Xanthanolides in Xanthium L.: Structures, Synthesis and Bioactivity

Xanthanolides were particularly characteristic of the genus Xanthium, which exhibited broad biological effects and have drawn much attention in pharmacological application. The review surveyed the structures and bioactivities of the xanthanolides in the genus Xanthium, and summarized the synthesis tactics of xanthanolides. The results indicated that over 30 naturally occurring xanthanolides have been isolated from the genus Xanthium in monomeric, dimeric and trimeric forms. The bioassay-guided fractionation studies suggested that the effective fractions on antitumor activities were mostly from weak polar solvents, and xanthatin (1) was the most effective and well-studied xanthanolide. The varieties of structures and structure-activity relationships of the xanthanolides had provided the promising skeleton for the further study. The review aimed at providing guidance for the efficient preparation and the potential prospects of the xanthanolides in the medicinal industry.


Introduction
The plants belonged to the genus Xanthium L. were found world wide, which showed important applications in traditional herbal medicine in many countries [1]. They were commonly used to treat nasitis, fever, arthritis, tumors, gastric ulcer and microbial infections [2]. In China, Xanthium sibiricum Patrin ex Widder was listed in the Pharmacopoeia for treating rhinitis, headache, rheumatism and many other diseases. However, the acute toxicity of Xanthium L. was also reported during the medicinal application and ingestion poisoning of live-stock [3]. The constituents of X. strumarium were responsible for the activity or toxicity of the species. To date, varieties of compounds have been isolated from the plants belonged to the genus Xanthium, including sesquiterpenoids, flavonoids, coumarins, steroids, phenylpropenoids, lignanoids, glycosides, anthraquinones, and naphthoquinones [1,4]. Some of these compounds had been proven to exhibit significant medicinal properties, such as diuretic, anthelmintic, antifungal, anti-inflammatory, antidiabetic, anticancer, and many other activities [5][6][7][8].
Xanthanolides were the characteristic ingredients of the genus Xanthium, which possess bicyclic sesquiterpene lactones with structural feature of five-membered γ-butyrolactone ring fused heptatomic carbocycle. The genus Xanthium is the richest in the limited plant species for the source of xanthanolides. Xanthanolides were reported to show various bioactivities, including antitumor, antifungal, anti-ulcerogenic, anti-ulcerogenic and insecticidal activity [5]. In recent years, xanthanolides have drawn increasing attention due to its promising skeleton and potential value [6][7][8][9][10]. In this paper, the structures, synthesis and bioactivities of the xanthanolides in the genus Xanthium L. were summarized for these unique structure's potential use in the pharmaceutical industry.

The Biological Activity of Xanthanolides
X. strumarium has been used for a long time in clinic as the traditional medicine due to its broad activity. The fruits of X. strumarium, named as "Cang-Er-Zi" in China, have been used to treat rhinitis, anti-inflammory and many other diseases. Xanthanolides, as the typical constitutes of the genus Xanthium, showed important medicinal values by exhibiting a broad spectrum of biological effects, including anti-tumour, antimalarial, antifungal, antiviral and anti-inflammatory activities.

Anti-Tumor Activity
The anti-tumor activities of some Xanthium species have been discovered for a long history, such as X. canadense, X. catharticum, X. chinense, X. echinatum, X. indicum, X. italicum, X. macrocarpum, X. orientale, X. spinosum and X. strumarium. [5,20] In our previous study, the leaf extract of X. italicum was found to exhibit significant cytotoxic activity against human tumour cell lines A549 and hep G2 [68]. To date, the crude extracts of many Xanthium species, including X. strumarium L., X. italicum and et al., exhibited antitumor acitivity. The bioassay-directed fractionation and cytotoxicity investigation revealed that the weak polar solvents, such as dichloro-methane or chloroform, were the most effective fractions. Our previous study conducted a bioassay-directed isolation of the crude extract of X. italicum, which led to the identification of two effective xanthanolides, xanthatin (1) and xanthinosin (22), from the chloroform fraction [68].
As the most acknowledged antitumor xanthanolides, the mechanism studies on the cytotoxicity of xanthatin (1) have been intensively studied in recent years. In 2012, Lu et al., reported that xanthatin (1) exhibited significant antitumor activity against non-small-cell lung cancer cells A549 through cell cycle arrest and apoptosis by disrupting NF-kappa B signaling [70]. In the following study, they found that xanthatin (1) could suppress DNA replication by triggering Chk1-mediated DNA damage [71]. In 2018, Fang et al., reported that xanthatin (1) could promote the oxidative stress-mediated apoptosis of HeLa cells via inhibiting thioredoxin reductase, which was supported by the research of Zhang et al., in 2021 [72,73]. In 2019, Yu et al., demonstrated that xanthatin could covalently bind to JAK and IKK kinases and inhibit the STAT3 and NF-κB signalling pathways to suppress the development of cancer and inflammatory diseases [74]. In 2020, Feng et al., reported that xanthatin (1) could activate endoplasmic reticulum stress-dependent CHOP pathway to induce the apoptosis of glioma cell and inhibit the growth [75]. In 2021, Jia et al., reported that xanthatin could selectively inhibit the proliferation of retinoblastoma cells by inhibiting the PLK1-mediated cell cycle [76].

