The Genus Echinops: Phytochemistry and Biological Activities: A Review

The genus Echinops belongs to the family of Asteraceae and comprises about 130 species. Many species belonging to the genus Echinops are traditionally used as medicinals mainly in Africa and Asia. The genus is reported to contain diverse secondary metabolites. The aim of this review is to critically evaluate the available research reports on the genus and systematically organize the findings. Information for this study was obtained using various search engines including PubMed and Google Scholar. This review revealed that the genus is used traditionally to treat pain, inflammation, respiratory diseases, diseases caused by different microorganisms, as an aphrodisiac, to fasten expulsion of placenta, and for removal of renal stones. More than 151 secondary metabolites have been reported from the genus in which thiophenic compounds held the biggest share. Various extracts, essential oils, and isolated compounds from members of this genus are shown to exhibit different biological effects mainly anti-microbial, anti-proliferative, and anti-inflammatory. However, there are a number of species in this genus that are claimed to have traditional medicinal uses but their biological effect not yet been evaluated.


INTRODUCTION
Echinops L., belongs to the family of Asteraceae, a family which is distributed all over the world except in Antarctica. Asteraceae is a monophyletic taxon distinguished by florets arranged on a receptacle in centripetal heads and bounded by bracts. It comprises 1,600−1,700 genera and 24,000−30,000 species (Funk et al., 2005). The genus Echinops belongs to the tribe Cardueae and is recognized by the presence of uniflowered capitula aggregated into second-order spherical or oval heads. This feature makes it unique within the tribe (Garnatje et al., 2005;Sánchez-Jiménez et al., 2010). It contains 120−130 species distributed across north and tropical Africa, the Mediterranean Basin, and central Asia. Members of this genus are mostly perennial with few annuals (Hedberg et al., 2004;Sánchez-Jiménez et al., 2010).
Many members of this genus are traditionally used to treat different diseases. Some are scientifically investigated for various biological activities and phytoconstituents. Previously, reviews that focus on single species, Echinops spinosus L. and E. echinatus Roxb. have been conducted (Bouzabata et al., 2018;Maurya et al., 2015). To the authors' knowledge, there is no study that reviewed the traditional use, phytochemistry, and biological activities of the whole genus. This review is aimed to critically evaluate available research reports on the genus and systematically organize and present the findings. It is attempted to include all articles published from 1990−2018 while some articles published before 1990 were included considering their significance. This review excluded unpublished findings and publications which were not available online and articles written in languages other than English. Chemical structures of only isolated and characterized compounds were provided while structures of compounds identified from essential oils and other chemical analysis were not. The main sources of the structures of isolated compounds were the research articles and these were confirmed using PubChem. Structures that were not available in the articles were obtained from theses, books, PubChem, and other reliable sources. Different search engines including PubMed and Google Scholar were employed to search literature using searching words such as Echinops, plant, phytochemical, phytochemistry, pharmacological activity, biological effect, and traditional use.

TRADITIONAL USeS
Ethnomedicinal claims on the genus Echinops to treat a number of ailments are depicted in Table 1. The common traditional uses can fall into three general groups. The frequently described application is to treat symptoms like inflammation, pain, and fever (Regassa, 2013;Rathore et al., 2015). The other common traditional use was to treat ailments related to respiratory tract including cough and sore throat (Ghasemi Pirbalouti et al., 2013;Sajjad et al., 2017). Members of the genus have been used as an aphrodisiac (Hamayun et al., 2006), facilitation of expulsion of retained placenta and delivery (Okello and Ssegawa, 2007;Qureshi and Bhatti, 2008), as an abortifacient (Abouri et al., 2012), treatment of uterus tumor (Abderrahim et al., 2013), and leucorrhoea (Wagh and Jain, 2018). Three species (E. bannaticus Rochel ex Schrad, E. cornigerus D.C., and E. polyceras Boiss.) reported to have been employed in the managment of kidney stones (Mustafa et al., 2012;Nawash et al., 2013;Kumar et al., 2018).
In addition to the traditional medicnal applications described in Table 1, the plants have nutritional value. In Iran, the bulb of E. viscidulus Mozaff is consumed as a vegetable (Ghasemi Pirbalouti et al., 2013). The roots of E. giganteus A. Rich. and E. spinosus are used as a spice in Morocco and Cameroon, respectively (Pavela et al., 2016;Tbatou et al., 2016). The use of E. giganteus might be attributed to the presence of nutrients including iron, phenols, carotenoids, and vitamins E and C in the plant (Abdou Bouba et al., 2012).

