The Effect of Extraction Technique on the Yield, Extraction Kinetics and Antioxidant Activity of Black Pepper (Piper nigrum L.) Ethanolic Extracts

Abstract

This study examines the effect of extraction techniques (maceration-M, reflux-RE, ultrasonic-UE, Soxhlet extraction-SE) on the yield of total extractive matter (TEM), extraction kinetics, and antioxidant activity of black pepper fruits ethanolic extracts (BPFEEs). The content of total phenols and flavonoids was determined by Folin-Ciocalteu and AlCl₃ methods, respectively. The antioxidant activity of the extracts was determined by five tests (DPPH, ABTS, FIC, FRAP, and ferricyanide assay), that react by different mechanisms. The highest yield of TEM was observed in the extract obtained by SE (18.77 g/100 g p.m.). Model Ponomarev and a non-stationary diffusion model through the plant material were used for modelling the extraction process. The extract obtained by UE showed the highest content of phenols (85.64 mg GAE/g d.e.), while the extract obtained by RE showed the best antioxidant activity according to DPPH, ABTS, FIC, and FRAP tests, while the extract obtained by UE showed the best activity according to the ferricyanide test. The study provides a comparative analysis of extraction techniques, highlighting their specific advantages, and guiding proper selection based on the desired goal (maximum TEM yield, highest phenolic and flavonoid content, and best antioxidant activity).

1. Introduction

Black pepper (BP) (Piper nigrum L.), the most known species from family Piperaceae, is a perennial plant, native to the tropics of India and Southeast Asia, and is grown commercially in many tropical areas. The fruits of BP are one of the most important spice, which is also used in traditional medicine [1].

The chemical composition of BP is diverse, and this plant, in addition to the alkaloid piperine, also contains essential oil, phenolic compounds, fatty oil (saturated and unsaturated fatty acids), starch, proteins, vitamins (A, C, E, K, vitamin B complex, etc.), minerals (calcium, potassium, copper, iron, magnesium, zinc: Ca, K, Cu, Fe, Mg, Zn, etc.) [2]. Black pepper extracts are important because of the variety of active compounds that contribute to its numerous biological activities such as antioxidant [3], antimicrobial [4], anticancer [5], hepatoprotective [6], anti-inflammatory [7], etc. Literature data have shown that BP extracts obtained with different solvents show good antioxidant activity [4,8]. Aqueous and aqueous-ethanolic extracts of BP are a good source of phenolic compounds that are a mixture of phenolic acid glycosides and flavanol glycosides. Some of the most common phenolic compounds in BP extracts are phenolic acids such as hydroxybenzoic acids (protocatechuic acid 4-O-glucoside, protocatechuic acid, p-hydroxybenzoic acid, etc.) and hydroxycinnamic acids (chlorogenic acid, p-Couraffeyl tyrosine, etc.). Flavones that can be found in pepper are: apigenin 6,8-di-C-glucoside, apigenin 6-C-glucoside, kaemferol-3-O-β-glucoside, etc. [8]. Due to the poor solubility of piper alkaloids in water, alkaloids are usually found in extracts obtained by organic solvents such as ethanol, methanol, chloroform, dichloromethane [4,9].

The use of synthetic antioxidants such as tert-butyl-4-hydroxyanisole (BHA), tert-butyl-4-hydroxytoluene (BHT), propyl gallate (PG), ascorbyl palmitate (AP) can have side effects. Good alternative to synthetic agents are extracts of spices and herbs which may be a potential replacement in food and pharmaceutical industries [10,11].

A tendency among modern society is the use of antioxidant agents obtained from natural sources, increased the interest in the research. Therefore, finding the most efficient extraction technique for isolation of bioactive compounds from natural sources, is a great challenge for researchers in the food, pharmaceutical and cosmetic industries. The amount of isolated bioactive components from plant material depends on various factors such as extraction technique, solvent, extraction time and temperature, ratio of plant material to solvent, etc. [12].

Considering that BP fruit is often used in traditional Serbian cuisine and food industry, it is important to explore widely its potential as a medicinal plant. Different solvents showed different selectivity to the compounds present in the BP fruit, and by analizing literature data [4,8], the choice in this investigation was primarly focused on safe, non-toxic solvent such as ethanol. Therefore, the aim of this investigation was to analyse the effect of extraction techniques (maceration-M, reflux extraction-RE, ultrasonic extraction-UE and Soxhlet extraction-SE) on the yield of total extractive matter (TEM), extraction kinetics, on the content of total phenols and flavonoids, and on the antioxidant activity of black pepper ethanolic extracts (BPFEEs) (DPPH, ABTS, FIC, FRAP, and Ferricyanide assays). Currently, there is no comprehensive study in the literature comparing the impact of different extraction techniques on the yield, kinetics, and antioxidant properties of ethanolic extracts from black pepper fruit. A contribution to this work was the use of extraction kinetic models that play an important role in understanding and optimizing the extraction process to obtain the desired compounds from natural sources.

Based on criteria such as the highest TEM yield, the highest content of phenols and flavonoids, the best antioxidant activity by different assays, a certain optimal BPFEE can be selected.

The main criterion according to which the optimal conditions of the extraction by the maceration technique was selected, was the maximum yield of TEM. For the extracts isolated by different extraction techniques, the content of total phenols and flavonoids was compared, as well as the antioxidant activity of BPFEEs according to different test mechanisms. In addition, the correlation of the antioxidant activity with total phenols and flavonoids was determined.

Therefore, this study can be of great importance when it comes to defining optimal extraction conditions, choosing the appropriate extraction technique for obtaining BPFEE with optimal antioxidant activity, that are potentially important source of nutrients for humans, natural raw materials for the development of dietary supplements and can be used in the industries of nutraceuticals, functional foods, agriculture and pharmaceutical products.

