Caryocar coriaceum Wittm. (Pequi) fixed oil presents hypolipemic and antiinflammatory effects in vivo and in vitro

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Caryocar coriaceum

Wittm. (Pequi)

_ﬁ

xed oil presents hypolipemic and

anti-in

_ﬂ

ammatory effects

in vivo

and

in vitro

Patrícia Rosane Leite de Figueiredo

a

, Isabella Bezerra Oliveira

a

, José Benício Santana Neto

a

,

Juliana Albuquerque de Oliveira

a

, Larissa Bernardo Ribeiro

a

,

Glauce Socorro de Barros Viana

a

_{, Talita Magalhães Rocha}

b

_,

Luzia Kalyne Almeida Moreira Leal

b

, Marta Regina Kerntopf

c

,

Cícero Francisco Bezerra Felipe

d

_{, Henrique Douglas Melo Coutinho}

e

_,

Irwin Rose de Alencar Menezes

c,n

a_{Laboratório de Bio}_ﬁ_{siologia e Farmacologia, Faculdade de Medicina Estácio de Juazeiro do Norte}

–Estácio/FMJ, Avenida Tenente Raimundo Rocha s/n, CEP

63040-360 Juazeiro do Norte, Brazil

b_{Laboratório de Farmacognosia, Universidade Federal do Ceará}

–UFC, Rua Capitão Francisco Pedro, 1210, Rodolfo Teóﬁlo, CEP 60430-370 Fortaleza, Brazil c_{Laboratório de Farmacologia e Química Molecular}

–LFQM, Universidade Regional do Cariri–URCA, Rua Cel Antônio Luiz, 1161, Pimenta, CEP 63105-000

Crato, Brazil

d_{Departamento de Biologia Molecular}

–DBM, Universidade Federal da Paraíba–UFPB, Campus I–Jardim Cidade Universitária, CEP 58059-900 João Pessoa,

Brazil

e_{Laboratório de Microbiologia e Biologia Molecular}

–LMBM Universidade Regional do Cariri–URCA, Rua Cel Antônio Luiz, 1161, Pimenta, CEP 63105-000,

Crato, Brazil

a r t i c l e

i n f o

Article history:

Received 21 January 2016 Received in revised form 3 June 2016

Accepted 13 June 2016 Available online 15 June 2016

Keywords:

Caryocar coriaceumwittm Pequi

Dyslipidemia

Monounsaturated fatty acids Inﬂammation

Myeloperoxidase

a b s t r a c t

Caryocar coriaceumWittm. (Pequi) is found in southern Ceará, where the fruit is used as food and in folk medicine as an anti-inflammatory, and to promote healing. However, little is known about the effects of repeated administration of its oil on the biochemical parameters of the blood. This work aimed to evaluate the effectsCaryocar coriaceumfixed oil (OFCC); on the lipid profiles of healthy mice, on dysli-pidemia induced by tyloxapol, and to study its anti-inflammatory effect bothin vivoandin vitro. The results revealed significant reduction in total serum cholesterol and triglycerides, and an increase in HDL-C. The paw edema (induced by carrageenan) and myeloperoxidase (MPO), in polymorphonuclear culture cells, was reduced at all dose levels. Results demonstrated thatCaryocar coriaceum'sfix oil present anti-inflammatory activity and, for thefirst time describe the hypolipidemic effects, supporting its traditional use and suggest that present a potential cardioprotective effect.

1. Introduction

The speciesCaryocar coriaceumWittm it's found in the north-ernmost part of the Northeast region in Brazil. This plant playing an important social and economic role in the round of Araripe Platue and in the states of Ceará, Pernambuco and Piauí (Júnior et al., 2013;Silva et al., 2015). Its fruit is popular in regional cui-sine, with economic importance (Cavalcanti et al., 2015), therefore, is a valuable food source for the low-income population and, in the off season, the extraction of oil from the pulp and almond occurs, which has greater commercial value. The pulp and almond (edible

oil portions of fruit) for its distinctive ﬂavor and be a source of lipids and antioxidant vitamins (A and E), are quite used as food and in the preparation of sauces and spices, following regional dishes and replacing other sources of lipid.

Inﬂammatory and Cardiovascular diseases (CVD)(s) present high prevalence, and are the principal causes of both morbidity and mortality in the world population (Kivimäki et al., 2011). Dyslipidemia is an important cardiovascular risk factor, char-acterized by low serum levels of high density lipoprotein (HDL-C), elevated serum levels of low-density lipoprotein (LDL-C), and elevated triglycerides (TG) (Kabakci et al., 2008).

