The Effects of Chinese Medicine Lianhua Qingwen on Inhibiting the

Replication of Covid-19 Virus

Hongzhu Ji

Fordham University FCLC, New York, U.S.A.

Keywords: Chinese Medicine, Lianhua Qingwen, Covid-19, Virus Replication.

Abstract: Lianhua Qingwen, a commonly used traditional Chinese medicine, has been used to treat fever and respiratory

infections for many years including SARS. Recent studies have shown that it can also be used as a COVID-

19 treatment. The goal of this work is to test if Lianhua Qingwen fights against coronavirus by reducing virus

replication? The antiviral activity of Lianhua Qingwen against SARS-CoV-2 was assessed in infected cells

from patients using CPE assay and plaque reduction assay. There are six possible results: they are based on if

Lianhua Qingwen shows positive, no, or negative inhibition on the CPE inhibition assay and weather it gives

us an inhibitory effect on the formation of plaque of the virus or not, and we will say Lianhua Qingwen

significantly inhibited SARS-CoV-2 replication in cells which partially supports our hypothesis, or it partly

supports our hypothesis and these results wouldn't give a clear answer to our research question, or it

contradicts our hypothesis. The result of our study will provide important information for how Lianhua

Qingwen helps to treat COVID-19, and it will help us with controlling the COVID-19 pandemic. Future

studies should focus on more potential effects of Lianhua Qingwen and vivo experiment is needed.

1 INTRODUCTION

COVID-19 is a highly contagious respiratory

infectious disease caused by severe acute respiratory

syndrome coronavirus‐2 (SARS‐CoV‐2)(Xia et

al. 2020). It's an animal transmission disease that is

likely originated from bats and spread dramatically in

humans, and now it has widely spread around the

world. By now there are more than 115 million cases

and 2.55 million deaths. This COVID-19 pandemic

not only severely damaged people's life and health,

but also harmed social, economic, and political. The

symptoms of it are fever, dyspnea, nasal congestion,

sore throat, asthenia, dry cough, multiple organ

dysfunction, and death. China has successfully

controlled the pandemic with its strict policy and

traditional Chinese medicine including Lianhua

Qingwen.

Additionally, by analyzing COVID-19 infected

patients’ clinical records from Wuhan Ninth Hospital

and CR & WISCO General Hospital, researchers

found the Lianhua Qingwen combination could allay

fundamental symptoms significantly and shorten the

progress and development of it (Li et al. 2020).

Lianhua Qingwen, the classic traditional Chinese

Medicine, is still commonly used today as a treatment

for inflammatory fever and respiratory infectious

diseases. It was used to fight against SARS in China

from 2002 to 2003, and it was included in the

Guideline for the Diagnosis and Treatment of Novel

Coronavirus (2019-nCoV) Pneumonia (On Trials, the

Fourth/Fifth/ Sixth/Seventh Edition) issued by the

Chinese government. Lianhua Qingwen consists a

total of 13 different herbs: Lian Qiao, 255 g; Ma

Huang, 85 g; Jin Yin Hua, 255 g; Ban Lan Gen, 255

g; Mianma Guanzhong, 255 g; Bo He, 7.5 g; Shi Gao,

255 g; Guang Huo Xiang, 85 g; Hong Jing Tian, 85

g; Yu Xing Cao, 255 g; Da Huang, 51 g; Ku Xing Ren,

85 g; and Gan Cao, 85 g (Hu 2020). Previous studies

had shown using conventional drugs with the addition

of Lianhua Qingwen is a treatment with bright

potential for both pneumonia and the COVID-19

virus, and by using Lianhua Qingwen the healing

period of COVID-19 is shortened and several

symptoms like fever, cough, fatigue, sputum, muscle

aches, difficulty breathing, chest tightness and

pulmonary imaging can be improved (Hu et al. 2020).

These studies have shown that Lianhua Qingwen

relieved symptoms of the COVID-19 remarkably, but

research about the fundamental mechanism is

elusive.

284

Ji, H.

The Effects of Chinese Medicine Lianhua Qingwen on Inhibiting the Replication of Covid-19 Virus.

DOI: 10.5220/0011292900003438

In Proceedings of the 1st International Conference on Health Big Data and Intelligent Healthcare (ICHIH 2022), pages 284-288

ISBN: 978-989-758-596-8

Therefore, my research question is: Is the use of

Lianhua Qingwen block the replication of the

COVID-19 virus? And my hypothesis is I predict that

Chinese Medicine Lianhua Qingwen helps to block

replication of COVID-19. I will treat lung cells from

COVID infected human patients with an increasing

amount of Lianhua Qingwen (0mg/mL which is the

buffer solution, 0.1 mg/mL, 0.3 mg/mL, 0.5 mg/mL,

0.7 mg/mL, and 1 mg/mL) for the various durations

(12h, one day, three days, and seven days) and

measure it by CPE inhibition assay and plaque assay

with 10 replicates.

