Mutagenicity Ames assessment of Artemisia annua and Artemisia afra

A Major Qualifying Project Report

Submitted to the Faculty of

WORCESTER POLYTECHNIC INSTITUTE

in partial fulfillment of the requirements

for the Degree of Bachelor of Science

By

___________________________________________

Jessica Marquez

Approved:

____________________________________________

Dr. Pamela J. Weathers, Advisor

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Acknowledgement I would like to thank Professor Weathers for giving me the opportunity to work on this project and for being my advisor. She has been incredibly kind and patient with me during this process and has given so many opportunities all while holding me to a high academic standard. I never could have finished this project without her help.

Many thanks to Melissa Towler for her guidance in the lab and patience throughout the course of this project.

A special thanks to the Weathers lab team for their feedback and help on this project.

Thank you to Tomás Ringer-Silva for all the support and feedback throughout this project.

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Table of Contents

Acknowledgement ........................................................................................................................................ 1

Table of Tables ............................................................................................................................................. 4

Table of Figures ............................................................................................................................................ 4

Abstract ......................................................................................................................................................... 5

1.0 Background ............................................................................................................................................. 6

1.1 Artemisia ............................................................................................................................................. 6

1.1.1 Artemisia annua ........................................................................................................................... 6

1.1.2 Artemisia afra ............................................................................................................................ 12

1.2 Artemisinin ....................................................................................................................................... 13

1.2.1 Artemisinin Derivatives ............................................................................................................. 14

1.2.2 Artesunate .................................................................................................................................. 14

1.2.3 Artemether ................................................................................................................................. 14

1.2.4 Dihydroartemisinin .................................................................................................................... 14

1.3 Ames Test ......................................................................................................................................... 14

1.4 Ames Test Process ............................................................................................................................ 15

1.5 TA Strains ......................................................................................................................................... 17

1.6 Ames mutagens ................................................................................................................................. 18

1.7 Multi-well Ames Test ....................................................................................................................... 19

1.8 Rationale ........................................................................................................................................... 19

2.0 Hypothesis and Objectives .................................................................................................................... 21

2.1 Hypothesis......................................................................................................................................... 21

2.2 Objectives ......................................................................................................................................... 21

3.0 Methodology ......................................................................................................................................... 22

3.1 Plant Materials .................................................................................................................................. 22

3.2 Bacterial strains and their cultivation ................................................................................................ 22

3.3 Protocol ............................................................................................................................................. 22

3.4 Analysis............................................................................................................................................. 23

3.5 Statistical analysis ............................................................................................................................. 23

4.0 Anticipated Results ............................................................................................................................... 24

4.1 Mutagenic ......................................................................................................................................... 24

4.2 Unanticipated Results ....................................................................................................................... 24

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5.0 Results ................................................................................................................................................... 25

5.1 Raw Data ........................................................................................................................................... 25

5.2 MR Ratio ........................................................................................................................................... 26

6.0 Discussion ............................................................................................................................................. 27

6.1 ANOVA ............................................................................................................................................ 28

6.1.1 Error and Uncertainty................................................................................................................. 29

7.0 Conclusion ............................................................................................................................................ 29

8.0 Cited references ....................................................................................................................................30

9.0 Appendices ............................................................................................................................................ 34

9.1 Appendix A: Recipes ........................................................................................................................ 34

9.1.1 Vogel-Bonner medium E (50x) .................................................................................................. 34

9.1.2 0.5 mM histidine/biotin solution ................................................................................................ 34

9.1.3 Sodium phosphate buffer, 0.1 mM, pH 7.4 ................................................................................ 35

9.1.4 Glucose solution (10% v/v) ........................................................................................................ 35

9.1.5 Ampicillin solution (0.8% w/v) .................................................................................................. 35

9.1.6 Minimal glucose plates .............................................................................................................. 36

9.1.7 Histidine/Biotin plates (Master plates for non R-factor strains) ................................................ 36

9.1.8 4-Nitroquinoline 1-oxide (4NQO) ............................................................................................. 37

9.2 Appendix B: Materials and Reagents ................................................................................................ 37

9.2.1 Materials .................................................................................................................................... 37

9.2.2 Reagents ..................................................................................................................................... 38

9.2.3 Equipment .................................................................................................................................. 38

9.3 Appendix C: Statistic Analysis of Results ........................................................................................ 39

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Table of Tables Table 1 Acute toxicities of A. annua SAM components ............................................................................... 8 Table 2 Flavonoid Subgroups ..................................................................................................................... 10 Table 3 Ames tested artemisinic derivatives ............................................................................................... 14 Table 4 The genotype of TA strains ............................................................................................................ 18 Table 5 TA100 SAM .................................................................................................................................. 25 Table 6 TA98 SAM .................................................................................................................................... 25 Table 7 TA98 SAM .................................................................................................................................... 26 Table 8 TA100 SEN .................................................................................................................................... 26 Table 9 TA98 SEN ...................................................................................................................................... 26 Table 10 Mutagenicity Assay Results (n≥3) ............................................................................................... 27 Table 11 - TA 100 w A. Annua .................................................................................................................. 39 Table 12 - TA 100 w A. Afra ...................................................................................................................... 39 Table 13 - TA 98 w A. Annua .................................................................................................................... 39 Table 14 - TA 98 w A. Afra ........................................................................................................................ 39

Table of Figures Figure 1 Leaves of A. annua ......................................................................................................................... 7 Figure 2 Basic structure of a flavonoid (open access) .................................................................................. 9 Figure 3 Camphor found in A. annua (open access) ................................................................................... 11 Figure 4 Structure of coumarin (open access) ............................................................................................ 11 Figure 5 Scopoletin found in A. annua (open access) ................................................................................. 12 Figure 6 Chlorogenic acid found in A. annua (open access) ...................................................................... 12 Figure 7 (a) Artemisinin, (b) Artesunate, (c) Artemether, and (d) Dihydroartemisinin.............................. 13 Figure 8 The overall process of the Ames test ............................................................................................ 17 Figure 9 4-Nitroquinoline 1-oxide (4NQO) ................................................................................................ 19

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Abstract

Artemisia annua and Artemisia afra are putative cost-effective antimalarial drugs. It is therefore necessary to understand the possible lasting genetic effects they could have on a patient and the patient’s children via testing. One of these tests is the mutagenicity assay, also called the Ames test. The Ames test is done by using genetically modified Salmonella typhimurium strains to determine if the test sample can restore the gene function of the modified strain through mutation, which allows the bacteria to grow. Colonies are counted and compared to negative controls to suggest the sample’s mutagenicity. In this assay for A. annua and A. afra, it was anticipated that the Ames test would validate that the plant extracts are non-mutagenic due to previous clinical trials that showed no negative effects. From this assay on A. annua and A. afra, there is implied non-mutagenicity. Further testing is required, but the assay suggests that further testing would show similar results and supports the promotion of future research into uses for A. annua and A. afra and their derivatives. Results of this assay also will impact a request for an Investigational New Drug FDA approval as the Ames test is one of the steps required to prove the infusions are non-mutative to patients.

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1.0 Background When marketing consumables such as medicine, rigorous testing is necessary to ensure the safety of those who take it. The US Food and Drug Administration (FDA) has requirements for the approval of consumables and one of these tests is mutagenicity assays. This assay will focus on the antimalarial plants, Artemisa annua and Artemisia afra. Malaria is a disease caused by mosquito-borne parasites which enter the blood cells and, if left untreated, may develop into severe complications or even death (CDC - Parasites - Malaria, 2020). This global issue is especially deadly in areas where medical treatment is more difficult to attain. Therefore, a solution that could be grown locally and used as treatment could be vital in lowering the death toll.

