Genus Echinophora—Biological Activity, Chemical Composition, and Future Perspectives

Species belonging to the genus Echinophora (Apiaceae) have been used by humanity since ancient times as flavoring agents, preservatives, and medicines for the treatment of a broad spectrum of conditions, including respiratory, digestive and kidney disorders, fungi infections, wounds, and gastric ulcers. Phytochemical studies on this botanical genus mainly investigate the essential oil composition and reveal the species as a rich source of volatile compounds, including monoterpenes and phenylpropanoids. Among the non-volatile secondary metabolites, flavonoids, coumarins, phenolic acids, phytosterols, and polyacetylenes have been identified. Pharmacological studies revealed Echinophora extracts and essential oils exhibit different biological properties, including antibacterial, antifungal, anticancer, insecticidal, anti-inflammatory, and hepatoprotective activities. However, compared to other genera, the biological activity and chemical diversity of this genus are not well studied. In future decades, it is highly likely that the small genus Echinophora will play an important role in drug discovery and drug development.

The name Echinophora originates from ancient Greek and is composed of the words echino (spine) and phora (leaf), which describe the spiny leaves of the species within the genus [2].The genus is distributed in regions of the Mediterranean and the Balkan Peninsula, Anatolia, eastward to Iran and Afghanistan [3].Three of the species, namely E. chrysantha, E. lamondiana, and E. trichophylla, are endemic to Turkey, while E. platyloba and E. cinerea are endemic to Iran [1,3].In Europe, the genus is represented by E. spinosa, E. tenuifolia, and E. tenuifolia subsp.sibthorpiana (or E. sibthorpiana) (Figure 1), the latter being the most widely distributed species from the genus that can be found in numerous countries, including countries of the Balkans (Greece, Bulgaria, and North Macedonia), Turkey, and Iran [1,[3][4][5][6].E. spinosa is a halophytic plant distributed in the sandy coastal regions of the Mediterranean, found in South Europe (including France, Italy, Montenegro, and Greece), as well as the coastal regions of Northern Africa, including Algeria, and more recently, Tunisia [7][8][9][10].and more recently, Tunisia [7][8][9][10].Since ancient times, Echinophora species have been used by humanity as flavor agents, preservatives, and medicines [1].Compared to other genera, the biological act ty and chemical diversity of Echinophora are not well studied.Currently, plants belo ing to this genus are considered promising candidates for novel phytopharmaceutic Some of the target directions of Echinophora species are oncology [11][12][13], cardiology [ gynecology [15], and gastroenterology [16].Previous studies established that Echinoph extracts contain molecules called echinophorins [2,17].It was found that these co pounds demonstrate significant selectivity toward the transient receptor potential ch nels of ankyrin type-1 (TRPA1).Echinophorins are considered to have a promising tential in the management of pain and inflammation [2].
The aim of this study is to review the phytochemistry, biological activity, pharmacologic properties of Echinophora species.

Secondary Metabolites in Echinophora Extracts
The Apiaceae family is characterized by the production of distinctive secondary tabolites such as coumarins and furanocoumarins, polyacetylenes, volatile phenylp penes, etc. [18].Phytochemical studies regarding the composition of Echinophora extr are limited.In general, the main compounds identified in extracts of this botanical ge are polyphenolic compounds, including phenolic acids, flavonoids, and coumarins well as polyacetylenes, fatty acids, and terpenoids, namely phytosterols [2,11,[19][20][21][22][23][24][25].overview of the secondary metabolites identified and isolated from extracts of E nophora species is depicted in Table 1.
The aim of this study is to review the phytochemistry, biological activity, and pharmacologic properties of Echinophora species.

