Assessment of Pyrethrin Novel Green Extraction Methods from Dalmatian Pyrethrum (Tanacetum cinerariifolium)

Abstract

Six novel green extraction techniques were evaluated and optimized to extract pyrethrin from dried Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir./Sch.Bip.). This approach offers a promising natural alternative to conventional chemotherapeutics. Four methods are presented for the first time in this study: microwave-assisted extraction (MAE), high-voltage electric discharge (HVED) extraction, subcritical water extraction (SWE), and deep eutectic solvent (DES) extraction, together with supercritical CO₂ extraction (SC-CO₂) and ultrasound-assisted extraction (UAE), for pyrethrin extraction from Dalmatian pyrethrum. The study revealed that supercritical CO₂ extraction was the most effective method for extracting all six pyrethrins, yielding the highest total amount of 124.37 ng/mg. This approach offers a “natural” insecticide produced with a clean, environmentally friendly technology that can contribute to the development of sustainable and effective insecticide strategies that are in line with environmental safety and organic production standards. In addition, this research highlights the potential application of pyrethrins as antiparasitic agents, emphasizing their role in environmentally friendly and ecological practices.

1. Introduction

Pyrethrins are active components of a flower extract from Dalmatian pyrethrum (Tanacetum cinerariifolium/Trevir./Sch.Bip.), which are used as natural insecticides, primarily in plant protection. In recent years, their use has expanded significantly in organic farming for protecting fruits and vegetables. This is due to their dual action, acting as insecticides and offering strong repellent effects against pests [1].

Due to the increasing usage of insecticides and to reduce costs, synthetic alternatives called pyrethroids have been developed [2]. However, pyrethroids come with significant environmental concerns, including harmful ecological effects, bioaccumulation in the food chain, and risks to consumer safety. Although pyrethroids degrade quickly in water, they can bind to organic materials, leading to aquatic pollution [3]. In contrast, natural pyrethrins present a more sustainable solution, offering an environmentally friendly alternative to synthetic chemicals for pest control. Given the ecological risks associated with pyrethroids, the development of natural insecticides is crucial for sustainable agriculture [4]. Natural pyrethrins, extracted from Dalmatian pyrethrum (Tanacetum cinerariifolium), are favored in organic farming due to their low toxicity to vertebrates, biodegradable nature, short half-life, and environmental persistence [5,6]. Unlike their synthetic counterparts, natural pyrethrins are biosynthesized in the plant’s flowers.

The Dalmatian pyrethrum plant (Tanacetum cinerariifolium/Trevir./Sch.Bip.) is distributed from Italy to the coastal mountains of the eastern Adriatic and its islands (Croatia, Albania, and Montenegro). The highest pyrethrin concentration (>93%) was found in the floral head, while lower amounts were present in the disc flower, ray flower, and receptacles [7,8]. Natural pyrethrin consists of a combination of six monoterpene esters, which are divided into two groups. The first group, called type I, includes pyrethrin I, cinerin I, and jasmolin I, which are closely related to the esters of chrysanthemic acid, while the second group, type II, consists of pyrethrin II, cinerin II, jasmolin II, which are related to the esters of pyrethric acid (Figure 1) [5,9,10]. The amount extracted depends on both the extraction method chosen and the conditions (type of solvent, temperature, etc.). Kolak et al. [11] and Casida and Quistad [12] found that the pyrethrin content in native populations can vary between 0.9 and 1.3% of the weight of the dried flowers. Morris et al. [8] found that in commercial varieties from Tasmania, levels ranged from 1.8 to 2.5%.

