Phytochemical Composition and Biological Activities of Scorzonera Species

The genus Scorzonera comprises nearly 200 species, naturally occurring in Europe, Asia, and northern parts of Africa. Plants belonging to the Scorzonera genus have been a significant part of folk medicine in Asia, especially China, Mongolia, and Turkey for centuries. Therefore, they have become the subject of research regarding their phytochemical composition and biological activity. The aim of this review is to present and assess the phytochemical composition, and bioactive potential of species within the genus Scorzonera. Studies have shown the presence of many bioactive compounds like triterpenoids, sesquiterpenoids, flavonoids, or caffeic acid and quinic acid derivatives in extracts obtained from aerial and subaerial parts of the plants. The antioxidant and cytotoxic properties have been evaluated, together with the mechanism of anti-inflammatory, analgesic, and hepatoprotective activity. Scorzonera species have also been investigated for their activity against several bacteria and fungi strains. Despite mild cytotoxicity against cancer cell lines in vitro, the bioactive properties in wound healing therapy and the treatment of microbial infections might, in perspective, be the starting point for the research on Scorzonera species as active agents in medical products designed for miscellaneous skin conditions.

This review aims to present the phytochemical composition of Scorzonera species, including the compounds characteristic for the genus, as well as novel compounds, which have not been previously isolated from Asteraceae. Extraction methods have been briefly summarized as well. A summary of available data regarding the use of Scorzonera in folk 20% aqueous methanol. The study by Bahadır-Acıkara et al. [27] includes a phytochemical analysis of n-hexane extracts of S. acuminata aerial parts and roots.
A 20% aqueous methanol extract of the aerial parts of S. acuminata was reported to contain chlorogenic acid, rutin, and cyranoside [9]. Another study reports the presence of α-amyrin, lupeol, and lupeol acetate in the n-hexane extract [27].

Scorzonera aristata Rameond ex DC.
Samples of S. aristata were collected in Northern Italy [26,55,56]. The leaves from the specimen in the study by Jehle et al. [56] were extracted with methanol and subsequently with a mixture of methanol, acetone and water (3/1/1, v/v/v), the roots were first treated with the mixture of methanol, acetone, and water (3/1/1, v/v/v), then were extracted with methanol. The subaerial parts of the samples in the study by Zidorn et al. [55] were mixed with a stock solution and sonicated with methanol, then HPLC was performed. A study by Granica and colleagues [26] involved extraction of aerial parts of the species with 50% aqueous methanol and the HPLC analysis of obtained extracts.

Scorzonera aucheriana DC.
Samples of S. aucheriana were collected in central Turkey and aerial parts were extracted with methanol at room temperature [7,29].

Scorzonera baetica (Boiss.) Boiss.
Aerial parts of plant samples, collected in Spain, were extracted with 50% aqueous methanol at room temperature and the extracts were analyzed using HPLC.
In the extract of the aerial parts, rutin and notable amounts of chlorogenic acid were reported present.
The presence of chlorogenic acid was detected in the root extract [9]. Plant samples were collected in central Turkey [9,27]. Aerial and subaerial parts were separated and extracted with n-hexane [27] and 20% aqueous methanol [9].
In the root aqueous methanol extracts, chlorogenic acid was found. [9]. There were also reports on the presence of α-amyrin. taraxasteryl acetate, lupeol, and lupeol acetate in the n-hexane extract [27].

Scorzonera cinerea Boiss.
Samples of S. cinerea collected in central Turkey were extracted with n-hexane at room temperature.

Scorzonera cretica Willd.
Samples of S. cretica were collected on Crete, Greece. The whole plant was subjected to extraction with dichloromethane and subsequently with methanol.

Scorzonera crispatula Boiss.
Plant samples (aerial parts) were collected in England [44] and Spain. Samples from Spain were extracted at room temperature with a mixture of methanol and water (1:1, v/v) and subjected to HPLC [26]. Samples collected in England were extracted with ethanol [44].

Scorzonera eriophora DC.
Samples of S. eriophora aerial and subaerial parts were collected in Turkey and extracted at room temperature with 20% aqueous methanol [9] and n-hexane [27].
Chlorogenic acid was detected in aqueous methanol extracts of both aerial parts and roots. The aerial part extract was also reported to contain luteolin and luteolin 7-glycoside [9]. The n-hexane extracts of aerial and subaerial parts both contained taraxasteryl acetate, lupeol, lupeol acetate Additionally, 3β-hydroxy-fern-7-en-6-one-acetate was reported to be present in the n-hexane root extract [27].

Scorzonera graminifolia L.
Aerial parts of the plant, collected in England, were extracted with ethanol and reported to contain quercetin and luteolin [44].

Scorzonera hieraciifolia Hayek
Samples of the plant were collected in central Turkey. Aerial and subaerial parts were separated, and aerial parts were extracted with ethanol at room temperature and fractioned. Then isolation of compounds was performed.

Scorzonera hirsuta L.
Samples of S. hirsuta aerial parts, collected from the University of Reading (Reading, UK), were extracted with alcohol and the extract was reported to contain flavonoid aglycones: kaempferol, luteolin, and quercetin [44].

