Understanding the Ongoing Threat of SARS-CoV-2
SARS-CoV-2, the virus responsible for COVID-19, emerged in late 2019 and quickly became a global health emergency. From 2020 to 2023, it caused widespread illness, death, and disruption across nearly every sector of society. The pandemic also highlighted both the potential and limitations of antiviral therapies. While some repurposed drugs like hydroxychloroquine and ivermectin were initially promising, they ultimately lacked clinical efficacy and are no longer recommended for treatment.
Currently available antiviral treatments include monoclonal antibodies and direct-acting drugs such as nirmatrelvir–ritonavir, remdesivir, and molnupiravir. However, each has limitations, including drug–drug interactions, the need for intravenous administration, and reduced effectiveness against emerging variants.
As the virus continues to evolve, the scientific community is under pressure to discover new antiviral agents that are effective, scalable, and adaptable to mutating viral strains. Among natural products, Artemisia annua L. has re-emerged as a candidate worth investigating.
The Role of Artemisia annua in Antiviral Research
A. annua has long been recognized for its medicinal properties. Best known as the plant source of artemisinin, a key antimalarial compound, it has also shown promise in antiviral research. During the 2002–2003 SARS outbreak, it was ranked second in efficacy among 200 tested herbal remedies.
Recent in vitro studies have demonstrated that ethanolic and hot water extracts of A. annua can inhibit several SARS-CoV-2 strains, including Alpha, Beta, Gamma, Delta, Kappa, and various Omicron sub-variants. However, until now, the exact compounds responsible for these effects remained unclear.
New Research Focus: Arteannuin B
A team of researchers from Bosnia and Herzegovina, Serbia, and the United States conducted a detailed investigation into the antiviral properties of two A. annua-derived compounds: artemisinin and arteannuin B.
Their goal was to isolate these sesquiterpenoids using high-speed countercurrent chromatography (HSCCC) and test their in vitro antiviral activity against a genomically characterized Alpha SARS-CoV-2 variant—specifically, the hCoV-19/Bosnia and Herzegovina/VFS-UNSA-LMGFI031/2021 strain.
This variant carries mutations associated with increased infectivity and transmissibility, including N501Y and deletions at spike protein positions 69-70 and 144.
Targeted Extraction and Identification
The team began by extracting A. annua leaves using two methods:
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Ultrasonic ethanol extraction
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Supercritical carbon dioxide (SC-CO₂) extraction
The SC-CO₂ method yielded significantly higher levels of artemisinin (14.65 µg/mg) than the ethanol method (6.87 µg/mg), confirming its superior efficiency.
HSCCC allowed the researchers to isolate arteannuin B and artemisinin in a single chromatographic step. Using techniques such as LC-ESI-QTOF-MS/MS and GC–MS, they confirmed the chemical identity and purity of each compound. Arteannuin B content was particularly high in both extracts—27.18% in the ethanol extract and 18.95% in the SC-CO₂ extract.
Notably, arteannuin B was effectively separated from artemisinin, enabling the creation of an artemisinin knockout fraction, free of artemisinin but rich in other sesquiterpenoids.
In Vitro Testing Against the Alpha Variant
The antiviral activity of the extracts and purified fractions was evaluated in Vero E6 cells infected with the Alpha SARS-CoV-2 variant. The researchers tested the following:
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SC-CO₂ extract
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Ethanol crude extract
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Artemisinin knockout fraction
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Purified arteannuin B fraction
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Pure artemisinin
Key Results (EC₅₀ – Half-maximal Effective Concentration)
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Arteannuin B fraction: 38.1 µg/mL
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SC-CO₂ extract: 93.7 µg/mL
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Ethanol extract: 173.5 µg/mL
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Artemisinin knockout fraction: 187.3 µg/mL
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Pure artemisinin: >100 µg/mL (not significantly active)
At concentrations of 50 and 100 µg/mL, the arteannuin B fraction significantly inhibited viral replication, reducing amplification of SARS-CoV-2 N and RdRp genes by 84% and 100%, respectively.
