Screening for natural and derived bio-active compounds in preclinical and clinical studies: One of the frontlines of fighting the coronaviruses pandemic

Background Starting December 2019, mankind faced an unprecedented enemy, the COVID-19 virus. The world convened in international efforts, experiences and technologies in order to fight the emerging pandemic. Isolation, hygiene measure, diagnosis, and treatment are the most efficient ways of prevention and intervention nowadays. The health organizations and global care systems screened the available resources and offered recommendations of approved and proposed medications. However, the search for a specific selective therapy or vaccine against COVID-19 remains a challenge. Methods A literature search was performed for the screening of natural and derived bio-active compounds which showed potent antiviral activity against coronaviruses using published articles, patents, clinical trials website (https://clinicaltrials.gov/) and web databases (PubMed, SCI Finder, Science Direct, and Google Scholar). Results Through the screening for natural products with antiviral activities against different types of the human coronavirus, extracts of Lycoris radiata (L'Hér.), Gentiana scabra Bunge, Dioscorea batatas Decne., Cassia tora L., Taxillus chinensis (DC.), Cibotium barometz L. and Echinacea purpurea L. showed a promising effect against SARS-CoV. Out of the listed compound Lycorine, emetine dihydrochloride hydrate, pristimerin, harmine, conessine, berbamine, 4`-hydroxychalcone, papaverine, mycophenolic acid, mycophenolate mofetil, monensin sodium, cycloheximide, oligomycin and valinomycin show potent activity against human coronaviruses. Additionally, it is worth noting that some compounds have already moved into clinical trials for their activity against COVID-19 including fingolimod, methylprednisolone, chloroquine, tetrandrine and tocilizumab. Conclusion Natural compounds and their derivatives could be used for developing potent therapeutics with significant activity against SARS-COV-2, providing a promising frontline in the fighting against COVID-19.


Introduction
Coronaviruses were isolated in 1965 from the respiratory tract of adult humans with common cold symptoms (Kahn and McIntosh, 2005). At the beginning, the endemic corona viruses such as: HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 caused mild upper respiratory disease in humans (Chan and Chan, 2013). In the past two decades coronaviruses have appeared on a large pandemic scale displayed by the appearance of the severe acute respiratory syndrome virus (SARS-CoV) followed by the other types of Coronaviruses like (MERS-CoV) (Chan-Yeung and Xu, 2003). The severe respiratory syndrome virus reached many countries and affected many people after crossover from animal (bats as a natural reservoir host) to human, causing high fatality rates (Yuan et al., 2017). The novel SARS-CoV-2 belongs to the coronavirus family that appears to have originated from bats with unknown intermediate host (s) .
Human coronaviruses (CoVs) are enveloped viruses, classified into various types namely HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2 (COVID-19) ( Fig. 1) (Corman et al., 2018;Pillaiyar et al., 2020). The first two CoVs were reported in 1960 ′ s, followed by the discovery of four other types in the 1970 ′ s (Lau et al., 2006). The first four have been accompanied by symptoms such as the common cold, cough, fever, nasal congestion, sore throat, sneezing, and neurological diseases (Walsh et al., 2013;Woo et al., 2005). Their distribution was related mainly to temperate climates during the winter and spring months but geographically they have been observed all over the world (Talbot et al., 2009). HCoV-229E entered cells via binding to the aminopeptidase N receptor (CD13) found on the surface (Yeager et al., 1992). The incubation period ranged between 2 and 5 days (Lessler et al., 2009). The HCoV-229E virus replicated in the epithelial cell line of the trachea (Shirato et al., 2017). The First isolation for the HCoV-NL63 type was observed in a 7-month-old baby in early 2004 (Fielding, 2011). Different clinical evidences were reported and showed that HCoV-NL63 induced similar symptoms to those associated with HCoV-229E and HCoV-OC43 (Talbot et al., 2009). Since HCoV-NL63 and HCoV-229E can share 65% of their amino acid identity, they have an identical mechanism of cellular entry (van der Hoek et al., 2004).
