Arctium Species Secondary Metabolites Chemodiversity and Bioactivities

Arctium species are known for a variety of pharmacological effects due to their diverse volatile and non-volatile secondary metabolites. Representatives of Arctium species contain non-volatile compounds including lignans, fatty acids, acetylenic compounds, phytosterols, polysaccharides, caffeoylquinic acid derivatives, flavonoids, terpenes/terpenoids and volatile compounds such as hydrocarbons, aldehydes, methoxypyrazines, carboxylic and fatty acids, monoterpenes and sesquiterpenes. Arctium species also possess bioactive properties such as anti-cancer, anti-diabetic, anti-oxidant, hepatoprotective, gastroprotective, antibacterial, antiviral, antimicrobial, anti-allergic, and anti-inflammatory effects. This review aims to provide a complete overview of the chemistry and biological activities of the secondary metabolites found in therapeutically used Arctium species. Summary of pharmacopeias and monographs contents indicating the relevant phytochemicals and therapeutic effects are also discussed, along with possible safety considerations.


INTRODUCTION Botanical and Ethnobotanical Aspects
The genus Arctium L. (Asteraceae/Compositae, tribe Cardueae, subtribe Carduinae), together with the related genera Cousinia Cass., Hypacanthium Juz. and Schmalhausenia C. Winkl, forms the so-called Arctium-Cousinia group (de Souza et al., 2004). The species of the Arctium genus, also known as 'burdock, ' comprise biennial herbs occurring in waste places, streams and roadsides, less hybridization giving rise to questions about their integrity (European Scientific Cooperative on Phytotherapy, 2003).
Greater burdock (A. lappa) has been traditionally used in both Asian and European medicines as depurative, diuretic, carminative, anti-inflammatory, and anti-tubercular agent (Zhao et al., 2014). For therapeutic purposes, its different parts such as roots, fruits, and leaves are used. The latter have been used to treat ulcers and fester wounds (Jaric et al., 2007). They are also applied externally on the forehead to cure headache and fever, on the scalp to treat bruises and hair loss, mixed with oil and honey and applied on the chest to heal cough. In addition, under infusion, they are taken orally to treat enuresis in children (Pieroni et al., 2011). Fruits of burdock (Arctii fructus) are used to purify the blood (Lans and Turner, 2011) and to treat respiratory and infectious diseases (Bai et al., 2016). In addition, A. lappa roots, together with aerial parts of Rumex acetosella L., leaves of Ulmus rubra Muhl. and rhizomes of Rheum officinale Baill., are used to make 'Essiac, ' a tea used by the Ojibwa tribe of Canada for the treatment of cancer (Leonard et al., 2006). In the veterinary medicine, the root is used to treat mastitis (Lans et al., 2007), whereas the whole plant is applied against endoparasites in poultry (Lans and Turner, 2011). Besides therapeutic uses, A. lappa is also appreciated as an edible plant. For the latter purpose, young leaves, and stalks are eaten raw or cooked (Pieroni et al., 2011).
Lesser burdock (A. minus) leaves are traditionally used externally to treat rheumatic pains, fever, sunstroke, wounds, general infections, skin and body inflammations, alopecia, and bladder diseases (de Souza et al., 2004;Erdemoglu et al., 2009;Neves et al., 2009). They are also disposed above the body of the patient, wetted with vinegar or milk, to stimulate sweating (Sezik et al., 2001). Roots and leaves, under infusion, are also used against snake and scorpion bites and to purify the blood (Mosaddegh et al., 2012). Basal leaves and stems are also eaten raw as a snack or stewed (Tardio et al., 2005). Due to their bitter taste, they are also used to stimulate the appetite and liver functions (Tardio et al., 2005).
Wooly burdock (A. tomentosum) leaves are used as vulnerary, to treat skin rash, ulcers, abscesses, mouth sores and against rheumatic pains, whereas root is applied against alopecia and to wash hairs (Sezik et al., 2004;Saric-Kundalic et al., 2010). Roots are also employed to make a tea used for digestive problems, ulcers, rheumatisms, to purify the blood and increase sweating and as diuretic (Saric-Kundalic et al., 2010).

