Comparative pharmacognostic and chromatographic assessment of COVID-ORGANICS (CVO) herbal product and Artemisia annua L.

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INTRODUCTION
Documentation on the use of traditional medicine to manage viral infections is common in literature. In China, traditional herbal medicine is often used with conventional medicine to treat SARS with most Chinese opting to receive traditional Chinese medicine for the treatment of Covid-19 (Yang et al., 2020). The use of traditional Chinese medicine in the treatment of SARS-CoV-2 is largely inspired by its earlier use in the *Corresponding author. E-mail: sadiqoyene@yahoo.com Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License treatment of SARS caused by SARS coronavirus (SARS-CoV) (Zhong, 2004). In a controlled clinical study, supplementary treatment with traditional herbal medicine resulted in marked improvement of symptoms of SARS and shortened the disease course (Hsu et al., 2006). Glycyrrhizin, a major active constituent of liquorice root, and baicalin another Chinese herbal compound both inhibited the replication of clinical isolates of SARS virus (Cinat et al., 2003;Chen et al., 2004). Reports have shown that the use of Artemisia annua L. along with conventional orthodox medicines can be beneficial in the management of viral infections (Karamoddini et al., 2011). A. annua L. known as sweet wormwood is one of the most commonly used species in the genus Artemisia, family Asteraceae (Compositae). It is native to many countries in Asia and Europe but has since been domesticated in the medicinal plant garden of the National Institute for Pharmaceutical Research and Development (NIPRD), Nigeria towards the production of Artemisinin-Based Combination Therapies (ACTs) against Malaria (Figure 1) (Jegede and Brisibe, 2007;Jegede et al., 2012). The aqueous, alcohol and non-polar solvent extracts of the aerial part of A. annua have been severally reported for their antimalarial, antifungal, antibacterial and antioxidant properties (Zheng, 1994;Allen et al., 1998;Tan et al., 1998). The antiviral properties of the methanol extracts of A. annua aerial parts against Herpes Simplex Virus type 1 (HSV1) have been reported (Karamoddini et al., 2011). A. annua has a well-established role in the management of tumors (Zhu et al., 2013). The ethanol extract of A. annua was reported by Li et al. (2005) to be active against SARS COV 1. The main compounds present in its essential oil appear to be camphene, β-camphene, isoartemisia ketone, 1-camphor, β-caryophyllene and β-pinene. In addition, other minor ingredients, such as artemisia ketone, 1, 8-cineole, camphene hydrate, and cuminal are also found in the volatile parts of A. annua (Verdian, 2009). Most common non-volatile compounds present in the aerial part are sesquiterpene lactone artemisinin and some of its biogenetic precursors arteannuin B and artemisinic acid, artemisin, dehydroartemisinin, artemisinol, scopoletin, chrysosplenetin, chrysosplenol D, casticin, dihdroxy-6-methoxyacetophenone, eupatin, sistosterol and coumarin 4-methylesculetin (Verdian, 2009;Zhu et al., 2013;Chougouo et al., 2016).
The herbal product named 'COVID-ORGANICS (CVO) Tambavy Tisane Herbal Tea' was produced by the Institute of Malagasy Recherches Appliquees, Ratsimamanga in Madagascar. The tea which is composed of A. annua (62%) and other medicinal plants (38%) was acclaimed by the Government of Madagascar to be effective in treatment and management of COVID-19 infection. The herbal tea is said to strengthen the immune system, treat viral infections, reduce fever and cure breathing difficulties.
The process of verification of claims is primarily aimed at substantiating claims of identity, purity, content, efficacy, and safety of herbal medicines and products.
Hence the aim of this study is to identify and confirm the presence of A. annua as a major component of CVO as well as compare CVO Herbal product with samples of A. annua cultivated in NIPRD Garden.

Sample collection and preparation
Two samples of the COVID-ORGANICS (CVO), Green and Orange pack designated as GPA and OPB respectively were used for the study. Two samples of A. annua cultivated in NIPRD Garden were collected at pre-flowering and flowering stages and designated as NFAa and FAa respectively.

Tests for identity and purity
All analyses were carried out according to guidelines specified in the WHO quality control guidelines for the assessment and evaluation of herbal medicines (WHO, 1992(WHO, , 2011.

Organoleptic, macroscopic and microscopic evaluation
Macroscopic and organoleptic properties, viz; appearance, colour, taste and odour of all samples were documented. A minimum of 2 g of each powdered sample was soaked and cleared in sodium hypochlorite (3.5% w/v) overnight. The cleared particles were mounted in a mixture of glycerol in water (1:1) and viewed under the microscope at different magnifications. Tests for the presence of different metabolites using appropriate stains and indicators were carried out. Photomicrographs were taken using the TUCSEN camera and ARE Capture software.