Cytotoxicity and Cell Viability
To ensure safety, a MTT assay was performed to assess cell viability after exposure to the extracts. No significant cytotoxic effects were observed below 200 µg/mL for any sample, and all test samples maintained cell viability above 85% at effective antiviral concentrations.
The IC₅₀ values for cytotoxicity were:
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SC-CO₂ extract: 191.8 µg/mL
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EtOH extract: 239.1 µg/mL
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Arteannuin B fraction: >200 µg/mL
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Artemisinin: >200 µg/mL
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Knockout fraction: >400 µg/mL
This confirms that arteannuin B exhibits strong antiviral activity at non-toxic concentrations, making it a promising candidate for further investigation.
Why Arteannuin B Stands Out
The research provides direct evidence that arteannuin B, not artemisinin, is likely the primary contributor to A. annua’s antiviral effects against SARS-CoV-2.
Interestingly, the artemisinin knockout fraction still showed measurable antiviral activity, supporting earlier findings that antiviral efficacy may correlate inversely with artemisinin content. Studies on Artemisia afra, which does not contain artemisinin, further reinforce this conclusion.
Mechanistically, arteannuin B is known to inhibit the main protease (Mpro) of SARS-CoV-2 through covalent bonding with its cysteine residue, potentially blocking viral replication pathways.
Implications and Future Directions
The study demonstrates the feasibility of using HSCCC and SC-CO₂ extraction for obtaining high-purity antiviral compounds from A. annua. This method is efficient, scalable, and avoids the use of toxic solvents.
Moreover, arteannuin B shows promise as a broad-spectrum antiviral candidate, particularly for SARS-CoV-2 variants carrying similar mutations. The researchers note:
“An important contribution of this study is the demonstration of the antiviral activity of arteannuin B against the Alpha variant of SARS-CoV-2, which is known to have increased infectivity and transmissibility.”
The results also underscore the importance of further studies in animal models to assess pharmacokinetics and clinical relevance.
Final Thoughts
This research builds a strong case for re-evaluating natural products like Artemisia annua as sources of antiviral agents. Through precise extraction and in vitro evaluation, the study identifies arteannuin B as a compound with strong potential to counter SARS-CoV-2, especially variants with increased transmission.
With growing interest in plant-based antivirals, this work highlights the value of integrating advanced chemical profiling with targeted bioassays. Future testing against Omicron sub-variants and other strains will be essential to establish the full antiviral spectrum of arteannuin B.
The translation of the preceding English text in Bosnian:
Razumijevanje stalne prijetnje virusa SARS-CoV-2
SARS-CoV-2, virus odgovoran za bolest COVID-19, pojavio se krajem 2019. godine i brzo je prerastao u globalnu zdravstvenu krizu. Od 2020. do 2023. godine izazvao je široko rasprostranjeno obolijevanje, smrtne ishode i poremećaje u gotovo svim sektorima društva. Pandemija je također ukazala na potencijal, ali i ograničenja antivirusnih terapija. Iako su neki postojeći lijekovi, poput hidroksihlorokina i ivermektina, u početku djelovali obećavajuće, na kraju nisu pokazali kliničku efikasnost i više se ne preporučuju za liječenje.
Trenutno dostupni antivirusni tretmani uključuju monoklonska antitijela i direktno djelujuće lijekove poput nirmatrelvir–ritonavira, remdesivira i molnupiravira. Ipak, svaki od njih ima određena ograničenja, uključujući interakcije s drugim lijekovima, potrebu za intravenskom primjenom i smanjenu efikasnost protiv novih varijanti virusa.
Kako se virus i dalje razvija, naučna zajednica je pod pritiskom da otkrije nove antivirusne agense koji su efikasni, mogu se proizvoditi u većim količinama i prilagodljivi su mutacijama virusnih sojeva. Među prirodnim proizvodima, biljka Artemisia annua L. ponovo je prepoznata kao kandidat vrijedan daljnjeg istraživanja.