2. Materials and Methods

2.1. Plant Material

The commercial sample of dried BP fruit from the Serbian market (Piper nigrum L.; country of origin: Vietnam; packed by “Solunac Gyros and top spices”, Ugrinovački put, part 25-no.32, Zemun, Altina) was used in this investigation. The plant material was protected from light and refrigerated in original package. Gravimetric analysis determined the moisture content in the BP fruit, which was 10.67 g/100 g of dry mass of the sample. Right before the analysis, the plant material was ground in an electric mill (laboratory electric mill “BRAUN AROMATIC KSM2”). The mean particle diameter (0.258 mm) of the grounded plant material was determined by the sieve analysis. For granulometric analysis, a set of laboratory sieves with a mechanical shaker (LABDEX LTD., London, UK) were used. The plant material was placed in a thin layer on the first sieve with the largest mesh size (1.32 mm) and covered with a lid. Below this, a series of sieves with progressively smaller mesh sizes (1 mm, 0.8 mm, 0.63 mm, 0.5 mm, 0.4 mm, and 0.1 mm) were arranged, with a collection pan at the bottom. The shaker was activated, and the sieving process was set for 15 min. After sieving, the mass of each fraction was measured, the mass fractions were calculated, and the mean particle diameter was determined by equation:

$${\displaystyle \frac{100}{{{\displaystyle d}}_{mean}}}={\displaystyle \sum \frac{\%massfraction}{{{\displaystyle d}}_i}}$$

(1)

where d_mean was the mean particle size of grounded plant material, %massfraction was mass or percentage of the sample that passes through each sieve and d_i was the sieve diameter.

2.2. Reagents and Chemicals

Ethanol 96% p.a. (Reahem d.o.o., Novi Sad, Serbia), rutin (Merck, WGK, Darmstadt, Germany), Folin-Ciocalteu reagent, gallic acid; aluminum (III) chloride hexahydrate, potassium acetate, sodium carbonate, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferrozine, sodium salt of 3-(2-pyridyl)-5,6-diphenyl-diphenyl-1,2,4-triazine-4′,4″-disulfonic acid (ferrozine); ethylene-diamino-tetra-acetic acid (EDTA), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), iron (III) chloride hexahydrate, potassium ferricyanide, butylated hydroxy-toluene (BHT) (Sigma Chemical Company, St. Louis, MO, USA), iron (II)-chloride tetrahydrate, trichloroacetic acid (TCA) (JT Baker, VA Deventer, The Netherlands). All other chemicals are of analytical reagent grade (p.a.).

2.3. Determining of Total Extractive Matter Content (TEM) in the Plant Material

Total extractive matter (TEM) content in the plant material was determined according to the method of Stanojević et al. with slight modification [13]. The measured quantity of the ground and homogenized plant material (25 g) was extracted with 250 mL of solvent (70% ethanol) in the Soxhlet apparatus. The extraction was performed at the boiling point temperature for 360 min (79.4 °C). The yield of TEM (dry extract) was determined by drying of analyzed extract (2 mL) at 105 °C to constant weight. The content of TEM in the plant material (q₀) was calculated according to dry residue and expressed per 100 g of plant material (g of TEM/100 g p.m.). The content of TEM in the plant material was 19.85 g/100 g p.m. All experiments were performed in triplicate.

2.4. Extraction Techniques

2.4.1. Maceration and Reflux Extraction

The influence of operating conditions on the yield of TEM. The influence of ethanol concentration and solvomodule (plant material/solvent ratio; m/V) on the yield of TEM during maceration process at room temperature (25 °C) was determined. After choosing the optimal concentration of ethanol and solvomodule during the maceration process, the influence of temperature and time extraction was further investigated.

The influence of ethanol concentration on the yield of TEM. The measured amount of ground and homogenized plant material (2 g) was poured over with a solvent (10%, 30%, 50%, 70%, and 96% v/v ethanol) in a ratio of 1:15 m/V and extraction of TEM was performed for 120 min at a temperature of 25 °C. The liquid extract was separated by filtration on a “Büchner” funnel. The yield of TEM was determined by drying the 2 mL of obtained extracts at 105 °C to constant weight. The TEM yield was calculated and expressed as g TEM/100 g p.m. Based on TEM yield, the optimal concentration of ethanol was determined to be 70% v/v.

The influence of solvomodule on the yield of TEM. The effect of solvomodule on TEM yield was examined at the optimal ethanol concentration and other unchanged operating conditions. The measured amount of ground and homogenized plant material (2 g) was poured over with 70% v/v ethanol at different solvomodule (1:5, 1:10, 1:15, 1:20 and 1:25 m/V) and TEM extraction was performed for 120 min at a temperature of 25 °C. The liquid extract was separated from the plant material according to the procedure described in the section “The influence of ethanol concentration” and TEM yield was determined. The optimal solvomodule of 1:10 m/V was determined based on the TEM yield,

The influence of temperature and time. With the optimal concentration of ethanol and solvomodule, and other operational conditions unchanged the effect of extraction temperature on the yield of TEM was investigated. The measured amount of ground and homogenized plant material (2 g) was poured over with 70% v/v ethanol at solvomodule 1:10 m/V and extraction of TEM was performed at reflux, for 180 min at different temperatures (40 °C, 50 °C and boiling point). The liquid extract was separated from the plant material according to the procedure described in section “The influence of ethanol concentration”, and TEM yield was determined. The boiling point temperature (80.2 °C) was found to be the optimal extraction temperature based on TEM yield. The extraction kinetics of TEM were monitored at predetermined time intervals ranging from 5 to 180 min.

2.4.2. Ultrasonic Extraction

The ground and homogenized plant material (2 g) was poured over with 70% v/v ethanol at a solvomodule of 1:10 m/V and the TEM was extracted for 60 min at room temperature in a thermostated ultrasonic bath (Sonic, Niš, Serbia; internal dimensions: 30 × 15 × 20 cm; total nominal power: 3 × 50 W; and frequency: 40 kHz). The extraction kinetics of TEM were monitored at specific periods (5–60 min). The liquid extract was separated from the plant material according to the procedure described in section “Influence of ethanol concentration”, and TEM yield was determined.