Medicinal Uses of Arctium Species in Pharmacopeias and Monographs
Burdock species and in particular A. lappa are used in traditional medicine for different purposes. The main traditional use of the roots of A. lappa in Europe comprises treatment of dermatological disorders (Saric-Kundalic et al., 2010;Miglani and Manchanda, 2014) whereas in other Eastern and Asian countries A. lappa fruits and roots are used as an antidiabetic remedy (Tousch et al., 2014;Xu et al., 2014Xu et al., , 2015Ahangarpour et al., 2017). In Traditional Chinese Medicine (TCM), apart from the antidiabetic activity, the roots of A. lappa are considered as a blood detoxifying agent (Qin et al., 2014). In the Japanese pharmacopeia, the fruit is included as a traditional herbal medicine with recent studies revealing the potential of its extracts in oncology (Ikeda et al., 2016). The leaves of A. lappa have also been reported as an anti-inflammatory agent to relieve gastrointestinal disorders in Brazilian traditional medicine (de Almeida et al., 2013).
According to international institutions that work in the validation of traditional herbal medicines, such as the European Medicines Agency (EMA) and the European Scientific Cooperative on Phytotherapy (ESCOP), A. lappa is recommended and approved for different indications. For example, the EMA monograph approves the use of roots of A. lappa, A. minus, and A. tomentosum as an adjuvant in minor urinary tract complaints, in temporary loss of appetite and for seborrheic skin conditions (European Medicines Agency, 2011). All these indications are based upon long-standing use. In 2016, ESCOP released a monograph where the roots of all the former three species are indicated to be internally and externally used for seborrheic skin, eczema, furuncles, acne, psoriasis and internally for minor urinary tract disorders (European Scientific Cooperative on Phytotherapy-The Scientific Foundation for Herbal Medicinal Products, 2016). For oral and internal administration, the herbal drug can be used as an infusion, extract, tincture or decoction but the fresh pulp of the roots or a decoction can also be directly applied to the skin. The later monograph reveals that A. lappa preparations should not be ingested during pregnancy, lactation, or in case of hypersensitivity to the Compositae and in patients with oedema due to impaired heart or kidney function. Although certain preclinical studies can be found in the literature, clinical trials are not available for these indications approved by ESCOP and EMA.

Non-volatile Compounds
Till date, more than two hundred non-volatile compounds have been isolated from Arctium genus. These chemical compounds include lignans, terpenoids, sterols, flavonoids, phenolics, lactones, polyacetylenes, quinic acids, and sugars (polysaccharides). In particular, lignans are the most characteristic components in the Arctium genus. The details of chemical compounds, their occurrence in different plant parts, and the analytical methods used for their quali-quantitative determinations are briefly summarized in Table 1 whereas their description is provided in this section. The chemical structures of some compounds from Arctium species are shown in Figure 2.

Phytosterols
Daucosterol, a natural phytosterol-like compound, was obtained from the seeds of A. lappa (Ahangarpour et al., 2017). The fruits of A. tomentosum are reported to contain 2 steroids, such as daucosterol and β-sitosterol. Using bioactivity-guided fractionation, daucosterol and β-sitosterol were recovered from the ethanolic extract (95%) of A. lappa seeds (Ming et al., 2004). Later, sitosterol-beta-D-glucopyranoside was found in the methanolic extracts of A. lappa (Miyazawa et al., 2005). Also, daucosterol and β-sitosterol compounds were detected from the chloroform extracts of A. lappa roots (Han et al., 2013). It was shown that phytosterol daucosterol inhibited cancer cell proliferation by inducing autophagy through reactive oxygen species-dependent manner , and exhibited immunoregulatory activity by inducing protective Th1 immune response .

