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Sandy Everlasting (Helichrysum arenarium (L.) Moench): Botanical, Chemical and Biological Properties

Sandy everlasting [Helichrysum arenarium (L.) Moench] is herbaceous perennial plant belonging to Asteraceae family and it is native to Europe, Central Asia, and China. It belongs to the section HELICHRYSUM (Asteraceae family, genus Helichrysum) along with H. plicatum DC. Prodr., which very similar phenolic profile and H. italicum (Roth), which is widely used for essential oil extraction. Its flowers have a long tradition in European ethnomedicine as a cholagogue, choleretic, hepatoprotective, and detoxifying herbal drug. The flowers are rich in phenolic compounds including flavonoids, chalcones, phenolic acids, coumarins, and pyrones. Apart from polyphenols, other compounds such as sterols, lignans, and glycosides of aromatic compounds have been also isolated from H. arenarium. The majority of authors confirm that the most important group of compounds responsible for biological activities is flavonoids. Moreover, significant activities of naringenin, one of the main flavonoids of H. arenarium, were reported. On the other hand, there are no clinical data about testing the extracts or preparations based on H. arenarium. Although H. arenarium is well known in phytotherapy for its potential in the treatment of gallbladder disease and are classified as endangered species in a number of European countries, very few data about its cultivation are available in the literature.


INTRODUCTION
Sandy everlasting [Helichrysum arenarium (L.) Moench] has a long tradition in European ethnomedicine as a medicinal plant which is attributed to cholagogue, choleretic, hepatoprotective, and detoxifying activities . It is herbaceous perennial plant belonging to Asteraceae family and it is native to Europe, Central Asia, and China (Anderberg and Anderberg, 2005;Erhardt et al., 2008;Yang et al., 2009). The origin of the genus name is derived from the Greek words helios, meaning sun, and chrysos, meaning gold, what refers to the shiny-golden color of inflorescence (Maznev, 2004). The main biologically active compounds of Helichrysi arenarii inflorescentia (syn. H. a. flos) are flavonoids, with chalcone isosalipurposide, and flavanones salipurposide, prunin and naringenin as dominant constituents, while other compounds present in remarkable amount are phtalides, carotenoids, essential oil and yellow pigments: α-pyron derivates such as arenol and homoarenol (Bryksa-Godzisz et al., 2006;Kurkina et al., 2012). Nevertheless, Smirnova and Pervykh (1998) reported that it is the sum of flavonoids that account for the cholagogue activity of the everlasting flower extracts.
Although Helichrysi arenarii flos has not been included in the European Pharmacopoeia, the State Pharmacopoeia of the USSR, Pharmacopoeia Helvetica and Polish Pharmacopoeia listed it as officinal drug (Ph. Helv. VII, 1987;Ph. USSR, 1990;Polish Ph VI, 2002;Ph. Eur. 7.0., 2011) for its choleretic and cholagogue activity. Furthermore, due to the long usage in traditional medicine as well as proven therapeutic properties, it has been included in the monographs of the World Health Organization, German Commission E, Physician's desk reference for herbal medicines and German pharmaceutical codex (Blumenthal et al., 1998;PDR, 1998;DAC, 2005;WHO, 2015).
Synthetic drugs which are used in the treatment of gallbladder, dyspeptic, and liver disorders are often inadequate and may sometimes lead to serious side-effects. Medicinal plants could be a fundamental source of potentially useful new compounds for the development of effective cure to fight a variety of gastroenterology problems. According to the WHO monographs (WHO, 2015), Helichrysi flos (biological source Helichrysum arenarium (L.) Moench) is used as a cholagogue, in the treatment of dyspeptic diseases, and in traditional medicine as choleretic, diuretic, mild spasmolytic, hepatoprotective agent, and for detoxification.
The main suppliers of sandy everlastings' flower heads on market are former USSR countries, Poland and Turkey (Bisset and Wichtl, 1994;WHO, 2015). There are three forms of the drug in the trade: dry flower heads (Helichrysi arenarii flos), fluid extract (Helichrysi arenarii extractum fluium), usually obtained by extraction with water-ethanol or glycerin and dry extract of flower heads (Extractum florum Helichrysi arenarii siccum), usually obtained from fluid extract (EMA, 2015). The flower heads are mainly used for decoctions, while dry extracts are widely used for the production of galenic preparations in the form of capsules and tablets. These forms on the market are mainly represented by Russian preparations where the most popular is "Flamin tablets" (Kurkina et al., 2012).
H. arenarium is fully protected in Sweden and Serbia, while in Denmark and Estonia the species is listed as "care demanding" (Lilleleht, 1998;Butorac, 1999;Olsson et al., 2005). In the 1970s the species received partial legal protection in Poland, which stopped the overexploitation of its natural resources (Sawilska and Jendrzejczak, 2013). Besides the unsustainable collection, the main risk factor that threatens species subsistence is the transformation of natural habitat to agricultural land (orchards, vineyards) and areas for the cultivation of fast-growing tree species (varieties of poplar, pine, and acacia) (Butorac, 1999). Protection regulations triggered a need to develop a new efficient growing technology for sandy everlasting, which could restore the species for it use in phytotherapy (Sawilska and Jendrzejczak, 2013). After first unsuccessful attempts of growing sandy everlasting in plantations, Sawilska et al. (2009) have explained the reason why those attempts failed and pointed out the influence of mycorrhizal fungi on the inflorescence yield. Small farms in Latvia also cultivate H. arenarium for local consumption, where the production area was estimated as less than 2 ha since marketing problems have affected the production of domestic medicinal plants in general (Olsson et al., 2005). This survey aims to systematize the published knowledge about sandy everlasting so far and to highlight the importance of knowledge of botanical, chemical and pharmacological properties of this herbal drug, together with overview of cultivation approaches.
Sandy everlasting (H. arenarium (L.) Moench) belongs to the section HELICHRYSUM (Asteraceae family, genus Helichrysum) along with H. plicatum DC. Prodr., which has according to Bigović et al. (2011) very similar phenolic profile and H. italicum (Roth), which is widely used for essential oil extraction. Botanical division of European taxa in genus Helichrysum Mill. is listed in Table 1.
The Global Biodiversity Information Facility has reported five H. arenarium subspecies with several synonyms listed in This species is broadly distributed in Europe, western Siberia, and central Asia (Kirpičnikov, 1959). Grows on dry sandy places, from Netherlands, Sweden, and Estonia, southwards to Germany, Bulgaria, and Kazakhstan (EMA, 2015). According to Euro+Med Plantbase reports the occurrence of H. arenarium is present from the Bay of Biscay to Ural mountain and from southern Scandinavia to the northern parts of Balkan peninsula (Greuter, 2006). Distribution of the species is also proposed graphically by Anderberg and Anderberg (2005) in Figure 1. In Serbia, H. arenarium area is limited only to two sandy sites Kladovska and Deliblatska peščara (Sarić, 1989).