Antimicrobial Activity
The antimicrobial activity of the Xanthium species has been reported and applied for a long time. Rodino et al., reported the the antimicrobial activity of extracts from Xanthium strumarium against phytophthora infestans [77]. Tsankova et al., investigated the antibacterial activity of the leaf extract of Xanthium italicum and isolated two xanthanolides: xanthinin (14) and xanthatin (1). The results showed that both the crude extract and isolated xanthanolides all exhibited significant activities against the Gram-positive bacterium Staphylococcus aureus [15].
Sato et al., isolated xanthatin (1) from the leaves of Xanthium sibiricum Patr er Widd and found that xanthatin could inhibit the growth of 20 strains of methicillin-resistant Staphylococcus aureus (MRSA) and 7 strains of methicillin-susceptible Staphylococcus aureus (MSSA) with the MICs of the range from 7.8 to 15.6 ug/mL. However, it showed no inhibitory effect on Escherichia coli [15].
Xanthatin (1) had been widely reported as the main antimicrobial component among all the xanthanolides, which had been recognized as an important precursor for the bio-mimic synthesis in exploring the novel antimicrobial agents. Zhi et al., synthesized a series of Michael type amino derivatives from xanthatin (1) and investigated their antifungal activities against several phytopathogenic fungi, and led to the finding of the more effective ompounds than xanthatin [78]. In 2009, Liu et al., reported that the water extracts of X. strumarium exhitbited antiviral activity against duck hepatitis B virus and xanthatin (1) exerted significant activity against influenza A virus [79]. Tsankova et al., found that the leaf extract exerted antiviral activity against the pseudorabies virus A-2 strain, while both xanthinin (14) and xanthatin (1) showed no antiviral activity. The further study showed that all the three samples above displayed no antiviral activity against influenza A/chicken/H7N7 (FPV) and Newcastle disease virus (NDV) [15]. The results indicated that the xanthanolides might have very weak anti-viral activity.

Anti-Inflammatory Activity
X. strumarium has long been used to inhibit various inflammatory diseases and its crude extracts, either water or methanol, have been confirmed to show effects via decreasing IFN-γ, NO production and TNF-α production. In 2005, Kim et al., investigated the antiinflammatory activities of the methanol extracts of X. strumarium, which indicated that the crude extract could down-regulate the mRNA express of NO, PGE 2 and TNF-α [80]. In 2008, Yoon et al., treated the cells with the ethyl acetate extract of X. strumarium, which was found to reduce the production of NO to 0.9 uM. With the further purification, xanthatin (1) and xanthinosin (22) were obtained and were proven to inhibit the production of NO and activated the microglia in a dose-dependent manner (IC 50 = 0.47, 11.2 and 136.5 mM, respectively) [12]. In 2022, Liu et al., investigated the anti-inflammatory effect of xanthatin (1) and found that xanthatin (1) could decrease the amount of nitric oxide (NO), reactive oxygen species (ROS) and down regulate the expression of inflammatory cytokines including COX-2, TNF-α, IL-β, and IL-6 and et al. The results demonstrated that xanthatin (1) could exhibit anti-inflammatory effects by down regulating NF-kappaB, MAPK and STATs signaling pathways [81].
Li et al., reported that isoxanthanol (17) could exhibited protective and anti-inflammatory effects on subchondral bone deterioration. The results indicated that isoxanthanol could inhibit the excessive release of interleukin-6, NO and PGE2 in a dose dependent manner both in vitro and in vivo [82].

Synthesis of Xanthanolides
Xanthanolides were the characteristic sesquiterpenoids from the genus Xanthium. The synthesis of xanthanolides are drawing increasing attention because of the unique structure, impressive biological profile and low content in plant.