1.1 Artemisia

This proposal aims to study the mutagenicity of Artemisia sp., specifically Artemisia Annua L. and Artemisia afra tea infusions. Artemisia sp. have been used throughout history to help fight malaria and many other illnesses (Newman et al., 2015), which indicates the importance of determining any possible mutagenicity of the plant. While there is not much mutagenicity testing done with the plants in this genus, the ethnopharmacological history of each species has not related any major ill effects despite centuries of use.

This proposal will focus on A. annua and A. afra as they have been tested directly in clinical trials as a low-cost cure for malaria. Both A. annua and A. afra infusions are polytherapies due to the seeming synergy of artemisinin with other phytochemicals within the plant that produces better outcomes than ASAQ against malaria (Munyangi et al., 2019; Mueller et al., 2004).

1.1.1 Artemisia annua

The first plant to be tested is Artemisia annua L. (Figure 1). Grown natively in Asia, A. annua has been used in Chinese medicine to treat fevers and inflammation for several millennia (Hsu, 2006). A. annua produces the sesquiterpene lactone, artemisinin, which is a potent antimicrobial used to treat a variety of infectious diseases, especially malaria (Weathers et al., 2014). In Figure 1, the woody stems and small, pointed leaves are shown. These leaves are picked and dried for medicinal use.

A. annua provides up to 1.5% artemisinin in dry plant material (Kumar et al., 2004) while A. afraonly contains trace amounts (Liu et al., 2009). Another aspect of A. annua is the flavonoids thatare reported to work synergistically with artemisinin to become more effective in the humanbody (Rasoanaivo et al., 2011).

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Figure 1 Leaves of A. annua

The flavonoids in A. annua leaves have been linked to suppression of cytochrome P450 (CYP450) enzymes that alter the liver metabolism of artemisinin in the body, which seems to allow artemisinin delivered through dried leaf annua to be distributed to tissues in higher concentrations in vivo (Desrosiers et al., 2020). These flavonoids are linked to a beneficial immunomodulatory activity in patients afflicted with parasitic and chronic diseases (Ferreira et al., 2010). A. annua contains artemisinin which, when combined with these flavonoids, is reported to have a synergistic reaction that allows for the plant to work more effectively in people (Ferreira et al., 2010). When tested individually, specific phytochemicals known to be part of the inhibition of artemisinin showed weaker results than the whole plant extract in preforming the same function (Desrosiers et al., 2020). The plant is often consumed by first drying the leaves and then steeping about 5 g/L in boiled water to make a tea infusion. In previous clinical trials testing the effects of this plant against malaria, patients were treated by drinking 0.33 L of an infusion of 5 g of dried leaves per L of hot water every 8 hours for 7 days (Munyangi et al., 2019). A. annua cured malaria faster and more effectively than artesunate-amodiaquine (ASAQ), another anti-malarial treatment, in clinical trials, and it also appeared that the tea infusion treatment was able to break the cycle of malaria by eliminating gametocytes in the parasitic life cycle (Munyangi et al., 2019).

Another aspect of the plant to consider is the individual components that make up the resulting infusion, as shown in Table 1. To the best of my knowledge, none of the phytochemicals shown in Table 1 are mutagenic.

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Table 1 Acute toxicities of A. annua SAM components

Components LD 50 (mg/kg)

Delivery Route

Species Source

Artemisinin 5,576 P.O. Rat http://datasheets.scbt.com/sc-202960.pdf

Dihydroartemisinic acid

N/A N/A N/A N/A

Arteannuin B 400 P.O. Rat https://books.google.com/books?id=iQlmDAAAQBAJ&dq=arteannuin+b+ld50&source=gbs_navlinks_s

Artemisinic acid N/A N/A N/A N/A α-Pinene 3,700 P.O. Rat http://datasheets.scbt.com/sc-

239166.pdf Eucalyptol (1,8 cineole)

2,480 P.O. Rat Jenner et al. 1964

Camphor 1,310 P.O. Mouse https://www.caymanchem.com/msdss/23175m.pdf

Chlorogenic acid 10 I.P. Mouse https://www.caymanchem.com/msdss/70930m.pdf

Rosmarinic acid 561 I.V. Mouse https://pubchem.ncbi.nlm.nih.gov/compound/Rosmarinic-acid#section=Toxicity

Scopoletin 3,800 I.P. Rat https://www.caymanchem.com/msdss/20042m.pdf

Artemetin N/A N/A N/A N/A Casticin N/A N/A N/A N/A Chrysoplenol-D N/A N/A N/A N/A Chrysoplenetin N/A N/A N/A N/A Eupatorin N/A N/A N/A N/A Kaempferol 980 P.O. Rat https://www.tandfonline.com/doi/p

df/10.3109/13880209.2014.982301

Luteolin 180 I.P Mouse https://www.caymanchem.com/msdss/10004161m.pdf

Myricetin 1410 I.P. Mouse Lewis (ed.) 2004 Quercetin 161 P.O. Rat http://datasheets.scbt.com/sc-

206089.pdf PO, Oral; IP, Intraperitoneal; IV, Intravenous; N/A, Not available

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Flavonoids

Flavonoids are a group of compounds with variable phenolic structures found in plants. These compounds are an important part of nutraceutical, pharmaceutical, medicinal, and cosmetic applications (Panche et al., 2016). These compounds are divided into groups depending on the C ring into the subgroups of flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins and chalcones (Panche et al., 2016).

Figure 2 Basic structure of a flavonoid (open access)

The C ring in this molecule could be altered in the following ways as described in Table 2. Flavones have a double bond between positions 2 and 3 and a ketone in position 4 of the C ring, and often have a hydroxyl group in position 5 of the A ring. Flavonols have a hydroxyl group in position 3 of the C ring. Compared to flavones, flavanones are only structural different by the saturated C ring. The flavanols hydroxyl group is always bound to position 3 of the C ring and there is no double bond between positions 2 and 3. Anthocyanins are defined by the hydroxyl groups on the A and B rings. Chalcones are known as the open-chain flavonoids as they are defined by a lack of C ring.

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Table 2 Flavonoid Subgroups

Subgroup Subgroup flavonoid structures featuring C ring

Examples verified in A. annua cv. SAM

Flavones Apigenin

Flavonols

Flavanones Not as yet detected.

Flavanols (aka catechins)

Kaempferol

Anthocyanins Not as yet detected.

Chalcones Not as yet detected.

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While flavonoids are not completely understood, these compounds have antioxidant effects and exhibit anti-inflammatory, anti-mutagenic and anti-carcinogenic properties (Panche et al., 2016). In A. annua the majority of phenolics extracted consist of flavonoids such as 10 species of flavones and 28 flavonols (Ferreira et al., 2010).

Monoterpenes

Monoterpenes are a terpene subgroup that contains two 5C isoprene units. Natural monoterpenes found in plants are associated with secretory or storage structures, e.g. glandular trichomes, and these compounds are usually formed in plastids (Goodger et al., 2009, p.). The over 20 monoterpenes found in A. annua show distinct roles of the products produced when tested through phytohormone and wounding treatments (Ruan et al., 2016). Camphor, shown in Figure 3, is one example of a monoterpene found in A. annua.

Coumarins

Coumarins are a family of benzopyrones found in nature of oxygen-containing heterocycles (Matos et al., 2015). They contain the basic framework found in Figure 3.