2.4.3. Soxhlet Extraction

Soxhlet extraction of TEM was performed under the optimal extraction conditions (solvent: 70% ethanol, solvomodule: 1/10 m/V, and boiling point temperature). The measured amount of ground and homogenized plant material (25 g) and 250 mL of 70% v/v ethanol were put into the Soxhlet apparatus and extraction of TEM was performed for 240 min. The TEM yield was determined according to the procedure described in the section “The influence of ethanol concentration”. The extraction kinetics of TEM were monitored at predetermined time intervals ranging from 15 to 240 min

2.5. Extraction Kinetics of TEM

Modeling of extraction kinetics from BP fruit was performed using the Ponomarev model (model A) [14] and the model based on non-stationary diffusion through plant material (model B) [15]. Models are based on the two-stage extraction mechanism and are two-parametric. For the purpose of mathematical modeling the kinetic equations are given by the following expressions:

Model A: (q₀ − q_i)/q₀ = b + k·t

(2)

Model B: q_i/q₀ = (1 − b)·e^−kt;    lnq_i/q₀ = ln(1 − b) − k·t

(3)

where q₀ represents the initial TEM content in the plant material; q_i—the content of the TEM in the plant material after the period t; b—coefficient of the fast extraction period; k (min⁻¹)—coefficient of the slow extraction period [13].

2.6. Determination of Total Phenols Content

The content of total phenols in the BPFEEs was determined spectrophotometrically (Cole Parmer spectrophotometer, Cole-Parmer, Chicago, IL, USA) according to the Folin-Ciocalteu method descibed by Stanojević et al. with some modifications [16]. The gallic acid was used as a standard (the concentration range was: 0.00625–0.2 mg/mL). The absorbance of galic acid at 765 nm was expressed in Equation (4):

A_765n = −0.0112 + 7.34857·cGAE (mg/mL), R² = 0.997

(4)

The content of total phenols was expressed as gallic acid equivalents per gram of dry extract, mg GAE/g d.e.

2.7. Determination of Total Flavonoid Content

The content of total flavonoids in BPFEEs was determined spectrophotometrically (Cole Parmer spectrophotometer, Cole-Parmer, Chicago, IL, USA) according to the method of Lin and Tang, with aluminum (III)-chloride with certain modifications [17]. The rutin was used as a standard (the concentration range was: 0.005–0.1 mg/mL). The absorbance of rutin at 415 nm was expressed in Equation (5):

A_415nm = 0.0232 + 12.3585·cRE (mg/mL), R² = 0.989

(5)

The content of total flavonoids was expressed as rutin equivalents per gram of dry extract, mg RE/g d.e.

2.8. Antioxidant Activity

2.8.1. DPPH Test

The antioxidant activity of BPFEEs was evaluated using several methods, including the DPPH test, following the procedure described by Stanojević et al. [18]. DPPH test was also performed for synthetic antioxidant BHT, under the same experimental conditions [18]. Ethanolic solutions of extracts were prepared in a series of different concentrations (0.0039–0.5 mg/cm³). To 2.5 mL of extract 1 mL of ethanolic solution of DPPH radical (3 × 10⁻⁴ mol/L) was added. The absorbance was measured on Cole Parmer Spectrophotometer at 517 nm after DPPH radical addition and after incubation at room temperature for 20 min (A_S). The absorbance was also determined for the ethanolic solution of DPPH radical (1 mL DPPH radical solution with 2.5 mL ethanol, A_C) and for the extracts without the DPPH radical (2.5 mL extract with 1 mL ethanol, A_B). The DPPH radical scavenging activity (in %) was calculated according to the equation:

$$\mathrm{DPPH} \mathrm{radical} \mathrm{scavenging} \mathrm{activity}=100-\left[\left({{\displaystyle A}}_S-{{\displaystyle A}}_B\right)\times {\displaystyle \frac{100}{{{\displaystyle A}}_C}}\right]$$

(6)

BHT was used as the positive control. All experiments were performed in triplicate and the results were expressed as the mean value ± standard deviation.

2.8.2. ABTS Test

Antioxidant activity of BPFEEs was determined also by ABTS test according to the method of Stanojević et al. [19]. ABTS test of synthetic antioxidant BHT was also performed, under the same experimental conditions [19]. The ABTS•⁺ radical cation was formed in reaction of ABTS (7 × 10⁻³ mol/dm³) with potassium persulfate (2.4 mM) in a 1:1 (v/v) ratio, during 12–18 h in the dark at +4 °C (stock solution). The working solution of ABTS was obtained by diluting the stock solution with ethanol to achieve an absorbance of 0.7 at 734 nm. For the assay, 1.8 mL of the ABTS working solution and 2.1 mL of ethanol were mixed with 0.1 mL of the extract (concentration range: 0.0156–10 mg/mL). The absorbance of the mixture was measured at 734 nm after 6 min of incubation in the dark at room temperature (A_S). Additional absorbance measurements were taken for a diluted ABTS working solution (1.8 mL ABTS solution mixed with 2.2 mL ethanol, A_C) and the extract diluted in ethanol (0.1 mL extract with 3.9 mL ethanol, A_B). Ethanol was used as the blank. The percentage of ABTS radical scavenging was calculated using the same formula as applied in the DPPH assay. The ABTS test was also performed using a standard synthetic antioxidant (BHT). All experiments were conducted in triplicate, and the results were expressed as the mean value ± standard deviation.

2.8.3. FIC (Ferrous Ion-Chelating) Test

The ability to chelate iron ions with BPFEEs was determined in accordance with the method described by Dinis et al. with certain modifications [20].

To the 1 mL of extract solution was added 3.75 mL of ethanol and 0.05 mL of 2 mM FeCl₂ × 4H₂O. The reaction was initiated by the addition of 0.2 mL of 5 mM ferrozine. The mixture was incubated at room temperature for 10 min and the absorbance was measured at 562 nm (A_s). A mixture of ethanol, ferrozine and FeCl₂ × 4H₂O without a test sample was used as a control (A_c), and a mixture of ethanol and a sample without ferrozine and FeCl₂ × 4H₂O as a blank (A_b). EDTA was used as a reference standard for the test [18].

