Phytochemical Profiling, Antioxidant and Anti-Inflammatory Activity of Plants Belonging to the Lavandula Genus

Lavender is a valuable medicinal plant belonging to the Lamiaceae family. Currently 39 species are known, but only Lavandula angustifolia is a pharmacopoeial raw material. Lavender has a long history of medicinal use and mainly exhibits antioxidant, anti-inflammatory, sedative, antidepressant, spasmolytic, anticholinesterases, antifungal and antibacterial properties. Used internally, it relieves symptoms of mental stress and insomnia and supports digestion. Topical use of lavender in aromatherapy, neuralgia and antiseptics is also known. The constant interest in lavender, and in particular in Lavandula angustifolia, in the field of medicine and pharmacy is evidenced by the growing number of publications. In view of so many studies, it seems important to review traditional and modern extraction techniques that determine the chemical composition responsible for the antioxidant and anti-inflammatory effects of various extracts from the species of the Lavandula genus.


Introduction
Lavender is a valuable medicinal plant belonging to the Lamiaceae family. It is native to the Mediterranean region and grows in natural sites of the lower parts of the mountains. Lavender is cultivated as an ornamental plant in many countries in Europe, north Africa, southwest Asia, western Iran, eastern India, China and Japan ( Figure 1). The Lavandula genus includes 39 species, but only Lavandula angustifolia is considered a pharmacopoeial raw material [1,2]. Lavender has a long history of medicinal use. In traditional medicine it is a popular herb used to treat multiple diseases. Lavender has antioxidant [3,4], antiinflammatory [5][6][7][8], sedative [9], antidepressant [10], spasmolytic, anticholinesterases [11], antifungal [8] and antibacterial [1] properties. Lavender is known as a medicinal product used internally to relieve symptoms of mental stress, insomnia and digestive disorders, and externally in aromatherapy, neuralgia and as an antiseptic. Lavender decoctions and hydrolates are applied as compresses that have a beneficial effect on the skin. Lavender infusions and lavender oil in the form of inhalation have sedative and anxiolytic effects that have been confirmed in both animal and human studies [9,10,12]. Lavender is a very popular aromatic plant and is commonly used in food and cosmetics thanks to its antibacterial, antifungal, antioxidant and anti-inflammatory properties. Lavender essential oil is present in eau de toilette, lotions, soaps, shampoos and household cleaners [13,14]. The essential oil of lavender can be added to cosmetics without the need for preservatives [1]. Because of its unique composition, pro-health benefits and attractive sensory attributes lavender can be used in the processing industry as a component of products with functional properties [3]. Owing to its medicinal activities, lavender, in particular Lavandula angustifolia, enjoys constant interest in the medical and pharmaceutical areas, as evidenced by an increasing number of publications in the last 25 years (Figure 2). 2

of 26
wound-healing properties of essential oil [9,10,12,17,18]. However, to the best of knowledge this work is the first attempt to review the data concerning the extraction a profile of other bioactive components of lavender, namely phenolic acids and flavono as well as the antioxidant and anti-inflammatory properties of lavender phenolic-r extracts.  In this review are presented the traditional and modern extraction techniques, chemical composition as well as antioxidant and anti-inflammatory activity of the extracts from different species of the Lavandula genus. The specific characteristics of each species are presented in Table 1 [15,16]. recent years, more and more species have been cultivated outside their natural The same species grown in different areas may show morphological difference color of leaves, flowers, plant density or seed weight. The morphological variabi thus the quantitative composition of individual compounds, is due to climatic co such as temperature, insolation, rainfall, humidity and altitude as well as enviro conditions such as fertilization, soil type and its pH level [16,19].

Extraction Process
Extraction of plant material is a process of separating bioactive compounds f sample by means of selective solvents and standard extraction procedures [20,21] extraction yield results from the appropriate selection of the solvent which sh suited to the nature of the compounds to be extracted [21,22]. The polarity of the compounds is especially important. For the extraction of non-polar compounds and chloroform are used [23]. Moreover, methanol, ethanol and acetone as we droalcoholic mixtures are the most generally used solvents for the extraction of po pounds [24]. Phenolic compounds are more stable at low pH, hence the acidified h coholic solvents are frequently used for their extraction [25]. The extraction proce fluenced by the physicochemical parameters of the solvents (boiling point, viscos sity, vapor pressure and solvent power), their cost, non-flammability and non [21,24], as well as sample preparation (drying, grinding and sample particle size) traction parameters (extraction time, temperature, number of extraction steps, rati vent to sample and use of co-solvent) [21,22,26,27].