Many studies have shown a positive association between ser-um TG and the risk of coronary artery disease (CAD), thru the formation of atherosclerotic plaques (Austin, 1991;Hokanson and Austin, 1996;Kabakci et al., 2008). Atherosclerosis results from the body's response to tissue injury, with an emphasized Contents lists available atScienceDirect

journal homepage:www.elsevier.com/locate/jep

Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2016.06.038

n

Correspondence to: Department of Chemical-Biology, Regional University of Cariri (URCA), 63105-000 Crato, Ceará, Brazil.

E-mail addresses:irwinalencar@yahoo.com.br,

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inﬂammatory process (due to endothelial dysfunction), that re-sults in the formation of oxLDL. Thus, anti-inﬂammatories and oral blood-lipid modulators are part of the therapeutic arsenal of pa-tients with cardiovascular disease, and represent a large share of the costs (Zimmet et al., 2001).

Medicinal plants having blood-lipid activity (after validation and safety determinations) have become low-cost alternatives for use in the health system, (Pereira et al., 2015). The“pequizeiro”is a native Brazilian tree, popularly known by the names of “pequi”,

“piqui”, “pequá”, “Thorn almond”, “horse bean” or “Brazilian al-mond”. From the genus Caryocar, the species: C. brasiliense, C. villosum, C. coriaceum, C. cuneatumand C. glabrum stand out in Brazil (Prance and da Silva, 2006). The Caryocar name comes from the Greekcaryon(¼_{core, walnut), and}kara(¼_{head), and refers to}

the globular fruit. The name“pequi”has an indigenous origin; py-chi, py(¼skin, bark),qui (¼thorn), meaning _“spiny skin_”, due to

the endocarp spines. The species Caryocar coriaceum Wittm., is found in the“Chapada do Araripe” where, fruit and woods were explored commercially and, plays an important socio-economic role in the region (Albuquerque et al., 2011; de Oliveira Campos et al., 2015;Júnior et al., 2013;Silva et al., 2015). Its fruit is used in food, and in folk medicine as an anti-inﬂammatory and to promote healing (Ribeiro et al., 2014). Theﬁxed oil ofCaryocar coriaceum

(OFCC), (as well as olive oil and other vegetable oils) is rich in oleic acid (Ramos and Souza, 2011;Sena et al., 2010), an unsaturated fat used as an adjunct in the treatment of hyperlipemias with po-tential cardioprotective effect (Salgado et al., 2008), anti-bacterial (Saraiva et al., 2011a,2011b), anti-inﬂamatory (de Oliveira et al., 2015; Saraiva et al., 2011a, 2011b) and wound effect (da Silva Quirino et al., 2009). Another parts of this plant were also studied and demonstrated an important therapeutic potential as

anti-in-ﬂammatory (Araruna et al., 2014) and anti-microbial (Araruna et al., 2013).

Despite being described for the treatment of a wide variety of diseases, there are no studies evaluatingCaryocar coriaceum's ef-fect on lipid profiles, nor its possible activity as anti-inflammatory agent and oral blood-lipid modulator. Thus, this study aimed to evaluate the influence of the daily administration of thefixed oil from the fruit pulp of Caryocar coriaceum WITTM., on the bio-chemical parameters of healthy rats, on dyslipidemia, and to analyze thein vivoandin vitroanti-inflammatory effects of the oil.

2. Material and methods

2.1. Animals

The Wistar Rats (Rattus norvegicus) used came from the animal facilities of the Faculty of Medicine of Estacio Juazeiro (Estacio/ FMJ). The animals were housed in polypropylene cages at room temperature (22–24°C) with a light/dark cycle of 12 h. They re-ceived standard feed and water ad libitum. All of the animal ex-periments obeyed the experimental protocols previously approved by the Commission of Experimentation and Animal Use at the Regional University of Cariri (CEUA/URCA), (see opinion No. 05/ 2012).

2.2. Drugs

All of the drugs and reagents used in this study were of ana-lytical grade, namely: Tiloxapols_{(Triton WR 1339, Sigma/Aldrich),}

and Gemﬁbrozil (Lopids

–Ache Laboratórios Farmacêuticos SA, São Paulo, SP).

2.3. Preparation of plant material to obtain OFCC

The fruits of C. coriaceum Wittm. were collected in October 2010 (ﬂowering period) in the community of Cacimbas in the Jardim-CE municipality (Latitude: 07°34′57″S Longitude: 39 17′53″

W Altitude: 648 m). A voucher specimen of the plant material was deposited in the Herbarium Caririense Dárdano de Andrade-Lima, of the Cariri Regional University (URCA) under the registration number 44,523. Thein naturapulp fruit ofC. coriaceumwas sub-jected to manual shelling and pulping; separating the inner me-socarp from the endocarp. The pulp obtained was subjected to intensive cooking with water, separating the supernatant oil. The oil was dried at low heat, using a metal container, until it lost opacity due to moisture. In this case, to obtain 1 l of oil, required 7400 g of Pequi pulp. The oil wasﬁltered throughﬁlter paper with anhydrous sodium sulfate.

