6.1.2. Antitumor Activity In Vivo and Clinical Trials

Artemisinin (**1**) and its derivatives (**1a**, **1b** and **1c**) are very potent anticancer compounds, highly selective on cancer cells with almost no side effects on normal cells. This specificity is due to certain tumor cell characteristics, such as increased metabolism and the high concentration of ferrous ion required to assist their rapid proliferation. There is indeed a high concentration of transferrin, an iron transporter protein situated on the surface cell, and also a susceptibility to reactive oxygen species (ROS) [225]. Furthermore, interest in artemisinin (**1**) and its derivatives resides in their minimal toxicity and adverse effects, which suggest the possibility of utilizing them as antineoplastic drugs [186].

Recent studies have shown that compound **1** and derivatives inhibit the growth of numerous types of neoplasm cells, including breast, ovarian, prostate, lung, colon, leukemia, pancreas, melanoma, renal, hepatic, gastric, and CNS cancer cells. Several reviews have been published in the last few years describing the outstanding antitumor activities of artemisinin (**1**) and derivatives upon different pathways in human cancer cells [186,198,226,227]. Therefore, we will only point to the most relevant aspects of the in vivo and clinical trials carried out.

The antitumor mechanism of artemisinin (**1**) is also based on cleavage of its endoperoxide bridge by the ferrous iron in cancer cells and formation of ROS. Such free radicals produce cell alterations such as apoptosis, DNA damage, autophagy and cell cycle arrest G0/G1 [227]. They can also inhibit angiogenesis by inhibiting the secretion of VEGF, VEGFR2, and KDR/flk-1 in tumors [228,229]. They also may affect signaling pathways and transcription factors associated with tumor growth, including the Wnt/β-catenin and AMPK pathways, nitric oxide signaling, NF-κB, CREBP, MYC/MAX, mTOR, and AP-1 [230].

Some derivatives have reached the phase of clinical trials against several cancers such as breast, cervical, hepatocellular carcinoma, non-small cell lung, squamous cell laryngeal [229,231,232]. However, insufficient large-scale clinical studies have been conducted on their applications in cancer therapy.

Artenusate (**1a**) was used in phase I clinical trial to treat metastatic breast cancer (ClinicalTrials.gov identifier: NCT00764036) and concluded that 200 mg per day are recommended for future trials [233]. This compound (**1a**) is also being tested regarding colorectal cancer phase I (ISRCTN registry: ISRCTN05203252) and its safety and efficiency in stage II/III to the same disease (ClinicalTrials.gov identifier: NCT02633098), as treatment in patients with cervical dysplasia (ClinicalTrials.gov identifier: NCT02354534) and as treatment of HPV-associated anal intraepithelial neoplasia (ClinicalTrials.gov identifier: NCT03100045). Recently, Trimble et al. [234] assess for the first time the safety and efficacy of intravaginal artesunate (**1a**) to treat cervical intraepithelial neoplasia 2/3 (CIN2/3) with good results which support continuing phase II clinical (ClinicalTrials.gov identifier: NCT04098744). A phase-one study was also conducted to evaluate its safety and pharmacokinetic properties when administered orally in patients with advanced hepatocellular carcinoma (ClinicalTrials.gov identifier: NCT02304289) [235]. One double-blind placebocontrolled trial consisted of giving human colorectal cancer patients oral compound **1a** prior to surgery. During a median follow-up of 42 months, 1 patient in the artesunate group had a recurrence of colon cancer compared to 6 patients in the placebo group [236].

Artemether (**1b**) has also been included in a phase I/IIa study to assess its potential use in treating subjects with advanced solid tumors (ClinicalTrials.gov identifier: NCT02263950) [235]. Another report describes beneficial improvement in a patient with pituitary macroadenoma treated with it for 12 months [237].