Extraction Process
Extraction of plant material is a process of separating bioactive compounds from the sample by means of selective solvents and standard extraction procedures [20,21]. A high extraction yield results from the appropriate selection of the solvent which should be suited to the nature of the compounds to be extracted [21,22]. The polarity of the targeted compounds is especially important. For the extraction of non-polar compounds, hexane and chloroform are used [23]. Moreover, methanol, ethanol and acetone as well as hydroalcoholic mixtures are the most generally used solvents for the extraction of polar compounds [24]. Phenolic compounds are more stable at low pH, hence the acidified hydroalcoholic solvents are frequently used for their extraction [25]. The extraction process is influenced by the physicochemical parameters of the solvents (boiling point, viscosity, density, vapor pressure and solvent power), their cost, non-flammability and non-toxicity [21,24], as well as sample preparation (drying, grinding and sample particle size) and extraction parameters (extraction time, temperature, number of extraction steps, ratio of solvent to sample and use of co-solvent) [21,22,26,27].

Traditional Extraction Techniques
The conventional methods include solid-liquid extraction, such as maceration, digestion, percolation, infusion, decoction and Soxhlet extraction [28]. They are the general techniques used for the extraction of medicinal plants and are mostly applied for galenical preparations. One of these is tincture which is made as a result of maceration or percolation of plant material with ethanol of a suitable concentration [2]. Maceration is based on soaking plant material in a solvent at room temperature for several hours up to several days. The process of maceration that takes place at elevated temperature is called digestion [29]. The use of repeated maceration, grinding of plant material, high temperature and stirring during the extraction process increases its efficiency [29,30]. Moreover, percolation is a continuous extraction method in which after 24 h maceration a fresh solvent flows through the comminuted plant material and thus allows it to be completely etched. Percolation usually takes less time than maceration and requires percolators, that is, vessels with a conical shape that facilitate the removal of the extracted raw material [28,29]. Infusion is obtained by macerating the ground plant material with cold or boiling water for 5 to 15 min. Decoction differs from infusion in that the raw material is boiled in parallel with water from 15 to 30 min. In both cases the thus-obtained extract is cooled and filtered [28,29]. One of the most widely used traditional methods for the extraction of heat-stable compounds of medicinal plants is Soxhlet extraction. It is a form of continuous hot extraction in which the target compounds are extracted from solids with repeated washing with organic solvents such as ethyl acetate or hexane [24]. This technique is often used in the industry, but it should be remembered that it is not environmentally friendly because it uses large amounts of toxic solvents. During the extraction of lavender essential oil with hexane, other substances such as waxes, pigments and albuminous materials are extracted in addition to volatile compounds. The hexane extracts obtained in this way can be purified, but this is time-consuming and contributes to yield loss [3]. Additionally, long-term extraction time and high solvent temperature may result in the decomposition of valuable substances [22]. The disadvantages of traditional extraction techniques are that they require long analytical times and large quantities of solvents [21,25,27,30], and also they may contribute to the degradation of thermolabile compounds [22,27], thus resulting in a lower extraction yield [21,27]. Moreover, these methods are characterized by low selectivity and reproducibility [25]. Despite numerous drawbacks, these methods are still used because they are simple, easy to implement and do not require specialized equipment [31].

Modern Extraction Techniques
Several modern extraction methods which are environmentally friendly and thus called green techniques, such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), accelerated solvent extraction (ASE) and supercritical fluid extraction (SFE), have recently been introduced for the extraction of compounds from plant materials [27,30]. These techniques are characterized by lower volumes of organic solvents (up to 100 mL), shorter extraction times (up to 1 h, as compared to even days for, e.g., maceration) and thus lower energy consumption [24]. By using these methods better reproducibility and selectivity as well as higher quality of the extract can be obtained than with traditional techniques [21,24,25].