Scorzonera hispanica L.
In the studies by Granica et al. [20,26], plant samples were collected in Germany, subaerial parts for the quantitative analyses were purchased in Austria and Warsaw. In the study by Zidorn et al. [32], plant samples from Belgium were used. In the study by Petkova [60], the plant was harvested in Bulgaria.
For the isolation and identification of major constituents of S. hispanica subaerial parts, maceration with ethyl acetate was carried out. The phenolic compounds in aerial and subaerial parts were quantified using a modification of a method described by Zidorn et al. [61] with a mixture of methanol/acetone/water (3:1:1) [20]. For the elucidation of inulin content, the roots were extracted with water via microwave-assisted extraction [60].

Scorzonera humilis L.
Samples of S. humilis were collected in Austria and subaerial parts of the plant were extracted with methanol [53][54][55].
From the methanol extract, tyrolobibenzyls were isolated via column chromatography [53][54][55]. Tyrolobibenzyls A, B, and C were isolated and identified in the study from 2000, together with a lignin-pinoresinol-1-yl β-D-glucopyranoside [54]. In the study from 2002, the structure of tyrolobibenzyl D was elucidated [53]. A year later, the structure of two novel tyrolobibenzyls (E and F) was identified and the presence of chlorogenic acid and 3,5-dicaffeoylquinic acid was detected [55].

Scorzonera incisa DC.
Samples of the plant were collected in Turkey and aerial and subaerial parts were extracted separately at room temperature with 20% aqueous methanol [5] and n-hexane [27].

Scorzonera judaica Eig.
The roots of the plant were collected in Jordan and subsequently extracted with nhexane, chloroform, a mixture of chloroform and methanol (9:1), and methanol. Then, the isolation of compounds was carried out.

Scorzonera laciniata L. ssp. laciniata
Plant samples were collected in the north-western [9,27] and eastern parts of Turkey. [22]. Aerial and subaerial parts were separated and extracted with 20% aqueous methanol [9] and n-hexane [27]. In the study by Erden et al. [22], the extraction was carried out using methanol, a mixture of hexane and isopropyl alcohol (3:2, v/v), water, and a mixture of HNO 3 :H 2 SO 4 :H 2 O 2 (10:1: In the aerial part 20% aqueous methanol extract, chlorogenic acid and luteolin 7glucoside, as well as trace amounts of rutin were reported [9]. Myricetin, kaempferol, and trace amounts of morin and quercetin were present in the methanol extract. Phytosterols (ergosterol, stigmasterol, and β-sitosterol) and vitamins D and K were identified in the hexane/isopropyl alcohol (3:2, v/v) extract and notable amounts of potassium were identified in the extract obtained with the mixture of HNO 3 :H 2 SO 4 :H 2 O 2 [22]. The presence of lupeol, lupeol acetate, taraxasteryl acetate, and α-amyrin in the n-hexane aerial part extract has been reported as well [27].

Scorzonera mirabilis Lipsch.
Samples of the plant were collected in the city of Van, eastern Turkey, aerial parts and roots were separated in extracted at room temperature with n-hexane.
In the aqueous methanol extract of aerial parts, chlorogenic acid, rutin, hyperoside, and cyranoside were detected [5]. The presence of taraxasteryl acetate, lupeol, lupeol acetate, and α-amyrin was reported in n-hexane extracts from both aerial and subaerial parts of the plant [27].
Aqueous methanol root extract was reported to contain chlorogenic acid [5].

Scorzonera papposa DC.
Samples were collected in Jordan. Aerial parts and roots were separated, dried, and sequentially macerated with n-hexane, chloroform, a mixture of chloroform and methanol (9:1), and methanol.
Aerial and subaerial parts of the plant, collected in central Turkey, were separated and extracted at room temperature using n-hexane [27] and 20% aqueous methanol [5].
In the roots, chlorogenic acid was detected in the aqueous methanol extract [5]. Taraxasteryl acetate, lupeol, and lupeol acetate were the main components of the n-hexane extract [27].

Scorzonera pseudodivaricata Lipsch.
Samples for the analysis were collected in Mongolia. Aerial and subaerial parts of the plant were separated, then aerial parts were macerated with methanol at room temperature.

Scorzonera pusilla Pall.
In the aerial parts, collected in Reading (England), the presence of quercetin and luteolin was reported [44].

Scorzonera pygmaea Sibth. and Sm.
Plant samples (subaerial parts) were collected in Turkey. The dried and powdered subaerial parts were macerated in ethanol.

Scorzonera radiata Fisch.
Samples of S. radiata aerial parts were collected in Mongolia and macerated with methanol at room temperature [12,13,15].

Scorzonera suberosa C. Koch ssp. suberosa
Samples of the plant were collected in the central part of Turkey. Aerial and subaerial parts were separated and extracted with n-hexane at room temperature [27]. In the study by Erden et al. [22], the solvents used for extraction were methanol, a mixture of hexane and isopropyl alcohol (3:2, v/v), water, and a mixture of HNO 3 :H 2 SO 4 :H 2 O 2 (10:1: Aerial parts and roots are reported to contain taraxasteryl acetate, lupeol, and lupeol acetate [27]. Myricetin, morin, and quercetin were found in the methanol extract, vitamins D, E, and K, retinol, and phytosterols: β-sitosterol, ergosterol, and stigmasterol were detected in the hexane/isopropyl alcohol extract. Sodium and potassium were also reported present (extraction using the mixture of HNO 3 :H 2 SO 4 :H 2 O 2 ) [22].