TRADITIONAL USES
Helichrysi flos (biological source H. arenarium) is well-known species in traditional medicine. It is used as a choleretic, hepatoprotective and detoxifying agent, diuretic, as a mild antimicrobial and spasmolytic agent (Cosar and Cubukcu, 1990;Czinner et al., 2000Czinner et al., , 2001Bigović et al., 2010Bigović et al., , 2011. The flowers contain antibacterial constituents and bitter substances, which may also promote gastric and pancreatic secretion (Amirdowlat Amasyaci, 1990). It is also indicated for indigestion as well as for loss of appetite (Turova and Sapozhnikova, 1984). The average daily dosage is 3 g of the drug or equivalent preparations (Blumenthal et al., 1998;WHO, 2015). In Russia, the inflorescence of H. arenarium has been applied in the form of infusions for stimulating gastric secretion, treating of gallbladder disorders as well as cystitis, rheumatism, arthritis, and gout (Shikov et al., 2014). Moreover, Flores Helichrysi arenarii is included in USSR Ph. USSR (1990). Recommended administration of decoction is 1:20, 100 mL 2-3 times per day. Furthermore, tablets "Flamin" containing purified flavonoids and studies showed that dose of 50 mg taken 3 times/day in a period of 40 days was safe (Sokolov, 2000).
In Serbian traditional medicine, galenic preparations with Helichrysi flos are prepared to diminish the concentration of bile acids, increase the content of bilirubin in bile and tonus of gallbladder and to promote the secretion of bile. Moreover, a spasmolytic effect on the sphincter of the gallbladder is stated. It is used in the form of tea as infusion or decoction (Tasić et al., 2009). According to European Medicinal Agency (EMA) in Austria Helichrysi flos is used in a form of tea mixtures

CHEMICAL CONSTITUENTS
The flowers of sandy everlasting (H. arenarium) are the rich source of phenolic compounds including flavonoids, chalcones, phenolic acids, phthalides, coumarins, and pyrones. Apart from polyphenols, other compounds such as sterols, lignans, and glycosides of aromatic compounds have been also isolated and identified from H. arenarium. Table 3 summarizes the chemical constituents that have been reported in the literature to date.