Severe Acute Respiratory Syndrome Coronavirus (SARS CoV) disproportionately infected adults, with notable severe symptoms i.e. fever, chills, myalgia, malaise, and headache, followed by a nonproductive cough and dyspnea 3-5 days later (Fowler et al., 2003;Tsui et al., 2003). SARS-CoV was also named atypical pneumonia and was recorded as the first pandemic virus in human history (Peiris et al., 2003). Its incubation period is 2-10 days (World Health Organization, 2003). Young children and infants infected with SARS-CoV suffered from less severe symptoms than youth and adults (Bitnun et al., 2003). Infection with SARS-CoV type started by the inoculation of respiratory tract mucosa through Angiotensin-converting enzyme 2 (Li et al., 2003). Middle East Respiratory Syndrome Coronavirus (MERS-CoV) rarely infected children, but it can cause severe infections for youth. Infected cases with MERS-CoV suffered from many symptoms i.e. fever, cough, chills, sore throat, myalgia, arthralgia, dyspnea, pneumonia, diarrhea and vomiting (Assiri et al., 2013). It was isolated for the first time in a Saudi Arabian patient in 2012, then in another case from South Korea in 2015. About 2500 fatality cases were reported in February 2020 (Gao et al., 2016;Hilgenfeld and Peiris, 2013;Ye et al., 2020). Its incubation period ranged from 2 to 13 days (Coleman and Frieman, 2013). MERS-CoV infection started with inoculation of the respiratory tract mucosa mediated by functional receptors such as dipeptidyl peptidase 4 (DPP4) (Ng et al., 2016). Then the world faced a new challenge in December 2019, starting with the recording of the first SARS-CoV-2 case in Wuhan, Hubei province, China . The statistics reported on the of 14 th July 2020, confirmed that the number of fatality cases reached 570,288 total cases worldwide (World Health Organization, 2020a). SARS-CoV-2 causes severe respiratory infection accompanied by different symptoms i.e. fever, cough, dyspnea, myalgia, and headache . SARS-CoV-2 is mostly less pathogenic, but spreads more compared to SARS-CoV and MERS-CoV (Zhou et al., 2020).

SARS-CoV-2 origin, taxonomy and structure
In Wuhan, the capital of central China's Hubei province, the Coronavirus  outbreak appeared at the end of 2019, where China's Infectious Disease Information System recorded the first case on December 8, 2019 Wu and McGoogan, 2020). The city's Huanan Seafood Market was the origin of the zoonotic virus SARS-CoV-2 which was transferred rapidly . The recent phylogenetic analysis detected that SARS-CoV-2 may be evolved from a strain found in bats, thus considered the natural reservoir host of SARS-CoV-2 (Chen et al., 2020a;Zhou et al., 2020). Till now, the researchers are not sure about the intermediate host (s) of SARS-CoV-2 or whether the infection can be transmitted directly from bat to human (Ye et al., 2020). It was then recognized as worldwide pandemic disease that can be transmitted rapidly between humans through droplets or direct contact .
2019-nCoV (COVID-19) also named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel member of the coronaviruses . Nevertheless, it has a specific gene sequence that differentiates it from previously sequenced coronaviruses (Zhou et al., 2020). Interestingly, it has an 88% shared identity with SARS-CoV and about 50% to MERS-CoV, indicating that it belongs to the same species (Chen et al., 2020a;Lu et al., 2020). The phylogenetic analysis done to classify SARS-CoV-2 revealed that it falls into the Sarbecovirus subgenus of the genus Betacoronavirus Zhu et al., 2020).
In general, HCoVs are long and positive single stranded RNA viruses characteristic of two groups of proteins; the first group is namely the structural proteins: Spike (S), Nucleocapsid (N), Matrix (M), and Envelope (E) (Fig. 2), while the second group is non-structural proteins: proteases (nsp3 and nsp5) and RdRp (nsp12) (Elfiky et al., 2017). In the case of SARS-CoV-2, the densely glycosylated spike protein found on the outer surface is arranged in this way in order to facilitate the recognition, attachment, and entry into the host cell (Ibrahim et al., 2020). Similarly, the genetic material of SARS-CoV-2 is a positive sense RNA strand (Chhikara et al., 2020). SARS-CoV-2 was described with a transmission electron microscope (TEM) where the double-wall surface of SARS-CoV-2 was seen, but it gives only a poor description of the different organelles in the cytoplasm (Chhikara et al., 2020;Walls et al., 2020). Thus, it was assumed that SARS-CoV-2 has distinct ultrastructural features similar to the coronavirus family such double-membrane vesicles, large granular areas of cytoplasm and nucleocapsid inclusions, in addition to viral proteins and genetic material RNA (Chhikara et al., 2020).  are similar in the mechanism of replication . Their replication begin with the binding of its spike protein (S) into the host's cell surface molecules (Sahin et al., 2020). Usually, (S) protein is divided functionally into the S1 domain, responsible for binding to human receptors, and the S2 domain is responsible for cell membrane fusion (He et al., 2004). SARS-CoV-2 enters the host cells through recognition of human receptor angiotensin-converting enzyme 2 (ACE2) (Zhou et al., 2020). RNA material of SARS-CoV-2 undergoes replication and transcription after the fusion with the plasma membrane. Replicase proteins of SARS-CoV-2 are generated from translation of positive sense RNA genomes through open reading frame 1a/b (ORF1a/b). These proteins use positive sense RNA as a template to generate full-length negative sense RNA. Then proteins are collected with new RNA genome assembly in the endoplasmic reticulum   Khalifa et al. Phytomedicine 85 (2021) 153311 (ER) and Golgi-apparatus ( Fig. 3)  . Biophysical and structural analysis confirmed that the S protein of SARS-CoV-2 has an affinity to bind with ACE2 about 10-20 times higher than the S protein of SARS-CoV (Wrapp et al., 2020). The high affinity of the S protein for human ACE2 can facilitate the spread of SARS-CoV-2 between humans (Zhou et al., 2020).

