The pandemic caused by the spreading of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the related disease named coronavirus disease 2019 (COVID-19), from China since late 2019 to most of the world since January 2020, proved unprecedented in many aspects. A combination of the high contagiousness rate (even from asymptomatically infected people), the long course of the disease, relatively high incidence of severe and lethal pneumonia (especially for elderly and immunosuppressed subjects) [1
], lack of effective therapies, and initial unpreparedness to cope with the pandemic threaten to overwhelm local and national health care infrastructures—intensive care units in particular [2
]. The consequent reaction concretized literally overnight, expanding to a remarkable fraction of the world population in total or partial lockdowns and social distancing prescriptions, which are still in force at the time of writing. While everyday chronicles proved the effectiveness of such measures in slowing down the infection rate, they are also likely to cause unpredictable economic havoc and endanger the livelihood of many people for a long time [4
While similar to other coronaviruses responsible for past epidemics, especially SARS-CoV (early 2000s), with respect to targeted receptors of human type II lung cells [5
], genetic mutations that occurred in SARS-CoV-2 made it more infectious and likely more effective in slowing the response by the immune system until the virus has spread in the lung cells and has started replicating [7
]. In particular, long spike glycoproteins that protrude from the SARS-CoV-2 particle latch on to the angiotensin converting enzyme-2 (ACE2), a protein located on the surface of type II lung cells [6
As with SARS-CoV, most of the damage in COVID-19 is caused by the immune system carrying out an overreaction to stop the virus from spreading. Upon entry into alveolar epithelial cells, SARS-CoV-2 replicates rapidly and triggers a strong immune response, resulting in cytokine storm syndromes, or hypercytokinemia, and pulmonary tissue damage. The uncontrolled production of proinflammatory cytokines (and chemokines) causes acute respiratory distress and multiple organ failure [8
], even possibly affecting the male gonadal function [10
]. Beyond the lethal cases, it is still unclear whether and at which extent these damages could be reversed in recovered subjects.
An unprecedented worldwide scramble is underway to search for effective vaccines [11
]. However, their very feasibility and effectiveness is still uncertain. Recent preliminary results point to insufficient development of SARS-CoV-2-specific neutralizing antibodies in a fraction of recovered patients, especially the younger or those affected by common or mild symptoms [12
]. The same finding, if confirmed, might reduce the expectancies about the perspective for herd immunity.
An intensive research also is underway to identify therapeutic drugs to be repurposed, a few of which could already have shown preliminary positive results, yet lack large and randomized verifications and consideration of possible harmful side effects [13
]. Natural bioactive compounds are also actively looked for, to assess their preventive or therapeutic activities, namely the ability to prevent the virus from binding to the ACE2 enzyme of the host cell, inhibit the virus replication after its penetration in the host cell, as well as restrain or counteract the proinflammatory overreaction of the immune system.
The frantic search for effective bioactive compounds is part of the general vision that perceives the boost to the individual immune system, and the effective inhibition of the infection by SARS-CoV-2 as the most effective shield against the onset and serious progress of COVID-19, protecting both individuals and society as a whole [14
]. Contributing to transform this vision into reality is also the goal of this study.
Past and recent studies proved that hesperidin, a citrus flavonoid abundant in citrus peel, which is a byproduct of the juice industry, as well as the major flavonoid in sweet orange and lemon, is endowed with plenty of beneficial biological activities, some of which are shared with other citrus flavonoids. Thus, it is not surprising that many food supplements and drugs containing hesperidin and other citrus flavonoids have been available since long. Hesperidin and its aglycone hesperetin were attributed particularly strong binding affinity to the receptors of SARS-CoV-2, along with remarkable anti-inflammatory activity, making these molecules attractive ingredients for preventive and therapeutic drugs.
In Section 2
, the sources and remarkable bioactive properties, including antiviral activity, of hesperidin and other citrus flavonoids are briefly reviewed, along with the challenging issue posed by generally low bioavailability. Section 3
focuses on the properties of hesperidin and other citrus flavonoids potentially relevant to the contrast to COVID-19. Section 4
reviews the extraction methods of the same compounds, pointing to controlled hydrodynamic cavitation (HC) as the most effective, efficient and scalable in the perspective of large-scale production. The discussion and conclusions in Section 5
highlights the immediate feasibility of mass production of hesperidin-rich products based on existing plants and their upscale and replication, also suggesting an affordable process line.
