Review of Literature on the anti-SARS2 Activity of Whole Leaf Artemisia annua

This review suggests the need for a new proposed testing method

This research is presented to the blogger and researcher DoorlessCarp in a research collaboration effort on the anti-SARS2 potential of the whole leaf of Artemisia annua.

Brief summaries of papers on A. annua regarding the effectiveness of A. annua in fighting malaria, SARS1, and SARS2 are included. The bioavailability of whole leaf A. annua versus chemical derivatives is briefly discussed. Press releases from the World Health Organization on A. annua are also included in this review.

Based on my review of these papers and topics, I propose a different testing method of the anti-SARS2 potential of whole leaf A. annua than the test methods used in the studies that I have reviewed.

REVIEW OF LITERATURE OF ANTIMALARIAL ACTIVITY OF ARTEMISIA ANNUA

In the 1960s, the Chinese military began a research project of ancient literature on herbal medicine to find a more effective treatment for malaria, which at the time were derivatives of cinchona bark (such as quinine, chloroquine, and hydroxychloroquine).   

Tu Youyou led the effort at the Institute of Chinese Materia Medica. She reviewed over 2,000 ancient Chinese herbal recipes. From 1969 until 1971, she tested over 100 chemical extracts on rodent malaria with poor results. She once again reviewed the literature and discovered the key that led to her Nobel Prize for Medicine in 2015.

Tu Youyou – Biographical - NobelPrize.org

In Ge Hong’s A Handbook of Prescriptions for Emergencies (肘后备急方), I noticed one sentence “A handful of Qinghao immersed in two liters of water, wring out the juice and drink it all” (青蒿一握, 以水二升渍, 绞取汁, 尽服之) when Qinghao was mentioned for alleviating malaria fevers. Most herbs were typically boiled in water and made into a decoction before taken by the patients.

This unique way of using Qinghao gave me the idea that heating during extraction might have destroyed the active components and the high temperature might need to be avoided in order to preserve the herb’s activity. Ge Hong’s handbook also mentioned “wring out the juice.” This reminded me that the leaf of Qinghao might be one of the main components prescribed. I redesigned experiments in which the stems and leaves of Qinghao were extracted separately at a reduced temperature using water, ethanol and ethyl ether.

We produced extracts from different herbs including Qinghao using the modified process and subsequently tested those ethyl ether, ethanol and aqueous extracts on rodent malaria. On October 4, 1971, we observed that sample number 191 of the Qinghao ethyl ether extract showed 100% effectiveness in inhibiting malaria parasites in rodent malaria. In subsequent experiments, we separated the extracts into a neutral portion and a toxic acidic portion. The neutral portion showed the same effect when tested in malaria-infected monkeys between December 1971 and January 1972.

Youyou's team later isolated artemisinin from their "cold" extract of A. annua. Many other chemical derivatives of A. annua have been subsequently developed.

THE WORLD HEALTH ORGANIZATION'S POSITION ON THE USE OF WHOLE LEAF AND CHEMICAL DERIVATIVES OF A. ANNUA

In November 2001, The World Health Organization met in Shanghai, China to discuss treatments of malaria using chemicals derived from A. annua.

The WHO report recommended that several chemicals derived from A. annua, such as artemether, artesunate, dihydroartemisinin, should be developed for with other drugs in combination to treat malaria. These combination drugs have come to be referred to as Artemisinin-based combination therapy (ACT). The WHO did not recommend that two chemicals derived from the same plant should be used in combination or that the antiviral effects of the whole plant should be researched.

Report: Meeting on Antimalarial Drug Development, Shanghai, China, November 16-17 2001.

Chinese scientists are now working on the development of several combinations of artemisinin drugs with longer-acting synthetic drugs. WHO considers such combinations very promising as they may provide affordable, short-course treatments that could prove useful for large-scale use, even in areas of intense malaria transmission. ... There is a need for more antimalarial combination treatments containing artemisinin derivatives.

Later on May 4, 2020, as SARS2 was reported to be spreading, the WHO Regional office of Africa released a statement with an extremely misleading headline:

WHO supports scientifically-proven traditional medicine.

"WHO recognizes that traditional, complementary and alternative medicine has many benefits and Africa has a long history of traditional medicine and practitioners that play an important role in providing care to populations. Medicinal plants such as Artemisia annua are being considered as possible treatments for COVID-19 and should be tested for efficacy and adverse side effects."

"Africans deserve to use medicines tested to the same standards as people in the rest of the world. Even if therapies are derived from traditional practice and natural, establishing their efficacy and safety through rigorous clinical trials is critical."

