Bioactivity of Secondary Metabolites and Extracts from the Leontopodium R.Br. ex Cass. Taxa with Targeted Medicinal Applications

Abstract

The Leontopodium R.Br. ex Cass. taxa have been extensively phytochemically researched for their beneficial properties by the pharmaceuticals industry. These species have been used as remedies since ancient times, and their usage in traditional medicine has been a source of inspiration for current research. As a result, a comprehensive review study concerning the bioactivities of secondary metabolites belonging to Leontopodium species would be of great interest. Our research shows that the majority of studies addressed the anti-inflammatory activity, and the most studied compound was leoligin, a lignan with cardioprotective properties. The diverse range of bioactivities were intricately linked to the abundance of secondary metabolites, which conferred effective antimicrobial activity, antioxidant properties, the anti-neurodegenerative potential due to improvements in cholinergic transmission, the anti-tumour effects on various cancer cell lines, particularly breast and lung cancer, and the hypoglycaemic and hepatoprotective properties. All these important bioactivities also recommend Leontopodium taxa as a valuable source for the discovery of new drugs.

1. Introduction

The biologic activity of compounds, known as bioactivity, refers to the ability to determine a positive or negative physiological response and to modify the metabolism at the cellular, tissue or organ level [1]. These compounds can have a synthetical origin or can be natural products synthesized by plants, fungi, bacteria or various living organisms.

The biological activity is usually measured using a bioassay test, generally in a dose-dependent manner, by registering the dose–response averages curves [2]. While compound bioactivity refers to the interaction or effect on any cell, tissue, organ or human body level, pharmacological activity is usually considered to describe the beneficial effects, i.e., the effects of drug candidates, as well as the toxicity of a substance [2].

A series of scientific studies conducted in the late 2000s demonstrated the exploitable bioactivities in the pharmaceutic industry of Edelweiss, i.e., Leontopodium alpinum Cass. or L. nivale subsp. alpinum (Cass.) Greuter, such as (a) strong anti-oedematous activity [3]; (b) anti-inflammatory properties revealed via the ex vitro inhibition of leukotriene synthesis [4]; (c) potent antioxidative capacity due to a newly discovered compound named leontopodic acid B, which even protects DNA against oxidative damage [5]; (d) having chemoprotective properties manifested by an increase in detoxifying enzymes, the main mechanism of antioxidant-mediated chemoprevention [6]; (e) antinociceptive effects associated with the inhibition of the prostanoid system or lipoxygenase/cyclooxygenase activity [3,4,7]; (f) antibacterial activity due to fatty acid constituents such as linolenic and linoleic acids [8]; (g) anti-neurodegenerative properties, which improve cholinergic transmission through the inhibition of acetylcholine esterase activity [9]; and (h) cardioprotective action exerted by inhibiting the intimal endothelial proliferation and reversed graft disease in pre-damaged vessels [10].

These findings were based on data from folk medicine, where Edelweiss has been used against abdominal pain, diarrhoea, dysentery and fever [7,11,12]. Aerial organs of Edelweiss have been used to attenuate inflammation and pain in the respiratory system (tonsillitis, bronchitis, and pneumonitis), gastrointestinal system (gastritis and colitis) [13], joints (rheumatoid arthritis) [14], and tumour-affected tissues (skin and connective tissues in breast cancer) [15]. The aerial parts of Leontopodium leontopodioides Beauv., a species mainly growing in the northeast and northwest regions of P. R. China, are commonly used to treat albuminuria, haematuria, acute or chronic nephritis, vaginitis, bronchopneumonia and gastric ulcer [13].

Due to the growing interest in its medicinal purposes, various biotechnologies have been developed to produce cellular mass rich in secondary metabolites. The most exploited in vitro culture techniques include the callus [12,14,16], cell suspensions [17], adventitious roots and hairy roots cultures [18,19,20].

For stimulating the production of certain compounds, different types of cultures were treated with various elicitors: physical, such as UV, LED light [16]; gamma radiation [21] or chemical, such as methyl jasmonate; silver nitrate or yeast extract [20]. The extracts from whole plants [22,23], roots [3,4,9,11,18], aerial parts [3,6,12,17], flowering aerial parts [5,7], leaves [24], stems [25] or commercial extracts [26,27] were used in various extraction procedures, while solvents included dichloromethane [3,4,11], ethanol [12,28,29] methanol [20,25], CO₂ [7], acetonitrile [20], 1,3-butanediol [14], hot water [16] or other types of solvents [30]. Also, different fresh [26,31,32] or dry plant materials [30,33] were analysed.

The experimental model used to highlight the bioactivities of these extracts was represented by the following: normal or immortalized keratinocyte cell lines [12,34], fibroblasts cell lines [14,15], macrophages’ cell lines [13,35], tumour cell lines [36,37], hepatocyte cell lines [38], dermal papilla cultures [24], dermal models [39], model animals [3,7] or human volunteers [24,34].

Considering the abundance of information on the secondary metabolites found in the Leontopodium taxa and their potential applications in medicine, we conducted an analysis of the significant bioactivities exhibited by various Leontopodium species. The aim of this study was to consolidate the findings from both older and more recent research, representing a novel contribution to the field. Each section of the research focused on the biological potential of secondary metabolites from the Leontopodium taxa, providing details regarding the type of explant, the species of origin, the experimental model used, the effects of the bioactive compounds and the specific tests employed to highlight these bioactivities.

2. Habitat, Distribution, Sozological Status and Cultural Value of Leontopodium Taxa

The Leontopodium taxa belong to the Gnaphalium genus, Asteraceae family, being perennial herbs, which naturally grow in arid grasslands, loess slopes, gravel, and mountain grasslands, on limy soils, stony meadows and even on rocks. These taxa are usually found at an altitude between 1000 and 3400 m but also to higher altitudes in the Tibet region [40,41,42,43,44,45].

Leontopodium species have a widespread distribution across temperate and subalpine regions of Eurasia. The main distribution of the genus is in Central and Eastern Asia, including Russia, Japan, South Korea, Mongolia, China and along the Himalaya to the borders of Afghanistan and Pakistan (Figure 1). The centre of diversity is southwestern China, where 15 to 18 different species can be found [46]. The genus occupies an extensive and largely continuous distribution in the mountains of Central, temperate South-eastern and Eastern Asia but also Central and Southern Europe. Some scientists believe that the Edelweiss plant originally grew in Asia but migrated to the Alps during the Ice Age [47]. The Leontopodium taxon (Asteraceae) comprises 58 species in Asia and Europe [48], with 41 species in China, including some presumed hybrids and 18 endemic species [49]. Only one species is native to Europe, L. nivale (Ten.) Huet ex Hand-Mazz. [46,50], but Blöch (2010) [51] considered that there are two European taxa, L. alpinum and L. nivale, that form a genetically distinct group. Some authors recognize just one species with two subspecies [52], Leontopodium nivale ssp. alpinum (Cass.) Greuter, which is native to the Pyrenees, the European Alps, the Tatra and the Balkan Mountains, and its endemic sister species, Leontopodium nivale (Ten.) Huet. ex Hand.-Mazz., which has a disjunct distribution in the Central Apennines in Italy and the Pirin Mountains in Bulgaria [6]. So far, the relationship between L. nivale and L. alpinum has not been entirely solved, with the literature containing an unfortunate combination of the two nomenclatures [53].

