Herbal medicine, a reliable support in COVID therapy

ABSTRACT

At present, specific therapies for COVID-19 are not well established, being certain only that the immune system plays a decisive role in the initiation and progression of the disease. Plants have given and continue to give compounds with great efficiency and low toxicity, some of them being a starting point for extremely effective synthetic substances. Although herbal remedies are used mainly for preventive purposes, there are also guidelines issued by some countries that indicate the use of traditional remedies for different stages of COVID-19 disease.Europe has a long and strong tradition of using medicinal plants for therapeutic purposes, but clinical trials for this type of approach are scarce, compared to Asia. In this regard, a bridge between tradition and science, would have a strong impact on the capacity for prevention and treatment of COVID-19.

The paper reviews compounds of plant origin that have previously proven effective in counteracting some coronaviruses but also some of their major effects - direct action on virus replicative apparatus (viral entry or replication, action on the viral enzymatic system), collateral action of natural compounds on the immune system and also the contribution of herbal medicine as vaccine adjuvants are tackled.

KEYWORDS:

• Medicinal plants
• traditional
• inflammation
• immune system

Introduction

At present, specific therapies for COVID-19 are still debatable, with the fact that only the immune system plays a decisive role in the initiation and progression of the disease. Most patients with COVID-19 have mild to moderate symptoms, but about 15% progress to severe pneumonia and about 5% eventually develop acute respiratory distress syndrome (ARDS), septic shock, and/or multiple organ failure.

Inborn uncontrolled inflammatory responses and impaired adaptive immune responses can lead to tissue damage both locally and systemically. In patients with severe COVID-19, but not in patients with mild disease, lymphopenia is a common feature, with a drastically reduced number of CD4 + T cells, CD8 + T cells, B cells, and natural killer (NK) cells, as well as a low percentage of monocytes, eosinophils, and basophils. An increase in the number of neutrophils and the neutrophil-lymphocyte ratio usually indicates a higher severity of the disease and a poor clinical outcome. In addition, depletion markers, such as NKG2A, on cytotoxic lymphocytes, including NK cells and CD8 + T cells, are regulated in patients with COVID-19. Most patients with severe COVID-19 have substantially elevated serum levels of proinflammatory cytokines, including IL-6 and IL-1β, as well as IL-2, IL-8, IL-17, G-CSF, GM-CSF, IP10, MCP1, MIP1α (also known as CCL3) and TNF, characterized as a cytokine fraction.^[1]

The involvement of the immune system can be divided as follows: the first phase of protection based on immune defense and the second harmful phase caused by inflammation,^[2] therefore therapeutic strategies should investigate an adequate inflammatory response to pathogen clearance.^[3,4]

Since the disease started in China, a place with strong roots in traditional medicine, it is understandable to approach natural therapy in this case as well. Although herbal remedies are used mainly for disease prevention,^[5] there are also guidelines issued by the Chinese and Korean ministries of health that indicate the use of traditional remedies for different stages of the disease.^[6] However, detailed clinical trials are needed to determine the effectiveness of these remedies.^[5] On the other hand, plants have given and continue to give compounds with great efficiency, some of them being a starting point for easy-to-obtain and highly effective synthetic substances. An example is chloroquine, a structural analog of quinine, originally extracted from the bark of cinchona trees, which has been shown to be effective in treating COVID-19.

By now, among the most used herbal species are Astragalus mongholicus Bunge, Glycyrrhiza glabra L., Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk., Atractylodes lancea (Thunb.) DC., Atractylodes macrocephala Koidz., Lonicera japonica Thunb., and Forsythia suspensa (Thunb.) Vahl. .^[7] A detailed analysis based on the benefits/risks assessment conducted by Silveira et al.,^[8] identified several herbal medicines as very likely to appeal to the COVID-19 patient – positive in 5 cases (Althaea officinalis, Commiphora molmol, Glycyrrhiza glabra, Hedera helix and Sambucus nigra) and promising in 10 cases (Allium sativum, Andrographis paniculata, Echinacea angustifolia, Echinacea purpurea, Eucalyptus globulus essential oil, Justicia pectoralis, Magnolia officinalis, Pelargonium sidoides, Salix sp, Zingiber officinale).^[8]

India has recommended certain preventive and medicinal plants for prevention and prophylactic of COVID-19 (Tinospora cordifolia, Andrograhis paniculata, Cydonia oblonga, Zizyphus jujube and Cordia myxa) and also for the symptomatic management of COVID-19 (Agastya Haritaki and Anuthaila (sesame oil drops).^[9] For prevention purposes, the most used plants (use frequency >40%) in Morocco were Allium Sativum (80.9%), Olea europaea (72.7%), Allium cepa (66.7%), Zingiber officinale (66%), Thymus maroccanus (59.2%), Eucalyptus globules (56.5%), Foeniculum vulgare (54.3%), Curcuma xanthorrhiza (50%), Phoenix dactylifera (50%), Rosmarinus officinalis (47.9%), Thymus satureioides (41.9%), Mentha pulegium (41.3%) and Pimpinella anisum (40%).^[10]