Scorzonera trachysperma Guss.
Aerial parts of S. trachysperma samples, collected in Italy, were extracted with 50% aqueous methanol and subjected to HPLC.

Scorzonera undulata ssp. alexandrina Boiss.
Samples of the plant were collected in Algeria and the whole plant was macerated with petroleum ether.

Scorzonera undulata ssp. deliciosa (Guss.) Marie
Plant samples were collected in Algeria and subaerial parts were macerated in dichloromethane, then the isolation of compounds was performed.

Scorzonera veratrifolia Fenzl.
The samples used for the studies on S. veratrifolia were collected in eastern Turkey. The subaerial parts were separated from the aerial parts and extracted with methanol at room temperature [30,64].

Scorzonera villosa Scop. ssp. villosa
Samples of S. villosa were collected in Slovenia. Aerial parts were extracted with 50% aqueous methanol at room temperature and HPLC analysis was performed.
Compounds present in aerial parts of species within the genus Scorzonera, together with the concentration in the dry plant matter and the solvent used in the process of extraction (if available in the literature) are listed in Table 1. The phytochemical composition of subaerial parts of species belonging to the genus, with concentrations and solvents used, is presented in Table 2. Compounds isolated from the whole plants are presented in Table 3. As was presented in Tables 1 and 2, subaerial parts of the species within the genus Scorzonera are reported to contain a greater diversity of triterpenoid ad phenolic acid derivatives. This could be explained by the fact that a larger range of solvents was used for the extraction of phytochemicals from subaerial parts. The aerial parts; however, are reported to contain notably more different flavonoids. This is an anticipated outcome because, as flavonoid compounds are involved in biochemical processes within the whole plant, they are significant for the activities related to exposure to external factors (e.g., UV radiation or attracting pollinators) [65,66]. Another reason is the fact that only aerial parts of Scorzonera species were thoroughly assessed for flavonoid content in a study by Granica et al. [26]. Based on the research included in this review, the steroid, coumarin, and dihydroisocoumarin content seems similar in both aerial and subaerial parts. The species that are most investigated in the greatest number of papers are S. divaricata [14,34,[57][58][59], S. hispanica [20,26,32,45,46,60], S. latifolia [3][4][5][6]27,47,[67][68][69][70][71], and S. radiata [12,13,15]. Dihydroisocoumarins and dihydroisocoumarins glycosides Flavonoid C-glycosides S. radiata 7.334 µg/g methanol [13] S. radiata 30.667 µg/g methanol [13] Apigenin 8-C-glucoside (vitexin) Luteolin 6-C-glucoside (isoorientin) S. aristata 24.815 µg/g methanol methanol:acetone:water   Flavonoid O-C-glycosides Macrolides Sacrolide A S. divaricata 0.6 µg/g methanol [34] Organic acids/Phenolic acids and their derivatives   Compound concentration was taken directly from literature or it was calculated, dividing the mass of the isolated compound by the mass of plant material used for extraction; N/D-no data was available in the literature.        Compound concentration was taken directly from literature or it was calculated, dividing the mass of the isolated compound by the mass of plant material used for extraction; N/D-no data was available in the literature. Compound concentration was taken directly from literature or it was calculated, dividing the mass of the isolated compound by the mass of plant material used for extraction; N/D-no data was available in the literature.

Biological Activity
The biological activity of species within the Scorzonera genus is the subject of research due to their presence in folk medicine in Eurasia and northern Africa. In Mongolia, S. divaricata and S. pseudodivaricata play a significant role in herbal therapy. S. divaricata is used to treat fever and poisonous ulcers or even malignant stomach neoplasia. S. pseudodivaricata is a folk remedy for digestive problems, parasites, or lung edema [14].

Biological Activity
The biological activity of species within the Scorzonera genus is the subject of researc due to their presence in folk medicine in Eurasia and northern Africa. In Mongolia, S divaricata and S. pseudodivaricata play a significant role in herbal therapy. S. divaricata i used to treat fever and poisonous ulcers or even malignant stomach neoplasia. S pseudodivaricata is a folk remedy for digestive problems, parasites, or lung edema [14].

Cytotoxic Activity
Cytotoxicity is the primary characteristic of compounds and substances in terms o their qualification as therapeutic agents and is the toxicity that a certain factor causes i live cells [76]. High cytotoxicity against rapidly dividing cancer cells in vitro is the basi for further research on their bioactivity (e.g., necrosis, autophagy, or apoptosis induction low cytotoxicity on the other hand is desired in the development of drugs that are no intended to induce death in cells. In this review, the cytotoxicity of extracts an compounds obtained and isolated from Scorzonera species against cancer cell lines wa presented.