The conservation status of L. alpinum is well known due to ample work on various aspects of its biology, ecology, chorology and secondary metabolites’ chemistry [40,43,44,54,55,56]; however, less is known about the Asian members of the genus [46].

In European countries, such as Switzerland and Austria, Edelweiss has been protected since 1878 and 1887 [57]. According to the IUCN Red List, this species is classified as least concern [45], but in each resident country or region of Europe, it has a different conservation status. It is critically endangered in Albania; regionally endangered in Austria, Bulgaria, and Germany [58]; near threatened in Slovakia; critically endangered and near threatened in some regions in Switzerland; and critically endangered in Ukraine [59]. Data regarding the endangered status for the Asian members of this genus were found only in Korea [60] and Japan [61]. For other countries, where Leontopodium species are native, these were not listed in the National Red Lists, or a database of endangered species was not identified in that country (Supplementary Material S1).

In Romania, L. alpinum was included as vulnerable and rare in the Red List of vascular plants [62] due to aggressive harvesting for ornamental purposes [63], despite the large habitat area. Currently, there is no national assessment of Edelweiss in Romania, but in current climatic conditions, the Edelweiss conservation strategy regarding human harvesting should be limited by raising awareness of the importance of these species within local communities and by monitoring and educating tourists [59]. At the European level, in order to reduce the pressure of over-collection for horticultural, pharmacological or cosmetic purposes, this species has been domesticated and introduced into small-scale cultures, since 1995 in Bruson, Valais, Switzerland at 1100 m altitude [64,65]. A series of propagation methods have also been developed through in vitro culture techniques, for ornamental multiplication [56], ex situ conservation [66] or biomass producing useful metabolites [17,18,19,20].

For people living in the European Alps, L. alpinum, which is known as the Alpine Edelweiss, is a very important part of their cultural heritage, being an iconic species for Austria, Switzerland and Germany, representing a symbol of the Alps in Central Europe. The scientific Latin name of the plant derives from the typical white hairiness (Greek: leon = lion, podion = small foot), while the folk name, Edelweiss, is a combination of “edel”, which means noble, and white in German [67]. Edelweiss is often called the queen of mountain flowers, bearing names, such as Glacier Queen, Alpes Star, Glacier Star, Snow Star, Silver Star, Beautiful Star, Starry Cotton, Snow Everlasting or Lion’s Foot, with reference to the external appearance [64,65] (Figure 2).

In Asian culture, the Edelweiss flower is primarily valued for its symbolism of purity and resilience, rather than being a deeply rooted traditional symbol like other flowers. Unfortunately, few studies exist regarding the cultural value of Leontopodium in Asian regions, with few exceptions, probably due to the linguistic barrier or inaccessible sources. The whole plant of Leontopodium dedekensii Beauv., commonly named Babongbin, is used as kindling and fuel by the Lhoba people in Milin County, Tibet [68]. Some taxa of this genus, such as L. artemisiifolium (Levl.) Beauv., L. calocephalum var. uliginosum Beauv., and L. leontopodioides (Willd.) Beauv., are commonly used as herbal remedies in China [48].

3. The Use in Traditional Medicine

Leontopodium taxa have been used in folk medicine for a long time [47], usually by boiling plant parts in wine or mixing with milk [69].

The first mentions date back to 1582, where Edelweiss was used to treat diarrhoea and dysentery [70], angina pectoris, diphtheria, coughs, bronchitis, abdominal pain and fever in humans and animals [65,69,71,72,73,74,75,76]. The old German names, “Strahlendes Ruhrkraut” (radiant dysentery herb) and “Bauchwehblume” (stomach ache flower), leave no doubt as to the traditional use of the plant to treat diarrhoea and dysentery [76]. In traditional Polish medicine, the tea was used for local applications against breast cancer, most probably due to the plant’s soothing properties [77]. In the Tyrol region, it was used as a traditional remedy for the treatment of rheumatic pains [65].

Traditional Chinese Medicine mentioned the use of L. leontopodioides as a remedy for urinary tract infections and traumatic bleeding in the Chinese Materia Medica Editorial Board of State Administration of Traditional Chinese Medicine of the People’s Republic of China 1999 [78]. It is generally used to treat influenza in Uygur medicine [79].

The use of this species over time in the treatment of a large number of diseases is explained by the presence of a very large number of secondary metabolites, which are synthesized as a defensive response to the action of adverse environmental factors in which the species grows (rocky area, stingy wind, low temperatures, bright sun, etc.). Thus, habitat and geographical distribution are factors that influence the growth, development and secondary metabolism of the taxon.

4. Secondary Metabolites Identified in the Leontopodium Taxa

Considering the traditional use of the Leontopodium herbs in the traditional medicine in Europe and Asia, several studies were conducted to identify the secondary metabolites responsible for the curative effects of these folk remedies. Many scientific articles investigated the content of secondary metabolites in Leontopodium species, the first papers indicating the presence of the main compounds from the phenylpropanoids’ class, such as chlorogenic and 3,4-di-O-caffeoylquinic acids [17,56,64] and flavonoid derivatives from luteolin [55,64,80], in the methanolic extracts from leaves and inflorescences. In the roots, the analysis of essential oils revealed, for the first time, the presence of terpenoids [56] and a chroman [18]. Other studies identified sitosterol [17] and different sesquiterpenes, such as isocomene and its derivatives, modhephene and an acetoxy derivative, along with a derivative of caryophyllene [19] in a root extract.

Subsequently, many phytochemical studies on L. alpinum succeeded in determining almost 50 different secondary metabolites, very well described in the review paper presented by Tauchen and Kokoska [53]. Roots and hairy roots cultures represent a valuable source of secondary metabolites, including sesquiterpenes, such as β-isocomene, silphinene, silphiperfolene acetate [9,11,49,81] and bisabolene derivatives [4,54], diterpenes derivates from the ent-kaurenoic acid [4], coumarins such as obliquin, its derivatives and one benzofuran [11], lignans such as leoligin and its methoxy-derivatives [4,10,11,20] and two polyacetylenes [4]. The aerial parts are a rich source of phenolic compounds, mainly caffeoyl-D-glucaric acid derivatives, such as leontopodic acid A [5], leontopodic acid B and 3,5-di-O-caffeoylquinic acid [82] and tetracaffeyol-D-glucaric acid derivative [37,83]. Flavonoids like apigenin, luteolin and their O-glycosylated derivatives but also chrysoeriol and quercetin glycosylated derivatives, were described in aerial parts [82,84], while some papers highlighted the presence of fatty acids, such as linoleic and linolenic acid [8,81].

In several Asian species, the presence of flavones derived from luteolin and apigenin was also described in aerial plant extracts [80,82]. Further phytochemical investigations have shown that flavones and flavonoid glycosides are the main components of L. leontopodioides whole plant extracts [22,28]. Among these, in addition to the previously identified compounds, a series of methoxylated O-glycosylated and/or acyl flavone derivatives from kaempferol [22,28] or luteolin [85], coniferin and its derivatives, as well as tetrahydroxy-3-methoxyflavone [23], dihydroxy flavanone, di/tetrahydroxy flavonol or dihydroxy flavanonol derivatives were also isolated [15]. Additionally, new secondary metabolites were discovered in aerial parts of the species L. leontopodioides: two new compounds, a neolignan and a benzofuran [15]; two new ent-kaurene diterpenoids, leontocin A and leontocin B; a new lignan, leontolignan [78]; nine isobenzofuranones, including six new ones, together with three known isobenzofuranones [79]. Other researchers isolated isotrichocarpin, leontopodioside D and E, along with nebrodenside A, pungenin, betulalbuside A and two glucopyranoside derivates in extracts from the L. leontopodioides whole plants [86] and five new isobenzofuranones named leontoaerialosides A, B, C, D and E [87].