2.4. Chemical proﬁle

The fatty acids determination was made indirectly by means of their respective methyl esters. The oil (0.2 g) was saponiﬁed for 30 min under reﬂux with a methanolic potassium hydroxide so-lution according to the methodology described by Hartman and Lake (1973). After treatment and pH adjustment, the free fatty acids were methylated with methanol in acid catalysis, to obtain the respective methyl esters.

The analyses of the volatile constituents were carried out on GC/MS Hewlett-Packard Model 5971 equipment, using a non-polar capillary column DB-1 fused silica (30 m0.25 mm id, 0.25 mm film); carried with helium gas;flow rate 0.8 ml/min, and in split mode. The temperature of the injector and detector were 250°C and 200°C, respectively. The column temperature was pro-grammed from 35°C to 180°C at 4°C/min, and then from 180°C to 250°C at 10°C/min. The mass spectra were recorded from 30 to 450m/z. Individual components were identified by matching their mass spectra at 70 eV, with a database using the library built by Wiley spectrometers, and two computers using retention rates for pre-selection (Alencar et al., 1984;McLafferty and Stauffer, 1994). Visual comparison of the fragmentation pattern with those re-ported in the literature was used as well (Horai et al., 2010).

2.5. Lipid proﬁle assessment in rats treated with OFCC

Different groups of animals were treated with OFCC 500, 1000 and 2000 mg/kg and 0.9% saline (p.o.) for 7, 15, and 30 days. At the end of each period, the animals were weighed and fasted for 12 h, for later blood collection. The blood samples (2 ml) were collected below the orbital plexus in 2 ml serum collection tubes with gel separator (VACUETTEs_{, São Paulo, SP, Brazil), and evaluated for the}

following biochemical parameters: total cholesterol, HDL C, LDL-C and triglycerides. The determination of each analyte was per-formed according to methods described by the manufacturer (Labtest).

2.6. Evaluation of OFCC's lipid-lowering potential in rats with Triton WR-1339 induced dyslipidemia

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collection tubes serum with gel separator (VACUETTEs_{, São Paulo,}

SP, Brazil), and were evaluated for the following biochemical parameters: total cholesterol, HDL-C, LDL-C and triglycerides. The determination of each analyte was performed according colori-metric methods described by the manufacturer (Labtest). For each animal, the aorta, heart and liver were rapidly dissected out and

fixed in buffered formalin (10%). Afterfixation, the stent fragments on the cut surface of each of the 5-mm segments were removed under a dissection microscope. These selected fragments were removed carefully and pulling them out of the paraffin-embedding block. The tissue processed in paraffin were cut at 5-

μ

m thickness and stained with hematoxylin-eosin and evaluated by optical microscopy.

2.7. Evaluation of the in vivo anti-inﬂammatory effect of OFCC

Different groups of animals were treated with OFCC at 500, 1000 and 2000 mg/kg, and 0.9% saline (p.o.) for 7, 15 and 30 days and at the end of each period, the initial volume (Vi) of the right hind paw was recorded using a plethysmograph (Ugo Basiles_).

After 1 h, each animal received an intra-plantar injection of car-rageenan 2% (w/v) in the right hind paw (0.2 ml/paw). The volume of the right hind paw of each animal was evaluated again by the plethysmograph at 1, 2, 3, and 4 h after injection of the phlogistic agent. The results were expressed as the difference between the

ﬁnal volume of the paw at each time (Vf) and the initial volume (Vi) represented by the formula: Ve¼Vf Vi, where Ve is the

edema volume (ml) (Winter et al., 1962). One group of animals was treated with Indomethacin 10 mg/kg, i.p. as a control for

anti-in-ﬂammatory effect.