Different methods of pyrethrum extraction from the dried flowers of the pyrethrum plant (T. cinerariifolium) were investigated. The most commonly used methods for pyrethrin determination include high-performance liquid chromatography (HPLC), matrix solid phase extraction (MSPD) [13,14], and reversed-phase high-performance liquid chromatography (RP-HPLC), while for separation, ultrasound extraction [15], ultrasound-assisted extraction (UAE) [15,16], and Soxhlet [15,17,18,19] extraction have been applied. Recently, some non-conventional methods, such as supercritical CO₂ extraction (SC-CO₂) [17,18,20,21] and matrix solid phase extraction (MSPD) [14,16], have been tested and compared for pyrethrin separation. In addition, different organic solvents have been used for pyrethrin extraction, including hexane [8,13,18,20,21], acetonitrile [19], and petroleum ether [9]. In order to find the most suitable method, some authors have compared the effectiveness of different methods and solvents. Grdiša et al. [16] compared the extraction efficiency of maceration, ultrasound-assisted extraction (UAE), and matrix solid phase extraction (MSPD). Ban et al. [15] compared Soxtec, RP-HPLC, and ultrasound extraction with three different solvents (hexane, ethanol, and petroleum ether). Gallo et al. [21] compared three extraction methods, including traditional maceration in n-hexane and ethyl alcohol, supercritical CO₂ extraction (SC-CO₂), and cyclic pressure extraction, known as rapid solid–liquid dynamic extraction (RSLDE). Traditional methods, such as maceration, Soxhlet extraction, and solvent extraction, have notable limitations, including prolonged extraction times, high solvent consumption, and the risk of thermal degradation of pyrethrins due to extended heat exposure. These approaches often yield lower extraction efficiencies compared to more advanced techniques like ultrasound-assisted extraction or supercritical CO₂ extraction. Furthermore, the reliance on large volumes of organic solvents raises environmental and safety concerns, making the development of more efficient and sustainable alternatives increasingly important [22].

To promote sustainable practices, novel green extraction techniques were used to maximize extraction efficiency for the target compound while minimizing the use of solvents, shortening the amount of time, and lowering the temperature to prevent thermal degradation of pyrethrins from different species [23,24]. However, some unconventional extraction techniques have not been tested for pyrethrin extraction, including microwave-assisted extraction (MAE), subcritical water extraction (SWE), high-voltage electric discharge (HVED) extraction, and deep eutectic solvent (DES) extraction.

Against this background, the innovative, non-conventional extraction techniques of microwave-assisted extraction (MAE), high-voltage electric discharge (HVED) extraction, deep eutectic solvent (DES) extraction, and subcritical water extraction (SWE) were evaluated for the extraction of pyrethrins from Dalmatian pyrethrum (Tanacetum cinerariifolium), together with previously investigated supercritical CO₂ extraction (SC-CO₂) and ultrasound-assisted extraction (UAE). A key novelty of this research lies in the application of extraction methods that have not yet been explored for pyrethrin extraction.

The objective was to evaluate the most effective green extraction techniques and to optimize the process conditions in order to obtain pyrethrin from the Dalmatian pyrethrum plant (Tanacetum cinerariifolium) as a natural alternative to synthetic chemotherapeutics.

2. Materials and Methods

2.1. Chemicals

All chemicals were sourced from commercial suppliers. The reagents used to prepare the deep eutectic solvents are all commercially available from Acros Organics (Waltham, MA, USA): choline chloride (99%), N-methylurea (97%), thiourea (99+%), acetamide (99%), butane-1,4-diol (99%), xylitol (99+%), oxalic acid (98%, anhydrous), levulinic acid (98+%), malic acid (99+%), and malonic acid (99%); Gram mol (Zagreb, Croatia): urea (p.a.), glycerol (redistilled, p.a.), D-(+)-glucose (p.a.), and citric acid (p.a.); Carlo Erba (Carlo Erba Reagents GmbH, Emmendingen, Germany): ethane-1,2-diol (p.a.); T.T.T. (T.T.T. d.o.o., Croatia): lactic acid (min. 88%, p.a.); and Fischer Scientific (Fisher Scientific UK Ltd., Loughborough, Leicestershire, UK): sorbitol (lab. reag. grade), D-(+)-fructose (lab. reag. grade), and acetonitrile. Pyrethrins (technical) were purchased from Dr. Ehrenstorfer, Augsburg, Germany.

2.2. Plant Material

The flower heads of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./Sch.Bip.) were harvested by hand when three-quarters of the disc flowers were open in their natural habitat on the island of Brač, Split-Dalmatia County, Croatia, in mid-June 2020. The flowers were then spread out in a thin layer and first air-dried for 12 h and then air-dried for two months in cool, dry, and dark conditions. The dry flowers were then pulverized and stored at 40 °C until further analysis.