2 METHOD

2.1 Materials

This experiment will use COVID-19 infected cells

(experiment cells) from patients, and the growth of

cells was supported at 37 °C by Dulbecco’s Modified

Eagle’s medium (DMEM) along with fetal bovine

serum at the concentration of 10 %. The infection

experiments were conducted in a biosafety level-3

laboratory for safety concerns. Lianhua Qingwen

capsules were obtained and the black powder of it

was removed from the capsules. The black powders

were dissolved in dimethyl sulfoxide first to 200

mg/mL. Next, at room temperature mix the solution

by shake or stir it for 30 minutes, and prepare the

stock solution by diluting the Lianhua Qingwen

solution with serum-free DMEM to the concentration

of 0.1 mg/mL, 0.3 mg/mL, 0.5 mg/mL, 0.7 mg/mL

and 1 mg/mL, and at the temperature of −20 °C

reserve it. Additionally, preparing Remdesivir

solution with a similar method by dissolved in

dimethyl sulfoxide and mix to the concentration of

100 mM and stored at −20 °C with the Lianhua

Qingwen solution, and the dilution buffer that used in

later experiments is prepared by DMEM with 2%

fetal bovine serum. Materials like 96-well plates,

0.6 % agar, timer, thermometer, and microscope are

also needed in the following experiment.

2.2 Cytopathic Effect (CPE) Inhibition

Assay

The cytopathic effect is observable abnormalities

such as structural changes in host cells that are caused

by the viral invasion, and different viruses infect

different types of cells will cause different cytopathic

effects. CPEs are important aspects of a viral

infection in diagnostics. The CPE assay is used to test

if a compound exhibits any antiviral efficacy or not.

CPE assay is a cost-effective and time-efficient assay

that we used to evaluate test articles' ability to inhibit

CPE. Many combinations of cells and viruses can be

used to measure interferon activity via CPE assay.

And by using the dose-response assay we are able to

tell the selected antiviral efficacy range (Britannica

2019).Put experiment cells in monolayers inside

separate 96-well plates. To explore the effectiveness

against experiment virus of Lianhua Qingwen, in the

experiment group, the infected cells were incubated

with Lianhua Qingwen solution at various

concentrations for different durations (12h, one day,

three days, and seven days). Then, the positive

control group was developed at the different

concentrations of Remdesivir solution mentioned

before. As for the negative control group, grow the

experiment cells with indicated concentrations of

solely buffer solution (which contains 0 mg/mL of

Lianhua Qingwen). After various times of incubation

in these different solutions, the infected experiment

cells will show full CPE under the microscope

(100%). Next, the CPE at different percentages in all

three groups with different solutions treated cells

were recorded. Also, 10 replicates will be done for

this experiment, and by using the Reed-Muench

method we will be able to calculate IC50 which is the

half virus-induced CPE of inhibition concentration

for all the experiments and the calculated IC50 will

be compared later (Li et al. 2020).

2.3 Plaque Reduction Assay

Plaque assay is a way of measuring virus quantity that

is used to test the concentration of virus’s dose of

infection, and it specifies the quantity of appeared

units of plaque in the test sample of viruses. A

monolayer of infected cells at different

concentrations and covered with a semi-solid

medium (we will use agar in this experiment), to

forestall the infection of virus from spreading

randomly. When a certain virus passes infection to a

cell monolayer the infected cell area will create a

plaque. The culture will then be stained with a dye,

which stains only viable cells. We will use 1% crystal

violet in this experiment. As a result, the plaque (the

dead cells) will appear unstained against the colored

background for researchers to count (Kaufmann,

Kabelitz 2002). The SARS-CoV-2 infected cells were

put in monolayers and were put in 96-well plates

separately. And for all three groups, the plates were

all covered with 0.6 % agar, 2% fetal bovine serum.

Next, for each of the individual groups, the plate was

covered with the different concentrations of either

The Effects of Chinese Medicine Lianhua Qingwen on Inhibiting the Replication of Covid-19 Virus

285

Lianhua Qingwen (0.1 mg/mL, 0.3 mg/mL, 0.5

mg/mL, 0.7 mg/mL, and 1 mg/mL), Remdesivir, or

buffer (0.6 % agar, 2% fetal bovine serum together

with either one of these three are all considered as

agar overlays). Then incubated all plates with 5%

CO2 at 37 °C for different durations (12h, one day,

three days, and seven days). Following, removed the

agar overlays for each plate. And, fixed the cell

monolayer with 10 % formalin, stained with 1%

crystal violet. This experiment will be done with 10

replicates. Lastly, the plaques for all groups were

counted carefully and photographed (Li et al. 2020).