Ultrasound is sound waves with frequencies from 20 kHz to 100 MHz [30]. The best extraction frequency for lavender is 20-40 kHz [17,[32][33][34]. The propagation of ultrasound waves in the liquid is related to changes in the acoustic pressure causing the cavitation phenomenon. Furthermore, cavitation leads to the formation and collapse of gas bubbles, thus resulting in mechanical disruption of the cell walls thereby causing the release of target compounds from plant material [21,35]. Ultrasound extraction is a simple extraction technique requiring only a water bath or an ultrasonic probe applied directly to the sample [30,36]. The application of ultrasound can be an alternative technique for the extraction of phenolic compounds due to the reduction in the extraction time and the amount of the solvent [22]. This technique enables the extraction of several samples at the same time in water bath; however, after the extraction process, filtration or decantation is required. The extraction efficiency is influenced by many factors such as sample size, temperature, frequency and sonication time, as well as the kind of solvent and its pH [24]. For instance, polyphenolic compounds show higher stability at a lower pH of the solvent [25].
Microwave radiation is electromagnetic radiation in the range of 300 MHz to 300 GHz [30]. Microwave power from 230 to 500 W is most often used to extract lavender [33,37,38]. Microwave-assisted extraction (MAE) can be applied both for polar and non-polar solvents; however due to the high dielectric constant, polar solvents are more often used [21]. Solvents absorb microwave radiation and are heated to a temperature above the boiling point, which results in the rapid isolation of target compounds and high extraction efficiency [22]. Generally speaking, microwave-assisted extraction could reduce extraction time and solvent consumption and also increase the purity of the obtained extract in comparison with conventional methods [22,30,36]. Additionally, this technique allows processing of several samples at the same time [39]. Extraction conditions, such as temperature and extraction time, are very important but they should be chosen with caution to avoid thermal degradation of phenolic compounds [40].
Accelerated solvent extraction (ASE), also called pressurized liquid extraction (PLE), is performed at an elevated pressure, thus resulting in a higher extraction yield in comparison with conventional techniques. The extraction efficiency is influenced by parameters such as temperature, pressure, extraction time, the nature and volume of the solvent used as well as the solid-to-liquid ratio [35,41]. The extraction is carried out at temperatures between 40 • C and 200 • C and pressures between 3.3 and 20.3 MPa, and takes up to 20 min [22,24]. Increased pressure allows one to keep the solvent in the liquid phase above its boiling point whereas high temperature accelerates the kinetics of extraction [22,29]. This leads to a reduction in surface tension and solvent viscosity, and thus to an increase in the solubility of the compounds [27]. In accelerated solvent extraction ethanol or water are used to extract polyphenols [35]. Accelerated solvent extraction can be an alternative to other techniques because of the reduction in solvent quantities and extraction time, automation of the process and the possibility of extracting samples with high humidity [24,30]. On the other hand, the use of high temperatures may contribute to the decomposition of thermolabile compounds [29].
Supercritical fluid extraction (SFE) is an attractive technique for the extraction of bioactive compounds present in medicinal plants. Supercritical fluids exhibit the characters of both a liquid and a gas at their critical point [40]. Their densities are similar to the density of a liquid whereas low viscosity and surface tension make them similar to a gas [29,30]. Owing to their high diffusivity, supercritical fluids increase the extraction rate. The extraction procedure usually consists of static and dynamic phases. In the first phase the vessel with sample is filled with supercritical gas and thermostated for 10-15 min.