The ability to chelate iron ions, FIC activity (%) was calculated using Equation (7):

$$\mathrm{Ferrous} \mathrm{ion} \mathrm{chelating} \mathrm{ability} (\%)=100-\left[\left({{\displaystyle A}}_S-{{\displaystyle A}}_B\right)\times {\displaystyle \frac{100}{{{\displaystyle A}}_C}}\right]$$

(7)

The extract concentration (EC₅₀) needed for the chelation of 50% of iron ions was determined. FIC test of standard metal chelator such as EDTA was performed and EC₅₀ value was calculated.

2.8.4. FRAP Test (Ferric Reducing Ability of Plasma)

The FRAP method described by Benzie and Strain [21] was used to determine the antioxidant activity of BPFEEs, with specific modifications according to Stanojević et al. [22]. The concentration of Fe²⁺ (mmol/L) in the BPFEEs and synthetic antioxidant BHT, was determined from the FeSO₄ × 7H₂O calibration curve equation. The results were expressed as concentration of Fe²⁺ equivalents per gram of dry extract (mg EFe²⁺/g d.e.)

The FeSO₄ × 7H₂O calibration curve was constructed based on known concentrations (0.2–1 mmol/L) and measured absorbance values. The absorbance at 593 nm was expressed in Equation (8):

A_593nm = 0.03521 + 0.5949·cFeSO₄ × 7H₂O (mmol/L), R² = 0.993

(8)

2.8.5. Ferricyanide Method

The reducing power of BPFEEs and gallic acid, as standard antioxidant component, was determined according to the method of Oyaize known as a “Prussian blue” method, with certain modifications [23].

To the 1 mL of phosphate buffer and 1 mL of K₃[Fe(CN)₆] was added 0.4 mL of extract. The mixture was incubated in the test tube for 20 min at 50 °C in the water bath. After the cooling reaction mixture, 1 mL of 10% TCA was added. Subsequently, 1 mL of distilled water and 0.2 mL of FeCl₃ × 6H₂O were added to the tube, and the reaction mixture was left to incubate at room temperature for 30 min. The reduction power of BPFEEs and ascorbic acid as standard was determined by measuring absorbance at 700 nm, in relation to the blank. The ability to reduce Fe³⁺ ions to Fe²⁺ was determined from the calibration curve of the standard gallic acid solution. The absorbance of gallic acid at 700 nm was expressed in Equation (9):

A_700nm = 0.03883 + 22.94176·cGAE, R² = 0.9989

(9)

The results were expressed as mg GAE/g d.e.

2.9. Statistical Analysis

All results were presented as mean value ± standard deviation based on three measurements. Statistical analysis were performed by one-way ANOVA (analysis of variance) followed by Tukey’s multiple comparison test by software JMP Pro 17 (SAS Institute Inc., Cary, NC, USA). Differences are reflected as significant at p < 0.05.

3. Results

3.1. Maceration and Reflux Extraction

3.1.1. Influence of Operating Conditions on the TEM Yield

Results of the analysis of the influence of ethanol concentration and solvomodule on the TEM yield (extraction time: 120 min; temperature: 25 °C) obtained by the maceration are shown in

Table 1. Influence of ethanol concentration on the total extractive matter yield (time of extraction: 120 min; temperature: 25 °C).

Solvomodule, m/V	Ethanol, %	TEM Yield, g/100 g p.m. *
1:15	10%	5.50 ± 0.294 bc
	30%	4.70 ± 0.431 cd
	50%	5.70 ± 0.510 b
	70%	8.42 ± 0.038 a
	96%	4.25 ± 0.260 d

* Results are expressed as the mean value of triplicate ± standard deviation. Column values (different small letters) are significantly different (ANOVA: Single Factor test, and Tukey’s HSD test, p < 0.05).

and

Table 2. Influence of solvomodule on the total extractive matter yield (time of extraction: 120 min; temperature: 25 °C; concentration of ethanol 70%).

Solvomodule, m/V	TEM Yield, g/100 g p.m. *
1:5	6.55 ± 0.053 c
1:10	8.40 ± 0.015 b
1:15	8.42 ± 0.038 b
1:20	8.67 ± 0.058 a
1:25	8.67 ± 0.072 a

* Results are expressed as the mean value of triplicate ± standard deviation. Column values (different small letters) are significantly different (ANOVA: Single Factor test, and Tukey’s HSD test, p < 0.05).

.

With the increase of the ethanol concentration, the TEM yield was increased to a certain limit (Table 1). The highest yield was obtained with 70% v/v ethanol and it was adopted as the optimal solvent (solvomodule 1:15 m/V). This research showed that with the increase of the solvomodule above 1:10 m/V, the TEM yield was a slighty increased (up to 3.21%), but no significant difference has been observed (Table 2). Therefore, due to the economy of the extraction process, solvomodule 1:10 m/V was used in subsequent experiments.

3.1.2. Influence of Temperature and Extraction Time on the TEM Yield

The influence of temperature (25 °C, 40 °C, 50 °C and boiling point) and extraction time (solvent: 70% v/v ethanol, and solvomodule 1:10 m/V) on the TEM yield obtained from the BP fruit, was monitored (

Table 3. Influence of extraction temperature and time on the total extractive matter yield (ethanol concentration: 70%; solvomodule: 1:10 m/V).

	Time of Extraction
	120 min	180 min
Temperature (°C)	TEM Yield, g/100 g p.m.*
25	8.40 ± 0.015	9.10 ± 0.058
40	9.83 ± 0.008	10.00 ± 0.252
50	12.10 ± 0.100	12.57 ± 0.173
Boiling point	14.37 ± 0.306	14.60 ± 0.300

* Results are expressed as the mean value of triplicate ± standard deviation.

).