The first discovered coumarins named leontonanin, together with six known compounds, umbelliferone, 3, 4′, 7-trihydroxyflavone, oleanolic acid, β-sitosterol, daucosterol and stearic acid, were described in Leontopodium nanum Hand.-Mazz [88]. In another Asian species, Leontopodium longifolium Ling., an obliquin derivative, a coumarin, and three new bisabolenes were identified in a root extract [38]. Further, a norsesquiterpene called longifolactone and other three ent-kaurenoic acid derivates were isolated from roots [35].

The major components of essential oil from aerial parts of L. leontopodioides were palmitic acid, n-pentadecanal, linalool, β-ionone, hexahydrofarnesyl acetone, bisabolone and β-caryophyllene [36], while the same type of essential oil extracted from L. longifolium had, as predominant compounds, α-bisabolol and α-bisabolol oxide [31].

The main classes of metabolites identified in Leontopodium sp. were terpenes and phenylpropanoid compounds (Supplementary Material S2). Among the terpenes, tricyclic sesquiterpenes and bisabolene sesquiterpenes, but also diterpenes, monoterpenes or sterols, were isolated, mostly present at the root level, with the exception of two ent-kaurenoic derivatives reported in the extracts from the aerial parts [78]. Among the identified phenylpropanoid compounds, the phenolic acids were found mainly in the aerial parts or in extracts from in vitro cultures, flavonoids in extracts from aerial parts, inflorescences or whole extracts; coumarins were reported exclusively in the roots, while lignans and benzofurans in both the aerial and underground parts. In L. alpinum, these two types of metabolites were extracted exclusively from the root level [4,11,33], while in L. leontopodioides, they were reported in the aerial parts [13,78,79].

Other studies which investigated the metabolic fingerprint in Leontopodium sp. and phylogenetic relationships between the species of this genus showed a species-specific occurrence of some metabolites. The European L. alpinum and eight Asian Leontopodium species showed significant differences in the bisabolenes and coumarins patterns in the root extracts. For example, Leontopodium franchetti Beauv. and Leontopodium sinense Hemsl. contained none of the known bisabolene derivatives [81], while the known lignan derivative occurred in all investigated species. Root extracts of L. alpinum, Leontopodium campestre (Ledeb.) Hand.-Mazz. and L. leontopodioides, three closely related species, showed a similar secondary metabolites pattern. The analysis of the aerial parts revealed no significant differences between the investigated species, leontopodic acids being present in all investigated samples [82]. Another study showed that among 11 different Leontopodium species, two species groups were distinguished, the first group having as discriminators fatty acids and sucrose and the second group differing from the first based on the content of ent-kaurenoic acids and their derivatives [33]. The authors identified two bisabolene derivatives responsible for the discrimination of different Asian Leontopodium taxa and one ent-kaurenoic acid derivative, which could divide the two taxa from the second group L. franchetti and L. sinense. Additionally, two morphologically related species, Leontopodium dedekensii Beauv. and L. sinense, could be distinguished based on ¹H NMR and LC–MS metabolic fingerprinting [33].

The peculiarities of the species, especially that concerning the content of secondary metabolites, which belong to diverse classes of compounds, explain the multitude of the extracts’ bioactivities. Sesquiterpenes could be involved in the anti-inflammatory activity through bisabolene and tricyclic sesquiterpene derivatives, having anti-oedematous effects [3] or by inhibiting the synthesis of leukotrienes [4]. Ester, tricyclic and bisabolene sesquiterpenes could have antimicrobial activity; however, the most potent activity was manifested by linoleic and linolenic fatty acids [8]. The anti-neurodegenerative activity is due to another sesquiterpene, isocomene, which inhibits acetylcholinesterase activity [9]. Diterpenes, ent-kaurenoic derivatives, have anti-inflammatory activity by decreasing NO levels [4] and inhibiting cyclooxygenases [35]. Phenylpropanoid compounds, caffeoyl derivatives of quinic and glucaric acids and flavonoids mainly showed anti-inflammatory and antioxidant activity [78], while flavonoids inhibited α-glucosidase activity [85] but would also be involved in antiparasitic activity [32]. Coumarins have anti-inflammatory [3] and antimicrobial activity [8], as well as sesquiterpenes. The most studied compounds, in terms of manifested bioactivities, are lignans, especially leoligin and its two derivatives, with proven activities on the cardiovascular system [10]. In addition, lignans and benzofurans have antidiabetic activity [87] and anti-inflammatory activity by decreasing nitric oxygen levels [13].

5. Bioactivity of Secondary Metabolites in the Leontopodium Taxa

Over time, methods have been described for extracting compounds with medicinal value from different parts of the plant [4,10,54]. Edelweiss extracts have shown antibacterial activity against some human pathogens, suggesting their potential use in folk medicine as respiratory and abdominal ailments [8]. Early studies have demonstrated the anti-inflammatory efficacy of Edelweiss extracts in mice and rats [7], in human keratinocytes and endothelial cells [12]. In addition, Edelweiss root extracts contain constituents that enhance cholinergic neurotransmission, indicating its potential use as anti-dementia agents [9], and possess angiogenic and anti-inflammatory properties that inhibit intimal hyperplasia of vein bypass grafts [10]. Some compounds are highly bioactive, with anti-inflammatory [4], angiogenic [89], antioxidant and DNA protective [5], chemoprotective [6,7] and antimicrobial effects [8].

Moreover, the extracts of L. leontopodioides, which contained phenolic acids, coumarins, flavonoids and steroids [11,13,85], have been reported to possess significant anti-inflammatory, antibacterial, anti-diabetics and hepatoprotective activities [90,91,92].

5.1. Anti-Inflammatory Activity

Inflammation is a protective response of a given organism’s immune system against harmful external agents [93] and includes the activation of macrophages by pro-inflammatory mediators, which triggered the production of various interleukins (ILs) [94,95].

Different illnesses, such as autoimmune disease, asthma, Alzheimer’s disease, Parkinson’s disease, inflammatory bowel disease, arthritis, and other more common disorders, such as cancer, cardiovascular disease, diabetes or hepatitis, are also associated with prolonged inflammation [96] and the overproduction of pro-inflammatory mediators [94,95]. The excessive aggregation of immune system cells and the production of immune mediators can determine tissue destruction; therefore, regulation of the immune response is necessary.

In general, the inflammatory reaction is an immune response to protect the body from external stimuli, and the treatments address the control of the inflammation by suppressing this inflammatory response [93].

The literature reported some targets of Leontopodium extracts or isolated secondary metabolites, which counteract inflammation, as pro-inflammatory mediators: lipopolysaccharide (LPS), interleukin-1b (IL-1), interferon-ϒ (IFN-ϒ) and the nuclear factor kappa B (NF-κB), tumour necrosis factor (TNF-α), prostaglandins (PGs), thromboxanes, leukotrienes (LTB), cyclooxygenase (COX), lipoxygenase (LOX), nitric oxide (NO) and reactive oxygen species (ROS) (

Table 1. Anti-inflammatory activities of different types of extracts from the Leontopodium taxa.