The combined use of anti-inflammatory and antiviral drugs may be more effective than using either method. Based on in vitro evidence for inhibition of human severe acute respiratory syndrome coronavirus (SARS-CoV) replication and blocking SARS-infection-induced pro-inflammatory cytokine production, Lianhua Qingwen, a traditional Chinese drug has demonstrated clinical efficacy in conjunction with conventional treatment, as a synergetic therapeutical strategy.^[1,11]

Of 90 studies on traditional Chinese medicine for human severe acute respiratory syndrome (SARS), they found positive but inconclusive results on the effectiveness of the combination treatment, using traditional Chinese medicine as complementary agent. Based on previous experience of treating SARS with traditional Chinese herbal medicines, the General Office of the National Health and Health Commission of China and the Office of the State Administration of Traditional Chinese Medicine have encouraged the integration of traditional Chinese medicine and Western medicine. Different prescriptions are recommended at different stages of the disease. Compared to chemical medicines, herbal medicines and natural plant products are less understood in terms of their mechanism of action, but more clinical investigations have been started to more accurately assess their effects. Like chloroquine phosphate, these herbal medicines are generally not very potent and therefore cannot be considered a treatment in themselves. However, as a complementary treatment, recovery rates may increase when combined with other treatments.^[12]

Currently, there are numerous ongoing clinical trials worldwide with a herbal compound/extract as the adjuvant component, most of which are performed in China – lopinavir/ritonavir + interferon-alpha + traditional Chinese medicine (TCM) preparations; lopinavir/ritonavir + TCM; dihydroartemisinin/piperachine + antiviral treatment (interferon-alpha + umifenovir), sodium escinate + standard treatment.^[13] Another herbal remedy recruited to control COVID-19 is diazotized glycyrrhizinate, extracted from licorice root. The species Glycyrrhiza glabra (licorice), has been used since ancient times against coughs and colds in both traditional Asian and European medicine. Diazotate glycyrrhizinate extracted from this species has anti-inflammatory activity and is used to treat liver damage caused by hepatitis B. A recent clinical study (13.02.2020) investigates the effect on COVID-19 of a treatment regimen based on entero-resistant capsules with glycyrrhizinate diazotate (oral, 150 mg, 3 times/day), vitamin C tablets (oral, 0.5 g, daily) and standard antiviral treatment (Chinese Clinical trial Registry, http://www.chictr.org.cn/).Studies on the effects of medicinal plants in COVID-19 therapy registered on the International Platform of the Clinical Trials Register of the World Health Organization (https://www.who.int/ictrp/en/) show the special interest only of Chinese and Iranian researchers on this subject. In Iran, clinical studies are being carried out to investigate the effect of licorice extract, licorice, and mallow (2.5 + 2.5 g), Fluherb (grass extracts, plantain, isop, licorice, and turmeric, suspension, 5 days 3 times/day), echinacea, and ginger, mixture with sage, fennel, and St. John’s wort (5 ml, 3 times/day), shock syrup.

A review by the British organization Cochrane that examined the results of alternative therapies used during the SARS epidemic suggested that in general combinations of herbal medicines complementary to conventional therapy do not lower mortality, but may improve quality of life, reduce the chances of deep lung infiltration. and the application of low doses of drugs such as corticosteroids.^[14]

The paper reviews compounds of plant origin that have previously proven effective in counteracting some coronaviruses but also some of their major effects, being able to constitute new prevention and/or treatment strategies.

Direct action on virus replicative apparatus

Viral entry or replication

Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses that include both human and zoonotic species. Four Coronaviruses genera are known: Alpha (Feline and Canine coronaviruses, Porcine epidemic diarrhea virus, Transmissible gastroenteritis coronavirus, etc.) and Beta (OC43, HKU1, SARS-CoV, SARS-CoV-2, MERS-CoV) genera descending from the bat gene pool^[15] and Gamma (Avian coronaviruses) and Delta genera derived from the avian and pig gene pools.^[16]

Phenolic compounds

Tannins such as phlorofucofuroeckol, dieckol act both by inhibiting both viral entry and replication.^[16] It was showed that in the case of Porcine epidemic diarrhea virus (PEDV) infection, ethanol extract from Ecklonia cava and isolated phenolic compounds belonging to tannins class – eckol, 7-phloroeckol, phlorofucofuroeckol, dieckol inhibit viral hemagglutination binding to SA receptors in the host cell.^[17] Some polyphenols, as are those isolated from pomegranates, are able to induce small changes in viral envelope glycoproteins, confirmed by the fact that the treatment decreased Ab binding to viral surface molecules.^[18]

Other phenolic compounds such as procyanidins, inhibit the infection, probably by interfering with the viral replication, but are not inhibiting the internalization. For instance, procyanidin A2 and procyanidin B1 showed moderate anti-wtSARS-CoV activity (IC(50S), 29.9±3.3 and 41.3±3.4 microM; SIs, 37.35 and 15.69, respectively).^[19] Q7R could be considered as a lead compound for development of anti-PEDV drugs and it may be used during the early stage of PEDV replication (IC(50)) of 0.014 microg/mL), with an efficiency higher than ribavirin, interferon-alpha, coumarin, and tannic acid.^[20] Its action is not simply due to a general action as an antioxidants and is highly specific, as several other antioxidants (NAC, PDTC, trolox) are inactive against PEDV infection.^[21]

Many extracts of plants acted against infectious bronchitis in chicken (IBV), a highly infectious avian pathogen which affects the respiratory tract, gut, kidney and reproductive system prior to and during infection. Lelesius et al., 2019^[22] revealed the efficiency of M. piperita, T. vulgaris and D. canadense hydroalcoholic extracts. Previously, other results demonstrated that Sambuccus nigra extract can inhibit IBV at an early point in infection, probably by rendering the virus noninfectious. Electron microscopy of virions treated with S. nigra extract showed compromised envelopes and the presence of membrane vesicles, which suggested a mechanism of action.^[23] Although both studies revealed the mechanism of action of crude extracts, the contribution of phenolic compounds to the pharmacologic effect is obvious, given the fact that all plants involved are widely used and known to contain high amounts of polyphenol derivatives.