Cytotoxic Activity
Cytotoxicity is the primary characteristic of compounds and substances in terms of their qualification as therapeutic agents and is the toxicity that a certain factor causes in live cells [76]. High cytotoxicity against rapidly dividing cancer cells in vitro is the basis for further research on their bioactivity (e.g., necrosis, autophagy, or apoptosis induction), low cytotoxicity on the other hand is desired in the development of drugs that are not intended to induce death in cells. In this review, the cytotoxicity of extracts and compounds obtained and isolated from Scorzonera species against cancer cell lines was presented.
The first reported attempt to evaluate the antineoplastic activity of Scorzonera species in vitro was made in 2000 by Zidorn and colleagues [54]. The biological activity of compounds isolated from a methanol extract from S. humilis subaerial parts (together with newly isolated tyrolobibenzyls) was assessed and none influenced the DNA biosynthesis in the GTB and HL60 human leukemia cell lines at the concentration range of 0.25-4.00 µM. In their further research, tyrolobibenzyls D was isolated from the extract. It was assayed for cytotoxicity against the P388 (mouse leukemia) cell line along with previously obtained tyrolobibenzyls A-C and their peracetyl derivatives. In the assay, only Tylorobibenzyl D exhibited low cytotoxic activity with IC 50 (half-maximal inhibitory concentration) of 25 µg/mL. The cytotoxicity of crude extracts was assayed as well and no activity was observed up to the point where the concentration reached 0.5 mg/mL. The EtOAc fraction of the crude extract exhibited cytotoxic properties with IC 50 value at the concentration of 95 µg/mL. In the research, the antimicrobial activity of tyrolobibenzyls and their derivatives was evaluated but none was active against neither bacteria nor fungi. The DPPH assay did not reveal any significant radical scavenging properties of tyrolobibenzyls and the compounds were able to inhibit the activity of COX-1 (cyclooxygenase 1) to an insignificant degree [53].
In the study from 2009, Wang and colleagues [31] obtained two triterpene fatty esters: erythrodiol and 3β-tetradecanoyl erythrodiol from a methanol extract of Scorzonera mongolica whole plants. The isolated compounds were then assayed for their cytotoxicity towards 3 cancer cell lines (P388 mouse leukemia cell line, A549 human lung cancer cell line, and Bel-7402 human hepatocellular carcinoma cell line) and both esters exhibited cytotoxic activity against A549 lung cancer cells (in the concentration of 50 µg/mL, the compounds induced the cell growth inhibition by 66.8% and 69.8%).
Two out of five congeners (Scorzodihydrostilbenes A and B) isolated from the methanol extract from the aerial parts of Scorzonera radiata Fisch. were tested in the MTT cytotoxicity assay but at a concentration of 10 µg/mL, neither displayed cytotoxic activity toward mouse lymphoma cell line (L5175Y) [15]. The concentration (10 µg/mL) converted to µM is 21.53 µM for Scorzodihydrostilbene A and 20.90 µM for Scorzodihydrostilbene B. When compared to a study from 2007, dihydrostilbenes isolated from a Bulbophyllum odoratissimum Lindl., low toxicity of Scorzonera radiata Fisch. is even more notable, compounds in the mentioned study were toxic towards SGC-7901 (human gastric cancer), KB (nasopharyngeal carcinoma), and HT-1080 (fibrosarcoma) cell lines with IC 50 values of 5.50-9.20 µM for SGC-7901 and KB lines and 25.50-40 µM for HT-1080 line [77].
A screening study on cytotoxic activity of several species of Asteraceae genus present in Hungary, including Scorzonera austriaca Willd, was carried out in 2009. In a cytotoxicity assay, the most active against human cell lines: A431 (skin epidermoid carcinoma), HeLa (cervix adenocarcinoma), and MCF-7 (breast adenocarcinoma) was a chloroform root extract of S. austriaca IC 50 values of the extract were: 4.71 µg/mL for A431 line, 6.42 µg/mL for HeLa line and 5.52 µg/mL for MCF-7 line. It was more active than the extracts obtained using other solvents, as well as leaf extracts from the same plant-at a concentration of 10 µg/mL the antiproliferative activity of S. austriaca root chloroform was at 86.32% for A431 cell line, 77.27% for HeLa cell line, and 83.79% for MCF-7 cell line. Other S. austriaca extracts obtained in the study did not influence the proliferation of those cell lines by more than 48.11% [78]. Those results can be compared to a 2018 research, in which leaf chloroform extract from another species within the Asteraceae family, Anvillea garcinii (Burm.f.) DC., exhibited antiproliferative properties against MCF-7 and HeLa cell lines with IC 50 of 24.50 µg/mL for MCF-7 and 12 µg/mL for HeLa [79]. Another Asteraceae family member, Pulicaria undulata (Forssk.) Oliver., was evaluated as a potential source of cytotoxic agents. Whole plant chloroform extract turned out to have cytotoxic properties with the IC 50 value of 16.4 µg/mL for MCF-7 cell line, 3.01 µg/mL for HepG2, and 7.4 µg/mL for HCT-116 cell lines. Those values were compared to cisplatin used as a positive control in the study (IC 50 of cisplatin was 3.68-4.51 µg/mL) [80]. However, in the study from 2011, Bader et al. isolated nine new phenolic compounds and nine known phenolic derivatives from Scorzonera judaica root extracts. The newly obtained compounds were assayed for their cytotoxic activity toward human lymphocyte T cells, as well as MCF-7 and HeLa cell lines. Compounds did not exhibit cytotoxic activity in concentrations below 100 µM [25].
In the study by Granica et al. [20], (-)-syringaresinol was isolated from the ethyl acetate extract of Scorzonera hispanica subaerial parts. It was then reported that (-)-syringaresinol was cytotoxic towards NCI and MMS-1 myeloma cell lines and exhibited moderate activity against SW-480 colon cancer cells. Moreover, the compound's cytotoxicity was reported in peripheral blood mononuclear cells. In the previous research on (-)-syringaresinol, it was reported to exhibit the ability to induce apoptosis and arrest the G 1 phase in the HL-60 human leukemia cell line [81]. Jeong et al. [82] found out that (-)-syringaresinol inhibits P-glycoprotein in MCF-7/ADR human breast cancer cell line and enhances the cytotoxic activity of daunomycin.