Chemical Synthesis
The seven-membered carbocycle fused γ-butyrolactone was the structural core of xanthanolides and the main difficulty to overcome. In 2003, Nosse et al., prepared the 5,7-fused xanthanolide core via the cyclopropanation of furan-2-carboxylic methyl ester [22]. In 2004, Vaissermann et al., prepared the 5,7-fused skeleton of xanthanolides via complexes of cycloheptatriene carboxylic acid esters and chromium tricarbonyl in three steps [83]. In 2021, Tang et al., reported the efficient synthesis of the α-methylene-β-lactones efficiently via dyotropic rearrangement, which realized the preparation of the building blocks of natrual xanthanolides [84].
The first total synthesized xanthanolide was 11, 13-dihydroxanthatin lacking the reactive exocyclic methylene reported by Morken et al., in 2005 [85]. In the same year, Martin et al., reported the first total synthesis of the (+)-8-epi-xanthatin starting from the commercially available ester in 14 steps with overall yield of 5.5% [86]. In 2008, Shishido et al., completed the enantioselective total synthesis of xanthatin by the construction of the vinyl functionality at C1 and the exo-methylene at C11 via the syn-elimination of selenoxides from an optically pure cis-fused bicyclic lactone, and then, they reported an enantioselective total synthesis of 8-epi-xanthatin (2) by developing a synthetic route without the stoichiometric selenium reagents in 2012 [87,88]. In 2012, Tang et al., developed a method to synthesize the enantiopure γ-butyrolactones via a controllable Wagner-Meerwein-type dyotropic rearrangement of cis-β-lactones and successfully applied it to prepare a number of xanthanolides [89]. In 2013, Kenji et al., reported the enantioselective total syntheses of xanthatin and 11, 13-dihydroxanthatin via stereocontrolled conjugate allylation to an optically pure γ-butenolide [90]. In 2017, Tang et al., improved the previous method and completed the synthesis of (+)-8-epi-xanthatin (2) through a CPA-catalyzed tandem allylboration/lactonization reaction and prepared a series of xanthanolides efficiently [9]. The representative tactics on the total synthesis of xanthanolides were shown in Scheme 1.

Biological Synthesis
Synthetic biology was proposed to be an alternative strategy to produce sesquiterpenoids and some outstanding achievements has been made in this field [91]. The biosynthetic strategy requires the fully identification of xanthanolides related metabolite genes of the plants and revelation of the enzymes involved in the biosynthesis. The skeletons of xanthanolides were proposed to be formed by the modifications on the backbone of germacranolide [5]. In recent years, the biogenesis of xanthanolides has been elucidated from the plant species. In 2010, a sesquiterpene synthase (STP) forming δ-guaiene was isolated from cultured cells of Aquilaria, which enabled the conversion of the common substrate farnesyl pyrophosphate (FPP) to form the specialized sesquiterpene skeletons [92]. In 2013, Lange and Turner proposed that the X. strumarium plant could biosynthesize xanthanolides in glandular cells and distribute them on the surface of plant organs [93]. Soetaert et al., conducted an RNA-sequencing strategy to elucidate the biosynthetic pathways of STLs and finally identified some key genes involved in STL biosynthesis [94]. In 2016, Zhang et al., successfully identified three X. strumarium sesquiterpene synthase genes, which provided the molecular basis for the biosynthesis of sesquiterpenoids in X. strumarium [95]. With more and more plant sesquiterpene synthases being isolated, engineering synthesis become possible to prepare xanthanolides in microorganisms [35]. However, due to the mechanism complexity of the synthases and the competitive pathways, the yield of biosynthesis was still at a low level, which limited the commercialization of these methods. To date, xanthanolides were still produced by plant extraction or chemical synthesis.

Conclusions
Xanthanolides were distinctive sesquiterpenoids exist in the genus Xanthium, which were responsible for the significant bioactivities of the Xanthium sepecies. Over 30 xanthanolides had been isolated from the species of Xanthium since the first discovery of xanthatin (1) and xanthinin (14), which have drawn much attentions in pharmacological research because of its broad activity and specific structures. Here in, the structures and biological activities of the xanthanolides in the genus Xanthium were summarized. Among all the xanthanolides, xanthatin, with α-methylene-γ-lactone ring as key active group, was recognized as a key molecule and had been studied in-depth against tumor and inflammatory diseases. Meanwhile, the total synthesis tactics of xanthanolides were also summarized for the efficient preparation of the xanthanolides. Above all, xanthanolides, as naturally occurring sesquiterpenoids, were thought to be promising in the medicinal industry and more studies should to be conducted for their potential application values.