"As efforts are under way to find treatment for COVID-19, caution must be taken against misinformation, especially on social media, about the effectiveness of certain remedies." The World Health Organization later announced that they had collaborated with Twitter, Facebook, Instagram, LinkedIn, Snapchat, Tiktok, Pinterest, and Youtube to "fight COVID19 misinformation." As one of the individuals who tried to spread the word of the anti-SARS2 potential of whole leaf A. annua on social media, I can attest that social media platforms did collaborate with the WHO to suppress the discussion of the potential of whole leaf A. annua to treat SARS2.

REVIEW OF SARS1 TESTING

In 2004, Li et al, of the Beijing Genomics Institute, set about to find natural herbs that had anti-SARS1 activity. 

Identification of natural compounds with antiviral activities against SARS-associated coronavirus

They screened 200 historically used herbs in China for virus treatments.  Extracts of these herbs were created by dissolving the leaves in ethanol and chloroform for 3 hours.  They tested the extracts against two strains of SARS1:  BJ001 and BJ006, which were added to Vero E6 and HepG2 cells. 

In conclusion, the compounds extracted from A. annua, L. radiata, P. lingua, and L. aggregata have been identified to show antiviral activity against SARS-CoV in Vero cell-based CPE/MTS screening

Of the 200 herbs, Lycoris radiata, Artemisia annua, Pyrrosia lingua, and Lindera aggregata all had strong anti SARS1 activity.  Artemisia annual was the second most active extract.  Lycoris radiata was first.  They developed "Lycorine" from Lycoris radiata.

REVIEW OF SARS2 TESTING

Three studies are reviewed that tested artemisia annual and derivatives for anti-SARS2 activity:  Gilmore et al (2020), Nair et al (2021), and Cao et al (2020).  I believe these three studies are sufficient to form a basis for future testing of the anti-SARS2 potential of A. annua. 

Cao tested nine chemical derivatives of A. annua.  Gilmore tested one dried leaf extract and three chemical derivatives of A. annua.   Nair tested dried seven leaf extracts of A. annua. 

All studies tested difference types of SARS2.  All studies reported anti-SARS2 activity of artemisia extracts and chemical derivatives.  Results varied widely.  Partial summaries follow.

NAIR STUDY

NAIR: Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants

Sscientists from Columbia University, the University of Washington, and the Worcester Polytechnic Institute tested seven extracts of artemisia annua sourced from multiple locations.  They created A. annua extracts by boiling dried leaves in water for 10 minutes, then straining the liquid.   They infected Vero E6 cells with SARS-CoV-2 isolate USA/WA12020. 

They also tested the absorption of artemisinin in the blood from a volunteer who consumed 3 grams of dried leaf A. annua. 36% of the original weight of the compound was detected at 2 hours, but dropped rapidly to .8% at 5 hours.

The author's principle conclusions were that artemisia annua had anti-SARS2 activity by an unknown compound or compounds acting together.  They reported that anti-SARS-2 activity was inversely correlated with artemisinin, which is not what one would have expected.   Unfortunately, their results are not as reliable as they would have been if they had not boiled the dried leaves of A annua, which likely damaged some compounds in the plant, as Tu Youyou described in her biography submitted to the Nobel Prize committee.

Nair et al said that there results on artemisinin were not consistent with Gilmore's testing. Nair et al also incorrectly stated the Gilmore study boiled their extracts:  

“Our hot water extracts are not directly comparable to those of Gilmore et al. because we did not dry, concentrate, and then weigh our extracts. Furthermore, we extracted for 10 min in boiling water, while they extracted for 200 min in boiling water. At present, it is not possible to compare our hot water extracts directly. In addition, different viruses were used in our study versus that of Gilmore et al., which could affect the inherent replication kinetics of the assay and in turn affect the specific IC50 numbers.”  

GILMORE STUDY

Gilmore: In vitro efficacy of Artemisinin-based treatments against SARS-CoV-2

Gilmore et al, of Max Planck, the University of Copenhagen, and Free University of Berlin, and the City University of Hong Kong tested one extract of artemisia annua grown in Kentucky, United States.  Gilmore et al infected Vero E6 cells and Huh7.5 with SARS-CoV-2 isolate human/Germany/BavPat 1/2020 and Danish SARS-CoV-2 isolate SARS-CoV2/human/Denmark/DK-AHH1/2020 for testing.  

Artemisia annua plants grown from a cultivated seed line in Kentucky, USA, were extracted using either absolute ethanol or distilled water at 50 °C (114 °F) for 200 min.

In addition to testing dried leaf extracts of A. annua, Gilmore et al also tested the A. annua derivatives artemisinin, artesunate and artemether. They determined that the whole leaf extracts had anti-SARS2 activity.  They determined that artesunate was the most potent of the derived A. annua compounds that they tested.  Artemesinin was also active against SARS2, but Artemether did not have a significant antiviral effect in their test results. The authors of the Gilmore study derived the artemisinin themselves but purchased the artesunate and artemether.  