Anti-Inflammatory Activity
For centuries, suppression of inflammatory response has been an observed effect of various medicinal plants. Ethnopharmacological reports give examples of plants extracts able to combat the process of inflammation in human bodies and; therefore, novel plantderived products are investigated for their anti-inflammatory activity [83].
A study carried out by Bahadır-Acıkara et al. in 2018 [27] revealed that n-hexane extracts from roots and aerial parts of eleven Scorzonera species (S. acuminata, S. cinerea, S. eriophora, S. incisa, S. latifolia, S. mirabilis, S. mollis ssp. szowitsii, S. parviflora, S. suberosa ssp. suberosa and S. tomentosa) contained significant amounts of triterpenes, including taraxasteryl acetate, lupeol, and lupeol acetate. In general, root extracts were notably richer in analyzed triterpenes, except for lupeol, whose concentration in aerial parts of S. incisa, S. latifolia, S. mirabilis, S. parviflora, and S. suberosa was approximately three to seven times higher than in root extracts (approximately 0.9-1.5 mg/g) . The results from the study correlate with previously observed anti-inflammatory and pain-relieving properties of lupeol [84,85].
The evaluation of the anti-inflammatory properties of Scorzonera pygmaea subaerial parts was conducted in 2018 by measuring COX (cyclooxygenase) inhibition. The inhibitory activity of ethanol extract and its fractions against COX-1 and COX-2 (cyclooxygenase 2) was low [35]. That is on the contrary to the study of Bahadır Acıkara et al. from 2015 [10], where it has been observed that extracts from other Scorzonera species (S. cana var. jacquiniana, S. cinerea, S. eriophora, S. incisa, S. latifolia, S. mollis ssp. szowitsii, S. parviflora, and S. tomentosa) have an inhibitory effect on pro-inflammatory cytokines (TNFα (tumor necrosis factor α) and IL-1β (interleukin 1β)) production and NF-κB (nuclear factor kappa B) nuclear translocation in macrophages. However, it might suggest that the anti-inflammatory activity of S. pygmaea could be evaluated by the measurement of the inhibitory activity against pro-inflammatory cytokines, as they induce COX production [35].

Analgesic Activity
Pain is an experience known to nearly every animal. There are cases where pain requires medical intervention. Although pain may indicate injury of organs, nervous systemderived pain should be relieved beforehand to prevent the deterioration of the quality of the patient's life. Morphine, a well-known analgesic, was isolated from opium 200 years ago [86]. Nowadays other natural products are assayed for their pain-relieving activity.
Scorzonera latifolia is a plant endemic to Turkey, whose roots are used as a pain-reducing and anthelmintic agent in Turkish folk medicine [4]. An in vivo study on the properties of a methanol extract from S. latifolia roots showed that n-hexane, chloroform, ethyl acetate, n-butanol, and water fractions indeed exhibit analgesic activity on mice in the dose of 50 mg/kg in the tail-flick test. Taraxasteryl mirystate and taraxasteryl acetate present in one of the extracts were active in the dose of 10 mg/kg in both writhing test and flick-tail test. The general antinociceptive properties of S. latifolia are reported to be significant. Such outcome of the study can be a result of the synergy of the extract's components [6]. The study was extended to four Scorzonera species in 2012. It was then reported that S. tomentosa, S. latifolia, and S. mollis ssp. szowitsii all possess analgesic properties in the writhing test and tail-flick test (the dose was 100 mg/kg) [70].

Hepatoprotective Activity
The liver plays a significant role in the metabolism and detoxication of the human body. Because of its importance, liver diseases are one of the greatest threats to people's lives. Herbal medicine has used plants as preventive agents for hepatic problems for ages [87]. Based on that knowledge, in vitro and in vivo investigations are conducted to assess the hepatoprotective potential of plants, including the ones with the Scorzonera genus.

