Ethnobotanical Uses, Phytochemical Composition, Biosynthesis, and Pharmacological Activities of Carpesium abrotanoides L. (Asteraceae)

Carpesium abrotanoides L. (Asteraceae) is a medicinal plant with immense therapeutic importance and bioactivities. It is commonly encountered in various Asian regions. It has numerous ethnomedicinal uses for curing diverse ailments such as toothache, stomach ulcer, boils, tonsillitis, bronchitis, bacterial infection, bruises, swelling, virus infection, fever, and amygdalitis, as well as an anthelmintic versus round-, tape-, hook-, and pinworms. Different classes of phytoconstituents such as sesquiterpenes, sesquiterpene dimers, monoterpenes, and nitrogenous compounds have been reported from this plant. These phytoconstituents have proved to possess anti-inflammatory, cytotoxic, antimicrobial, and insecticidal capacities. The present review aims to summarize all published data on C. abrotanoides including traditional uses, phytoconstituents, bioactivities, and toxicological aspects, as well as the synthesis and biosynthesis of its metabolites through an extensive survey on various databases and various publishers. These reported data could draw the attention of various natural-metabolite-interested researchers and medicinal chemists towards the development of this plant and/or its metabolites into medicine for the prevention and treatment of certain illnesses. Despite the diverse traditional uses of C. abrotanoides, there is a need for scientific evidence to support these claims. Clinical trials are also required to further assure these data and validate this plant utilization in treating several diseases.


Introduction
Medicinal plants have remarkable participation in the separation and discovery of new drugs. Obviously, there is increasing interest in using medicinal plants as part of various traditional medicines for treating various ailments [1][2][3]. The side effects, high cost, and therapeutic limitations of available medications are principal factors that encourage the revivification of herbal treatment [4]. It was also reported that more than 80% of people worldwide rely on traditional medicines as a primary source of healthcare, involving the utilization of plant extracts or their phytoconstituents [4]. Recently, plantderived metabolites have become greatly important due to their structural diversity and promising bioactivities [5][6][7].
Breast cancer represents one of the most prevalent malignant tumors in women with low survival rates. Chai et al. evaluated the anticancer capacity of root pet. ether fraction (Conc. 50 and 100 µg/mL) versus MDA-MB-231 and MCF-7 using MTT assay. The fraction was found to noticeably suppress proliferation and migration and cause apoptosis in both cells [17]. C. abrotanoides exerted its potential via prohibiting glycolysis-linked genes, GLUT1 (glucose transporter-1), LDHA (lactate dehydrogenase A), and HK2 (hexokinase-2) and down-regulating PKM2 (pyruvate kinase M2). PKM2 is closely linked to the angiogenesis, migration, and proliferation of cancer cells, as well as autophagy inhibition [23]. Therefore, the plant was established as PKM2/HIF-1α (hypoxia-inducible factor-1α) axis inhibitor, revealing its potential versus breast cancer [17]. Additionally, the cytotoxic capacity of the essential oil on various hepatic cancer cells (Hep3B, HepG2, Huh7, and SMMC-7721) and LO-2 normal liver cell line was investigated by Wang et al. in the MTT assay. The oil prohibited the proliferation of all cancer cells (IC 50 s from 41.28 to 130.36 µg/mL), with little cytotoxicity of L-O2 cells (IC 50 > 300 µg/mL). Furthermore, it caused apoptotic morphological changes and G2/M and S phases cell cycle arrest in HepG2 cells. Additionally, it reduced Bcl-2/Bax ratio and raised caspase-3 and -9 activation, suggesting the involvement of mitochondria-induced apoptosis in this effect [15].