2.8. Evaluation of the in vitro anti-inﬂammatory effect OFCC

Polymorphonuclear cells, mainly neutrophils (80–90%) were ob-tained from human blood donated by the Hemato-therapy and He-matology Center of Ceara. The cells were isolated according to the method described by Henson (1971), and later modiﬁed by Lucisano and Mantovani (1984). A neutrophil suspension (5_{106 cells/ml)}

was incubated withCaryocar coriaceumﬁxed oil (OFCC; 1, 10, 50, 100 and 200

μ

g/ml), indomethacin (35.7 mg/ml, standard), and Vehicle (DMSO (Dimethylsulfoxide) 1% in water) or Hanks solution, and after 15 min, the material was centrifuged. The supernatant obtained, rich in enzymes released by white blood cell degranulation, was used to measure myeloperoxidase using the methodology described by Ubeda et al. (2002). To the supernatant were added PBS (phosphate buffered saline solution), phosphate buffer, and H2O2. After 15 min at

37°C was added 3.3′, 3.5′-tetramethylbenzidine (TMB), and the re-action was stopped by adding sodium acetate. The absorbance at 620 nm was determined. The construction of a standard curve by adding increasing amounts of MPO (0.125–3 U/ml) allowed relating the absorbances obtained to the enzymes in units/ml (De Young et al., 1989). The readings were done in replicate (three to six), and repeated for three consecutive days.

2.9. Statistical analysis

Results were expressed as mean7standard error of the mean (SEM) analyzed by one-way or two-way ANOVA, and as apost hoc,

followed respectively by Student-Newman-Keuls or Dunnett tests. Results with values of po0.05 were considered statistically signiﬁcant.

3. Results

In this study we obtained an average of 4.5 g of pulp oil with an average yield of 45%. Using GC/MS analysis it was possible to

identify the predominant presence of unsaturated (64.2%), as compared to saturated fatty acids (35.8%), as shown inTable 1. During daily treatment with OFCC at doses of 500 mg/kg, 1000 mg/kg and 2000 mg/kg OFCC, no behavioral changes, nor physical changes, nor deaths in the experimental groups were observed. There were no significant weight gains (Table 2), The animals' lipid profiles' (Table 3), at 30 days of treatment, revealed that total serum cholesterol levels had decreased by 16% in the group treated with OFCC 2000 mg/kg as compared to the saline control. Similarly, serum triglyceride levels also decreased by 23% in the group treated with OFCC 2000 mg/kg as compared to the controls. On the other hand, treatment with OFCC significantly increased HDL-C levels in animals treated for 15 days [500 mg/kg (35%), 1000 mg/kg (41%) and 2000 mg/kg (39%)], and at 30 days [500 mg/kg/day (44%), 1000 mg/kg/day (62%), and 2000 mg/kg/ day (71%)]. No changes in lipid profile were observed with OFCC treatment for seven days.

The hypertriglyceridemia induced by Tyloxapol was reduced by OFCC at all three doses tested in the experiment when compared to the control treated with saline (Table 4). This, for both thefirst 24 h [500 mg/kg (37%), 1000 mg/kg (96%) and 2000 mg/kg (83%)] as well as at 48 h [500 mg/kg (68%), 1000 mg/kg (93%) and 2000 mg/kg/(83%)]. Similarly, OFCC reduced hypercholesterolemia for both the first 24 h [500 mg/kg (25%), 1000 mg/kg (79%), and 2000 mg/kg (76%)] as well as at 48 h [500 mg/kg (53%), 1000 mg/ kg (82%), and 2000 mg/kg (71%)]. Further, the OFCC caused an increase in serum HDL-C at 24 h [1000 mg/kg (26%)] and at 48 h [1000 mg/kg (100%)] after dyslipidemia induction. Importantly, OFCC had an effect similar to Gemfibrozil which itself however, was unable to increase plasma HDL-C levels as did OFCC within this protocol.

Treatment with OFCC for 7 days significantly reduced the paw edema induced by carrageenan (T0-T24), at doses of 500 mg/kg [in T3 (21%)], at 1000 mg/kg [in T3 (31%)] and at 2000 mg/kg [in T2 (31%) and T3 (38%)] with respect to the group treated with saline. Indomethacin (10 mg/kg, p.o.) reduced the amount of edema sig-nificantly compared to the control group [in T3 (30%)], but there were no differences as compared to animals treated with OFCC. Treatment with OFCC for 15 days significantly reduced the paw edema induced by carrageenan at doses of 500 mg/kg [in T3 (31%) and T4 (33%)] 1000 mg/kg [in T4 (21%)] and 2000 mg/kg [in T3 (31%) and T4 (29%)] compared to the group treated with saline. Indomethacin (10 mg/kg, p.o.) reduced the amount of edema sig-nificantly compared to the control group [in T2 (38%), T3 (46%) and T4h (41%)], but with no differences as compared to animals treated with OFCC. Treatment for 30 days with OFCC also significantly reduced the paw edema induced by carrageenan at doses of 500 mg/kg [in T2 (46%), T3 (46%) and T4 (48%)], at 1000 mg/kg [in T2 (28%), T3 (23%) and T4 (16%)] and 2000 mg/kg [in T2 (42%), T3 (34%) and T4 (36%)] as compared to the saline control. Similarly, indomethacin (10 mg/kg, p.o.) significantly reduced the volume of the edema compared with the group treated with saline [in T2 (36%), T3 (29%) and T4 (28%)] (Table 5), with no differences in relation to groups of animals treated with OFCC (Fig. 1).