In Vitro Assays
A study from 2016 investigated the hepatoprotective properties of Scorzonera austriaca. The plant is used in folk medicine to treat hepatitis B in China. Xie et al. [24] isolated flavonoid glycosides and their derivatives from Scorzonera austriaca herb ethanolic extract. Having measured the concentration of ALT (alanine aminotransferase) in CCl 4 -treated rat hepatocytes, it was reported that two flavonoid glycoside derivatives, 5,7,41-trihydroxyflavone

In Vivo Assays
The in vivo assays of a Scorzonera alexandrina hydroethanolic extract from aerial and subaerial parts revealed that the extract caused a reduction in glucose concentration in rat's blood, as well as the ALT, and total protein levels in doses of 200 and 400 mg/kg. The extract also exhibited hepatoprotective, and anti-ulcerogenic effects in rats [75].
Hepatoprotective activities of the roots of several Scorzonera species (S. cana var. jacquiniana, S. latifolia, S. mollis ssp. szowitsii, S. parviflora, S. tomentosa), together with compounds isolated from the S. latifolia root extract (chlorogenic acid, hydrangenol-8-O-βglucoside, and scorzotomentosin-4 -O-β-glucoside) were evaluated in a preclinical in vivo study from 2017. The tests were aiming to elucidate the extract's influence on counteracting CCl 4 -induced liver damage in rats. Although the influence of the extract and compounds on the ALT and AST (aspartate transaminase) levels was insignificant, the histological condition of animal livers was notably better in most samples (except for hydrangenol-8-Oβ-glucoside and scorzotomentosin-4 -O-β-glucoside-treated groups). What is interesting in terms of future clinical research is the fact that chlorogenic acid was the most active compound in the treatment of acute carbon tetrachloride-induced liver toxicity [73].

Antimicrobial Activity
Folk medicine has been treating microbial infections for centuries. Along with the decrease in bacteria's susceptibility to antibiotics, the need for novel antimicrobial drugs is increasing. Plants have been a source of folk medications in the treatment of infectious diseases before the concept of infectious agents emerged [88]. The activity against microbes could also be used to substitute synthetic additives in food to prevent foodborne diseases induced by pathogenic bacteria [89] Several species within the genus Scorzonera have been investigated as a source of products with antimicrobial potential.
Volatile oil distilled from aerial parts of Scorzonera undulata ssp. deliciosa was assessed as an antibacterial agent against Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus) and Gram-negative (Salmonella typhimurium, Escherichia coli, and Pseudomonas aeruginosa) bacteria strains and it was reported more active towards Grampositive strains with MIC (minimal inhibitory concentration) values of 0.5 mg/mL for S. aureus and M. luteus and 0.8 mg/mL for S. epidermidis, S. typhimurium, and E. coli. MBC (minimal bactericidal concentration) was not determined for any Gram-negative strain. The authors suggest that greater activity against Gram-positive bacteria strains could be caused by easier penetration through the lipophilic cell membranes by hydrophobic ingredients of the oil. Unfortunately, no reference compound was assessed together with the oil, thus it is difficult to compare those results with any known antibacterial substances [90].
Antibacterial properties of S. undulata were assayed in 2010 by Abdelkader and colleagues [91]. The study showed that ethyl acetate fraction of the aerial part methanol extract exhibited antibacterial properties against P. aeruginosa, S. aureus, E. faecalis, C. freundei, and P. mirabilis with MIC exceeding 1 mg/mL. The petroleum ether fraction; however, was active against P. aeruginosa, S. aureus, and C. freundei. Fractions obtained from the roots had a narrower spectrum of activity, but the petroleum ether fraction exhibited a stronger antimicrobial potential against S. aureus with a MIC of 500 µg/mL.
A comprehensive study on endemic to Lebanon species Scorzonera mackmeliana in terms of the plant's antibacterial and antibiofilm properties was carried out by Sweidan and colleagues in 2020 [92]. The authors conducted a phytoanalysis of the constituents of water and ethanol extracts of the whole plant as well as its particular parts (flowers, stems, leaves, roots) and determined their activity against Gram-positive (Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacterial strains. The highest activities in inhibition of the bacteria strains were observed in two water extracts: the ones obtained from the stems and the whole plant. For the stem water extract, the inhibitory effect was observed in four out of five tested strains, the lowest MIC values were reported in P. aeruginosa (48.98 mg/mL), S. aureus (48.98 mg/mL), and S. epidermidis (48.98 mg/mL). For the water extract from the whole plant, it was proven active against S. epidermidis, P. aeruginosa, and E. coli in the concentration of 122.25 mg/mL for each strain. MBC was determined for only two extracts and those were root ethanol extract (for S. epidermidis MBC = 284.35 mg/mL) and water flower extract (for P. aeruginosa, MBC = 160.85 mg/mL). Those values; however, are notably high, compared to the literature data available for other plant water and ethanol extracts. MIC and MBC values for Cinnamomum impressicostatum stem bark extract against MRSA strain were 19.53 and 39.06 µg/mL, respectively. For Cinnamomum porrectum stem bark extract, it was 2.5 (MIC) and 5 mg/mL (MBC) [93]. In the Asteraceae family, water extracts from Sonchus erzincanicus aerial parts exhibit antibacterial properties against Staphylococcus aureus, Escherichia coli, and Proteus mirabilis with the MIC value of 1.25 mg/mL [94]. In the mentioned study on S. mackmeliana; however, an interesting pattern of the activity against biofilm formation was observed-with the concentration decrease, greater destruction of the biofilm occurred. It was also observed that flower and stem water extracts and ethanol leaf, flower, and whole plant extracts exhibited the most potent activity in eradicating bacterial biofilm, with MBEC (minimal biofilm eradication concentration) values of 0.1-2.2 mg/mL, causing 84-98% biofilm eradication. For water extracts, the presence of coumarin was suspected to be the active factor and in ethanol extracts, terpenoids were major constituents and; therefore, they are thought to be responsible for the antibiofilm effect [92].
The antibacterial and antifungal activity of the aerial parts and root extracts from S. papposa was investigated in a recent study by Mohammed and colleagues [95]. The extracts were toxic for bacteria at the concentrations of 50-800 µg/mL and their antifungal effectiveness was observed at 50-100 µg/mL. The reference compounds (ampicillin, amikacin, ciprofloxacin, fluconazole, and amphotericin B) on the other hand were active at notably lower concentrations (1.56-3.12 µg/mL). In general, samples collected in Turkey were slightly more effective against both bacteria and fungi (50 µg/mL against Pseudomonas aeruginosa), although all tested extracts can potentially be used as mild nature-derived antimicrobial agents.

