, Brazil), and were comminuted using a knife mill (Marconi®, model ma 048, China). The humidity was then determined according to

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Jacobs immiscible solvent distillation method [31]. The particle size was analyzed using Tyler sieves (Bertel, Brazil) ranging in mesh size from 20 to 60, and the geometric mean diameter was determined according to the method recommended by the American Society of Agricultural Engineers (ASAE) [32]. The real density of the particles was determined with a helium pycnometer at the Analytical Center of the Chemistry

Institute of the University of Campinas (Unicamp), while the apparent bed density was calculated using the bed volume and the mass of the solid feed accommodated inside it. The bed porosity was calculated using the mathematical relation of the real and apparent densities.

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2.2. Supercritical fluid extraction (SFE)

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The extractions were carried out using the Spe-edTM SFE (Applied Separations,

model 7071, USA). This equipment has previously been described by Botelho et al. [33] and Oliveira et al. [34]. A cosolvent pump (LabAlliance, model 1500, USA) was

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coupled, the volume capacity of the extractor vessel used was 1 × 10-4 m3 (height of

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0.1254 m and internal diameter of 0.0320 m), the solvents used were CO2 (99.9%,

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White Martins®, Brazil) and EtOH (99.8% Neon, Brazil), and the sample mass was

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0.010 kg. The assays were performed based on a response surface methodology (RSM)

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with an experimental Box–Behnken design, in order to determine the best operating condition of the extraction. The independent variables were: pressure (200 to 400 bar)

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temperature (40 to 60 °C), and percentage of cosolvent (EtOH) (0 to 10%) (Table 1). The corresponding responses were: global yield, total phenolic compounds, total

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flavonoids, and antioxidant activity. The constant operating parameters were the 0.5 h static extraction time, the 3 h dynamic extraction time, and the 4.52 g/min CO2 flow

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rate. The extracts obtained with CO2 and CO2 + EtOH were collected in glass flasks until the end of each extraction. The obtained extracted mass was measured in an analytical balance (Quimis, model AS 210, Brazil) and the result was used to calculate the global yield. However, the extracts obtained using CO2 + EtOH were evaporated under vacuum at 40 °C using a CentriVap (Labconco®, USA) in order to evaporate the

EtOH before weighed. The extracts were then stored, protected from light, oxygen and temperatures below 5 °C until their analyses. The experimental data were analyzed using Statistica® 7.0 with 0.05 as the significance level. The assays were performed in duplicates with randomized execution.

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2.2.1. Global yield (X0)

The global yield was calculated on a dry basis (db), from the mathematical ratio

the extraction cell (mraw material), in accordance to Eq. (1).

𝑚𝑟𝑎𝑤 𝑚𝑎𝑡𝑒𝑟𝑖𝑎𝑙

× 100

(1)

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𝑚𝑒𝑥𝑡𝑟𝑎𝑡

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𝑋0 𝑑𝑏 (% ) =

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between the mass of the extract (mextrat) and the mass of the dried raw material fed into

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2.2.2. Density

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The density of CO2 and the mixtures CO2 + 5 and 10% EtOH under the selected

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operating conditions were calculated using the Peng–Robinson equation of state [35] in

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the Aspen Hysys simulator (an integral part of the Aspen One 8.6).

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2.3. Conventional extraction by Soxhlet

The conventional extraction to obtain the hexanic extract (HE) and ethanolic

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extract (EE) was performed according to the method described in the Brazilian Pharmacopoeia [36] using EtOH (99.5%, Dinamica®, Brazil) and hexane (95%, Vetec®, Brazil) in a solute–solvent analogy (C. sicyoides:solvent) of 1:10 (m/v), maintained at reflux for 3 h in a 500 ml Soxhlet apparatus. The solvents were removed by evaporation under vacuum at 40 °C using a CentriVap (Labconco®, USA). The assays were performed in triplicate. The yield of the extract was calculated by Eq. (1).

2.4. High performance thin layer chromatography (HPTLC)

The phytochemical screening of C. sicyoides extracts was performed by highperformance thin layer chromatography (HPTLC) according to the method of Wagner

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and Bladt [37] and Srivastava [38], using a CAMAG–TLC/HPTLC (CAMAG,

Switzerland) that was equipped with an automatic TLC sampler (ATS 4), an automated

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multiple development (AMD 2), and a TLC visualizer. The extracts were dissolved in

dichloromethane (99.9%, Tedia®, Brazil) at a 5000 ppm concentration. A volume of approximately 15 µl was applied in band form on silica gel plates 60 Å (SiliCycle Inc,

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Canada). It used dichloromethane (99.9%, Tedia®, Brazil) as the mobile phase (see the

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Supplementary material S1) and methanol (99.9%, Tedia®, Brazil) acidified with 1%

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formic acid (85%, Isofar®, Brazil) at a proportion of (95:4:1) (v/v/v). The plates were