Macro-and Micro-elements
Very little attention has been paid to the content of macro-and microelements in Helichrysi flos herbal drugs and extracts. To the best of our knowledge, only one report done by Lemberkovics et al. (2002) partly studied this subject. Reported values of the observed elements in their highest levels were: Al (353 mg/kg), Cr (6 mg/kg), Cu (19 mg/kg), Mn (349 mg/kg), and P (2907 mg/kg) in the cultivated drug sample from Hungary, while the concentration of Ba (19 mg/kg), Ca (7575 mg/kg), Fe (159 mg/kg), and Zn (59 mg/kg) was highest in a commercial sample from Poland. Macro-and microelements are critical components  for a number enzymatic and nonenzymatic processes involved in antioxidant defense of the human body, and a deficiency of any of these essential elements may impair the function of the overall antioxidant system (Zidenberg-Cherr and Keen, 1991).

PHARMACOLOGICAL PROPERTIES
The literature data related to the chemical profile of the H. arenarium inflorescences are very different, but the majority of authors confirms that the most important group of compounds responsible for biological activities are flavonoids, which can occur in aglycone and glycoside forms (Czinner et al., 1999Wichtl, 2001;Lemberkovics et al., 2002;Olsson et al., 2005;Kurkina et al., 2012;WHO, 2015). Among flavonoid components in the inflorescence extracts, chalcone derivative isosalipurposide has been reported as the most abundant compound, and it has been indicated as responsible for the yellow color of the involucral bracts and for the hepatoprotective activity of the drug (Hänsel et al., 1960;Skakun and Stepanov, 1988;Czinner et al., 1999;Bryksa-Godzisz et al., 2006;Kurkina et al., 2012;Jarzycka et al., 2013;WHO, 2015).
According to the WHO (2015), the only use of Helichrysi arenarii flos described in pharmacopeias and well-established documents are the treatment of dyspeptic disorders. On the other hand, choleretic, cholagogue, hepatoprotective and detoxifying activity of the inflorescence of H. arenarium has been recognized for a long time in Europe (Kroeber, 1951;Szadowska, 1962;Wagner, 1993;Shikov et al., 2014). Furthermore, Szadowska (1962) has reported mild choleretic and spasmolytic effect of this drug observed on rats. In this research intravenous administration of three flavonoids from sandy everlasting (kaempferol-3-glycoside, naringenin-5-glycoside and apigenin) has been applied in a dose 4 mg/100 g vs. positive control (Decholin, deoxycholic acid) as well as negative control (isotonic NaCl). Increase of bile secretion was 180, 185, and 160%, in comparison to baseline (100%) after 15 min. Increase (135%) was also obtained with the ether extract of sandy everlasting applied in dose of 5 mg/100 g with a maximum after 30 min. Szadowska (1962) also conducted experiments on antispasmodic activity on the smooth muscle isolated from rabbit and rat intestines and on gall-bladders isolated from guinea pigs and rabbits. Apigenin and the ether extract of H. arenarium that contains mainly apigenin, had the strongest antispasmodic activity on smooth muscles and isolated gall bladders ex vivo. Infusions and decoctions of H. arenarium flowers had weak spasmolytic activities.The drug is therefore mainly used as an adjuvant in the treatment of cholecystitis and cramp-like gallbladder disorders (Wichtl, 2001). In Europe, the therapeutic application includes treatment of various health issues such as cystitis, arthritis, rheumatism, and gout as well as for stimulating gastric secretion and for the treatment of gallbladder disorders (Shass, 1952;Vereschagin et al., 1959;Shikov et al., 2014).
Recently, Mao et al. (2017) reported anti-atherosclerotic activities of flavonoids (i.e., narirutin, naringin, eriodictyol, luteolin, galuteolin, astragalin, and kaempferol) isolated from the flowers of H. arenarium. They supposed that the main mechanism of the activity is through the pathway of antiinflammation, especially by reduction of the expression of C-Reactive Protein (CRP), inhibition of the activities of the c-Jun NH 2 -terminal kinases (JNK2) and p38, and the mitogenactivated protein kinase (MAPK) pathway suppression.
Moreover, significant activities of naringenin, one of the main flavonoids of H. arenarium, were reported. Pafumi et al. (2017) found naringenin as the inhibitor of Two-Pore Channel 2 (TPC2) -mediated signaling, a key therapeutic step in a number of pathological conditions including the progression and metastatic potential of melanoma, Ebola virus infection, and Parkinson's disease. Park et al. (in press) suggest naringenin as a potential therapeutic molecule with anti-cancer effects on choriocarcinoma cells acting by inducing generation of ROS and activation of the MAPK pathways, while Liang et al. (2017) reported that naringenin protect keratinocytes from apoptosis and oxidative stress injury through inhibition of the NOD2mediated NF-κB pathway. Furthermore, taking into account that naringenin and naringenin-5-O-glucoside are among the dominant compounds in H. arenarium, it is worth noted that Agus et al. (2017) reported that naringenin-rich fraction of pigeon pea leaves (Cajanus cajan) extract showed fairly well inhibitory effect toward Salmonella thypi in comparison with chloramphenicol.