Wound Healing Activity
Wounds defined as a disruption of tissue do not pose a threat unless the blood loss is significant. They can; however, be a gateway for pathogenic infections which is much more dangerous for the patient. The acceleration of wound recovery includes infection prevention and the promotion of the natural healing process. Medicinal plants are suspected to possess both those qualities and are considered potentially effective in the therapy of wound healing [96].

In Vitro Assays
A study carried out by Küpeli Akkol and colleagues in 2019 [4] reports that ethyl acetate and chloroform fractions of a methanol extract from aerial parts of S. latifolia have wound healing properties in vitro, which are a result of the inhibitory effect on collagenase and elastase enzymes activity. None of the fractions influenced the activity of hyaluronidase to any considerable degree.

In Vivo Assays
In 2011, a study by Küpeli Akkol et al. [5] on the promotion of the process of wound healing in mice took place. Researchers obtained hydroethanolic extracts from the aerial parts and roots of several Scorzonera species (S. cinerea, S. latifolia, S. incisa, S. mobilis, S. mollis ssp. szowitsii, S. tomentosa). Most promising results in the assays carried out with wound models were observed with ointments made with the extracts obtained from S. latifolia, S. mollis ssp. Szowitsii, and S. tomentosa aerial parts. Those three extracts were the most active in the hydroxyproline level enhancement as wells as in terms of skin remodeling. Moreover, the S. latifolia aerial part extracts were reported to have anti-inflammatory properties in vivo, with an inhibitory value of 23.5% at the dose of 100 mg/kg.
A study from 2012, carried out on mice, reports wound healing properties of several Scorzonera species. Aqueous methanolic extracts from the aerial parts of S. cana var. jacquiniana, S. eriophora, and S. acuminata caused the contraction of wound area by up to 46.27% on day 12 in the circular excision wound model. Ointments containing extracts of S. cana (C.A. Mey.) Hoffm. var. jacquiniana (W. Koch) Chamb. and S. eriophora DC. aerial parts, when applied topically on the linear incision wound models, caused an increase in the activity of anti-hyaluronidase and significant enhancement of hydroxyproline level in the regenerated tissue [9]. Figure 2 summarizes literature data regarding the biological activity of Scorzonera species in vivo.

Antioxidant Capacity
Exposure to reactive species in the environment may have a negative impact on humans and animals. The balance between oxidants and antioxidants is becoming more difficult to maintain [97].; therefore, the need for antioxidant agents is growing and, because of the potentially harmful effect of synthetic antioxidants, the attention seems to be currently directed towards naturally occurring antioxidants found in plants [98]. The products from species belonging to the genus Scorzonera have been assayed as antioxidant agents as well.