Figure 4 Structure of coumarin (open access)

These compounds are medical candidates for drugs with strong pharmacological activity, low toxicity and side effects, less drug resistance, high bioavailability, broad spectrum, better curative effects, etc., (Matos et al., 2015). Coumarin derivatives have also been applied to address many pharmacological targets in a selective way, such as selective enzyme inhibitors, and more recently, a number of selected targets (multitarget ligands) involved in multifactorial diseases, such as Alzheimer’s and Parkinson’s diseases (Stefanachi et al., 2018). Figure 5 shows scopoletin, a coumarin commonly found in A. annua.

Figure 3 Camphor found in A. annua

(open access)

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Figure 5 Scopoletin found in A. annua (open access)

Polyphenolic acids

Polyphenols are composed of two groups tannins and flavonoids. While flavonoids have previously been discussed, tannins can also be broken down into hydrolysable and condensed tannins (Ferreira et al., 2010). A total of 40 phenolic compounds were identified or tentatively characterized in the methanol extract of A. annua (Han et al., 2008). These phenolic acids include chlorogenic acid as seen in Figure 5, quinic acid, and coumaric acid (Ferreira et al., 2010). More specifically, in the A. annua SAM cultivar, chlorogenic acid has been identified. Chlorogenic acid is an ester formed from cinnamic acids and quinic acid and is also known as 5-O-caffeoylquinic acid (Clifford, 2000).

Figure 6 Chlorogenic acid found in A. annua (open access)

1.1.2 Artemisia afra

Common to South Africa, A. afra is also a known ethnopharmacological plant. This species has been used for years as a treatment for bronchial issues, diabetes, colds, headaches, and swelling (Liu et al., 2009). While only the rare cultivar has at most trace amounts of artemisinin, A. afra was still effective against malaria as shown in a recent clinical trial (Munyangi et al., 2019). In that clinical trial, A. afra infusions were prepared similar to A. annua; 5 g of dried leaves and twigs were steeped in 1 L of boiling water. A. afra performed similarly to A. annua in that, even though A. afra contains only trace amounts of artemisinin, which is considered the main

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antimalarial compound, almost all patients who were treated with A. afra had the same therapeutic response as those treated with A. annua (Munyangi et al., 2019). Gruessner et al. (2020) discussed those levels of artemisnin in A. annua and A. afra used int hat study and argued that there was more than enough bioavailable artemisnin to treat malaria the lowest antimalarial threshold of which is ~10 µg/L.

1.2 Artemisinin

The chemical derivative found in A. annua, artemisinin (Figure 2), and its derivatives are reported to be active against a range of diseases such as many parasites, some viruses, Pnuemocystis carinii, and a number of human cancer cell lines (Efferth, 2007).

To enhance its bioavailability, artemisinin has been chemically modified into several derivatives including artesunate (AS), artemether (AM), and dihydroartemisinin (DHA) (Figure 2). While those derivatives are used in combination with other antimalarial drugs to treat malaria and other diseases, the focus of this study will primarily be A. annua and A. afra.

Despite low bioavailability of pure AN, it is highly bioavailable when delivered orally as dried leaves of the plant (Weathers et al., 2011; Desrosiers et al., 2020).

Figure 7 (a) Artemisinin, (b) Artesunate, (c) Artemether, and (d) Dihydroartemisinin

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1.2.1 Artemisinin Derivatives

Artemisinin is a natural part of A. annua; its multiple semi-synthetic derivatives are semi-synthetic and not naturally found in the plant. Consequently, AS, AM, and DHA will not be tested in this mutagenicity assay because they have already been analyzed via the Ames test as shown in Table 2 (Tu, 2017).

Table 3 Ames tested artemisinic derivatives

Artemisinin Derivative Mutagenicity Results AS negative AM negative

DHA negative

1.2.2 Artesunate

Artesunate chemically is composed of three main rings similar to artemisinin, one of which is a seven membered ring that contains a peroxide bridge. The structure of the ring is slightly different from artemisinin also due to the oxygen in the ring that is in the same plane as the rest of the molecule structure. Artesunate is synthesized from DHA. After the oxygen attached to the carbon 10 from the artemisinin molecule has been substituted for a hydroxide to make DHA, a base is switched for the hydroxide and the ring opens to create the final AS.

1.2.3 Artemether

When the lactone in artemisinin has been converted to the corresponding lactol methyl ether the product formed is artemether (Artemether (CHEBI:195280), n.d.). It is used in combination with lumefantrine as an antimalarial for the treatment of multi-drug resistant strains of falciparum malaria as the artemether component is absorbed rapidly and biotransformed to dihydroartemisinin (White et al., 1999).

1.2.4 Dihydroartemisinin

Dihydroartemisinin is the active metabolite of all artemisinin compounds. The proposed mechanism of action of artemisinin involves cleavage of endoperoxide bridges by iron in blood cells, producing free radicals which damage biological macromolecules (Cumming et al., 1996).

1.3 Ames Test

The Ames test is a bioassay done in vitro to understand the risk of mutagenicity of drugs, reagents, and other substances. While a mutagenic sample may directly cause cancer in a patient,

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it could also lead to possible unknown defects in later generations. Therefore, mutagenicity testing is an essential step before releasing a drug for commercial use.

The Ames test, published by Bruce Ames’s laboratory in 1973, provides a procedure for rapid and simple chemical mutagenicity testing and supplies information on the metabolism of non-mutagenic chemicals to their potentially DNA-reactive forms (Zeiger, 2019). Following the original publication, many tests demonstrated the efficacy of the Ames test and its validity in predicting the likelihood of a chemical being carcinogenic.

The test works by using genetically modified Salmonella typhimurium strains to determine if the sample can restore the gene function of the modified strain through mutation, which allows the bacteria to grow. The S. typhimurium strains each have a slightly different modified histidine operon that prevents it from synthesizing histidine; strains are grown on an agar plate with minimal histidine to allow for a few replications. Only the bacteria that revert to synthesizing histidine for itself will be able to thrive on the plate. The control plates are necessary for comparing the test plate with growth of spontaneous natural revertants, which tend to be low in number and relatively constant (Mortelmans & Zeiger, 2000).

1.4 Ames Test Process

The original Ames test has gone through alterations that made it easier to gather necessary information, quicker, and with better similarity to in vivo human processes. Unlike animal and human biology, bacteria do not use cytochrome P450s to metabolize chemicals, therefore, liver S9 mix later was added to the Ames test to gather additional information on how the tested substance would interact and metabolize in the human body (Mortelmans & Zeiger, 2000). The S9 mix is a specific fraction of homogenized liver and more recently is now prepared from a pool of human liver microsomes.

To perform the Ames test, Typhimurium Ames (TA) strains should be prepared ahead of the test along with fresh mutagen for each experiment along with the positive and negative controls. While the negative controls will always be autoclaved water, the positive controls may differ depending on whether S9 mix has been added for metabolic activation.

The positive controls without the S9 mix include sodium azide, 2-nitrofluorene, and mitomycin. Sodium azide (NaN3) is acutely toxic and influences the bacterial strains by inhibiting cell division with a greater inhibitory effect on DNA synthesis (Cieśla et al 1974). Sodium azide targets the central nervous system in humans and it is also a cytochrome oxidase inhibitor which is a nitriding agent and an inhibitor of terminal oxidation (PubChem, n.d.).