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derivatized with a) vanillin–sulfuric acid (VS) (Merck®, Brazil) specific for the

Sigma-Aldrich®,

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terpenes, steroids, and fatty acid identification; with b) ferric chloride (FeCl3) (97%, Brazil)

for

phenolic

compound

identification;

with

c)

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diphenylboryloxyethylamine (NP) (Sigma-Aldrich®, Brazil) and polyethylene glycol 4000 (PEG) (Sigma-Aldrich®, Brazil) (NP/PEG) for flavonoids identification; and with

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d) a methanolic solution (99.9%, Tedia®, Brazil) of the radical 2,2-diphenyl-1picrylhydrazyl (DPPH•) (Sigma-Aldrich®, Brazil) for qualitative evaluation of the

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antioxidant activity. The plates were visualized under visible light and under UV light at a wavelength λ of 366 nm.

2.5. Total phenolic compounds (TPC)

The determination of total phenolic compounds was performed using the Folin– Ciocalteu method according to the methodologies described by Singleton et al. [39] and Georgé et al. [40]. The extracts of C. sicyoides were dissolved in EtOH (96%, Dinamica®, Brazil) at 7% (v/v) at a concentration of 140 mg/l. A volume of 0.5 ml of this solution was subjected to the reaction with 2.5 ml of Folin–Ciocalteu (Tedia®,

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Brazil) at 10% (v/v) and 2 ml of sodium carbonate solution (99.5%,Vetec®, Brazil) at

7.5% (m/v). After 1 h incubation at room temperature in dark conditions, absorbance

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measurements were performed at 760 nm on a spectrophotometer UV–VIS (Thermo Scientific, model Evolution 60, USA). The content of phenolic compounds was expressed as milligram gallic acid equivalent (GAE) (98%, Vetec®, Brazil) per gram of

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extract on a dry basis (db) (mg GAE/g extract) using the calibration curve equation

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(y=0.0099x–0.0277; R2=0.9971) (see the Supplementary material S2). The analysis was

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2.6. Total flavonoids (TF)

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performed in triplicate.

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The determination of total flavonoids (TF) was performed using the procedure described by Dowd [41] and Meda et al. [42]. The C. sicyoides extracts were dissolved

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in EtOH (96%, Dinamica®, Brazil) at a concentration of 140 mg/l. A sample solution volume of 0.5 ml was mixed with 0.5 ml of ethanolic solution of aluminum chloride

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(AlCl3) (99%, Fluka, Germany) at 2% (m/v). After 10 min of incubation at room temperature in the dark, the absorbance was measured at 425 nm using a spectrophotometer UV–VIS (Thermo Scientific, model Evolution 60, USA). The total flavonoid content was expressed as milligram quercetin equivalent (QE) (95%, SigmaAldrich®, Brazil) per gram of extract on dry basis (db) (mg QE/g extract) using the

calibration curve equation (y=0.0355x+0.0127; R2=0.9983) (see the Supplementary material S2). The analysis was performed in triplicate.

2.7. Antioxidant activity (AA)

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The quantitative determination of the antioxidant activity (AA) of C. sicyoides extracts by the DPPH method was performed according to the method proposed by

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Brand–Williams et al. [43] and Sánchez-Moreno et al. [44]. A DPPH solution (2,2diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, Brazil) was first prepared at 60 μM in EtOH (96%, Dinamica®, Brazil). Next, 3.9 ml of this solution was added in 0.1 ml of

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the extract at different concentrations (10, 7, 6, 5, and 4 mg/ml). These solutions were

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kept at room temperature and in dark conditions. After the stabilization of tEC50 (time

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required to reduce the initial amount of the radical DPPH• by 50%), the absorbance was

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measured at 515 nm using the spectrophotometer UV–VIS (Thermo Scientific, model

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Evolution 60, USA). The value of EC50 was then calculated (amount of antioxidant needed to reduce the initial amount of the radical DPPH• by 50%) and the antioxidant

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activity was expressed as gram of C. sicyoides extracts per gram of DPPH on a dry basis (db) (EC50 expressed in g of extract/g of DPPH). The analysis was performed in

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triplicate.

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2.8. Neuroprotective and anti-inflammatory effects of the extract obtained by SFE

2.8.1. Animals and surgical procedures Eight adult male Wistar rats were used, five of which were ischemic animals, and three control animals, weighing approximately 0.250 kg, The animals were socially

housed in standard cages with food and water provided ad libitum, ceded by the Central Laboratory of the Federal University of Para (Brazil). All experimental procedures were in compliance with the standards suggested by the Society for Neuroscience, National Institute of Health (NIH, USA), National Council for Control of Experimental Animals, and the Ethics Committee on Animal Research of the Federal University of Para

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(CEPAE–UFPA: 137–13).