SARS
The studies conducted on the COVID-19 infection mechanism showed an increase in leukocyte levels, plasma pro-inflammatory cytokines i.e. IL-6, and blood C-reactive protein values from the normal range . Lower numbers of T and B lymphocytes in peripheral blood are observed and coagulation parameters i.e. D-Dimer raise abnormally . The main pathogenesis of the COVID-19 infection is described as extreme pneumonia, RNA aemia, and acute cardiac injury . The high rate of renal failure was observed in patients with COVID-19, suggesting the development of renal dysfunction .
The human-to-human spread is now the primary way of transmission of the infection; from both symptomatic and asymptomatic individuals. The transmission from symptomatic patients occurs to nearby individuals but does not occur through air. Transmission from asymptomatic individuals occurs through direct contact, handshaking (Kam et al., 2020), touching contaminated surfaces  or droplets spread by coughing or sneezing then touching the mouth, nose or eyes (Pan et al., 2020b). Therefore, the human-to-human   Khalifa et al. Phytomedicine 85 (2021) 153311 transmission depends on proximity and increased population density. Also, Leung et al. (2020) proved that inadequate use of masks could lead to increased chances of infection transmission (Leung et al., 2020). A study conducted by Chen et al. (2020a) on confirmed pregnant women cases, demonstrated no evidence of trans-placental transmission. These women underwent cesarean sections, so the transmission by vaginal birth is still under debate (Chen et al., 2020a).
In the previous years, SARS-CoV (2003) infected about 8098 individuals in 26 regions around the world with a mortality rate of 9% (World Health Organization, 2003). On the other hand, SARS-CoV-2 has infected roughly 7,690,708 individuals in more than 200 countries until the 14 th of June 2020 (World Health Organization, 2020b). Meaning that the transmission of SARS-CoV-2 is higher than SARS-CoV. Li et al., 2020a proved that the high spread of COVID-19 is due to the genetic recombinant S protein . Initial reports identified two species of snakes that may be a reservoir of SARS-CoV-2 (Ji et al., 2020;Rothan and Byrareddy, 2020). Mammals or birds are the only evidenced reservoirs of SARS-CoV-2 (Bassetti et al., 2020). Genomic sequence analysis of SARS-CoV-2 showed an 88% identity share with two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses Lu et al., 2020;Wan et al., 2020b). Despite the fact that the genetic material of SARS-CoV-2 is compatible with other types of coronavirus, its gene sequences remarkably differ from previous sequences of other CoV types (Zhou et al., 2020). The genetic studies of SARS-CoV-2 showed that its sequence shared 79% identity with the other CoVs types .

Drugs management in preclinical studies (in vivo, in vitro)
Despite huge efforts, the world has been unable to discover potential therapies or vaccines against COVID-19, thus most of the scientific efforts are now allocated towards trying to locate a proper medication from the old conventional drugs applied earlier for previous coronavirus varieties, among them are HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 which caused mild upper respiratory disease in humans, as well as severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV (Pillaiyar et al., 2020). Natural products, mainly plants, remain as a rich source of novel therapeutic agents for the treatment of different human illnesses (Yang et al., 2020b) . In this context, many natural product derivative therapeutic agents have been reported to inhibit entry and replication of many coronaviruses; SARS, MERS, etc. (in vitro and in vivo) (Tables 1 and 2 ; Glycyrrhizin (34) is an active terpenoid saponin compound isolated from liquorice roots (Glycyrrhiza glabra L.). Liquorice roots have been used in traditional medicine as prophylactic agent for gastric and duodenal ulcers, anti-inflammatory, contraceptive, laxative, antiasthmatic, galactagogue, and antiviral, and expectorant (Damle, 2014;Sato et al., 1996). Cinatl et al. demonstrated that glycyrrhizin inhibited SARS virus replication by inhibition of adsorption. The penetration of the virus at early steps of the replicative cycle was stopped at both concentrations of (EC 50 of 300 mg/l) and CC 50 value of >20 000 mg/l in comparison to ribavirin (positive control) when greater than >1000 that could not be tolerated by the target tissue or organs (Cinatl et al., 2003).