Table 1

Chemical constituents identiﬁed in OFCC.

Fatty acids Retention time (min) (%)

Palmitic (C16:0) 25.5 34.1

Stearic (C18:0) 29.1 1.7

Palmitoleic (C16:1) 26.7 0.3

Oleico (C18:1) 29.5 55.8

Linoleic (C18:2) 29.9 1.8

Heptadecenoic (C17:1) 30.6 5.9

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The histopathologic analysis of the aorta and heart did not reveal changes in the tissues of the structure pattern after ad-ministration of 2000 mg/kg, 1000 mg/kg and 500 mg/kgﬁxed oil (OFCC) in rats for 7, 15 and 30 days (Figs. 2,3and4). But, in liver

was demonstrated focal steatosis only in two of ten animals treatment for 30 days with a dose of 2000 mg/kg (Fig. 5).

Finally, the OFCC showed a biphasic effect on myeloperoxidase activity. At low concentrations [0.05

μ

g/ml (47.872.3), 0.1

μ

g/ml (56.472.3), and 1

μ

g/ml (41.272.5)] the oil was able to inhibit MPO activity similarly as compared to indomethacin 37.7

μ

g/ml (47.674.6). On the other hand, at higher concentrations [10

μ

g/ml

(20.272.5), 50

μ

g/ml (22.872.3), 100

μ

g/ml (23.671.6) and

200

μ

g/ml (15.072.2)], there was a loss of MPO enzymatic activity inhibition (Fig. 1).

4. Discussion

Caryocar coriaceum(OFCC)ﬁxed oil was chemically character-ized; both saturated and unsaturated fatty acids were identiﬁed, the major component being oleic acid, followed by linoleic acid. Similar results have been found in previous studies conducted in our laboratory with the OFCC (Ramos and Souza, 2011; Saraiva et al., 2008).

Oral administration of OFCC caused no change worthy of note, as the little affected weight gains of the treated animals demon-strate. After six weeks of diet with pequi oil, there were no dif-ferences in weight gain between the groups (Aguilar et al., 2012). The hypolipidemic activity of OFCC was observed to commence after 15 days of oil administration. However, the best results were obtained at 30 days of treatment with a reduction in serum tri-glycerides and total cholesterol associated with an increase in HDL-C. A histopathological examination of the liver (data not shown) indicated the occurrence of micro-goticular steatosis in two of the animals treated for 30 days with OFCC at 2000 mg/kg. This effect was not observed in the other groups. In recent dec-ades, signiﬁcant effort has been channeled into the development of non-pharmacological approaches which reduce the risk of cor-onary heart disease. (Balligand, 2001; Jaye and Krawiec, 2004;

Shahar et al., 2003).

The beneﬁcial effects of monounsaturated fats of vegetable origin have been found in olive, canola, nuts, and avocados, which reduce the risk of developing cardiovascular disease. An isocaloric replacement of a diet of saturated fatty acids with polyunsaturated fatty acids reduces plasma total cholesterol (TC), and LDL-c ( Jack-son et al., 2006). Monounsaturated fatty acids have the same ef-fects on cholesterolemia (Sposito et al., 2007). For instance, oleic acid is a better substrate for lecithin-cholesterol acyltransferase (LCAT) in the liver; this might be one of the possible reasons why it does not raise LDL-c counts. Excess cholesterol in free form is ra-pidly esteriﬁed, not inducing LDL receptor suppression. Further, as compared to polyunsaturated fatty acids, oleic fatty acid induces less endogenous cholesterol synthesis (Sposito et al., 2007).

Since the treatment for 15 days showed no signs of toxicity (macrogoticular steatosis); did not affect triglyceride levels or total cholesterol; and increased HDL-c levels; this same protocol was used to test the effect of OFCC facing Triton WR-1339-induced hyperlipidemia. The intraperitoneal administration of Triton

Table 2

Body weight gain in rats treated daily with OFCC.

0 Day 07 Days Gain (%) 15 Days Gain (%) 30 Days Gain (%)

Control 192.876.7 196.976.6 2.1 210.376.2 9.1 241.278.2 25.1#

OFCC 500 199.176.0 202.976.2 1.9 208.975.9 4.9 237.275.0 19.1#

OFCC 1000 196.676.7 201.475.9 2.4 209.478.4 6.5 236.976.8 20.5#

OFCC 2000 194.276.7 198.776.1 2.3 204.777.4 5.4 234.876.5 20.9#

Values represent mean7S.E.M., (n¼10). Results were analyzed by ANOVA two way and Dunnet test as apost hoctest.