A bacterial mutagen, 2-nitrofluorene, and mitomycin, a chemotherapy drug, will modify the Salmonella strains to induce reversion via frameshift mutations. (Molina & Peterson, 1980). Mitomycin is a highly reactive bis-electrophilic intermediate when converted upon reduction and this allows it to alkylate cellular nucleophiles (Paz et al., 2012). However, due to the decision to

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focus on TA98 and TA100 strains for this project, mitomycin will not be used as it has no effect on these strains (Hamel et al., 2016). When ingested, 2-nitrofluorene can take a minor metabolic pathway, which results in the formation of hydroxylated nitrofluorenes; these chemicals, although not detected individually, appear to be the mutagenic metabolites of 2-nitrofluorene (Humans, 2014). Then the 2-anthramine mutagen is used for the positive controls with S9 mix, as this mutagen stimulates mutations in the TA strains and allows the cultures to grow.

Following controls, preparation of minimal glucose agar plates occurs just before they are needed for culture growth. During experimental procedure mixtures are added to each plate of TA culture, histidine/biotin (his/bio) solution, test sample or control sample, and S9 mix, as shown in the diagram of the Ames test in Figure 3 below.

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Figure 8 The overall process of the Ames test

The plates are then covered with sterile aluminum foil to protect the light sensitive biotin in the samples and the plates are incubated for 48 hours at 37° C. After 48 hours, natural revertants are visible to the naked eye and can be counted and easily compared with test samples. Once the procedure is finished, the number of colonies on the test plates are compared to those on the negative control and analyzed as described in section 3.4.

1.5 TA Strains

Each strain of TA has different histidine mutations that allow them to detect a range of mutagenic agents with different mechanisms of action. These mutations make the TA more susceptible to mutations that ease the testing process such as uvrB deletion, that with the exception of TA102, codes for the DNA repair system (Mortelmans, 2019). These histidine auxotrophic mutants should be analyzed for their spontaneous mutation rate before use, and strains should be checked before each experiment as each strain can react differently. For example, TA102 is biotin independent and will grow regardless of biotin levels (Mortelmans, 2019).

There are also deep rough (rfa) marker mutations for strains that cause a defective lipopolysaccharide layer of the bacterial surface, which allows larger MW chemicals to readily permeate the membrane of these strains (Mortelmans & Zeiger, 2000). In TA1535 and TA1538, the introduction of plasmid pKM101 results in the corresponding isogenic strains of TA100 and TA98 also with strains TA97, TA102, and TA104 (Mortelmans & Zeiger, 2000). This mutation also enhances chemical mutagenesis due to the increase in a recombinational DNA repair mechanism, which often results in errors.

Table 3 shows common TA strains and their specific histidine mutation. With each mutation is a reversion event that explains how the mutagen would have to affect the bacteria in order to revert the bacteria to its his+ form.

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Table 4 The genotype of TA strains

Strain Histidine

Mutation

Reversion Event Target DNA Plasmid R-factor

TA98 D3052 Frame shift

CGC GCG C GCG

CG

CGCGCGCG pKM101 +R

TA100 G46 Base Pair Substitution

GAG/CTC

GGG/CCC

GGG pKM101 +R

TA102 G428 Transition/Transversion

TAA TTA

TAA pKM101,

pAQ1

+R

One of the differences between TA102 and the others is that its target DNA has AT base pairs instead of GC. The mutation resides on the multi-copy pAQ1 in TA102 and this becomes a good marker for the presence of plasmid due to the tetracycline resistance it confers (Mortelmans & Zeiger, 2000).

For this study testing Artemisia extracts and pure artemisinin, TAs with a base pair substitution and frameshift will be chosen that also have pKM101 plasmid mutations as shown in Table 3. TA100 and TA98 strains were shown sufficient in identifying about 90% of the mutagens in a selection of chemicals where 36% were mutagenic (Zeiger et al., 1985). Therefore, TA100 and TA98 will be used.

1.6 Ames mutagens

Mutagens used for Ames testing depend on the bacterial strain used, therefore in this project the mutagen 4-nitroquinoline 1-oxide (4NQO) will be used as it has a strong effect on both TA100 and TA98 (Hamel et al., 2016). This chemical is highly mutagenic and carcinogenic and is able to mimic the effects of ultraviolet light on numerous living organisms by binding covalently to cellular macromolecules (Ikenaga et al., 1975).

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This molecule becomes mutagenic when metabolized by the bacteria into 4-hydroxyaminoquinoline 1-oxide, which forms large purine adducts that can be corrected by nucleotide excision repairs. In TA100, 4NQO completes a base pair substitution at the histidine

mutation site G46. For TA98, this compound makes a frame shift on D3052.

The molecular formula for this compound is C9H6N2O3 and is a quinoline N-oxide that carries a nitro substitution at position 4 as seen in Figure 4 (PubChem, n.d.). This compound is a yellow solid and does not solubilize easily in water and therefore when the compound is prepared for testing as the positive control it needs to be dissolved in DMSO before being added to the mix.

1.7 Multi-well Ames Test

Along with the plate tests, there is another option for a multi-well system, which although more costly for materials, allows quicker results with less waste. This test follows the same logic as the normal Ames test by finding mutagenic properties in tested substances by finding reversions in Salmonella samples. The major difference with this test is that the data collection at the end of the incubation process is done by counting how many wells have changed color via a pH indicator rather than counting colonies on plates.

A drop in pH during this test is a metabolic signal of replicating bacteria, which would only occur if the bacterial strain mutated to his+. Due to the ability to easily replicate tests with this process, it is faster for comparing a variety of TA strains against possible mutagenic samples.

1.8 Rationale

Due to the relevancy of A. annua and A. afra as cost-effective antimalarial drugs across the globe, it is necessary to understand the possible lasting genetic effects they could have on a person or their offspring. Results of this assay also may impact a request for an Investigational New Drug (IND) FDA approval.

In a double-blind, randomized clinical trial with over 900 patients, A. annua and A. afra were found to be more effective and with far fewer adverse side effects than one of the several the medications currently used to treat malaria, artesunate-amodiaquine (Munyangi et al., 2019).

Figure 9 4-Nitroquinoline 1-oxide (4NQO)

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With broad availability and efficacy, these two plant species could cost effectively treat millions of people, so it is important to be sure of possible long-term effects before using it on large numbers of ill people.

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2.0 Hypothesis and Objectives

2.1 Hypothesis

A. annua and A. afra have been used throughout history and have a good reputation as herbal medicines. They have also been used in multiple clinical trials with no negative outcomes and often performed better than the synthesized artemisinin derivatives. Additionally, the Ames results for artemisinin and its derivatives were negative, which supports the probability of both Artemisia species also not being mutagenic. Therefore, I anticipate that the Ames test will validate that the plant extracts are non-mutagenic.

2.2 Objectives

1. Determine the concentration of A. annua and A. afra teas that are non-mutagenic and safe for human consumption by measuring the extremes of toxicity for both plant species.

2. Compare mutagenic results of both Artemisia sp. with artemisinin.

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3.0 Methodology

3.1 Plant Materials

Samples of Artemisia annua SAM cultivar (voucher MASS 00317314) garden harvested in 2018 and A. afra SEN cultivar (voucher LG0019529) harvested August of 2016 will be used to make tea for this test with a range of dilutions. Following the drug administration in the large scale clinical trial to cure malaria, the standard dilution will be 5 g of dried plant matter per 1 L of water (Munyangi et al., 2019). Along with this dilution, samples at 10 and 15 g/L of water will also be tested and depending on the results, the range of dilutions may change during experimentation to find possible upper and lower extremes of toxicity.

3.2 Bacterial strains and their cultivation

TA strains were chosen as shown in section 1.3 and they can be purchased either in kits for the multi-well assay or separately from companies such as MolTox at http://moltox.com/index.php?main_page=index&cPath=5_27. It should be noted that these strains of Salmonella are very sensitive and accidental contamination can result in skewed mutagenic results or loss of the bacterial strain.