2.3. Extraction Procedure

The extraction of pyrethrins from the Dalmatian pyrethrum plant (Tanacetum cinerariifolium/Trevir./Sch.Bip.) (Figure 2) was carried out using microwave-assisted extraction (MAE), high-voltage electric discharge (HVED) extraction, deep eutectic solvent (DES) extraction, subcritical water extraction (SWE), supercritical CO₂ extraction (SC-CO₂), and ultrasound-assisted extraction (UAE) techniques. The applied extraction techniques and the obtained extract are summarized in Figure 3.

2.3.1. Microwave-Assisted Extraction (MAE)

The MAE of pyrethrins was conducted following the experimental plan with different solvents (water, acetone, and methanolic and ethanolic aqueous solutions), a solid/solvent ratio of 1:20 g/mL, and at temperatures of 30, 50, 70, 100, and 120 °C. A closed-vessel system (Milestone flexiWAVE Microwave apparatus) from Milestone Srl, Sorisole, Italy, was used for the extraction, with the maximum microwave power set to 500 W and 800 W. The power was not delivered continuously during the experiment in order to maintain a constant temperature. The preheating time was 2 min, and the cooling time was 10 min. After the completion of the extraction, the vessel was removed from the device, the resulting mixture was filtered, and the pyrethrin content was measured.

2.3.2. Ultrasound-Assisted Extraction (UAE)

An ultrasonic bath (Elmasonic P 70 H, Elma Schmidbauer GmbH, Singen, Germany) was used to generate the ultrasound effect. The temperature was controlled from 50 to 70 °C. Different solvents (water, acetone, and methanolic and ethanolic aqueous solutions) were used, and the solid/solvent ratio (1:20 g/mL), the treatment time (30 min), and the frequency (37 kHz) were constant.

2.3.3. High-Voltage Electric Discharge (HVED)-Assisted Extraction

The high-voltage electric discharge (HVED) device used in this study was developed by Ingeniare CPTS1, which is available at the Faculty of Food Technology in Osijek, Croatia, and is described in detail in Barišić et al. [25]. HVED-assisted extraction was performed with different frequencies (40, 70, 100 Hz), different solid/solvent ratios (1:100 and 1:300 g/mL), different solvents (methanolic aqueous solution and water), and a fixed treatment time (30 min). The HVED apparatus comprised a magnetic stirrer, a high-voltage pulse generator with an output power of 30 kV, a treatment chamber with two electrodes in the form of cylindrical stainless-steel needles (2.5 mm diameter), and a plate (45 mm diameter) for the ground electrode.

2.3.4. Deep Eutectic Solvent (DES) Extraction

Prior to extraction, the DESs were prepared according to Molnar et al. [26]. In brief, the components of the DESs were mixed in the exact molar ratio indicated in

Table 1. List of deep eutectic solvents (DESs) used for extraction.

	DES Combination	Molar Content
ChClU	Choline chloride: urea	1: 2
ChClmU	Choline chloride: N-methylurea	1: 3
ChCltU	Choline chloride: thiourea	1: 2
ChClG	Choline chloride: glucose	1: 1
ChClF	Choline chloride: fructose	1: 1
ChClX	Choline chloride: xylitol	1: 1
ChClS	Choline chloride: sorbitol	1: 1
ChClB	Choline chloride: butane-1,4-diol	1: 2
ChClE	Choline chloride: ethane-1,2-diol	1: 2
ChClGl	Choline chloride: glycerol	1: 2
ChClA	Choline chloride: acetamide	1: 2
ChClM	Choline chloride: malic acid	1: 1
ChClC	Choline chloride: citric acid	1: 1
ChClMa	Choline chloride: malonic acid	1: 1
ChClO	Choline chloride: oxalic acid	1: 1
ChClLa	Choline chloride: lactic acid	1: 2
ChClL	Choline chloride: levulinic acid	1: 1

. The mixtures were stirred and heated to 80 °C until a clear liquid was formed, which was used for the extraction process. Then, 50 mg of plant material was mixed with 1 mL of the solvent (all DESs were mixed with water to obtain a final mixture containing 20% water). The extraction was performed at 50 °C in a water bath for 30 min. The mixtures were centrifuged, decanted, and diluted with methanol before HPLC analysis.

