Bioactive compounds and biological properties of oils from three Camellia species

Salinero C.¹, García-Sartal C.¹, Tolentino G.², Estevinho L.M.²

¹ Estación Fitopatolóxica de Areeiro, Deputación de Pontevedra, Subida a la Robleda s/n, 36153 Pontevedra, Spain. carmen.salinero@depo.es
² Departamento de Biologia e Biotécnologia, Escola Superior Agrária de Bragança.
Campus de Santa Apolónia–Apartado 1172, 5301-588 Bragança, Portugal. leticia@ipb.pt

Introduction

     Camellia oil has been traditionally used for cooking and as a protective cosmetic to maintain the health of skin and hair in Asian cultures (Jung et al., 2007). Moreover, camellia oil is often the target for adulteration or mislabelling because it is a high-priced product with high nutritional and medical values (Wang et al., 2006).
     Camellia oil has been recognized as a source of vitamins A, B and E, essential fatty acids, primarily as oleic acid, and also polyunsaturated fatty acids such as omega-6 linoleic acid (Li et al., 2012).  In a previous work, Salinero et al. (Salinero et al., 2011) have characterized the triglyceride composition of oil from C. reticulata and C. japonica harvested in Galicia (North Western Spain) by nuclear magnetic resonance (NMR). Furthermore, other functional components, such as camellia saponins, squalene or polyphenolic compounds have been found in camellia oil.  All these compounds appear to be very promising for possible pharmaceutical exploitation since they confer anti-tumour effects, blood lipid reduction, protection of liver and heart, antisepsis and anti-inflammation effects, atherosclerosis delaying, anti-oxidation activities and immune function regulation on the population (He et al., 2011).
     Even though Camellia spp. have been used in ethnomedicine, there is relatively little information regarding the biological activity of Camellia species’ oils, except for tea oil (Feás et al., 2013).
     This work investigates and establishes for the first time, the physicochemical characterization, bioactive compounds, antimicrobial and antioxidant properties of Galician camellia oils from 8 different Camellia japonica cultivars, 1 C. sasanqua cultivar and 1 C. grijsii.

Materials and Methods

     All the chemicals and solvents used were of analytical grade.

     Camellia seed oils

     Seeds from 10 different camellia specimens, 8 Camellia japonica cultivars, 1 C. sasanqua cultivar and 1 C. grijsii, were harvested in the Galician region (North-western Spain). Table 1 shows the label, camellia specie and origin of the samples under study.

Table 1. Sample name, camellia specie and location of the samples under study

     The seeds were cleaned by hand to remove foreign materials and then washed with tap water. Cleaned seeds were dried at room temperature.  After these treatments, the seeds were ground in a mill. Camellia oil samples were obtained by cold-pressed extraction.  About 200g. of crushed seeds are loaded into a hydraulic press and then they are pressed by mechanical means to extract the oil. The crude oil portions belonging to a given camellia cultivar were blended and decanted and filtered. Finally, the oils were stored in amber coloured glass bottles and kept at 15°C until further analysis.

     Chemical and physical methods

Moisture and volatile matter determination
     Moisture and volatile matter was determined gravimetrically according to the UNE-EN ISO 662-1998 standard method (ISO 662-1998, 2001) as the mass loss suffered by five millilitres of oil sample after heating in an oven at 103 ± 2°C. Determinations were performed in duplicate. Results were expressed as mass percentage.

Density determination
     The density of the samples, expressed as the average value of six replicates, was determined as the measure of the oil mass per unit volume at 25°C.

Acid value
     The acid value (AV) was determined by a standard titration procedure according to the UNE-EN ISO 660:2009 (ISO 660:2010, 2010).  An amount of 10g. of camellia oil was dissolved in a 50:50 mixture of ethanol (purity 96%) and diethyl ether (purity 99.9%). Finally, the acids in solution were titrated with 0.1 M KOH prepared in ethanol solution. Results were expressed as grams of oleic acid per 100g. of oil. These determinations were carried out in triplicate.