PHYTOCHeMICALS
As presented in Table 2 and Figure 1, 151 compounds have been isolated and characterized using different spectroscopic/ spectrometric techniques. Members of the genus Echinops contain primarily thiophenes and terpenes. Flavonoids and other phenolic compounds, alkaloids, lipids, and phenylpropanoids were also reported. The root of the plant is the main source of the thiophenes while most of the terpenes and flavonoids were isolated from the aerial part/the whole plant. The genus is also known for essential oil content and all morphological parts of the plants are reported to contain some of the essential oils. Around 53 of the isolated and characterized compounds are reported to have different biological activities. The structural formulae of isolated and characterized compounds are given in Figure 1.
The biological activities of thiophenes were evaluated mainly in vitro and they have an insecticidal, anti-proliferative, and antifungal potential effects.

Terpenoids
Sesqui-and triterpenoids were reported mainly from the whole plant and aerial parts of the genus Echinops. Most of the sesquiterpenoids contain lactones. Sesquiterpene lactones are also the most prevalent secondary metabolites in the family of Asteraceae (Chadwick et al., 2013). Most triterpenoids exist in various forms including lactones, esters, and sterols along with their glycosides. The common sesquiterpenoid reported was costunolide (61), which was isolated from three species whereas lupeol (86) and lupeol acetate (94) were the common triterpenoids. Many sesquiterpenoids were also detected from the essential oils of the genus.

Flavonoids and Other Phenolic Compounds
Flavonoids from the genus Echinops were mainly flavones and mostly isolated from the whole plant and aerial parts of the members. Apigenin (105) Table 2). In addition to flavonoids, phenolic compounds including coumarins, phenylpropanoids, and lignans were reported (Tene et al., 2004;Dong et al., 2008a;Senejoux et al., 2013).

Alkaloids
The first alkaloids isolated from the genus Echinops were echinopsine (139), echinozolinone (140), and echinopsidine (141) from the aerial parts of E. echinatus (Chaudhuri, 1987). Later on, another alkaloid, 7-hydroxyechinozolinone (142), was isolated from the flowers of the same plant (Chaudhuri, 1992). Additional four alkaloids of which two were in glycosidic form were reported ( Table 2). The alkaloids were mainly isolated from the aerial parts of the plants. The predominant alkaloid, which was isolated from four different species, was 1-methyl-4quinolone (139).