In 2005, Li and his co-authors tested around 200 Chinese medicinal plants against Severe Acute Respiratory Syndrome associated coronavirus (SARS-CoV) using MTS assay. The results demonstrated that 4 of the 200 plants extracts, namely L. radiata (L'Hér.), A. annual ., P. lingua Mirb. and L. aggregate Sims, showed superior activity at EC 50 of 2.4, 34.5, 43.2 and 88.2 µg/ml respectively compared with interferon alpha as a positive control (EC 50 = 660.3 IU/ml). Four potent extracts recorded CC 50 values ranged between 886.6 ± 35.0 to 2378.0 ± 87.3 µg/ml. CC 50 was determined based on reducing cell viability by using different concentration of extracts. As L. radiata (L'Hér.) was the most active one, it was subjected to fractionation and purification to find the most active compound, identified as lycorine (45). Lycorine is an alkaloid compound that gave EC 50 of 15.7 nM and CC 50 value of 14,980.0 nM against SARS-CoV (Li et al., 2005). Lycorine have been investigated later against the other types of coronaviruses, HCoV-OC43, HCoV-NL63, MERS-CoV and MHV-A59 to give EC 50 values ranging from 0.15 µM to 1.63 µM (Shen et al., 2019).
Emetine is a natural plant alkaloid of ipecac (Carapichea ipecacuanha L.), emetine dihydrochloride (31), a derivative of emetine inhibiting the viral protein synthesis of coronaviruses, with an EC 50 value of 0.12 to 1.43 µM. Emetine hydrochloride revealed the strongest anti-CoV activities against MERS-CoV and SARS-CoV with the lowest EC 50 of 0.014 and 0.05 µM, whilst the activity against HCoV-NL63, HCoV-OC43, MERS-CoV and MHV-A59 showed EC 50 values of 1.43 µM, 0.30, 0.34 and 0.12 µM respectively via blocking of viral propagation at an early stage of infection, thereby minimizing the chance of the virus to adapt and acquire drug resistance. It also has CC 50 values of 3.63, 2.69, 3.08 and 3.51 µM respectively (Dyall et al., 2017;Shen et al., 2019).
Toona sinensis Roem, is a Chinese traditional plant belonging to the Meliaceae family. In the folk medicine, the leaves of the plant were used to treat gastric ulcers, enteritis, dysentery, cerebrovascular, and cardiovascular diseases (Kakumu et al., 2014). TSL-1, a fraction of the aqueous extract of the plant leaves showed anti-viral activity against SARS-coronavirus with EC 50 of 30 µg/ml and CC 50 value > 500 µg/ml via inhibition of the viral replication. Determination of CC 50 takes place by decreasing the cell viability by 50% (Chen et al., 2008). Another study was conducted by Wen et al., where 200 plants were tested and only five of them, namely Gentiana scabra, Dioscorea batatas, Cassia tora, and Taxillus chinensis, exerted survival inhibition of SARS-CoV (Wen et al., 2011). The plants possess a wide range of folk applications including liver disorder, inflammation and pneumonia treatment Oh et al., 2004;Pawar and D'mello, 2011;Zhang et al., 2013). The potent inhibition of SARS-CoV viral enzymatic activity (SARS-CoV 3CLprotease) occurred between 25 and 200 µg/ml and the most potent plants were C. barometz and D. batatas with IC 50 value of 39 and 44 µg/ml respectively. The six plant extracts recorded the same CC 50 value of > 500 µg/ml in comparison with valinomycin (positive control) that has value of 75.01 µg/ml. The determination of CC 50 showed that reduction of cell viability by 50% and there was no effect on the growth of host cells, indicating the effectiveness and safety of these extracts (Wen et al., 2011).