CULTIVATION APPROACHES
Production of sandy everlasting for therapeutic use could be carried out by small farms as a niche product and since the sandy soil is needed, sandy terrains could be utilized for the production of this medicinal plant (Olsson et al., 2005). Although the sandy everlasting is very interesting for the pharmaceutical industry, and also classified as endangered species in a number of European countries, very few data about its cultivation are available in the literature. First attempts of growing H. arenarium date back to the mid of the 1970s (Fijalkowski and Seroczynska, 1974;Moroz et al., 1976;Pacholak and Zalecki, 1979;Sawilska et al., 2009), but none of them was completely successful in terms of surviving ratio and growth (Buchwald, 1992). Most of the authors concluded that this plant is inconvenient for cultivation since experimental trials yielded only slightly higher amounts of inflorescence biomass than those from natural stands. Tyszynska-Kownacka (1972) even suspected that this species might not be suitable for growing. Later cultivation trials have only explained the reason why those attempts failed (Sawilska, 2006(Sawilska, , 2007(Sawilska, , 2008. The main flaws of the attempts at growing sandy everlasting in the previous century were neglecting the clonal character of its growth and ignoring its mycorrhizal associations. Sawilska et al. (2009) took step forward in the further explanation on the necessity of mycorrhizal associations of arbuscular fungus Glomus intraradices with plant roots, but, although feasible, soil vaccination with mycorrhizal inoculum did not much influence the growth and flowering of single shoots. Sawilska and Jendrzejczak (2013) suggested in vitro method of propagation, which seems to be more profitable as it allows the acquisition of a potentially unlimited number of shapely plants that flower at the same time. Each method of cultivation proved to be feasible, as each yielded a particular amount of valuable, raw herbal material.
Similarly to the most of perennial species from Asteraceae family, the flowering biology of H. arenarium implies that the plant has to spend at least one year in rosette phenophase before it enters the generative phenophase (Pacholak and Zalecki, 1979;Sawilska et al., 2009). For the more detailed study of the species researchers tend to develop basic cultivation models with the use of agro-technical analysis methods, which could provide a study of the elements for industrial cultivation technology, in particular, to clarify the optimal methodology for seedlings production, time of planting, sowing rate, and maintains of established plantations.
The most comprehensive research that covers direct sowing and seedling production as plantation establishment methods has been published by Esmagambetova and Ahmetzanova (2006). In this work, authors reported that optimal plant density of 5-6 plants per 1 m 2 was achieved by sowing rate of 2.5-3.0 kg/ha, and emphasized that although laboratory germination was 87%, the field of germination at sowing time was low, not exceeding 10.3%. Possible reason for this huge germination rate discrepancy could be in very small dimensions of H. arenarium achenes, where weak seed-soil contact could provoke germination without radicle rooting. Thousand seed weight (TSW) was estimated to about 0.06 g. Furthermore, observed field self-propagation by seeds in following 3 years of cultivation was very low (0.41%). Such a low realization of seed reproduction potential is common for wild-growing plants with small seeds, which have a high ratio of accidental fatality of seeds and seedlings associated with a limited supply of nutrients in the seed (Šohina and Valuckaya, 1984). This disadvantage could be overcome by increasing the sowing rate, which would consequently increase plant density and therewith competition between them, which leads to mutual inhibition of their development (Esmagambetova and Ahmetzanova, 2006). In the same report, authors suggested that mutual inhibition could be avoided after the thinning of plants in rows, which again leads to additional investments in both labor and material costs.

CONCLUSIONS
Helichrysi flos (biological source Helichrysum arenarium (L.) Moench) is a well-known herbal drug in traditional medicine and it is used as a cholagogues, choleretic, diuretic, as mild spasmolytic, as a hepatoprotective agent and for detoxification.
Recent studies pointed out significant biological activities of H. areanarium together with its main compounds such as flavonoids. On the other hand, there are no clinical data about testing the extracts or preparations based on H. arenarium. Although the sandy everlasting is classified as endangered species in a number of European countries and also very interesting for the pharmaceutical industry, none of the reported cultivation approaches proved to be successful. Additional efforts regarding cultivation methods, such as fertilization or inoculation with the range of mycorrhizal fungi, should be made to achieve sustainable agricultural production of this crop.

AUTHOR CONTRIBUTIONS
DP took the lead in writing the manuscript and together with TJ conceived the main subject of this review. DP, TJ, DB, and KŠ wrote the Introduction section. DP summarized botanical description, taxonomy, and distribution of the species. TJ and DB contributed to chemical constituents of the species, while KŠ and SJ summarized reported knowledge about traditional uses and cultivation approaches of the species.