3. Early Evidence of Potential Activity against SARS-CoV-2
According to recent studies, hesperidin showed remarkable binding affinity to the three main protein receptors of SARS-CoV-2, i.e., the SARS-CoV-2 protease domain, the receptor binding domain of the spike glycoprotein (RBD-S), and the receptor binding domain of the ACE2 at the protease domain (RBD-ACE2), responsible for cell infection and virus replication [58
]. The above-mentioned remarkable binding affinity to the three main targets was considered representative of the inhibitory activities of hesperidin against viral infection, by either inhibiting the latching of the virus to the ACE2, or inhibiting the virus replication in the cells. Thus, hesperidin could be a promising active substance for drugs potentially useful to prevent or treat COVID-19, possibly along with other citrus flavonoids.
In a molecular docking study, scholars in Indonesia found that hesperidin had the highest affinity to bind all three receptors (lowest docking score), thus inhibiting the proteins responsible for viral infection and virus development [58
]. Hesperidin outperformed lopinavir, a repurposing drug involved in clinical trials for COVID-19, as well as nafamostat, a reference compound for RBD-S binding. Moreover, hesperidin outperformed several other natural molecules. In the same study, other citrus flavonoids also abundant in citrus peel, namely tangeretin, nobiletin and naringenin, as well as hesperetin that derives from hesperidin in the intestine after ingestion, showed excellent affinity to the selected receptors, suggesting that all these citrus flavonoids might contribute to inhibit the viral infection and replication.
Hesperetin was the only citrus flavonoid among the flavonoids investigated in an another study [64
]. It showed high binding affinity to ACE2 enzyme, similar to other flavonoids typical of Chinese medicine, present in various herbs, roots, and soybean.
In another study, scholars in China reached similar conclusions [59
]. In detail, the team analyzed all the proteins encoded by SARS-CoV-2 genes, compared them with other coronaviruses, such as SARS-CoV and MERS-CoV, and modeled the protein structures using said structures along with those of human relative proteins (human ACE2 and type-II transmembrane serine protease enzymes) as targets to screen three databases of approved drugs. These databases were the following: the database of traditional Chinese medicine and natural products (including reported common antiviral components from traditional Chinese medicine), the database of commonly used antiviral drugs (78 compounds), and the ZINC drug database of the Food and Drug Administration of the USA by virtual ligand screening method. The method clearly showed that hesperidin was the only compound that could target the binding interface between the spike protein and human ACE2, so that by superimposing the RBD–ACE2 complex to the hesperidin–RBD complex, a distinct overlap of hesperidin with the interface of ACE2 was observed. This suggests that hesperidin may disrupt the interaction of ACE2 with RBD and prevent the virus from entering the cell.
In a further study, a molecular model was built of the 3-chymotrypsin-like protease (Mpro
) structure of the SARS-CoV-2, which is vital to virus replication (as it was for SARS-CoV) and is considered as a promising drug target [60
]. The study carried out virtual screening to identify readily usable therapeutics derived from the previous progress about specific inhibitors for the corresponding SARS-CoV enzyme [42
], which can be conferred on its SARS-CoV-2 counterpart. Results showed that the flavonoid glycosides diosmin (a preapproved drug) and hesperidin (an approved drug) obtained from citrus fruits fitted very well into and blocked the substrate binding site, resulting as the top scorers. In particular, hesperidin hits showed up multiple times, suggesting it has many modes of binding. Both hesperidin and diosmin were attributed only mild, occasional and reversible adverse reactions.
Another computational and in vitro and in vivo study found that multiple flavonoids abundant in citrus peels have the potential to cooperate to prevent the SARS-CoV-2 infection and restrain its harmful consequences [65
]. In particular, simulated molecular docking showed that naringin, hesperetin and naringenin, in descending order, have strong binding affinity with the RBD–ACE2 receptor, at a level similar to chloroquine and higher than hesperidin. Moreover, in vitro and in vivo experiments showed the potential of naringin for inhibiting or restraining the expression of the proinflammatory cytokines induced by different disorders through the overreaction of the human immune system, thereby suggesting that naringin could have a potential in preventing cytokine storms associated with severe forms of COVID-19. It appears that integral flavonoids-rich extracts from citrus peels could show simultaneously multiple activities against COVID-19.
