Antioxidant and antimicrobial potential of essential oils of different types of pepper ( Piper sp. ) Antioksidativni i antimikrobni potencijal eteričnih ulja različitih

Essential oils are mixtures of potent compounds with biological effects, such as those with antioxidant, anti-microbial, antitumor and/or anti-inflammatory effects. In this paper, the antioxidant and antimicrobial activity of essential oils of different types of commercial pepper was tested. Essential oils are prepared by hydrodisti-llation. Polyphenol content, reduction potential and DPPH radical inhibition were analyzed. Antimicrobial ac - tivity was tested on reference strains using the diffusion technique. The analysis showed that the essential oil of black pepper has a significantly higher antioxidant potential compared to the essential oils of green and white pepper. High antimicrobial activity was also found for the black pepper essential oil sample, except in the case of Pseudomonas aeruginosa .

through several studies (Wang et al., 2021;Zhang and Xu, 2015). The aim of this study was to evaluate the antioxidant and antimicrobial activity of the essential oils obtained by hydrodistillation of three commercially available pepper samples.

Materijal i metode
All pepper seed samples were commercially available. White pepper originates from India, while green and black pepper originate from Brazil. Demineralized water was used to prepare the solution for spectroscopic measurements. Folin-Ciocalteu reagent for testing polyphenol content and sodium carbonate were purchased from Semikem, Bosnia and Herzegovina. Methanol HPLC grade, 2,4,6-Tripyndyl-s-triazine, iron(III) chloride, hydrochloric acid, sodium acetate and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (USA). Spectroscopic measurements were performed on a Perkin Elmer λ25 spectrophotometer.

Hydrodistillation
Five hundred grams of finely ground pepper samples were subjected to hydrodistillation using Clevenger-type apparatus for 5 h according to the European Pharmacopoeia. Essential oils were dried over sodium sulfate anhydrous and the sample was stored at 4°C before use.

Determination of total phenolic content (TPC)
Total phenolic compounds present in the essential oil were quantified spectrophotometrically through the Folin-Ciocalteu test following the protocol (Singleton et al., 1999). 200 μL of essential oil was mixed with 2.54 mL of 10% Folin-Ciocalteu reagent. After 5 min 420 μL of 10% sodium carbonate was added. 910 μL distilled water was added to each sample prior to measuring. The absorbance of the resulting blue-coloured solution was measured at 765 nm.

Examination of the reducing ability (FRAP method)
The test of the reducing ability of the essential oil was tested using the FRAP (ferric reducing antioxidant power) method, according to the published procedure (Benzie and Strain, 1999). 3 mL of prepared FRAP reagent was mixed with 100 μL of essential oil. Absorbance was measured at 593 nm after 30 min incubation at 37°C.

Inhibition of DPPH radicals
A DPPH radical inhibition assay was performed according to a published method (Horozić et al., 2019). Essential oils were mixed with absolute methanol and then mixed with DPPH radical solution. Absorbance measurements were performed at 517 nm, after which DPPH radical inhibition was calculated according to the equation: where As is the absorbance of the solution containing the sample at 517 nm, and Ac is the absorbance of the DPPH solution.

Analysis of antimicrobial activity
The in vitro antibacterial activities of the essential oils were investigated using Gram-positive bacteria (B. subtilis and S. aureus) and Gram-negative bacteria (E. coli and P. aeruginosa). C. albicans was used to test the antifungal activity of the isolated oils. From the microorganisms strains of overnight cultures, suspensions of 0.5 McFarland turbidity were prepared (density 10 7 -10 8 CFU/mL). The strains were then placed on the surface of the nutrient substrate Mueller-Hinton agar, and dispersed in sterile Petri dishes. Substrate thickness was 4 mm. In the agar, sterile drill-shaped holes were made ("wells"), into which 100 μL of essential oil was poured. After the plates were left at room temperature for 15 minutes,

RESULTS -Rezultati
Hydrodistillation obtained essential oils with a particularly strong aroma, with a good yield. Table 1 shows the yield of isolated oil depending on the type of pepper.  Table 2 shows the results of the content of polyphenols, as well as the values of reduction potential and DPPH radical inhibition in in vitro conditions. Vitamin C was used as a reference. Figure 1 shows graphs of the dependence of DPPH radical inhibition on concentration, through which the IC50 value for essential oil samples was calculated.  Table 3 shows the results of testing antimicrobial activity in in vitro conditions.

DISCUSSION -Diskusija
The content of polyphenols in essential oils of commercial peppers is relatively low, and is correlated with antioxidant capacity. The highest total phenol content was measured in the black pepper sample which also showed the highest antioxidant potential. Li et al. (2020) recently studied black and white pepper and they found that black pepper fruit was slightly better than white pepper fruit in terms of antioxidant activity. From Table  2 it can be seen that the effective concentration (EC50) value of the black pepper fruit was 7.67 mg/mL, which is in good agreement with the previous study (Wang et al., 2021). However, these values obtained for white pepper are significantly lower than those reported previously (Zhang and Xu, 2015). The DPPH radical neutralization efficiency as well as the reduction potential of all tested essential oils is significantly lower than the values obtained for vitamin C, well known antioxidant which was used as a control.
Pepper essential oils generally showed a better antimicrobial effect against gram-positive bacteria B. subtilis and S. aureus, with an inhibition zone in the range of 8-15 mm. The highest sensitivity was confirmed with the essential oil of black and green pepper. White pepper essential oil showed extremely weak antimicrobial activity in in vitro conditions. All tested samples showed antifungal activity. Through research conducted in the last few years, the antibacterial effect of the essential oil of black (Morsy and El-Salam, 2017;Hikal, 2018), white (Singh et al., 2013) and green pepper (Myszka et al., 2019) has been confirmed. In the aforementioned studies, larger zones of inhibition were recorded, which can be explained by the different origin of the samples, and the way the samples were treated and stored.
The presence of terpenoids in pepper essential oil is responsible for its antimicrobial potential (Menon et al., 2003). An important characteristic of essential oil components is their hydrophobicity, which enables them to partition into the lipids of the bacterial cell membrane, disturbing the cell structures, rendering them more permeable, and leading to lysis and leakage of intracellular compounds (Gill and Holley, 2006;Bajpai et al., 2013).
This study on the antioxidant and antimicrobial activities of essential oil from black, green and white pepper fruit proves that pepper essential oils exhibit antioxidant and antimicrobial effects owing to their active components. Black pepper essential oil was more effective than white and green pepper essential oil which is caused by differences in essential oil compositions. From the result of this study, it can be concluded that pepper essential oils have great potential as a natural source of therapeutic agent for bacterial infection. Further studies regarding the chemical composition of essential oils should be performed. Bajpai, V.K., Sharma, A., Baek, K.H. (2013). Antibacterial mode of action of Cudrania tricuspidata fruit essential oil, affecting membrane permeability and surface characteristics of food-borne pathogens. Food Control, 32 (2)