During the dynamic phase the supercritical fluid is continuously passed through the sample for about an hour and then the extract is collected in a receiver [3,42]. Solvents such as carbon dioxide, water, ethane, propane and dimethyl ether could be used in supercritical extraction [43,44]. However, the most commonly used supercritical fluid is carbon dioxide, which is considered safe for humans and the environment due to its non-toxicity, nonflammability and non-explosiveness. It enables the recovery of thermolabile compounds owing to its low critical temperature (31.1 • C) and pressure (7.38 MPa) [43,45]. Carbon dioxide is mainly used to extract carotenoids, lipids and essential oils due to its nonpolarity [22,30]. The addition of up to 15-20% of co-solvent (ethanol, methanol, water) to the supercritical fluid extraction process enables the extraction of polyphenolic compounds, which are polar [46]. The total use of carbon dioxide during extraction depends on its pressure, flow rate, temperature, sample size and extraction time. In the study of Woźniak et al., 2017 [42] the CO 2 consumption was 120-130 g for a 10 g sample and a CO 2 flow of 1.8 g/min. The major advantage of this method is the possibility to modify the solubility of individual compounds by changing the extraction parameters such as temperature and pressure [44]. Optimal extraction conditions to obtain the maximum yield and polyphenol content from lavender flowers were a temperature of 54.5 • C, a pressure of 29.79 MPa and an extraction time of 45 min [47]. Further advantages of supercritical fluid extraction are the lack of oxygen which prevents unfavorable oxidation processes [30] and the ease of separating the extractant from the product [22,45]. Various methods used for the extraction of secondary plant metabolites are presented in Table 2.
As can be seen, in the case of lavender the majority of studies used traditional methods, i.e., maceration, infusion or digestion with water, alcohol or hydroalcoholic mixtures (17 studies). Among them the best results in terms of total yield were obtained for maceration [11,48]. As for the modern techniques, the most often used (five studies) was ultrasound-assisted extraction. This is probably due to the relatively easy access to ultrasound devices in scientific laboratories. From the analysis of various approaches to the extraction of bioactive compounds other than the essential oil components from lavender, the best method of extraction seems to be ultrasound-assisted extraction with a hydroalcoholic solvent, which decreases the extraction time compared with traditional methods. As the phenolics in lavender are not very thermolabile, an elevated temperature (up to 60 • C) can be used, increasing the yield. Unfortunately, in many cases the yield of extraction could be determined because no dry extract was obtained [37,[49][50][51][52], and in some of the papers the mass of the extract and the extraction yield were not given.  14.17 ± 9.09 9.00 ± 3.00 6.56 ± 1.13 [49]    ND-no data. * denotes analyzes performed for flowers.
An equally important group of compounds present in lavender flowers are polyphenols. They are secondary plant metabolites with various biological properties. They are found in various parts of plants: flowers, leaves, stems, fruits and seeds [63][64][65]. So far, more than 8000 polyphenolic compounds have been identified. In terms of chemical structure, they are characterized by the presence of one or more aromatic rings in a molecule and different numbers of hydroxyl groups [63]. Polyphenols are biosynthesized through the shikimic acid pathway [66]. They can be divided into several different groups: phenolic acids, flavonoids, coumarins, stilbenes and lignans [65,66]. Most phenolic compounds are found in combination with sugars, organic acids and esters [63,64,67].
The presence of phenolic compounds in plant tissues protects them against adverse environmental conditions, such as high and low temperature, ultraviolet radiation, drought and salinity, and also against attack by herbivores, insects and microorganisms [64,66,69]. Besides biotic and abiotic stresses, the geographical area [16] and environmental factors such as soil composition, mineral fertilization, rainfall or temperature exert a notable effect on the content of polyphenolic compounds [49,58,64]. On the other hand, a study by Costa et al. [11] showed that the cultivation method affects the level of phenolic compounds. In vitro cultures of L. viridis were characterized by a higher content of phenolic compounds than wild plants, which was caused by differences between the growing conditions. The content of polyphenolic compounds also depends on the species, cultivars and selection of parts of the plant material. In the study of Blažeković et al. [60] the extracts prepared from the inflorescence stalk were characterized by a lower content of total polyphenols (3.09% and 4.54% for Lavandula x intermedia 'Budrovka' and Lavandula angustifolia, respectively) than lavender flower extracts (6.65% and 8.46%). However, the highest content of polyphenols was found in leaf extracts (7.05% and 9.20%). Likewise, Adaszyńska-Skwirzyńska and Dzięcioł [70] obtained the highest total polyphenol content for leafy stalk extracts (4.06 and 3.71 mg GAE/g d.m. for Lavandula angustifolia 'Blue River' and Lavandula angustifolia 'Ellagance Purple' extracts, respectively), and much lower for flower extracts (1.13 and 1.12 mg GAE/g d.m.). Moreover, the study by Bajalan et al. [71] showed that population variability has a significant effect on the variation in the content of phenolic compounds. Additionally, a positive correlation was found between the content of those compounds and the content of phosphorus in the soil.