Animals were anesthetized intraperitoneally with an injection of a mixture of

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ketamine hydrochloride (Vetanarcol®, Köning. 72 mg/kg) and xylazine hydrochloride

(Kensol®, Köning. 9 mg/kg). After confirmation of the loss of reflexes, they were placed on the stereotaxic apparatus (Insight® EFF–336) under a thermal blanket. The

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following coordinates were obtained from the bregma: mid–lateral: 2.3, anteroposterior:

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1.2, and dorsoventral: 0.5 [45]. Craniotomy was performed to expose the cortical

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surface, and endothelin–1 (ET–1) (E7764 Sigma-Aldrich, USA) was injected at the

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concentration of 80 pmol, and was diluted in 1 μl of colanyl blue dye [46, 47].

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After the surgical procedures, the animals remained housed in standard cages with food and water supplied ad libitum, temperature controlled at 24 °C, using circadian

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cycle 12/12 h light–dark conditions during the seven-day survival. They were then reanesthetized intraperitoneally and perfused for exposure of the pericardial sac with 0.9%

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heparinized saline solution followed by 4% paraformaldehyde.

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2.8.2. Microtomy and immunohistochemical analysis The brains were cryoprotected at different concentrations with glycerol-based

solution that contained 30% sucrose and sectioned using a cryostat (Zeiss HM505E) at a thickness of 50 μm. The lesion area and inflammatory cells were identified using the cresyl violet dye [48, 49].

2.8.3. Treatment with extract of C. sicyoides The C. sicyoides extract obtained with SC–CO2 + 10% EtOH at 400 bar and 50 °C was administered intraperitoneally at a daily concentration of 100 mg/kg shortly after the injection of ET–1, and the dose was applied in accordance to the 12/12 h cycle

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during the survival period. The control group consisted of ischemic animals, treated

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only with a physiological solution with 5% tween.

3. Results and discussion

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3.1. Characterization of the raw material

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The raw material had a humidity of 7.75±0.31%. The particle’s geometric mean

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diameter (dgm) was 0.50±0.07 mm, the apparent density (ρa) was 0.27±0.02 g/cm³, the

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real density (ρr) was 1.32±0.02 g/cm³, and the bed porosity (ε) was 0.80±0.02. These parameter values are suitable for the SFE and are considered to be some of the main

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parameters that directly influence the mass transfer rate of the process. Therefore, the raw material must be pretreated or wrapped correctly to increase the extraction rate [28,

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29].

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3.2.Global yield (X0)

The global yield of extracts obtained by SFE ranged from 1.27 to 3.21% (db), and the lowest yield was obtained with an SC–CO2 at 200 bar, 50 °C, and a density of 760.9 kg/m3, while the highest yield was obtained with an SC–CO2 + 10% EtOH at 400 bar,

50 °C, with a mixture density of 953.0 kg/m3 (Table 2). The yield of the extract obtained by Soxhlet was 5.06% (db) for the hexanic extract and 11.02% (db) for the ethanolic extract, which respectively represent yields that were approximately 1.57 (hexanic extract) and 3.43 (ethanolic extract) times the value of the extract in the case of the higher SFE yield. These differences in yields are justified since the Soxhlet

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extraction is exhaustive and capable in depleting the solute contained in the vegetal

matrix. However, the method has a low selectivity, and can obtain extracts using several

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undesirable substances. In contrast, the yield of extracts with SFE may be increased by increasing the percentage of cosolvent or reducing the particle size, which causes an increase in the contact surface between the solute and the solvent, raising the extraction

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rate.

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The regression coefficients of the independent variables as a function of the

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response (global yield) for SFE are listed in Table 3. It can be seen that the pressure and

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the percentage of cosolvent (EtOH) were influenced significantly (p≤0.05) the answer in

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its linear form. Temperature was not significant, yet it was considered significant because it presented a value that was close to alpha (0.05). However, the interactions

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between the variables under consideration were not significant (p˃0.05). The coefficient of determination (R2) was 0.97, thus indicating that 97% of the response variation can

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be explained by the model. The lack of fit was not significant, which allows the use of the mathematical model. This means that the global yield is maximized when the

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pressure, temperature, and percentage of cosolvent (EtOH), are at their highest levels (400 bar, 60 °C, and 10% EtOH) (see the Supplementary material S3). The analysis of variance results (ANOVA) are not presented herein. Similar results similar have already been reported in the literature for SFE from other plants using SC–CO2 + EtOH, such as the boldo leaves (Peumus boldus M.) [50], ingá-cipó leaves (Inga edulis), and nó-de-

cachorro (Heteropterys afrodisíaca) [51]. The global yield was influenced directly by the pressure, temperature, and the addition of cosolvent, thus indicating that these variables affect the extraction process. The increase in pressure increases the density of the supercritical fluid, thus increasing its solvation power. In regard to the temperature, there is an influence on the solvent and solute properties. Increasing temperatures

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increase the vapor pressure, thus raising the solubility of the supercritical fluids [29, 30,

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52].