The pilot clinical phase I/II trial of dihydroartemisinin (**1c**) [238] against advanced cervical carcinoma indicates that after three weeks of treatment in ten women, the majority showed improvement in the signs and symptoms. This included the vaginal discharge and pain, with no evidence of severe toxicity. These patients had a lower expression of epidermal growth factor receptor (EGFR) and Ki-67 oncogenes.

Furthermore, with the goal of increasing the antineoplastic effect of these drugs, the combination of the usual chemotherapy with artemisinin or its derivatives has been investigated, showing that their multifactorial action on various pathways may improve overall activity [232]. Interestingly, Wang et al. [239], demonstrated that dihydroartemisinin (**1c**) improves the anticancer effect of gemcitabine, a drug used in pancreatic cancer, which develops resistance over time. They confirmed by in vitro and in vivo analysis that compound **1c** induced increased growth inhibition and apoptosis 4- and 2-fold, using both drugs and alone, respectively.

Tilaoui et al. [240] observed a synergistic effect when they used vincristine and artemisinin (**1**), in combination, against murine mastocytoma (P815) cells. A randomized controlled trial with artesunate (**1a**) combine with a chemotherapy regime using vinorelbine plus cisplatin, in patients with advanced non-small cell lung cancer (NSCLL) shown that this treatment can raise the short-term survival rate and prolong the progression time, without extra side effects [241]. Liu et al. [242] found that a combination of artesunate (**1a**) with lenalinomide, commonly used for the treatment of multiple myeloma, caused an impressive enhancement of antineoplastic activity in polyploid cell lines [243], while Singh and Verna [244] described significant improvement and 70% reduction in tumor size after 2 weeks of treating a patient diagnosed with stage II cancer of the larynx with the same compound **1a** (50 mg). Additionally, the first long-term treatment of two patients with metastatic uveal melanoma with artesunate (**1a**), in combination with standard chemotherapy, was reported [245]. The standard therapy alone was ineffective in stopping tumor growth, while the disease was stabilized after adding compound **1a** to this chemotherapy, followed by objective regressions of spleen and lung metastases [245].

Another characteristic of tumors and cancer cells is their ability to develop resistance to chemotherapy due to their rapid cell-division rate and genetic mutations [246]. In this context, Reungpatthanaphong et al. [247] report that artemisinin (**1**), artesunate, and dihydroartemisinin (**1c**), combined with doxorubicin and pirarubicin, increased the cytotoxic effect induced by pirarubicin or doxorubicin only in MDR cell lines. They proposed that artemisinin and its derivatives reverse the MDR phenomenon at the mitochondrial level.

Currently, another series of derivatives are being explored that present a longer plasma life and are more powerful and effective at lower concentrations. These include artemisinin dimers and trimers, hybrid compounds, and tagging of the compounds to molecules that are involved in the intracellular iron-delivery mechanism. These compounds are promising potent anticancer agents that produce significantly less side effects than conventional chemotherapeutic agents [226].

#### *6.2. α-Santonin and Its Derivatives*

Santonin (C15H18O3) (**2**) (Figure 1) is an eudesmanolide sesquiterpene lactone first isolated from A. santonicum by Kahler in 1830 [248], but it is much less known than artemisinin (**1**). Santonin exists in two isomeric forms, α-santonin and β-santonin [249], with α-santonin being the most studied form due to its higher stability [250].

Its isolation and characterization proved an arduous and grueling task for chemists at the time [251]. Its mevalonoid biosynthesis pathways has long been studied, revealing that it is formed by a methylene reduction, C-1 hydroxylation and C-3 oxidation of the precursor costunolide, sharing an identical initial pathway [252,253]. The synthesis of santonin (**2**) was first described by Marshall and Wuts in 1978 [250] and involved the reduction-alkylation of m-toluic acid with lithium in ammonia.