3.3 Protocol

For each assay, a set of diluted A. annua and A. afra teas was prepared at the concentrations of 5 g, 10 g, and 15 g/L each. Each experiment had at least three replicate plates and would be compared to literature for mutagenicity of artemisinin samples. Since full liter samples were not needed, 200ml of tea infusion samples were prepared and frozen at 4°C for use.

Prior to beginning the assay, preparations of the TA strains are necessary. A fresh colony of each of the S. typhimurium strains TA 98 and 100 were inoculated into nutrient broth 15-18 hours prior to performing the experiment. Each strain was grown separately in 10 ml Erlenmeyer flasks. These samples of liquid culture were incubated at 37°C for at least 12 hours in an incubator shaker at 120 rpm for aeration to achieve 1 x 109 bacterial cells. Samples were taken and measured for their optical density at 600nm and 650nm to reach between 0.8 and 1 OD, respectively.

Fresh mutagen solutions should be prepared for each experiment. These solutions are used for the positive controls in the experiments without S9 mix and requiring 1 µg/ml 4NQO. The recipe for this mutagen is listed in Appendix A, section 9.1.8.

Following this, preparation of the minimal glucose agar plates is done just before use with 900ml of distilled water, 15g of agar, 20ml of 50x VB salts, and 50ml of 10% glucose all per liter of the final solution as shown in Appendix A section 9.1.6. Of this solution, 25 ml should be poured

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into each Petri dish and once each gel cools and solidifies, the plates can be stored in sealed plastic bags at 4°C for several weeks and be allowed to warm to room temperature before use. Also note that if the plate shows excess moisture before use, they can be left in a stationary incubator at 37°C overnight.

In labeled, sterile glass tubes, 0.5 ml of sodium phosphate buffer, 0.05 ml test sample or control sample, and 0.1 ml TA culture were added and mixed. If working with S9, the metabolic activation mix would be added in place of the sodium phosphate buffer. The 0.05ml of positive control is made up of 0.045ml of distilled water, and 5µl of 4NQO solution. However, because the 4NQO solution is composed of mostly DMSO as shown in the recipe in Appendix A, another control was made for DMSO, which comprised of 45.5µl distilled water and 4.5µl of DMSO to reach the same final concentration of DMSO as the positive control.

Each test tube was mixed and then its contents incubated at 37°C for 20 minutes. Then, 2ml of melted top agar maintained between 43°C and 48°C is mixed into each test tube and poured onto a Petri plate and spread across the surface using an L-shaped spreader. Once the agar solidified (~ 3 minutes), the plates were inverted and left in the incubator for 48 hours at 37 °C. At this point, revertant colonies are visible and can be counted and compared easily.

3.4 Analysis

After incubation for 48 hours, the number of colonies on the test plates were compared to those on the negative control plates. If there is mutagenic activity, the concentration of the tested sample should increase in a dose-dependent manner, and a mutagenicity ratio (MR) is calculated (Vijay et al., 2018).

The number of revertant colonies per plate are counted and then applied to the formula below to find the MR of the test sample (Maron & Ames, 1983)

𝑀𝑀𝑀𝑀 =𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑀𝑀𝑇𝑇𝑅𝑅𝑇𝑇𝑅𝑅𝑇𝑇𝑅𝑅𝑅𝑅𝑇𝑇𝑇𝑇𝑁𝑁𝑇𝑇𝑁𝑁𝑅𝑅𝑇𝑇𝑁𝑁𝑅𝑅𝑇𝑇 𝑐𝑐𝑐𝑐𝑅𝑅𝑇𝑇𝑅𝑅𝑐𝑐𝑐𝑐

A positive MR > 2.0 implies a mutagenic result, and a negative MR < 2.0 implies a non-mutagenic result (Vijay et al., 2018).

3.5 Statistical analysis

Once colonies were counted and recorded for each section, statistical analysis was performed using the ANOVA method with a significance of p ≤ 0.05. The averages of each section for a test where calculated and from these values, the sums of squares, degrees of freedom, mean squares, between treatments, and errors were determined. The F statistic was computed by taking the ratio of the between treatment variability to the error variability and determines the validity of the

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hypothesis. Here ANOVA was used to determine how similar each test would be to the negative control.

4.0 Anticipated Results

4.1 Mutagenic

A. annua and A. afra contain flavonoids that are harmful when ingested in high concentrations such as camphor, therefore, the higher the concentration of tea being tested the more likely there may be an increase in mutagenicity. This experiment will most likely result in showing a slow upward curve of mutagenicity for all tested extracts with most of the dilutions showing little to no mutagenicity at all.

Considering that the plant has been used for centuries with no reported long-lasting negative effects, it is expected there will be no mutagenicity of the plant extracts. If there is no mutagenicity detected in this assay, these results will be able to support the promotion of future research into safe uses for A. annua and A. afra and their derivatives.

4.2 Unanticipated Results

There is still a possibility that this mutagenicity assay will show that the teas are mutagenic in nature, which may cause detrimental side effects in people. If the Artemisia samples do show mutagenicity it will most likely be at higher concentrations, which would reveal that there is an upper limit to the concentration allowed for use before it becomes harmful through its potential mutagenicity.

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5.0 Results

While this assay was cut short due to the coronavirus pandemic, assays were completed at least once for each combination of TA strains and test compound. The raw data collected each have an average number of colonies on the test sections that were similar if not equal to the collected data for the negative control, therefore, this suggests there was no mutagenicity before any analysis or calculations are done.

5.1 Raw Data

As stated in the methods, each test should have at least three replicates per section. To save space and time the number of replicates was reduced to three from the original four replicates of the first test. In each table the bacterial strain and either A. annua SAM or A. afra SEN cultivar are labeled at the top. The sections are divided up by the contents of each plate and then counted for the number of colonies on each plate.

For the positive control plates, the plate was divided into four quarters and one quarter was counted before being multiplied by four. This is acceptable due to the fact that there were large numbers of colonies (too numerous to fully count), and colonies tended to grow evenly across the plates. Furthermore, the specific number of colonies per positive control plate was not necessary information for MR calculations. Rather, the positive control is a test to compare the high extreme of what a highly mutagenic compound would produce and ensure that implied non mutagenicity by the test compounds is not a result of defective bacteria that could not be mutated.

Table 5 TA100 SAM

Negative Control (ct)

1g/200mL tea (ct)

2g/200mL tea (ct)

3g/200mL tea (ct)

Positive Control (ct)

DMSO Control (ct)

9 9 12 7 319 10 9 10 11 12 372 12

10 6 12 8 320 10 15 17 9 11 440 N/A

Table 6 TA98 SAM

Negative Control (ct)

1g/200mL tea (ct)

2g/200mL tea (ct)

3g/200mL tea (ct)

Positive Control (ct)

DMSO Control (ct)

4 7 3 4 375 4 12 8 7 8 320 11 7 7 13 11 423 3

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Table 7 TA98 SAM

Negative Control (ct)

1g/200mL tea (ct)

2g/200mL tea (ct)

3g/200mL tea (ct)

Positive Control (ct)

DMSO Control (ct)

7 6 5 8 440 8 8 13 11 10 318 7 8 7 7 4 483 4

Table 8 TA100 SEN

Negative Control (ct)

1g/200mL tea (ct)

2g/200mL tea (ct)

3g/200mL tea (ct)

Positive Control (ct)

DMSO Control (ct)

8 6 4 5 324 4 8 6 7 6 419 8 14 7 11 12 380 9

Table 9 TA98 SEN

Negative Control (ct)

1g/200mL tea (ct)

2g/200mL tea (ct)

3g/200mL tea (ct)

Positive Control (ct)

DMSO Control (ct)

8 9 11 13 389 7 13 12 16 14 415 16 12 8 5 11 355 11

5.2 MR Ratio

The mutagenicity ratio discussed in section 3.4 was used to complete ratios for each test. First the average of all the plates per tea section of one test repetition was taken, then each dilution colony average was compared to the negative control average of that test repetition. If each test dilution showed no implied mutagenicity, the test was averaged as non-mutagenic. Table 9 shows the results of these tests as all MRs were calculated to be less than two and often determined to be very close to a MR of one which suggests that there is no difference in mutagenicity between the tea dilutions and distilled water.

