key: cord-0833677-iod38u7q
authors: Dogan, Kubra; Erol, Ebru; Didem Orhan, Muge; Degirmenci, Zehra; Kan, Tugce; Gungor, Aysen; Yasa, Belkis; Avsar, Timucin; Cetin, Yuksel; Durdagi, Serdar; Guzel, Mustafa
title: Instant determination of the artemisinin from various Artemisia annua L. extracts by LC‐ESI‐MS/MS and their in‐silico modelling and in vitro antiviral activity studies against SARS‐CoV‐2
date: 2021-09-28
journal: Phytochem Anal
DOI: 10.1002/pca.3088
sha: 7db302b01632deae431e8408f015fbc2118f891e
doc_id: 833677
cord_uid: iod38u7q

INTRODUCTION: Numerous efforts in natural product drug development are reported for the treatment of Coronavirus. Based on the literature, among these natural plants Artemisia annua L. shows some promise for the treatment of SARS‐CoV‐2. OBJECTIVE: The main objective of our study was to determine artemisinin content by liquid chromatography electrospray ionisation tandem mass spectrometry (LC‐ESI‐MS/MS), to investigate the in vitro biological activity of artemisinin from the A. annua plants grown in Turkey with various extracted methods, to elaborate in silico activity against SARS‐CoV‐2 using molecular modelling. METHODOLOGY: Twenty‐one different extractions were applied. Direct and sequential extractions studies were compared with ultrasonic assisted maceration, Soxhlet, and ultra‐rapid determined artemisinin active molecules by LC‐ESI‐MS/MS methods. The inhibition of spike protein and main protease (3CL) enzyme activity of SARS‐CoV‐2 virus was assessed by time resolved fluorescence energy transfer (TR‐FRET) assay. RESULTS: Artemisinin content in the range 0.062–0.066%. Artemisinin showed significant inhibition of 3CL protease activity but not Spike/ACE‐2 binding. The 50% effective concentration (EC(50)) of artemisinin against SARS‐CoV‐2 Spike pseudovirus was found greater than 50 μM (EC(45)) in HEK293T cell line whereas the cell viability was 94% of the control (P < 0.01). The immunosuppressive effects of artemisinin on TNF‐α production on both pseudovirus and lipopolysaccharide (LPS)‐induced THP‐1 cells were found significant in a dose dependent manner. CONCLUSION: Further studies of these extracts for COVID‐19 treatment will shed light to seek alternative treatment options. Moreover, these natural extracts can be used as an additional treatment option with medicines, as well as prophylactic use can be very beneficial for patients.

SARS-CoV-2 virus, which emerged in China in late 2019 and spread all over the world, caused a global pandemic . 1 Until now, the total number of cases in the world is 203 million while the number of deaths is 4.3 million. There are unfortunately no specific antiviral drugs for the treatment of the COVID-19 pandemic that affects the whole world. Although there is more than one vaccine with emergency use authorisation in the world, most of these vaccines seem to be not very effective for the variants. In addition, the right of access to the existing vaccines is not equal in every country, and there are delays in production due to the demand. Moreover, a certain majority reject the vaccination. Considering all these reasons, studies with natural products for the tr eatment of COVID-19 are very important.