3.3. High performance thin layer chromatography (HPTLC)

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HPTLC phytochemical screening of C. sicyoides extracts obtained by supercritical

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extraction, as presented in Table 2 (assays 1 to 15), and hexanic extract (HE) and

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ethanolic extract (EE), are shown in Fig. 1. Plates were derivatized with different

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reagents that produce a characteristic color for specific classes of chemical compounds.

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Thus, the plaque derivatized with VS (Fig. 1A) shows the formation of bands with purple/lilac staining indicating the presence of terpenes [37, 53]. According to the

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results, it was observed that the extracts obtained with SC–CO2 (assays 5–6 and 9–10), and the hexane extract, presented a similar phytochemical profile, thus verifying

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notoriously the presence of terpenes. It is thus possible to establish that this class of compounds is preferably extracted using SC–CO2 and hexane as the solvent. The plaque

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derivatized with FeCl3 (Fig. 1B) indicates the presence of phenolic compounds owing to the orange–brown coloration of the bands [37]. Thus, the supercritical extracts present a phytochemical profile similar to that for the hexane extract. However, in the ethanolic extract, a brown color is observed from the extract application point until the retention factor (Rf) of other extracts. Correspondingly, this has become possible by the presence

of highly polar phenolic compounds in the extract. On plates derivatized with NP/PEG (Fig. 1C) and visualized with UV light with a λ of 366 nm, the compounds present in the extracts become fluorescent upon their excitation with this radiation, and there is the possible presence of flavonoids justified by the yellow–green coloration of the bands [37, 53]. Thus, in both the supercritical extracts and in the hexanic extract yellow bands

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with the same Rf are visualized. In contrast, in the ethanolic extract there is a better

identification with a notorious band of yellow, thus corroborating that the EtOH is the

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suitable solvent needed to extract this class of compounds. In the plaque derivatized with DPPH (Fig. 1D), the presence of yellow spots on the purple bottom of the plaque

was identified that resulted from the reduction of the DPPH• radicals [54]. This is

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because DPPH is reduced in the presence of antioxidant substances, thus losing its

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purple coloration [43, 55]. Therefore, the results evidenced the presence (positive

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reaction) of chemical moieties characteristic of this plant, such as terpenes, phenolic

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compounds, and flavonoids, with antioxidant activity. Similar results had already been

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found by Viana et al. [10] in aqueous C. sicyoides extracts analyzed by TLC.

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3.4.Total phenolic compounds (TPC)

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The analysis of total phenolic compounds (TPC) provides an estimate of the

contents of all the compounds that belong to the subclasses of phenolic compounds

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present in the extracts. In Table 2 it is observed that the extract with a lower total phenolic compound content (37.83 mg GAE/g extract) was obtained with SC–CO2 at 200 bar, 50 °C, and a density of 760.9 kg/m3. Additionally, the extract with the highest content (65.20 mg GAE/g extract) was obtained with SC–CO2 + 10% EtOH at 400 bar, 50 °C, and with a mixture density of 953.0 kg/m3. These results demonstrate how the

process operating conditions and the use of the cosolvent influence the selectivity and obtaining the compounds of interest of the vegetal matrix. However, the ethanolic extract obtained by Soxhlet presented the highest total phenolic compound content (150.19 mg GAE/g extract) in comparison to the extract obtained with SFE, while the hexane extract yielded a similar value (60.51 mg GAE/g of extract to the content of the

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extract obtained with SC–CO2 + 10% EtOH at 400 bar and 50 °C. These differences are owing to the polarity of the solvents used. Chipiti et al. [56] estimated the phenolic

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compound content (136.1±6.7 mg GAE/g extract) in the ethanolic extract of cipó-uva stem (Cissus cornifolia) similar to the ethanolic extract of C. sicyoides. These results indicate that the content of the total phenolic compounds may vary according to the

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solvent and the extraction method used. Generally, they are extracted using EtOH,

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methanol, propanol, acetone, ethyl acetate, and their combinations thereof [57, 58].

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It can be observed in Table 3 that the variables that influenced significantly

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(p≤0.05) the total phenolic compound content were the pressure that yielded a negative,

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linearly dependent effect, and the percentage of cosolvent (EtOH) that led to a positive, linearly dependent effect. However, the extraction temperature and the interactions of

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the variables under investigation were not significant (p˃0.05). The coefficient of determination (R2) was 0.84, thus indicating that only 84% of the response variation can

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be explained by the model. This shows that the lack of fit was not significant, which allows the use of the mathematical model. This means that the total phenolic compound

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content is maximized when the pressure and percentage of cosolvent (EtOH) are at their highest levels (400 bar and 10%) (see the Supplementary material S3). This behavior was similar to those reported by Martinez-Correa et al. [59] in pitanga leave extracts (Eugenia uniflora L.) (Eugenia uniflora L.) and by Dias et al. [60] in jambu leaf extracts (Spilanthes acmella) obtained with SC–CO2 + EtOH.