Antioxidant Capacity
Exposure to reactive species in the environment may have a negative impact on humans and animals. The balance between oxidants and antioxidants is becoming more difficult to maintain [97].; therefore, the need for antioxidant agents is growing and, because of the potentially harmful effect of synthetic antioxidants, the attention seems to be currently directed towards naturally occurring antioxidants found in plants [98]. The products from species belonging to the genus Scorzonera have been assayed as antioxidant agents as well.
In the DPPH radical scavenging assay compounds isolated from the ethyl acetate fraction of the methanol extract from Scorzonera divaricata and Scorzonera pseudodivaricata aerial parts (ferulopodospermic acid A and B) did exhibit a strong antioxidant activity, more potent than chlorogenic acid used as a reference compound in the study. The IC50 values ferulopodospermic acid A and B were 36.36 and 34.24 μmol/mL, respectively, compared to the IC50 of chlorogenic acid, which was 67.92 μmol/mL [14]. The study was continued with five compounds obtained from a Scorzonera radiata aerial part MeOH extract were tested for their radical-scavenging activity in the DPPH assay. Scorzodihydrostilbenes A and E exhibited a higher activity level than resveratrol, well-known for its anti- In the DPPH radical scavenging assay compounds isolated from the ethyl acetate fraction of the methanol extract from Scorzonera divaricata and Scorzonera pseudodivaricata aerial parts (ferulopodospermic acid A and B) did exhibit a strong antioxidant activity, more potent than chlorogenic acid used as a reference compound in the study. The IC 50 values ferulopodospermic acid A and B were 36.36 and 34.24 µmol/mL, respectively, compared to the IC 50 of chlorogenic acid, which was 67.92 µmol/mL [14]. The study was continued with five compounds obtained from a Scorzonera radiata aerial part MeOH extract were tested for their radical-scavenging activity in the DPPH assay. Scorzodihydrostilbenes A and E exhibited a higher activity level than resveratrol, well-known for its antioxidant activity, used as a reference in the study. The IC 50 values were 105.51 µM for scorzodihydrostilbene A, 102.60 µM for scorzodihydrostilbene B, and 149.52 µM for resveratrol [15]. Although the difference in the scale of IC 50 values might seem interesting, as IC 50 in S. divaricata and S. pseudodivaricata was given in µmol/mL, whereas for S. radiata it was presented in µM (µmol/L), more informative is how those results correspond to reference compounds used in both studies.
In the assessment of antioxidant activity, acteoside isolated from a methanolic extract from Scorzonera undulata ssp. deliciosa roots was reported to possess similar antiradical power to Trolox used as a standard in the DPPH test (IC 50 values were 0.16 ± 0.02 mg/mg DPPH for acteoside and 0.2 ± 0.01 mg/mg DPPH for Trolox). The Trolox Equivalent Antioxidant Capacity (TEAC) value of acteoside in the DPPH assay was 1.25. In the CUPRAC (cupric reducing antioxidant capacity) assay acteoside was slightly less active than the reference compound, rutin (TEAC = 3.16 for rutin compared to TEAC = 2.4 for acteoside) [49].
Nasseri et al. [52] evaluated the chemical composition and the radical scavenging activity of Scorzonera paradoxa root and leaf ethanol/water extracts. Leaf extracts turned out to be a more potent antioxidant with an IC 50 value of 18.81 mg/mL, compared to the roots (IC 50 = 88.9 mg/mL). This may be due to higher levels of phenolic compounds, flavonoids and tannins reported in the study. The authors also made an assessment of the fatty acids composition of the plant samples and based on the data obtained in the study of chemical composition and antioxidant properties, it was suggested that S. paradoxa might be successful as an antidiabetic agent. Those results and IC 50 values correspond with a study from 2013, when Erden and colleagues [22] investigated the antioxidant properties of methanol extracts obtained from the leaves of three Scorzonera species (S. suberosa, S. laciniata, and S. latifolia). Those properties were examined in the DPPH assay and exhibited a concentration-dependent antioxidant activity with IC 50 values of 29.36 mg/mL for S. latifolia, 42.33 mg/mL for S. suberosa, and 77.07 mg/mL for S. laciniata.
In 2013 Milella et al. [62] measured the antioxidant properties of pure compounds isolated from methanol extracts from aerial parts and roots of Scorzonera papposa. The authors assessed the antioxidant activity of the compounds obtained from S. judaica in Bader's previous study from 2011 [25] as well. Four out of nine compounds isolated from S. papposa extracts were previously unknown. In the study, the antioxidant activity was measured in four different assays: the DPPH assay, the FRAP (ferric reducing antioxidant power) assay, the BCB (β-Carotene bleaching) assay, and the TPC (total phenolic content) assay. It has been observed that the antioxidant capacity of particular compounds depends on the method. The authors suggested that the antioxidant activity of the compounds found in the extract is a result of the synergistic effect of their combination. In the study, a new concept for presenting the antioxidant capacity of compounds-Relative Antioxidant Capacity Index (RACI)-Was applied [62]. Briefly, the parameter is used to integrate the data from several methods for the assessment of the antioxidant activity, where each method is assigned equal weight. RACI can take positive or negative values [99].
A recent study on Scorzonera papposa was a comparison of the antioxidant and antimicrobial activity of ethanol extracts from aerial parts and roots of S. papposa from Iraq and Turkey. It has been observed that samples from Iraq exhibited a higher level of TAS (Total Antioxidant Status) and a lower level of TOS (Total Oxidant Status) than samples collected in Turkey. Therefore, the OSI (Oxidative Stress Index-The TAS/TOS ratio) parameter in the samples from Iraq was lower than in the samples from Turkey. Compared to the reported TAS and TOS values of other plant species (i.e., Calendula officinalis L., Rhus coriaria L. var. zebaria, Shahbaz and Mentha longifolia L.), the extracts from the aerial parts of S. papposa obtained in the study exhibited a notable antioxidant activity [95].

Conclusions
Aerial and subaerial parts of species within the Scorzonera genus have been the subject of research regarding their phytochemical composition as well as their therapeutic potential. In many European and Asian cultures, Scorzonera species are commonly used in folk medicine,; therefore, modern phytoanalyses and biological studies have been carried out to verify the bioactive activities of the plants. Due to the presence of numerous bioactive compounds, including flavonoid aglycones and glycosides, triterpenoids, sesquiterpenoids, quinic acid, and caffeic acid derivatives, in the studied plant material, Scorzonera species are considered a potential source of antioxidant agents. Although the reported cytotoxicity of Scorzonera extract against cancer cell lines so far was insignificant, they exhibit other bioactive properties, potentially applicable not only in the therapy of pain, inflammation, and microbial infections, but also as an enhancement of the effectiveness of the wound healing process. It should be pointed out that a promising direction of further research on the genus Scorzonera is the investigation of their activity towards normal cell lines, especially skin cells, to assess their potential as wound-healing and skincare active agents.