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Citrus fruits are rich in flavonoids for immunoregulation and potential targeting ACE2

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The most recent outbreak of 2019 novel coronavirus, named as COVID-19, caused pneumonia epidemic in Wuhan with 2,121 deaths cases as of February 20 th 2020. Identification of effective antiviral agents to combat the novel coronavirus is urgently needed. Citrus fruit peel or wild citrus are rich in flavonoid, and is clinically documented for roles in relief of cough and promotion of digestive health. Therefore, citrus fruits are assumed to possess antivirus activities or enhance the host immunity. A previous study found that hesperetin could act as a high potent inhibitor of SARS-CoV 3CLpro. We determined six flavonoid compounds content of in three citrus species by using LC-MS technique. The content of naringin and naringenin was at higher levels in pummelo. Hesperetin and hesperidin were highly accumulated in mandarin and sweet orange. The subsequent in vitro and in vivo experiments indicated that naringin could inhibit the expression of the proinflammatory cytokines (COX-2, iNOS, IL-1β and IL-6) induced by LPS in Raw macrophage cell line, and may restrain cytokine through inhibiting HMGB1 expression in a mouse model. The results revealed that naringin may have a potential application for preventing cytokine storm. We simulated molecular docking to predict the binding affinity of those flavonoids to bind Angiotensin-converting enzyme 2 (ACE 2), which is a receptor of the coronavirus. Consideration of the potential anti-coronavirus and anti-inflammatory activity of flavonoids, the citrus fruit or its derived phytochemicals are promising in the use of prevention and treatment of 2019-nCoV infection.
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Citrus fruits are rich in flavonoids for immunoregulation and
potential targeting ACE2
Liping Cheng1, Weikang Zheng1, Ming Li1,Jie Huang2,Shuzheng Bao3, Qiang
Xu1, Zhaocheng Ma1
1. College of Horticulture and Forestry Sciences, Key Laboratory of Horticultural
Plant Biology, Huazhong Agricultural University, Ministry of Education, Wuhan
430070, China.
2. Network of Aquaculture Centres in Asia-Pacific, Bangkok 10903, Thailand.
3. China Association of Agricultural Science Societies, Beijing 100125, China.
These authors contributed equally to this study
Corresponding: Dr. Zhaocheng Ma: mzhaocheng@mail.hzau.edu.cn
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
© 2020 by the author(s). Distributed under a Creative Commons CC BY license.
Abstract
The most recent outbreak of 2019 novel coronavirus, named as COVID-19,
caused pneumonia epidemic in Wuhan with 2,121 deaths cases as of February 20th
2020. Identification of effective antiviral agents to combat the novel coronavirus is
urgently needed. Citrus fruit peel or wild citrus are rich in flavonoid, and is clinically
documented for roles in relief of cough and promotion of digestive health. Therefore,
citrus fruits are assumed to possess antivirus activities or enhance the host immunity.
A previous study found that hesperetin could act as a high potent inhibitor of
SARS-CoV 3CLpro. We determined six flavonoid compounds content of in three
citrus species by using LC-MS technique. The content of naringin and naringenin was
at higher levels in pummelo. Hesperetin and hesperidin were highly accumulated in
mandarin and sweet orange. The subsequent in vitro and in vivo experiments indicated
that naringin could inhibit the expression of the proinflammatory cytokines (COX-2,
iNOS, IL-1β and IL-6) induced by LPS in Raw macrophage cell line, and may
restrain cytokine through inhibiting HMGB1 expression in a mouse model. The
results revealed that naringin may have a potential application for preventing cytokine
storm. We simulated molecular docking to predict the binding affinity of those
flavonoids to bind Angiotensin-converting enzyme 2 (ACE 2), which is a receptor of
the coronavirus. Consideration of the potential anti-coronavirus and
anti-inflammatory activity of flavonoids, the citrus fruit or its derived phytochemicals
are promising in the use of prevention and treatment of 2019-nCoV infection.