With the increase of the temperature, the TEM yield increases, as can be seen from Table 3. Therefore, the TEM yield of the extract obtained at boiling point temperature was 41.54% and 37.67% higher than TEM yield of the extract obtained by maceration at 25 °C, for the time of 120 and 180 min, respectively. The extraction efficiency improves as the extraction duration is extended within a specific time frame. The TEM yield increased with the extraction time regardless of the applied extraction temperatures (Table 3). In the period of 120–180 min, the TEM yield was increased at all tested temperatures (2–3%). Further increasing the extraction time would probably not bring about any significant changes in the TEM yield, after the equilibrium of the solute was reached inside and outside the solid material, so the time of 180 min was adopted as the optimal extraction time [12].

3.2. The Influence of Extraction Technique on the TEM Yield

The variation of TEM yield obtained from BP fruit during the different extraction techniques with the optimal ethanol concentration of 70% v/v and optimal solvomodule (1:10 m/V), is shown at Figure 1. Comparison of TEM yield obtained from BP fruit by different extraction techniques was presented at

Table 4. Comparison of total extractive matter yield obtained from black pepper fruit by different extraction techniques.

Extraction Technique *	TEM Yield, g/100 g p.m.
Maceration, 180 min	9.10 ± 0.058
Reflux extraction (40 °C), 180 min	10.00 ± 0.252
Reflux extraction (50 °C), 180 min	12.57 ± 0.173
Reflux extraction (boiling point), 180 min	14.60 ± 0.300
Soxhlet extraction, 240 min	18.77 ± 0.115
Ultrasonic extraction (25 °C), 60 min	9.80 ± 0.200

* ethanol concentration 70%, solvomodul 1:10 m/V; results are expressed as the mean value of triplicate ± standard deviation.

.

Figure 1a shows that reflux extraction at boiling point temperature yielded the highest content of TEM (14.60 g/100 g p.m.) compared to maceration and reflux extraction at 40 °C and 50 °C. Figure 1b shows that Soxhlet extraction yielded the highest content of TEM (18.77 g/100 g p.m.) compared to other techniques. The TEM yield achieved after 60 min of ultrasonic extraction at 25 °C was 9.80 g/100 g p.m. (Table 4). In relation to maceration, the effect of ultrasound increases the TEM yield (by about 15%), and the duration of the extraction process is greatly reduced (from 180 min (maceration) to 60 min (ultrasonic extraction)–Figure 1c and Table 4).

3.3. Extraction Kinetics

Two extraction periods are noticeable on the kinetic extraction curves (Figure 2a–c and Figure 3a–c) [24]. In the extraction equations, washing coefficient b is characteritic for period of fast extraction, and slow extraction coefficient k for the period of slow extraction. The values of coefficients b and k in the kinetics equations for the TEM extraction, the fast extraction time (FET) and extraction levels for TEM in the period of fast extraction (EL, %) by different techniques with the optimal ethanol concetration (70%) and optimal solvomodule (1:10 m/V), are given in the

Table 5. The fast extraction time (FET), extraction level (EL) in the fast extraction time and the values of b and k coefficients in the equations of the extraction kinetics of TEM from BP fruit according to Ponomarev’s equation (Model A) and non-stationary diffusion equation (Model B).

Extraction Technique	FET, min	EL, %	Model A		Model B
			b	k·104, min−1	b	k·103, min−1
Maceration, 180 min	90	41.60	0.367	5.30	0.362	0.95
Reflux extraction (40 °C), 180 min	30	41.70	0.396	6.60	0.392	1.23
Reflux extraction (50 °C), 180 min	30	52.90	0.514	7.33	0.510	1.77
Reflux extraction (boiling point), 180 min	30	66.30	0.657	5.00	0.655	1.69
Soxhlet extraction, 240 min	150	85.10	0.811	5.56	0.687	7.09
Ultrasonic extraction (25 °C), 60 min	15	45.20	0.437	9.33	0.470	1.31

.

The linear dependencies (q₀ − q_i)/q₀ and ln q_i/q₀ on time exist in the later extraction period (slow extraction), in contrast to the initial period where the deviation of the experimental points from the straight line is observed.

During the period of fast extraction, 41.6–85.10% of TEM was extracted from the surface of destroyed plant material cells by rinsing and dissolving extractive matter (Table 5). This shows the high degree of cell destruction increases the surface from which extractives are washed and dissolved in a fast period and thus provides a high degree of their extraction in this period. The extraction level in the period of fast extraction by maceration was the lowest 41.6% for the period of 90 min. With the increasing of temperature the extraction level in the period of fast extraction increased for the reflux extraction (40 °C, 50 °C, boiling point), 41.70%, 52.90%, 66.30% for the period of 30 min. The extraction level in the period of fast extraction by Soxhlet extraction was the highest, 85.10%, for the period of 150 min. Ultrasonic extraction achieves an extraction level of 45.20% in 15 min during the fast extraction period. This extraction technique achieves a higher TEM yield in the fast extraction period (15 min) than the yield achieved in the fast maceration extraction period (90 min) under the same other operating conditions. The value of the coefficient b was higher for ultrasonic (0.437-Table 5) than for classical maceration extraction (0.367-Table 5). The reason may be the facilitated penetration of the solvent into the particles of plant material, the increased rate of mass transfer and the destruction of plant cells under the influence of ultrasound. The coefficients of slow extraction k according to the Ponomarev model, depending on the extraction technique, were of the order of 5.00–9.33 × 10⁻⁴ min⁻¹ (Table 5). The coefficients k in the equations of extraction kinetics according to the model B are higher than the coefficients k in the equations of extraction kinetics according to the model A. Based on the results shown in Table 5, it can be seen that the coefficients b in the TEM extraction kinetics equations according to model A and B were slightly different. These results are in the agreement with previous research [25,26].

3.4. Content of Total Phenols and Flavonoids

Since the focus of research in this paper was primarily on the choice of extraction technique with the highest yield of TEM, in further experiments extracts obtained by reflux extraction at 40 °C and 50 °C, will be neglected, considering that reflux extraction showed the highest yield with extraction at boiling point temperature. Extracts obtained by other extraction techniques will be compared on the basis of their composition (phenol and flavonoid content) and antioxidant activity.

The content of total phenols and flavonoids in the obtained BPFEEs is shown in the

Table 6. Content of total phenols and flavonoids in BPFEEs obtained by different extraction techniques.