Secondary Metabolites in Echinophora Extracts
The Apiaceae family is characterized by the production of distinctive secondary metabolites such as coumarins and furanocoumarins, polyacetylenes, volatile phenylpropenes, etc. [18].Phytochemical studies regarding the composition of Echinophora extracts are limited.In general, the main compounds identified in extracts of this botanical genus are polyphenolic compounds, including phenolic acids, flavonoids, and coumarins, as well as polyacetylenes, fatty acids, and terpenoids, namely phytosterols [2,11,[19][20][21][22][23][24][25].An overview of the secondary metabolites identified and isolated from extracts of Echinophora species is depicted in Table 1.
Characteristic bioactive chemical compounds for the Apiaceae family are polyacetylenes, mainly represented by the falcarinol-type C 17 -polyacetylenes [27].The archetypal C 17polyacetylene falcarinol is an aliphatic alcohol biosynthesized from fatty acids, the structure of which contains two triple bonds and two double bonds (Figure 2A) [2,27].A less common class of polyacetylenes, the C 14 -polyacetylenes, have been described in extracts of different plant species, including Codonopsis pilosula [28], Coreopsis tinctoria [29,30], and species from the genus Lobelia and the genus Siphocampylus [31].Unique among the C 14polyacetylenes are the C 14 -polyacetylenes containing α-pyrone moiety, first described in Mediasia macrophylla [32] and consequently found in the Iranian Echinophora species E. cinerea and E. platyloba [2,17,20].The echinophorins echinophorin A, echinophorin B, and echinophorin C, a type of α-pyrone containing C 14 -polyacetylenes named after the genus, were first isolated from acetone extract of E. cinerea [17].Echinophorin A and echinophorin B were later isolated from the dichloromethane fraction of E. platyloba extract in addition to one new echinophorin-echinophorin D [2].To date, no other echinophorins have been described.Similar to other polyacetylenes like falcarinol, the structure of echinophorins includes two or three conjugated triple bonds, but it differs by the presence of an α-pyrone ring, which is a unique feature among this class of compounds [2].The structures of the echinophorins, isolated from E. cinerea and E. platyloba, are presented in Figure 2B.Besides echinophorines, the C 17 -polyacetylene falcarinol was identified as one of the main constituents of the essential oil isolated from the roots of E. spinosa, as well as in essential oil from the roots of E. orientalis [7,[33][34][35].

Country of Origin Main Phytochemicals
Refs.

Volatile Constituents of Echinophora Essential Oils
One of the main features of the large Apiaceae family, to which the genus Echinophora belongs, is the production of volatile terpenoids, and many of the species within this family are excellent sources of essential oils (EOs) [36].Most of the phytochemical studies of the species within this genus are focused on the composition of their EOs.The major volatile constituents identified in the isolated EOs from Echinophora species are summarized in Table 2.
The chemical composition of E. tenuifolia subsp.sibthorpiana EO varies depending on different factors such as the vegetative stage of the plant, the drying conditions, the

Volatile Constituents of Echinophora Essential Oils
One of the main features of the large Apiaceae family, to which the genus Echinophora belongs, is the production of volatile terpenoids, and many of the species within this family are excellent sources of essential oils (EOs) [36].Most of the phytochemical studies of the species within this genus are focused on the composition of their EOs.The major volatile constituents identified in the isolated EOs from Echinophora species are summarized in Table 2.
The chemical composition of E. tenuifolia subsp.sibthorpiana EO varies depending on different factors such as the vegetative stage of the plant, the drying conditions, the harvest location, the year of collection of the plant, and storage conditions [38,40,42,43].Şanlı et al. compared the composition of the EO changes depending on the growth stage of the plant (rosette, vegetative growth, flowering, or fruit-ripening) and the drying conditions (fresh plant, shade-dried, and sun-dried) and found that the content of methyl eugenol decreased from the rosette stage to the fruit-ripening stage, being the highest in the rosette stage, while the content of α-phellandrene increased, being the highest at the flowering stage [38].Moreover, the content of methyl eugenol was the highest in the EO isolated from fresh plants and decreased during drying (shade and sun drying) in all flowering stages, whereas the content of α-phellandrene increased significantly during drying.Özcan and Akgül compared the chemical composition of the EOs from plants collected in different months (April, May, and June)-the quantity of both methyl eugenol and α-phellandrene in the EOs increased during the period of the study, being the highest in June (25.96%and 29.96%, respectively) [40].Additionally, the yield and composition of the EO are significantly affected by the cultivation of the plant [44].The cultivated E. tenuifolia subsp.sibthorpiana showed higher EO yield in addition to changes in the EO composition-the content of the main constituent in the wild plant δ-3-carene decreased dramatically in the cultivated plant, while that of α-phellandrene increased [44].
Apart from methyl eugenol, four other members of the phenylpropanoid class of volatile compounds have been detected in considerable amounts in species of the Echinophora genus, specifically myristicin in E. spinosa and E. orientalis [7,8,35,47] and asarone, anethole, and eugenol in E. platyloba [48].Myristicin, terpinolene, and the polyacetylene (Z)-falcarinol are the dominant compounds in the root EO of E. spinosa [7,33].Myristicin was the principal compound in the root EO (47.4%) of E. spinosa growing in Corsica Island, France, while its content in the aerial parts EO was notably lower (2.7%) [7].In addition to myristicin, the root EO contained significant amounts of terpinolene and (Z)-falcarinol and negligible amounts of p-cymene (0.3%), which was the most abundant compound of the aerial part EO (35.8%) along with α-phellandrene (21.7%) [7].Notable differences between the composition of the root and aerial parts EOs from E. spinosa growing on the Island of Sicily were observed as well [33].While the root EO was characterized by the predominant compounds myristicin, terpinolene, and (Z)-falcarinol, the major constituents of the aerial parts EO were α-phellandrene and p-cymene [33].Interestingly, the EO from roots of E. orientalis from Iran showed a similar profile to the E. spinosa root EO, with myristicin, terpinolene, and falcarinol being the major compounds identified [35].In the aerial parts and ripe fruit EOs of E. spinosa from central Italy, the content of myristicin was 16.5% and 15.3%, respectively [8].The main constituent of the aerial parts EO was β-phellandrene (34.7%), while the greatest fraction of the fruit EO was represented by p-cymene (50.2%) [8].The principal constituent of aerial parts E. spinosa EO from Montenegro was δ-3-carene (60.86%), whereas the content of myristicin was low (1.04%) [47].
The main volatile constituent detected in the EO of the endemic Iranian species E. cinerea is α-phellandrene, with α-pinene, p-cymene, and β-phellandrene being found in significant amounts [49][50][51][52][53].In a comparative study of E. cinerea EOs isolated from plants in different growth stages (early flowering stage and full flowering stage) from different populations in Iran, it was found that the yield of the EO and the content of α-phellandrene was the highest during the full flowering stage of the plant [49].In another study comparing the method of extraction of the EO, the content of α-phellandrene in the EO extracted via headspace solvent microextraction (HD-SME) was significantly higher than that extracted with conventional hydro-distillation [53].