Celastrus orbiculatus Thunb. family (Celastraceae), a Chinese herbal plant, traditionally used in the treatment of fever, chills, edema and bacterial infection (Wu et al., 2004), showed significant effect via inhibition of the 3CL pro enzyme. Three different extracts (EtOH, EtOAc and water fraction) have been tested against SARS-CoV infection, and exhibited IC 50 values of 19.4, 17.8 and 38.7 μg/ml respectively . Similarly, hydroethanolic extraction of Echinacea purpurea plant roots showed potent activity against HCoV-229E, SARS-CoV and MERS-CoV with IC 50 values of 3.2, 50 and 50 µg/ml respectively (Signer et al., 2020).
Furthermore, the essential oils of many plants play an important role against viral diseases i.e. Laurus nobilis, Juniperus oxycedrus ssp. oxycedrus, Thuja orientalis, Cupressus sempervirens ssp. pyramidalis, Pistacia palaestina, Salvia officinalis, and Satureja thymbra. The essential oils were investigated against SARS-CoV and HSV-1 viruses. The potent activity of L. nobilis oil was reported against SARS-CoV with IC 50 value of 120 µg/ ml. The plant oil is characterized by the excitation of β-ocimene, 1,8cineole, α-pinene, and β-pinene as the principle constituents. These plants act via the inhibition of viral replication (Loizzo et al., 2008).
Mycophenolic acid (MPA) (75) was first isolated from Penicillium spp. fungi by Bartolomeo Gosio in 1893 and was reported to have an immunosuppressive activity against psoriasis (Sollinger, 2004). Recently, mycophenolic acid was investigated against different types of coronavirus using a high-throughput screening approach, where the MPA showed potent anti-MHV-A59 activity in vitro at an EC 50 value of 0.17 µM compared to of HCoV-OC43, HCoV-NL63 and MERS-CoV (EC 50

Curative efficacy of clinical studies and approved drugs
For a long time, natural products and their molecular frameworks constitute valuable starting points or sources for drug discovery (El-Seedi et al., 2019;Rodrigues et al., 2016). In line with Newman and Cragg, the number of antiviral drugs approved by the FDA in the period between 1981 to 2019 are 186 drugs, among them 87 are vaccines like FluMist®, Invivac®, Bilive®, Anflu®, Afluria® and Optaflu®, and are used against the influenza virus, 26 are synthetic but the pharmacophores are natural products, 17 are biological sources like peptides and proteins and 6 are natural products derivatives such as Tamiflu®, Zanamivir® and Virreal®, 21 are synthetic drugs (NP pharmacophore)/ mimics of natural products and other 19 compounds are synthetics (Newman and Cragg, 2020).
Nowadays, there are around 80 running and pending clinical trials in China in a serious attempt to find a potential treatment for COVID-19 (Maxmen, 2020). In 2020, several compounds isolated from natural products ( Fig. 7; Table 3) were tested against coronavirus (COVID-19), i. e. Xiyanping (Mix of 78 and 79). Xiyanping, is a semi-synthetic product derived from the active components of the Andrographis paniculata plant, and licensed in China as an anti-inflammatory agent (Chong et al., 2013;Xiao et al., 2013). Xiyanping injection was investigated on 426 patients diagnosed with moderate to severe SARS-CoV-2 infection, every patient was injected with 10-20 ml of Xiyanping at 500 mg per 20 ml daily plus lopinavir/ritonavir tablets and α-interferon nebulization. The stated drug (lopinavir/ritonavir) has the ability to inhibit protease and CYP3A metabolism thus showing antiviral properties (Driggin et al., 2020) (ClinicalTrials.gov; NCT04275388) Fingolimod (FTY720) (80), another compound derived from myriocin (ISP-1), is a metabolite of the Isaria sinclairii fungus. Fingolimod has been approved by the regulatory authorities of the US, EU, Australia, and Russia, to treat the relapsing remitting multiple sclerosis. Fingolimod consequently represents the primary oral drug for treatment of this central nervous autoimmune disease (Ingwersen et al., 2012). In China, the Fingolimod drug was tested with 30 patients infected with COVID-19. Every patient was given 0.5 mg of Fingolimod orally daily, for three consecutive days. Fingolimod was used in the current study as an effective agent against COVID-19 acting via an immunology modulation of phingosine-1-phosphate receptors (ClinicalTrials.gov; NCT04280588) (Driggin et al., 2020).