The increase in pressure, density, and the use of cosolvents, in addition to the improvement of the extraction yield, increased the content of total phenolic compounds in C. sicyoides extracts. These results indicate that SC–CO2 is capable of extracting polar compounds, and its low polarity can be improved by using low amounts of polar cosolvents (5 to 10%), thus increasing the solvation power of polar bioactive

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intermolecular interactions between solute/cosolvent/solvent [29, 30, 52].

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compounds as the phenolic compounds. This effect has been attributed to the specific

3.5. Total flavonoids (TF)

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Total flavonoid (TF) analysis is an estimate of the content of all subgroups of this

N

class of compounds, such as flavonols, flavanones, isoflavones, anthocyanins, and

A

flavanols that are present in the extract. Table 2 shows that the extract with the lowest

M

total flavonoid content (0.14 mg QE/g extract) was obtained with SC–CO2 at 400 bar,

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50 °C, and a density of 951.2 kg/m3, and that the extract with the highest content (13.44 mg QE/g extract) was obtained with SC–CO2 at 300 bar, 40 °C, using 10% EtOH, and

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with a mixture density of 953.9 kg/m3. The regression coefficient of the independent variables as a function of the response (total flavonoids) for SFE presented in Table 3

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shows that the variable that influences this coefficient significantly (p≤0.05) was the percentage of cosolvent (EtOH) thereby exhibiting a positive effect in a linear form and

A

the interaction between the temperature and the percentage of cosolvent, thus exhibiting a negative effect on the response. However, the pressure, extraction temperature, and all remaining interactions between the variables under investigation were not significant (p˃0.05). The coefficient of determination (R2) was 0.85, showing that 85% of the response variation can be explained by the model. The lack of fit was not significant,

which allows the use of the mathematical model. This means that the total flavonoid content is increased when the percentage of cosolvent (EtOH) is at its highest level (10%) (see the Supplementary material S3). These results follow the behavior presented by Costa et al. [61] in copaiba leave extracts (Copaifera langsdorffii) obtained by their extraction with SC–CO2 + EtOH. The use of cosolvent in SFE increased the total

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flavonoid content in the extracts from 0.14 to 13.44 mg QE/g of extract, as obtained

with SC–CO2 and SC–CO2 + 10% EtOH, respectively. These results were in agreement

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with the previously reported phenolic compounds since the flavonoids are a specific class of these compounds, which have reduced solubility in SC–CO2 [29, 52].

As observed in Table 2, for the results of the extracts obtained by Soxhlet

U

extraction using organic solvents, the hexanic extract had a lowest value in total

N

flavonoids (6.97 mg AGE/g extract). This value is similar to that obtained in the SFE

A

(CO2 + 5 and 10% EtOH), thus showing that the modified SC–CO2 can have a solvation

M

power similar to hexane. In contrast, the ethanolic extract had the highest value (53.94

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mg QE/g extract) representing approximately 4.01 times the value of the extract with the highest content of phenolic compounds obtained with SC–CO2 + 10% EtOH. The

PT

difference is due to the high polarity, amount, recycling, and/or solvent reflux used (EtOH), which increased the solubility of these compounds and consequently improved

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their extraction.

Given that this is an extraction of a medicinal extract, and in view of the

A

characteristics of each solvent, the use of the ethanolic extract is not considered adequate. Vasconcelos et al. [62] reported the presence of hepatic changes in rats after administration of the hydroalcoholic C. sicyoides extract, while Dias et al. [63] reported increased toxicity of the hydroalcoholic extract. Therefore, the application is restricted

to the use of the extract obtained with SC–CO2 + 10% EtOH, thus allowing a small residual percentage of EtOH in the final product.

3.6. Antioxidant activity (AA)

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Table 2 shows EC50 values of extracts obtained by SFE, where the extract with the

lowest EC50 value (379.50 g extract/g of DPPH) was obtained with SC–CO2 + 10%

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EtOH at 400 bar, 50 °C, with a mixture density of 953.0 kg/m3, while the highest value

(899.14 g extract/g of DPPH) was obtained with SC–CO2 at 200 bar, 50 °C, and a density of 760.9 kg/m3. However, the antioxidant potential of the extract is inversely

U

proportional to the EC50 value. That is, the lower the EC50 value elicited by the extract

N

is, the lower is the sample amount (extract) needed to reduce the initial concentration of

A

DPPH to lead to 50% of antioxidant activity. The ethanolic extract yielded an EC50

M

value (325.67 g extract/g DPPH) that was similar to the extract obtained by SC–CO2 +

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10% EtOH, whereas the hexanic extract yielded the highest value of EC50 (876.99 g extract/g of DPPH) associated with lower antioxidant activity. The results of the

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antioxidant activity of the extracts are related to the results elicited by the total phenolic compounds and their extraction yields. This confirms that the antioxidant activity of