2.3.5. Subcritical Water Extraction (SWE)

SWE was performed in a high-pressure batch-type extractor (Parr Instrument Company, Moline, IL, USA), and the extraction procedure and apparatus have been described previously [27]. Extractions were performed at 125, 150, and 175 °C with a reaction time of 15 min, and the ratio of solid/solvent was 1:20 g/mL. After extraction, the extracts obtained were filtered through filter paper under vacuum and analyzed.

2.3.6. Supercritical CO₂ Extraction (SC-CO₂)

The SC-CO₂ extraction was carried out in a customized extraction system, explained in detail in [28]. An amount of 100 g of the plant material was placed in the extraction vessel, and the separation of the extract was carried out at 15 bar and 25 °C. The extraction was performed at a pressure of 300 bar, a temperature of 40 °C, and a CO₂ flow rate of 2 kg/h, with a time of 60 min. For this type of extraction and according to the configuration of the device itself (pilot scale), a much larger amount of material was required compared to other extraction types, which is why only one extraction was performed for screening.

2.4. Chemical Characterization of the Target Groups of Bioactive Components (Pyrethrins)

The pyrethrin content was determined by HPLC (Agilent 1260 Infinity II, Santa Clara, CA, USA). The device is equipped with a quaternary pump, an autosampler, a thermostatted column, and a diode array detector (DAD). COSMOSIL 5C18-MS-II (particle size 250 mm × 4.6 mm, 5 μm) was used as the column, and chromatography was performed at 25 °C. The pyrethrin content was determined under the following conditions: mobile phase water (A)/acetonitrile (B) (0–5 min 42:58 v/v; 5–50 min 58–75 v/v B; 50–51 min 75–100 v/v B), injection volume 20 µL, flow rate 1 mL/min, and analysis time 51 min. The analysis was monitored at 230 nm with a DAD detector.

The compounds analyzed by HPLC were pyrethrin esters—pyrethrin I and II, cinerin I and II, and jasmolin I and II. A total of six compounds were separated by HPLC and identified by comparing the retention time with the technical standard and published work [13,15], and they were quantified using the calibration curve of the components specified in the technical standard.

Standard stock solutions for the technical standard of pyrethrin I and II, cinerins I and II, and jasmolin I and II were prepared in methanol, and calibration was performed at five concentrations, ranging from 0.9 to 46.8 mg/L. The retention time for pyrethrin I was 33.793 min, for pyrethrin II 14.900 min, for cinerin I 33.149 min, for cinerin II 14.571 min, for jasmolin I 42.668 min, and for jasmolin II 20.070 min. The prepared extracts were analyzed in duplicate, and the results are presented as ng/mg.

2.5. Statistical Analyses

Data processing was performed using the statistical software package Statistica version 14.0.1.25 (1984–2020 TIBCO Software Inc., Hamburg, Germany). For comparison between the different extraction methods, the Kruskal–Wallis ANOVA test was used. All statistical tests were conducted with a significance level of p = 0.05.

3. Results

In this study, the effects of various extraction techniques and process conditions on pyrethrin extraction efficiency were examined. The goal of the green extraction process is to maximize extraction efficiency for the targeted compounds while using the least amount of solvent, the shortest amount of time, and the lowest temperature possible to prevent any thermal degradation of the compounds under investigation. Therefore, six extraction methods, namely supercritical CO₂ extraction (SC-CO₂), subcritical water extraction (SWE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), high-voltage electric discharge (HVED) extraction, and deep eutectic solvent (DES) extraction were employed in order to obtain pyrethrins of Dalmatian pyrethrum. Besides the extraction technique used, the type of solvent plays a crucial role in the MAE. The results obtained (

Table 2. Pyrethrin content (ng/mg ±SD) of Dalmatian pyrethrum extracts obtained with MAE.