Tetrandrine (58) represents the predominant constituent of the Stephania tetrandra plant, a Chinese traditional medicinal plant. Tetrandrine is bisbenzylisoquinoline alkaloid and recommended in the Chinese Pharmacopoeia as an analgesic and diuretic agent and also for treatment of hypertension and various other ailments like asthma, tuberculosis, dysentery, hyperglycemia, malaria, cancer and fever. It was also used against the Ebola virus infection (Bhagya and Chandrashekar, 2016). In the clinical studies, tetrandrine has been proven effective against COVID-19 via reducing the clinical progress, improving the prognosis, reducing the incidence of pulmonary fibrosis during rehabilitation, and improving patients' quality of life (ClinicalTrials.gov; NCT04308317). Tetrandrine has shown potential in decreasing the entry of SARS-CoV-2 S pseudovirions (Ou et al., 2020). Tocilizumab (82), is a humanized monoclonal antibody that interacts with the interleukin-6 receptor (IL-6R) and is nowadays approved by China's National Health  Chen et al., 2008 Commission for the treatment of inflammation in patients with COVID-19 (Cron and Chatham, 2020). Previously, Tocilizumab (Actemra®) was permitted by means of the FDA as a drug used for inflammatory bowel disease treatment (Newman and Cragg, 2020).
Recently, some physicians in Italy (Pascale Hospital, Naples) claimed that Actemra® (Tocilizumab) succeeded in treating severely ill patients via blocking the inflammatory molecule interleukin-6, decreasing systemic inflammation, improving survival rate, adjusting hemodynamic and relieving the respiratory distress (Day, 2020). Streptokinase and Heparin were investigated as a treatment for patients infected by Severe Acute Respiratory Syndrome (SARS) and Severe Acute Respiratory Distress Syndrome (ARDS), the first one is an enzyme isolated from the Streptococci bacteria and the other is a glycosaminoglycan derived from dog liver (Anderson et al., 1948;McLean, 1959). The two compounds had been administrated in 40 patients infected with ARDS. The first 20 patients were treated with heparin (10.000 IU) and the other 20 were treated with Streptokinase (250.000 IU). Each drug was prepared in a 3 ml volume of distilled water and nebulized for a period of 15 min every 4 h. The outcome of the study revealed an improvement of the hypoxemia as determined by PaO 2 /FiO 2 ratio >100, an improvement of the pulmonary compliance of the patient defined as dynamic compliance >50 ml/cm H 2 O, and a decrease of the occurrence of complications such as bleeding or coagulopathy within 72 h of initiation of therapy. Streptokinase and Heparin may prevent /or  Khalifa et al. Phytomedicine 85 (2021) 153311 dissolve intra-alveolar fibrin clots respectively helping alveolar re-expansion (ClinicalTrials.gov; NCT03465085).
Methylprednisolone (81) is a natural products derivative (prednisolone derivative glucocorticoid) implemented by the FDA as an anticancer drug (Feinberg et al., 1957;Hall, 1992;Newman and Cragg, 2020;Ravina, 2011). In early 2020, two clinical studies had been conducted on methylprednisolone against SARS-CoV-2 with 86 and 80 participants respectively. In both studies, all participants recovered except for one case that died in the first study (ClinicalTrials.gov; NCT04273321; NCT04244591).
Chloroquine (27) is a synthetic drug but its pharmacophore is a natural product origin (Quinin isolated from Cinchona spp. plant) (Oliveira et al., 2009), and approved antiphrastic (anti-malaria) drug as per the FDA (Newman and Cragg, 2020). In the 1960 ′ s, it was investigated as an antiviral in vitro for the first time (Touret and de Lamballerie, 2020). Chloroquine was able to inhibit MERS-CoV replication at a very early stage of infection with EC 50 of 3.0 µM, and it was reported as a potent anti-viral agent against flavivirus, influenza virus, HIV, Ebola virus, and Nipha-Hendravirus (Pillaiyar et al., 2020). Nevertheless, many clinical studies were conducted on chloroquine as an anti-COVID-19 virus agent (ClinicalTrials.gov; NCT04303507), and its efficacy and safety remain unclear. It is proven highly effective in blocking viral replication in other infections including the SARS-associated coronavirus (CoV) (Cortegiani et al., 2020). However, in a recent observational study, hydroxychloroquine treated patient was more seriously ill than those who hadn't received hydroxychloroquine (Geleris et al., 2020).

