Chemotaxonomic Study of the Covid-Organics of Madagascar Based on the Chemical Composition of Their Essential Oils ()
1. Introduction
Since the end of December 2019, a new coronavirus (Covid-19) with human-to-human transmission and serious human infection was identified. This virus affected many people in a very short time [1] [2] . On January 30, 2020, the Director-General of the World Health Organization declared the Covid pandemic a public health emergency of international concern and issued temporary recommendations under the International Health Regulations [3] .
This pandemic was still ongoing, so there was an urgent need to find new preventive and therapeutic agents as soon as possible. Although specific vaccines and antiviral agents are the most effective methods to prevent and treat viral infection, there are not yet effective treatments that target Covid-19. The development of these treatments could take months or years, which means that a more immediate treatment or control mechanism should be found if possible [4] . Medicinal plants present a potentially valuable resource for this purpose [5] .
Thus, in April 2020, the Malagasy Institute for Applied Research developed an herbal tea composed of 62% Artemisia annua and two other medicinal plants whose names were not revealed that grow in Madagascar (Covid-Organics), claiming preventive and curative properties against Covid-19 [6] . Artemisia annua L. (Asteraceae) is native to China, well known as the source of artemisinin and used in the treatment of the malaria. In addition, its essential oils have several therapeutic properties [7] [8] [9] [10] [11] .
In May, Senegal placed its order for “Covid-Organics”, but the National Scientific Committee demanded that we first carry out in-depth scientific examinations on this product made in Madagascar before possibly using it on patients with coronavirus because reliable botanical, chemical, toxicological and pharmacological data were lacking.
Therefore, the aim of this study was to investigate the chemical composition of the essential oils of “Covid-Organics” to identify its constituent species.
2. Experimental
2.1. Plant Material
Samples (two remedies in the form of herbal teas: curative and preventive) were provided by the Pharmacy and Drug Directorate. The two remedies were composed of 62% Artemisia annua and two other medicinal plants whose names were not revealed. The only difference was the dosage: the curative remedy was administered 3 times/day, while the preventive was administered 2 times/day.
2.2. Extraction of Essential Oils
Samples were hydrodistilled (6 h) using a Clevenger-type apparatus according to the method recommended in the European Pharmacopoeia [12] . The yields of essential oils (w/w, calculated on dry weight basis) were given in Table 1.
2.3. Chemical Compositions
The chromatographic analyses were carried out using a Perkin-Elmer Autosystem XL GC apparatus (Walthon, MA, USA) equipped with dual flame ionization detection (FID) system and fused-silica capillary columns, namely, Rtx-1 (polydimethylsiloxane) and Rtx-wax (poly-ethyleneglycol) (60 m × 0.22 mm i.d;
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Table 1. Chemical composition of the essential oils of the two remedies.
aOrder of elution is given on apolar column (Rtx-1); bRetention indices of literature on the apolar column (lRIa); cRetention indices on the apolarRtx-1 column (RIa); dRetention indices on the polar Rtx-Wax column (RIp).
film thickness 0.25 μm). The oven temperature was programmed from 60 to 230˚C at 2˚C/min and then held isothermally at 230˚C for 35 min: hydrogen was employed as carrier gas (1 mL/min). The injector and detector temperatures were maintained at 280˚C, and samples were injected (0.2 μL of pure oil) in the split mode (1:50). Retention indices (RI) of compounds were determined relative to the retention times of a series of n-alkanes (C5-C30) by linear interpolation using the Van den Dool and Kratz (1963) equation with the aid of software from Perkin-Elmer (Total Chrom navigator). The relative percentages of the oil constituents were calculated from the GC peak areas, without application of correction factors.
Samples were also analysed with a Perkin-Elmer Turbo mass detector (quadrupole) coupled to a Perkin-ElmerAutosystem XL, equipped with fused-silica capillary columns Rtx-1 and Rtx-Wax. The oven temperature was programmed from 60 to 230˚C at 2˚C /min and then held isothermally at 230˚C (35 min): hydrogen was employed as carrier gas (1 mL/min). The following chromatographic conditions were employed: injection volume, 0.2 μL of pure oil; injector temperature, 280˚C; split, 1:80; ion source temperature, 150˚C; ionization energy, 70 eV; MS (EI) acquired over the mass range, 35 - 350 Da; scan rate, 1 s. Identification of the components was based on: (a) comparison of their GC retention indices (RI) on non-polar and polar columns, determined from the retention times of a series of n-alkanes with linear interpolation, with those of authentic compounds or literature data; (b) on computer matching with commercial mass spectral libraries [13] [14] [15] and comparison of spectra with those of our personal library; and (c) comparison of RI and MS spectral data of authentic compounds or literature data.
3. Results and Discussion
The essential oil yields of the two remedies (curative and preventive) were 0.16% and 0.2%, respectively.
Chemical analysis by Gas Chromatography (GC) showed that the two essential oils of the two remedies (curative and preventive) have identical chromatographic profiles. This analysis allowed the identification of 32 compounds representing 64.9 and 76.7%, respectively, of the total chemical compositions. This confirms that the two remedies are made from the same plants. These essential oils (curative and preventive) were mainly dominated by camphor (17.9% and 11.9%, respectively), spathulenol (4.8% and 11.8%, respectively), α-acorenol (4.3% and 3.7%, respectively), (E)-β-caryophyllene (3.4% and 4.2%, respectively), 1,8-cineole (3.1% and 3.6%, respectively), Hexadecanoïc acid (3.8% and 3.2%, respectively) and caryophyllene oxide (3.4% and 2.4%, respectively). Camphor is a characteristic compound of the essential oil of A. annua and its content can be up to 50% in A. annua [7] [8] [16] [17] . It was the main compound of the essential oil of A. annua grown in Senegal [17] . 1,8-cineole was found at significant levels in both essential oils (3.1% and 3.6%, respectively) along with sabinene. These two compounds constitute the chemotype of Ravintsara (Cinnamomum camphora, ct 1,8-cineole and sabinene) from Madagascar [18] [19] [20] [21] [22] . These results show that both remedies contain A. annua and C. camphora. However, we were unable to identify the third species. This hypothesis was confirmed by the capsule form (Artemisinin and essential oil of C. camphora) [23] .
Ravintsara oil is distilled from the leaves of C. camphora belongs to the family Lauraceae in Madagascar. True Ravintsara essential oils contain at least 45% of 1,8-cineole [19] [20] [21] [22] . During the recent outbreak of coronavirus (SARS-CoV-2), Ravintsara oil is found to inhibit the coronavirus in vitro and in vivo due to its antiviral properties [24] [25] . Ravintsara oil is an excellent germ fighter and has antiviral, anti-inflammatory, and anti-spasmodic properties which can calm muscles and soothe coughs [19] [20] [21] [25] .