Run	Temperature (°C)	Power (W)	Time (min)	Solvent	Solid/Solvent Ratio (g/mL)	Cinerin II ng/mg	Pyrethrin II ng/mg	Jasmolin II ng/mg	Cinerin I ng/mg	Pyrethrin I ng/mg	Jasmolin I ng/mg	Total ng/mg
1	30	500	5	Water	1:20	-	-	-	-	-	-	-
2				50% ethanol	1:20	-	16.20 ± 0.00	-	-	-	-	16.20 ± 0.00 *
3				80% ethanol	1:20	-	16.60 ± 0.01	0.96 ± 0.03	-	-	-	17.56 ± 0.04 *
4				50% methanol	1:20	0.0	17.70 ± 0.11	1.08 ± 0.08	1.64 ± 0.02	0.0		20.42 ± 0.21 *
5				Acetone	1:20	-	-	-	1.86 ± 0.00	0.0	0.0	1.86 ± 0.00 *
6	50	500	5	Water	1:20	-	-	-	-	-	-	-
7				50% ethanol	1:20	-	-	-	-	-	-	-
8				80% ethanol	1:20	-	17.00 ± 0.01	0.10 ± 0.00	-	-	-	17.10 ± 0.02 *
9				50% methanol	1:20	-	-	-	-	-	-	-
10				Acetone	1:10	-	10.36 ± 0.10	0.57 ± 0.06	-	-	-	10.93 ± 0.16 *
11	70	500	5	Water	1:20	-	-	-	-	-	-	-
12				50% ethanol	1:20	-	16.70 ± 0.56	0.98 ± 0.08	-	-	-	17.68 ± 0.64 *
13				80% ethanol	1:20	0.0	18.90 ± 0.35	1.22 ± 0.04	2.05 ± 0.06	0.0	0.0	22.17 ± 0.45 *
14				50% methanol	1:20	-	-	-	-	-	-	-
15				Acetone	1:20	-	15.60 ± 0.07	-	-	-	-	15.60 ± 0.07 *
16	100	800	5	Water	1:20	-	-	-	-	-	-	-
17				50% ethanol	1:20	-	16.90 ± 0.24	0.99 ± 0.00	-	-	-	17.89 ± 0.27 *
18				80% ethanol	1:20	-	17.30 ± 0.15	1.01 ± 0.00	-	-	-	18.31 ± 0.18 *
19				50% methanol	1:20	0.0	18.30 ± 0.10	1.15 ± 0.00	1.72 ± 0.00	0.0	-	21.17 ± 0.00 *
20				Acetone	1:20	-	17.00 ± 0.01	1.00 ± 0.00	-	-	-	18.00 ± 0.01 *
21	120	800	5	Water	1:20	-	-	-	-	-	-	-
23				80% ethanol	1:20	0.0	18.9 ± 0.02	1.15 ± 0.00	2.08 ± 0.00	0.0	0.0	22.13 ± 0.02 *
24				50% methanol	1:20	0.0	18.1 ± 0.20	1.16 ± 0.02	1.76 ± 0.02	0.0	0.0	21.02 ± 0.24 *
25	50	500	5	Acetone	1:20	0.0	14.7 ± 0.03	3.86 ± 0.02	2.15 ± 0.00	0.0	0.0	20.71 ± 0.05 *
26				Acetone	1:20	0.0	18.9 ± 0.10	1.15 ± 0.00	1.89 ± 0.01	0.0	0.0	21.94 ± 0.11 *
27				Acetone	1:20	0.0	19.1 ± 0.01	1.16 ± 0.01	1.90 ± 0.01	0.0	0.0	22.16 ± 0.01 *

* Statistically significant differences (p = 0.006).

) show significant differences between the different solvents (p = 0.006). Water is not a suitable solvent for pyrethrins, while aqueous solutions of ethanol and methanol, as well as acetone, can successfully extract pyrethrins. When using 50 and 80% ethanol and acetone as solvents, an increase in total pyrethrins with increasing temperature is observed, whereas these phenomena were not observed with 50% methanol. The content of certain pyrethrins obtained by MAE depends on the extraction conditions. Cinerin II and pyrethrin I were not extracted at all with the above-mentioned solvents, while jasmolin II was extracted only with 50% ethanol at 120 °C. However, in all water extracts, no pyrethrins were extracted with MAE, regardless of the temperature applied. The highest pyrethrin II content was extracted with 50% ethanol at 120 °C and acetone at 50 °C, while the highest jasmolin II content was extracted with acetone at 120 °C (Table 2).