Resveratrol decreases the expression of nucleocapsid (N) protein essential for Middle East respiratory syndrome coronavirus (MERS-CoV) replication, inhibiting MERS-CoV infection, and prolonging cellular survival after virus infection.^[24] MERS is a viral respiratory disease caused also by a coronavirus first identified in 2012, with a clinical spectrum similar cu human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ranging from no symptoms (asymptomatic) or mild respiratory symptoms to severe acute respiratory disease and death. Recently, it was found that more research on prenylated flavonoids like kuwanon G can result in a more potent inhibitor able to stop the newly emerged SARS-CoV-2 strain of HCoV.^[25]

Saponins

Saikosaponins, a group of oleanane derivatives, isolated from medicinal plants such as Bupleurum spp., Heteromorpha spp. and Scrophularia scorodonia have been reported to possess various biological activities. Saikosaponin B2 has potent anticoronaviral activity and that its mode of action possibly involves interference in the early stage of viral replication, such as absorption and penetration of the virus.^[26] Also, oleanane triterpenes isolated from the flowers of Camellia japonica showed inhibitory effects on PEDV replication by interacting with PEDV genes encoding GP6 nucleocapsid, GP2 spike, and GP5 membrane protein synthesis based on RT-PCR data. Western blot analysis also demonstrated their inhibitory effects on PEDV GP6 nucleocapsid and GP2 spike protein synthesis during viral replication.^[27] Escin derivatives isolated from Japanese horse chestnut (seed of Aesculus turbinata) showed strong inhibitory activities on PEDV replication with lowered cytotoxicity and structure-activity relationship (SAR) studies have revealed that acylations at C-21 and C-22 with angeloyl or tigloyl groups were important for their cytotoxic effects.^[28] In another study, the importance of the friedelane skeleton as a potential scaffold for developing new anti-HCoV-229E drugs was suggested.^[29] A number of bioactive components such as nigellimine isolated from Nigella sativa share structural similarities with chloroquine and hydroxychloroquine and it was proposed that this compound could aid Zn entry in pneumocytes, boosting the immune response during viral infection.^[30]

Lectins

The plant lectins possessed marked antiviral properties against both coronaviruses with EC(50) values in the lower microgram/ml range (middle nanomolar range), being nontoxic (CC(50)) at 50–100 microg/ml. The strongest anti-coronavirus activity was found predominantly among the mannose-binding lectins but also, a number of galactose-, N-acetylgalactosamine-, glucose-, and N-acetylglucosamine-specific plant agglutinines exhibited anti-coronaviral activity. Two targets of possible antiviral intervention were identified in the replication cycle of SARS-CoV – early in the replication cycle, most probably viral attachment, and at the end of the infectious virus cycle.^[31]

Essential oils

Enveloped viruses are known to respond sensitively to essential oils but the majority of claims for eugenol, menthol, and carvacrol and their potential role of in the treatment of COVID-19 are based on data obtained from computer-aided docking and preliminary in vitro studies.^[32]

Essential oils have been evaluated for their inhibitory activity against SARS-CoV replication in vitro by visually scoring of the virus-induced cytopathogenic effect post-infection. Laurus nobilis oil, characterized by the presence of beta-ocimene, 1,8-cineole, alpha-pinene, and beta-pinene as the main constituents exerted an interesting activity against SARS-CoV with an IC(50) value of 120 microg/ml and a selectivity index (SI) of 4.16.^[33] Also, thymoquinone, the main compound of Nigella sativa essential oil, may block the SARSCoV-2 entry via ACE2 in pneumocytes.^[30]

Alkaloids

Lycorine, an alkaloid mainly found in Amaryllidaceae species has an anti-SARS-CoV EC50 value of 15.7 ± 1.2 nM. This compound has a CC50 value of 14980.0 ± 912.0 nM in cytotoxicity assay and a selective index (SI) greater than 900.^[34]

Tylophorine compounds, including naturally occurring and synthetic phenanthroindolizidines and phenanthroquinolizidines are potent in vitro inhibitors of enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) with EC₅₀ values ranging from 8 to 1468 nM. Analysis of the structure-activity relations indicated that the most active tylophorine analogs were compounds with a hydroxyl group at the C14 position of the indolizidine moiety or at the C3 position of the phenanthrene moiety and that the quinolizidine counterparts were more potent than indolizidines. The cytopathic effect induced by SARS CoV) in Vero 76 cells was reduced by tylophorine compounds with EC₅₀ values ranging from less than 5 to 340 nM. Moreover, a pharmacokinetic study demonstrated high and comparable oral bioavailabilities of 7-methoxycryptopleurine (52.7%) and the naturally occurring tylophorine (65.7%) in rats.^[35]