Antioxidant Activity
Free radicals are formed as a result of endogenous processes, i.e., enzymatic and nonenzymatic reactions in the cells, as well as due to exogenous factors such as environmental pollution, cigarette smoke, ionizing radiation, ultraviolet radiation, industrial solvents and pesticides. Free radicals contain at least one unpaired electron on the valence shell and react readily with the molecules in their vicinity, acting as prooxidants.
According to their structure, prooxidants can be classified into radical reactive species (superoxide anion radical, hydroxyl radical, peroxyl radical, nitric oxide radical) and nonfree radical reactive species (peroxynitrite, singlet oxygen, hydrogen peroxide). Their overproduction leads to imbalance in the organism and damage to lipids, proteins and DNA due to their high reactivity. The ROS and RNS thus contribute to premature skin aging and the development of multiple diseases such as diabetes mellitus, hypertension, atherosclerosis, cardiovascular disease, liver diseases, renal failure, arthritis, cancer, as well as Alzheimer's and Parkinson's disease [74][75][76].
The antioxidant activity of polyphenols results from their ability to prevent the formation of free radicals or scavenge the reactive oxygen species. They can donate a hydrogen atom or an electron showing reducing properties [74]. These compounds can prevent oxidation processes by inhibition of xanthine oxidase, induction of antioxidant enzymes such as superoxide dismutase, glutathione dismutase, glutathione peroxidase and catalase, and chelating capacity due to metal ions [67,75]. Besides binding ferrous and copper ions, phenolic compounds also absorb UV radiation [77].
The antioxidant activity of phenolic compounds is associated with the presence and position of hydroxyl groups in their molecule. Stronger antioxidant properties were found for hydroxycinnamic acids than for hydroxybenzoic acids. Reports indicate that the highest antioxidant activity is demonstrated by rosmarinic acid, followed by chicoric acid and caffeic acid [78,79]. In addition, the presence of a methoxy group increases the antioxidant activity of phenolic acids. Ferulic acid having one group attached to the benzene ring is a less effective antioxidant than synapic acid which has two methoxy groups [80]. On the other hand, the antioxidant properties of flavonols are the consequence of the presence of a hydroxyl group at the C3 position of the flavonoid skeleton [81].
The methods of measuring antioxidant capacity can be described according to the reaction mechanism, namely SET (single electron transfer) or HAT (hydrogen atom transfer) mechanism, or both. The SET mechanism is based on donating one electron, whereas the HAT mechanism is based on the hydrogen atom transfer by the antioxidant [77,82].
The SET methods based on the reduction of ions include the FRAP and CUPRAC assays. The FRAP (Ferric Reducing Antioxidant Power) assay measures the reduction of ferric ion (Fe 3+ ) to ferrous ion (Fe 2+ ) through the donation of an electron. This reaction is carried out in an acidic medium (pH 3.6) to maintain the solubility of iron and leads to the formation of an intensely blue ferrous-tripyridyl-S-triazine (TPTZ) complex with an absorption maximum at 593 nm. TPTZ is the most popular iron-binding ligand used in the FRAP assay. The absorbance value of the sample is directly proportional to the concentration of the antioxidant. Moreover, in the CUPRAC (Cupric Ion Reducing Antioxidant Capacity) method, the reduction of Cu(II) ions to Cu(I) ions is used to measure the antioxidant capacity. The Cu(I) ions form an orange-yellow complex with neocuproin with an absorption maximum at 450 nm [37,77].
The ORAC assay (Oxygen Radical Absorbance Capacity) is one of the methods based on the HAT mechanism. It uses the process of deactivating peroxide radicals. It is based on measuring the decrease in the fluorescence intensity of fluorescein-a molecular probe that is oxidized by peroxide radicals. In this assay, AAPH (2,2 -azobis (2-amidinopropane) dihydrochloride) is mostly used as a source of free radicals. The degradation of fluorescein is slower when there are more antioxidants in the sample [48,82].