Ethnobotanical Importance and Ethnomedicinal Use
The species within the genus Echinophora have a long history of traditional use, including ethnomedicinal use, specifically E. platyloba in Iran [66] and E. tenuifolia subsp.sibthorpiana in Turkey [67].The ethnomedicinal use of these plants is summarized in Table 3.
E. platyloba, also known as "Khosharizeh" or "Khosharouzeh" in Persian, is used in the cuisine of Iran as a spice, food flavoring agent in dairy products, and as a preservative in tomato paste and pickles [58,66,68].Moreover, E. platyloba has been reported in various ethnobotanical and ethnomedicinal studies for its use as a medicinal plant in many different regions of Iran.It is used for relieving the symptoms of respiratory conditions like cough and common cold, digestive disorders like ileus, flatulence, diarrhea, and hemorrhoids, in the treatment of kidney stones and kidney pain, joint pain, mouth ulcers, and as an antifungal agent [68][69][70][71][72][73][74].E. cinerea, also known as "Khosharizeh-e-Kohestani" in Persian, is used in the folk medicine of Iran as a stimulant and an invigorator of the stomach [49].
E. tenuifolia subsp.sibthorpiana (E.sibthorpiana), also known as tarhana herb or "Çörtük" in Turkish, is a popular aromatic plant in Turkey, the fresh or dried leaves of which are used as a flavoring agent in different foods such as meat and meatballs, pickles, and soups [67,75].The leaves of the plant are used in pickles to prevent foaming and improve the shelf life of pickled products [76].In some regions of Turkey, the young stems of E. sibthorpiana and E. tournefortii are eaten as a snack after pealing the outer part off the plant [77,78].One of the main culinary uses of E. sibthorpiana is its use as a flavoring agent in the fermented cereal food tarhana, which is produced by mixing flour, yogurt, yeast, different vegetables (tomatoes, red peppers, etc.), and herbs (thyme, dill, tarhana herb, etc.), followed by fermentation for several days, drying and grinding, and then prepared as a soup [79][80][81].Apart from flavoring, it has been found that the addition of appropriate ratios of E. tenuifolia subsp.sibthorpiana in tarhana could aid the fermentation process by preventing the decrease in yeast and lactic acid bacteria and improve the nutritional properties [67,82].For medicinal purposes, the plant has been employed in the treatment of various conditions for its antispasmodic, digestive, galactagogue, and wound-healing properties [83][84][85].Moreover, it is used orally in respiratory conditions like shortness of breath and externally for perspiration in case of the common cold [86,87].Along with E. tournefolii (also known as "Dikenli Çörtük" in Turkish), it is used in the treatment of gastric ulcers in the form of 5% infusions [61,88].
While there is no record of traditional medical use of E. spinosa, the leaves and roots of the plant are recognized as edible, and the young shoots and leaves without thorns can be consumed pickled, in salads, or cooked [7].The roots of E. orientalis are used as a flavoring agent and to impart tenderness to the traditional Turkish dessert "halva" (or "helva") [65,78].

Pharmacological Activities of the Echinophora Genus
Extracts and EOs isolated from this genus exhibit a wide range of biological and pharmacological activities, among which antioxidant, antibiotic and antifungal, antiinflammatory, anticancer, and insecticidal and larvicidal activities.An overview of the results from studies investigating these activities is presented in Table 4.