Flavonoids
The reported flavonoids include flavonols, flavones, and their glycosides. Two major constituents, namely rutin and isoquercetin, along with few other minor flavonoids including kaempferol-3-O-rhamnoglucoside, quercimeritrin and astragalin were identified in the ethanolic extracts of A. minus leaves (Saleh and Bohm, 1971). Likewise, the occurrence of quercetin-3-O-rhamnoside was reported from the leaves of A. lappa. Later, the presence of phenolic compounds such as quercetin, quercitrin, rutin, and luteolin have been reported in seeds, fruits, leaves and roots of A. lappa (Saleh and Bohm, 1971;Tamayo et al., 2000;Yu et al., 2003;Lou et al., 2010aLou et al., ,b, 2016Predes et al., 2011;Liu et al., 2012;Tang et al., 2014). Also, few isoflavone derivatives including genistein, nobilein, biachanin A and tangeretin have been detected in A. lappa roots (Eberding et al., 2007). A comparative study has shown the existence of chemical differences within the A. lappa organs (Ferracane et al., 2010). According to them, luteolin and quercetin rhamnoside were detected in roots whereas rutin, quercetin, quercitrin and luteolin in leaves. On the other hand, no flavonoids were found in the seeds of A. lappa. Two more flavonols, namely quercetin 3-O-glucuronide and quercetin 3-vicianoside, were identified by Rajasekharan et al. (2015) in the root extracts of A. lappa.

Others
From the concentrated sap obtained from A. lappa roots (A. lappa and A. tomentosum), β-asparagine was isolated for the first time (Boldizsar et al., 2010). The carotenoid crocin was reported to occur in the leaves of A. lappa (Lou et al., 2016).

Volatile Compounds
A total of 101 volatile chemical constituents were identified in A. lappa. The details of these compounds are partially summarized in Table 2

BIOACTIVITIES OF Arctium SPECIES
Arctium lappa is widely used as an ethno-medicinal plant especially in North America, Asia and Europe, and is applied to treat various diseases including diabetes, gout, rheumatism, and skin problems (Chan et al., 2011;Azizov et al., 2012). A. lappa roots have been used as a vegetable in Japanese (referred to as 'gobo') and Korean cuisine. Its root has been used to treat constipation, mercury poisoning, upper respiratory infections, inflammation and oxidative stress in patients with knee osteoarthritis (Maghsoumi-Norouzabad et al., 2016), while the leaves were efficacious in healing burns, rashes, and applied in women with labor condition (Force, 2001;Lewis and Elvin-Lewis, 2003;Amish Burn Study et al., 2014). A. lappa has also been found for the treatment of alopecia (loss of hair) among adults (Amish Burn Study et al., 2014). In Western countries, burdock is used as a remedy for several ailments ranging from arthritis, chronic inflammation, and various skin problems (e.g., scaly skin conditions such as psoriasis and eczema) to cancer treatment (Wu et al., 2010;Amish Burn Study et al., 2014).
Studies on the biological activities of extracts of different parts of A. lappa and compounds isolated thereof, were carried out and revealed antipyretic, antimicrobial, diuretic, diaphoretic, hypoglycaemic, antioxidant, anti-inflammatory, anti-hepatotoxicity, antiulcer, antimutagenicity, and antitumour activities.

Anticancer Effects
Arctium lappa fruit has been used in traditional medicine, and it is popular for its various anticancer effects. Arctigenin (ATG), a natural lignan product extracted from the seeds of Arctium lappa, has been shown to have estrogenic properties, that reduced the risk of osteoporosis, heart disease, and menopausal symptoms (Maxwell et al., 2017). It was found to possess antitumor effect by modulating the protein kinase activation pathway and hence rendering the tumor cells susceptible to effects of the nutrient-deprived environment (Awale et al., 2006). Later on, ATG was shown to induce apoptosis (programmed cell death) of estrogen receptor-negative cancer cells (MDA-MB-231) through the ROS/p38 MAPK pathway and epigenetic regulation of Bcl-2 by upregulating trimethylation of histone H3K9 (Hsieh et al., 2014). It was reported that ATG was able to inhibit cell proliferation and may induce apoptosis and cell cycle arrest at the G0/G1 phase in glioma cells (Maimaitili et al., 2017). In more detail, it was found that ATG increased the expression levels of p21, retinoblastoma and p53 proteins, and significantly decreased the expression levels of cyclin D1 and CDK4 proteins (Maimaitili et al., 2017). Furthermore, ATG was able to induce apoptosis in glioma cells, coupled with increased expression levels of cleaved caspase-3 and the pro-apoptotic BCL2-associated X protein (Maimaitili et al., 2017). ATG-induced apoptosis was significantly suppressed by the pretreatment of cells with Z-DEVD-FMK, a caspase-3 inhibitor (Maimaitili et al., 2017). More recently, study by Lou et al. (2017) demonstrated ATG to significantly inhibit in vitro migration and invasion of human breast cancer cells (MDA-MB-231) by downregulation of MMP-2, MMP-9 and heparanase (Lou et al., 2017).
Extracts from A. lappa also showed selective antiproliferative activity against certain human cancer cell lines including K562, MCF-7 and 786-0 (Predes et al., 2011). Lappaol F, a novel natural product isolated from the seeds of A. lappa, was found to suppress cancer cell growth in a dose-dependent manner in various human cancer cell lines through induction of G1 and G2 cell-cycle arrest. This effect was associated with strong induction of p21 and p27 and suppression of cyclin-dependent kinase 1 (CDK1) and cyclin B1 (Sun et al., 2014).
A. lappa is one of the herbs widely used by cancer patients in some Canadian populations to improve quality of life (QOL) and prevent cancer progression. A. lappa is one of the herbs constituting the two proprietary herbal products: Flor-Essence R and Essiac R suggested for prolong survival and the improvement of QOL among cancer patients (Tamayo et al., 2000).