Keywords: Citrus flavnoids, Naringin, Immunoregulation, ACE2, 2019-nCoV
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
Introduction
The number of pneumonia cases caused by a novel coronavirus (2019-nCoV)
continues to rise in China. Thus far, there are tens of thousands of confirmed cases by
the hospital agency. Common symptoms of patients infected with 2019-nCoV are
fever, cough and myalgia or fatigue. The severe cases have high amounts of cytokines,
such as TNF-α, IL-1β, IL-10, IFNγ and MCP-1, suggesting that the cytokine storm is
associated with disease severity (Huang et al., 2020). Currently, there is no specific
anti-viral treatment against the new coronavirus. Identifying effective antiviral agents
to combat the disease is urgently needed. Commercial antiviral agents and chemical
compounds extracted from traditional Chinese medicinal herbs were screened (Liu
and Wang, 2020). Remdesivir and chloroquine were found to be highly effective in
the control of 2019-nCoV infection in vitro (Wang et al., 2020). Some Chinese herbal
compounds including baicalin, scutellarin, hesperetin, nicotianamine and glycyrrhizin
were predicted to have a capacity for binding ACE2 with potential anti-2019-nCoV
effects (Chen and Du, 2020). The existing safe host-directed therapies were
repurposed to treat COVID-19 infection (Zumla et al., 2020). Traditional Chinese
medicine was used to treat the novel coronavirus-infected pneumonia and proved to
have a higher effect in the relief of cough and fever-reducing(Yao et al., 2020). In
addition, corticosteroids were used frequently for severe cases treatment to reduce
inflammatory-induced lung injury. Citrus is rich in bioactive compounds and some
varieties are used as Chinese folk medicine, such as Zhiqiao and Zhishi (Sour orange,
Citrus aurantium) or its hybrids, Huajuhong (Citrus grandis), and Chenpi (Citris
reticulata). They have been clinically documented for roles in the relief of cough and
the promotion of digestive health. Flavonoid compounds are expected to be developed
as anti-viral drugs. Hesperetin was found the high potent inhibitor of SARS-CoV
3CLpro (Lin et al., 2005). Meanwhile, nutrient supplements could reduce the host
immune responses. Therefore, we try to identify effective antiviral and
anti-inflammation compounds from citrus flavonoids and give a nutritional
recommendation for the prevention and treatment of COVID-19.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
Results
Flavonoid profiling in citrus species
Figure 1. Citrus fruits (A) and chemical structure of naringenin, naringin, hesperetin, hesperidin,
neohesperidin and nobiletin (B).
Citrus fruits are a rich source of vitamins, flavonoids and alkaloids. These
phytochemicals have been reported to benefit human health, used to prevent and treat
some diseases (Dall'Asta et al., 2013; Feng and Wang, 2018). To assess the potentials
of six flavonoid compounds (Figure 1) variation in citrus, different cultivars were
collected from three major species of mandarin (Citrus reticulata), pummelo (Citrus
maxima) and sweet orange (Citrus sinensis) for targeted metabolic profiling. We have
been detected and quantitated 459 known metabolites in the flesh, including segment
membrane and juice sacs of 16 cultivars using LC-MS/MS. Among 459 known
metabolites, we mainly analyzed the above six flavonoid compounds. The intensity of
metabolic profiling signals for total ions reflected substantial qualitative and
quantitative difference in different species. The relative content was represented by
the average of relative contents of mandarins, pummelos, sweet oranges, respectively.
Sixteen cultivars divided into mandarin, pummelo, sweet orange, were compared with
each other. As showed in Figure 2A, the contents of naringin and naringenin were at
higher levels in pummelo. On the other hand, mandarin and sweet oranges had higher
hesperetin and hesperidin contents compared to pummelo. In general, the contents of
neohesperidin and nobiletin were lower than the other four compounds in citrus.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
Meanwhile, these six flavonoids were detected in selected 8 citrus cultivars, including
‘Kao Pan’ pummelo, ‘Majiayou’ pummelo, ‘Wanbai’ pummelo, ‘Oukan’ mandarin,
Satsuma mandarin, Clementine mandarin, ‘Ponkan’ mandarin, ‘Newhall’ navel orange.
Notably, the content of naringin was higher in ‘Kao Pan’ pummelo and ‘Wanbai’
pummelo than other cultivars. It was also found high content of naringin in herbal
medicine Huajuhong.Hesperetin and hesperidin accumulated higher in ‘Ougan’,
Satsuma mandarin and ‘Newhall’ navel orange. Herbal medicine Chenpi, Zhiqiao and
Zhishi had high contents of these two compounds. Vitamins, rutin and herbal
medicine Chenpi, Zhiqiao and Huajuhong were used in the treatment of 2019-nCoV
infection. In order to prevent and decrease plasma cytokines levels of TNF-α, IL-1β,
IL-10, IFNγ in COVID-19, citrus fruit or its derived phytochemicals should be an
option to reduce the host immune responses and prevention of infection.