Owing to the natural and widespread distribution of Artemisia annua L., also known as Sweet Wormwood, in wide and various geographies, and usage patterns of numerous cultures different health problems are revealed. 2 Artemisia annua has antimalarial properties and has been used frequently in traditional Chinese medicine in the treatment of malaria and in high fever since ancient times. In extended ethno-botanical studies and archaeological studies, it was revealed that A. annua was used as fresh plant water. Artemisinin, the active ingredient of the A. annua plant, has passed World Health Organisation's (WHO's) recommendations as the first drug of choice in malaria resistant to chloroquine and other treatments or showing cerebral involvement. 3 Studies show that A. annua has a strong antiviral effect. Li et al. tested the ethanolic extract of A. annua and determined that it was effective against coronavirus associated with SARS at a 50% effective concentration (EC 50 ) dose of 34.5 ± 2.6 μg/mL and reported that it could be developed as an antiviral agent in the treatment of coronavirus. 4 In addition, Karamoddini et al. reported that A. annua has the most intense anti-herpetic effect among the methanolic extracts homogenised of various Artemisia species in vitro. 5 Inflammatory response is extremely important in SARS-CoV-2 infection as in many diseases. It has been suggested that a severe increase in inflammatory response is observed in patients with SARS-CoV-2 infection due to viral replication in the lungs, and the resulting cytokine storm may be closely related to the severity of the disease. 6, 7 To reduce the inflammatory response shaped in SARS-CoV-2 infection, it is recommended in different treatments besides classical antiinflammatory drugs. 8 It is emphasised that artemisinin and its analogues have antiviral, antifungal, anticancer and anti-inflammatory properties in addition to their antimalarial activities. 9 Macrophages play an extremely important role in initiating and regulating the immune response. They control various cytokines they produce in the inflammatory response process through NF-κB (nuclear factor kappalight-chain-enhancer of activated B cells). 10 Artemisinin, by regulating transcriptional signalling pathways in macrophages, consequently, reduce cytokine release by macrophages. 11 It has been reported that artemisinin in human monocytes suppresses matrix metallopeptidase 9 (MMP-9), tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) release by regulating NF-κB release. 12, 13 In addition, artemisinin has been shown to have an anti-inflammatory effect in phorbol 12-myristate 13-acetate (PMA)-treated human THP-1 cells. 13 Based on the overwhelmingly studies of A. annua extracts in the literature and the recent pandemic conditions we have been inspired to study this plant extract further. To investigate its potential antiviral properties since it is commonly grown in our region and the plant is used as a medicinal tea, especially for treating malaria. Nair et al., researched antiviral activity of dried leaf extracts of seven cultivars of A. annua against SARS-CoV-2. They used hot-water leaf extracts based on artemisinin. Artemisinin has alone an antimalarial effect with an estimated 50% inhibitory concentration (IC 50 ) of approximately 70 μM, while the derivatives of artemisinin, artesunate, artemether and dihydro artemisinin, are inactive or cytotoxic at high micromolar concentrations. 14 Many extraction methods have been investigated in the literature to obtain the artemisinin-rich fractions. However, the plant used in these studies is A. annua, collected from different places.

One of the crucial parameters that affect the active substance (secondary metabolite) content, is the location where the plant is collected. Therefore, it was more meaningful to compare the efficiency of the extraction methods in the literature. Hence, in our study we selected the most accurate and efficient extraction method which is aimed to be applicable to the industrial scale. This research was later supported by in silico molecular modelling as well as in vitro biological activity studies. Our aim and objective were to identify the best extraction method for artemisinin and subsequently investigate its antiviral properties with pseudoneutralisation assay and compare artemisinin binding motifs with known antivirals which have not appeared in the recent literature.

The novelty and advantage of our research from the literature is: (vi) comparative in silico molecular modelling studies of artemisinin with three known antiviral agents (manidipine, lercaidipine, and efonidipine).

The chemicals used in the experiments listed as follows: isopropanol, ethanol, methanol, n-hexane, chloroform, acetone, dichloroethane, dichloromethane, propylene glycol methyl ether and chloroform were supplied from Merck (Darmstadt, Germany). All chemicals used in this study were of analytical reagent grade. HPLC grade acetonitrile was purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Figure S1 ). Necessary permissions have been obtained from Bursa Regional Directorate of Forestry, Non-Wood Products and Services Branch Office for the supply of raw materials to be used in our studies.

Soxhlet extraction, ultrasonic assisted maceration and infusion methods were used to obtain the extract with the highest artemisinin content. Initially, the shade dried and powdered aerial parts of the A. annua were kept in an ultrasonic assisted maceration for 15 minutes and then macerated at room temperature for 24 hours (Table 1) . Then, extracts (AA-2, AA-3, AA-5, AA-6, AA-7, AA-8 AA-11, AA-12 and AA-13) were concentrated in vacuo at 40 C. The highest artemisinin content was obtained by ultrasonic assisted maceration using hexane (AA-5), ethanol (AA-7) and dichloromethane (AA-12) solvents. Therefore, extraction was performed by a Soxhlet apparatus using hexane (AA-4), dichloromethane (AA-16) and ethanol (AA-17).