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plants is dependent on the amount of phenolic compounds [57, 58]. The regression coefficient of the independent variables as a function of the

A

response (antioxidant activity) for SFE presented in Table 3 shows that the variable trends that were influenced significantly (p≤0.05) were the positive effect of the pressure and the negative effect of the percentage of cosolvent (EtOH). However, the extraction temperature and the interactions among the variables under investigation were not significant (p˃0.05). The coefficient of determination (R2) was 0.99, thus

showing that 99% of the response variation can be explained by the model. The lack of fit was not significant, which allows the use of the mathematical model. Therefore, this means that the percentage of cosolvent (EtOH) at its highest level (10%) contributes directly to a decrease in the EC50 value (see the Supplementary material S3). These results confirm the results reported in the literature for SFE obtained from other plants

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using SC–CO2 + EtOH del Valle et al. [50] for boldo leaves (Peumus boldus M.) and

Costa et al. [61] on extracts obtained from copaiba leaves. The results of this study

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suggest that the methodology of extraction with SC–CO2 + EtOH as the cosolvent was selective in obtaining the extracts with antioxidant activity from C. sicyoides leaves and

stems. Correspondingly, this compound serves as a potential source of natural

U

antioxidants that can be useful in the prevention of diseases associated with oxidative

N

stress. Thus, the antioxidant activity can be attributed to the phenolic compounds

A

present in the extracts. These results confirm those reported by Khalil et al. [64] for

M

aqueous C. sicyoides extracts obtained by decoction, and for plant extracts belonging to

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the Vitaceae family, as reported by. Prabhavathi et al. [65] for Cissus quadrangulares, or those reported by Akomolafe et al. [66] for Cissus populnea, and Chipiti et al. [56]

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for Cissus cornifolia.

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3.7. Process optimization of SFE

A

In the response surface methodology (RSM), based on the Box–Behnken design,

the independent variables (pressure, temperature, and cosolvent percentage) were adjusted at three levels only (-1, 0 and +1). Thus, to obtain a better understanding of the influences of the independent variables and their interactions on the response variables, the desirability function was used to optimize the process to obtain a higher extraction

yield, total phenolic compounds content, total flavonoid content, and a lower value of EC50 (greater antioxidant activity). Thus, the optimum condition corresponds to a pressure of 400 bar, temperature of 40 °C, and 10% EtOH, and reaches a value of D=0.87 (see the Supplementary material S4), which is considered an optimum value since it is close to unity (1) [67]. The optimum condition was tested by performing three

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experiments. Table 4 shows the values of the evaluated responses (predicted and

experimental), and it is observed that the experimental values were similar to the values

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predicted by the model. It should be noted that the coefficient of determination (R2) and

the adjusted coefficient of determination (R2adj) of the models (Table 3) show very similar values for the overall yield (0.97 and 0.91, respectively) and antioxidant activity

U

(0.99 and 0.98 , respectively), indicating a good fit. However, these coefficients (R2 and

N

R2adj) yielded different values for the total phenolic compounds (0.84 and 0.54,

A

respectively) and total flavonoids (0.85 and 0.58, respectively), thus indicating that the

M

model is not as accurate for these responses. In addition, the lack of the fit was not

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significant (p>0.05) for all study responses, thus revealing that the model is statistically

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significant.

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3.8. Neuroprotective and anti-inflammatory effects of the extract obtained by SFE

Microinjections of ET–1 into the rat motor cortex induced focal ischemia with

A

histopathological features, including tissue pallor, cell loss, and inflammatory response, as revealed by cresyl violet staining seven days post injury (Fig. 2A–C). There was a remarkable infarct area (Fig. 2A), as reported in our previous investigations [33]. Inflammatory mononuclear cells were present at high densities inside and around the lesion core (Fig. 2B–C).

Treatment of ischemic animals with C. sicyoides extract (100 mg/kg) during the first week post-injury reduced the infarct area (Fig. 2D) and inflammation (Fig. 2E–F) compared to control animals treated with 5% twee (Fig. 2A–C). The amount of mononuclear cells in the lesion site was clearly reduced in animals treated with C. sicyoides (Fig. 2D).

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In our previous studies, we provided evidence that SFE extracts obtained from Amazon plants have considerable anti-inflammatory and neuroprotective effects in

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experimental models of excitotoxicity [26, 49] and stroke [33]. Herein, we provide new information on the potential anti-inflammatory and neuroprotective effects of C. sicyoides. Reduction of exacerbated inflammatory response in the central nervous

U

system is of considerable importance as this pathological phenomenon is believed to

N

contribute to tissue damage, although may also induce neuroprotection [68].

A

The mononuclear cells present in the lesion site are mainly comprised of brain

M

macrophages derived from both resident microglia and blood-borne cells [69]. There is

ED

considerable evidence that exacerbated microglia activation contributes to secondary damage after stroke. Inhibition of microglia activation with minocycline reduces the

72].