Santonin is the most abundant sesquiterpene lactone in *Artemisia* cina Berg ex Poljakov and was isolated from several *Artemisia* species [254–256], including some edible species such as *A. absinthium*, *A. frigida*, *A. tridentata* [34,255]. *Artemisia* santonicum is commonly referred to as "wormseed", a name historically attributed to the plants' anthelmintic ("worms") activity [251]. Indeed, santonin was one of the most frequently used treatments for intestinal nematode infections up until the 1970s [257]. Nowadays santonin has fallen out of use due to a wealth of better anthelmintic therapeutics [258]. Nevertheless, santonin

has been revealed to have many other bioactivities worthy of investigating. In a recent publication [259] santonin proved to have potent insect growth inhibitory effect on the cotton bollworm, *Helicoverpa armigera*, a widespread pest of significant agricultural and economic impact. In this work, the researchers showed that a 2 mg/mL dose of santonin in wet feed would cause a significant (~80%) decrease in larval weight compared to the control. This is explained by the results obtained from the excised midguts, which revealed that trehalase activity had decreased to 32% of the control in treated larva. Trehalase is an important enzyme in the metabolism of trehalose, an abundant simple sugar in plants. This enzyme inhibitory activity could also be allied with santonin's effect of rupturing insect midgut cell lining [260]. Overall, this shows there is clear signs of a potential value for the compound in possible environmentally friendly pesticidal formulations, as well as a continued interest in the compound. We would also like to note the high quality of this publication, particularly in its clarity of language and presentation of data, something which is sorely missed in similar publications.

In modern times attention has mostly been devoted to α-santonin derivatives as opposed to the actual compound. This is because α-santonin's chemical structure lends itself very well to modifications, the compound a cheap and easy to use platform for drug synthesis [261]. Indeed, two reviews were recently published [256,261] about α-santonin derivatives and how different structural changes affect their bioactivities. In vitro results are much more abundant for santonin derivatives, mainly detailing the synthesis of novel cancer therapeutic agents. Santonin tumor inhibitory derivatives have included a diacetoxy acetal form [262]; spiro-isoxazoline and spiro-isoxazolidine derivatives [263,264]; cinnamic acid derivatives [265], immunosuppressants [266]; anti-inflammatory bromoketone [267–269]. Given the scope of the previously cited reviews, here it will be highlighting the main recently published derivatives showing potent in vivo effects.

Regarding in vivo effects, a recently published work [270] presents an α-santonin derivative (a benzyl ether derivative containing *o*-bromine named (3a*S*,9b*R*)-8-((2-bromobenzyl) oxy)-6,9-dimethyl-3-methylene3a,4,5,9b-tetrahydronaphtho[1,2-b]furan-2(3*H*)-one) (compound **2a**, Figure 1) with potent anti-inflammatory bioactivity. In this work [270], the α-santonin derivative **2a** was synthesized and screened for in vitro activity where it was the most potent one and selected for further in vivo assays. This compound was administered orally to an arthritis rat model with very similar characteristics to rheumatoid arthritis human patients. Rats were treated with doses of 5 and 20 mg/kg (*w*/*w*) per day of the selected derivative **2a** and monitored for arthritic disease progression. Results showed a significant improvement of arthritis symptomatology in the treated rats, with ~40% lower clinical disease scores (associated with the degree of limb swelling) and significant reduction of hind paw volume. It is also worth noting that the response seems to not be dose-dependent, since both doses tested revealed almost identical activity. After mechanistic studies, the authors attribute this effect to the selective bonding of the derivative with the active site of UbcH5c, a key enzyme for ubiquitination during TNF-α-triggered activation of the NF-κB inflammatory pathway. These results seem to indicate the potent anti-inflammatory bioactivity of this novel derivative **2a**, with potential future applications in the development of a new anti-rheumatoid arthritis (RA) drug.

However, some criticism can be levelled at the work of Chen et al. [270], primarily for the way the researchers measure disease progression; their score system seems simplistic and overly reliant on somewhat subjective and qualitative observation (swelling vs. no swelling), rather than more quantitative records and measures, rendering it quite unwieldy and difficult to understand. Another addition to this work we consider beneficial would have been to include a positive control. By including a known and widely used antirheumatic in this in vivo assay, we could assess the bioactivity of this novel compound by comparison with a known therapeutic. We believe more studies are required in order to better understand this new agent, and hope to see clinical trials in the near future.