Extraction Technique *	Total Phenols, mgGAE/g d.e.	Total Flavonoids, mgRE/g d.e.
Maceration, 180 min	69.54 ± 0.680	59.66 ± 0.234
Reflux extraction (boiling point), 180 min	79.29 ± 0.393	97.56 ± 0.234
Ultrasonic extraction (25 °C), 60 min	85.64 ± 0.393	73.15 ± 0.330
Soxhlet extraction, 240 min	74.75 ± 0.393	53.26 ± 0.117

* ethanol concentration 70%, solvomodul 1:10 m/V; results are expressed as the mean value of triplicate ± standard deviation.

.

The highest content of total phenols was determined in the extract obtained by ultrasonic extraction, expressed as 85.64 mg GAE/g d.e. which was the highest of all other extraction techniques. Ultrasonic extraction yielded a 19% higher yield of phenols for three times shorter time compared to maceration (69.54 mg GAE/g d.e.) under the same other conditions. The flavonoid content was the highest in the extract obtained by reflux extraction at boiling point temperature, expressed as 97.56 mg RE/g d.e., which is 45% higher than the content of flavonoids in the extract obtained by Soxhlet extraction (53.26 mg RE/g d.e.).

3.5. Antioxidant Activity

The

Table 7. EC₅₀ values, FRAP values and reducing power (RP) of BPFEEs determined by DPPH, ABTS, FIC, FRAP, Ferricyanide method and correlation with content of total phenols and flavonoids.

Sample	EC50, DPPH mg/mL	EC50, ABTS mg/mL	EC50, FIC mg/mL	FRAP Value, mg EFe2+/g d.e.	RP (Fe3+–Fe2+), mg GAE/g d.e.
Maceration, 180 min	0.152 ± 0.001	1.730 ± 0.008	1.353 ± 0.0006	64.67 ± 0.35	25.10 ± 0.22
Reflux extraction (boiling point), 180 min	0.112 ± 0.001	1.010 ± 0.002	1.146 ± 0.0155	67.82 ± 0.08	27.28 ± 0.22
Soxhlet extraction, 240 min	0.120 ± 0.0007	1.110 ± 0.004	1.327 ± 0.0099	63.72 ± 0.05	22.27 ± 0.22
Ultrasonic extraction (25 °C), 60 min	0.142 ± 0.0004	1.010 ± 0.005	1.646 ± 0.0046	66.64 ± 0.05	31.20 ± 0.22
BHT [18,19]	0.021 ± 0.001	0.081 ± 0.001	/	810.92 ± 40.546	/
EDTA	/	/	0.0517 ± 0.0006	/	/
Ascorbic acid	/	/	/	/	176.93 ± 0.333
Correlation between total phenolic content and antioxidant activity (R2)
Total phenols	0.0448	0.6765	0.2442	0.4337	0.6526
Total flavonoids	0.2281	0.2341	0.1307	0.9253	0.3020

* Results are expressed as the mean value of triplicate ± standard deviation.

shows EC₅₀ values, FRAP values and reducing power (RP) of extracts obtained by different extraction techniques. The correlation of antioxidant activity and phenol and flavonoid content was also showed in the Table 7.

3.5.1. DPPH Assay

The degree of neutralization of DPPH radicals in the extracts increases with increasing concentration of incubated samples. The antioxidant activity of the BPFEEs decreases in the following order: RE at boiling temperature > SE > UE > M. Table 7 shows the EC₅₀ values of BPFEEs obtained by different extraction techniques under optimal ethanol concentration (70%) and optimal solvomodule (1:10 m/V). The lower the EC₅₀, the less the concentration of extract is required to produce 50% of maximum effect and the higher the potency [22]. As can be seen from the Table 7, extracts obtained by RE at boiling point temperature (lowest EC₅₀ value) and SE showed better antioxidant activity compared to extracts obtained by maceration and ultrasonic extraction.

The EC₅₀ value of synthetic antioxidant BHT was determined under the same experimental conditions (Table 7) [19]. The lowest EC₅₀ values by RE at boiling temperature and SE extracts wasn’t in correlation with the results of total phenols (R² = 0.0448) and flavonoids content (R² = 0.2281) (Table 7).

3.5.2. ABTS Assay

The degree of ABTS radical neutralization in the extracts increases with increasing concentration of all samples incubated with the radical for 6 min. The antioxidant activity of BPFEEs decreases in the following order: RE at boiling point temperature > UE > SE > M.

As can be seen from the Table 7, extract obtained by RE at boiling point temperature showed better antioxidant activity compared to extracts obtained by maceration and ultrasonic extraction. The dependence is the similar as with the DPPH test. What differs is the degree of neutralization of ABTS radicals by the ultrasonic extract. The extract obtained by UE showed the highest amount of total phenols so it can be assumed that the role of phenols in the neutralization of ABTS radicals is greater than that of DPPH radicals. This is evidenced by the correlation between antioxidant activity and phenol content (R² = 0.6765), which has been shown to be better compared to the correlation with flavonoid content (R² = 0.2341) (Table 7). ABTS test of synthetic antioxidant BHT was also performed and EC₅₀ value was determined under the same experimental conditions (Table 7) [19].

3.5.3. FIC Assay

The EC₅₀ values of BPFEEs under the optimal ethanol concentration (70%) and optimal solvomodule (1:10 m/V) are shown in Table 7. The EC₅₀ value for EDTA was also determined (Table 7). The antioxidant activity of BPFEEs decreases in the following order: RE at boiling temperature > SE > M > UE. The chelating effect of EDTA was significantly better than obtained extracts. There is a match with the results of the DPPH and ABTS test for the best antioxidant activity, which is not the case when it comes to the lowest antioxidant activity. In this paper, there is no proper correlation in the content of phenolic compounds and chelating properties (Table 7) (for phenols R² = 0.2442; for flavonoids R² = 0.1307). The lowest antioxidant activity showed the extract obtained by UE, although contained the highest amount of phenols.