Uloga biljke Artemisia annua u antivirusnim istraživanjima
Artemisia annua je već dugo poznata po svojim ljekovitim svojstvima. Najpoznatija je kao izvor artemisinina, ključnog antimalarijskog jedinjenja, ali je također pokazala potencijal u antivirusnim istraživanjima. Tokom epidemije SARS-a 2002–2003. godine, zauzela je drugo mjesto po efikasnosti među 200 ispitanih biljnih preparata.
Nedavne in vitro studije pokazale su da etanolni i ekstrakti u toploj vodi iz A. annua mogu inhibirati više sojeva virusa SARS-CoV-2, uključujući Alfa, Beta, Gama, Delta, Kapa i različite Omikron podvarijante. Međutim, do sada nije bilo jasno koja tačno jedinjenja su odgovorna za te efekte.
Novi fokus istraživanja: Arteanuin B
Tim istraživača iz Bosne i Hercegovine, Srbije i Sjedinjenih Američkih Država proveo je detaljnu studiju antivirusnih svojstava dvaju jedinjenja iz A. annua: artemisinina i arteanuina B.
Cilj im je bio da izoliraju ove seskviterpenoide pomoću hromatografije u protustruji velikom brzinom (HSCCC) i ispitaju njihovu in vitro antivirusnu aktivnost protiv genomskim analizama potvrđene Alfa varijante SARS-CoV-2 – konkretno soja hCoV-19/Bosnia and Herzegovina/VFS-UNSA-LMGFI031/2021.
Ova varijanta nosi mutacije povezane s povećanom zaraznošću i prenosivošću, uključujući N501Y i delecije na pozicijama 69-70 i 144 spike proteina.
Ciljana ekstrakcija i identifikacija
Tim je započeo ekstrakciju listova A. annua koristeći dvije metode:
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Ultrazvučna ekstrakcija etanolom
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Superkritična ekstrakcija ugljičnim dioksidom (SC-CO₂)
SC-CO₂ metoda dala je značajno viši nivo artemisinina (14,65 µg/mg) u poređenju s etanolnom metodom (6,87 µg/mg), potvrđujući njenu veću efikasnost.
HSCCC je omogućio istraživačima da u jednoj hromatografskoj fazi izoliraju arteanuin B i artemisinin. Upotrebom tehnika poput LC-ESI-QTOF-MS/MS i GC–MS, potvrđeni su hemijski identitet i čistoća svake supstance. Sadržaj arteanuina B bio je posebno visok u oba ekstrakta – 27,18% u etanolnom ekstraktu i 18,95% u SC-CO₂ ekstraktu.
Važno je napomenuti da je arteanuin B efikasno odvojen od artemisinina, što je omogućilo stvaranje frakcije bez artemisinina, bogate drugim seskviterpenoidima.
In vitro testiranje protiv Alfa varijante
Antivirusna aktivnost ekstrakata i pročišćenih frakcija ispitana je u Vero E6 ćelijama zaraženim Alfa varijantom SARS-CoV-2. Testirani su sljedeći uzorci:
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SC-CO₂ ekstrakt
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Sirovi etanolni ekstrakt
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Frakcija bez artemisinina
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Pročišćena frakcija arteanuina B
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Čisti artemisinin
Ključni rezultati (EC₅₀ – koncentracija potrebna za 50% maksimalnog efekta):
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Frakcija arteanuina B: 38,1 µg/mL
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SC-CO₂ ekstrakt: 93,7 µg/mL
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Etanolni ekstrakt: 173,5 µg/mL
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Frakcija bez artemisinina: 187,3 µg/mL
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Čisti artemisinin: >100 µg/mL (nije pokazao značajnu aktivnost)
Na koncentracijama od 50 i 100 µg/mL, frakcija arteanuina B značajno je inhibirala replikaciju virusa, smanjujući umnožavanje SARS-CoV-2 N i RdRp gena za 84%, odnosno 100%.