Novel immunosuppressant drugs are currently in very high demand due to the high cost and serious side effects of currently used therapeutics [271]. A very interesting

research was published in 2017 [272], exhibiting a novel α-santonin derivative with in vivo immunosuppressant properties. In this paper [272] the authors describe the synthesis of several O-aryl/aliphatic ether, ester and amide α-santonin derivatives and the in vitro assay for immunosuppressant bioactivity, showed one particular compound exhibiting ~75–80% proliferation suppression rates for B and T lymphocytes. This derivative, a trimethyl acetate ester α-santonin analogue (compound **2b**, Figure 1), was used for in vivo immunosuppression testing using BALB/c mice. Rats were injected with 6.25 mg/kg (*w*/*w*), 12.5 mg/kg (*w*/*w*) and 25 mg/kg (*w*/*w*). Rat humoral immune response was assessed by quantification of post-challenge antibody production and cell mediated immune response was assayed by post-challenge left hind footpad thickness measurement. Results were impressive: humoral response was suppressed by 28% with the lowest dose (6.25 mg/kg (*w*/*w*)), and 41% at the highest dose (25 mg/kg (*w*/*w*)); and cell mediated immune response was suppressed by ~30% in the medium and highest doses. These results were comparable to the positive control cyclophosphamide regarding humoral response but fell considerably shorter of the positive control in the cell mediated response assay. Nevertheless, these results show that the novel derivative synthesized is capable of both humoral and cell mediated immune suppression, with reasonable potency, making it a prime candidate for future drug development. More tests with this compound, particularly exploring the mode of action, which was not presented by Dangroo et al. [272] and future clinical trials are expected.

From these studies and cited reviews, it is concluded that santonin and its derivatives are highly interesting, with new papers and bioactivities being constantly researched and published. It is expected to see some of these compounds enter pre-clinical testing soon.

#### *6.3. Achillin*

Achillin (**3**) (Figure 1) a sesquiterpene lactone (C15H18O3) of the guaianolide class, first identified in *Achillea millefolium* L. (syn *Achillea lanulosa* Nutt.) in 1963 by White and Winter [273] and synthesized in 1967 [274].

Although achillin *(***3**, Figure 1*)* is mainly extracted from plants of the *Achillea* genus, it has also been identified in edible *Artemisia* species such as *A. capillaris* [275], *A. frigida* [276], *Artemisia feddei* H. Lév. & Vaniot [277] and *A. ludoviciana* [278]. It has also been identified in plants of other species, such as *Taraxacum platycarpum* [279] and *Anthemis scrobicularis* [280]. Achillin biosynthesis pathway starts with the eudesmane skeleton of α-santonin, and involves the hydrolysis of an acetate precursor, followed by epimerization at C-11 and finishing with an allylic oxidation [281].

The first bioactivity for achillin (**3**) described it as a strong antifeedant agent against two grasshopper species [282]. This study showed that a concentration of only 0.5% (% dry weight) of compound **3** was enough to repell *Melanoplus sanguinipes* from feeding. This antifeedant effect was measured qualitatively rather than quantitatively, so it is difficult to accurately assess the full extent of the antifeedant bioactivity. Nevertheless, the rather limited preliminary study [282] was enough to show there was potential application to achillin (**3**). Subsequent in vitro assays followed, showing it had anti-allergic effect (IC50 = 100 μM) [279]; increasing chemosensitivity to paclitaxel, with potency comparable to known therapeutics at 100 μM concentration [283]; and antitumor against endocervical cell lines (IC50 = 160.3 μg/mL after 72 h [48].