CAO STUDY

CAO: Anti-SARS-CoV-2 Potential of Artemisinins In Vitro

Cao et al of the Beijing Institute and the Wuhan Institute of Virology tested nine chemical derivates of A. annua against SARS2. Compounds tested were Arteether, Artemether, Lumefantrine, Artemisinin, Artemisone, Dihydroartemisinin, Artesunate, Arteannuin B, Artemisic Acid.

They used CoV-2019BetaCoV/Wuhan/WIV04/2019 in Vero E6 cells.

"Artesunate, arteannuin B, and lumefantrine showed promise as anti-SARS-CoV-2 agents in vitro. Combined with the safety and potential immunoregulatory activities of artemisinins, we believe that artemisinin might represent a potential medical countermeasure against COVID-19."

The authors reported that Artemisinins reduce the production of SARS-CoV-2 protein. They reported that Arteannuin B and Lumefantrine block SARS-CoV-2 infection at the post-entry level. Arteannuin B had the most anti-SARS activity in their test results.

SUPERIORITY OF DRIED LEAF EXTRACT OVER CHEMICAL DERIVATIVES

This section is a brief review of a few studies that compared blood samples after ingestion of whole leaf A. annua and pure artemisinin.

WEATHERS ET AL 2011: Artemisinin production in Artemisia annua: studies in planta and results of a novel delivery method for treating malaria and other neglected diseases.

Weathers et al (2011) measured the amount of artemisinin in the blood of mice following the administration of pure artemisinin and the dried leaves of A. annua. "There was 45 times more pure artemisinin fed to the mice than the amount fed via A. annua leaves, yet almost the same amount of (artemisinin) appeared in the bloodstream."

ELFAWAL: Dried Whole Plant Artemisia annua as an Antimalarial Therapy

Elfewal et al (2012) tested the effectiveness of dried leaf A. annua against pure artemisinin. "We found conclusive evidence that orally ingested, powdered dried leaves of whole plant A. annua kills malaria parasites (in mice) more effectively than a comparable dose of pure drug."

IN CONCLUSION

The language in countless studies of A. annua is universally positive and hopeful for the development of drugs from A. annua to treat Malaria, SARS, SARS2 and other viruses. Available information strongly suggests that the whole leaf of the plant should be tested more scientifically.

In order to perform a statistically correct and medically correct study of A. annua extracts in vitro, I suggest the following. The essential oils of multiple and varying samples of A. annua should be created by a cold press method to preserve the delicate compounds in the plant.

A complete chemical analysis should be done of every sample of the dried leaf matter. A complete chemical analysis should be done of every sample of cold press extracts of the dried leaf matter. These results should be compared to determine what compounds were removed or altered in the cold press extraction process.

After the compounds in the multiple samples of cold press A. annua extracts have been quantified, they may be tested in Vero E6 and Huh7.5 cells infected with all available strains of SARS-CoV-2 to determine the micrograms per milliliter (µg/mL) necessary to achieve EC50 (half maximal effective concentration) responses.

The EC50 values can be described as a "function" of the quantity of the individual compounds in the extracts. The effectiveness of the individual compounds can be sorted using ordinary least squares (OLS) multiple linear regression (MLR).

This test method should greatly increase the understanding of the effectiveness of the individual compounds of A. annua in their natural state in fighting SARS2. It should give a coefficient of effectiveness of every individual compound in A. annua against all SARS2 strains that are tested in vitro.

This method is clearly superior as it captures any synergistic effects, while at the same time testing every individual compound. The test methods used in the reviewed study treat A. annua as a whole substance, and test individual compounds, one at a time. This method also preserves the natural structure of the compounds in the plant.

A similar method could be used for testing infected mice. This method should should also capture how the individual compounds can activate various immune functions in mammals. The dry leaf can also be tested in this manner, as it is not necessary to use extracts.

As for human testing, determining the EC50 values SARS2 in humans will prove more difficult because it has proven elusive for scientists to isolate SARS2 virus in the blood of humans. Nevertheless, various testing methods can still be used, such as measuring a reduction in the symptoms of volunteers who wish to try the most promising samples. I am open for suggestions on how to measure SARS2 in humans.

The image I used for this Substack came from Manfred Taege’s: Artemisia annua (Sweet wormwood) – from sowing to harvest a life-saving plant. The scientific study of compounds in their natural state will provide growers with the information that they need to selectively breed the plant for desired effect, thus bypassing the pharmaceutical industry.

Charles Wright