Using various UAE conditions, a yield of total pyrethrins of up to 104.65 ng/mg was obtained (

Table 3. Pyrethrin content (ng/mg ± SD) of Dalmatian pyrethrum extracts obtained with UAE.

Run	Temperature (°C)	Time (min)	Solvent	Solid/Solvent ratio (g/mL)	Cinerin II ng/mg	Pyrethrin II ng/mg	Jasmolin II ng/mg	Cinerin I ng/mg	Pyrethrin I ng/mg	Jasmolin I ng/mg	Total ng/mg
1	50	30	Water	1:20	-	-	-	-	-	-	-
2	50	30	50% Ethanol	1:20	-	8.25 ± 0.04	-	-	-	-	8.25 ± 0.04
3	50	30	50% Methanol	1:20	-	18.67 ± 0.10	0.91 ± 0.02	0.80 ± 0.01	80.72 ± 0.12	-	101.10 ± 0.25
4	50	30	Acetone	1:20	-	18.05 ± 0.00	0.15 ± 0.00	2.60 ± 0.03	72.66 ± 0.06	0.03 ± 0.03	93.49 ± 0.09
5	50	30	80% Ethanol	1:20	-	-	-	0.96 ± 0.02	76.33 ± 0.18	-	77.29 ± 0.20
6	70	30	Water	1:20	-	-	-	-	-	-	-
7	70	30	80% Ethanol	1:20	-	27.67 ± 0.10	1.52 ± 0.00	2.41 ± 0.02	73.05 ± 0.40	-	104.65 ± 0.53
8	70	30	80% Methanol	1:20	0.01	20.89 ± 0.01	1.49 ± 0.01	2.66 ± 0.08	72.49 ± 0.51	-	97.53 ± 0.61

). The extracts obtained by UAE at different temperatures and with different solvents show different concentrations. Similar to MAE, no pyrethrins were extracted with water. Cinerin II was extracted only with 80% ethanol at 70 °C and jasmolin I with acetone at 50 °C. In contrast to MAE, pyrethrin I was extracted with all solvents except water and 50% ethanol using UAE. The highest content of pyrethrin II and jasmolin II was extracted with 80% ethanol at 70 °C, and the highest content of cinerin I and pyrethrin I with 80% methanol at 70 °C and 50% methanol at 50 °C, respectively.

The high-voltage electric discharge (HVED)-assisted extraction, deep eutectic solvent (DES) extraction, and subcritical water extraction (SWE) of pyrethrins from Dalmatian pyrethrum were not suitable for the extraction of pyrethrins. None of the targeted compounds were detected in these extracts.

The application of SC-CO₂ is suitable for the extraction of all pyrethrins tested, with the most abundant being pyrethrins I and II. SC-CO₂ is the only one of the techniques tested that was able to extract all the pyrethrins listed, with the largest amount of 124.37 ng/mg of total pyrethrins extracted being obtained as the sum of six pyrethrins (

Table 4. Pyrethrin content (ng/mg ±SD) of Dalmatian pyrethrum extracts obtained with SC-CO₂.

Run	Cinerin II ng/mg	Pyrethrin II ng/mg	Jasmolin II ng/mg	Cinerin I ng/mg	Pyrethrin I ng/mg	Jasmolin I ng/mg	Total ng/mg
1	10.30 ± 0.01	48.22 ± 0.13	5.33 ± 0.16	9.95 ± 0.18	46.72 ± 0.46	3.85 ± 0.13	124.37 ± 1.07

).

Considering the results obtained, a statistically significant difference (p = 0.00005) in the total pyrethrin extracts (ng/mg) from Dalmatian pyrethrum is observed between the three techniques that proved successful in extraction: supercritical CO₂ extraction (SC-CO₂), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE).