Anti-Microbial Activity
The genus Echinops is traditionally used to treat different infectious diseases including trachoma, sepsis, typhoid, gonorrhea, and ulcerative lymphangitis. It is also used to treat different ailments that might be caused by bacterial/fungal infections including fever, respiratory diseases, toothache, leucorrhoea, and earache. Thus, they have been investigated for their anti-microbial activities. Anti-bacterial and antifungal activities of extracts from the genus with their respective minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBI), minimum fungicidal concentration (MFC), and zone of inhibitions are presented in Table 3. These studies showed that both Gram-positive and Gram-negative bacteria were sensitive to the extracts/isolated compounds obtained from the genus.
Out of the tested strains, M. tuberculosis (H37Rv) showed higher sensitivity to the ether root extract of E. giganteus and methanolic extract of E. amplexicaulis Oliv. with MIC of 12 µg/ mL and 32 µg/mL, respectively (Tekwu et al., 2012;Kevin et al., 2018). The methanolic root extract of E. amplexicaulis also showed a promising effect against a multidrug-resistant strain of M. tuberculosis with a MIC of 50 µg/mL (Kevin et al., 2018). The ethanolic root extract and essential oils obtained from E. kebericho Mesfin showed relatively strong effect against Staphylococcus aureus (Ameya et al., 2016) and Klebsiella pneumoniae (Belay et al., 2011). These results might justify the traditional application of E. kebericho in treating respiratory disease, toothache, and fever. The essential oil from E. ritro L. exhibited anti-bacterial effect and antibiofilm and disruption of the bacterial membrane were suggested as mechanisms of actions (Jiang et al., 2017).
Different extracts from members of the genus having antibacterial effect were analyzed for their chemical constituents. The unsaponifiable matter from the hexane extract of E. spinosissimus contained mainly taraxasterol, lupeol, pseudotaraxasterol, α-amyrin, β-amyrin, pseudotaraxasteryl acetate, lup-22(29)-en-3-yl acetate, β-sitosterol, and stigmasterol. The hexane extract showed anti-bacterial activity with MIC values of less than 125 µg/ mL against different bacterial strains (Bacillus amyloliquefaciens, Micrococcus luteus, Bacillus subtilis, and Salmonella enteric) (Bouattour et al., 2016). Thiophens (31, 46, 54, and 59) isolated from the root of E. ritro possessed anti-bacterial effect against S. aureus with a MIC value of 8 µg/mL. This was similar to the effect observed for the positive control, levofloxacin. The anti-bacterial effects of thiophenes 31, 46, 55, 57, and 59 against Escherichia  5-(penta-1,3-diynyl)-2-(but-3-en-1-ynyl)-thiophene     5-(penta-1,3-diynyl)-2-(3-acetoxy-4-hydroxy-but-1-ynyl)thiophene   coli with a MIC of 64, 32, 64, 64, and 8 µg/mL, respectively, were also described (Li et al., 2019). In addition to those described in Table 3, the root extract of Echinops spp from Ethiopia showed anti-bacterial activity through growth inhibition (Ashebir and Ashenafi, 1999). The study did not delineate the specific name of the plant, MIC/MBC, and zone of inhibitions which makes it challenging to compare with other study results. Methanolic extract of the whole plant of E. polyceras improved the effect of tetracycline on resistant strains of Pseudomonas aeruginosa (Aburjai et al., 2001). The effect of the plant without tetracycline however was not studied. The leaf and flower extracts of E. viscosus subsp. bithynicus were described to have anti-bacterial properties against E. coli, Micrococcus luteus, S. aureus, Mycobacterium smegmatis, P. aeruginosa, Enterobacter cloacae, and Bacillus megaterium. Even though the concentration of the extracts is not well defined in the study, the flower extract of E. microcephalus has been reported to have greater zone of inhibition than the standard drug, vancomycin (30 µg/disc) (Toroğlu et al., 2012).
Most of the anti-fungal studies on the genus revealed that the extracts/isolated compounds were effective mainly against Candida albicans with the most potent effect observed for the root methanolic extract of E. kebericho (MIC = 3.12 µg/mL) (Ameya et al., 2016).

effect on Cancer Cell Lines
The traditional use of the genus Echinops in the treatment of cancer is not common nevertheless the species in this genus were explored for cytotoxic activity. The methanolic extract of E. kotschyi Boiss. against MOLT-4 and K562 cancer cell lines (Afshaki et al., 2012) and essential oils obtained from E. kebericho, which consist of 43 compounds predominantly dehydrocostus lactone, showed cytotoxic activity against human monocytic leukemia cell line (THP-1) with an IC 50 value of 0.4 µg/L .
Even though the extracts and isolated compound from the genus showed promising effects against different cancer cell lines, the effects are ought to be further investigated using in vivo models.