Antidiabetic Effects
Root of A. lappa root has been found to mediate hypoglycemic activities making it a popular choice to be used as a traditional medicine in diabetes. Oral administration of burdock root ethanolic extract in streptozotocin-induced diabetic rats significantly lowered blood glucose and increased insulin level in the diabetic rats compared to the control diabetic group (Cao et al., 2012). Additionally, treatment with A. lappa extract also reduced the levels of serum total cholesterol (TC), triglycerides (TG) and low density lipoprotein (LDL), whereas high density lipoprotein (HDL) level was higher in the control rats. More recently in a similar study, Ahangarpour et al. (2017) investigated the antidiabetic and hypolipidemic properties of the root extract of A. lappa on nicotinamide-streptozotocin (NA-STZ)-induced type 2 diabetes in mice (Ahangarpour et al., 2017). The results show that root extract of A. lappa displays anti-diabetic effect at certain doses. It exerts its effects through hypolipidemic and insulinotropic properties and hence the root extract could serve successfully in treating patients with type 2 diabetes in the future. Moreover, sitosterol-β-D-glucopyranoside from burdock's root acts as a potent inhibitor of alpha-glucosidases, thereby having the potential to reduce glycogenolysis and help to decrease blood glucose level (Tousch et al., 2014). In addition, Zhao and Zhou (2015) demonstrated that trace elements (e.g., Na, K, Mn, Fe, and Mg) present in the root and fruit extracts of A. lappa exhibit antidiabetic effects. While A. lappa constituents do reduce absorption of glucose, they also elevate inulin content in blood and slow digestion of carbohydrates to confer its anti-diabetic activities. The pharmacological mechanisms of A. lappa roots are slightly different from other classes of oral antihyperglycemic agents such as metformin. Metformin decreases hepatic glucose production, decreases intestinal absorption of glucose, and improves insulin sensitivity by increasing peripheral glucose uptake and utilization (Dumitrescu et al., 2015).

Anti-oxidant, Hepatoprotective and Gastroprotective Activities
It is believed that lignans and caffeoylquinic acids from A. lappa are of value because of their antioxidant capacity (Maruta et al., 1995;Mkrtchian et al., 1998;Jaiswal and Kuhnert, 2011) by which they can scavenge free radicals that are thought to play an important role in many diseases.
The hydroalcoholic extracts of burdock roots possess significant antioxidant potential as seen by the application of various assays. Very recently, Fierascu et al. (2018) quantified antioxidant potential of burdock extracts using DPPH (2,2diphenyl-1-picrylhydrazyl) and phosphomolybdate assays to demonstrate that burdock extracts have very high antioxidative activities, presumably due to the high content of polyphenols (Fierascu et al., 2018). The potent antioxidative property makes these extracts effective inhibitors of lipid peroxidation in rat liver homogenate in vitro (Duh, 1998) and an excellent hepatoprotective agent in vivo and in vitro (Lin et al., 2000). Due to its radical scavenging ability, A. lappa is also used to treat gastrointestinal ulcers (da Silva et al., 2013).