Taxon	Extract/Bioactive Compound[s]	Experimental Model	Effects/Applied Tests	Ref.
L. leontopodioides	Extract	Rat paw oedema induced by reverse passive Arthus reaction	- suppressed the swelling of hind paws, - inhibited cutaneous haemorrhage of animals, - the disruption of lysosome membranes, - the migration of leukocytes.	[97]
	Aerial parts extract, phenolic acids (caffeoyl derivatives of quinic and glucaric acid)	Ex vivo inhibition assay	- inhibited COX 1 and COX 2	[78]
	Caffeoyl derivatives of quinic and glucaric acid and flavonoids	LPS-activated Raw 264.7	- inhibitory activity on chemokine CCR2b receptor	[97]
	Chlorogenic acid, ferulic acid	LPS-activated Raw 264.7	- inhibited IL-1, IL-6 and LTB4	[98]
	Neolignan and benzofuran derivatives	LPS-activated Raw 264.7	- decreased intracellular NO level	[13]
	Methanol extract from whole plant/stem	LPS-activated HK-2 line LPS-induced sepsis in male mice	- reduced the IL-6 and TNF-α expression, - decreased IL-6, TNF-α, and IL-1β, - diminished oxidative stress and apoptosis, - affected inflammatory factors and proteins expression.	[25]
L. alpinum	Lipophilic extracts of the aerial plant parts (dichloromethane and CO2-extract)	Rat paw oedema assay	- reduced the oedema by swelling reduction by 72% (CO2-extract) and 80% (dichloromethane extract)	[7]
	Aerial parts extract, fatty acids, root extract, bisabolene, tricyclic sesquiterpenes and coumarins	Animal model- mice with ear dermatitis induced by Croton oil	- anti-oedema activity by reduction of polymorphonuclear neutrophil leukocytes and the in vitro chemotaxis of inflammatory cells	[3]
	Bisabolene derivative, lignan, ent-kaurenoate acid	Ex vivo inhibition assay	- inhibited leukotriene synthesis	[4]
	Callus culture extract	UV irradiated HaCaT and PH	- inhibited iNOS expression, - decreased COX-2 expression.	[12,34]
	Exosomes derived from callus cultures	LPS-activated Raw 264.7	- decreased IL6 production	[99]
	Ethanolic extract of callus culture and/or leontopodic acid	PHK treated with TNF-α and IFN-ϒ, HUVECs exposed to oxLDL or LPS	- inhibited GM-CSF, IL-8, MCP-1, IP-10, - inhibited IL-6 and VCAM-1 expression.	[12]
	Alpaflor® Edelweiss commercial product	RHE irradiated with UVA + UVB	- inhibitory effect on pro-inflammatory markers from LOX 5 cascade, psoriasin chemokine [CXC motif] ligand 5	[39]
L. longifolium	Two ent-kaurenoic acid derivates	LPS-activated Raw 264.7	- decreased intracellular NO level	[35]
L. coreanum	Cells culture extract	LPS-activated Raw 264.7	- decreased intracellular NO level	[100]

). The anti-inflammatory activity was highlighted in experimental systems consisting of human keratinocyte cultures (HaCaT and PHK), mouse macrophage cultures (Raw 264.7), human vascular endothelial cells (HUVECs), human kidney cells lines (HK) or reconstructed human epidermis (RHE) subjected to stress with pro-inflammatory stimuli, the most used stimulus being UV radiation [13,34], but also bacterial lipopolysaccharides (LPS), human oxidized low-density lipoprotein (oxLDL) or pro-inflammatory mediators [12].

One of the first studies showed that the L. leontopodioides extract suppressed the swelling of hind paws in normal or adrenalectomized rats, induced by the reverse passive Arthus reaction [97]. This extract strongly inhibited the cutaneous haemorrhage of animals, the disruption of lysosome membranes and the migration of leukocytes. Subsequent studies using a similar animal model system showed that the different types of extracts (dichloromethane, methanolic and CO₂) from the aerial parts and roots of L. alpinum can reduce the inflammatory response [7]. The best results, in the rat’s paw oedema assay, were obtained in the case of lipophilic extracts of the aerial plant parts, exhibiting a swelling reduction of 72% for the CO₂ extract and 80% for the dichloromethane extract. Other authors analysed the anti-inflammatory activity of the aerial parts and roots of L. alpinum through the in vivo topical application on ear dermatitis in mice, induced by Croton oil [3]. The dichloromethane extract had a dose-dependent effect on reducing the oedema, being more active than other extracts, and the aerial parts proved to be more active than roots. Two bisabolene derivatives caused a reduction in the polymorphonuclear neutrophil leukocytes’ accumulation in the inflamed tissue, while a silphiperfolene-type sesquiterpene and a coumarin derivative inhibited the in vitro chemotaxis of these inflammatory cells [3].

Another study reported that some constituents of this L. alpinum roots’ extract were potent inhibitors of cyclooxygenase isoenzymes COX-1 and COX-2 [4]. The authors showed that the bisabolene derivates, one lignan and ent-kaurenoate acid, exhibited the highest inhibitory activity on the ex vivo LTB synthesis. In the same manner, other authors linked the anti-inflammatory activities in extracts from the aerial organs of L. leontopodioides to phenolic acids with caffeoyl moieties, such as 2,3,4,5-tetracaffeoyl-D-glucaric acid, 3,4-O-dicaffeoylquinic acid and 3, 5-O-dicaffeoylquinic acid [78], which were previously identified in the Leontopodium genus [82,83].

Some compounds identified in L. leontopodioides were tested as antagonists for C-C chemokine receptor 2b (CCR2b). This receptor is part of the G protein-coupled receptor family, the largest class of cell surface receptors, representing the most important targets for marketed anti-inflammatory drugs [97]. From all tested compounds, tetracaffeoyl-D-glucaric acid, 3,4 di-caffeoylquinic acids and apigenin 3-O-β-glucoside showed antagonism to the CCR2b receptor, suggesting that L. leontopodioides could be used for the treatment of CCR2b-related diseases [101].

In the case of using UV irradiation as a pro-inflammatory trigger, intracellular NO levels were increased without activating the expression of inducible NO synthase (iNOS) but with the transient activation of COX-2 expression [12]. Other authors signalled an increased expression of the two pro-inflammatory marker genes, COX-2 and iNOS [34]. Treatment with L. alpinum callus extracts determined the decrease in COX-2 and iNOS mRNA transcription, with results comparable to dexamethasone. It also had no inhibitory effects on the transcription of other markers specific to the UV-induced inflammatory response, such as TNF-α, IL-6 and IL-1α [12]. Transcriptomic analysis of RHE irradiated with UV showed that the Alpaflor^® Edelweiss product was able to rebalance the expression of some pro-inflammatory markers corresponding to the LOX 5 cascade, psoriasin (S100 calcium binding protein A7) and chemokine (CXC motif) ligand 5, which is the key mediator in sunburn signalling [39].