The combined use of Galanthus nivalis agglutinin GNA and nelfinavir has therapeutic potential in the prophylaxis and treatment of cats with early-diagnosed FIP by a synergistic antiviral effect defined by complete blockage of viral replication.^[36]

Polysaccharides

The avian coronavirus causes infectious bronchitis (IB), which is one of the most serious diseases affecting the avian industry worldwide. Astragalus polysaccharides inhibited IBV replication in vitro, associated with reduced mRNA levels of the cytokines IL-1B, IL-6, IL-8, and TNF-α.^[37] In the case of PEDV infection, polysaccharides from Ginkgo biloba exocarp inhibit the virus attachment and entry to the Vero cells.^[38]

Antraquinones

Older studies showed that pseudohypericin and hypericin interfere with retroviral infection and/or spread by direct inactivation of the virus or by preventing virus shedding, budding, or assembly at the cell membrane.^[39] It was suggested that antiretroviral activity of hypericin and pseudohypericin could be attributable to the inhibition of some phosphorylation involved by protein kinase C during infection of cells with retroviruses.^[40]

Rocaglates, a class of natural compounds isolated from plants of the genus Aglaia, are potent inhibitors of translation initiation. They are proposed to form stacking interactions with polypurine sequences in the 5ʹ-untranslated region (UTR) of selected mRNAs, thereby clamping the RNA substrate onto eIF4A and causing inhibition of the translation initiation complex. rocaglate CR-31-B (-) inhibited the replication of corona-, Zika-, Lassa-, Crimean Congo hemorrhagic fever viruses.^[41]

Action on the viral enzymatic system

Two viral proteases of SARS-CoV, a chymotrypsin-like protease (3 CL(pro)) and a papain-like protease (PL(pro)) are attractive targets for the development of anti-SARS drugs.

3 CLpro

The 3 C-like protease (3 CL(pro)) of SARS-CoV is one of the most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle.

Phenolics

Recent studies of molecular docking showed that 5,7,3′,4′-tetrahydroxy-2ʹ-(3,3-dimethylallyl) isoflavone, myricitrin, and methyl rosmarinate may inhibit SARS-CoV-2 3CL^pro and serve as potential anti-COVID-19 drug compounds.^[42]

Quercetin (IC(50) = 73 μM), epigallocatechin gallate (IC(50) = 73 μM) and gallocatechin gallate (GCG) (IC(50) = 47 μM) displayed good inhibition toward 3CLPro. GCG showed a competitive inhibition pattern with K (i) value of 25 ± 1.7 μM. In molecular docking experiments, GCG displayed a binding energy of −14 kcal mol(−1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.^[43] Apigenin moiety at position C-3ʹ of flavones appeared to be more effective as suggested Ryu et al., 2010.^[44] The research team found 3CL(pro) inhibitory effect of amentoflavone (IC(50) = 8.3 μM), apigenin (IC(50) = 280.8 μM), luteolin (IC(50) = 20.2 μM), and quercetin (IC(50) = 23.8 μM but also of the ethanol extract of Torreya nucifera leaves traditionally used as a medicinal plant in Asia (62% at 100 μg/mL).^[44] Previous studies revealed that also quercetin-3-beta-galactoside is a potent inhibitor of the protease and further structure-activity relationship of the new compounds were proposed: (1) removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivatives; (2) acetoxylation of the sugar moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4) replacement of the galactose moiety with other sugars does not affect inhibitor potency.^[45]

Phlorotannins isolated from the edible brown algae Ecklonia cava possess SARS-CoV 3CL(pro) inhibitory activities in a dose-dependently and competitive manner. Of these phlorotannins, two eckol groups with a diphenyl ether linked dieckol showed the most potent trans/cis-cleavage inhibitory effects (IC(50)s = 2.7 and 68.1 μM, respectively). Dieckol exhibited a high association rate in the SPR sensorgram and formed extremely strong hydrogen bonds to the catalytic dyad (Cys145 and His41) of the SARS-CoV 3CL(pro).^[46]

An alkylated chalcone, containing the perhydroxyl group, isolated from Angelica keiskei exhibits potent competitive inhibition with 3CL(pro) and noncompetitive inhibition with PL(pro) (IC(50)s = 11.4 and 1.2 μM, respectively).^[47]

Hesperetin, the 4ʹ-methoxy derivative of flavanone eriodictyol, dose-dependently inhibited cleavage activity of the 3CLpro (IC(50) = 8.3 μM).^[48]

A docking analysis showed the inhibition potential of several compounds, ranked by affinity (ΔG); nelfinavir > lopinavir > kaempferol > quercetin > luteolin-7-glucoside > demethoxycurcumin > naringenin > apigenine-7-glucoside > oleuropein > curcumin > catechin > epigallocatechin > zingerol > gingerol > allicin. Kaempferol, quercetin, luteolin-7-glucoside, apigenin-7-glucoside, naringenin, oleuropein, demethoxycurcumin, curcumin, catechin, and epigallocatechin were the most recommended compounds found in medicinal plants as potential inhibitors of COVID-19 Mpro.^[49]