The most commonly used methods based on both mechanisms are the DPPH and the ABTS assays. DPPH (2,2-diphenyl-1-picrylhydrazyl) is a stable radical that can accept an electron or a hydrogen atom [83]. The DPPH alcoholic solution is dark purple in color with an absorption maximum at 517 nm. As a result of the reaction with phenolic compounds the ethanolic solution of the DPPH radical changes its color to light yellow [77]. The decrease in absorbance of the solution or signal intensity of electron paramagnetic resonance spectroscopy (EPR) is proportional to the amount of the reduced DPPH form that was formed during the reaction [74,84]. On the other hand, the ABTS method uses the ABTS radical cation (2,2 -azobis (3-ethylbenzothiazoline-6-sulfonate) which is formed as a result of chemical or enzymatic reactions. The ABTS radicals produced during the reaction with potassium persulfate are blue-green in color and have an absorption maximum at 734 nm. In the presence of antioxidants, the radical cation is reduced, resulting in discoloration of the solution [58,79], proportional to the antioxidant content in the sample [50]. This assay enables the measurement of the antioxidant activity of hydrophilic and lipophilic compounds due to the solubility of the ABTS radical in both aqueous and organic solvents [58]. Antioxidant activity of the extracts is usually expressed as Trolox equivalents, the synthetic vitamin E derivative [48,58,79]. In addition, Relative Antioxidant Capacity Index (RACI) can be used to comprehensively determine the total antioxidant capacity of the samples as determined using various methods, including ABTS, DPPH and ORAC [85].
As a different approach, the TBARS method (Thiobarbituric Acid Reactive Substances Assay) is used to measure lipid peroxidation products. It is based on the spectrophotometric measurement of malondialdehyde (MDA) produced during lipid peroxidation. As a result of the reaction of thiobarbituric acid (TBA) with MDA, a pink complex is formed that absorbs at a wavelength of 532-535 nm. The absorbance value is proportional to the concentration of MDA, a compound that is commonly used as an oxidative stress marker. In the presence of antioxidants the formation of MDA is inhibited [82]. The results of the antioxidant activity assessment of lavender extracts are reported in Table 2. Significant differences in antioxidant activity can result from the sample origin, the extraction method used, as well as the differences between species. Unfortunately, the way in which the results of antioxidant activity are presented by different scientific teams is difficult to compare due to different conversions for extracts, different units and substances as equivalents. In addition, the extraction conditions such as different concentrations of reagents, solvents and temperature are responsible for different obtained values even for the same substance marked with the same test. We have made an effort to standardize these values; however, the publications lack many details required for such conversion.
Robu et al. [4] performed a comparative study of antioxidant activity for various cultivars of Lavandula angustifolia with Lavandula hybrida. The highest result was obtained for Lavandula hybrida (IC 50 = 49.90 and 73.53 µg/mL for ferrous ion chelating and DPPH assay, respectively), followed by Lavandula angustifolia 'Hidcote Blue' (IC 50 = 49.93 and 96.53 µg/mL), and Lavandula angustifolia 'Munstead' (IC 50 = 50.60 and 101.40 µg/mL). Whereas, in the study by Blazeković et al. [60] in most of the tests a slightly higher antioxidant activity was observed for Lavandula angustifolia extracts as compared with Lavandula x intermedia 'Budrovka' extracts, which could be due to their higher polyphenolic contents. However, in both studies all Lavandula extracts showed a concentration-dependent antioxidant activity, wherein the strongest DPPH-radical-scavenging and iron-chelating activity were observed at a higher concentration of the extracts. On the other hand, in the study by Ahn et al. [49] the aqueous-methanolic leaf extract of Lavandula angustifolia 'Lady' afforded an over twice as high DPPH value (14.17 µmol TEAC/g DW) than the Lavandula latifolia extract (6.56 µmol TEAC/g DW). In another study, the antioxidant activity of methanolic extracts of different species and cultivars of lavender was analyzed [68]. The ability to scavenge free radicals of plant extracts decreased in the following order: Lavandula viridis (99.47% of inhibition), Lavandula stoechas (95.18%), Lavandula angustifolia 'Rosea' (93.92%), Lavandula lanata (92.78%), Lavandula angustifolia 'Afropurpurea' (92.09%) and Lavandula angustifolia (91.51%). The highest inhibition of the Lavandula viridis extract can be due to the presence of ferulic acid which was found only in this sample.