Iodine value
     The iodine value (IV) was calculated according to the standard method UNE-EN ISO 3961:2009 (ISO 3961:2009, 2012). Data were obtained from the analysis of each sample oils prepared in triplicate. About 0.20g of sample oil was dissolved in a mixture of cyclohexane and glacial acetic acid (50:50). Finally, the excess of iodine generated after adding Wijs solution, potassium iodide and deionized water, was titrated with sodium thiosulphate. Results were expressed as grams of iodine per 100 grams of oil.

Peroxide value
     To determine the peroxide value (PV), a portable HANNA instruments® photometer HI 83730 (Guipúzcoa, Spain) equipped with a commercially available kit of reactive was used. Results, expressed as milliequivalents of oxygen per kilogram of oil, were calculated as the average value of three replicates of the same sample.

Spectrophotometric determination of polyphenols
     The determination of the polyphenols was carried out according to Capannesi et al. (2000). Galic acid standard solutions using methanol were used for constructing the calibration curve (y = 0.3442x + 0.0062; R² = 0.9977).
     A 50ml solution was prepared containing: 1 ml of a standard solution of gallic acid, 6 ml of methanol, 2.5 ml of the Folin±Ciocalteau reagent and 5 ml of 7.5% Na₂CO₃. The solutions were stored overnight and the spectrophotometric analysis was performed at   λ=765 nm.
     The determination was carried out by diluting 2.5g. of camellia oil in 2.5 ml of n-hexane. Then, the mixture was extracted three times by 5 min centrifugation (5000 rpm) with CH₃OH:H₂O (80:20 v/v). The extract was added to 2.5 ml Folin±Ciocalteau reagent, 5 ml of Na₂CO₃ (7.5%), in a 50 ml volumetric flask to which purified water was added. The samples were stored overnight, and the spectrophotometric analysis was performed at λ=765 nm.

DPPH Scavenging Activity
     Various concentrations of oil extract (300 µL) were mixed with 2.7 ml of MeOH solution containing DPPH•, at a concentration of 6 × 10-5 mol/L. The mixtures were shaken vigorously and left 60 min in the dark, until stable absorption values were obtained. The reduction of the DPPH• was measured by continuous monitoring of the decrease of absorption at 517 nm. The radical-scavenging activity (RSA) was calculated as a percentage of DPPH discoloration using the equation: %RSA = [(A_DPPH - A_S)/A_DPPH] × 100, A_S being the absorbance of the solution when the extract oil was added at a particular level and A_DPPH the absorbance of the DPPH solution (Morais et al., 2011).

ß-Carotene Bleaching (BCB) Assay
     This assay has been previously described by Feás et al. (2013). In brief, 2 ml of ß-carotene solution (2 mg ß-carotene/10 mL chloroform) were added to 40 mg of linoleic acid and 400 mg of Tween 20. The mixture was shaken and portions of 4.8 ml were transferred into different test tubes containing different oil concentrations (200 µL). After that, these mixtures were incubated in a water bath at 50°C for 60 min.  As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm. Absorbance readings were then recorded at 20-min intervals until changes were observed in the colour of the control sample. A blank, in the absence of ß-carotene, was prepared for background subtraction. Lipid peroxidation inhibition (LPO) was calculated using the equation: LPO = (ß-carotene content after 2 h of assay/initial ß-carotene content) × 100.

Microbial Strains
     In the present study, microorganisms isolated from biological fluids were used, which were collected in the Northeast Hospital Centre (Bragança, Portugal) and identified in the Microbiology Laboratory of Escola Superior Agrária de Bragança, using molecular biology techniques.
     The isolates were stored in Muller-Hinton medium with 20% glycerol at -70ºC. The inoculum for the assays were prepared by diluting cell mass in 0.85% NaCl solution adjusted to 0.5 MacFarland scale confirmed spectrophotometrically at 580 nm for bacteria and 640 nm for yeasts. Cell suspensions were finally diluted to 10⁴ CFU/ml.