Figure 2. The content of six compounds were analyzed by LC-MS/MS (Shimadzu LCMS-8060) in
different citrus species and cultivars. The data were analyzed by software, LabSolution Insight
LCMS. Peak area of ions signal repreasent relative content. (A) Distribution of naringenin,
naringin, hesperetin, hesperidin, neohesperidin and nobiletin in different citrus species. (B) The
content of six flavonoid compounds in different cultivars.
Anti-inflammation of citrus naringin in vitro and in vivo
Cytokine storm was observed in most severe COVID-19 patients with increased
plasma concentrations of TNF-α, IL-1β, IL-10, and IFNγ. Corticosteroids were used
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
frequently for severe cases of treatments to reduce inflammatory-induced lung injury.
Therefore, treatment with anti-inflammatory approach is critical to alleviating clinical
symptoms related to COVID-19. Lipopolysaccharide (LPS), a bacterial
Gram-negative endotoxin, can induce cytokine storm with the increase of cytokines,
such as IL-1β, TNF-α, IFNγ, IL-6 and MCP-1 (Ramos-Benitez et al., 2018). In
addition, macrophage, one kind of the immune cells, can be caused by LPS to
overzealously product inflammatory cytokines in immune response. The
anti-inflammatory effect of citrus naringin (extracted from Citrus wilsonii Tanaka) on
inflammatory cytokines, COX-2, iNOS, IL-1β and IL-6 were determined at mRNA
levels in LPS-induced RAW 264.7 macrophages shown in Figure 3 A-D. The results
demonstrated that the COX-2,iNOS,IL-1β and IL-6 mRNA expression levels in
LPS-treated macrophage cells were increased compared to the control group.
Application of naringin (10, 20, 40 μg/mL) significantly diminished the effects of
LPS induction of COX-2,iNOS, IL-1β and IL-6 expression. Myocardial
ischemia-reperfusion injury in rats were used to further examine the
anti-inflammatory activity of naringin in vivo. The expression of high mobility group
box 1 (HMGB1) level and the phosphorylation p38mitogen activated protein kinase
(MAPK) level related to inflammation in the myocardium were measured to further
investigate the anti-inflammatory activity of naringin. As shown in Figure 3 F-H, I/R
caused a significant increase in expression levels of HMGB1, and p-p38 proteins
compared to the Sham group. The naringin pretreatment significantly reversed the I/R
effects on the expression of HMGB1 and p-p38 proteins.
Naringin exhibits a potent anti-inflammatory activity in the present and previous
studies (Chtourou et al., 2016). It could inhibit the expression of the proinflammatory
cytokines (COX-2, iNOS, IL-1β and IL-6) induced by LPS in vitro in the present
study. Moreover, HMGB1 is a ubiquitous DNA-binding nuclear protein and can be
released actively by immune cells, such as macrophages and monocytes, following
inflammatory stimulation (Ulloa and Messmer, 2006; Wang et al., 2004). HMGB1
also acts as a pro-inflammatory cytokine and regulates cytokine storm, up-regulating
cytokines such as TNF-α, IL-6, IL-1β, and IL-8 (Huang et al., 2010). It is further
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
demonstrated that naringin could restrain cytokine storm to a certain extent through
inhibiting HMGB1 expression. Additionally, it was found that p38 MAPK has an
important role in HMGB1-mediated production of proinflammatory cytokines (Park
et al., 2003), and previous studies indicated inflammation is related to the
phosphorylation level of p38 MAPK (Kim et al., 2019; Wu et al., 2019). Naringin
pretreatment could attenuate p-p38 MAPK level in this study. These results revealed
that naringin has potential use for preventing cytokine storm of COVID-19.