The above studies showed a significant antiradical activity of lavender extracts. On the other hand, a study by Celik et al. [37] showed that the antioxidant activity of the microwave-assisted lavender extract is significantly lower than that of other plants of the Lamiaceae family. The highest TAC values evaluated by the CUPRAC assay were obtained for Origanum majorana (0.66 mmol TR/g) and Mentha pulegium (0.58 mmol TR/g) extracts whereas the lowest ones were obtained for the Lavandula officinalis extracts (0.39 mmol TR/g). Likewise, Nicolai et al. [86] observed that ultrasound-assisted ethanolic extracts of Melissa officinalis, Salvia officinalis, Hypericum perforatum and Rosmarinum officinalis afforded the strongest DPPH-radical-scavenging activity of 95.2%, 94.7%, 92.7% and 72.5%, respectively. In contrast, the Lavandula angustifolia extract had a significantly lower value equal to 17.7%.

Anti-Inflammatory Activity
Inflammation is a protective response elicited by numerous biological (bacteria, fungi, viruses, endo-and exotoxins), chemical (acids, bases, carrageenan) and physical (mechanical factors, ultrasonic waves, ionizing radiation, magnetic field) agents. Each of these factors triggers a body defensive reaction [87]. Inflammation can be acute or chronic, and each type is related to different effects. In the acute phase, lasting from several minutes to a few days, neutrophils migrate from dilated blood vessels to the site of infection, causing redness, swelling and pain. Moreover, the state of chronic inflammation can lead to the development of multiple diseases such as rheumatoid arthritis, gout, cardiovascular diseases, diabetes, bowel diseases, Alzheimer's disease, cancer and depression.
For testing anti-inflammatory activity several animal experimental models are used (Table 5) such as the carrageenan-induced paw edema in mice [5] and in rats [92], the formalin test in mice [93], the croton-oil-induced ear edema in mice [7], the TPA-induced ear edema in mice [18], and the cell line stimulated with LPS [16]. In these studies antiinflammatory drugs such as dexamethasone [16,93] or the nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin [7,93], aspirin [6], ibuprofen [18] and diclofenac [5] are used as a positive control. NSAIDs work by inhibiting cyclooxygenase, the enzyme responsible for eicosanoid synthesis [94]. Cyclooxygenase (COX) converts arachidonic acid to prostaglandin G2, then into prostaglandin H2, and finally to prostaglandins (PGF2 α and PGE 2 ), prostacyclin (PGI 2 ) and thromboxane A2 (TXA 2 ) [91]. There are three cyclooxygenase isoforms. COX-1 is a constitutive form that plays a role in normal physiological processes. COX-2 is an inducible form involved in inflammation, whereas the least known COX-3 is associated with the central nervous system. The majority of NSAIDs inhibit both COX-1 and COX-2 [91,95]. On the other hand, lipoxygenase (5-LOX) metabolizes arachidonic acid to 5-hydroxyeicosatetraenoic acids and leukotrienes (LTs) [88,96]. Research has confirmed that the anti-inflammatory activity of lavender is due to the presence of essential oil components, non-volatile terpenoids and polyphenols. In the study by Carrascoet al. [13], thymol, fenchone and camphor from Lavandula stoechas essential oil showed an inhibitory effect on lipoxygenase (LOX). Luo et al. [18] observed that essential oil of Lavandula angustifolia reduced the expression of inflammatory mediators such as IL-6, NF-κB and TNF-α in the TPA-induced ear edema model in mice. Furthermore, in the study by Sosa et al. [7] the higher inhibition of croton-oil-induced ear edema in mice was observed for ethanolic extracts of Lavandula multifida than for aqueous extracts, but in both cases the antiphlogistic activity was dose-dependent. The terpenoids ursolic acid, oleanolic acid and maslinic acid and phenolic monoterpene carvacrol were obtained as a result of fractionation of the ethanolic extract. Numerous scientific reports [5,6,16,92] indicate that plants of the Lavandula genus are a source of polyphenolic compounds with anti-inflammatory activity. One of many antiphlogistic mechanisms of polyphenols is their ability to inhibit enzymes including iNOS, LOX and COX [92,96]. The anti-inflammatory activity of the Lavandula extracts was assessed using a fluorimetric test based on the detection of prostaglandin G2 generated by the COX enzyme. Cyclooxygenase