3.5.4. FRAP Assay

The concentration of Fe²⁺ equivalents (FRAP value) in the extracts was read directly from the calibration curve of FeSO₄ × 7H₂O, on the basis of which the concentration of Fe²⁺ in the samples was determined and converted to the mass of the extract (Table 7).

The results in Table 7 show that the highest FRAP value, and thus the best reducing ability, i.e., antioxidant activity, is shown by the extract obtained by RE at boiling point temperature. The antioxidant activity of the BPFEEs decreases in the following order: RE at boiling temperature > UE > M > SE. There is an agreement with the results of DPPH, ABTS, FIC test for the best antioxidant activity. This is not the case when it comes to the lowest antioxidant activity (SE). It must also be taken into account that there wasn’t a big differences in activities between obtained extracts, but it is a proper dependence of flavonoid content and reducing ability of extracts (for phenols R² = 0.4337; for flavonoids R² = 0.9253; Table 7). The antioxidant activity of synthethic antioxidant BHT showed about twelve times higher FRAP value than BPFEEs (810.92 mg EFe²⁺/g d.e.—Table 7).

3.5.5. Ferricyanide Method

Values corresponding to the amount of reduced iron (Fe³⁺) in iron (Fe²⁺) were read from the gallic acid calibration curve and are presented in Table 7 as mg GAE/g d.e.

The reducing power of the BPFEEs decreases in the following order (mg GAE/g d.e. ± sd.): UE > RE at boiling temperature > M > SE. This comparison of the reduction power or antioxidant activity of BPFEEs shows a similar dependence as in previous tests, with minor deviations. The best reducing power is attributed to the extract obtained by UE (31.2 mg GAE/g d.e.). The greatest congruence was noticed with the FRAP test, and lies in the fact that the extract obtained by SE showed also the lowest reducing power, which can be attributed to the similarity of test mechanism. In this work, there is good correlation in the content of total phenols and reducing power, while the correlation with flavonoid content is less significant (for phenols R² = 0.6526, for flavonoids R² = 0.302) (Table 7). The ascrobic acid showed better antioxidant activity than all BPFEEs with value 176.93 mg GAE/g d.e.

4. Discussion

The amount of TEM from the BP fruit may depend on structural factors of bioactive compounds, such as the number of phenolic hydroxyl or methoxyl groups, keto groups, free carboxyl groups and other structural properties. Aqueous-ethanol mixtures are most often used due to low toxicity and higher polarity than the monocomponent systems, which is reflected by the extraction of wider class of compounds and increased extraction yields [27,28]. The effect of the solvomodule has been reported for different plant materials [13,25]. It was noticed that the higher the ratio, the higher was the total amount of TEM, which is in accordance with mass transfer principles. The mass transfer process occurs faster due to the concentration gradient between the plant material and the volume of the solvent, which is greater when a higher ratio is used [27]. Temperature and time are the dominant factors that work cooperatively, influencing the efficiency and selectivity of the extraction process [25]. The TEM increased by the increase of the temperature, and this was the consequence of enhanced solubility of the TEM in the extracting solvent due to decrease of its viscosity at higher temperatures. Also, with increasing temperature, there is an increased mass transfer of TEM from interior of plant material into the solution [26]. The results can be also conditioned by the nature of the components present in the analyzed plant material, i.e., their higher solubility in the solvent (70% v/v ethanol) at boiling point temperature. There are no data in the literature that compare the content of TEM yield of BP fruit at different extraction temperatures and times.

The highest TEM content was obtained using the Soxhlet extraction method as a consequence of the circulation of the solvent until the complete depletion of the plant material [26]. The Soxhlet extraction method combines the benefits of reflux extraction and maceration, employing the principles of reflux and siphoning to continuously extract plant material using fresh solvent [12]. Classical maceration takes place by the mechanism of normal diffusion through cell walls and this process requires a significantly longer extraction time [14]. The reduction in the duration of ultrasonic extraction can be attributed to faster destruction of cell walls under the action of ultrasound, reduction of particle size and better transfer of TEM mass from plant cells [26].

In the first period, fast extraction occurs where the extracted substances are washed with a solvent from the surface of the ground plant material. In the second period, there is a slow molecular diffusion of matter extracted from the inner part of the porous plant material (slow extraction). The coefficient of fast extraction increases with increasing temperature, under the influence of ultrasound, due to better solubility of extractives and increasing diffusion coefficient, which is explained by the increase in the degree of degradation of plant material cells. The coefficient of slow extraction also increases with increasing temperature, due to the increase of the diffusion coefficient (shorter path of diffusion of extractive substances through the particles of plant material to the solution) and the solid-liquid contact surface [15]. Taking into account the obtained results, it can be concluded that both kinetics models can be used for modeling the extraction of TEM from BP fruit by 70% v/v ethanol as solvent.

Although the content of phenolic compounds is expected to increase with increasing temperature, this only happens to a certain extent, and the results can be explained by the structural changes of certain bioactive components under the action of high temperatures over a long period of time [29]. There are many papers that discuss the content of phenols and flavonoids in BP extracts [3,30,31,32]. It is important to emphasize that there are no data in the literature comparing the content of phenols and flavonoids in BP extracts obtained by different extraction techniques under the defined conditions as in this investigation. In the study of Akbar et al. the content of total phenols in BP ethanolic extract obtained with 96% ethanol, was 140.5 mg GAE/g, and the difference from the current study may lie in the use of a different concentration of ethanol for extraction [31]. A study by Andrade and Ferreira showed that the amount of phenols in ethanolic extracts obtained by Soxhlet extraction and ultrasonic extraction was about 3 times lower than the amount of phenols in this study, which can be affected by the quality of plant material [33]. Also, it was noticed that white pepper ethanolic extract showed higher amount of total phenols than BP ethanolic extract, probably like the result of the different processing of the BP fruit [31,32,34]. In the study of Zarai et al. the highest content of phenols and flavonoids showed extracts of BP obtained with ethanol and chloroform, respectively. This indicates that phenolic and flavonoid compounds of the different extracts are attributed to the polarities of compounds present in the plant material [4].