Achillin (**3**) has proven to possess interesting and potent bioactivities tested in vivo. Firstly, the work of Rivero-Cruz et al. [278] showed compound **3** had potent antinociceptive effect in mice assessed using the formalin test. This work started by focusing on the effects of *A. ludoviciana* (edible species), showing it had strong in vivo analgesic and anti-inflammatory effects. Subsequent analysis by HPLC attributed the bioactivity to the presence of two sesquiterpene lactones being one of them achillin and the other dehydroleucodin. Regarding achillin (**3**), it was individually tested using the formalin assay and showed significant activity causing near 50% reduction in formalin wound time with a dose of 17.7 mg/kg (*w*/*w*). Researchers did not specify a mode of action for this

activity but cited a previous work [284] attributing the effect to the NF-κB inhibition by sesquiterpene lactones. Another point of critique to the work of Rivero-Cruz et al. [278] would be the heavy emphasis on assaying the plant extract as opposed to the isolated compounds. More extensive testing with the purified compounds would be desirable, producing far more relevant and compelling results. Nevertheless, this work proved achillin possesses interesting antinociceptive bioactivity, which should be studied further, particularly in finding out its mode of action.

Another highly relevant study with achillin (**3**) in animal model was its effect as an inhibitor of meiosis in toad (*Rhinella arenarum*) oocytes was carried out by Zapata-Martínez et al. [285], where denuded toad oocytes were exposed to different concentrations of achillin prior to the necessary hormonal stimulus necessary for meiotic resumption. Results showed a marked overall decrease of germinal vesicle breakdown (GVBD) in occytes with exposure to **3**. It is also worth noting that the response was dose-dependent; a 6 μM concentration of achillin (**3**) showed a ~10% reduction of GVBD compared the control, whereas a 36 μM concentration reduced GVBD by ~35% more than control oocytes. So, it appears achillin (**3**) has meiosis inhibiting potential, which the authors [285] attribute to the covalent bonds formed between achillin's partially electrophilic center and the nucleophilic center of target molecules. The meiotic inhibitors have been shown to improve human embryonic development in in-vitro fertilization procedures by allowing the embryo to have enough time to finish cytoplasmic maturation [286].

Finally, we present a very recent 2020 paper by Arias-Durán et al. [287] showing achillin's potential as a smooth muscle cell relaxant. The authors used an ex vivo rat trachea model, where the relaxing effects of increasing concentrations of achillin (**3**) were measured in the rat trachea rings. The results showed achillin (**3**) exhibited almost identical activity to theophylline, a widely used medicine for asthma and chronic obstructive pulmonary disease. The effect seems to be mainly due to a release of nitric oxide and calcium channel blockade influx into the smooth muscle cells of the tracheal rings [286]. This result indicates the great potential as a smooth cell muscle relaxant, with possible applications in the treatment of asthma, bronchospasm and chronic bronchitis.

In conclusion, achillin (**3**) exhibits very interesting bioactivities, mainly anti-inflammatory, meiotic inhibitor, and tracheal relaxant. It is also worth noting that this compound has also been identified as the possible bioactive agent behind interesting *Artemisia* extracts bioactivities, such as allelopathic [276] and anticarcinogenic [275]. It would be very interesting to follow-up on these results with more assays involving the purified compound, although such would possibly require biosynthetic methods, since it is reportedly very difficult to obtain large quantities of purified achillin (**3**) [275]. Nevertheless, there is great potential for its future drug development, but further research is needed.

#### *6.4. Tehranolide*

Tehranolide (C15H22O6) (**4**) (Figure 1), also sometimes referred to as artediffusin, is a sesquiterpene lactone first isolated from the aerial parts of *Artemisia diffusa* Krasch. ex Poljakov (edible) [288]. Currently, based on The Plant List database, *A. diffusa* name is a synonym of *Seriphidium diffusum* (Krasch. ex Poljak.) Y.R. Ling.