Patents of natural products prescriptions against COVID-19 pandemic
As discussed earlier, small-molecule, approved drugs, and natural products are promising entities to combat COVID-19 depending on various mechanisms of action. Traditional Chinese medicine (TCM) has a synergistic effect that plays a vital role in resisting the virus and resisting inflammation of the lung and thus some TCM formulas have been proposed and approved as patents against coronaviruses. Our review devotes to patents from natural product sources (Table 4).
One prescription for treating pneumonia caused by the new coronavirus infection was inspired by the TCM composition and discloses. The prescription raw materials combine, parts by weight, of the following TCM materials: 9 parts of Ephedra, 6 parts of honey-fried licorice root, 9 parts of almond, 15-30 parts of Gypsum, 9 parts of Cassia  Twig, 9 parts of Rhizoma Alismatis, 9 parts of Frifola, 9 parts of bighead Atractylodes rhizome, 15 parts of Poria cocos, 16 parts of Radix Bupleuri, 6 parts of Scutellaria baicalensis, 9 parts of ginger processed Pinellia tuber, 9 parts of ginger, 9 parts of aster, 9 parts of Flos Farae, 9 parts of Blackberry lily, 6 parts of Asarum, 12 parts of Chinese yam, 6 parts of immature bitter orange, 6 parts of dried orange peel and 9 parts of wrinkled Gianthyssop herb. The effective rate of treatment was reported to 95%. The percent of effective rates was calculated based on the number of confirmed diagnosis and the number of treated patients from different places. This patent was documented in patent office CN with publication number (CN110870402A) (Ge, 2020).
Another TCM prescription is composed of the following raw materials in parts by weight: 40 parts of Folium isatidis, 40 parts of the wild Chrysanthemum flower, 20 parts of Coptis chinensis, 30 parts of sweetsop seed, 20 parts of wrinkled Gianthyssop herb, 20 parts of Rhizoma Atractylodis, 20 parts of Radix Bupleuri, 2 parts of Calculus Bovis Factitius, 20 parts of Houttuynia cordata, 5 parts of dandelion root, 30 parts of honeysuckle, 30 parts of Fructus Forsythiae, 20 parts of Scutellaria baicalensis, 20 parts of blackberry lily, 15 parts of Salvia miltiorrhiza, 15 parts of Bulbus Fritillariae Cirrhosae, 10 parts of Saussurea involucrate, 60 parts of Astragalus mongholicus, 20 parts of Cordyceps sinensis, 20 parts of Codonopsis. Observation of clinical experiments shows that the patent boosted the pneumonia symptoms caused by the new SARS-CoV-2 coronavirus. This patent was documented in patent office CN with publication number (CN111150792A) .
Tripterygium wilfordii is the main formula for one of the TCM patents. The composition of this patent has the following raw materials as parts by weight: 3-15 parts of Tripterygium wilfordii, 10-50 parts of Gypsum, 8-24 parts of raw Chinese yam, 5-15 parts of Radix Scrophulariae, 4-12 parts of Fructus Forsythiae, 4-10 parts of Periostracum cicada, 4-12 parts of Codonopsis pilosula, 3-8 parts of mint, 3-10 parts of burdock and 4-20 parts of raw ochre. This patent was claimed suitable for the severe stage of COVID-19 pneumonia diseases, regulating immunity and decreasing the fever. The administration rate of the prescription (3-5 times) can decrease body temperature to the normal range. Tripterygium wilfordii relates to Chinese herbal medicine, and used in traditional medicine for promoting blood circulation, killing parasites, regulating immunity, and has a good medicinal effect when utilized in treating 2019 new coronavirus pneumonia. This patent was documented in patent office CN with publication number (CN111184805A) (Lei and Yang, 2020).