Glucosinolate

Sinigrin, a natural aliphatic glucosinolate present in plants of the Brassicaceae family (IC50: 217 microM) was more efficient in blocking the cleavage processing of the 3CLpro than Isatis indigotica root extract (IC50: 752 μM) and beta-sitosterol (IC50: 1210 μM) in the cell-based assay.^[48]

Hydroxanthracene derivatives

Fractionation of Rheum palmatum L. revealed that some components had a high level of anti-SARS-CoV 3CL protease activity with IC(50) of 13.76 ± 0.03 microg/mL and an inhibition rate up to 96%.^[50] Previously, it was showed that emodin, the main anthracene derivative of this species, dose-dependently inhibited cleavage activity of the 3CLpro with an IC50 of 366 μM.^[48]

Terpenes

Betulinic acid and savinin, abietane-type diterpenoids and lignoids, are competitive inhibitors of SARS-CoV 3CL protease with Ki values = 8.2 ± 0.7 and 9.1 ± 2.4 microM, respectively.^[51] Tanshinones, abietane diterpene compounds derived from Salvia miltiorrhiza were found to be specific and selective inhibitors for both main SARS-CoV proteases. In a detail kinetic mechanism study, all of the tanshinones except rosmariquinone were identified as noncompetitive enzyme isomerization inhibitors. Tanshinone I exhibited the most potent nanomolar level inhibitory activity toward deubiquitinating (IC(50) = 0.7 μM) and the inhibition is selective because these compounds do not exert significant inhibitory effects against other proteases including chymotrysin, papain, and HIV protease.^[52]

Quinone-methide triterpenes, celastrol (IC50: 10.3 μM), pristimerin (IC50: 5.5 μM), tingenone (IC50: 9.9 μM) and iguesterin (IC50: 2.6 μM) isolated from Triterygium regelii also showed potent inhibitory activities on 3CLPro and it was suggested the importance of quinone-methide moiety in A-ring and a hydrophobic E-ring in exhibiting potent activity.^[53]

PLPro

The MeOH extracts of the fruits of the Paulownia tree yielded many small molecules capable of targeting PLpro in a dose-dependent manner with IC50’s raging between 5.0 and 14.4 μM Five of these compounds were new geranylated flavonoids, tomentin A, B, C, D, and E containing a 3,4-dihydro-2 H-pyran moiety, a very rare chemotype derived from cyclization of a geranyl group with a phenol functionality.^[54]

Cinnamic amides isolated from the methanol extract of Tribulus terrestris fruits displayed significant inhibitory activity on PLPro with IC50 values in the range 15.8–70.1 µM.^[55]

The ethanol extract of the seeds of Psoralea corylifolia showed high activity against the SARS-CoV PLpro with an IC₅₀ of value of 15 µg/ml. Flavonoids such as bavachinin, neobavaisoflavone, isobavachalcone, 4ʹ-O-methylbavachalcone, psoralidin and corylifol isolated from the ethanol extract of the seeds of Psoralea corylifolia inhibited PLpro in a dose-dependent manner with IC₅₀ ranging between 4.2 and 38.4 µM.^[56]

Broussonetia papyrifera-derived polyphenols such as broussochalcones A and B, 4-hydroxyisolonchocarpin, papyriflavonol A, 3ʹ-(3-methylbut-2-enyl)-3ʹ,4,7-trihydroxyflavane, kazinol A, B, F, and J, broussoflavan A are more potent against papain-like protease (PL^pro) than against 3-chymotripsin-like protease (3CL^pro); among them, apyriflavonol A was the most potent inhibitor of PL^pro with an IC₅₀ value of 3.7 μM.^[57]

Hirsutenone, a diarylheptanoid isolated from Alnus japonica, showed the most potent PL(pro) inhibitory activity (IC(50) = 4.1 μM). Structure-activity analysis showed that catechol and α,β-unsaturated carbonyl moiety in the molecule were the key requirement for SARS-CoV cysteine protease inhibition.^[58]

Collateral action of natural compounds on the immune system

Several natural compounds are implicated in combating viral-mediated cytokine production (cytokine storm), excessive inflammatory infiltrates, and tissue destruction. Inflammatory reactions through lung deficiencies can damage other organs through a large amount of inflammatory substances released. Above a certain critical point, the kidney-lung intersection could lead to an irreversible self-amplification storm of cytokines that quickly induces failure and death for multiple organs.^[59,60] In this regard, omics profiling using whole-genome, epigenome, transcriptome, proteome, and metabolome would be of maxim importance, offering detailed information of the human body facing viral infection in an integrative manner.^[61]

Phenolics

Among phenolics, isoliquiritigenin inhibits the expression of inflammatory cytokines induced after the infection of mice with influenza virus, dependent on the activation of the peroxisome proliferator-activated receptor gamma pathway.^[62] Resveratrol and emodin increase the expression of interferon-beta (IFN-β) through Toll-like receptor 9 (TLR9) and it was suggested that IFN-β likely acts synergistically with resveratrol to inhibit H1N1 replication.^[63]