A biosynthesis pathway has been suggested by [289], involving the oxidative cleavage of the Δ<sup>4</sup> bond of a eudesmanolide derivative, followed by an internal aldol condensation, rearrangement by hydroxy addition and ending with acetal formation. Chemical synthesis has not been described for compound (**4**) which explains why work done with this compound is predominantly centered in a certain few labs and research groups geographically close to the relatively narrow *A. diffusa* natural distribution. There is continued interest in the eudesmanolide chemical synthesis and its derivatives [290,291].

Structurally, tehranolide (**4**) exhibits great similarity to artemisinin (**1**), including the presence of an endoperoxide (C-O-O-C) bridge common to both compounds. This is very noteworthy, since the endoperoxide group is reported to be vital to artemisinin antimalarial activity [202]. This established connection between structure and function lead to

the hypothesis that compound **4** could have antimalarial activity, which was investigated by Rustaiyan et al. [292–294], with in vivo results using the purified compound. In this work, the authors use NMRI infected with *Plasmodium berghei*, a widely used malaria animal model organism. The mice were injected daily with doses ranging from 1.7 g to 17 mg (total dose) of HPLC purified tehranolide (**4**). Results showed the lowest dose (17 mg) significantly reduced *P. berghei* parasitemia by ~10% (compared to negative control) 2 days after infection while the highest dose (1.7 g) significantly reduced it by ~14%. It is important to note that, even though dosages varied by a factor of 100, differences between doses were relatively small, and became almost imperceptible 10 days after infection (while still maintaining ~10% difference to negative control after 10 days). We can then conclude that although some dose-dependence effect was exhibited, it was relatively small, and that over time all doses showed almost identical effect when compared between each other.

Some criticism can be made to this work, particularly the fact that total doses were uniformly used instead of doses adjusted to mouse weight. This means that mouse weight fluctuations translated into differing compound systemic concentrations (i.e., "lighter" mice will exhibit higher compound concentrations than "heavier" mice, which could result in exacerbated effect). This ended up not being very relevant, since the results showed relatively small difference between doses, but could have definitely cast doubt upon the results if compound dose-dependency was more exuberant and should be avoided in future assays. Other points open to criticism could be the use of a relatively small sample size (*n* = 5, in triplicate), and the lack of usage of a positive control, in order to compare compound **4***-* s efficacy against known used therapeutics. Nevertheless, the work serves to prove that the in vivo anti-malarial bioactivity.

The mode of action for this compound has not been specifically described but, it is thought to be very similar to artemisinin's (**1**), due to the similar structure [288]. Mechanistically, the antimalarial bioactivity results from the presence of haem or Fe2+ resulting from the *P. falciparum* hemolysis. The Fe2+ functions as a catalyst to the opening of the peroxide bridge of the compound, which leads to the formation of free radicals, alkylating *P. falciparum* proteins and eventually causing parasite death.

The bioactivity presented above exhibited by tehranolide (**4**) is very interesting and relevant to current society when consider the ever-increasing *Plasmodium falciparum* antimalarial drug resistance [295]. There is indeed an urgent need for novel antimalarial compounds which exhibit potential as a possible therapeutic and the compound **4** was considered a very promise antimalaria agent [296]. Given these facts, it would be expected to see more work done with this compound, hopefully aiming at pre-clinical testing.

Tehranolide (**4**) has also proven to be a very promising anticancer agent, with several recent publications supporting in vivo effect. Much like before, the initial hypothesis was derived from the structural similarity between compound **4** and artemisinin (**1**), whose anticancer activity is discussed above. The earliest account of **4** anticancer bioactivity was described by Noori et al. [297,298]. These papers are arguably some of the most important works on this subject, due to the in vivo nature of the assays and the immunomodulatory insights provided. In summary, a total dose of 5.64 μg of tehranolide (**4**) per mouse was injected intratumor in Balb/c breast cancer mouse models. Results showed this dose significantly inhibited tumor growth by ~75% compared to negative control. Treated animals also exhibited ~2.5× increase in lymphocyte proliferation, as well as ~12% decrease in CD4+CD25+Foxp3+ regulatory T cells, an important factor in tumor tolerance [299], when compared with negative control. These results showed compound **4** had potent in vivo anticancer activity and great potential as an immunotherapeutic regulator. Subsequent follow-up work by the same research group [300] with similarly mice treated (same dose and conditions) revealed a ~80% increase in apoptosis index of tumor cells compared to untreated mice. This result confirms compound **4** indeed possesses in vivo antitumor activity by apoptosis induction (attributed to a tumor cell selective G0/G1 cycle arrest. Finally, Noori et al. [301] describe in detail the mechanism behind compound **4** anticancer activity as being linked to calmodulin and phosphodiesterase type 1 inhibition, as well