Naringin, among all the flavonoids available in the traditional Chinese herb Exocarpium Citri grandis
, was attributed the greatest potential for application in alleviating the respiratory symptoms caused by COVID-19 [66
]. This role was due to several properties, such as antitussive, regulation effect on both mucus and serous components in sputum, improvement of lung function and regulation of pulmonary secretion, and inhibition of the secretion of pulmonary inflammatory factors, thus alleviating acute lung injury. Moreover, naringin was shown to possess therapeutic effects in attenuating pulmonary fibrosis and enhancing the antiviral immune response.
In a further study, a library of phenolic natural compounds (80 flavonoids) was investigated by in silico based screening method against the crystallized form of SARS-CoV-2 main protease (Mpro
]. The importance of Mpro
derives from its key role in the self-maturation and processing of viral replicase enzymes, thus in virus replication. Hesperidin exhibited the highest binding energy at the active site of SARS-CoV-2, and revealed as the best potential inhibitor of Mpro
by using a molecular docking approach, closely followed by rutin and diosmin (another citrus flavonoid). Moreover, both hesperidin and diosmin showed a better binding affinity to Mpro
than nelfinavir, an antiviral widely used in the treatment of HIV, as well as one of the early candidates for the treatment of COVID-19 [67
Later studies, published in April 2020, provided important confirmation to the potential role of hesperidin against COVID-19. Joshi and coauthors performed an extensive molecular docking study with over 7000 molecules from different classes such as flavonoids, glucosinolates, antitussive, anti-influenza, antiviral, terpenes, terpenoids, alkaloids and other predicated anti-COVID-19 molecules [62
]. The three docking targets were Mpro
, involved in virus replication; RNA-dependent RNA polymerase (RdRp), which carries out the synthesis of viral RNA from RNA templates and is involved in the replication and transcription of viral genome; and human ACE2, which is the entry point of the virus. Based on the finding in a previous study [59
] that effective molecules should target multiple key proteins, out of all the considered molecules, only 29 were predicted as potentially effective against COVID-19, and among these, several flavonoids showing better binding affinities to the three targets than existing synthetic antiviral drugs. Out of the predicted molecules, hesperidin showed the second highest average binding score across the targets, as well as by far the highest binding score with human ACE2, thus potentially representing one of the most promising molecules for any stage of the infection, as well as the most promising for prevention purposes.
The latter result concerning hesperidin was confirmed in another independent study by Indian authors [68
]. They concluded that hesperidin, showing the lowest binding energy with the spike protein fragment of SARS-CoV-2 and its human host ACE2 receptor, can be considered as the most suitable ligand across several phytochemicals typical of Indian medicinal plants.
In another study, plant bioactive compounds were assessed based on their binding affinity with Mpro
and spike glycoprotein of SARS-CoV-2, by means of a molecular docking approach [63
]. The well-known drugs, namely nelfinavir, chloroquine and hydroxychloroquine sulfate, which were widely recommended for clinical trials against COVID-19, were used as a comparison. Hesperidin turned out to have the highest binding score towards both targets, outperforming also the above-mentioned drugs, and significantly, chloroquine and hydroxychloroquine. Other bioactive compounds from citrus fruits, such as rhoifolin, nobiletin, tangeretin, and chalcone, showed good binding affinity.
In a later important development, the Qingfei Paidu Decoction, a formula consisting of 21 components including both herbs and mineral drugs, well known in traditional Chinese medicine, was analyzed in order to explain the mechanisms underlying its observed success in treating COVID-19 patients [69
]. It was found that such natural drug contains plenty of bioactive compounds, including hesperidin, neohesperidin, naringin and rutin, and is particularly effective in regulating the innate immune system and preventing cytokine storms through the regulation of the toll-like signaling pathway. Synergistic effects were preliminarily revealed, although requiring further investigation along with the possibility of additional actions against COVID-19.
5. Discussion and Conclusions
Hesperidin, a flavonoid abundant in citrus peels, was identified as a potentially very interesting molecule in the fight against COVID-19. Its antiviral activity was proven for other viruses, in particular SARS-CoV, thus it could reveal useful also in case of further mutations of SARS-CoV-2.
In the therapeutic use, hesperidin has the advantage of strong binding affinity to all the main viral and cellular targets, outperforming not only other natural molecules, but also antiviral drugs recommended for clinical trials on COVID-19 inpatients. These targets correspond to different stages of the infection, from the entry of the virus into the host human cell, to the transcription of viral genome and virus replication.
