Antibacterial Properties of Pequi Pulp Oil (Caryocar coriaceum – WITTM.)

Abstract

Pequi fruits have a high nutritional value, and they are rich in protein and vitamins. This work reports the results of the chemical characterization and antibacterial activity of “pequi” fruit pulp oil of Caryocar coriaceum (Wittm). Chemical composition was assessed by GLC and the antibacterial activity was assayed by the disk diffusion method. GLC indicated the following fatty acid content: oleic (55.79 g/100 g oil), palmitic (34.18 g/100 g oil), heptadecenoic (5.86 g/100 g oil), linoleic (1.80 g/100 g oil), stearic (1.73 g/100 g oil), eicosenoic (0.37 g/100 g oil), and palmitoleic (0.27 g/100 g oil). The oil showed antibacterial activity, inhibiting the growth of Salmonella cholerasuis (MIC < 1.25%), Staphylococcus aureus (MIC 1.25%), Pseudomonas aeruginosa (MIC 1.25%) and Streptococcus pneumoniae (MIC 5%). There is great interest in extending the use of this natural product so that derivatives with combined social, economic and therapeutic value can be produced.

Keywords:

• Caryocar coriaceum
• Oil composition
• Antibacterial activity
• Oleic acid
• Pequi

INTRODUCTION

The genus Caryocar is widespread in South America and comprises more than twenty species. In Brazil, considering an area including the states of Amazonas, Pará, Maranhão, Piauí, Goiás, Bahia, Ceará, São Paulo and Minas Gerais, the main species are Caryocar brasiliense (Cambess.) and Caryocar coriaceum (Wittm.).[1,2] Caryocar coriaceum (Wittm.), popularly known as “piqui” or “pequi,” is the only representative of the family Caryocaraceae in Ceará State, and is one of the most noticeable agrestal plants around Araripe.[3] It has drupaceous fruits that are orange-sized, globe-shaped, with yellowish-green peel and a large and fleshy nut; a whitish, butyraceous mesocarp is protected by a ligneous endocarp possessing thin and sharp erect spines.[4,5] The fruit pulp is an excellent nutritional source, and is largely used by the population of the Cariri region, in Ceará State, and of neighboring cities of Pernambuco and Piauí states.[3,4] Besides being a nutritional source, the handmade oil extracted from the pulp or the nut is widely employed to treat respiratory tract infections, muscle pain, and chronic arthritis.[6] It also plays a part in popular medicine preparations used by farmers for wounds, contusions, peelings and swelling in animals.[3]

The diet of the Brazilian population is extremely lacking in lipids, especially in the North and Northeast regions of the country. Such low-calorie diets, together with the lack of the main groups of nutrients, results in chronic conditions that impair health.[5] The abundance of native plants of the species C. coriaceum (which is a rich source of essential fatty acids) on the Araripe plateau, and its observed therapeutic potential, suggest that it is a promising species for the food and pharmaceutical industries.[7,8] However, the scarcity of studies regarding this species is evident, and a thorough study of the physicochemical and microbiological properties of pequi fruit pulp is justified. In this work, chemical characterization and microbiological studies of the pulp oil of C. coriaceum are presented, in order to present an outlook of a potential source of unsaturated fatty acids and its antibacterial properties.

MATERIALS AND METHODS

Plant Material

Ripe fruits of C. coriaceum were collected on the Araripe plateau (line D of the National Araripe Forest) in the city of Crato, Ceará State, Brazil, and the exocarp (peel) was separated from the inner mesocarp plus endocarp (oily pulp with drupe).

Oil Extraction

The oil was extracted from 752 g of pequi pulp, carried out at 60°C with hexane by the continuous technique in a Soxhlet extractor for 8 h. Afterwards hexane fraction was dried with Na₂SO₄ and the solvent was concentrated in a rotary evaporator under reduced pressure.

Fatty Acids Determination

Fatty acids were determined indirectly using their corresponding methyl esters. The oil (0.2 g) was saponified for 30 min under reflux with potassium hydroxide solution in methanol, following the method described by Hartman and Lago.[9] After adequate treatment and pH adjustment, the free fatty acids were methylated with methanol by acid catalysis in order to obtain the respective methyl esters.

Gas Liquid Chromatography (GLC) Analysis

The analysis of volatile constituents was carried out in a Hewlett-Packard GC/MS, model 5971, using the non-polar fused silica column DB-1 (30 m × 0.25 mm i.d., 0.25 ྒྷm film), eluted with helium gas at 8 mL/min with split mode. Injector and detector temperatures were set to 250°C and 200°C, respectively. The column temperature was programmed from 35°C to 180°C at 4°C/min, and then from 180°C to 250°C at 10°C/min. Mass spectra were recorded from 30 to 450 m/z, with an electron beam energy of 70 eV. The individual components were identified by computer MS library searches, using retention indices as a pre-selection routine, and visual inspection of the mass spectra from the literature for confirmation,[10,11] as well as by visually comparing standard fragmentation to that reported in the literature.[12,13]

Antibacterial Assays

Antibacterial activity and minimum inhibitory concentration (MIC) were assessed using the agar diffusion method.[7] The microorganisms considered in the experiments were obtained from the American Type Culture Collection (ATCC), namely: Shigella flexineri (ATCC12022), Salmonella choleraesuis (ATCC13314), Staphylococcus aureus (ATCC12692), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC15442), Klebsiella pneumoniae (ATCC10031) and Streptococcus pneumoniae (ATCC6314). Using a sterile swab, bacteria were replicated in Petri dishes previously prepared with Müller-Hilton agar. Paper disks, 6 mm in diameter, were impregnated with 20 μL of oil solution at concentrations of 10, 5, 2.5, and 1.25%, and placed in the center of the agar plate. Essays were carried out in triplicate, using ampicillin (AMP, 100 μg/disk) and chloranphenicol (CLO, 100 μg/disk) as positive controls, which were obtained from LABORCLIN(Brazil), and Tween 80 and distilled water as negative controls. The dishes were incubated at 37°C for 24 h. MIC was regarded as the lowest oil concentration which completely inhibited bacterial growth, causing the visualization of an inhibition zone.