Hepato-Protective and Anti-Oxidant Activities
Members of the genus Echinops were also shown to have hepatoprotective and anti-oxidant activities. Most of the studies were conducted in carbon tetrachloride (CCl 4 )-induced liver damage, in which biomarkers of liver function like aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured.
The methanolic root extract, as well as n-butanol and aqueous fractions of E. grijsii, showed hepatoprotective activity in CCl 4 -induced liver damage in rats. The effect was prominent in the aqueous and butanol fractions, at a dose of 300 mg/kg, that markedly decreased AST and ALT levels (Lin et al., 1993). A study conducted by Eram et al. (2013) in CCl 4 -intoxicated rabbits justified the traditional claim of E. echinatus to treat jaundice (Gupta et al., 2010). The ethanolic aerial parts extract of E. echinatus at 500 and 750 mg/kg resulted in a significant decrease of ALT and AST, of which the lower dose (500 mg/kg) showed a higher effect (Eram et al., 2003). As presented in Table 1, flavonoids were isolated from the root of E. grijsii and the whole plant of E. echinatus. These might be responsible for the hepatoprotective effects of the extracts Zang et al., 2017) and further investigations are required on phytoconstituents of the plants.
The hepatoprotective effect of compounds isolated from members of the genus Echinops was also investigated along with crude extracts. The protective effects of E. galalensis Schweinf. as well as isolated compounds β-sitosterol (89), apigenin-7-O-β-D-glucoside (109), 3β-acetoxy-taraxast-12,20(30)diene-11α-21α-diol (100), α-amyrin (101), erythrodiol (102), lup-20(29)-ene-1,3-diol (103), and dicaffeoyl-quinic acid derivatives (135-138) on human hepatoma cell line (Huh7) have also been established. The highest protection was exhibited by 100, 102, and 103 and they significantly decreased the level of ALT. Except for the crude extract, all the tested samples decrease the level of AST and 89, 101, and 135 showed the highest effect . According to Abdallah et al. (2013), the protective effect of the extract and isolated compounds was suggested to be partly due to anti-oxidant effects of the samples. Methotrexate-induced hepatotoxicity was also used to evaluate the hepatoprotective effect of some of the plants. Using this model, the protective effect of ethanolic aerial part extract and flavonoid fraction of E. heterophyllus P.H. Davis was established in rabbits. The crude ethanolic extract (250 mg/kg) significantly decreased the serum proteins, liver enzymes, and oxidative stress markers than the flavonoid fraction (Abdulmohsin et al., 2019).
Active cell cultures of human peripheral blood mononuclear cells were also used to evaluate the anti-oxidant effect of aqueous methanolic extract of E. albicaulis aerial parts. The study showed that the active oxygen species (ROS) generation in the cells was significantly reduced at concentrations of 1, 20, and 50 mg/mL of the extract; however, the extract induced overproduction ROSs at higher concentrations (Kiyekbayeva et al., 2017).
Regardless of the effects described, the anti-oxidant activity evaluations are not still sufficient. In most of the reports the IC 50 value for the in-vitro anti-oxidant effect are not mentioned. No single in vivo anti-oxidant model was employed. In some of the hepatoprotective effect studies standard drugs were not utilized and comparison was made only with the negative control ( Table 5). The hepatoprotective effect of traditionally used plant, E. spinosus L. (Akdime et al., 2015), has not been scientifically investigated yet.