Bacterial LPSs are substances that cause inflammatory reactions and were applied in studies using the Raw 264.7 line to study their involvement in pro-inflammatory cascades. A newly discovered neolignan and luteolin isolated from the aerial parts of L. leontopodioides exhibited inhibitory activities on NO production in this experimental model, being more potent inhibitors than N-monomethyl-L-arginine used as a positive control [13]. In the same model system, two ent-kaurenoic acid derivatives isolated form L. longifolium roots inhibited the NO production at 63% and 61%, respectively, after treatment with a 5 mg/mL concentration [35]. Extracts of Leontopodium coreanum Nakai appeared to have inhibitory effects on NO production [100], while those of L. alpinum reduced cytokine production (IL-6) with better efficiency than dexamethasone [99]. The chromatographic fingerprints of different L. leontopodioides extracts were used in acute inflammatory studies. The HPLC data showed that six compounds were associated with anti-inflammatory activities on IL-1, nine compounds on IL-6 and two compounds on LTB4 [98], with only chlorogenic acid and ferulic acid inhibiting the production of IL-1 and IL-6.

The effect of the L. leontopodioides methanolic extract was studied using HK-2 cells, a proximal tubular cell line derived from the human kidney, and the results showed a significant decrease in the IL-6 and TNF-α expression in HK-2 cells and in a culture medium [25]. In the animals’ model with acute kidney injury, sepsis induced uncontrolled systemic inflammatory response syndrome, but the extract remarkably improved oxidative stress and inhibited apoptosis in the kidney tissue by reducing the protein expression related to the NF-κB signalling pathway [25].

In chronic inflammatory skin diseases, such as atopic dermatitis or psoriasis, epidermal keratinocytes respond to pro-inflammatory cytokines released by leukocytes, such as TNF-α and IFN-ϒ, through a sustained expression of numerous inflammatory mediators, which maintain the cycles of chronic inflammation [102]. This chronic inflammation was induced in primary human keratinocyte cultures by administering combinations of such pro-inflammatory molecules [12] and treatments with extracts derived from callus cultures or leontopodic acid, combined with the administration of TNF-α and IFN-ϒ cocktails, showing inhibitory effects on the growth factor GM-CSF and cytokine IL-8. When using only TNF-α as a stimulus, the chemokines IL-8, MCP-1, IP-10 and the transcription factor GM-CSF were inhibited in a dose-dependent manner, similar to the corticosteroid triamcinolone. This study assumed that other major compounds from the callus extracts, such as chlorogenic and 3,5-dicaffeoylquinic acids, can exhibit anti-inflammatory activity by inhibiting the overexpression of some chemokines, as previously reported in other works on these compounds [103].

A special type of inflammatory process is atherogenic inflammation. Endothelial cells exposed to pro-inflammatory stimuli can accumulate inflammatory mediators (chemokines, early pro-inflammatory cytokines, intercellular adhesion molecules), resulting in damage to the vascular tissue, followed by an accelerated proliferation of vascular cells [104]. To stop this destructive cascade, inhibiting the production of pro-inflammatory mediators at the level of endothelial cells would represent the first line of attack in the treatment of atherosclerosis [105]. In HUVEC cultures treated with LPS or oxLDL, a trigger for atherogenic inflammation, the treatments with L. alpinum callus extract remarkably reduced the expression of vascular cell adhesion molecules (VCAM-1), which are characteristic in atherogenic inflammation [12]. VCAMs play a key role in initiating inflammatory responses in the vascular system. The extract also displayed inhibitory activity on LPS-induced IL-6 transcription. These results could provide a basis for the development of new vascular anti-inflammatory drugs derived from Edelweiss callus culture extract.

5.2. Angiogenic and Cardioprotective Bioactivity

The recovery after myocardial infarction (MI) is affected by insufficient angiogenesis and arteriogenesis. The myocardium has only limited regenerative abilities, and, in MI, the lost myocardial mass is replaced with fibrous tissue. As a compensatory mechanism for the loss of muscular mass through cardiomyocyte necrosis and apoptosis, the remaining myocardium increases its mass through cardiomyocyte hypertrophy and tissue remodelling processes (e.g., left ventricular dilatation). In allopathic therapy, complications associated with coronary arterial bypass grafts (neointima formation, hyperplasia and atherosclerosis) are treated with chemotherapeutics or immunosuppressive agents but with partial efficiency and adverse effects [106]. As an alternative treatment, natural compounds able to stimulate angiogenesis can be used. One of the promising compounds used for an improvement in ventricular function and the stimulation of angiogenesis and arteriogenesis was leoligin, a furan-type lignan identified in L. alpinum root extract [10,11].

In one of the first studies on this subject, the inhibitory effect of leoligin on the endothelial intimal hyperplasia after arterial bypass grafting was studied using mice or in vitro human saphenous vein organ cultures as model systems [10] (

Table 2. Angiogenic and cardioprotective activity of leoligin and 5-metoxileoligin isolated in L. alpinum roots.

Bioactivity	Taxon/Etract/ Bioactive Compound(s)	Experimental Model	Effects/Applied Tests	Ref.
Angiogenic	Roots extract Leoligin or 5-metoxileoligin	mice HUVEC lines in vivo rat model with MI chicken chorioallantoic membrane assay	- inhibited proliferation of vascular smooth muscle cells, - inhibited intimal hyperplasia.	[10]
				[89]
		Rabbit and human plasma	- angiogenic activity - inhibited CETP	[108]
Cardioprotective activity		Hyperlipidaemic ApoE-/-mice	- inhibited 3-hydoxi-methyl-glutaryl-CoA-reductase, - decreased cholesterol level.	[107]
		THP-1-derived macrophages cell culture	- promoted activity cholesterol efflux, - upregulated ABCA1 and ABCG1.	[109]

). Leoligin presented anti-hyperplasic activity, inhibiting the proliferation of vascular smooth cells through the induction of cell cycle arrest in the G1 phase, but did not induce cell death in smooth muscle and endothelial cells [107].

A leoligin derivative, 5-methoxyleoligin, was able to induce angiogenesis by upregulating CYP26B1, which encodes a cytochrome P450 protein, in HUVECs and stimulated arteriogenesis in an in vivo rat model with myocardial infarction [89]. The same authors demonstrated that the compound 5-methoxyleoligin stimulated capillary tube formation, in vitro angiogenic sprouting and angiogenesis in a chicken chorioallantoic membrane assay and induced a higher regeneration rate of arterioles in the peri-infarction and infarction areas, reducing myocardial muscle loss and promoting a significant increase in the left ventricular function, in a study using an animal model [89].

Another activity of leoligin on the cardiovascular system showed that higher doses than 1 mM inhibited the cholesteryl ester transfer protein (CETP) [10]; therefore, this compound could be used against coronary artery disease.

A series of subsequent studies on lipoprotein metabolism proved that leoligin could be a promising candidate for counteracting atherosclerosis. Leoligin affected the CETP level in rabbit and human plasma [108], and through the inhibition of 3-hydroxy-methyl-glutaryl-CoA reductase, cholesterol levels can decrease in hyperlipidaemic (ApoE^-/-) mice [105]. Another study showed that leoligin induced cholesterol efflux in THP-1-derived macrophages (a human leukaemia monocytic cell line) by upregulating ABCA1 and ABCG1 expression [109]. These genes encode for receptor-dependent cholesterol efflux through ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), which are the predominant transporter-mediated cholesterol in human macrophages. Also, proteome analyses revealed the modulation of protein expression fingerprints in the presence of leoligin.