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Table 10 Mutagenicity Assay Results (n≥3)

Treatment Set Average MR ± SE Mutagenicity

TA 98 w A. annua 1.05 ± 0.09 None

TA 98 w A. afra 1.03 ± 0.12 None

TA 100 w A. annua 0.98 ± 0.07 None

TA 100 w A. Afra 0.71 ± 0.05 None

As expected and discussed in section 4.1, these findings imply that A. annua and A. afra are non-mutagenic.

6.0 Discussion

This study suggested that Artemisia annua and A. afra are not mutagenic However, more tests need to be done to ensure non-mutagenicity, especially regarding how the infusions could react once metabolized by liver microsomes. While the phytochemicals extracted in A. annua infusions inhibit the liver CYP450 enzymes that metabolize artemisinin, almost nothing is known about its tissue distribution and elimination (Desrosiers et al., 2020). Further investigation on these findings would likely support an absence of mutagenicity although FDA approval likely would require mutagenicity testing with liver microsomes. When completing a mutagenicity test to the standards of the FDA as a decision marker for safety of human consumption, more than two strains of bacteria should be used. For the test to be completed fully, S9 liver microsomes are needed to show how it would possibly interact with CYP450s. However, further testing should also show no mutagenicity based on the results of this project. These additional steps will be necessary for these affordable and accessible anti-malarial treatments to gain approval for widespread use.

Further investigation should be made with the liver S9 mix, which is liver tissue homogenate in which S9 hepatocytes minimize the mammalian metabolic activation formed in the liver that accounts for how drug interactions with the liver can make the drug more mutagenic (Vijay et al., 2018). The addition of this mix with TA strains shows how a substance might be carcinogenic in humans.

As the individual compounds found in both species of Artemisia have shown no mutagenicity, the further testing of all compounds through these infusions has implied that synergistically they

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also would not show any mutagenic effects as well. Even with the discussed LD50s of each compound in the plant, when ingested they would not approach toxic concentrations.

While there are certain phytochemicals in both Artemisia sp. which are toxic in high concentrations, such as camphor, within the tea infusions they do not reach harm inducing levels. For example, the LD50 of camphor is 1310 mg/kg when delivered orally to a mouse, whereas concentration of camphor in A. annua leaves obtained from various places ranged from 100 mg/kg of leaf to 600 mg/kg (Rosine Desiree et al., 2016). Because doses are made at 5g of leaves per liter of water in clinical trials, the concentration of camphor stays far below dangerous concentrations.

Similarly, with chlorogenic acid, which is commonly found in black and green teas (Camellia sp.), the levels of concentration necessary to reach health threats would not be attained through the Artemisia sp. tea infusion doses. The LD50 as stated in section 1.1.1, is 10 mg/kg via intraperitoneal injection in rats. For higher concentrations of tea, this might be a factor in mutagenicity. In a regular dose, this would not reach high enough concentrations to become an issue. One might consider multiple doses could reach potentially harmful levels, however that is unlikely as chlorogenic acid, found at high levels in black tea, is processed a few hours in the liver.

One monoterpene, thujone, found in A. afra is toxic in high amounts and has a LD50 of 192 mg/kg in rats from a α- and β-thujone mixture. In A. annua, thujone is not found unless extracts are cross contaminated with A. afra.

While the basic ratio of mutagenicity is optimistic, further analysis is necessary to prove that this possible conclusion is viable. In order to determine whether the data collected is enough to suggest non-mutagenicity the Analysis of Variance or the ANOVA method was used.

6.1 ANOVA

The ANOVA method is used when the hypothesis is about a difference in means between more than one type of treatment. This method is the correct choice because this project is testing for a lack of effect. In other words, the null hypothesis for this project is that the means of the counts in the different treatments are different. If that null hypothesis is rejected, then the alternative hypothesis that the means are the same can be accepted.

The procedure for the ANOVA method is similar to other variance methods but is made more difficult because there are several different treatments that need to be tested. This means that the calculations need to consider sizes, standard deviations, and means for each comparison group and then between every comparison group.

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The ANOVA method was used with a 95% confidence interval. This method uses sum-of-squares to determine variance, so it is distributed in an F-distribution. Because of this, the ANOVA method uses an F score to reject or accept the null hypothesis.

The ANOVA tables for the four different treatments are shown below, in Tables 10, 11, 12, and 13. The F-values on the far right are compared to a critical F value that is calculated separately.The claims made in Table 9 that none of the treatments resulted in mutagenicity is derived fromthe relationship between the F value and the critical value. Increasing the number of data points,the confidence interval could be improved.

6.1.1 Error and Uncertainty

The error and uncertainty of this project in part came from the bacteria itself. The TA strains of bacteria are destabilized to allow mutations to easily occur. This is what allows the negative control plates to still have colonies living on the plate from mutating on their own. This mutation is somewhat at random but occurs in low amounts which is how the test matter can be tested for both the spontaneous revertants that happen naturally and the induced revertants which are created from the test materials mutagenic influence.

7.0 Conclusion

No mutagenicity of either A. annua or A. afra tea infusions however further studies are needed. This study found no mutagenicity in either A. annua or A. afra. Limitations in sample size and sampling precision mean that more studies will be needed in order to conclusively determine the mutagenicity of A. annua and A. afra. In order to meet FDA approval for medicinal use of A. annua and A. afra, studies will need to adhere to specific FDA guidelines around mutagenicity assays. Because this study was able to demonstrate the statistical significance of its results, it preliminary evidence of safety and can be considered a good model for further studies.

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8.0 Cited references Artemether (CHEBI:195280). (n.d.). Retrieved June 6, 2020, from

https://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:195280

Artemether (CHEBI:195280). (n.d.). Retrieved June 6, 2020, from https://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:195280

Cieśla, Z., Mardarowicz, K., & Klopotowski, T. (1974). Inhibition of DNA synthesis and cell division in Salmonella typhimurium by azide. Springer-Verlag. https://doi.org/10.1007/BF00271148

Clifford, M. N. (2000). Chlorogenic acids and other cinnamates – nature, occurrence, dietary burden, absorption and metabolism. Journal of the Science of Food and Agriculture, 80(7), 1033–1043. https://doi.org/10.1002/(SICI)1097-0010(20000515)80:7<1033::AID-JSFA595>3.0.CO;2-T

Cumming, J. N., Ploypradith, P., & Posner, G. H. (1996). Antimalarial Activity of Artemisinin (Qinghaosu) and Related Trioxanes: Mechanism (s) of Action. In J. T. August, M. W. Anders, F. Murad, & J. T. Coyle (Eds.), Advances in Pharmacology (Vol. 37, pp. 253–297). Academic Press. https://doi.org/10.1016/S1054-3589(08)60952-7

Desrosiers, M. R., Mittleman, A., & Weathers, P. J. (2020). Dried Leaf Artemisia Annua Improves Bioavailability of Artemisinin via Cytochrome P450 Inhibition and Enhances Artemisinin Efficacy Downstream. Biomolecules, 10(2), 254. https://doi.org/10.3390/biom10020254