Antimicrobial Effects
Extracts of different parts of A. lappa have been investigated for their microbial-modulatory properties by many researchers. An organic extract from A. lappa has shown inhibiting properties toward the growth of Pseudomonas aeruginosa, Escherichia coli, Lactobacillus acidophilus, Streptococcus mutans, and Candida albicans residing in the teeth of the oral cavity (Gentil et al., 2006). Pereira et al. (2005) further reported potent growth inhibiting activities of A. lappa extract against a broad spectrum of oral microorganisms, specifically those associated with teeth infections, namely Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Candida albicans (Pereira et al., 2005). Very recently, Fierascu et al. (2018) investigated antifungal potential of hydroalcoholic extract of burdock roots and observed that it is active against the fungal lines Aspergillus niger ATCC 15475 and Penicillium hirsutum ATCC 52323 (Fierascu et al., 2018). Fruit extract of A. lappa was tested (Dias et al., 2017) for in vitro antiviral properties against Herpes simplex virus type-1 (HSV-1) and was found to decrease viral load significantly at all concentrations tested (400, 50, and 3.125 µg/mL). At 400 µg/mL concentration, it showed comparable antiviral activity as acyclovir (50 µg/mL). Arctigenin, one of the key constituents of A. lappa extract, has shown potent activities against human immunodeficiency virus type-1 (HIV-1) both in vivo and in vitro presumably by increasing the expression of Heme oxygenase-2 (HO-2) and blocking HIV-1gag proteins (Schroder et al., 1990).

Anti-inflammatory Effects
Various parts of A. lappa demonstrated anti-inflammatory effects (Liu et al., 2005). Burdock extract is known to alleviate wound irritation and swelling and therefore has been traditionally used for healing burn wounds. This effect might be mediated through the inhibition of the cyclooxygenase-2 (COX-2) enzyme. Cyclooxygenase is a lipid metabolizing enzyme that catalyzes the oxygenation of polyunsaturated fatty acids. This process forms prostanoids, specifically eicosanoids, which are known to be potent cell signaling molecules connected to inflammatory processes (Charlier and Michaux, 2003). Phenolic compounds present in burdock extract (e.g., arctigenin, lappaol F, diarctigenin) are inhibitors of this enzyme (Zhao et al., 2009;Lee and Kim, 2010), thereby suppressing lipopolysaccharide (LPS)-stimulated NO production  and pro-inflammatory cytokines secretion (including TNF-α and IL-6) in a dose-dependent manner (Zhao et al., 2009;Kwon et al., 2016). Arctigenin also strongly inhibited the expression of iNOS (Inducible Nitric Oxide Synthase) and its enzymatic activity (Wang et al., 2007;Zhao et al., 2009). Moreover, it induced endothelial nitric oxide synthase (eNOS) and supressed in a rat model subarachnoid hemorrhage-induced vasospasm by regulation of the PI3K/Akt signaling pathway (Chang et al., 2015). Among the studied phenolic compounds, diarctigenin was found to inhibit the DNA binding ability of NF-κB and to inhibit NF-κB-regulated iNOS expression (Kim et al., 2008), thereby overall targeting NF-κB-activating signaling cascade directly to confer anti-inflammatory response. Luteolin, an important flavonoid from burdock was also reported to possess significant anti-inflammatory properties (Ferracane et al., 2010;Nabavi et al., 2015).

Effects Against Skin Conditions
Leaves of Arctium species have been used in traditional medicinal practices in various skin conditions (e.g., rashes, boils, eczema, ichthyosis, acne, psoriasis, and abscesses) presumably due to the presence of various phenolic compounds. The potent antioxidant and anti-inflammatory properties of these compounds serve to detoxify and mediate healing action (Chan et al., 2011). Several hydroxycinnamic acids which are among the active phytochemicals in the A. lappa extracts Tousch et al., 2014) have been found to act as free radical scavengers and possess antioxidant activities, which confer them potential to serve as skin protectors and wound healers (Graf, 1992;Phan et al., 2001;Taofiq et al., 2017). In addition, hydroxycinnamic acid derivatives also display anti-collagenase, anti-inflammatory, antimicrobial and anti-tyrosinase activities, as well as ultraviolet (UV) protective effects, suggesting that they can be exploited as anti-aging and anti-inflammatory agents, preservatives and hyperpigmentationcorrecting ingredients (Ahangarpour et al., 2017). These bioactivities are the reason why burdock extracts find their use in various commercial cosmetic products.