Antibacterial Activity
The antibacterial activity of the aerial parts EO and different extracts (methanol, ethanol, and aqueous) from aerial parts and roots from E. tenuifolia subsp.sibthorpiana was investigated against eight pathogenic bacteria [6].The EO demonstrated the highest antibacterial activity, specifically against S. typhimurium (MIC of 0.34 mg/mL, MBC of 1.35 mg/mL), B. cereus (MIC of 0.45 mg/mL, MBC of 1.35 mg/mL), and P. aeruginosa (MIC of 0.34 mg/mL, MBC of 2.70 mg/mL) [6].In addition, strong antibacterial activity was observed with the ethanol root extract, which showed high activity against B. cereus (MIC of 0.45 mg/mL), as well as activity against E. cloacae, E. coli, M. flavus, P. aeruginosa, and S. typhimurium (with MIC of 1.5 mg/mL for all strains), while the aqueous extracts were the least active [6].
The EO isolated from E. tenuifolia subsp.sibthorpiana leaves was screened for antibacterial activity against thirteen bacterial strains, including Gram-positive (S. aureus, S. epidermidis, S. warneri, S. hominis, B. cereus, E. faecalis, Streptococcus spp.) and Gram-negative bacteria (E.coli, S. flexneri, Enterobacter spp., Salmonella spp., Klebsiella spp.) [39].In general, the EO was more effective against the Gram-positive pathogens and demonstrated good antibacterial activity against B. cereus (MIC 62.5 µg/mL) and moderate activity against S. epidermidis and S. aureus (MIC 125 µg/mL for both strains), while the activity against the tested Gram-negative bacteria was weak [39].Moreover, according to the composition of the studied EO, it could have a great potential for skin recovery effects [39].One of the main compounds identified in the EO is α-phellandrene.This compound promotes the wound healing processes, accelerates the wound closure, and acts as an adhesive of primary intention [89].α-Phellandrene contributes to collagen deposition as well [89].The EO is rich in other compounds that are associated with wound-healing activity.These effects, in addition to the antibacterial activity, make the EO worth studying as a skin recovery therapy in surgical procedures or other interventions [89][90][91][92].Currently there are no human studies in surgery for the wound healing activity of the EO isolated from Echinophora species.
Hashemi et al. investigated the antimicrobial activity of E. platyloba aerial parts EO and methanolic extract against food-borne pathogenic bacteria, including S. aureus, L. monocytogenes, S. typhimurium, and E. coli [57].The results of the study revealed that while both the EO and the extract possessed antibacterial activity, the activity of the EO was higher than that of the methanolic extract [57].The EO showed the highest activity against L. monocytogenes (MIC = MBC = 6250 ppm), as well as activity against S. aureus (MIC 12,500 ppm and MBC 25,000 ppm) and E. coli (MIC 50,000 ppm), whereas the extract was active only against L. monocytogenes and S. aureus (MIC 25,000 ppm for both strains).Neither the EO nor the extract showed activity against S. typhimurium [57].
Ghasemi Pirbalouti and Gholipour reported the antibacterial activity of EO isolated from different populations of E. cinerea collected during different harvest times (early and full flowering stage) on five pathogenic microorganisms, including the Gram-positive bacteria B. cereus and L. monocytogenes and the Gram-negative bacteria P. vulgaris and S. typhimurium [49].The EOs showed moderate-to-good antibacterial activity, especially against B. cereus, with the lowest MIC (32 µg/mL) for EO isolated from plants collected during the full flowering stage [49].The same EO also showed highest activity against L. monocytogenes and P. vulgaris with MIC = 62 µg/mL [49].
The antibacterial activity of methanol extracts from different parts (leaves and ripe fruits) of E. spinosa against B. subtilis, S. aureus, E. coli, P. mirabilis, and P. aeruginosa was investigated by Ghadbane et al. [9].The highest activity demonstrated the ripe fruit extract compared to leaves extract and gentamicin against B. subtilis, E. coli, and P. mirabilis with inhibition zones of 23.67 ± 1.53 mm, 30.50 ± 0.50 mm, and 20.50 ± 0.50 mm, respectively [9].Neither the leaves nor ripe fruit methanol extracts showed activity against S. aureus and P. aeruginosa [9].In addition, the EO isolated from ripe fruits of E. spinosa exhibited higher antibacterial activity compared to aerial parts EO in a study conducted by Fraternale et al. [8].Both EOs inhibited the growth of potentially pathogenic Gram-positive gastrointestinal bacteria, including C. difficile, C. perfringes, E. faecalis, and E. limosum, with MIC values for the ripe fruit EO of 0.13% (v/v) [8].Moreover, the MIC values were significantly higher for the valuable intestinal microflora tested, including two strains of Bifidobacterium and two strains of Lactobacillus (>4.00%) [8].