as cAMP-dependent protein kinase A activity. These processes allow tehranolide (**4**) to selectively inhibit tumor proliferation and induce tumor cell apoptosis.

In conclusion, compound **4** has proven to have great potential as an antimalarial but mainly as anticancer therapeutic. Both of these bioactivities are extremely relevant and important in the modern medical/scientific paradigm, because of the need for novel antimalarials as previously mentioned, as well as rising cancer rates [302]. We hope to see tehranolide (**4**) progress into pre-clinical stages of research with regards to both of these activities.

#### **7. Hotpoint Research:** *Artemisia* **Species and Its Constituents as Strategy to Treat COVID-19 Infection**

Caused by a member of the Coronavirus family (CoV), SARS-CoV-2 (COVID-19) has recently posed a potential threat to the survival of human beings on Earth and was declared a global health emergency by the WHO [303]. The therapeutic strategy to treat infection by this coronavirus has used the knowledge and experience acquired in the previous epidemics caused by SARS-CoV-1 and MERS-CoV. To date, there are no vaccines or specific antiviral agents against coronavirus infections, so it is a great challenge for scientists to find treatments for them. Repositioning of drugs already in clinical use is being studied, as a quick response to provide effective treatments in humans and assess other compounds that may be effective against the virus [304]. The WHO proposed *A. annua* as a possible treatment to be considered for COVID-19 treatment, however its efficacy and side-effects must be determined. Additionally, *A. annua* is one of the of Jinhua Qinggan granule ingredients, one of the Traditional Chinese Patent Medicines recommended in 13 therapeutic regimens of COVID-19 in China [305]. The *A. argyi* it was also mentioned as one of the plants that can be used by aromatherapy method of Traditional Chinese Medicine with effects of contagion prevention [306]. Scientific evidence supporting this proposal is partly based on bioactive compounds present in the plant with antiviral effects against hepatitis B, bovine viral diarrhea, and Epstein–Barr virus [307]. It also contains compounds with antioxidant, anti-inflammatory and immunomodulatory properties [176], which would play an important role in controlling the acute inflammatory process triggered by Covid-19 infection. Another research line requiring further attention in the context of acute Covid-19 is the efficacy of the artesunate to ameliorate bleomycin-induced pulmonary fibrosis pathology in rats, possibly by inhibiting profibrotic molecules [308]. Other promising pointers are that *A. annua* showed significant activity in vitro against SARS-CoV-1-2002 (IC50 = 34.5 ± 2.6 μg/mL) [309,310]. Tea infusions of *A. annua* and *A. afra* were found to be highly active against HIV virus, although the role of artemisinin is rather limited [311], as were various *Artemisia* species against Herpes simplex virus type 1 [312] and *A. capillaris* against Hepatitis B [169]. In a recent preliminary in silico study, Rolta et al. [313] evaluated the possibility of binding artemisinin (among other compounds) to the cellular ACE-2 receptors via the spicules of the SARS-CoV-2 membrane, as well as ADMET prediction and toxicity analysis. The results obtained support the possibility that artemisinin can act as an antiviral by means of a predictable binding to the receptor, also being non-carcinogenic, non-cytotoxic and safe to administer.