COX-2 was more strongly inhibited by lavender extracts than cyclooxygenase COX-1. Shaikh et al. [6] observed that the ethanolic fraction of Lavandula bipinnata obtained by Soxhlet extraction inhibited COX-2 by 50%, compared to COX-1 by 19%. A high-performance thin-layer chro-matography (HPTLC) analysis showed that the extract was rich in flavonoids. Likewise, Husseini et al. [93] demonstrated that the anti-inflammatory activity of hydroalcoholic macerate of Lavandula officinalis was associated with the inhibition of COX-2 by 45% and COX-1 by 33%. In addition, the anti-inflammatory effect of COX-2 increased with the increasing concentration of the extract. Researchers also showed that the extract inhibited the chronic (inflammatory) phase of formalin-induced pain in mice, whereas it had no effect on the acute (neurogenic) phase. Moreover, in the study of Algieri et al. [5], Lavandula stoechas and Lavandula dentata hydromethanolic extracts, obtained using accelerated solvent extraction (ASE), showed anti-inflammatory activity against carrageenan-induced paw edema in mice. The hydromethanolic extract of Lavandula stoechas at a dose of 100 mg/kg significantly decreased the expression of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α and the enzymes iNOS, COX-2 and MMP-9, whereas the extract of Lavandula dentata decreased the expression of only iNOS, COX-2 and IL-1β. The qualitative analysis of the extracts performed using RP-HPLC-DAD-MS showed that they contained the phenolic acids hydroxybenzoic acid, hydroxycinnamic acid and their derivatives as well as flavones which are a subclass of flavonoids. Similarly, Yassine et al. [92] observed the anti-inflammatory activity of the extracts of Lavandula stoechas on carrageenan-induced paw edema in rats. A significant inhibition of edema was found in the case of hydroethanolic extract obtained using ultrasound-assisted extraction and two fractions rich in flavonoids and mucilages, whereas that effect was not observed for the tannin fraction. Furthermore, Lopes et al. [16] used the mouse macrophage-like cell line RAW 264.7 stimulated with LPS to study the antiinflammatory activity of the extracts of Lavandula pedunculata. The hydroethanolic extracts displayed a higher anti-inflammatory potential through inhibition of NO production than the aqueous extracts (infusions).

Conclusions
According to Pharmacopoeia XI, only Lavandula angustifolia is currently recognized as a medicinal raw material. However, in recent years other species such as Lavandula stoechas, Lavandula intermedia, Lavandula latifolia, Lavandula dentata and Lavandula luisieri have also gained increasing interest in the medical and pharmaceutical areas. Many of these species are a rich source of phenolic compounds such as phenolic acids and flavonoids, thus resulting in antioxidant and anti-inflammatory properties. However, in order to obtain a high-quality extract, it is crucial to select the appropriate extraction method.
In this review are presented both the traditional and modern extraction methods, also called green techniques. These techniques are characterized by lower consumption of the solvents and shorter extraction times, which means lower energy consumption. By using these methods better reproducibility and selectivity as well as higher quality of the extracts can be obtained than by traditional techniques. Each of the presented methods has its advantages and disadvantages. Which method a given laboratory chooses depends on many factors, such as the availability of equipment, solvents and trained staff, as well as maintenance costs.
However, since in the analyzed studies there was a great variation in both extraction protocols and plant material, there is a need for a systematic study comparing various extraction techniques of the same plant material to reliably recommend the optimal approach. Further studies are needed with special attention paid to the optimization extraction and activity-guided extraction. Additionally, a key issue is to standardize the units and substances as equivalents in which the obtained results of activity determination are presented. There is also a gap concerning the extraction and analysis of lavender flower residues left after the distillation of essential oil-currently the main product of lavender-which could be a valuable source of phenolics exhibiting antioxidant and anti-inflammatory properties.