Five different tests were used to determine the antioxidant activity of the obtained BPFEEs. As expected, antioxidant tests contain a lot of chemistry. Therefore, it must be taken into account that antioxidant methods are not specific, have no similarities with biological systems, sometimes have an inexplicable correlation with the bioactive compounds present in the sample, and other types of compounds that are not important may react with oxidant leading to overestimation [35].

The extracts obtained by RE and SE showed the best antioxidant activity by DPPH assay, which is most likely a consequence of the application of higher temperatures, which increases the solubility of bioactive components and better depletion of plant material. These results were compared to phenolic and flavonoid contents, which indicate that its antioxidant activity may be not directly related to its content, but can be a consequence of synergistic effect of phenolics with other extracted compounds, as shown by examples from the literature [3,36].

When it comes to ABTS assay, results are similar to those obtained by DPPH assay. It is assumed that in the reaction of ABTS radicals with large polyphenolic molecules, the reaction may be slower than with simpler ones, because the former must be reoriented before the reaction. Hagerman et al. concluded that high molecular weight phenols have a higher ability to remove free radicals (ABTS•⁺) and that their efficiency depends on both the molecular weight and the number of aromatic rings and the position of hydroxyl groups, which may be crucial for strong antioxidant activity [37]. The ABTS assay was done for samples such as aqueous, ethanolic, methanolic extracts and oleoresins of BP [38,39]. Olalere et al. showed that 0.09471 mg/mL of BP extract obtained by microwave RE, was required to scavenge half of the stable ABTS free radicals, which in relation to this study represents a much higher antioxidant activity [38]. The study of Krishna et al. showed that BP oleoresin showed better radical scavenging activity compared to BP methanol extract, which may be due to the presence of essential oil in the oleoresin [39]. However, it is important to note that there are no data for BPFEEs obtained according to defined extraction conditions as in this investigation.

FIC (Ferrous ion-chelating) activity was used as a test for antioxidant activity of BPFEEs and showed their measure of reducing the absorbance of iron (II) and ferrozine complexes to 562 nm. The results obtained by this assay show that exact mechanism of chelating properties of different phenolic compounds is unknown due to difficulties of creation iron-antioxidant complexes with multiple iron-binding sites [40]. The chelating ability of metal ions shown by phenolic compounds is usually a function of their characteristic structure, number and position of hydroxyl groups [41]. The fact that the piperamides which can be present in the extracts can also contribute to the chelating activity of the extracts should not be overlooked. There was also a scientific evidence of a positive correlation between the ability to chelate iron ions and the content of total phenols, which cannot be said entirely for results from this work. Also, ethanolic extract of BP from study of Akbar et al. showed better chelating ability at concentration of 0.5 mg/mL (65.9%), than all extracts from this investigation [31]. Metal chelating ability of water and ethanolic extract of BP in concentration of 0.075 mg/mL was reported as 84% and 83%, respectively [34]. Our research showed a weaker metal chelating ability at higher concentrations. The study of Sruthi and Zachariah showed that the best ability to chelate ferrous ions appear to be methanolic extract of BP with 245.5 mg EDTA/g of extract [42]. The literature clearly shows that the binding capacity of transition metals is influenced not only by the chemical components in the extracts but also by matrix effects, such as pH and redox potential [43].

The FRAP method is based on the reduction of the [Fe³⁺–TPTZ] complex to an intense blue (absorption maximum 593 nm) [Fe²⁺–TPTZ] complex in an acidic medium [21]. Literature data for the FRAP test applied to BP fruit extracts were found [31,44], but results for extracts obtained with 70% ethanol using various techniques were not found.

The study by Akbar et al. supports the present investigation, because the ethanolic extract of BP showed better antioxidant activity than ethanolic extracts of white pepper, lower phenol content, but higher flavonoid content, which proves a good correlation between flavonoid content and antioxidant activity. Thus, the antioxidant activity of BPFEEs may be due to the presence of phenolic compounds [31]. Based on the results of Kittisakulnam et al. the aqueous extract of BP showed the higher activity in the FRAP test than ethanolic extracts (50% and 95%). The aqueous pepper extract in the mentioned study had a lower content of phenols and flavonoids, and the results may suggest that not only the phenolic compounds, but also a synergistic effects of phenolic and non-phenolic compounds play a major role in its antioxidant activity [44].

The ferricyanide method is based on the reduction of iron (Fe³⁺) to iron (Fe²⁺), in the presence of antioxidants (extracts). In the case of potassium ferricyanide, cyanide (CN-) is considered as a strong field ligand that causes a big splitting. The reducing ability or antioxidant activity of BPFEEs shows a similar dependence as in previous tests, with minor deviations. In the work of Zarai et al. the reducing ability of ethanolic extract of BP and purified piperine increased with increasing concentration. Since the extract showed a better reducing ability than piperine, it was assumed that the compounds present in the extract act in synergy and thus increase the antioxidant capacity of the extract [4]. There are no literature data of antioxidant activity determined by ferricyanide method, for BPFEEs obtained by different techniques under defined conditions in the present work.

5. Conclusions

Applied extraction techniques have an impact on TEM yield, kinetics and activity. The extract obtained by Soxhlet extraction yielded the highest total extractive matter (TEM), due to its continuous solvent circulation. Ultrasonic extraction significantly reduced time by disrupting cell walls and enhancing mass transfer. The application of kinetic extraction models as a novelty when it comes to ethanolic extracts of black pepper was significant for this research. The extract obtained by reflux extraction at boiling point temperature showed the highest antioxidant activity according to all applied tests, except for the reducing power method, where the extract obtained by ultrasonic extraction showed the higher activity. The correlation between phenolic and flavonoid content and antioxidant activity was presented for each test, leading to the conclusion that the antioxidant activity of BPFEEs does not only originate from these compounds but is also a result of synergistic effects with other extracted components. Therefore, future analyzes will focus on determining the chemical composition of BPFEEs, in order to better understand the antioxidant activity of the obtained extracts.