Effect on Potency and Fertility
Diabetes mellitus induces many complications among which dysfunctions male reproductive system is worth mentioning. Glucose metabolism plays an important regulatory role on the production or development of mature spermatozoa (spermatogenesis) as well as on maintaining specific functions, such as motility and fertilization ability in mature sperm cells. Therefore, it is not surprising that A. lappa root extract, which has hypoglycemic and antioxidative properties, would have beneficial effects on male potency and fertility. Ahangarpour et al. (2015) investigated the effect of A. lappa root extract on gonadotropin, testosterone, and sperm parameters in nicotinamide/streptozotocin-induced diabetic mice (Ahangarpour et al., 2015). The root extract led to increased level of luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone as well as enhancement in sperm viability only in diabetic mice compared with the control group, indicating A. lappa root extract to be a potentially effective treatment for male sterility arising from diabetic conditions.

Effect on NO Production
It was reported that arctigenin inhibited NO release by IFN-γ signal, whereas it significantly enhanced lipopolysaccharidetriggered NO production in RAW264.7 cells, suggesting that arctigenin may regulate immune responses in activated macrophages and lymphocytes including TNF-α and NO production and lymphocyte proliferation (Cho et al., 1999). Another study shows that arctigenin suppressed the overproduction of NO through down-regulation of iNOS expression and iNOS enzymatic activity in LPS-stimulated macrophage (Zhao et al., 2009). Besides, lappaol F and diarctigenin from Arctium lappa were shown to significantly inhibit NO production in the LPS-stimulated RAW264.7 cells with IC 50 values of 9.5 and 9.6 µM, respectively .

Safety Considerations on Arctium Species
Several adverse effects have been reported in literature stemming from long-term use of A. lappa. For example, contact dermatitis might develop after several days of applying a burdock root plaster to a wound, or even as fast as within 12 h in some cases (Rodriguez et al., 1995). In one instance, anticholinergic poisoning has been reported upon oral consumption of A. lappa extract (Force, 2001). However, this poisoning later turned out to be caused by products that have been contaminated with root of belladonna (deadly nightshade). The latter herb contains the poisonous chemical atropine. Long-term consumption of burdock also has led to anaphylaxis in one case (Chan et al., 2011). Root oil made from A. lappa was also found to cause unfavorable physiological effects such as redness, and anaphylactic shock (Rodriguez et al., 1995;Lewis and Elvin-Lewis, 2003;Sasaki et al., 2003). Caution is advised for pregnant or nursing women to consume burdock or its extract, as it might have detrimental effects on the fetus (Chan et al., 2011). Burdock can also interfere with blood clotting. People who are already on blood thinning medications are advised not take it without approval from their doctors. Even though burdock is considered a 'safe' food, consuming it in large amounts should be avoided due to lack of large amount of safety studies on burdock. More in vivo studies are in particular needed on A. lappa to further evaluate its therapeutic potential and safe application window. Due to the presence of sesquiterpene lactones, the use of Arctium species should be avoided in patients with hypersensitivity to Asteraceae/Compositae (Chan et al., 2011).

SUMMARY
In summary, the volatile and non-volatile secondary metabolites present in different parts of Arctium species showed pharmacological potential in the treatment of various diseases. The literature existing on extracts of different parts of A. lappa and isolated compounds demonstrates antipyretic, antimicrobial, diuretic, diaphoretic, hypoglycaemic, antioxidant, anti-inflammatory, anti-hepatotoxicity, antiulcer, antimutagenicity, and antitumour activities. Hence, Arctium species display a broad therapeutic potential but further studies are needed on potential risks associated with their application.