Due to the spectacular pharmacological profile of this lignan and its methoxy derivative, recent studies aimed to create synthetic derivatives with improved bioactive properties, such as NF-kB inhibitory agents [110,111], selective inhibitors of the proliferation of vascular smooth muscle cells [110,112], antagonists for the Farnesoid X receptor to block cholesterol uptake in intestinal cells [113] or synthetic ligands with the potential to promote cholesterol efflux from macrophages [114]. Leoligin, known to inhibit intimal hyperplasia and the regrowth of endothelial cells, was exploited along with several structural analogues in stent drug-releasing experiments [115], as a continuation of its pharmaceutical potential in cardiovascular diseases.

5.3. Antimicrobial Activity

The usefulness of Leontopodium sp. extracts for antimicrobial therapy has shown promising results since ancient times. Some of the antibacterial, antifungal or antiparasitic activities exhibited by representatives of the Leontopodium genus are presented in

Table 3. Antimicrobial activities of extracts and isolated compounds from different Leontopodium taxa.

Bioactivity	Taxon/Extract/Bioactive Compound(s)	Experimental Model	Effects/Applied Tests	Ref.
Antibacterial activity	L. alpinum - extract: linoleic and linolenic acids	Multiresistant strain Staphylococcus aureus DSM 1366	- growth inhibition/MIC assay	[8]
	- crude dichloromethane extracts from aerial parts and roots: ester sesquiterpene and coumarin	B. subtilis., E. coli, P. aeruginosa, S. aureus, and S. pyogenes strains.
	- 15 compounds isolated	E. faecalis, E. faecium, and S. aureus ATCC 25923 strains
	- bisabolene sesquiterpenes, tricyclic sesquiterpenes	E. faecium, S. aureus, S. pneumoniae and S. pyogenes strains
	L. leontopodioides - ether and ethylacetate fractions - n-butanol and water fractions	S. aureus E. coli, Salmonella sp.	- disc agar diffusion assay	[92]
	L. longifolium - essential oil from aerial parts - α-bisabolol	S. aureus strain	- disc agar diffusion assay	[31]
Antifungal activity	L. alpinum - ethanolic extracts from different plant parts	Candida glabrata	- low inhibition on biofilm formation	[29]
	L. leontopodioides - crude extract from whole plant	Spore suspension of Trichophyton mentagrophytes	- low anti-dermatophyte activity	[116]
Antiparasitic activity	L. campestre - root extracts-flavonoids, flavones and lignans	Human fibroblasts cells (HFF) infected with tachyzoites from Toxoplasma gondii	- anti-Toxoplasma activity - (IC50 40.39 μg/mL)	[32]
		or synchronous cultures with parasite Plasmodium falciparum 3D7 strain	- anti-Plasmodium activity - (IC50 19.705 μg/mL)

. Fifteen antimicrobial compounds isolated from L. alpinum and extracts obtained from roots and aerial parts were tested against bacterial and fungal pathogens [8]. The antimicrobial bioactivity of extracts seemed to depend on the type of solvents (ethanol, methanol, dichloromethane). Thus, the crude dichloromethane extracts exhibited significant growth inhibition on different strains of Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes, while methanol and methanol/water extracts exhibited only weak antimicrobial activity. Some of the isolated compounds from L. alpinum, such as different bisabolene sesquiterpenes and other tricyclic sesquiterpenes, showed significant antibacterial activity against E. faecium, S. aureus, S. pneumoniae and S. pyogenes strains, whereas other sesquiterpene esters and coumarin selectively inhibited the growth of S. pyogenes and S. pneumoniae strains [8].

Linolic and linolenic fatty acids had the most important antimicrobial effect, with an MIC of up to 4 mg/mL, showing activity even against the multiresistant S. aureus strain DSM 13661 [8].

Another study using different types of L. leontopodioides extracts indicated that the petroleum ether-soluble and ethyl acetate-soluble fractions of the alcoholic extract strongly inhibited S. aureus, while the n-butanol and water-soluble fractions had potent antibacterial activity against E. coli and Salmonella sp. [92].

A study reported the selective antibacterial activity of essential oil (sesquiterpeineol) from L. longifolium against some bacterial strains, such as S. aureus ATCC 6538, E. coli ATCC 25922, B. subtilis ATCC 6633 and P. aeruginosa ATCC 27853, from a total of eight tested microorganisms [31]. The S. aureus strain was the most sensitive to the essential oil, while E. coli was the least inhibited.

The extracts and isolated compounds obtained from L. alpinum were tested on Candida albicans and C. parapsilopsis, Aspergillus flavus and A. fumigatus; however, no antifungal activity was identified [8]. Moreover, the essential oil obtained from L. longifolium did not inhibit fungal species, such as Candida albicans, Aspergillus favus, Mucor mucedo or Phytophthora parasitica [31]. Weak antifungal activity on Candida and Pichia sp. and a low inhibition of Candida glabrata biofilm formation were detected using essential oil from L. nivale [29]. Likewise, the crude extract from the whole plant of L. leontopodioides showed low antifungal activity against dermatophytes, such as Trichophyton mentagrophytes [116].

The crude extracts from L. campestre were found to be effective against parasites, such as Plasmodium falciparum and Toxoplasma gondii, which cause malaria and toxoplasmosis, important public health diseases affecting millions of people and animals. Roots’ phenolic compounds, such as flavonoids, flavones and lignans, from L. campestre have proven their antimalarial activities [32]. Some of these compounds are expected to be therapeutic agents against malaria parasites, having low cytotoxicity for humans and pharmacological application [117,118].

5.4. Antioxidant Activity

The antioxidant activity can be described as the property of a given compound to inhibit or decrease the oxidation of its substrate in low concentrations [119]. An antioxidant acts on free radicals that negatively affect biological systems by neutralizing them through electron donation or by breaking down processes [120].

In this respect, different ex vitro tests, such as the Briggs–Rauscher oscillating reaction or antioxidant capacity assay using different synthetical substrates, highlighted the antioxidant activity of L. alpinum extracts or the isolated compounds. Among them, antioxidant activities were estimated using the Trolox equivalent antioxidant capacity (TEAC) method [5,7] or by using the DPPH synthetic radical [35,39]. Studies have also confirmed the antioxidant efficacy of leontopodic acid and chlorogenic acid, major compounds of Leontopodium cell culture extracts. These compounds seemed to protect cellular DNA against oxidative damage induced in vivo, in a 3D assay, a method based on the measurement of the degree up to which a particular compound can protect DNA from free radicals [5,116]. In essential oils from the aerial part of two other species, L. leontopodiodides and L. longifolium, only weak DPPH scavenging activity was detected [34,37].

The ability of callus culture extracts and the main compounds, such as leontopodic and chlorogenic acids, to reduce the levels of reactive oxygen species (ROS) has also been demonstrated in in vitro model systems (

Table 4. Antioxidant activity of different types of L. alpinum extracts shown by in vitro assay tests.

Taxon/Extract/Bioactive Compound(s)	Experimental Model	Effects/Applied Tests	Ref.
Callus culture extract	HFF line induced with blue light	- anti-ROS production	[14]
	HaCaT line treated with H2O2	- decreased intracellular ROS	[15]
Extracellular vesicles derived from callus elicitated with different LED light	HaCaT line induced with UVB		[34]
Extracts from flowering parts - leontopodic acid	LLC-PK1 line treated with ochratoxin A, SH-SY5Y line exposed to β-amyloid aggregates.	- decreased induced ROS formation	[121]
	U937 line treated with deoxynivalenol	- increased the activity of glutathione peroxidase	[6]

), mostly represented by cultures of human fibroblasts and keratinocytes but also the other cell cultures as histiocytic lymphoma line (U937), pig kidney cells line (LLC-PK1) or human neuroblastoma cells line (SH-SY5Y).