Antiparasitic Activity
Paralichthys olivaceus (Olive flounder) is an important South Korean fish species; however, there are serious industrial losses owing to diseases influencing this fish [24]. Scuticociliatosis is one of the prevalent parasitic diseases caused by Miamiensis avidus, resulting in high fish mortality [25]. Additionally, this parasite infects various fish species such as turbot, sharks, adult kingfish, and black rockfish [24]. Hydrogen peroxide and formalin are the approved antiparasitic agents for scuticociliatosis in P. olivaceus [26]. However, this treatment is non-effective as they cannot enter the internal organs and formalin over-use is carcinogenic. Therefore, effective and safe natural antiparasitic agents versus M. avidus are needed.
The fruit EtOH extract (conc. 50,70, and 100% v/v) demonstrated antiparasitic potential versus M. avidus as evident by the induced morphological alterations, including cell shrinkage, reduced motility, and lysis, whereas 100% cell lysis was noticed after 2 h treating with 100% extract [24]. Additionally, the 70% and 100% extracts following concanavalin A pretreatment increased CD8-α, IL-8, IL-1β, TNF-α, UGT2B19, CYP-1A, CYP-1B, and CYP-3A4 expression; however, their expression reduced with 50% extract. Furthermore, they reduced HINAE (hirame natural embryo) cell viability with % between 67 and 80% and IC 50 s from 38.39 to 102.3 mg/L, revealing the less cytotoxic potential of 50% EtOH extract than 70% or 100% extract, while still exhibiting antiparasitic activity against M. avidus [24]. Therefore, C. abrotanoides had potential as an antiparasitic agent versus M. avidu,s supporting its utilization as an anti-parasite in the aquaculture industry [24]. However, the main components accountable for this influence need to be specified and in vivo investigations should be carried out to assess its usage in aquaculture.
Taenia asiatica cysticerci was in vitro cultured with C. abrotanoides H 2 O decoction (Conc. 20, 40, and 60 mg/mL). It was found that the decoction had a prominent killing capacity on the cysticerci with the highest %mortality (95.0%) at conc. 60 mg/mL. The dead cysticercus had shrunk with degenerated sucker tissue and enlarged scolex [27].

Insecticidal Activity
Plants are a substantial pool for discovering novel and safe insect repellents, especially to protect from blood-sucking insect pests such as bed bugs and mosquitoes. Spodoptera exigua Hübner is one of the major pests that occurs in fields, vegetables, and flower crops [28]. The repeated use of chemical pesticides resulted in the development of S. exigua resistance to different insecticides, leading to difficulty in controlling this pest [29]. A growing interest has been focused on the discovery of phytochemicals with insecticidal potential [30]. Since C. abrotanoides has been traditionally utilized as an insecticide in Chinese and Korean medicines. Feng et al. assessed the insecticidal potential of the fruit EtOH extract versus S. exigua. The results revealed that the fruit extract displayed a promising antifeedant potential versus the S. exigua larvae with FDI (Feeding-deterrence index) of 48.32% when the larvae fed on treated leaves with 10 mg/mL extract [30].
Aedes aegypti (L.) is one of the most threatening pests for humans globally [31]. Ae. aegypti was reported to have a potential in the transmission of dengue and zika viruses in humans [32]. Various insecticides such as organophosphate (pirimiphos-methyl and malathion), organochlorines (DDT), carbamate (bendicarb and propoxur), and pyrethroids (lambda cyhalothrin, permethrin, and deltamethrin) were used for controlling Ae. Aegypti. [33]. However, their repeated use results in increasing resistance to Ae. aegypti versus these insecticides in addition to their hazardous effect on humans that necessitates the discovery of natural alternative agents [34].

Antidiabetic Activity
Diabetes is a chronic worldwide disease that influences millions of people [35]. It was estimated to be the ninth reason for death with 1.5 million deaths in 2019 [35]. One of its therapeutic approaches for treatment is the carbohydrate absorption suppression after food intake which is aided by the prohibition of α-glucosidase and α-amylase enzymes [36,37]. The main goal of its treatment is to keep the blood glucose levels near normal in both the postprandial and fasting conditions [38]. The aerial parts' 80% MeOH extract were found to contain remarkable amounts of total phenolics (88 mg gallic acid/g extract) and flavonoids (2 mg quercetin/g extract). It also revealed non-competitive α-glucosidase inhibitory effectiveness (IC 50 44.22 µg/mL), compared to acarbose (IC 50 2.5 µg/mL) [39].

Anti-Inflammatory Activity
Jeong et al. assessed the influence of the aerial parts' EtOH extract on the COX-2 (cyclooxygenase-2) expression by various TLRs (Toll-like receptors) agonists in murine macrophages. TLRs and their signaling components could be a potential therapeutic target for chronic inflammation disorders. C. abrotanoides repressed COX-2 expression caused by LPS (lipopolysaccharide, TLR4 agonist), macrophage-activating lipopeptide 2-kDa (TLR6 and TLR2 agonist), and polyriboinosinic polyribocytidylic acid (TLR3 agonist). This indicated its capacity to regulate the TLRs TRIF (Toll-interleukin-1 receptor domain-containing adapter inducing interferon-β)-and MyD-88 (myeloid-differential-factor-88)-dependent pathways, resulting in reduced expression of inflammatory genes; therefore, it could be beneficial for treating chronic inflammatory disorders [41]. In another study, Lee et al. also revealed that the 70% EtOH extract of aerial parts suppressed iNOS (inducible nitric oxide synthase) expression boosted by these TLRs agonists in murine macrophages [42]. Additionally, the blooms' MeOH extract (dose 10 mg/kg BW) exhibited anti-inflammatory capacity by reducing the elevated IL-6 and IFNγ levels induced by LPS in mice. It also attenuated IL-6, IFNγ, and IL-4 levels in a Con-A inflammation model [43].