Determination of the antimicrobial activity
     Antimicrobial activity was carried out according to Morais et al. (2011), using Nutrient Broth (NB) or Yeasts Peptone Dextrose (YPD) on microplate (96 wells).
     The extracts were diluted in dimethylsulfoxide (DMSO) and transferred into the first well and serial dilutions were performed. The inoculum was added to all wells and the plates were incubated at 37°C. Fluconazol and gentamicine were used as controls. A positive control (inoculated medium), a negative control (medium) and a DMSO control (DMSO with inoculated medium) were used. Antimicrobial activity was detected by adding 20 µL of 0.5% TTC solution. The Minimum Inhibitory Concentration (MIC) was considered to be the lowest concentration of the tested sample able to inhibit the growth of bacteria after 24 h and fungi after 48 h, as indicated by the TCC staining (dead cells are not stained by TTC). All the tests were performed in triplicate (n = 3) and the results are expressed as mg/ml.

Statistical analysis
     Results are shown as mean values ± standard deviation. In each parameter the differences between the samples were analysed using one-way analysis of variance (ANOVA) followed by Tukey's HSD Post-hoc test, in which p≤0.05 were considered significant.

Results and Discussion

     The percentage of extraction of the C. grijsii (7.93±1.48%) was significantly lower than that obtained from the other species. The density of the three types of oils was equal to 0.90g/ml. The percentage of moisture in the Camellia japonica’s oil (0.23±0.13%) differed significantly from that determined for the oil of Camellia sasanqua (0.03±0.02%) and Camellia grijsii (0.04±0.03%). Significant differences were also obtained regarding the iodine index, which ranged from 80.06±0.59 (C. grijsii) to 85.66±0.26g/100g (C. sasanqua). Concerning the acidity and peroxide index, no significant differences were obtained between the three types of oil under study. As it can be seen, Table 2 shows the physical characteristics of the oils.

Table 2. Physical characteristics of the oils. For a given physical parameter, mean values with the same letters (a or b) were similar and no statistically significant differences were observed between samples (p<0.05)

     Regarding the antimicrobial activity, all the oils efficiently inhibited the microorganisms under study. The most sensitive species was Klebsiella ESA53, especially when Camellia grijsii oil was used, being the minimum inhibitory concentration equal to 5.11±2.05 mg/mL. The minimum inhibitory concentration of the three oils against Salmonella varied between 10.84±4.06 mg/mL (C. grijsii) and 12.91±4.18 (C. japonica). Regarding S. epidermides the most efficient oil was, again, that obtained from Camellia grijsii and the least efficient was that extracted from C. sasanqua. Cryptococcus neoformans was the most resistant microorganism and the three minimum inhibitory concentrations did not differ significantly (p><0.05). Table 3 shows the antimicrobial activity of the three types of oil under study.

Table 3. Antimicrobial activity of the three types of Camellia spp. oil. For a given specie, mean values with the same letters (a or b) were similar and no statistically significant differences were observed between samples (p<0.05)

     Concerning the phenolic compounds, the concentrations were between 0.02±0.001 and 0.04±0.00, for Camellia japonica and for both C. sasanqua and C. grijsii, respectively. Antioxidant activity was higher in C. grijsii, independent of the used methodology. Results are shown in Table 4. Significant differences were observed for the bioactive compounds and antioxidant activity between the oils.  In addition, the species with higher concentration of polyphenols were the most efficient in the neutralization of free radicals.

Table 4. Polyphenolic compounds and antioxidant activities of the Camellia spp. oils. For a given assay, mean values with the same letters (a or b) were similar and no statistically significant differences were observed between samples (p<0.05)

Conclusions

     The results obtained demonstrate the antioxidant and antimicrobial activities of the three Camellia spp. oils, Klebsiella being the most sensitive to the inhibitory effect.
     The findings hereby reported open new possibilities for future therapeutic applications of the Camellia oils, even though further studies are needed to analyse the potential of in vivo use.

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