#_p_o_{0.0001 vs control 0 day.}

Table 3

Lipid proﬁle in rats treated daily with OFCC.

07 Days 15 Days 30 Days

Triglycerides (mg/dL)

Control 132.8711.5 122.778.6 139.176.8

OFCC 500 135.177.4 102.876.9 131.277.9

OFCC 1000 133.675.4 128.474.4 123.576.5

OFCC 2000 121.376.7 143.6714.1 106.874.2a Total Cholesterol (mg/dL)

Control 108.073.7 121.078.0 133.975.3

OFCC 500 114.375.5 108.074.4 127.3714.3

OFCC 1000 103.574.3 120.979.6 136.574.7

OFCC 2000 107.672.0 139.379.7 111.776.8a HDL-c (mg/dL)

Control 67.2075.3 86.677.5 49.671.9

OFCC 500 74.1675.1 117.176.9a _71.4₇_2.7a OFCC 1000 65.4375.5 122.078.5a _80.5₇_2.2a OFCC 2000 80.6274.6 120.477.6a _84.8₇_4.7a

Values represent mean7S.E.M., (n¼10). Results were analyzed by ANOVA and Student-Newman-Keuls test as apost hoctest.“a”vs Control when po0.05.

Table 4

Lipid Proﬁle of OFCC rats treated for 15 days and subjected to dyslipidemia induced by Triton WR 1339.

24 h 48 h

Triglycerides (mg/dL)

Control 103.5710.8 79.576.8

Triton WR 1339 2276731.52a ₁₂₅₄₇₁₀₆a GenﬁbrozilþTriton WR 1339 73.776.37b _62.6₇_6.83b OFCC 500þTriton WR 1339 14417216.9a,b,c _395.5₇_{3 8.6}a,b,c OFCC 1000þTriton WR 1339 81.9711.5b _89.2₇_11.6b OFCC 2000þTriton WR 1339 397.5795.7b _215.5₇_63.1b Total Cholesterol (mg/dL)

Control 8272.1 81.574.2

Triton WR 1339 536.6714.3a ₃₉₂₇_28.3a GenﬁbrozilþTriton WR 1339 73.776.3b _74.5₇_5.8b OFCC 500þTriton WR 1339 400.775 7.0a,b,c ₁₈₆₇_17.9a,b,c OFCC 1000þTriton WR 1339 112.178.8b _71.2₇_8.3b OFCC 2000þ_{Triton WR 1339} 130.379.5b _114.3₇_11.7b HDL-c (mg/dL)

Control 28.972.4 25.773.7

Triton WR 1339 19.672.5a _11.7₇_2.1a GenﬁbrozilþTriton WR 1339 19.471.0a ₁₀₇_0.7a OFCC 500þTriton WR 1339 16.971.2a _12.8₇_1.0a OFCC 1000þTriton WR 1339 24.771.2 23.671.5

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WR-1339, a non-ionic detergent, induces marked hypertriglycer-idemia in normolipemic rats. Maximum peak levels of cholesterol, triglycerides, and phospholipids occur at around 20 h and then

decrease (Frantz and Hinkelman, 1955; Schurr et al., 1972). The agent blocks bothclearance of lipoproteins and intravascular de-gradation; while stimulating hepatic lipoprotein synthesis. After Tritons

injection, some studies have shown increased acetate-based hepatic cholesterol synthesis (Frantz and Hinkelman, 1955) and increasing HMG-CoA reductase activity (Goldfarb, 1978). In our study, after administration of Tyloxapol detergent, increased serum cholesterol and triglyceride levels were observed; corro-borating the literature. On the other hand, OFCC administration reduced both hypercholesterolemia and hypertriglyceridemia, and increased CDH-c serum levels. The OFCC lipid-lowering effect in this model may be related to inhibition of HMG-CoA reductase (a cholesterol biosynthesis limiting enzyme, which is inhibited by statins such as gemﬁbrozil), or by activation of LPL (an enzyme related to VLDL triglyceride hydrolysis). However, more studies are needed to conﬁrm these hypotheses.

Table 5

Effect of daily treatment with OFCC on carrageenan induced edema.