At 100 and 200 mg/kg, HCF elevated the levels of interferon-β in lungs and also decreased the expression of TLR3/4/7 and level of NF-κB p65(p) in lung tissues.^[64] A similar result was recorded for the Shixiangru (Mosla chinensis Maxim) total flavonoids (STF) mainly containing luteolin and apigenin. Higher doses (288 or 576 mg/kg) were administrated to alleviate pathological changes of lung in the viral pneumonia mice and it was associated with reduced expression levels of TLR3, TLR7, MyD88, TRAF3, and NF-κB p65 of the TLR pathway. The up-regulation (IL-6, TNF-α, IFN-γ, and NO) and down-regulation (IL-2, SOD, and GSH) of inflammatory cytokines and anti-oxidant factors were associated with higher clearance of virus and reduction of inflammatory lung tissue damage.^[65]

The expression level of the inflammatory cytokine IL-6 during virus infection, expression of which may result in serious tissue injury and apoptosis is decreased by theaflavin derivatives from black tea,^[66] (2 R,3 R)-7-O-galloylplumbocatechin A, (-)-5,3ʹ,4ʹ,5ʹ-tetrahydroxyflavan-7-gallate, (+)-3,5,3ʹ,4ʹ,5ʹ-penta-hydroxyflavan-7-gallate and (-)-7,4ʹ-di-O-galloyltricetiflavan isolated from Pithecellobium clypearia Benth^[67] and hydro-alcoholic extracts from bark of Uncaria guinanensis selectively containing the flavonoid kaempferitrin.^[68]

The bee pollen, which is the bee product with high amounts of quercetin,kaempferol, and its derivates,^[69] can be a promising alternative to fight against COVID-19 as well as other bee products – propolis and royal jelly that in combination to standard medication could deal with immunologic complications and bacterial co-infections.^[70]

Propolis is frequently used in prevention or treatment of COVID-19 (500 mg/day would be equivalent to 30 drops of propolis extract (with 11%w/v of dry matter), 3 to 4 times a day, diluted in about 100 ml of water), but it could be applied also in more severe cases of COVID-19, with dosages higher than 500 mg/day. A promising pharmacological approach is targeting the downstream effectors, such as p21-activated kinases (PAKs). Caffeic acid phenethyl ester (CAPE), one of the most important constituents of propolis, has shown the property to downregulate RAC (a signaling protein found in human cells), therefore acting as a RAC/CDC42-activated kinase 1 (PAK1) blocker, stoping or inhibiting coronavirus-induced fibrosis in the lungs.^[71]

In this case, the vegetal source is of extremely importance as propolis from Europe and temperate Asia, usually made by bees from resins collected from Poplar trees, has predominantly flavonoid compounds, while green propolis (from Baccharis dracunculifolia), a propolis exclusively found in Brazil, has various kinds of flavonoids and prenylated phenylpropanoids, such as artepellin C, baccharin and drupanin.^[72]

Polysaccharides

The polysaccharides extracted from Radix isatidis, a traditional Chinese herbal medicine strongly inhibited the protein expression of TLR-3 induced by PR8, suggesting that they impair the upregulation of pro-inflammatory factors induced by influenza A virus (human seasonal influenza viruses -H1N1 and H3N2 and avian influenza viruses -H6N2 and H9N2) by inhibiting activation of the TLR-3 signaling pathway. The polysaccharides significantly reduced the expression of pro-inflammatory cytokines (IL-6) and chemokines (IP-10, MIG, and CCL-5) stimulated by A/PR/8/34 (H1N1) at a range of doses (7.5 mg/mL, 15 mg/mL, and 30 mg/mL).^[73]

Echinaforce (EF) prevented the super-expression of inflammatory cytokines by suppressing the expression of NFκB and possibly TLR-4. These results indicate that Echinaea purpurea has the potential to reduce the risk of respiratory complications by preventing virus-induced bacterial adhesion and through the inhibition of inflammation super-stimulation (cytokine storms).^[74] Alkamides, caffeic acid derivatives, and polysaccharides have been considered important constituents of the plant.^[75] The same mechanism was described also for Sophora subprosrate polysaccharide which antagonizes inflammatory responses induced by Porcine circovirus 2.^[76]

A mechanism associated with inhibition of inflammation, protection of intestinal barrier and regulation of mucosal immunity, which may be related to the regulation of gut-lung axis was suggested for Hottuynia cordata polysaccharides (HCP). The plant is an important traditional Chinese medicine for the treatment of respiratory diseases including bacterial and viral infections. HCP markedly decreased the concentration of pulmonary proinflammatory cytokines/chemokines and the number of intestinal goblet cells, and strengthened the intestinal physical and immune barrier, according to the increase of sIgA and tight junction protein (ZO-1) in intestine. At the same time, the inhibition of inflammation in lung and gut was related to the suppressing of the expression of TLR4 and p-NFκB p65 in lung.^[77]

Quinones

PMM-034, a shikonin ester derivative (belonging to naftoquinones class) suppresses the expressions of pro-inflammatory cytokines in RD cells, exhibiting antiviral activity against Human enterovirus 71, as evidenced by the reduced VP1 mRNA and protein levels in PMM-034-treated cells.^[78]