The especially great binding affinity with the human ACE2, thus the potential to prevent the virus to spread into the cells, could suggest a special role of hesperidin in prophylaxis. On the other hand, the regular and prolonged administration of hesperidin for prophylaxis would be allowed by its safety, short lifetime in the body, and the absence of cytotoxicity up to high doses.
Other flavonoids, coexistent with hesperidin in citrus peels, also showed good binding affinity to one or more targets, especially hesperetin (the aglycone of hesperidin) and naringin. The latter flavonoid also showed the ability to restrain the proinflammatory overreaction of the immune system, which could help fighting the severe forms of COVID-19.
In this study, we call for the urgent uptake of HC-based processes applied to citrus peels, for the efficient and green industrial production of aqueous extracts and pectin tablets rich in hesperidin. The extraction process takes no longer than 10–15 min, however, including all the necessary steps such as grinding the citrus peels before the inlet to the processing unit, separating the solid residues, and discharging and packaging the aqueous extract, the overall process for a plant with a nominal capacity of about 2000 L could require up to 2 h. Based on the figures exposed in Section 3
, undertaking the processing of 500 kg waste citrus peel (as such) in 1500 L water, the process would be able to extract 3 kg of hesperidin per cycle, hence at least 36 kg of hesperidin per day (in 12 cycles). The only additional technological components next to the industrial scale HC-based extractor would be a grinder, a filter/separator, and a lyophilizer, such as those commonly operated at pharmaceutical companies, where they are used to remove solvent from a frozen product by sublimation. After the lyophilization, IntegroPectin tablets containing the required dose of hesperidin and other flavonoids could be readily produced, due to the low density and open, porous structure of the pectin.
Lyophilization, or freeze-drying, is the main drying technique already adopted both in the pharmaceutical and nutraceutical industries, since it removes the water from sensitive products without damaging them. The operational cost of lyophilization is tightly related to the cost of electricity, which is very low in large countries with important manufacturing bases, such as China, India and Russia. In other countries, pharmaceutical and nutraceutical companies today are virtually all equipped with several hundred kW, or even MW solar photovoltaic rooftops which have cut their electricity bills by 30%–50%.
Under the laboratory conditions of the studies mentioned in this article [78
], batch drying of IntegroPectin took up to 4 days. However, continuous lyophilization techniques, developed for the coffee industry in the 1960s and reducing drying time and electricity consumption, are in the process of being extended to the pharmaceutical and nutraceutical industries by the booming lyophilization equipment industry [91
In particular, new continuous freeze-dryers for suspended vials were proposed and demonstrated for pharmaceutical applications, which are cheaper and 6–8 times smaller than conventional batch lyophilizers, based on comparison done on constant throughput [92
]. They improve heat transfer uniformity and reduce the primary drying time by 3–4 times, while improving vial-to-vial and intravial homogeneity. The total cycle time was estimated approximately 6 times shorter, because some time-consuming operations, such as filling, are carried out in parallel to the process and do not contribute to increase the cycle time. Such equipment would be particularly well suited for the production of IntegroPectin. However, we acknowledge that further research is recommended, aimed at optimizing and streamlining the industrial-scale IntegroPectin production process, from HC-based extraction and continuous freeze-drying to the manufacturing and packaging of tablets.
shows the main technological components of the proposed process based on the experience gained by authors, although variants are easy to set up, such as replacing the centrifugal pump and the Venturi-shaped reactor with a rotor–stator arrangement, according to specific and local expertise or whatever preference. Moreover, the Venturi-shaped reactor could be realized in accordance with long established rules for circular-section ones [81
], or in the form of generally more performing slit Venturi [72
], as well as optimized by numerical simulations [93
]. Other emerging setups could be used too, such as based on vortex diode [94
]. The dosing pump is optional and could be useful for introducing any natural or technical additives. Minor components such as a thermometer and a pressure gauge can be applied to the working vessel and are not shown.