RESULTS AND DISCUSSION

On average, 40 mL of oil were obtained from the pulp. After the identification of the methyl esters, it was possible to assess the percent chemical composition of the major fatty acids from the lipid fraction of C. coriaceum pulp. The unsaturated fatty acids are predominant over the saturated ones, with 64.09% of the former and 35.91% of the latter. This species, as well as others of the same genus spread over other regions (e.g., C. brasiliense), shows fatty acid content variability (Table 1), which has been attributed mainly to geographic location and climate conditions. The following fatty acid constituents were identified: oleic acid, palmitic acid, heptadecenoic acid, linoleic acid, and stearic acid. The main fatty acid found in the pulp oil was oleic acid, followed by palmitic acid, as occurs in C. brasiliense.[14,15] Previous work reported a high oleic acid content (64.21%) and absence of heptadecenoic acid, which was found to be 5.86% in our investigation.[16]

Table 1 Methyl esters from C. coriaceu m pulp compared to the ones from C. brasiliense

	Content (g/100 g oil)
Fatty Acid	C.coriaceum ^a	C.coriaceum ^b	C.brasiliense ^c	C.brasiliense ^d
Palmitic (C16:0)	34.18	31.65	34.4	40.2
Palmitoleic (C16:1)	0.27	–	2.1	1.4
Estearic (C18:0)	1.73	1.83	1.8	2.3
Oleic (C18:1)	55.79	64.21	57.4	53.9
Linoleic (C18:2)	1.80	2.29	2.8	1.5
Linolenic (C18:3)	–	–	1.0	0.7
Araquidic (C20:0)	–	–	–	0.2
Heptadecenoic (C17:1)	5.86	–	–	–
Eicosenoic (C20:1)	0.37	–	–	–
^aPresent research;
^bFigueiredo et al. (1989);
^c Brasil (1985);
^dFacioli et al. (1998).

Oleic acid, found in several oils such as olive oil, has shown an interesting anticancer activity, demonstrated alone or combined with trastuzumab against colorectal and breast cancer cell lines, respectively.[17–19] Antibacterial essays in vitro have shown that C. coriaceum pulp oil can act as a growth inhibitor against almost all of the bacteria tested, with exception of K. pneumonia and E. coli (Table 2). The best results were obtained against S. choleraesuis and S. aureus, with inhibition halos of 15 and 13 mm, respectively, the former also accounting for the lowest MIC of 1.25%.

Table 2 Antibacterial activity and Minimum Inhibitory Concentrations (%) of the lipidic fraction from “pequi” pulp towards bacterial lineages. Inhibition halo in mm

	Oil concentration (%)
Microorganism	10	5	2.5	1.25	CLO	AMP
S. flexneri	ND	ND	ND	ND	18.66±0.5	20.66±0.9
S. choleraesuis	15 ± 0.2	10 ± 0.70	10.2 ± 1.2	9.3 ± 0.8	20.7 ± 0.9	19.3 ± 0.9
S. aureus	13.7 ± 1.0	10 ± 0.7	10.7 ± 1.2	ND	25.3 ± 0.5	15.3 ± 0.8
E. coli	ND	ND	ND	ND	22.7 ± 0.5	25.3 ± 0.5
P. aeruginosa	10.3 ± 0.5	10,7 ± 0.05	8 ± 0.8	ND	12.3 ± 0.5	14.3 ± 1.0
K. pneumoniae	ND	ND	ND	ND	15.1 ± 0.1	12.2 ± 0.3
S. pneumoniae	7.7 ± 0.03	ND	ND	ND	19.3 ± 0.5	17.7 ± 0.5
ND: No Detected; CLO: chloranphenicol (30 μg/disk); and AMP: ampicilin (10 μg/disk).

Fatty acids are the main elements of antimicrobial food additives used to inhibit unwanted microorganisms,[20] with several reports about their antibacterial activity, mainly with regard to the unsaturated fatty acids, which inhibit fatty acid synthesis.[21] Hinton and Ingram[22] observed that oleic acid reduces the populations of the bacterial flora on poultry skin. On the other hand, plants rich in this oil are used as traditional medicines to inhibit the growth of infectious agents, as observed in circumcision rites. In these rites, Helichrysum pedunculatuam, a plant rich in oleic and linoleic oils are used to prevent infections.[23]

The best antibacterial activity was observed against Gram-positive bacteria. This result indicates the involvement of the outer membrane of Gram-negative bacteria, as a barrier against hydrophobic substances.[21–23] Many natural products, used as foods or otherwise, have been evaluated worldwide not only for direct antimicrobial activity, but also as resistance-modifying agents, due to the growing incidence of infections resistant to antibiotics.[24–29]

CONCLUSIONS

In conclusion, the oil from pequi pulp was studied by a variety of techniques and characterized with regard to its composition and antibacterial properties. These results are unparalleled in the literature for the C. coriaceum species, and provide a valuable source of data for characterizing the oil. Its composition was determined by GLC, and confirmed by spectroscopic data. The observed properties place pequi oil as a valuable nutritional source of unsaturated fatty acids, with a high energy content, and potential application against some bacterial infections.

ACKNOWLEDGMENTS

To Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico – FUNCAP and to Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq for financial support and to Instituto Nacional de Controle de Qualidade em Saúde/FIOCRUZ.