Efferth, T. (2007). Willmar Schwabe Award 2006: Antiplasmodial and Antitumor Activity of Artemisinin - From Bench to Bedside. Planta Medica, 73(04), 299–309. https://doi.org/10.1055/s-2007-967138

Ferreira, J. F. S., Luthria, D. L., Sasaki, T., & Heyerick, A. (2010). Flavonoids from Artemisia annua L. as Antioxidants and Their Potential Synergism with Artemisinin against Malaria and Cancer. Molecules, 15(5), 3135–3170. https://doi.org/10.3390/molecules15053135

Goodger, J. Q. D., Cao, B., Jayadi, I., Williams, S. J., & Woodrow, I. E. (2009). Non-volatile components of the essential oil secretory cavities of Eucalyptus leaves: Discovery of two glucose monoterpene esters, cuniloside B and froggattiside A. Phytochemistry, 70(9), 1187–1194. https://doi.org/10.1016/j.phytochem.2009.06.004

Gruessner, B.M., Cornet-Vernet, L., Desrosiers, M.R., Lutgen P., Towler, M.J., Weathers P.J. It is not just artemisinin: Artemisia sp. for treating diseases including malaria and schistosomiasis. Phytochem Rev (2019) 18:1509-1527.

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Hamel, A., Roy, M., & Proudlock, R. (2016). Chapter 4—The Bacterial Reverse Mutation Test. In R. Proudlock (Ed.), Genetic Toxicology Testing (pp. 79–138). Academic Press. https://doi.org/10.1016/B978-0-12-800764-8.00004-5

Han, J., Ye, M., Qiao, X., Xu, M., Wang, B.-R., & Guo, D.-A. (2008). Characterization of phenolic compounds in the Chinese herbal drug Artemisia annua by liquid chromatography coupled to electrospray ionization mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 47(3), 516–525. https://doi.org/10.1016/j.jpba.2008.02.013

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It is not just artemisinin: Artemisia sp. For treating diseases including malaria and schistosomiasis | Request PDF. (n.d.). ResearchGate. Retrieved July 8, 2020, from https://www.researchgate.net/publication/335946329_It_is_not_just_artemisinin_Artemisia_sp_for_treating_diseases_including_malaria_and_schistosomiasis

Kumar, S., Gupta, S., Singh, P., Bajpai, P., Gupta, M., Singh, D., Bajpai P., Gupta M. M., Singha D., Gupta A.K., Ram G., Shasany A.K., Sharma S. (2004). High yield of artemisinin by multi-harvest of Artemisia annua crops. Industrial Crops and Products, 19, 77–90. https://doi.org/10.1016/j.indcrop.2003.07.003

Liu, N. Q., Van der Kooy, F., & Verpoorte, R. (2009). Artemisia afra: A potential flagship for African medicinal plants? South African Journal of Botany, 75(2), 185–195. https://doi.org/10.1016/j.sajb.2008.11.001

Maron, D. M., & Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research/Environmental Mutagenesis and Related Subjects, 113(3), 173–215. https://doi.org/10.1016/0165-1161(83)90010-9

Matos, M. J., Santana, L., Uriarte, E., Abreu, O., Molina Pérez, E., & Yordi, E. (2015). Coumarins—An Important Class of Phytochemicals (pp. 113–140). https://doi.org/10.5772/59982

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Molina, N. C., & Peterson, J. W. (1980). Cholera Toxin-Like Toxin Released by Salmonella Species in the Presence of Mitomycin C. INFECT. IMMUN., 30, 7.

Mortelmans, K. (2019). A perspective on the development of the Ames Salmonella/mammalian-microsome mutagenicity assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 841, 14–16. https://doi.org/10.1016/j.mrgentox.2019.04.004

Mortelmans, K., & Zeiger, E. (2000). The Ames Salmonella/microsome mutagenicity assay. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 455(1), 29–60. https://doi.org/10.1016/S0027-5107(00)00064-6

Mueller, M. S., Runyambo, N., Wagner, I., Borrmann, S., Dietz, K., & Heide, L. (2004). Randomized controlled trial of a traditional preparation of Artemisia annua L. (Annual Wormwood) in the treatment of malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 98(5), 318–321. https://doi.org/10.1016/j.trstmh.2003.09.001

Munyangi, J., Cornet-Vernet, L., Idumbo, M., Lu, C., Lutgen, P., Perronne, C., Ngombe N., Bianga J., Mupenda B., Lalukala P., Mergeai G., Mumbal D., Towler M., Weathers, P. (2019). Artemisia annua and Artemisia afra tea infusions vs. Artesunate-amodiaquine (ASAQ) in treating Plasmodium falciparum malaria in a large scale, double blind, randomized clinical trial. Elsevier GmbH, Volume 57, Pages 49-56. https://doi.org/10.1016/j.phymed.2018.12.002

Newman, D. J., Cragg, G. M., & Kingston, D. G. I. (2015). Chapter 5 - Natural Products as Pharmaceuticals and Sources for Lead Structures**Note: This chapter reflects the opinions of the authors, not necessarily those of the US Government. In C. G. Wermuth, D. Aldous, P. Raboisson, & D. Rognan (Eds.), The Practice of Medicinal Chemistry (Fourth Edition) (pp. 101–139). https://doi.org/10.1016/B978-0-12-417205-0.00005-5

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Paz, M. M., Zhang, X., Lu, J., & Holmgren, A. (2012). A new mechanism of action for the anticancer drug mitomycin C: Mechanism-based inhibition of thioredoxin reductase. Chemical Research in Toxicology, 25(7), 1502–1511. https://doi.org/10.1021/tx3002065

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Rosine Desiree, N.-C., Jonas, K., Johnson, T., FOTSOP, D., HANSEN, G., Lutgen, P., FLIES, M., FISHER, M., SVEN, S., KOUEMENI, L., Tene, M., Wouessidjewe, D., TEKAM, J., Kaptue, L., & Tane, P. (2016). COMPARATIVE STUDY OF CHEMICAL COMPOSITION OF ARTEMISIA ANNUA ESSENTIAL OIL GROWING WILD IN WESTERN CAMEROON AND LUXEMBOURG BY μ-CTE/TD/GC/MS. North Asian International Research Journal of Multidisciplinary, Vol. 2, 12–31.

Ruan, J.-X., Li, J.-X., Fang, X., Wang, L.-J., Hu, W.-L., Chen, X.-Y., & Yang, C.-Q. (2016). Isolation and Characterization of Three New Monoterpene Synthases from Artemisia annua. Frontiers in Plant Science, 7. https://doi.org/10.3389/fpls.2016.00638

Stefanachi, A., Leonetti, F., Pisani, L., Catto, M., & Carotti, A. (2018). Coumarin: A Natural, Privileged and Versatile Scaffold for Bioactive Compounds. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(2). https://doi.org/10.3390/molecules23020250

Tu, Y. (2017). From Artemisia annua L. to Artemisinins: The Discovery and Development of Artemisinins and Antimalarial Agents. Academic Press.