The antioxidant activity of callus culture extracts was also demonstrated using the cytotoxicity test of hydrogen peroxide applied in the HaCaT line [34]. The viability of cells treated with 1% extract was comparable to that of N-acetyl cysteine used as a control antioxidant. The decrease in intracellular ROS was promoted, also, by extracellular vesicles derived from callus cultures treated with LED light, using the same cell line and UVB irradiation [18].

Regarding the antioxidant activity of leontopodic acid, two studies demonstrated its cytoprotective activity against mycotoxins, and only one highlighted its antioxidant activity. The first showed that leontopodic acid increased the activity of glutathione peroxidase in U937 cells treated with deoxynivalenol [6]. In the second study, leontopodic acid reduced ROS formation in LLC-PK1 cells exposed to ochratoxin A and in the SH-SY5Y line exposed to amyloid β aggregates [121].

In recent years, the antioxidant activity of the extracts and compounds’ extracts at the biological level has recently been demonstrated, also, against blue light irradiation in the context of overexposure to electronic screens with LED illumination or fluorescent lamps. Although exposure to the radiation emitted by blue light with wavelengths between 400 and 450 nm, visible radiation with increased energy, is not as dangerous as solar radiation, it can penetrate the skin much deeper than UVA and UVB radiation [122]. Two new studies showed an increased ROS production in the HFF line irradiated with blue light and a positive effect of callus cultures extracts or extracellular vesicles derived from L. alpinum [14,15].

5.5. Anti-Neurodegenerative Activity

Aging is commonly associated with an increased incidence of cognitive impairment, the most common being Alzheimer’s disease, Parkinson’s disease and others [123]. Although few studies exist on this subject, several Leontopodium species have been tested, which led to the discovery of secondary metabolites that could easily pass the blood–brain barrier and either increase acetylcholine (ACh) levels or inhibit the expression of acetylcholinesterase (AChE) in the brains of affected individuals (

Table 5. Other bioactivities exhibited by different types of Leontopodium taxa extracts and/or bioactive compounds.

Taxon/Extract/Bioactive Compound(s)	Experimental Model	Effects/Applied Tests	Ref.
Anti-neurodegenerative activity
L. alpinum - roots extract - isocomene	Sprague-Dawley rats	- enhanced the cholinergic transmission activity by increasing ACh level, - positive effect on memorizing and learning.	[9]
L. dedekensii L. subulatum L. franchetti - methanolic extracts - sesquiterpene	Ex vitro assay of AChE activity	- inhibition of AChE	[124]
Anti-tumoral activity
L. longifolium - roots extract - bisabolene sesquiterpenes	SMMC-7721 and HL-60 lines	- a moderate cytotoxic effect on the HL-60 cell line (MTT test)	[38]
- longifolactone	HepG2 and HeLa lines	- weak effect	[36]
L. leontopodioides - aerial parts extract - essential oils	HepG2 and MCF-7 lines	- cytotoxic effect on HepG2 and MCF-7 cell lines	[35]
L. alpinum - extracts from callus culture	Tumor cell lines of breast (MCF-7, MDA-MB-231, MDA-MB-468, HS578T), prostate (22RV1 and LNCAP), colon (DLD-1, HCT-116) and lung (H1792, SK-MES-1) cancers	- cytotoxic effects on 7 cancer cell lines, - significantly reduced viability of SK-MES-1, MDA-MB-23, H1792 and HS578T cell lines, - cytotoxic effect (100 µg/mL extract) for SK-MES-1, MDA-MB-231 and HS578T lines, - inhibited the development of new colonies in all tumour lines.	[37]
Antidiabetic activity
L. leontopodioides - whole plants decoction	Alloxan or adrenaline-induced diabetic Kunming mice	- hypoglycaemic effect	[91]
	HepG2 line	- reduced the levels of blood glucose, cholesterol and triglycerides and increased the insulin level	[125]
- ethanolic extract	Hyperglycaemic mice	- reduced the levels of blood glucose	[126]
- luteolin derivative and lignan glucoside	Ex vitro assay of α-glucosidase activity	- potent inhibitors	[85]
- aqueous and hydroalcoholic extracts, - leontoaerialosides D and E.	Rats with type 2 diabetes	- insulin-like effect	[87]
Hepatoprotective/Chemoprotective activity
L. leontopodioides - aqueous extract	D-GalN-induced liver injury in mice HL-7702 line	- positive effects on AST, ALT levels and histopathological alterations	[127]
L. alpinum - callus culture extract - leontopodic acid	PHK with sirtuin-induced senescence and restored by trichostatin A (inhibitor of sirtuin) treatment	- inhibited cell cycle, - proliferation and apoptosis, - inhibited expression of p53 - and caspase 3.	[12]

).

The crude dichloromethane extract of L. alpinum roots inhibited ex vitro AChE activity and promoted an increase in the ACh level in an animal model [9]. A concentration of 1 mg/mL inhibited 78.79 ± 2.59% of the enzyme’s activity, while the application of an extract injection in the nucleus accumbens of rats caused a high increase in extracellular ACh by 2.5–3-times over the basal level. Further, this root extract was fractioned, and the effects of the obtained subfractions were determined on the brain ACh level and on the ability to memorize and learn in rats. The most potent compound proved to be isocomene, which caused an increase in extracellular ACh at a low concentration (2 µmol/dose) and had a generally positive effect on learning and memory in normal and impaired rats.

The anti-neurodegenerative potential of dried roots and aerial parts of several Leontopodium taxa has also been tested. Greater AChE inhibition was exhibited by the methanolic extracts, in comparison to dichloromethane extracts, with L. dedekensii showing the best inhibition (55.5 ± 15.2%), followed by Leontopodium subulatum Beauv. (39.4 ± 10.5%) and L. franchetti (37.4 ± 9.5%). In another in vitro AChE inhibition test, the most potent metabolite was a sesquiterpene, which inhibited 61.8% of the enzyme activity at a 100 μM concentration [124].

5.6. Anti-Tumoral Activity

The cytotoxic activities of three new bisabolene sesquiterpenes from L. longifolium roots extracts were assessed against two tumour cell lines: SMMC-7721 (human hepatoma) and HL-60 (human promyelocytic leukaemia) [38]. The results showed that two of the three bisabolene derivatives exerted moderate cytotoxic effects (IC₅₀ = 81.2 µg/mL and IC₅₀ = 88.6 µg/mL, respectively) but only on the HL-60 cell line.

The effects of the essential oils from aerial parts of L. leontopodioides were investigated on HepG2 (liver hepatocellular) and MCF-7 (human breast adenocarcinoma) cell lines using the MTT assay [36]. The essential oil extract had a cytotoxic effect on HepG2 (IC₅₀ = 67.44 ± 5.08 µg/mL) and MCF-7 (IC₅₀ = 70.49 ± 3.8 µg/mL) cell lines after 72 h exposure.

A novel norsesquiterpene, longifolactone, was isolated from L. longifolium roots, and its cytotoxicity was assessed against two human cancer lines, HepG2 and HeLa [35]. The results of this study showed that this compound exhibited weak cytotoxic effects on the tested human cancer cell lines.