Clearly, greater scientific attention is placed and needed toward *A. annua* and its bioactive compound/derivatives in addressing the treatment of COVID-19 and they need to be further assessed in clinical trials. Kapepula et al. recently published a review drawing attention to the path to be followed and the errors to avoid [314]. And this assessment already started. The phase II clinical study is currently underway (ClinicalTrials. gov identifier: NCT04530617, recruitment), aiming to evaluate the efficacy of *A. annua* and camostat to inhibit viral entry or replication of SARS-CoV-2 virus and their toxicity, administered immediately after COVID-19 positive testing, in mild to moderate disease patients and with high-risk factors such as diabetes, hypertension and obesity, among others.

#### **8. Conclusions**

This review starts to present an overview of *Artemisia* species traditional use as food, spices, condiment and beverage. The plants are mainly used in salads and tea, as well as to flavoring food and beverages. The leaves are the most used edible part, but the aerial parts, mentioned as "herb", is also widely used. The nutritional value of *Artemisia* species is also presented and discussed, based on the fatty acid, proteins, sugars, minerals, and vitamin contents reported in the literature.

Studies already published show that the use of *Artemisia* plants is not risk-free. The allergic reactions, mainly allergic rhinitis caused by pollens and skin dermatitis caused by the presence of sesquiterpene lactones, are the most frequently reported and studied adverse effects. Absinthe drinks are reported as causing some adverse side-effects, so its content is legislated. Some *Artemisia* species cause reduction of fertility, so its use is not recommended during pregnancy.

The evaluation, based on clinical studies, of the *Artemisia* formulations effectiveness remains a hot topic. The application of integral plants or extracts as anti-inflammatory agents is deepened and the spectrum of applications broadened to, for example, the preventive effect on hepatitis B cirrhosis, treatment of malaria, anti-allergenic and glycemic control.

Concerning the *Artemisia* constituents, clinical and in vivo studies involving artemisinin and its derivatives show them as efficient antimalarial and anticancer agents. Additionally, the additive or synergistic interactions of artemisinin and derivatives in combination with a wide array of clinically established drugs to combat different cancer are highlighted. The high therapeutic potential is evident in the WHO proposal to investigate artemisinin and derivatives as well as *A. annua* to the treatment of Covid-19 infection. In addition to artemisinin and its derivatives, other sesquiterpene lactones isolated from different species of *Artemisia*, such as santonin, achillin and tehranolide, have been the target of further studies with a view to the development of new derivatives and their application as medicines. These compounds exhibit very interesting activities, in in vivo models, such as immunosuppressant and anti-inflammatory and potent antinociceptive effect. Achillin acts as a meiotic inhibitor and smooth muscle cell relaxant, properties very relevant to improve human embryonic development in-vitro fertilization procedures and to treat asthma and chronic obstructive pulmonary disease, respectively.

Nevertheless, although this review shows the great potential for *Artemisia* species as dietary supplements, functional foods, source of new and more efficient and safe drugs, further research on action mechanism and involving clinical trials on toxicity, adverse side-effects, efficacy and health care uses continue to be needed.

**Author Contributions:** Conceptualization, A.M.L.S. and A.T.; writing—original draft preparation, A.M.L.S., A.T., L.M.M., P.M.C.S.; writing—review and editing, A.M.L.S., A.T. and L.M.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by FCT–Fundação para a Ciência e a Tecnologia, the European Union, QREN, FEDER, COMPETE, by funding the cE3c centre (UIDB/00329/2020) and the LAQV-REQUIMTE (UIDB/50006/2020) research units; Funded by RTI2018-094356-B-C21 Spanish MINECO project, co-funded by European Regional Development Fund (FEDER); And funded by the project EthnoHERBS (H2020-MSCA-RISE-2018, No. 823973).

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Thanks are due to the University of Azores, University of La Laguna and Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

1. The Plant List, a Working List of All Plant Species. Available online: http://www.theplantlist.org/tpl1.1/search?q=Artemisia (accessed on 24 June 2020).


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