*3.3. DPPH Free Radical Scavenging*

Antioxidant activity was determined using the DPPH reagent and showed as DPPH radical scavenging percent. Free radical scavenging effect was determined using the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent. 1 mL of the extract was added to 2 mL DPPH. The samples were gently mixed and left to stand in the darkness for 60 min. Absorbance was read at 517 nm using spectrophotometer. A control sample was prepared by mixing DPPH with distilled water. The ability of extracts to scavenge DPPH free radicals was calculated according to the following equation:

$$\text{Radical saving acting activity } [\text{\textquotedblleft}] \text{ AA} [\text{\textquotedblright}] = \frac{\text{Abs}\_{\text{contr}} - \text{Abs}\_{\text{sample}}}{\text{Abs}\_{\text{contr}}} \times 100$$

The values are presented as the means of triplicate analyses.

#### *3.4. Piperine Assay*

Crushed black pepper was extracted with ethanol (97% or absolute), and the obtained extracts were determined spectrophotometrically at a wavelength λ = 342 nm using UV-VIS spectrophotometer Unicam. The determination was made in three repetitions [115].

#### *3.5. Essential Oil Content*

The determination of essential oil content in crushed black pepper was made by steam distillation in a Dering apparatus [116]. The essential oil content was expressed as mL/100 g d.m. Determinations were performed in triplicate.

#### *3.6. Coumarin Content*

The separation of coumarin was obtained in ground cinnamon by the HPLC method using RP-Nova Pack C18 (240 × 4.6 mm, 5 μm) column. A mobile phase was composed of acetonitrile water with a gradient elution at a flow rate of 1 mL/min. The identification of coumarin in the cinnamon samples was conducted based on their retention time compared with the retention time of the standard. The HPLC method was validated in term of linearity, limit of detection, limit of quantification, precision and accuracy.

#### *3.7. Statistical ANALYSIS*

Results were presented as the mean and standard deviation. The experimental designs and calculations were conducted using the Software Package Statistica 10.0 (StatSoft Inc., Tulsa, OK, USA).

The experiments were evaluated using analysis of variance (ANOVA) to find the impact of the type and quantity of spices on the evaluated parameters. Statistical hypotheses were verified at a significance level of *p* values < 0.05.

## **4. Results and Discussion**

The analyses of selected bioactive compounds content and antioxidant activity were carried out in samples of black pepper and cinnamon available on the Polish retail market. These studies will allow to assess the amounts of bioactive substances which Polish consumers will find in these spices. The obtained results were compared with the other authors' studies.

#### *4.1. Analysis of Black Pepper (Piper nigrum L.)*

Black pepper samples (25) were encoded, the total content of polyphenols, antioxidant activity, the content of essential oil and piperine were determined. The analyses were performed in three repetitions and the results are presented in Table 5.

**Table 5.** Antioxidant activity (TP, AA), piperine and essential oil content in black pepper.


*Source: own study.*

#### 4.1.1. Antioxidant Properties of Black Pepper (*Piper nigrum* L.)

The research showed different antioxidant activity of the tested black pepper samples available on the Polish market. The content of total polyphenols ranged from 9.75 to 32.13 mg GAE/g. The results of the statistical analysis confirmed that the pepper origin (brand) significantly influences the total content of polyphenols (KW-H(24) = 114.126, (*p* = 0.000)). The antiradical activity of the tested samples was at a high level, ranging from 63.93% to 85.42%. Statistical analysis showed that the brand of pepper influences the ability to scavenge DPPH free radicals (KW-H(24) = 114.63, *p* = 0.001).

The obtained results of our own research showed that the content of essential oils in pepper significantly influences the antioxidant activity measured with the use of DPPH radicals. A statistically significant, positive weak correlation between these parameters was found *r* = 0.251 (*p* = 0.045). It means that as the content of essential oil increases, the ability of the spice to scavenge DPPH radicals also increases. It should also be noted that pepper oil has a moderate ability to scavenge free radicals compared to other oils [117]. Gülçin points to the strong antioxidant properties of water and ethanolic black pepper extracts. The total content of polyphenols was determined at the level of 54.3 mg GAE in aqueous extracts [118]. The total content of polyphenols determined by Andradea and Ferreira, depending on the modification in the extraction, ranged from 14 to 27 mg GAE/g [119]. Ahmad determined the total content of polyphenols in the methanol extracts of black pepper purchased in Delhi, at the level of 172.8 mg GAE/100 g [120]. Nagy et al., determined the content of polyphenols in black pepper at the level of 338 ± 1.41 mg GAE/100 g, and the scavenging capacity of DPPH free radicals in methanolic solutions of pepper at the level of 13.28% [121].

The ability to scavenge free radicals of aqueous pepper extracts in the studies by Nahak and Sahu was for cubeb pepper (*Piper cubeba*) from 35.38 to 45.84% and for black pepper from 28.15 to 39.92%. In ethanol solutions it increased to 77.61% for cubeb pepper and 74.61% for black pepper [122]. The DPPH scavenging capacity of 35.20% was determined for an aqueous solution of black pepper by Gupta [123].

The polyphenol content of various pepper extracts was determined by Sruthi and Zachariah. Pepper extracts in chloroform and methanol showed the highest content of polyphenols compared to those in which n-hexane and water were used. In the case of black pepper, the total content of polyphenols in the aqueous solution was at the level of 3.84 μg GAE/g, and in the case of *P. longum* pepper, 2.16 μg GAE/g [124].

The high total polyphenol content in black pepper grown in Korea was determined by Lee et al., at the level of 1421.95 ± 22.35 mg GAE/100 g [125]. In another study, m, determined the total content of polyphenols in ethanol extracts of whole Korean peppercorns and after debarking, and it was 1046 ± 22 and 797 ± 28 mg GAE/100 g, respectively [26]. One of the lowest total polyphenols contents in black pepper from Bhubaneswar, India was shown by Mallick et al., and it was at the level of 11.9 ± 0.1 mg GAE/100 g [126].

In this context, the results obtained by Trifan et al., in different extracts are very interesting. The scavenging capacity of DPPH extracts with hexane, dichloromethane, 50% aqueous methanol and methanol was 18.77 ± 0.24, 19.56 ± 0.59, 45.41 ± 0.03, and 32.41 ± 0.07 mg Trolox equivalents (TE)/g, respectively. The presented research results indicate the importance of the analytical technique used in the extraction of the research material, and specifically in the type of polar and non-polar solvent used for the extraction of the spice [127].

In their research, Suchaj et al., showed a statistically significant effect of black pepper irradiation on the increase in antioxidant activity determined with the use of DPPH radicals after 2 months of storage. After 4 months of storage, these changes ranged from 4 to 9% [128].