2.4 Statistical Analysis

The statistical significance of all numerical data

gathered through the Reed-Muench method will be

analyzed using the student’s T-Test on GraphPad

Prism® at (p <0.05).

3 RESULT

SARS-CoV-2 infected cells from patients were

supported by DMEM as specified in the method part.

To conduct an inquiry into the effectiveness of

Lianhua Qingwen against the virus, the cells were

incubated with Lianhua Qingwen at different

concentrations for about 72 h. And the positive and

negative control groups followed a similar method

with Remdesivir which is the positive control group

and buffer solution (DMEM with 2% fetal bovine

serum) as the negative control. Calculating the IC50

number for the virus-induced CPE of these three

experiments by the Reed-Muench method compared

the results to test our hypothesis. For the Cytopathic

effect inhibition assay, the positive control

experiment with Remdesivir would show positive

inhibition and the negative control experiment with

the buffer (0% of Lianhua Qingwen solution) would

show negative inhibition. Lianhua Qingwen could

show positive inhibition, no inhibition, or negative

inhibition. If the experiment with Lianhua Qingwen

solution shows positive inhibition or no inhibition,

we will conduct that Lianhua Qingwen inhibited the

tested virus replication in experiment cells (because

the negative control will give negative results of virus

inhibition) which agrees with our hypothesis.

Lianhua Qingwen inhibits SARS-CoV-2 replication

significantly in infected cells will be represented as a

"+" sign in Table#1. For the Plaque reduction assay,

after counting the plaques for all groups Remdesivir

would show less plaque which indicates positive

inhibition and the negative control experiment with

the buffer would show more plaque which shows

negative inhibition. And if Lianhua Qingwen inhibit

or reduced effects of the plaque appearing of the

virus, it will be represented as a "+" sign in Table#1,

and it agrees with our hypothesis.

There are six possible results: (1). If Lianhua

Qingwen shows positive inhibition on the CPE

inhibition assay and it prevents the plaque formation

of the virus, we will say Lianhua Qingwen

appreciably prevent the COVID-19 virus replication

in experiment cells which agree with our hypothesis.

(2). If Lianhua Qingwen shows no inhibition on the

CPE inhibition assay and it gives us the same effect

as the first result, and we also say the replication of

the virus in cells was invited by Lianhua Qingwen

remarkably which agree with our hypothesis. (3). If

Lianhua Qingwen shows negative inhibition on the

CPE inhibition assay and inhibitory effect was given

to us on the formation of plaque in cells, the results

of the CPE and plaque reduction assay contradicted

each other, which partly proves our hypothesis and

these results wouldn't give a clear answer to our

research question. (4). If Lianhua Qingwen shows

positive inhibition on the CPE inhibition assay and it

gives us no inhibitory effect on plaque formation of

the SARS-CoV-2 virus, we will the results of the CPE

and plaque reduction assay contradicted each other,

which partly proves our hypothesis and these results

wouldn't give a clear answer to our research question.

(5). If Lianhua Qingwen shows no inhibition on the

CPE inhibition assay and it shows no inhibitory effect

on plaque formation of the SARS-CoV-2 virus, we

say the results of the CPE and plaque reduction assay

contradicted each other, which partly supports our

hypothesis and these results wouldn't give a clear

answer to our research questions. (6). If Lianhua

Qingwen shows negative inhibition on the CPE

inhibition assay and it gives us no inhibitory effect on

plaque development of the virus, which means

Lianhua Qingwen did not significantly inhibit the

tested virus replication in cells and it disagrees with

our hypothesis.

4 DISCUSSION

Previous studies had shown Lianhua Qingwen

together with drugs that are currently used is a

potential bright treatment for COVID-19, and by

using it the healing period of COVID-19 is shortened

and improves several symptoms. Research has also

shown Lianhua Qingwen inhibits virus replication in

both Vero E6 and Huh-7 cells, and it has a dose-

dependent effect of inhibition on the formation of

ICHIH 2022 - International Conference on Health Big Data and Intelligent Healthcare

286

plaque. This study will use infected lung cells from

patients to verify if Lianhua Qingwen fights against

coronavirus by reducing virus replication. Our

hypothesis is consistent with previous studies and it

predicts that Lianhua Qingwen helps to block

replication of COVID-19.

Table 1: All Possible Results.