Emodin (6-methyl-1,3,8-trihydroxyanthraquinone) restored the cell viability in poly I: C-induced RAW 264.7 at concentrations of up to 50 μM, inhibiting significantly the production of nitric oxide, IL-1α, IL-Ιβ, IL-6, GM-CSF, G-CSF, M-CSF, MCP-1, MIP-1a, MIP-Ιβ, MIP-2, RANTES, and IP-10 as well as calcium release and mRNA expression of signal transducer and activated transcription 1 (STAT1).^[79]

Alkaloids

An in silico study developed by Koshak et al., showed that nigellidine had high binding energy with human receptors, inflammatory signal molecules, and other proteins such as human IL1R (1itb), TNFR1 (1ncf), and TNFR2 (3alq), being proposed as a potential phytotherapy for coronavirus disease 2019.^[80]

Terpenoids

Pterodontic acid, an eudesmane-type sesquiterpene compound isolated from Laggera pterodonta, was demonstrated to have a broad-spectrum effect against different influenza A viruses, including human (H1N1) and avian (H9N2) influenza viruses, with a half-maximal inhibitory concentration value of 19.9–91.4 µg/ml. It was showed that in vitro antiviral activity of pterodontic acid is most probably associated with inhibiting the replication of influenza A virus by blocking nuclear export of viral RNP complexes, and attenuating the inflammatory response by inhibiting activation of the NF-κB pathway. Western blot analysis revealed that this compound inhibits TLR7, myeloid differentiation primary response protein 88, and tumor necrosis factor (TNF) receptor-associated factor 6 expression, in addition to p65 phosphorylation, at a concentration of 100 or 150 µg/ml while immunofluorescence assays demonstrated that it may inhibit p65/NF-κB nuclear translocation. Additionally, C8 prevented an increase in cytokine mRNA expression, including interleukin (IL)-1β, IL-6, IL-8, and C-C motif chemokine 2 (MCP-1). Furthermore, the Bio-Plex assay results indicated that the protein expression of IL-6, IL-8, TNF-α, C-X-C motif chemokine 10, MCP-1, and C-C motif chemokine 5 was inhibited.^[81]

A similar mechanism was described for methanol extract of Boswellia serrata oleo-gum-resin (BSE) and one of its major constituent β-boswellic acid (a well-known pentacyclic terpenoid) that potently inhibited wild-type and a clinical isolate of Herpes Simplex Virus-1 (EC₅₀ 5.2–6.2 and 12.1–14.63 μg/ml), with nearly complete inhibition (EC₉₉) at 10 and 30 μg/ml, respectively, with significant down-regulation of NF-κB, and p38 MAP-kinase activation.^[82]

Counteracting the host excessive immune response induced by viral infection was described also for 14-deoxy-11,12-dehydroandrographolide, a major diterpenoid of the traditional Chinese medicine Andrographis paniculata. This compound is able to impair H5N1 replication at least in part by restraining nuclear export of vRNP complexes, and the inhibition of viral replication leading to a subsequent decrease of the intense proinflammatory cytokine/chemokine expression (TNF-α, IL-6, IL-8, IFN-α, IL-1β, and IFN-β) and chemokines (CXCL-10 and CCL-2) stimulated by H5N1.^[83]

Ginsenosides are triterpenic saponins and main active compounds of ginseng, with many pharmacological effects. In a study conducted by Yoo et al., ^[84] naive mice that were infected with influenza virus mixed with red ginseng extract showed significantly enhanced protection, lower levels of lung viral titers and interleukin-6, but higher levels of interferon-γ compared with control mice having virus infections without red ginseng extract, indicating an antiviral effect of ginseng.^[84]

Herbal medicine as vaccine adjuvants

In recent years, there have been a number of reports on the herbal extracts/isolated compounds as adjuvant agents for enhancing efficiency of weakly immunogenic vaccines. Up to now, most of these combinations are applied în veterinary medicine, but human clinical trials are ongoing.

Immune-enhancing properties in chickens immunized with ND vaccine were recorded after administration of ginseng stem-and-leaf saponins (5 mg/kg for 7 days),^[85] Astragalus, Achyranthes and Isatis root polysaccharides,^[86] Epimedium polysaccharide and propolis flavone.^[87] Propolis is considered a safe immunostimulant and a potent vaccine adjuvant that induces an earlier immune response and provides a longer protection period. It is also included as an adjuvant ingredient in traditional Chinese medicine, improving the humoral and cellular responses in mice inoculated with inactivated virus vaccines.^[72]

Astragalus polysaccharides were employed as an adjuvant for an avian infectious bronchitis virus (IBV) vaccine, increased the titer of IBV-specific antibodies, lymphocyte proliferation, and IL-1β, IL-2, IL-8, and TNF-α mRNA expression levels.^[88]

Protein and peptide-based vaccines provide rigorously formulated antigens. However, these purified products are only weakly immunogenic by themselves and therefore require the addition of immunostimulatory components or adjuvants in the vaccine formulation. Various compounds derived from pathogens, minerals, or plants, possess pro-inflammatory properties which allow them to act as adjuvants and contribute to the induction of an effective immune response. The results presented by Castro-Diaz et al., 2012^[89] demonstrate the adjuvant properties of novel saponins derived from the Spanish saffron Crocus sativus. These saponins were indeed able to increase both humoral and cellular immune responses to protein-based vaccines, ultimately providing a significant degree of protection against tumor challenge when administered in combination with a tumor antigen.^[89]

Saponin-based adjuvants are promising adjuvants that enhance both humoral and T-cell-mediated immunity. Vaccination with a live bivalent vaccine of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) is a routine practice in poultry industry in China. Ginseng stem-leaf saponins (GSLS) in combination with selenium (Se) promoted significantly higher NDV- and IBV-specific antibody responses.^[90] One of the most used natural products as vaccine adjuvants are Quillaja saponaria bark saponins and its fraction named Quil A®, a complex mixture of 25 different saponin molecules with a triterpenoid backbone in common able to induce a balanced immune response.