Anti-Inflammatory, Analgesic, Anti-Pyretic, and wound Healing Activities
Traditionally, members of the genus Echinops are documented to have been used to treat inflammation, pain, and fever.
Accordingly, several species have been explored for antiinflammatory, analgesic, and anti-pyretic activities.
The whole plant extract of E. heterophyllus and the alkaloidal faction facilitated epithelialization and left no scars in rabbits (Abdulrasool et al., 2013). This is the only wound healing activity reported on members of this genus although the dose, vehicle, and the standard drug are not described.
The in vivo anti-inflammatory effects of the genus seemed to be not promising since the plants resulted in an inhibition of edema at higher doses. In spite of the studies stated above, scientific data justifying the traditional claim of E. bovei (Boiss.) Maire., E. cornigerus, E. kebericho, E. longifolius A. Rich., E. macrochaetus, and E. spinosissimus to treat rheumatism and pain are not provided yet. Anti-Protozoal and Anti-Helmentic Activities As presented in Table 3, E. hoehnelii Schweinf. and E. kebericho have been used in traditional treatment of malaria. These plants along with other species showed anti-malarial activity. Aqueous extract of the aerial parts of E. polyceras exhibited strong (96%) in vitro growth inhibitory activity against Plasmodium falciparum. Nevertheless, the concentration of the extract used for the test and the standard drug used as positive control has not been reported (Sathiyamoorthy et al., 1999). A study on 70% ethanolic root extract E. kebericho resulted in an inhibition of parasitemia by 57.3% at a dose of 500 mg/kg in mice against Plasmodium berghei (Toma et al., 2015). A recent study conducted on the 70% methanolic extract from roots of E. kebericho exhibited 49.5% of inhibition at 1000 mg/kg in mice (Biruksew et al., 2018). This might suggest that the potency of E. kebericho extract could be dependent on the extraction solvent.

effects on Insects and Termites
The leaves of Echinops spp, which are commonly known as "Kebericho" in Ethiopia, had a mosquito repellant effect against   Anopheles arabiensis with the effectiveness of 92.47% as a smoke (Karunamoorthi et al., 2008). The activity of thiophenes (2, 8, 9, 10, 14, 15, 39, 51, and 52) isolated from E. transiliensis Golosk. against Aedes aegypti was reported and the toxic effect increased with the number of thiophene moieties in the molecule. Strong activity was observed for 2''-terthiophene (2) with an LC 50 value of 0.16 µg/ mL (Nakano et al., 2014). Similarly, the root extract of E. grijsii possessed significant larvicidal activity against Aedes albopictus, Anopheles sinensis, and Culex pipienspallens with LC 50 values of 2.65, 3.43, and 1.47 µg/mL, respectively.

effects on the Reproductive System
A number of species have been used for the management of various reproductive health problems ( Table 1). In spite of the traditional claims, only E. echinatus has been evaluated for these biological activities.
Corresponding to its traditional use, the terpenoidal fraction from E. echinatus displayed anti-fertility properties at doses of 30 and 60 mg/kg in male rats (Padashetty and Mishra, 2007). Earlier studies also indicated that the root ethanolic extract of E. echinatus has anti-fertility properties through decrement in sizes of testes, epididymis, ventral prostate, vas deferens, and seminal vesicle at doses of 50, 100, and 200 mg/kg. In addition, the extract also decreased sperm motility and density with an inhibition of sepermatogenesis in rats (Chaturvedi et al., 1995). The butanol fraction of the root extract demonstrated a protective effect on testosterone-induced prostatic hyperplasia at a dose of 100 mg/ kg in rats. The butanol fraction also showed better 5α-reductase inhibitory effect (IC 50 = 0.22 mg/mL) than of the crude extract and other fractions followed by the water soluble fraction (IC 50 = 0.43 mg/mL) (Agrawal et al., 2012). Similarly, the root petroleum ether extract of E. echinatus inhibited 5α-reductase. The enzyme plays an important role in the pathogenesis of benign prostatic hyperplasia (BPH), prostatic cancer, acne, alopecia, baldness in men, and hirsutism in women (Nahata and Dixit, 2014).