Antiviral activity of Lianhua

Qingwen on SARS-CoV-2 in

vitro

Result 1 Result 2 Result 3 Result 4 Result 5 Result 6

Cytopathic effect (CPE)

inhibition assay

positive

inhibition

(

)

inhibition

(

)

negative

inhibition

(

)

positive

inhibition

(

)

inhibition

(

)

negative

inhibition

(

)

Plaque reduction assay

positive

reduction

(+)

positive

reduction

(+)

positive

reduction

(+)

reduction

(-)

reduction

(-)

reduction

(-)

Agree or disagree with the

hypothesis

agree agree

partly

agree

partly

agree

partly

agree

disagree

Possible results one and two are consistent with

previous studies investigating Lianhua Qingwen, and

they are most likely to happen. Both of these results

show inhibition on the CPE inhibition assay and it

gives us an negative growth effect on plaque

formation of the virus, and they agree with our

hypothesis. The difference between these two results

is result 1 shows a stronger ability of the inhibition on

coronavirus. In result 2, Lianhua Qingwen shows no

inhibition on the CPE inhibition assay but it gives us

an inhibitory effect on plaque formation. This result

is still significant because showing no inhibition still

means Lianhua Qingwen succeeded in slowing or

even stopping the virus replication. This is essential

in controlling the pandemic and it could help save

thousands of lives. Based on previous research,

between these two results, result 1 is even more likely

to happen. These results show how Lianhua Qingwen

helps to treat COVID-19 by inhibiting the rapid virus

replication, and future studies should focus on more

potential effects of Lianhua Qingwen and vivo

experiment is needed. Research could also be done

about the differences between recovered cells that

were treated with different drugs (for example

shapes, size, viability, etc.). Also, even these results

prove Lianhua Qingwen inhibits virus replication,

studies could also find other possible mechanisms

that Lianhua Qingwen may use to fight the COVID-

19 virus.

Possible results three, four, and five partly prove

our hypothesis and these results are controversial and

wouldn't give a clear answer to our research question.

These results show contradictions between the CPE

inhibition assay and the plaque reduction assay, and

if there are no errors in operation that caused these

contradictions we should design more experiments on

the effects of Lianhua Qingwen on virus replication.

Future studies should focus on why these two assays

give different results and find more ways to test the

inhibition effects of Lianhua Qingwen on virus

replication. Also, studies that find other possible

ways that Lianhua Qingwen could use to fight the

COVID-19 virus.

The possible results six contradicts with the

current understanding of Lianhua Qingwen's effects

on the SARS-CoV-2 virus, this result indicates that

Lianhua Qingwen did not inhibit SARS-CoV-2

replication in cells dramatically and it disagrees with

our hypothesis. This result is unlikely to happen since

there has already been much research that proves

relief of clinical symptoms of coronavirus and the

inhibition effect that Lianhua Qingwen has on the

virus. However, since the mechanism of antiviral

effects on coronavirus is new and hard to find, there

could be other ways Lianhua Qingwen helps treat

coronavirus. Future studies should review the

existing researches that are not consistent with this

result and focus on finding other mechanisms that

Lianhua Qingwen could help fighting coronavirus.

5 CONCLUSIONS

This study explores the effect of Lianhua Qingwen in

The Effects of Chinese Medicine Lianhua Qingwen on Inhibiting the Replication of Covid-19 Virus

287

the inhibition of the COVID-19 virus. The result of

our study will indicate whether or not Lianhua

Qingwen fights against coronavirus by reducing virus

replication. The possible results that are likely to

happen prove our hypothesis that Lianhua Qingwen

inhibits virus replication which supports that

combines Lianhua Qingwen with existing therapies

to help to treat COVID-19 in clinical. The possible

controversial results on the infected lung cells from

patients will also indicate the inhibition effects of

Lianhua Qingwen on coronavirus, and we need to

find out what caused these two assays to give us

contradicted results. Future studies need to be

investigated in clearer details and find other

mechanisms that Lianhua Qingwen could help

fighting coronavirus and trying to validate this further

by conducting vivo experiments.

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Compared with Conventional Drugs in the Treatment

of COMMON Pneumonia and COVID-19

PNEUMONIA: A Meta-Analysis.

www.hindawi.com/journals/ecam/2020/5157089/.

Kaufmann, S.H.; Kabelitz, D. (2002). Methods in

Microbiology, Immunology of Infection. Academic

Press., ISBN 0-12-521532-0.

Li, Runfeng, et al. (2020) “Lianhuaqingwen Exerts Anti-

Viral and Anti-Inflammatory Activity against Novel

Coronavirus (SARS-CoV-2).” Pharmacological

Research, Published by Elsevier Ltd.,

www.ncbi.nlm.nih.gov/pmc/articles/PMC7102548/.

Xia, Qi‐Dong, et al. (2020) “Network Pharmacology and

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Capsule Indicate Akt1 Is a Potential Target to Treat and

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