Figure 3. Anti-inflammation of naringin in vitro and in vivo. (A-D) Effect of naringin on
LPS-induced mRNA expression of COX-2, iNOS, IL-1β, and IL-6 in RAW 264.7 macrophages.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
The concentration of LPS is 1 μg/mL. (A) COX-2 mRNA expression; (B) iNOS mRNA
expression; (C) IL-1β mRNA expression; (D) IL-6 mRNA expression. Different letters show
significant differences (p < 0.05). (E-G) Anti-inflammation effect of naringin on myocardial
ischemia/reperfusion injury in rats. Sham-operated control group (Sham): the rats were treated
with solvent for naringin; Ischemia/reperfusion group (I/R): the rats were treated with solvent for
naringin and subjected ischemia and reperfusion; Naringin + I/R group (Nar-I/R ): the rats were
treated with naringin (4 mg/kg per rat) and subjected ischemia and reperfusion. (E) Representative
images of the Western blot results. (F) Expression levels of HMGB1 protein. (G) Expression
levels of p-p38 protein. * p < 0.05 vs. the Sham group; # p < 0.05 vs. the I/R group.COX-2,
cyclooxygenase-2; HMGB1, high mobility group box 1 protein; IL-1β, interleukin 1β; IL-6,
interleukin 6; iNOS, inducible nitric oxide synthase; I/R, ischemia/reperfusion; LPS,
lipopolysaccharide; Nar, naringin.
Molecular docking result of citrus flavonoids to ACE2 enzyme, a
receptor of the coronavirus
In the last few weeks, rapid progress have been made in identification of viral
etiology, since one genome sequence (WH-Human_1) of the 2019-nCoV released on
Jan 10, 2020. Based on the computer-guided homology modeling method, it is found
that 2019-nCoV S-protein and SARS-CoV S-protein shared an almost identical 3-D
structure in the RBD domain and has a significant binding affinity to human ACE2
(Lu et al., 2020; Xu et al., 2020). ACE2 is widely expressed in the kidney, lung, brain,
digestive tract and is considered to be critical for the coronavirus to enter host cells
(Kuhn et al., 2006; Letko and Munster, 2020). Traditional herbal medicine was
suggested to be promising for inhibiting coronavirus. Citrus showed broad
pharmacological effects, including anti-obesity, anti-oxidant and anti-inflammation
(Feng and Wang, 2018). To investigate whether citrus flavonoid compounds have the
potential to anti-2019-nCoV, we simulated the molecular docking of the six
compounds to predict their capacity for binding ACE2 (Figure 4). The interaction
between citrus flavonoids and ACE2 were evaluated by binding energy. The docking
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
result showed that naringin may have highest binding activity to the ACE2 enzyme
with estimated docking energy of -6.85 kcal/mol, with the potential binding site at
TYR-515, GLU-402, GLU-398, and ASN394 (Figure 4A). Naringenin could bind to
ACE2 with estimated docking energy of -6.05 kcal/mol, with binding site PRO-146,
LEU-143, and LYS-131 (Figure 4B). As shown in Figure 4C, the stimulated result
showed that hesperidin had the potential binding to ACE2 with docking energy of
-4.21kcal/mol, with binding sites ASN-277, ARG-273, and HIS-505. The molecular
docking of hesperetin to the ACE2 enzyme showed that hesperetin had the potential
binding to ACE2 with docking energy of -6.09 kcal/mol, with binding sites LYS-562,
GLU-564, GLY-205 (Figure 4D). Neohesperidin could bind to ACE2, with docking
energy of -3.78 kcal/mol, with binding sites at TRP-349, ALA-348, TRP-69 (Figure
4E). Nobiletin could bind to ACE2 enzyme, with docking energy of -5.42 kcal/mol,
and the potential binding site at TRP-69, LEU-351, ASP-350 (Figure 4F). These
results suggested that among the six citrus flavonoids, the energy required for the
binding between naringin and ACE2 was the lowest, followed by hesperetin,
narigenin, indicating that they were easier to binding ACE2.
In addition, chloroquine and baicalin had been reported as potential inhibitors of
SARS coronavirus infection in vitro test(Chen et al., 2004; Vincent et al., 2005). The
cell surface expression of under-glycosylated ACE2 was inhibited by chloroquine,
resulting in its poor affinity to SARS-CoV spike protein. We used molecular docking
to calculate ACE2 binding energy of chloroquine and baicalin. As shown in Figure 4H,
chloroquine had the potential binding to ACE2 with docking energy of -5.70 kcal/mol,
and binding sites LEU-95, GLN-58, GLN-102. Baicalin could bind to ACE2 emzyme,
with docking energy of -4.70 kcal/mol, with the potential binding site at HIS-374,
HIS-378, and ALA-348 (Figure 4G). The docking energy of naringin, hesperetin and
narigenin binding to ACE2 were comparable with chloroquine. It’s worthwhile to
conduct further experiments to verify whether these citrus flavonoids could target
ACE2 and prevent the infection of 2019-nCoV in cell culture models and laboratory
animals.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
Figure 4. Molecular docking results of naringin (A), naringenin (B), hesperidin (C), hesperetin (D),
neohesperidin (E), nobiletin (F), baicalin (G), and glycyrrhizin (H) to ACE2 enzyme (PDB code:
6ACG). The AutoDock 4.2 software was selected for the docking study using a hybrid
Lamarckian Genetic Algorithm (LGA).