Antifungal Activity
Aerial parts EO from E. tenuifolia subsp.sibthorpiana demonstrated strong antifungal properties against A. versicolor, P. funiculosum, P. ochrochloron with MIC values of 0.17 mg/mL, and A. fumigatus, A. ochraceus, T. viride with MIC values of 0.34 mg/mL, while its activity against C. albicans was lower (MIC 2.70 mg/mL) [6].The antifungal activities of the root and aerial parts ethanol, methanol, and aqueous extracts of the same plant were significantly lower than that of the EO, with aqueous extracts exhibiting the lowest antifungal activity [6].Methanol extract from the ripe fruits of E spinosa demonstrated better activity than leaf extract against C. albicans with an inhibition zone of 19.67 ± 1.53 mm [9].
Out of all Echinophora species, the most promising antifungal activity has been reported for E. platyloba, which has been an object of numerous in vitro studies demonstrating its antifungal potential, linking it to its ethnomedicinal use [66,94].
The antifungal activity of ethanolic extract of E. platyloba was investigated against the dermatophytes T. schenlaini, T. verucosum, T. rubrum, M. gypsum, T. violaseum, T. mentagrophytes, M. canis and E. flucosum and the extract exhibited the highest activity against T. schenlaini and T. verucosum at concentrations as low as 35 mg/mL [66,94].Consequently, the activity of the extract was screened against C. albicans.The extract showed significant anti-Candida activity at different concentrations, the lowest concentration inducing inhibition of the growth being 2 mg/mL [95].
Ethanol and ethyl acetate extracts from E. platyloba leaves showed good antifungal activity against nine clinically isolated strains of C. albicans, with the lowest observed MIC (12.5 mg/mL) for the ethyl acetate extract [96].Moreover, extract from E. platyloba not only inhibited the growth of clinical strains of C. albicans resistant to fluconazole, but it also reduced the expression of the CDR1 and CDR2 genes, which play an important role in the development of azole drug resistance [97].
In a study investigating the synergistic anti-Candida activity of ethanolic extract of E. platyloba and azole antimycotics, it was found that the E. platyloba extract exhibited synergism with fluconazole and itraconazole and antagonism with clotrimazole and miconazole against clinical strains of C. albicans [98].The activity of the combination of fluconazole with E. platyloba extract was assessed in a randomized double-blind clinical trial, which included sixty women with recurrent candidal vaginitis [99].The participants were randomized into two groups, one of which was treated with fluconazole alone and the other treated with fluconazole and cream containing E. platyloba extract.The group receiving fluconazole and E. platyloba cream showed a statistically significant decrease in positive culture results after fourteen days of treatment and a decrease in the frequency of recurrence of the vaginitis [99].
In addition to fluconazole and itraconazole, the extract showed synergism with amphotericin B [94].The combination of 5% ethanolic extract with amphotericin B showed an increase in the zone of inhibition by 22% compared to amphotericin B alone (22 mm for the combination compared to 18 mm for amphotericin B alone) and a decrease in the MIC by 50% (1 mg/mL for the combination compared to 2 mg/mL for amphotericin B alone) [94].

Anti-Parasitic Activity
Ngahang Kamte et al. examined the activity of the EOs isolated from nine Apiaceae species, including E. spinosa root and aerial parts EOs, and their major compounds against the protozoan Trypanosoma brucei, the causative agent of African sleeping sickness [33].The E. spinosa root EO demonstrated the highest trypanocidal activity among the tested EOs (EC 50 value of 2.7 ± 0.6 µg/mL), while the highest activity out of all tested samples was observed with terpinolene (EC 50 value of 0.035 ± 0.005 µg/mL), which was found to be one of the major constituents of the EO [33].
Ngahang Kamte et al. examined the activity of the EOs isolated from nine Apiaceae species, including E. spinosa root and aerial parts EOs, and their major compounds against the protozoan Trypanosoma brucei, the causative agent of African sleeping sickness [33].The E. spinosa root EO demonstrated the highest trypanocidal activity among the tested EOs (EC50 value of 2.7 ± 0.6 μg/mL), while the highest activity out of all tested samples was observed with terpinolene (EC50 value of 0.035 ± 0.005 μg/mL), which was found to be one of the major constituents of the EO [33].