In addition to this, considering the artemisinin content in AA-4 after 

Spike and ACE2 binding inhibition were assessed by using SARS- 

HEK293T is widely used for retroviral production and highly transfectable cell lines. The pseudovirus involving SARS-CoV-2 S was prepared as described by the previous study. 22 Briefly, HEK293T cells were cultured (5 Â 10 5 cells/well) in the six-well plates. stored at À80 C until they were used for the infection assay. incubated at 37 C and 5% CO 2 for 20 hours. Thereafter these treatments, the cell culture supernatants were collected and analysed to determine the cytokines production using enzyme-linked immunosorbent assay (ELISA) kit as described by the manufacturer's instructions.

The level of the each examined cytokine in each treatment was quantified using a standard curve generated with the given standards in each ELISA kit. 

The statistical analysis of all assays was evaluated by using GraphPad In the literature, artemisinin has been conventionally isolated from A. annua with organic solvents such as toluene, 23, 24 n-hexane, 18, 25, 26 petroleum ether, 27,28 chloroform, 29 95% ethanol, 16 dichloromethane, 15, 22 propylene glycol methyl ether, 19 isopropanol, 15 1-butanol, 30 and water. 14, 31, 32 Since the amount of artemisinin found in A. annua is extremely low, different combined systems for increasing the extraction efficiency of this compound, e.g. Soxhlet, ultrasound, microwave assisted extraction and supercritical fluid extraction (SFE) (usually supercritical CO 2 extraction) have been used. In recent years, ultrasound-assisted extraction has been used to predict the best extraction parameters among extraction methods using various computational tools and mathematical modelling. 30 Ultrasonic extraction, known as a non-thermal extraction method, shortens the processing time, therefore, provides a higher purity product, reduces energy consumption and this extraction method is an environmentally friendly technology with less solvent usage. Most of the studies have been conducted with ultrasonic probe extraction.

Some researchers claim that heat treatment, although commonly used, has some negative effects on the nutrient content. Ultrasoundassisted extraction uses ultrasound or ultrasonic agitation to increase the extraction efficiency from a solid matrix using a solvent or solvent mixture. There are studies using ultrasound-assisted artemisinin isolation. 16, 19, 24, 30, 33 Zhang et al. extracted artemisinin from A. annua using a series of mono ether-based solvents with an ultrasound-assisted extraction system. Propylene glycol methyl ether (PGME) was found to be the most suitable as it is safe and of low toxicity. 19 

The regression equation of the calibration curve was y = 53952.0x + 21331.4, with R 2 = 0.9998605 ( Figure S2 ). Slope (S) and standard deviation (δ) of the response were determined for the calibration curve. LOQ and LOD were calculated. 3 injections were made from each analytical portion. The analysis method has been validated according to a single laboratory validation approach. 21 The LOD, LOQ, and recovery for artemisinin were 1.3 ng/mL and 4.2 ng/mL (Tables 3 and 4 ).

The extraction solvent is a very important factor in the recovery of artemisinin from A. annua so extraction is performed in solvents of different polarity as no single solvent may be reliable. Hexanes, 95%

ethanol and isopropanol were identified as the most effective solvents for the extraction, resulting in the highest artemisinin content in the range 0.062-0.066% (Table 1) .

Although water as the extraction solvent provided the highest extraction yield, artemisinin content was not determined. In a previous report, water used as solvent in the extraction of A. annua plant did not extract artemisinin, confirming the low solubility of artemisinin in water. 31 In another study, 11.6 mg of artemisinin/g of the feed had been Table 5 .