Among the curative effects of L. alpinum plants, ethnobotanical treatises mention using them against breast cancer [65], but the first research which highlighted the antiproliferative activity of a methanolic extract of L. alpinum callus culture on ten tumour cell lines characteristic of breast (MCF-7, MDA-MB-231, MDA-MB-468 and HS578T lines), prostate (22RV1 and LNCAP lines), colon (DLD-1, HCT-116 lines) and lung cancer (H1792, SK-MES-1 lines) was quite recent [37]. The viability evaluation via the MTT assay showed significant effects of the highest used extract concentration (400 µg/mL) on seven cancer cell lines, except for hormonal-dependent cancers, such as the breast (MCF-7) and prostate (22RV1, LNCAP) tumour lines. The significantly reduced viability ranged from less than 50% in one lung (SK-MES-1) and one breast (MDA-MB-231) cancer line to 58.1 ± 13.4% and 66.2 ± 13.8% in another lung (H1792) and breast (HS578T) cancer line, respectively. A cytotoxic effect was registered from the 100 µg/mL extract concentration for SK-MES-1, MDA-MB-231 and HS578T lines. The extract caused considerable effects on the cell population in the two colon cancer lines and one breast tumour line (MDA-MB-231) and significantly affected the nuclear fragmentation in one prostate tumour line (22RV1). The impact of the methanolic extract on cytoskeleton fragmentation was considerable in one lung cancer line (H1792), while significantly deformed cells were observed in one colon (HCT-116) and both lung cancer lines. The metastatic capacity was studied, and the results showed that treatment with the 400 µg/mL dose significantly inhibited the development of new colonies in all tumour lines, but the strongest effect was observed in the case of H1792, HCT116 and MDA-MB-231 lines.

5.7. Anti-Metabolic Disorder Activity

L. leontopodioides, known as common Edelweiss in China, was reported for the first time to have a hypoglycaemic effect in alloxan or adrenaline-induced diabetic Kunming mice [91]. The authors investigated the effects of treatments with two doses of L. leontopodioides decoction on blood glucose concentrations. Their results showed that administration for 10 consecutive days of the 30 g/kg dose generally decreased the concentration of blood glucose in normal mice in glucose-treated mice but also in alloxan-induced diabetic mice and in mice with increased blood glucose levels caused by the exogenous administration of adrenaline. Moreover, the decoction extract prepared from whole plants could significantly reduce the levels of blood glucose, cholesterol and triglycerides and increase the insulin level in serum of rats with type 2 diabetes, using as control Sitagliptin, an anti-diabetic drug [125]. Another study claimed, also, that the ethanolic extract from this species can remarkably reduce the blood glucose level in hyperglycaemic mice [126]. In fact, hypoglycaemic and hypocholesterolemic effects were further demonstrated to be due to some chemical constituents with inhibitory activity on α-glucosidase and lipase. Thus, two acyl flavone derivates from luteolin 4-O-β-D-glucopyranoside and one lignan glucoside were identified as potent inhibitors for α-glucosidase [85], and four compounds (nebrodenside A, pungenin, betulalbuside A and geranyl O-β-D-glucopyranoside) exhibited lipase inhibitory activity, which suggested participation in the reductive effect of this herb on triglyceride absorption [86].

Other studies showed an increased glucose uptake effect of the different extracts and individual compounds isolated from L. leontopodioides whole plants in the cytoplasm of human hepatoma cells (HepG2 line) [87]. The aqueous and hydroalcoholic eluates and certain compounds (leontoaerialoside D and E) had an insulin-like effect with enhanced glucose uptake (>11%) at a concentration of 30 µM.

Chemoprotective activity was described in a study investigating the hepatoprotective effect of an aqueous extract of L. leontopodioides against D-galactosamine (D-GalN)-induced hepatocyte injury using in vitro primary cultures of neonatal mice and HL-7702 hepatocytes [127]. The in vivo protective effect of this extract on D-GalN-induced liver injury was evaluated in mice by examining some parameters characteristic to hepatocyte injury, such as serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and histopathological alterations [127].

5.8. Toxicity

The cytotoxic activity of extracts from in vitro cell cultures such as cell suspensions, calli or adventitious roots was investigated using human fibroblast cultures (CCD-986-Sk and Detroit 551) but also the HaCaT line. Apparently, at concentrations between 0.1 and 1%, the viability of keratinocytes and fibroblasts was insignificantly reduced [34], while concentrations between 0.5 and 5% did not affect the viability of the CCD-986-Sk line [128]. In contrast to these results, other authors found significantly reduced inhibitory activity at a high concentration of 400 µg/mL, i.e., 0.04%, using extracts of L. alpinum callus and human epithelial fibroblast (BJ line) [37]. Cytotoxicity tests using extracts of L. coreanum were also performed on dermal papillae cell cultures, for which no significant changes in cell viability were observed [100].

These results demonstrated that Edelweiss extracts did not exert significant cytotoxicity on the normal cell lines, and transcriptomic studies supported these findings. Thus, the genes known to be induced by different types of stress, such as oxidative stress, stress determined by zinc, copper or cadmium ions, hypoxic stress or genes encoding proteins involved in antiviral immunity, were downregulated, which led the authors to conclude that the L. alpinum callus culture extract was not responsible for the observed stress in human keratinocytes [34].

6. Conclusions and Future Directions

The Leontopodium taxa, through their most widely known representatives, L. alpinum in Europe and L. leontopodiodides and L. longifolium in the Asian flora, are extensively researched for their phytochemistry and bioprospected for useful medicinal activities.

The multitude of bioactivities displayed by the different types of extracts, as well as the compounds derived from them, is closely related to the living environment characteristic of these species. The difficult conditions present in their habitat, generally high altitudes of over 1800 m, exposure to wind, increased UV radiation and rocky substrate, explain the abundance of secondary metabolites isolated from them.

A key part of the bioactivity of the Leontopodium sp. extracts was the significant anti-inflammatory effect, achieved through various mechanisms, such as inhibiting NO production, inducing iNOS expression, suppressing cyclooxygenase activity and leukotriene synthesis, restoring the balance of certain lipoxygenase 5 cascade members, and influencing the expression of cytokines, growth factors, chemokines and cell adhesion factors that play roles in the transduction of pro-inflammatory signals.

The compounds that have been extensively researched for their pharmacological benefits in post-myocardial infarction recovery, exhibiting angiogenic properties, and in atherosclerosis treatment by reducing circulating cholesterol levels include leoligin, along with its derivative 5-methoxy, extracted from the roots of L. alpinum.

The effectiveness of the antimicrobial activity relied on the specific extract type and the metabolites it contained. Also noteworthy was the antimicrobial effects on some resistant strains, especially S. aureus but, also, on certain parasites.

In addition to the mentioned effects, some works also referred to the potential anti-neurodegenerative impact by enhancing acetylcholine levels in the brain, the anti-tumour effects on various cancer cell lines, particularly breast and lung cancer, as well as the hypoglycaemic and hepatoprotective properties.

The diverse range of bioactivities exhibited by L. alpinum, as well as the other representatives with similar chemical compositions from the Gnaphalium genus, represents valuable resources for new drug discovery studies.

Nevertheless, this comprehensive evaluation of knowledge about the bioactivities of Leontopodium taxa extracts or isolated compounds showed gaps in clinical studies, pharmacological studies that assessed effective and safe doses, pharmacokinetics and toxicological studies.