T0 T1 T2 T3 T4 T24

07 Days

Control 1.0970.05 0.5470.03 1.0370.07 1.7470.09 1.6370.10 1.3570.10

Indo 10 1.1670.01 0.4870.03 1.0970.08 1.3270.07a _1.58₇_0.10 _0.54₇_0.06ª

OFCC 500 1.1270.04 0.4970.04 0.9670.11 1.4170.10ª 1.7270.10 1.6470.29

OFCC 1000 1.0270.04 0.4370.04a _0.81₇_0.11 _1.13₇_0.12ª _1.56₇_0.15 _0.95₇_0.14a OFCC 2000 1.1370.04 0.3270.03a _0.74₇_0.08a _1.11₇_0.12ª _1.30₇_0.09a _0.86₇_0.09ª 15 Days

Control 1.1870.04 0.5670.07 1.0970.12 1.5470.15 1.7470.22 0.7970.12

Indo 10 1.1470.05 0.3370.04a _0.65₇_0.06a _0.80₇_0.13a _1.00₇_0.07a _0.76₇_0.13 OFCC 500 1.0970.03 0.3770.05a _0.76₇_0.09a _0.98₇_0.10a _1.08₇_0.13a _0.86₇_0.14

OFCC 1000 1.1470.05 0.5470.09 0.9670.15 1.2070.13 1.3370.13 0.8270.15

OFCC 2000 1.1070.04 0.4570.06 0.8470.08 0.9970.08ª 1.1570.08a _1.22₇_0.20 30 Days

Control 2.4870.40 1.2870.23 2.3570.36 2.9870.39 3.4270.49 1.7970.19

Indo 10 2.5170.44 0.9070.14a _1.53₇_0.24a _2.14₇_0.35a _2.50₇_0.44a _0.64₇_0.13a OFCC 500 2.5670.44 0.7170.16a _1.30₇_0.22a _1.65₇_0.27a _1.85₇_0.30a _0.48₇_0.08a OFCC 1000 2.4270.39 0.8070.13a _1.64₇_0.28a _2.23₇_0.34a _2.81₇_0.40 _1.00₇_0.14a OFCC 2000 2.4270.40 0.7470.11a _1.33₇_0.18a _1.91₇_0.23a _2.14₇_0.28a _0.65₇_0.11a

Values represent mean7S.E.M., (n¼10). Results were analyzed by ANOVA and Student-Newman-Keuls test as apost hoctest. The values were considered signiﬁcant when po0.05.“a”vs Control when po0.05.

Fig. 1.Effect of OFCC on Myeloperoxidase (MPO) enzymatic activity. The bars re-present mean7S.E.M., (n¼10). Results were analyzed by ANOVA and Student-Newman-Keuls test as apost hoctest.“a”vs INDO 37.5 when po0.05.

Fig. 2.Histologically normal heart (left ventricle–the left (*VE)/Right ventricle (RV*) – right). Treatment with OFCC for 30 days at a dose of 2000 mg/Kg. Mi¼myocardium; N¼endocardium; P¼epicardium.

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In a recent study byAguilar et al. (2012), a diet supplemented for 6 weeks with intake of Pequi oil (Caryocar brasiliense) revealed atherosclerotic lesions in the aortic root, and a more atherogenic lipid proﬁle.Caryocar brasilienseCambess (Caryocaraceae) though a widely distributed tree is mainly found in theCerrado(or Bra-zilian savanna). Its oil product has fat-soluble vitamins and both saturated and monounsaturated fatty acids in high levels. Similar to Caryocar coriaceum, the presence of high amounts of car-otenoids and phenolic acids is responsible for its antioxidant properties (FDA, 1995). Although oleic acid, a monounsaturated fat is also present in the two species in similar concentrations (54.28%

vs. 55.79%), palmitic acid (saturated fat), is present in Caryocar brasiliensein signiﬁcantly higher concentrations than inCaryocar coriaceum (41.78%vs. 34.1%) (Aguilar et al., 2012). Palmitic acid, (predominant inC. brasiliense) is one of the most atherogenic fatty acids, and is widely linked to hypercholesterolemia, and hyper-triglyceridemia (Khouri et al., 2007). Thus, differences in chemical composition explain the difference observed in the animals' lipid proﬁles.

The carrageenan used in the paw edema model as a phlogistic agent, increases vasodilation, blood permeability, and pain. One hour after carrageenan administration, due to the release of

histamine, and serotonin, and other early inﬂammatory mediators, an increase in paw volume was observed. The peak maximum swelling occurs at 3–4 h, coinciding with the increase in

pro-in-flammatory eicosanoids (late mediators) (Di Rosa et al., 1971). Treatment with OFCC for 7, 15 and 30 days significantly reduced the paw edema induced by carrageenan at all of the doses tested. In the literature, some studies have shown that the presence of fatty acids in OFCC may be related to anti-inflammatory activity. The oleic acid protection mechanism against inflammation has been attributed to a decrease in the linoleic acid content of LDL (Covas et al., 2006). Oleic and palmitic fatty acids were shown to moderately inhibit platelet aggregation as induced by arachidonic acid, and to slightly inhibit formation of thromboxane (Hohlfeld et al., 2008).