Insecticidal and Larvicidal Activity
The EOs isolated from Echinophora species demonstrated promising insecticidal and larvicidal properties underlying their potential use as biopesticides and insect repellents (Figure 3) [7,45,59,62,65].In a study conducted by Papanikolaou et al., the biopesticide potential of microemulsion formulations of six EOs, including E. tenuifolia subsp.sibthorpiana EO, was assessed against two beetle species (Trogoderma granarium and Tribolium castaneum) in different development stages (larvae and adult) [45].The microemulsion of the E. tenuifolia subsp.sibthorpiana EO showed significant activity against T. castaneum larvae with a mean mortality rate of 90.0% fourteen days post-exposure, while its activity against T. castaneum adults and T. granarium was lower [45].Additionally, in a study by Evergetis et al., E. tenuifolia subsp.sibthorpiana EO demonstrated good activity against larvae of the Culex pipiens mosquitoes with LC 50 values of 59.46 mg/L [46].
The insecticidal and larvicidal activity of E. spinosa EO was investigated in two studies by Pavela et al. [7,34].In the first study, the larvicidal activity of the EOs isolated from aerial parts and roots of E. spinosa was assessed against the larvae of the medically important vector Cx.quinquefasciatus.The root EO demonstrated high efficacy against the Cx.quinquefasciatus larvae with LC 50 values of 18.9 µL/L, which was significantly higher than that of the aerial parts EO (LC 50 of 40.5 µL/L) [34].These results were confirmed in a second study in which the root EO showed higher activity than the leaves and stems EO against Cx.quinquefasciatus larvae (LC 50 of 15.7 µL/L for the root EO compared to LC 50 of 41.3 µL/L for the aerial parts EO) [7].Moreover, the root EO demonstrated relevant insecticidal and larvicidal activity against Musca domestica adults (LD 50 of 38.3 µg/adult) and Spodoptera littoralis larvae (LD 50 of 55.6 µg/larvae) [7].
Comparison between the larvicidal and biting deterrent activities of EOs isolated from different parts (stems, leaves, and flowers) of E. lamondiana and their major constituents (δ-3carene, α-phellandrene, and terpinolene) was conducted by Ali et al. [65].The leaf EO was significantly more toxic than the stem and flower EOs and exhibited the highest larvicidal activity against An.quadrimaculatus larvae with LC 50 of 26.5 ppm, whereas its activity against Ae.aegypti was lower (LC 50 of 138.3 ppm) [65].In addition, the flower and leaf EOs showed biting deterrent activity against Ae.aegypti and An.quadrimaculatus at 10 mg/cm 2 , similar to the insect repellent N,N-diethyl-meta-toluamide (DEET) at 25 nmol/cm 2 [65].
The EO obtained from aerial parts of E. chrysantha showed dose-and time-dependent contact insecticidal activity on Rhyzopertha dominica and Tribolium confusum [62].The highest activity was observed against T. confusum, with a mortality rate of 37.9% after 48 h of exposure, which was higher than that of R. dominica (25.8%) [62].
The fumigant and contact toxicity of EO from the aerial parts of E. platyloba on three beetle species (T.castaneum, R. dominica, and Callosobruchus maculatus) was demonstrated in a study by Sharifian and Darvishzadeh [59].The EO showed significant dose-and time-dependent fumigant toxicity against R. dominica and C. maculatus with LC 50 values of 5.66 and 3.835 µL/250 mL air after 24 h, respectively, while the highest contact insecticidal activity was observed with R. dominica with mean LC 50 values of 9.712 µL/39 cm 2 [59].In both cases, the most resistant species was T. castaneum [59].Additionally, the aerial parts EO of E. platyloba showed very low toxicity on Encarsia formosa, a parasitic wasp used in the biocontrol of the pest Trialeurodes vaporariorum (greenhouse whitefly) [100].
Methanolic extracts of E. platyloba showed promising anticancer activities on various cancer cell lines, including prostate adenocarcinoma, fibrosarcoma, breast cancer, and leukemia cell lines [12,[101][102][103].The methanolic extract from aerial parts of E. platyloba decreased cell viability and induced apoptotic cell death in malignant prostate adenocarcinoma (PC 3) cell line with IC 50 values of 236.136 ± 12.4, 143.400 ± 7.2, and 69.383 ± 1.29 µg/mL after 24, 36, and 48 h, respectively, while it demonstrated no significant activity on non-malignant Human Umbilical Vein Endothelial Cells HUVEC cell line [12].Moreover, apoptotic cell death and suppression of cell proliferation were induced by E. platyloba extract in mouse fibrosarcoma cell line (WEHI-164) [101].The effects of E. platyloba methanolic extract on a human breast cancer cell line (MDA-MB-231) were investigated by Birjandian et al. [102].The extract decreased the viability and induced apoptotic cell death on MDA-MB-231 cells, with the highest activity being observed in a concentration of 534.6 ± 7.2 µg/mL after 24 h [102].The methanol leaf extract of E. platyloba revealed an antimutagenic effect and significantly inhibited the proliferation of Acute Promyelocytic Leukemia cell line (NB4) with the highest activity of 87.35% at a concentration of 500 µg/mL after 24 h [103].
Akşit et al. screened the antiproliferative activity of E. chrysantha ethanol extract on various malignant cell lines, including brain cancer, gynecological cancer, and colon cancer cell lines [24].The extract demonstrated the highest activity against HT-29 colon cancer cells with IC 50 of 4.07 ± 0.2 µg/mL and HeLa gynecological cancer cells with IC 50 of 1.41 ± 0.1 µg/mL, while the observed cytotoxicity against normal cell lines (lung, retinal, and skin) was low [24].
In a study by Amirghofran et al., the activity of methanol extract from the aerial parts of E. cinerea was screened against four cancer cell lines, including lung carcinoma (A549) cell line, bladder carcinoma (Fen) cell line, myelogenous leukemia (K562) cell line, and T cell leukemia (Jurkat) cell line [104].The extract exhibited strong antiproliferative effects on the Jurkat T cell leukemia cell line (IC 50 of 6.9 µg/mL), as well as significant antiproliferative effects on the K562 myelogenous leukemia cell line [104].
The anticancer effects of Echinophora species on different cancer cell lines are summarized in Figure 4.
parts of E. cinerea was screened against four cancer cell lines, including lung carcinom (A549) cell line, bladder carcinoma (Fen) cell line, myelogenous leukemia (K562) ce line, and T cell leukemia (Jurkat) cell line [104].The extract exhibited strong antiprolife ative effects on the Jurkat T cell leukemia cell line (IC50 of 6.9 μg/mL), as well as signif cant antiproliferative effects on the K562 myelogenous leukemia cell line [104].
The anticancer effects of Echinophora species on different cancer cell lines are sum marized in Figure 4.