The product, either in the form of aqueous extracts or pectin tablets, could undergo in vitro, in vivo and clinical trials aimed at assessing the prophylactic or therapeutic activity against COVID-19 and the respective effective doses. As a reference, about one month after the outbreak of COVID-19, Chinese scholars were able to assess the specific antiviral activities of the well-known broad-spectrum antiviral drug remdesivir, and the long-known antimalarial drug chloroquine, against SARS-CoV-2 infecting Vero E6 cells in vitro, including the IC50
and the level of the 90% inhibitory concentration (IC90
]. Remdesivir showed IC50
= 0.77 μM and IC90
= 1.76 μM, while chloroquine showed IC50
= 1.13 μM and IC90
= 6.90 μM, the latter level clinically achievable as demonstrated in the plasma of rheumatoid arthritis patients who received 500 mg administration. The selectivity index was also reasonably high, about 130 for remdesivir and 88 for chloroquine, suggesting a good safety level.
Since chloroquine is relatively toxic with high doses and prolonged use, as well as its production largely discontinued following the introduction of other antimalarial drugs, about two months later, the same authors proposed hydroxychloroquine as a further potential therapeutic agent against COVID-19 [96
]. Hydroxychloroquine was synthesized long ago by introducing a hydroxyl group into chloroquine, and is still widely used in the treatment of inflammatory rheumatic diseases. It is about 40% less toxic than chloroquine, but shows a selectivity index about one third lower and requires higher doses to achieve comparable effectiveness, although still clinically achievable. Finally, hydroxychloroquine shares with chloroquine a good potential to attenuate the inflammatory response, thus potentially offering a broad-spectrum protection from COVID-19.
Despite lower toxicity, one of the main drawbacks of hydroxychloroquine is the well-known side effect of retinopathy, especially in case of prolonged use such as for treating rheumatic disorders and also due to its long half-life and accumulation in tissues and blood, leading to the recent widespread recommendation for lower doses [97
]. While this could not be such a big issue for therapeutic use against COVID-19, it could jeopardize the use of hydroxychloroquine as a preventive drug. However, the neuroprotective activities attributed to hesperidin, mentioned in Section 2.1
], might suggest an integrated approach against COVID-19, with hesperidin-rich products and hydroxychloroquine administered together, at respective doses yet to be defined, for both therapy and prevention.
As recalled in Section 2.4
, the bioavailability issue after oral administration could impair the performance of hesperidin-rich products during in vivo and clinical trials, which is also the case with research about COVID-19 [63
]. Based on the past experience with the HC processing of WOP and WLP [78
], as well as with other raw materials such as grains [87
] and biochar [88
], it could be hypothesized that the HC processing relieves the bioavailability issue both for the integral aqueous extracts and the IntegroPectin obtained after freeze drying the extract.
It is likely that the HC processing forms solid fine dispersions, associated with larger available surface and more rapid wetting and dissolution, so that the bioactive compounds can be released efficiently as very fine colloidal particles [46
]. If further research will confirm this hypothesis, the integral aqueous extracts could show remarkable bioavailability.
The polyphenols originally contained in the lemon peel were observed to migrate to the surface of the freeze-dried pectin as a result of the HC-based extraction [79
]. Thus, it can be hypothesized that the HC processing produces a conjugation of the polyphenols (including hesperidin) onto the pectin macromolecules, resulting in a product similar to that obtained by means of covalent conjugation via a proven preparation method involving epichlorohydrin chemistry [53
]. In the same way as these conjugates, IntegroPectin is likely to show remarkably higher solubility in water in comparison with neat pectin and polyphenols, along with similar or higher retention of the original biological properties of the conjugated polyphenols. The latter was proven by the exceptional antioxidant activity shown by the IntegroPectin [79
These topics are recommended for further research, along with the ability of IntegroPectin to regulate the release of hesperidin to the target cells, similarly to the hydrogel matrix obtained after mixing amide pectin with hesperidin and chitosan in a hydroalcoholic solution [52
]. In the case of positive results, IntegroPectin, also in the form of tablets, could be considered for in vivo and clinical trials and, eventually, for mass production.
Finally, since the primary targets of hesperidin and other citrus flavonoids as potential anti-SARS-CoV-2 agents are lung cells, the possibility of formulating such bioactive compounds as an inhalable dry powder (i.e., the IntegroPectin as such) could be considered, similarly to the formulation of naringin by spray-drying with certain amino acids [57
]. Further research is recommended on this topic, in particular aimed at investigating whether a substantial fraction of the obtained particles showed aerodynamic diameters <5 μm, thus being able to reach the lung region during inhalation therapy.