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
Conclusion
We determined the contents of six flavonoid compounds in three citrus species
by using LC-MS technique. The contents of naringin and naringenin were at higher
levels in pummelo. On the other hand, mandarin and sweet oranges had higher
hesperetin and hesperidin contents compared to pummelo. The contents of
neohesperidin and nobiletin were lower than other four compounds in citrus. Our data
also showed that naringin could inhibit the expression of the proinflammatory
cytokines (COX-2, iNOS, IL-1β and IL-6) induced by LPS in vitro. It was further
demonstrated that naringin could restrain cytokine through inhibiting HMGB1
expression in a myocardial ischemic/reperfusion injury model. The results suggested
that naringin could have a potential in preventing cytokine storms of COVID-19. The
molecular docking result predicted that naringin and hesperetin had stronger binding
affinity the ACE2. We suggested that these two phytochemicals, e.g., naringin and
hesperetin are most potential compounds targeting ACE2 receptor, which could
prevent coronavirus infection. Chinese traditional medicine is playing an important
role in the treatment of COVID-19. We should pay more attention to natural
compounds form citrus and other herbal medicine to combat coronavirus in the future.
Funding
The study was supported by the National Key Research and Development
Program (2018YFD1000200), the Ability Establishment of Sustainable Use for
Valuable Chinese Medicine Resources (2060302).
Acknowledgments
We thank Dr. Shan Li, Dr. Guiqing Peng, Dr. Ping Yin for their good advice. We
thank Dr. Dingbo Lin for editing the manuscript. Wuhan fighting.
Conflict of interest
The authors declare no conflict of interest.
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Objective: The industrial production and combustion of coal can produce silica nanoparticles (nano-SiO2). It enters the human body mainly through the respiratory tract and exerts a toxic effect. However, whether nano-SiO2 can increase the IL-1β-induced inflammatory expression in A549 cells has not been tested. Therefore, the synergistic toxicity of nano-SiO2 and IL-1β to A549 was observed in our study. Materials and methods: We exposed A549 cells to nano-SiO2 (0, 100, 500, and 1000 μg/ml) for 12 and 24 h. The effect of nano-SiO2 on the viability of A549 cells was observed by the CCK-8 method. The A549 cells were exposed to nano-SiO2 (1 mg/mL) and cytokine IL-1β (10 ng/mL) for 4 h, and we detected the expression of IL-1β and IL-6 cytokines by real time quantitative polymerase chain (RT-qPCR) and enzyme linked immunosorbent assay (ELISA). The expression of β-Actin, I-κB, phospho-ERK1/2 (P-ERK1/2), total-ERK1/2 (T-ERK1/2), phospho-JNK (P-JNK), total-JNK (T-JNK), phospho-P38 (P-P38), and total-P38 (T-P38) in A549 cells was detected by the Western Blot method. Results: The nano-SiO2 treatment resulted in a time-dependent decrease in the viability of A549 cells. The synergistic effect of nano-SiO2 and IL-1β was observed on the new production of IL-1β and IL-6 in A549 cells. The Western blot results showed that nano-SiO2 can increase the expression of IL-1β and IL-6 by promoting the phosphorylation of ERK1/2 and elevating the phosphorylation of I-κB by IL-1β. IL-1β and IL-6 were induced by nano-SiO2, and the IL-1β treatment with 20 μM of I-κBα phosphorylation inhibitor (PD98059) and 20 μM of ERK1/2 inhibitor (BAY11-7082) for 1 h was significantly lower than that of the control group in A549 cells. Discussion and conclusion: These results indicated that nano-SiO2 had a toxic effect on A549 cells, and this effect could increase IL-1β on the A549 cell-induced inflammatory response. The results suggested that the release of IL-1β and IL-6 in A549 was enhanced by the synergistic IL-1β-induced phosphorylation of ERK1/2 and I-κB. This process is similar to a snowball, and it is possible that IL-1β is continuously produced and repeatedly superimposed in A549 cells to produce an inflammatory effect; then, a vicious circle occurs, and an inflammatory storm is accelerated.
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