Other Activities
A study showed that 5-(penta-1,3-diynyl)-2-(3,4-dihydroxybut-1-ynyl)-thiophene (14), isolated from the root of E. grijsii, has an induction effect on nicotinamide adenine dinucleotide phosphate (NAD(P)H): quinone oxidoreductase1 (NQO1), an enzyme that is involved in detoxification of toxic quinones. The induction effect was dose-dependent and the maximum effect was observed at a concentration of 40 μM and it was 3.1 folds of the control, 4'-bromoflavone (Shi et al., 2010). Similarly, compounds 5, 9, and 14, from the root of E. grijisii, had a strong NQO1-inducing effect and the concentrations that caused a twofold induction were 1.86, 2.58, and 2.87 μg/mL, respectively. Compounds 5 and 14 were found to have an alkylating effect on cysteine residues in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) (Zhang and Ma, 2010).
The 70% hydro-alcoholic root extract of E. echinatus was reported to have significant anti-diabetic activity on alloxaninduced diabetic rats. The the extract treated animals (200 mg/ kg) showed lower blood glucose level (164 mg/dL) compared to the negative control (277.6 mg/dL) after 21 days of treatment. In addition, the extract exhibited the ability to regenerate pancreatic islet cells and normal structure of glomeruli and proximal and distal convoluted tubules in kidneys (Fatima et al., 2017). Similarly, the methanolic root extract of E. echinatus exhibited a significant anti-diabetic effect at doses of 100 and 200 mg/ kg on alloxan induced diabetic rats. The extract was also able to decrease serum cholesterol, serum triglyceride, serum lowdensity lipoprotein, serum very low-density lipoprotein, and serum alkaline phosphate significantly while it increased highdensity lipoproteins (Sarvaiya et al., 2017).
The molluscicidal activities of 80% methanolic root extracts of E. ellenbeckii and E. longisetus with a 100% mortality rate at 20.25 and 45 µg/mL, respectively, was described (Hymete et al., 2005a). The pancreatic amylase inhibitory activity (> 75%) of aqueous root extract of E. giganteus was reported although the exact concentration of the extract was not mentioned (Etoundi et al., 2010). The latex of E. persicus at 500 mg/kg resulted in lower number and level of stomach ulcer compared to the negative control in rats (Rad et al., 2010). The methanolic extract of root and aerial parts of E. echinatus significantly increased urine volume and excretion at doses of 250 and 500 mg/kg (Patel et al., 2011a). The immunomodulating activity of aerial parts methanolic extract of E. lasiolepis Bunge has been reported. The extract at different concentrations (0.1, 1, 10, 100, and 200 µg/ mL) inhibited peripheral blood mononuclear cells (PBMCs) proliferation of which 1 µg/mL showed optimum proliferation (30.66%) (Asadi et al., 2014).
Biological effects evaluated on genus Echinops and the doses with maximum effect are summarized in Tables 3-5.

CONCLUSION
The genus Echinops is well known for its use to treat pain and respiratory manifestations. The traditional claims were justified by different biological evaluations. Findings from in vitro studies indicated that members of the genus have a potential effect against different cancer lines, microbial strains, and insects. They also showed significant in vivo anti-inflammatory, analgesic, and hepatoprotective activities. Some of the extracts and isolated compounds showed promising effects. This includes the anticancer activity of compounds 5 and 14, antioxidant potential of 109, anti-leishmanial and anti-helmentic effects of E. kebericho, and the larvicidal effect of compound 1. The safety and efficacy of secondary metabolites responsible for the in vitro effects of extracts/fractions should further be investigated in in vivo models. The most abundant bioactive secondary metabolites in members of the genus are thiophenes and terpenoids which are also mentioned as responsible for the cytotoxic effect observed. In the current review, it has been observed that the potential uses of the species in the removal of kidney stones and use to solve nerve-related problems have not been scientifically addressed yet. Investigation of the anti-microbial activity of isolated compounds seems to be limited. We believe this review will provide summarized information to the scientific community working on the genus.

AUTHOR CONTRIBUTIONS
HB developed concept of the review, conducted the literature review, extracted relevant information to the study, and drafted the manuscript. AH guided the literature search and edited the manuscript. Both authors have read and approved the manuscript.