Vijay, U., Gupta, S., Mathur, P., Suravajhala, P., & Bhatnagar, P. (2018). Microbial Mutagenicity Assay: Ames Test. BIO-PROTOCOL, 8(6). https://doi.org/10.21769/BioProtoc.2763

Weathers, P. J., Arsenault, P. R., Covello, P. S., McMickle, A., Teoh, K. H., & Reed, D. W. (2011). Artemisinin production in Artemisia annua: Studies in planta and results of a novel delivery method for treating malaria and other neglected diseases. Phytochemistry Reviews : Proceedings of the Phytochemical Society of Europe, 10(2), 173–183. https://doi.org/10.1007/s11101-010-9166-0

Weathers, P. J., Towler, M., Hassanali, A., Lutgen, P., & Engeu, P. O. (2014). Dried-leaf Artemisia annua: A practical malaria therapeutic for developing countries? World Journal of Pharmacology, 3(4), 39–55.

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9.0 Appendices

9.1 Appendix A: Recipes

Recipes from Mortelmans, K., & Zeiger, E. (2000) 9.1.1 Vogel-Bonner medium E (50x)

For GM agar plates (Recipe 6.1.9)

Ingredients Per liter

Warm distilled H2O (about 50oC) 650 ml

Magnesium sulfate (MgSO4·H2O) 10 g

Citric acid monohydrate 100 g

Potassium phosphate, dibasic, anhydrous (K2HPO4) 500 g

Sodium ammonium phosphate (Na2NH2PO4·4H2O) 175 g

1. Salts are added to the warm water in a flask. Make sure each salt is fully dissolved before adding the next.

2. After each salt dissolves entirely, adjust volume to one liter and transfer the solution into glass bottles and autoclave for 30 min at 121 °C

3. When the solution gets cool, cap the bottle tightly and store at room temperature in the dark

9.1.2 0.5 mM histidine/biotin solution

For top agar

Ingredients Per liter

Distilled H2O 1000 ml

D-biotin (F.W. 247) 124 mg

L-Histidine·HCl (F.W. 191.7) 96 mg

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1. Dissolve the biotin in boiling distilled water. Add the histidine once all biotin is dissolved.

2. If not used immediately for top agar, the solution is autoclaved for 20 min, at 121 °C and then stored at 4 °C

9.1.3 Sodium phosphate buffer, 0.1 mM, pH 7.4

For testing chemicals in the absence of metabolic activation

Ingredients Per liter

0.2 M sodium phosphate dibasic (NaH2PO4·H2O) 30 ml (14.2g/l)

0.2 M sodium phosphate monobasic (Na2HPO4) 120 ml (13.8g/l)

1. Mix together and adjust pH to 7.4 using dibasic sodium phosphate. 2. Sterilize the buffer by autoclaving for 30 min at 121 °C 3. Store bottles at room temperature in the dark

9.1.4 Glucose solution (10% v/v)

For carbon source in GM agar plates.

Ingredients Per liter

Distilled water 700 ml

Dextrose 100 g

1. Add dextrose to water in a 3-l flask and stir until solution is clear. 2. Add additional water to bring final volume to 1000 ml. 3. Autoclave for 20 min at 121 °C and store at 4°C.

9.1.5 Ampicillin solution (0.8% w/v)

For testing the presence of TA100 and TA98 strains before assay

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Ingredients Per 100 ml

Distilled water 100 ml

Ampicillin 800 mg

1. Ampicillin is dissolved in warm (65°C) water. 2. Filter sterilize using a 0.45 µm filter and store at 4°C.

9.1.6 Minimal glucose plates

Used in mutagenic bioassay

Ingredients Per liter

Distilled H2O 900 ml

Agar 15 g

VB salts (50x) 20 ml

Glucose (10% v/v) 50 ml

1. Add agar to water and autoclave for 30 min at 121°C. Allow to cool for 45 min to reach about 65°C.

2. After cooling, add the VB salts and mix thoroughly. 3. Once the solution is fully dissolved, add the glucose and stir again. 4. Dispense the agar medium in 100 x 15 mm petri dishes (approximately 25 ml/plate). 5. Once solidified, the plates can be stored at 4°C for several weeks if placed in sealed bags.

Before use, the plates should be warmed to room temperature and if there is excess moisture present, they should be incubated at 37°C overnight prior to use.

9.1.7 Histidine/Biotin plates (Master plates for non R-factor strains)

Used in tests for histidine requirement

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Ingredients Per 500 ml

Agar 7.5 g

Distilled H2O 457 ml

50x VB salts 10 ml

40% glucose 25 ml

Sterile histidine (2 g per 400 ml H2O) 5 ml

Sterile 0.5 mM biotin 10 ml

40% glucose 3 ml

1. Dissolve agar in the given concentration in distilled water. 2. Autoclave each solution separately for 20 min. 3. After cooling of solution, add each salt gently

9.1.8 4-Nitroquinoline 1-oxide (4NQO)

Used in tests for positive control

Ingredients Per 500 ml

Distilled H2O 45µl

4NQO 5µl

This solution should be made fresh before each test. 4NQO is a carcinogen and appropriate precaution should be taken.

9.2 Appendix B: Materials and Reagents

Materials and reagents list taken from Vijay et al. 2018

9.2.1 Materials

1. Tips (1,000 µl, 200 µl, 10 µl)

2. Sterile Petri plates

3. Erlenmeyer flask and beaker (10 ml, 250 ml, 500 ml)

4. Eppendorf tubes (1.5 ml, 2.0 ml)

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5. Metal loop holder

6. L shaped spreader

9.2.2 Reagents

1. Oxoid nutrient broth No. 2

2. 70% ethanol

3. Magnesium sulphate heptahydrate (MgSO4·7H2O)

4. Citric acid monohydrate

5. Potassium phosphate, dibasic (K2HPO4) (anhydrous)

6. Sodium ammonium phosphate tetrahydrate (NaNH4HPO4·4H2O)

7. D-biotin

8. L-histidine

9. Hydrochloric acid (HCI)

10. Potassium chloride (KCl)

11. Magnesium chloride hexahydrate (MgCl2·6H2O)

12. Sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O)

13. Disodium hydrogen phosphate (Na2HPO4)

14. D-glucose-6-phosphate (monosodium salt)

15. Ampicillin trihydrate

16. Sodium hydroxide (NaOH)

17. Agar-Agar

18. Nutrient broth

19. Vogel-Bonner medium E (50x) (see Recipe 9.1.1)

20. 0.5 mM histidine/biotin solution (see Recipe 9.1.2)

21. 0.2 M sodium phosphate buffer, pH 7.4 (see Recipe 9.1.3)

9.2.3 Equipment

1. Spectrophotometer

2. Orbital shaking incubator

3. Laminar Flow hood

4. Pipettes (1,000 μl; 200 μl; 10 μl)

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5. Vortex mixer

6. Hot water bath

7. Autoclave

8. pH meter

9.3 Appendix C: Statistic Analysis of Results

Table 11 - TA 100 w A. Annua

Source of Validation Sums of Squares (SS) Degrees of Freedom (df) Means Squares (MS) F Between Treatments 5.188 4 1.297 0.161 Error (or Residual) 112.750 14 8.054

Total 117.938 18

Table 12 - TA 100 w A. Afra

Source of Validation Sums of Squares (SS) Degrees of Freedom (df) Means Squares (MS) F Between Treatments 21.667 4 5.417 0.694 Error (or Residual) 78.000 10 7.800

Total 99.667 14

Table 13 - TA 98 w A. Annua

Source of Validation Sums of Squares (SS) Degrees of Freedom (df) Means Squares (MS) F Between Treatments 3.000 4 0.750 0.104 Error (or Residual) 181.000 25 7.240

Total 184.000 29

Table 14 - TA 98 w A. Afra

Source of Validation Sums of Squares (SS) Degrees of Freedom (df) Means Squares (MS) F Between Treatments 14.000 4 3.500 0.398 Error (or Residual) 88.000 10 8.800

Total 102.000 14