The IC 50 value of artemisinin using MCF-231 cell line was reported as 177 μM after 72 hour exposure. 40 As demonstrated in T A B L E 3 Liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) parameters of artemisinin and 48B, 43 respectively. These studies demonstrate that artemisinin caused differential cytotoxic effects depending not only on the concentration and time of exposure but also on the specific target cells. cells. 51 In our study, the immunosuppressive effects of artemisinin on IL-1ß production of J774a.1 macrophages were examined but there was no significant inhibition observed (data not shown here). The reason for the differences in the results from these studies could be due to the applied different macrophage cell lines. The elevated levels of IL-1β, IL-6, IL-8, and TNF-α have been detected in COVID-19 patients during the pandemic. 49 Therefore, the use of immunosuppressive drugs against cytokine and chemokine storm has been very crucial for F I G U R E 5 Immunomodulatory effects of artemisinin on proinflammatory cytokine production on LPS or SARS-CoV-2 S pseudovirionstimulated THP-1 and J774a.1 macrophage cells. Human THP-1 differentiated into the macrophages and mouse J774a.1 cells were seeded into the 96 well plates in triplicates (2 Â 10 4 /well) and incubated for 24 hours. Later, they were treated with artemisinin at the indicated concentrations for 4 hours, then LPS (μg/ml) or SARS-CoV-2 S pseudovirion was added. (A) After 20 hours, WST-1 calorimetric agent was added and incubated at 37 C and 5% CO 2 for 2 hours and the cell viability was measured at 450 nm with a microplate reader. (B) The cell culture supernatant was collected from each treatment and each cytokine level was determined by ELISA. The results from one representative experiment of three independent experiments were presented as means ± standard deviation (*P < 0.01; **P < 0.001) the treatment of COVID-19. Artemisinin has been reported being used in a combinational approach along with other antiviral drugs designed to evaluate the neutralisation efficacy of SARS-CoV-2.

Artemisinin is one of the currently considered therapeutic agents as potential candidate/employed for ongoing trials and the treatment of COVID-19 disease due to its anti-inflammatory and immunomodulatory actions. 49 16.2 uM, and 38.5 uM, respectively. 52 Thus, we docked these known main protease inhibitors with the same docking protocol used in the docking of artemisinin. Top-docking scores were found as À4.88 kcal/ mol, À5.72 kcal/mol, and À5.03 kcal/mol for manidipine, lercanidipine, and efonidipine, respectively. Docking scores of known main protease inhibitors and artemisinin have similar values. Figure 6 shows two-dimensional (2D) ligand interaction diagrams of these three main protease inhibitors. Top-docking positions of artemisinin shows that Asn142 forms hydrogen bonds with the ligand at the binding pocket of the main protease. Other residues in contact with the ligand within 3 Å were Thr25, His41, Met49, His163, Glu 166, and Gln189 ( Figure 7) . Corresponding residues at the Spike/ACE-2 were Glu23, Lys26, Thr27, and Asp30 from ACE-2 region and Lys417, Tyr421, Phe456, Arg457, and Tyr473 from Spike region (Figure 8 ). In order to compare the docking score of artemisinin at the Spike/ACE-2 interface, its docking score is also compared with the known inhibitor.

In the report of Bojadzic et al., it is stated that a drug-like compound DRI-C23041 inhibits the interaction of hACE-2 with Spike protein. 53 Thus, DRI-C23041 is docked with the same protocol of artemisinin at the Spike/ACE-2 interface. Its docking score was found as À3.69 kcal/mol which is lower than the corresponding docking score of artemisinin. Figure 9 shows a 2D ligand interaction diagram of DRI-C23041 at the Spike/ACE-2 interface. Since experimental studies show promising results for IL-6 and IL-8 targets, we also docked artemisinin to these targets and docking scores were found as À3.63

and À4.75 kcal/mol, respectively.

There have been tremendous efforts worldwide to find possible cures for COVID-19. Despite huge worldwide efforts to produce a vaccination to stop it, this deadly pandemic is still a threat to mankind.

Thus, the use of prophylactically potential herbal medicines is another approach to prevent this virus spreading. In the future, effective herbal medicines will be very attractive sources as food supplements for healthy lifestyle and free of virus infections. Artemisinin can be a good candidate to develop common immunity since it can grow in most countries in the world. Moreover, this herbal extract can provide extra antiviral properties once used with common antiviral agents as an additional synergetic natural product. As a result of this study, we have demonstrated that local A. annua extracts can be considered a potential antiviral. Further studies need to be conducted especially in vivo SARS-CoV-2 challenge experiments will be the following goals to carry out this research on a higher level, which will be reported in due course.

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