Henry et al. (2002) observed that oleic acid shows moderate inhibitory activity for COX-1 (25%) (Henry et al., 2002). Oleic and palmitic acids, being major components of OFCC, were found in the chemical composition, and with oral administration may partly explain decreases in inflammatory activity. Linoleic acid, a poly-unsaturated fat found in OFCC, has also been identified in the literature as anti-inflammatory. This study was showed that lino-leic acid (LA) can inhibit COX-1 and COX-2in vitro, and that human monocytic THP-1 cells treatedin vitrowith LA showed significant reductions in secretion of IL-6, IL-1

β

, TNF-

α

, and transcription factor NF-kB (Zhao et al., 2005).

Myeloperoxidase (MPO) is a protein with a molecular weight of 144 kD, consisting of two dimers, containing one subunit with light and heavy chains, and with functionally identical heme groups that are joined together by a disulfide bridge. This enzyme is the major constituent of the azurophil granules of neutrophils, it is also found in certain monocytes and tissue macrophages. It is derived from leukocytes and catalyzes the formation of numerous reactive oxidant species, contributing to the body's innate immune response, and to tissue damage during inflammation (Fu et al., 2001). In our work, OFCC at low concentrations reduced the re-lease of MPO from polymorphonuclear cells. This result corrobo-rates thein vivoanti-inflammatory effect of OFCC, discussed ear-lier. It is also known that MPO participates in pro-thrombotic biological processes related to the development of cardiovascular disease, including initiation, propagation and acute complications of atherosclerosis (Hansson, 2005).

Studies have demonstrated the role of MPO as a central parti-cipant link between inﬂammation and cardiovascular disease. In addition, the participation of MPO in the lipid load composition of atherosclerotic plaque, activation of proteases and in vasocon-striction and thrombosis mechanisms evidences its involvement in the development of atherosclerotic disease and thrombotic com-plications (Nicholls and Hazen, 2005;Spagnoli et al., 2007). Data suggest that the MPO activity may serve as a marker of cardio-vascular disease, causing potential instability and progression of atherosclerotic plaques in the ischemic stage.

Histopathological analysis also revealed no signiﬁcant toxicity or changes in the liver, heart and aorta of animals treated with OFCC for 7 and 15 days in doses of 2000, 1000 and 500 mg/kg. In the liver, steatosis microgoticular it was observed in two animals for 30 days treatment at a dose of 2000 mg/kg, an effect not ob-served in the other experiments. As well as in the heart, aorta and there was no evidence of fat deposit or atherosclerotic disease signs.Almeida et al. (2012)investigated thein vivogenotoxicity of

Caryocar villosumand his antigenotoxicity of potential data in DNA induced by doxorubicin (DXR) (Almeida et al., 2012). Phytochem-ical analysis revealed the presence of carotenoids, phenolic com-pounds includingﬂavonoids; tannins and

α

-tocopherol in pequi pulp. There were no statistically signiﬁcant differences in the parameters evaluated, demonstrating the absence of cytotoxic and genotoxic effects of“Pequi”at all doses tested. In the kidney, liver,

Fig. 4.Liver histologically normal. *Space-door Representation in the center of the Figure. Treatment with OFCC for 30 days at a dose of 2000 mg/kg. Ah¼branch of the hepatic artery; Vp¼portal vein; Db¼duct bilífero; He¼hepatocyte; Si: sinusoid.

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heart and bone marrow cells was significantly reduced the DNA damage induced by DXR dose-response (Almeida et al., 2012). The chemical characteristics and fatty acid composition of Coconut oil show presence of lauric acid content (46.64–48.03%) (Marina et al., 2009). Coconut oil is said to have a functional food due to its perceived health benefits (Shankar et al., 2013), as potential ben-eficiary effect of virgin coconut oil in lowering lipid levels in serum and tissues and LDL oxidation by physiological oxidants (Nevin and Rajamohan, 2004).

5. Conclusion

Taken together,Caryocar coriaceum's(in vivoandin vitro) anti-inflammatory and hypolipidemic effects recommend its potential cardioprotective effect; since repeated administration of the plant's oil improved the lipid profiles of animals with dyslipide-mia, and reduced inflammation, events commonly associated with cardiovascular disease. However, further research is needed to provide conclusive evidence on its clinical applications.

Conﬂict of interest

All authors wish to confirm that there are no known any actual or potential conflicts of interest, including any personal or others with other persons or organizations associated with this publica-tion and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgement

This research was supported by FUNCAP/BPI, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Con-selho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq).

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