4. Discussion

In this study, the effects of different non-conventional extraction techniques and process conditions on the efficiency of pyrethrin extraction were investigated. After the extraction of pyrethrins from the Dalmatian pyrethrum plant (Tanacetum cinerariifolium) using six novel green extraction techniques, it was found that the amounts of extracted pyrethrins obtained with MAE are very similar between the solvents and between the different extraction temperatures, and the highest amount of total pyrethrins was extracted with 80% ethanol at 70 °C, which is in accordance with previously published results [15]. Ethanol suitability is ambiguous and can be explained with similarity–intermiscibility theory or with suitability for microwave irradiation. According to this theory, ethanol is a suitable solvent because the pyrethrins mentioned are soluble in ethanol, and the more similar the polarity of the solvent and the solute, the faster the dissolution of solute from plant material [29]. Since pyrethrin compounds have a bipolar character, it cannot be claimed that the polarity of the solvents predominantly influences the extraction efficiency. However, the polarity of ethanol influences molecular dipole rotation to a greater extent, which means that it can absorb microwave radiation more efficiently and produce a thermal effect [30,31]. In previous studies, ethanol was shown to be efficient in extracting pyrethroids from litchi fruit [29] and pyrethrins from Dalmatian pyrethrum flowers [15].

By applying UAE conditions, the amounts of total pyrethrins extracted were up to five times higher than with MAE, with a significant difference (p < 0.05) between the two, i.e., the largest amount of pyrethrins was extracted with 80% MeOH at 70 °C and then with 80% EtOH, which is consistent with the work of Ban et al. [15], where ultrasound extraction with EtOH was found to be the most suitable for pyrethrin extraction. When comparing these two techniques (MAE and UAE), UAE is more efficient, considering that this technique extracted pyrethrin I and had the highest content of total extracted pyrethrins.

HVED-assisted extraction was not efficient in the extraction of pyrethrins since water is not an efficient solvent. The solubility of pyrethrins is due to their physical and chemical properties, including low vapor pressure, low Henry’s law constants, and large octanol/water coefficients. Apart from their interaction with polarized light, the enantiomers’ physical properties are all the same, except in the case of optical rotation, as the diastereomers have different physical properties (e.g., boiling point, melting point, solubility, etc.) [32]. Although HVED-assisted extraction can change some physical properties of the solvent, this was not sufficient for the successful extraction of the pyrethrins due to their negligible water solubility. The DES proved to be unsuitable for the extraction of pyrethrins, too. This could be due to the fact that the DESs used are very hydrophilic, which is not conducive to the extractability of pyrethrins, as shown in previous techniques. Water as a solvent was not suitable for the extraction of pyrethrins, even at high temperatures, which influenced the change in polarity of the water itself during SWE. Nevertheless, the application of such high temperatures does not have a positive effect on the extraction of pyrethrins. The same was observed in the MAE, where pyrethrins were not extracted in any of the water extracts, regardless of the temperature applied. This emphasizes how crucial the choice of solvent is during extraction and how much the solvent itself influences the selectivity of the process and, ultimately, the extraction of the target component.

It is well known that the most active components of pyrethrum extract are pyrethrin I and pyrethrin II. Pyrethrin I alone is toxic and acts within minutes, while pyrethrin II alone anesthetizes insects within seconds, so it has a high knockdown effect, although insects readily metabolize pyrethrin II and recover within a few hours [2]. When SC-CO₂ was used, the total amount of pyrethrins extracted was 124.37 ng/mg. According to the obtained results, the pyrethrin content was found to be higher in SCO₂, a non-polar solvent, than in polar solvents (particularly in water). The study by Kiriamiti et al. [18], as well as Pan et al. [20], confirmed the feasibility of the SC-CO₂ extraction of pyrethrins from pyrethrum flowers, which was confirmed on Dalmatian pyrethrum as well. Otterbach and Wenclawiak [17] compared pyrethrum extracts obtained by ultrasonic extraction, Soxhlet extraction using hexane, and SC-CO₂ extraction and found that the SC-CO₂ method gave better quality in terms of pyrethrin content.

5. Conclusions

From the obtained results, it is clear that the extraction efficiency does not depend on the use of a particular solvent or a particular extraction method, but it depends on the use of a proper solvent for a certain method. Among the used green extraction methods, SC-CO₂ extraction proved to be the most effective, as it successfully extracted all six pyrethrins tested. Therefore, this type of extraction provides a “natural” insecticide extracted with a clean, green technology and contributes to the development of sustainable and effective insecticide strategies by providing a natural alternative to synthetic chemicals while promoting the use of natural products. By optimizing pyrethrin extraction processes, this research can facilitate the potential application of pyrethrins as an insecticide and for parasite control while maintaining environmental safety and ecological production standards.