Anti-Inflammatory Activity
The in vitro anti-inflammatory activity of E. tenuifolia inflorescence methanol extra was demonstrated in a study by Marrelli et al. [19].The n-hexane and dichloromethan fractions of the extract exhibited significant anti-inflammatory activity by inhibiting th lipopolysaccharide-induced production of NO in macrophages (RAW 264.7 cell lin with IC50 values of 17.04 ± 1.37 μg/mL and 39.97 ± 3.16 μg/mL, respectively, which we lower than that of the positive control indomethacin (58.00 ± 0.9 μg/mL) [19].

Anti-Inflammatory Activity
The in vitro anti-inflammatory activity of E. tenuifolia inflorescence methanol extract was demonstrated in a study by Marrelli et al. [19].The n-hexane and dichloromethane fractions of the extract exhibited significant anti-inflammatory activity by inhibiting the lipopolysaccharide-induced production of NO in macrophages (RAW 264.7 cell line) with IC 50 values of 17.04 ± 1.37 µg/mL and 39.97 ± 3.16 µg/mL, respectively, which were lower than that of the positive control indomethacin (58.00 ± 0.9 µg/mL) [19].
Chianese et al. reported the modulation of six transient receptor potential (TRP) proteins by the polyacetylenes echinophorin A, echinophorin B, and echinophorin D isolated from E. platyloba extract.The three polyacetylenes exhibited anti-inflammatory activity by selective action toward the TRP ankyrin 1 (TRPA1) cation channel, an ion channel that plays a role in the mediation of neuropathic and inflammatory pain [2].

Analgesic Effects
The analgesic effects of E. platyloba leaf methanolic extract were investigated in vivo on male Wistar rats using three different tests (tail flick, rating, and formalin tests) in a study by Asgari Nematian and Mohammadi [105].In both acute and chronic phases, the extract produced significant analgesic activity and reduced the pain peripherally and centrally, the highest activity being observed with the highest tested dose of 300 mg/kg [105].Furthermore, low doses of the ethanolic extract from the aerial parts of E. platyloba induced rewarding effects in vivo in female albino mice, most likely due to effects on the opioid receptors, and inhibited the rewarding effects of morphine [106].
In a single-blind clinical study involving sixty women with dysmenorrhea, E. platyloba extract showed considerably higher analgesic activity compared to placebo [107,108].

Hormonal Effects
The effectiveness in the treatment of moderate-to-severe premenstrual syndrome (PMS) with E. platyloba extract was assessed by two clinical studies [15,109].The first study, a single-blind randomized clinical trial, included ninety women with moderate-to-severe PMS, who were randomized into three groups: a group receiving E. platyloba extract, a group receiving fennel extract, and a placebo group.A reduction in the intensity of the PMS symptoms was observed in all three groups, yet the extracts showed a more significant reduction in the severity of the PMS [15].In a second single-blind randomized clinical trial, sixty women with moderate-to-severe PMS were randomized into two groups: a group receiving E. platyloba extract and a placebo group.There was a significant reduction in the PMS symptoms in the E. platyloba group after the treatment, especially in the anxiety and depression symptoms [109].
In an in vivo study on male Wistar rats, the effects of E. platyloba aerial parts alcoholic extract on the levels of prolactin and on the activity of the pituitary-gonadal axis were described [110].In the animals treated with the E. platyloba extract, a significant increase in the levels of testosterone and prolactin levels was observed, in addition to a significant decrease in cholesterol levels [110].