Phytochemicals and Their Pharmacological Activities
In total, 118 metabolites have been separated from various parts of C. abrotanoides using various chromatographic tools, including various terpenoids, sterols, and aliphatic and nitrogenous compounds ( Figure 1). Additionally, some volatile compounds have been characterized from the plant essential oil by GCMS. These metabolites have been assessed for diverse bioactivities ( Figure 2). It is obvious that most of the reported metabolites have been evaluated for their cytotoxic potential. Furthermore, the reported compounds along with their molecular weights/formulae, part of the plant/fraction from which they were isolated, and the place of location are listed in Table 1.
Bradysia odoriphaga is one of the main insect pests influencing Northern China's Chinese chive that results in 30-80% loss in production and attacks 20-30% of Chinese chives [74]. It also feeds on other plant species such as garlic, Welsh onion, cucumber, Chinese cabbage, and lettuce, as well as mushroom sheds causing production losses. Its larvae gather in the stems and roots of the plant making its control hard with common strategies. Generally, organophosphates are the principal tool for controlling Chinese chives pests [75].
It was postulated that the cyclopentane unit in 64 and 65 could be generated from a [3+2] cyclo-addition reaction beginning from a three-membered ring in one molecule and a double bond in the other (Scheme 5) through radical intermediates. The cleavage of the bond between C-5 and C-1 forms a radical-intermediate C having C-5 already radical that is then trapped with the double bond in D. Due to steric hindrance in the structure, C-1 firstly forms a new bond with C-13, and then C-5 forms another bond with C-11. Hence, a five-membered ring fuses the two monomers C and D. Additionally, the free rotation of the side chain around C-1 resulted in two possible configurations at C-1 [64]. In 2020, Yang et al. purified five uncommon C15/C17 dimeric sesquiterpenes, carabrodilactones A-E (67-71), along with formerly reported C15/C15 dimeric analogs, 72, 73, 75, and 76, from the EtOAc fraction of 70% EtOH of the whole plant (Figures 11 and 12). Compounds 67-71 displayed tailed acetyl linked to the C-13 position and flexible C-13 /C-11-linked single bond among the two sesquiterpene moieties [65]. They were evaluated for their cytotoxic potential versus MDA-MB, A549, BEL7404, and HCT116 cells using the MTT assay. Among them, 67 had potent effectiveness versus these cell lines (IC 50 ranged from 3.08-8.05 µM); however, the others possessed weak potential (IC 50 27.70-101.34 µM). It was demonstrated that the α-ethylene-γ-lactone group's exo-ethylene, C-4 carbonyl of carabrone unit, and two sesquiterpene units' spatial orientation were crucial for the activity. Moreover, 75 and 76 were found to have cytotoxic potential versus K562, CCRF-CEM, HCT116, and HL-60 cells (IC 50 ranged from 2.63 to 8.50 µM) in the MTT assay [70]. Biosynthetically, these C15/C17 metabolites were assumed to be generated through Stetter and Michael's addition reactions. Firstly, the acetaldehyde anion is formed by umpolung of pyruvic acid through catalysis by thiamine. After that, it attacks the first C15 sesquiterpene lactone exoethylene to generate the C17-enolate anion at C-11 by Stetter reaction. Thenceforward, the Michael addition reaction of enolate with the α,β-unsaturated bond of the other lactone moiety yields four possible C17/C15 stereo-isomeric dimers with a C-13 /C-11 C-C bond among the two sesquiterpene moieties [65] (Scheme 7). bond is cleaved to form a radical intermediate, and the C-5 radical is then trapped with the double bond. Due to steric hindrance in the structure, C-1 forms a new bond with C-13 first, and subsequently C-5 forms another bond with C-11. Thus, a cyclopentane ring is introduced to link the two monomers [63].