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infarct area and inflammation after focal ischemia in both the cortex and striatum [70–

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The mechanisms underlying the anti-inflammatory and neuroprotective effects of

C. sicyoides are unknown. Nevertheless, modulation of microglia activation may be

A

involved. It has been shown that microglia display both detrimental (M1) and beneficial (M2) phenotypes after stroke [73]. It is possible that C. sicyoides contains compounds with immunomodulatory properties, thus inducing a shift toward a preferential M2 phenotype in the ischemic environment. In addition, the neuroprotective effects of C. sicyoides might be related to its antioxidant activity and the presence of phenolic

compounds and its composition [5, 8, 12]. A plant extract with increased antiinflammatory and anti-oxidant effects possesses a great potential as a neuroprotective agent considering that oxidative stress and exacerbated inflammation are the main components of the stroke pathophysiology [22, 68].

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4. Conclusion

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The highest global yield for the SFE process from C. sicyoides leaves and stems was obtained with SC–CO2 + 10% EtOH at 400 bar, 50 °C, with a density of 953.0 kg/m3. With this condition, the extract with a high content of total phenolic compounds

U

and antioxidant activity was also obtained. The RSM that was based on the statistical

N

design of Box–Behnken determined that the pressure and the percentage of cosolvent

A

(EtOH) added to the SC–CO2 flow were the factors that most influenced SFE. The

M

optimal condition of the process corresponded to a pressure of 400 bar, temperature of

ED

40 °C, and 10% EtOH. In terms of antioxidant activity, both extraction methods (SFE and Soxhlet) yielded extracts with low values of EC50, indicative of increased

PT

antioxidant activity. However, in the SFE, the obtained extracts are free of organic solvents, and this represents an advantage over the extracts obtained with these solvents.

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In addition, the high selectivity of the process is a determining factor for obtaining and increasing the yield of phenolic compounds and flavonoids.

A

Phytochemical screening of extracts by HPTLC evidenced the presence of

terpenes, phenolic compounds, flavonoids, and antioxidant activity. The preliminary in vivo assay in the focal cerebral ischemia model demonstrated that the extract obtained by SFE has a neuroprotective and anti-inflammatory effect. These effects are associated with the presence of phenolic compounds and the increased antioxidant activity of the

extract. Finally, extracting C. sicyoides with SFE using low amounts of cosolvents (5 and 10%) represents a great opportunity to obtain polar bioactive compounds without the drawbacks associated with conventional extraction processes.

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Acknowledgements

M.A.R. Salazar thanks to Partnerships Program for Education and Training

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between the Organization of American States and the Coimbra Group of Brazilian Universities and CAPES - PROPESP/UFPA - Brazil for the master’s scholarship.

N

J.A. Lombardi, Vitaceae: Gêneros Ampelocissus, Ampelopsis e Cissus, Flora

J. Drobnik, A.B. De Oliveira, Cissus verticillata (L.) Nicolson and C.E. Jarvis

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[2]

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Neotropica. 80 (2000) 1–250.

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IP T SC R U N A M ED PT CC E A Fig. 1. HPTLC of C. sicyoides extracts (assays 1 to 15) supercritical extracts; (HE) hexanic extract; (EE) ethanolic extract; derivatized plaques with: (A) VS; (B) FeCl3; (C)

NP/PEG; (D) DPPH; mobile phase: dichloromethane, methanol and formic acid

A

CC E

PT

ED

M

A

N

U

SC R

IP T

(95:4:1).

IP T SC R U N A M ED PT

Fig. 2. Gross histopathology revealed by cresyl violet staining: (A˗C) control group;

CC E

(D˗F) treated group; (*) asterisks point to the lesion center; () arrows for mononuclear cells (macrophages); (C) motor cortex and (CC) corpus callosum; with increase: A, D x

A

40; B, E x 100; C, F x 400.

Table 1. Independent variables with their actual and coded values, used in the Box– Behnken design. Levels 1

0

+1

bar

X1

200 300 400

Temperature

°C

X2

40

50

60

Cosolvent (EtOH)

%

X3

0

5

10

A

CC E

PT

ED

M

A

N

U

SC R

Pressure

IP T

Independent variables Unit Notation

I N U SC R

Table 2. Box–Behnken experimental design, with the independent variables on the response variables evaluated for C. sicyoides extracts obtained by SFE, compared to conventional extraction (Soxhlet).

M

Temperature

Cosolvent

Density

Global yield

TPC(mg GAE/g

TF (mg QE/g

AA (EC50: g of

(bar)

(°C)

(EtOH) (%)

(kg/m3)

(%)

of extract)a

of extract)b

extract/ g of DPPH)c

1

2

+1

(200) (400)

1

(40)

0

(5)

865.6

1.84

52.43

3.85

596.34

1

(40)

0

(5)

984.1

2.08

53.21

4.43

595.62

3

1

(200)

+1

(60)

0

(5)

759.3

1.94

51.12

3.70

610.38

4

+1

(400)

+1

(60)

0

(5)

927.7

2.69

56.08

8.06

597.71

A

CC E

1

ED

Pressure

PT

Exp. no.