Physicochemical evaluation
Test for physicochemical properties of the samples as crude drugs, viz; moisture content, ash content, sulphated ash, water and alcohol extractives, were carried out as described in the WHO guidelines for methodologies for research and evaluation of herbal medicines.

Extraction
Extracts were prepared as stated in the instructions for use of the CVO (as stated on the packs) and as indicated in USP method for identification of artemisinin, a major bioactive component of A.
annua (International Pharmacopoeia, 2019). Three different extraction methods were thus employed to obtain extracts from the four dried powdered samples.

Method 1:
The method prescribed on the leaflet for preparation of Madagascar COVIDORGANICS dried powdered sample was employed for the extraction of all samples. 1000 mg of each sample was extracted by infusion with 60 mL of boiling water and allowed to stand for 15 min, filtered and dried. Within 24 h, dried extracts were reconstituted in 20 mL of ethanol, filtered through a 0.45 µm syringe filter. Samples were transferred to 1.8 mL vials and used for TLC and HPLC analyses.
Method 2: Artemisinin was extracted from 1000 mg of CVO dried powder and NIPRD A. annua dry leaf powder by refluxing with 50 mL of hexane (45°C) for 2 h, transferred to beakers and left to dry overnight in a fume hood. Within 24 h, samples were reconstituted in 20 mL of acetonitrile (two washes of 10 mL each) to obtain 50 mg per mL solution, filtered through a 0.45 µm nylon syringe filter. Samples were transferred to 1.8 mL vials and used for TLC and HPLC analyses (WHO, 2019).

Method 3:
Artemisinin was extracted from 1000 mg of CVO dried powder and NIPRD A. annua dry leaf powder by maceration with 20 mL of ethanol (95% v/v) for 2 h with ultra-sonification to obtain 50 mg per mL solution filtered through a 0.45 µm nylon syringe-top filter. Samples were transferred to 1.8 mL vials and used for TLC and HPLC analyses. (https://www.mmv.org/docs).

Thin layer chromatography (TLC) analysis
Thin-layer chromatography analysis was performed using silica gel precoated aluminum sheet as stationary phase and a mixture of hexane and ethyl acetate (4:1) as the mobile phase. Spotting was done by the application of 10 µL of each of the test samples and artemisinin reference standard (0.1 mg/mL) separately. The spotted sheet was placed in the chromatographic chamber and the solventfront was allowed to run up to 60 mm from the origin. The sheet was air-dried and sprayed with freshly prepared anisaldehyde/ sulfuric acid solution and heated at 105°C for about 7 min. The chromatogram was examined in daylight. The artemisinin spot obtained with sample solution corresponds in position and appearance (pink) with that obtained with artemisinin reference standard solution (WHO, 2019).

High performance liquid chromatography (HPLC) analysis
High-performance liquid chromatography was performed using a stainless-steel ODS column (15 cm × 4.6 mm, particle size: 5 µm). The mobile phase consisted of a 50:50 mixture of acetonitrile and water. Artemisinin reference standard solution (0.1 to 20 mg per mL) was used. The HPLC system was operated at a flow rate of 1.0 mL per minute. The ultraviolet detector was set at a wavelength of 210 nm. 20 µL of sample and reference solutions were injected separately. The run time was set to 15 min. The areas of the peak responses obtained in the chromatograms were documented and the percentage composition of each peak was calculated based on the total peak areas (WHO, 2019).

Organoleptic and macroscopic properties
The macroscopical and organoleptic features of the samples of CVO and A. annua are presented in Table 1. Light to dark green colour was observed for all the samples while sweet-bitter taste was noted for CVO samples and bitter astringent taste for A. annua samples (Table 1). CVO samples (OPB and GPA) had similar appearance and odour. Their appearance differed slightly from those of A. annua (NFAa (Pre-flowering) and FAa (Flowering) collected from NIPRD garden. NFAa and FAa had a bitter astringent taste which is a characteristic of A. annua (Verdian, 2009) while the two CVO samples had a sweet-bitter taste indicating the presence of a sweetener in the product. GPA was sweeter and lighter in colour than OPB. Other parameters observed from the macroscopy include bark or root fragments, red and green leaf fragments bigger in size than the green leaf fragments of A. annua (Table 1).

Qualitative microscopy
Chemomicroscopic properties are shown in    (Table 3 and Figures 2 to 9). The relatively higher abundance of A. annua features in the CVO indicates that it is the major component of the product.