Organoprotection and Cytoprotection
Heidarian et al. investigated the hepatoprotective effect of E. platyloba leaf ethanolwater extract in an in vivo acetaminophen-induced hepatotoxicity model in rats [111].The E. platyloba extract showed excellent hepatoprotective effects characterized by a reduction in the serum levels of hepatic enzymes like the transaminase enzymes aspartate transaminase (AST) and alanine transaminase (ALT), induction of the liver catalase (CAT) activity and antioxidant capacity of the liver and histopathological improvements [111].These results support the traditional medicinal use of E. platyloba as a hepatoprotective agent [70].
Ethanolic extract from leaves of E. cinerea demonstrated promising cardioprotective effects [14].The effect of the extract on cardiac function was assessed in vivo in rats with aluminum phosphide-induced poisoning.The E. cinerea extract improved the hemodynamic and the electrocardiogram parameters caused by the poisoning, including an increase in the arterial systolic blood pressure, prevention of the decline in heart rate, and increase in the corrected QT interval [14].
In a study by Shokoohinia et al., two compounds isolated from acetone extract of E. cinerea, namely quercetin-3-O-b-D-glucopyranoside and osthol, showed in vitro protective effects against cisplatin-induced cytotoxicity in rat pheochromocytoma-derived PC12 cell line, often used as a model for dopaminergic neurons [20].The two molecules exerted their effect by inhibiting apoptosis.The results from the study demonstrate the potential use of E. cinerea in the prevention of cisplatin-induced neurotoxicity [20].In addition, quercetin-3-O-b-D-glucopyranoside suppressed the generation of reactive oxygen species and exhibited cytoprotective effects on the PC12 cell line in H 2 O 2 -induced cytotoxicity [21].

Materials and Methods
The search strategy was to screen for studies regarding the occurrence, isolation, and identification of phytochemical bioactive compounds in Echinophora species and their ethnomedicinal use and pharmacological activities.The studies were obtained from scientific electronic databases, including PubMed, Google Scholar, Scopus, Springer Link, and Sci-enceDirect.The keywords included in the search were: "bioactive compounds", "Apiaceae", "Echinophora", "Echinophora extract", "Echinophora essential oil", "Echinophora platyloba", "Echinophora cinerea", "Echinophora chrysantha", "Echinophora spinosa", "Echinophora tenuifolia", "Echinophora lamondiana", "Echinophora tournefortii", "Echinophora tenuifolia subsp.sibthorpiana", "ethnomedicinal use", "animal studies", "cell culture studies".The keywords were used separately or in combination, depending on the database.Articles not in English and not relevant to this review were excluded.A total of 113 articles were included in the review, 57 of which concerned the pharmacological and biological activities of the genus.

Conclusions
The species within the genus Echinophora are valued for their edible and medicinal properties, often used as flavoring agents, preservatives, and in the treatment of various health conditions.Although the genus is associated with various applications and safety, the scientific studies on Echinophora species remain limited.Plants belonging to this genus are rich in phenolic compounds, including flavonoids, phenolic acid, and coumarins, as well as volatile and non-volatile terpenes and polyacetylenes.The extracts and the EOs derived from Echinophora species are associated with significant therapeutic potential and have demonstrated a wide range of biological activities, including anticancer, antibacterial, antifungal, anti-inflammatory, hepatoprotective, and insecticidal.Among the Echinophora species, E. platyloba demonstrated the greatest antifungal activity.Studies about E. platyloba also reported excellent hepatoprotective, analgesic, and anticancer activities.Most of the studies related to the genus Echinophora were performed in preclinical settings (in vitro and in vivo), apart from four clinical studies with humans.
However, there is limited information on the secondary metabolites produced by these species and their pharmacological effects.Further research is required on the phytochemistry and pharmacology of the extracts and isolated chemical compounds from these plants.Metabolomic studies could be useful for gaining new insights into the phytochemistry, while more comprehensive in vitro and in vivo studies, as well as molecular docking 13, x FOR PEER REVIEW 2

Figure 4 .
Figure 4. Anticancer effects of Echinophora extracts on different cancer cell lines.Downward arrows indicate inhibition, while upward arrows indicate stimulation.

Figure 4 .
Figure 4. Anticancer effects of Echinophora extracts on different cancer cell lines.Downward arrows indicate inhibition, while upward arrows indicate stimulation.

Table 2 .
Comparison of the main volatile compounds in EOs of Echinophora species.

Table 4 .
Biological and pharmacological activities of the Echinophora genus.