A

Supercritical fluid extraction (SFE)

5

1

(200)

0

(50)

1

(0)

760.9

1.27

37.83

1.08

899.14

6

+1

(400)

0

(50)

1

(0)

951.2

1.80

44.09

0.14

897.36

I (200)

0

(50)

+1

(10)

845.1

2.37

57.38

10.97

415.34

8

+1

(400)

0

(50)

+1

(10)

953.0

3.21

65.20

9.01

379.60

9

0

(300)

1

(40)

A

N U SC R

1

1

(0)

926.8

1.52

43.82

0.28

896.28

10

0

(300)

+1

(60)

1

(0)

829.0

2.03

41.74

0.72

898.50

11

0

(300)

(40)

(10)

935.9

2.58

49.04

13.44

460.81

12

0

13

0

14 15

ED

+1

(300)

+1

(60)

+1

(10)

884.6

2.76

47.21

3.12

440.88

(300)

0

(50)

0

(5)

906.6

2.35

38.61

3.70

609.80

0

(300)

0

(50)

0

(5)

906.6

2.16

43.56

4.50

598.23

0

(300)

0

(50)

0

(5)

906.6

2.38

40.96

5.52

599.33

-

5.06

60.51

6.97

897.99

1

PT

CC E A

M

7

Conventional extraction by Soxhlet HEd

-

-

-

-

-

-

I -

-

-

-

-

-

11.02

150.19

53.94

325.67

Total phenolic compounds; b total flavonoids; c antioxidant activity, effective concentration EC50/DPPH; d hexanic extract; e ethanolic extract.

A

CC E

PT

ED

M

A

a

-

N U SC R

EEe

I N U SC R

Table 3. Regression coefficients for the variables responses evaluated for C. sicyoides extracts obtained by SFE. TF (mg QE/g of

AA (EC50: g of extract/ g

extract)b

extract)c

of DPPH)d

P

Regression

ED

Regression

M

(%) Effecta

TPC(mg GAE/g of

A

Global yield

coefficients

coefficients

P

Regression

P

coefficients

Regression

P

coefficients

2.1742

0.0003

49.9292

0.0002

4.9000

0.0029

640.6633

0.0000

X1

0.2950

0.0198*

2.4775

0.1055

0.2550

0.5120

-6.3638

0.1062

X1 2

0.0548

0.2196

-4.4598

0.0202*

-0.3368

0.2919

7.1746

0.0497*

X2

0.1750

0.0535*

-0.2938

0.7691

-0.8000

0.1313

-0.1975

0.9383

X2 2

0.0248

0.5083

-1.6235

0.1279

0.1183

0.6675

-5.9542

0.0698

X3

0.5375

0.0061*

6.4188

0.0181*

4.2900

0.0056*

-236.8313

0.0001*

A

CC E

PT

Mean/Intercept

I N U SC R

0.0123

0.7304

-0.5810

0.4623

-0.0267

0.9208

-29.8779

0.0031*

X1*X2

0.1275

0.1660

1.0450

0.4875

0.9450

0.1741

-2.9875

0.4482

X1*X3

0.0775

0.3235

0.3900

0.7826

-0.2550

0.6324

-8.4900

0.1171

X2*X3

-0.0825

0.3008

0.0625

0.9643

-2.6900

0.0276*

-5.5375

0.2250

ED

M

A

X3 2

0.97

0.84

0.85

0.99

R2adjusted

0.91

0.54

0.58

0.98

0.3578

0.1312

0.0748

0.0684

a

CC E

Lack of fit

PT

R2

X1: Pressure, X2: temperature, X3: cosolvent (EtOH); b total phenolic compounds; c total flavonoids; d antioxidant activity, effective

A

concentration EC50/DPPH; * significant values.

I N U SC R

Table 4. Responses values (predicted and experimental) for C. sicyoides extract obtained by SFE under optimum conditions: 400 bar, 40 °C and

Values

M

A

10% cosolvent (EtOH).

Global yield (%)

ED

Response variables

PT

TPC(mg GAE/g of extract)a

CC E

TF (mg QE/g of extract)b

AA (EC50: g of extract/ g of DPPH)c

Goals

Low

Medium

High

Desirability

Predicted

Experimental

Maximize

1.2700

2.240

3.2100

1.0000

2.803

3.04 ± 0.10

Maximize

37.830

51.515

65.200

0.9734

62.845

63.55 ± 0.75

Maximize

0.1400

6.790

13.44

1.0000

11.898

12.13 ± 0.29

Minimize

379.600

639.370

899.14

0.9708

416.810

404.81 ± 2.78

A

c compounds; b total flavonoids; c antioxidant activity, effective concentration EC50/DPPH.

a

Total

phenoli