Physicochemical properties
Air-dried A. annua samples (NFA and FA) had a moisture content of 9.8 and 8.5%w/w respectively, while CVO (GPA and OPB) samples had a moisture content of 10.3 and 10.4%w/w respectively. The presence of a sweetener and other plant materials which may be root or      stem-bark in the product might be responsible for the higher moisture content in GPA and OPB. While the extractive values of CVO (GPA) were higher than those of all the other samples, those of OPB were similar to those of A. annua (NFAa and FAa). The ash content of GPA, OPB, NFAa and FAa were 9.4 ± 0.4%w/w, 12.1 ± 0.1%w/w, 9.1 ± 0.3%w/w and 7.8 ± 0.5%w/w respectively. Slight variations observed could be due to difference  in geographical location and collection sources, however very noticeable are the higher extractive values. The higher extractive value of GPA relative to OPB might be indicative of the presence of more sweetener in the former especially as GPA was sweeter than OPB.

Thin layer chromatography
TLC fingerprint of the hot water infusion, n-hexane and 95% ethanol extracts are shown in Figure 10. The fingerprint indicated the presence of artemisinin in all four samples (OPB, GPA, NFAa and FAa (Figure 10).

High performance liquid chromatography
Percentage composition of chemical constituents determined by HPLC of the four samples extracted with 95% ethanol, hot water and n-hexane are presented in Tables 5 to 7  For the hexane extract, GPA and OPB had 16 and 17 peaks respectively, while FAa and NFAa had 21 and 19 peaks respectively. The number of peaks that cut across the four samples were 14 in number. The number of peaks with same retention time and comparable percentage composition in GPA and OPB were 18 in number while in FAa and NFAa, they are 19. Since hexane has been reported to be a suitable solvent for extraction of artemisinin from A. annua, the percentage composition of the peak corresponding to artemisinin can be taken as a good indication of the relative artemisinincontents of the samples. For the hot water infusion extract, GPA and OPB had 13 and 10 peaks respectively, while FAa and NFAa had 13 peaks each. 4 peaks had the same retention time across the four samples while for GPA and OPB had 5 peaks with the same retention time. However, a single peak was responsible for 99 and 60% composition in GPA and OPB, respectively indicating that they were the major components in the hot water infusion of the two CVO samples. The number of peaks with the same retention time and comparable percentage composition in FAa and NFAa were 7 in number. The major components in FAa and NFAa were 37.01 and 55.48% respectively. The number and correspondence of profile peaks in in these samples is a pointer to the similarities or disparity in the chemical profiles of the extracts, which is an indication of the variation in the medicinal plants' composition of the samples. The peak corresponding to artemisinin was present in all the samples except the water extract of OPB (Figures 12, 14 to 23). This could be because the artemisinin concentration in the samples was below machine sensitivity since artemisinin is known to be poorly extracted in water. While the HPLC analysis confirmed the presence of artemisinin in the CVO samples (GPA and OPB), the water infusion prescribed in the use of the product does not extract sufficient artemisinin. Rather, the major components in the water extracts were at peaks retention time of 1.73 to 1.85. More work needs to be done to isolate and characterize these major compounds in the infusion which may be a source of major bioactivity.
This study has shown that CVO contains A. annua and other plants. A. annua has been reported to contain bioactive compounds with potential antiviral properties (Karamoddini et al., 2011). It has a history of being safe and easily available for therapies and has shown significant activity against SARS coronavirus. The ethanol extract of the whole plant of A. annua has been reported to have a significant inhibition against two strains of SARS-CoV (BJ001, BJ006) in Vero cell-based CPE/MTS screening (Shi et al., 2005). A. annua derivative, artesunate, is also a promising novel drug to treat pulmonary fibrosis by inhibiting pro-fibrotic molecules associated with pulmonary fibrosis. Pulmonary fibrosis is observed in SARS coronavirus-2 (SARSCoV-2) infection with increased severity, mediated by Interleukin-1 (Conti et al., 2020).

Conclusion
This study shows that the COVID-ORGANICS samples contain A. annua as the major component. They also contain other plant materials possibly including a sweetener. The two products had similar pharmacognostic and phytochemical characteristics as A.
annua grown by NIPRD in Nigeria. There was low artemisinin content in the hot water infusion of the products prepared following the instructions by the manufacturer with one of the samples having undetectable levels. Despite other limitations, the inherent potentials of A. annua as a phytomedicinal remedy for several conditions have been identified. This  is indicative of the considerable potential that exists in the sector. It is therefore critical that government and other stakeholders prioritise the development of the phytomedicinal sector.      of the NIPRD Artemisia annua samples.