Toxicity Studies
Toxicity evaluation is substantial to assess the untoward effects of plants on biological systems. It is noteworthy that systemic toxicity and safety assessments are not extensively performed on this plant. Additionally, C. abrotanoides is formally reported to have slight human toxicity [77]. Thus, there is little evidence of this plant's toxicity. In Chinese pharmacopeia, this plant was recorded as a mild toxic medicine [14]. C. abrotanoides is safe for health and its commercial preparations are commonly used for years [10]. It was reported that carpesia lactone is the main poisonous ingredient that has irritant properties and a bitter taste [78]. Its ingredients could produce certain symptoms [78] such as asdizziness, tinnitus, nausea, diarrhea, abdominal pain, colonic spasm, and weakness [79]. In Western medicine, the reported methods of its poisoning treatment include promoting vomiting, stomach washing out, grape sugar supplies, and symptomatic treatment. In Korean medicine, the herbs such as Glycyrrhizae Radix and Phaseoli Radiati Semen are used to counteract its poison [78]. Furthermore, Shi et al. reported a 96 h acute toxicity (LC 50 2.60 g/L) of C. abrotanoides extracts in goldfish [80]. The fruit extract also had a 24 h LC 50 0.76 g/L versus Penaeus monodon [81]. However, a study by Xia et al. proved that the fruit (Conc. 140 to 230 mg/L) had a developmental toxic influence on zebrafish larvae/embryos. It noticeably altered the embryos' hatching rate and time and raised malformations, where the heart was the target organ. Moreover, it reduced caspase-3 potential, changed the defense enzymes (CAT, SOD, and GPX), and elevated MDA content, indicating that apoptosis and oxidative stress might be accountable for its toxic effects [82]. In clinical practice, Carpesii Fructus is mainly used as antiparasitic drugs and is usually combined with other drugs since it displayed poor efficacy as a single drug [78]. Therefore, this plant is contraindicated during pregnancy, not suitable for long time usage, and should be taken with caution for debilitated patients [79]. However, further safety studies must be carried out on C. abrotanoides. Future investigations are required to explore the mechanism of toxicity and to analyze and specify the extract components relevant to toxicity.

Conclusions
In this review, the traditional uses, phytoconstituents, and pharmacological activities of C. abrotanoides have been highlighted. The current work revealed that 118 metabolites have been purified from different parts of the plant, among them monoterpenes and sesquiterpenes are the main constituents. It is obvious that more research studies have been carried out on the aerial parts; therefore, the other plant parts should be further investigated.
It was found that the synthesis and structure modifications of certain metabolites produced more bioactive derivatives as in 13 and 33. Hence, these constituents could be prominent candidates for developing effective and novel pharmaceutical agents. Additionally, this research direction should be further investigated by medicinal and organic chemists. These metabolites and plant extracts have been assessed for various bioactivities. Some metabolites demonstrated potent cytotoxic capacities (e.g., 11, 13, 19, 23, 24, 47, 53, 57, and 60) similar or more than positive controls. Furthermore, the main reported mechanisms of their cytotoxic capacity included the induction of autophagy and apoptosis. These studies suggested that the plant and its sesquiterpenes could serve as promising chemotherapeutic agents for various cancer types. Additionally, compound 31 exhibited marked antiH1N1 potential. However, future in vitro and in vivo studies for chemo-preventive effects are essential to confirm this efficacy and to identify the compounds responsible for this chemo-preventive potential. Moreover, studies on the structure-activity relationships and/or derivatization of these metabolites should be carried out.
The anti-inflammatory potential of the plant/metabolites could support its usage as an alternative medicine for relieving and retarding inflammatory responses. Based on its powerful antioxidant and α-glucosidase inhibitory capacities, the plant could have remarkable potential for diabetes treatment. However, further studies for separating the active phytoconstituents accountable for these activities along with in vivo experiments and mechanistic studies are needed. Most of the reported activities of the plants have been attributed to its sesquiterpene contents. C. abrotanoides extracts and some metabolites have been established to display insecticidal capacity versus various pest species verifying their potential use in developing botanical insecticides. Comparing the results of biological studies with the plant traditional uses, it is obvious that only a few of the traditional uses have been scientifically verified. Moreover, the study of bioactivities of aqueous extracts is substantially needed for confirming the data of traditional use of decoctions and infusions. Some of the reported constituents are either not tested or possessed no notable effectiveness in the evaluated activities; therefore, an in silico screening for other potential bioactivities could be a clearly goal for future research studies. Most of the natural metabolites are frequently produced by the plants in small amounts; therefore, securing sufficient quantities of them for further clinical applications has become a persisting concern [83]. The technological progress, including genome sequencing and development of molecular biology and genetic techniques engineer the biosynthesis of many valuable natural metabolites [84]. In this review, highlighting the biosynthesis of these metabolites could draw the attention of molecular biologist-and genetic-interested researchers for isolating the genes accountable for the biosynthesis of these interesting metabolites and discovering the detailed mechanisms of their formation by various enzymes. This could allow the preparation of these metabolites and their analogs by engineering their biosynthetic pathways. Although the reported studies of C. abrotanoides revealed great promise, most of its activities have only been assessed in vitro. Accordingly, it is of the uttermost importance that the investigation of C. abrotanoides extracts and its constituents' safety and efficacy testing should be carried out in in vivo studies. Lastly, clinical trials must be implemented to prove if the prominent activities of C. abrotanoides can result in clinical usability in an effective and safe manner and to completely certify its renowned use in the traditional medicines of various countries.