AS01_B is an adjuvant suspension used with the antigen component of Shingrix vaccine, the recombinant zoster vaccine recommended for persons aged 50 years or older. AS01_B is made of up of monophosphoryl lipid A (MPL), an immune-boosting substance isolated from the surface of bacteria, and QS-21, a natural compound extracted from the above mentioned Quillaja saponaria Molina. AS01_B is also a component of vaccines currently being tested in clinical trials, including malaria and HIV vaccines. Derivatives of this compound were also tested. A semi-purified aqueous extract and a previously little characterized saponin-enriched fraction (QB-80) from Q. brasiliensis as vaccine adjuvants and an inactivated virus (bovine viral diarrhea virus, BVDV) antigen were co-formulated in experimental vaccines in mice model.^[91]

Subcutaneous immunization of mice with an immunostimulating complex containing Glabilox and H7N1 influenza virus antigens stimulated high levels of humoral and cellular immunity. Vaccination of chickens with the same immunostimulating complex protected 100% of the animals after experimental infection with a homologous virus. Comparative studies showed that the immunogenic and protective activity of immunostimulatory complexes containing Quil-A and immunostimulatory complexes containing Glabilox are comparable to each other. The results of these studies indicated that Glycyrrhiza glabra saponins show great promise as safe and effective adjuvants.^[92]

In recent years, there have been a number of reports on the successful use of immunostimulatory complexes with saponins and viral glycoproteins as veterinary vaccines and in clinical trials for human medicine. The saponins Algiox, Sapanox and Pangisan were isolated and purified by HPLC from Allochrusa gypsophiloides, Saponaria officinalis and Gypsophila paniculata plants in Kazakhstan and they proved to have low toxicity in experiments with mice, chickens, and chicken embryos. Algiox, Sapanox, and Pangisan can be used to create immunostimulatory complexes (ISCOMs) similar to saponin-Quil-A-containing ISCOMs both in structure and in immunostimulatory efficiency. The adjuvant effect of the obtained saponins was studied by subcutaneous injection of mice with ISCOMs containing these herbal saponins and lipids and glycoproteins of H7N1 influenza virus.^[93]

Administering QR448(a) (oleoresines and essential oils) to chickens at a 1:20 dilution by spray, 2 h before challenge with IBV was determined to be the most effective treatment. Treatment decreased the severity of clinical signs and lesions in the birds, and lowered the amount of viral RNA in the trachea. Treatment with QR448(a) protected chickens for up to 4 days post-treatment from clinical signs of disease (but not from infection) and decreased transmission of IBV over a 14-day period. Anti-IBV activity of QR448(a) was greater prior to virus attachment and entry indicating that the effect is virucidal.^[94]

MF59 is the adjuvant contained in Fluad (an influenza vaccine licensed for adults aged 65 or older) is an oil-in-water emulsion composed of squalene, a naturally occurring oil found in many plant and animal cells, as well as in humans. MF59, used in flu vaccines in Europe since 1997 and in the United States since 2016, has been given to millions of people and has an excellent safety record.

Conclusions

Herbal medicine could be applied as diet or supplement to prevent infection and strengthen the immunity^[95] or as supportive therapy in combination with validated anti-COVID drugs to modulate the cellular and humoral immune responses, to limit co-infections or even to reduce virus titers. Since some natural products with IC50 below 10 µM could be also considered as promising anti-SARS-CoV-2 agents, but this effect must be evaluated through prospective and interventional studies paying attention to the specificity of the action exerted by such products, sustainable sourcing of the species, doses range used, or the use of appropriate controls.^[7]

Besides prevention, other ways of action for herbal medicine in supporting COVID therapy were also proposed: use as an antiviral agent by coating on masks; use as an air-disinfectant to stop aerosol transmission; and use as a surface sanitizing agent to provide a disinfected environment.^[96]

Europe has a long and strong tradition of using medicinal plants for therapeutic purposes, but clinical trials for this type of approach are scarce, compared to Asia. In this regard, a bridge between tradition and science, would have a strong impact on the capacity for prevention and treatment of COVID-19.

Additional information

Funding

This research was funded by the Romanian Ministry of Education and Research, The Executive Unit for Financing Higher Education, Research, Development and Innovation (UEFISCDI), in the framework of: Program 1—The Improvement of the National System of Research and Development, Subprogram 1.2—Institutional Excellence—Projects of Excellence Funding in RDI, Contracts No. 7PFE/2018 and PCCDI 1/2018 and by grants PN 19.29.01.04, PN.19.41.01.01; grant COP A 1.2.3.-G: P_40_406/2015, grant COP A 1.2.3.-G: P_40_197/2016, grant COP-A.1.1.4-E, Dia-Cure P_37_794.