Citotoksičnost i održivost ćelija
Kako bi se osigurala sigurnost, izveden je MTT test za procjenu održivosti ćelija nakon izlaganja ekstraktima. Nisu zabilježeni značajni citotoksični efekti ispod 200 µg/mL ni za jedan uzorak, a svi su uzorci zadržali održivost ćelija iznad 85% na efektivnim antivirusnim koncentracijama.
IC₅₀ vrijednosti za citotoksičnost bile su:
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SC-CO₂ ekstrakt: 191,8 µg/mL
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Etanolni ekstrakt: 239,1 µg/mL
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Frakcija arteanuina B: >200 µg/mL
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Artemisinin: >200 µg/mL
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Frakcija bez artemisinina: >400 µg/mL
Ovi rezultati potvrđuju da arteanuin B pokazuje snažnu antivirusnu aktivnost pri netoksičnim koncentracijama, čineći ga obećavajućim kandidatom za daljnja istraživanja.
Zašto se arteanuin B izdvaja?
Istraživanje pruža direktan dokaz da je arteanuin B, a ne artemisinin, vjerovatno glavni nosilac antivirusnog efekta A. annua protiv SARS-CoV-2.
Zanimljivo je da je i frakcija bez artemisinina pokazala mjerljivu antivirusnu aktivnost, što podržava ranije pretpostavke da antivirusna efikasnost može biti obrnuto proporcionalna sadržaju artemisinina. Studije na Artemisia afra, biljci koja ne sadrži artemisinin, dodatno potvrđuju ovu tvrdnju.
Na molekularnom nivou, poznato je da arteanuin B inhibira glavni proteazni enzim (Mpro) virusa SARS-CoV-2 kovalentnim vezivanjem za njegovu cisteinsku reziduu, potencijalno blokirajući putanje virusne replikacije.
Implikacije i budući pravci
Studija pokazuje da je moguće koristiti HSCCC i SC-CO₂ ekstrakciju za dobijanje visoko čistih antivirusnih jedinjenja iz A. annua. Ova metoda je efikasna, može se primijeniti u većem obimu i izbjegava korištenje toksičnih rastvarača.
Također, arteanuin B pokazuje potencijal kao antivirusni agens širokog spektra, naročito protiv SARS-CoV-2 varijanti koje nose slične mutacije. Istraživači navode:
„Važan doprinos ove studije je dokaz antivirusne aktivnosti arteanuina B protiv Alfa varijante virusa SARS-CoV-2, za koju je poznato da ima povećanu infektivnost i prenosivost.“
Rezultati također naglašavaju potrebu za daljim istraživanjima na životinjskim modelima kako bi se procijenila farmakokinetika i klinička relevantnost.
Zaključak
Ovo istraživanje daje snažan argument za ponovno razmatranje prirodnih proizvoda poput Artemisia annua kao izvora antivirusnih agenasa. Kroz preciznu ekstrakciju i in vitro testiranja, studija identifikuje arteanuin B kao jedinjenje s velikim potencijalom za suzbijanje SARS-CoV-2, posebno varijanti s povećanom prenosivošću.
Uz sve veći interes za biljne antiviruse, ovaj rad ističe važnost integracije naprednog hemijskog profiliranja s ciljanom biološkom evaluacijom. Buduća testiranja protiv Omikron podvarijanti i drugih sojeva biće ključna za utvrđivanje punog antivirusnog spektra djelovanja arteanuina B.
Reference:
Irma Gušić, Ilma Terzić, Toni Eterović, Adis Softić, Šejla Goletić, Teufik Goletić, Dejan Nikolić, Emina Korić, Katarina Bijelić, Haris Nikšić, Senka Vidović, Kemal Durić
Profiling of sesquiterpenoid fractions from Artemisia annua L. and testing their in vitro anti-SARS-CoV-2 activity.
Biomol Biomed [Internet]. 2025 Apr. 25 [cited 2025 Aug. 15];
Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/12052
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