One more patent that also relates to TCM, is possessing a formula for treating or preventing both novel coronavirus pneumonia and viral influenza. This formula combines raw materials, parts by weight, as a following: 15-45 parts of prepared aconite, 10-30 parts of dried ginger, 30-60 parts of honey-fried licorice root, 15-30 parts of American ginseng, 10-30 parts of Ephedra, 15-30 parts of Cassia twig, 5-10 parts of Asarum, 10-70 parts of honeysuckle flower, 10-30 parts of Fructus Forsythiae, 10-30 parts of Isatis root, 10-30 parts of reed rhizome, 15-30 parts of Folium Isatidis, 20-240 parts of Gypsum, 10-30 parts of lalang grass rhizome, 10-15 parts of Rhizoma Paridis, 5-15 parts of wrinkled Gianthyssop herb, 5-10 parts of Eupatorium, 5-10 parts of safflower, 5-15 parts of Cortex Moutan, 5-15 parts of Rheum officinale, 15-45 parts of ginger, 10-30 parts of Chinese date and 5-9 parts of musk. This patent has a corollary effect on ventilating lungs and improving human immunity. It was used for coronavirus, influenza, and pneumonia prevention or treatment. The findings showed that 24   patients out of 30 were cured and that this formula was helpful in treating the new coronavirus pneumonia effectively. The cure rate was estimated to 80%. This patent was documented in patent office CN with publication number (CN111110819A) . Another formula was studied in vitro against common coronavirus and new coronavirus. The formula composed of the following raw materials, as parts by weight: 40 parts of wrinkled Gianthyssop herb, 40 parts of wild Chrysanthemum flower, 24 parts of Chinese mosla herb and 40 parts of sweet wormwood herb. This formula has a potent effect on inhibiting cytokine expression i.e. TNF-α and IL-6 produced by SARS-Cov-2. The formula was used earlier as a therapeutic agent against pneumonia, nephritis, hepatitis. It has very good prospects for clinical application and thus it was approved from CN patent office under publication number (CN111150755A) (Pan et al., 2020a).

The protective role of plants and their constituents against COVID-19
The consumption of healthy diets is one way to boost the immune system and in return help in fighting off viruses (Galanakis, 2020). There is a wide range of plants that contribute to boosting the immune system given their high content of a wide variety of nutrients and biological active compounds (Sultan et al., 2014). A strong immune system is essential for fighting against COVID-19. COVID-19 seems to be hazardous for people with comorbidities and those have a weaker immune system than healthy ones Jiang et al., 2020). Garlic stimulates the immune system as it is rich with a wide plethora of nutrients, vitamins, and sulfur-containing compounds (Iciek et al., 2009). Certain vegetables such as broccoli, spinach and sweet potatoes are abundant with vitamin A which include retinol, retinoic acid and β-carotene. These fat-soluble compounds participate in enhancement of the immune function. Additionally, isotretinoin (vitamin A derivative), inhibits ACE2 and the entry of SARS-COV-2 into the body thereby lower the susceptibility to infections (Galanakis, 2020). Citrus fruits are one of the best sources for vitamin C. Vitamin C increases the production of white blood cells providing a key role in fighting infections (Calder et al., 2020). Vitamins are recommended for the protection against coronavirus as they increase patients' resistance against infection (Basiri, 2020).

Conclusions
The outbreak of COVID-19 has swept across the world rapidly spreading in a very alarming pace invading more than 200 countries, territories, areas leaving a trail of victims behind. Although SARS-CoV-2 is accompanied by symptoms similar to that of SARS-CoV-2 i.e. fever, cough, and fatigue, SARS-CoV-2 is far more contagious. It can be Fumigation bath preparation for preventing and treating pestilence, and its application method and application/ (CN111184823A)/ CN SARS-CoV-2/ 90% (80%)/ Improve human immunity and has prevention and treatment effects on new coronavirus pneumonia Yang et al., 2020a Chinese name: 陈文才 English name: Chen Wencai 20-100 g of Laggera pterodonta, 10-50 g of honeysuckle, 10-30 g of dried ginger, 10-40 g of divaricate saposhnikovia root, 10-50 g of Officinal magnolia bark and 10-60 g of pilose asiabell root/ liquid Laggera pterodonta composition for treating new COVID-19 and application thereof/ (CN111166862A)/ CN SARS-CoV-2/ Not reported/ Preventing and treating infectious plague and common respiratory diseases, resisting virus and enhancing immunity Chen, 2020 transmitted by droplets or close contact. Given the affinity by which SARS-CoV-2 protein S can bind to ACE2 receptors, human to human transmission seems to spread much faster than the common flu or even the previous coronaviruses. Thus, containing the outbreak is a burning issue requires extensive measures and developing specific safe drugs. Natural products have so far proven to be promising for the development of effective and less toxic antiviral agents. Owing to the genetic resemblance between coronaviruses, the drugs and antiviral agents which are usually effective against the other types of coronaviruses could be used to treat COVID-19. Some of the reported antivirals showed a promising antiviral activity against at least four types of human coronaviruses with significant EC 50 and CC 50 values. Other therapeutic agents have also been reported and are already moving into clinical trials including Xiyanping, fingolimod, methylprednisolone, streptokinase, and heparin.

Funding
This work was supported by the Swedish Research Council Vetenskapsrådet (grants 2015-05468 and 2016-05885)

Declaration of Competing Interest
The authors declare no conflict of interest.