The effect of Psidium guajava Leaves’ extract for mild and asymptomatic corona virus Disease-19

COVID-19 is an emerging disease that attacks the respiratory and systemic systems. Various treatments have been used for COVID-19, but no antiviral agents seemed to be efficacious. Many medicinal plants are commonly used for viral infections in Indonesia, including the guajava leaf. The study aimed to determine the effects of Psidium guajava extract supplementation on inflammation markers of asymptomatic and mild COVID-19 patients. The conversion time of PCR results was also evaluated. This study was an experimental, single-blinded, randomized clinical trial (ClinicalTrials.gov: NCT04810728) comparing the efficacy of P. guajava extract at the dose of 1000 mg/8h on top of standard treatment with the standard treatment only for asymptomatic and mild COVID-19 subjects. The primary endpoints were neutrophil and lymphocyte percentages as well as the neutrophil/lymphocyte ratio (NLR) on day 7 of the treatment. The secondary endpoints were high-sensitivity c-reactive protein (hs-CRP) level, PCR-based conversion time, and recovery rate at weeks 2 and 4. A total of 90 subjects were enrolled, and there were 40 subjects in the P. guajava (experimental) group and 41 subjects in the control group who completed the research. On day 7, significantly lower percentage of neutrophil (52.4% vs 58.9%, p = 0.002), higher lymphocyte percentage (35.5%, vs 29.7%, p = 0.002), and lower NLR (1.5 vs 2.1, p = 0.001) were demonstrated in the experimental versus control group. The PCR-based conversion time was shorter (14 days vs 16 days, p < 0.001), and recovery rate at 2 and 4 weeks were higher (49% vs 27%, p = 0.03 and 100% vs 82%, p = 0.003, respectively) in the experimental group. There were no differences in the baseline characteristics. P. guajava extract supplementation reduced neutrophil and increased lymphocyte percentages which led to reduced NLR, accelerated PCR-based conversion time, and increased recovery rate in subjects with mild and asymptomatic COVID-19 infection.\


Introduction
At the end of 2019, the world was shocked by the emergence of a new virus that attacks the respiratory system. The virus originating from Wuhan City in the Hubei Province of China is an RNA virus from the genus Coronavirus and the family Coronaviridae. World Health Organization has named this virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and named the disease Coronavirus disease-19 (COVID-19) (WHO, 2020).
COVID-19 patients have a variety of clinical variations, ranging from asymptomatic, mild, and moderate to severe symptoms. Various treatments have been used for COVID-19, but no antiviral agents seemed to be efficacious (Cao et al., 2020;Borba et al., 2020). Several traditional alternative medicines had been investigated. The empirical study in Wuhan reported the use of traditional Chinese medicine in cases of pneumonia COVID-19. The use of this traditional medicine increased the patient's leucocyte, lymphocyte, and neutrophil levels within the normal range. The composition of the drug consists of saponin, oleanolic acid, xylopyranoside, flavonoid, quercetin, arabinopyranoside dan guaijavarin, and others .
In for various diseases, including viral infections. A standardized extract derived from Psidium guajava, which is known as a guava leaf extract, contains chemical substances, such as saponin, oleanolic acid, xylopyranoside, flavonoid, quercetin, arabinopyranoside dan guaijavarin (Khamath et al., 2008;Naseer et al., 2018). An in vitro study of dengue virus type 2 found that quercetin significantly inhibited the activity of the DEN-2 virus, while other flavonoids showed weaker activity (Zandi et al., 2011).
Several studies show the benefits of guava leaf to inhibit the viral activity. Ortega et al. found the benefits of flavonoid Psidium guineense, guava leaf from Brazil, in inhibiting HIV-1 viral activity (Ortega et al., 2017). Pamungkas et al. in their literature study found that quercetin in Psidium guava inhibits RNA polymerase, which is important in dengue virus replication. In addition, quercetin can inhibit the NS2B-NS3 protease enzyme, helicase domain, and the viral ATPase enzyme (Pamungkas et al., 2020). Research shows quercetin inhibits the production of reactive oxygen species and the secretion of interleukin 6 in oxLDL-stimulated macrophages. Quercetin also inhibits the production of interleukin 1b, (Lara-Guzman et al., 2012) a proinflammatory cytokine. This antiviral and anti-inflammatory effect is expected to improve the condition that occurs in patients with Covid-19 infection. There were antiviral effects of Psidium guajava based on limited in vitro and literature studies, but whether it is effective against COVID-19 has not been known. The single-blinded, randomized clinical trial was conducted to determine the effects of Psidium guajava extract supplementation on inflammation markers of asymptomatic and mild COVID-19 patients, which is a source of high virus transmission to people around them.

Study design and subjects
This study was an experimental, single-blinded, randomized clinical trial registered on ClinicalTrials.gov with the number NCT04810728. Subjects were divided into two groups, the experimental group and the control group according to the randomization prepared by a biostatistician. Researchers did not know subject allocation because drug administration and blood sampling were performed by third parties. Subjects were recruited from the quarantine locations belonging to West Sumatera Provincial Government. Subjects aged from 18 to 59 years old with asymptomatic and mild symptoms were recruited for this study. All subjects were showing positive RT-PCR SARS-Cov-2 results at the time of recruitment and had no comorbidity. The mild symptoms patients have criteria according to the COVID-19 management guidelines issued by the Indonesian Ministry of Health: patients without evidence of viral pneumonia or hypoxia. Patients also have symptoms such as fever, cough, fatigue, anorexia, shortness of breath, and myalgia. Other non-specific symptoms include sore throat, nasal congestion, headache, diarrhea, nausea and vomiting, loss of smell (anosmia), or loss of taste (ageusia). This study used primary data and applied an intervention to research subjects. Therefore, the informed consent process was made very clear. The study was conducted following the ICH-GCP standard procedure and the study protocol was approved by the Ethics Committee of the Faculty of Medicine, Universitas Andalas (No. 283/KEP/ FK/2020), before study commencement.

Data collection procedure
Subjects in the experimental group received standard treatment and Psidium guajava extract (Psidii Ò ) at the daily dose of 1000 mg/8h on top of standard treatment for 7 days, while the control group received standard treatment only. Psidii Ò produced by Dexa Laboratories of Biomolecular Science, has been established and has a distribution permit from the National Agency of Drug and Food Control of Indonesia. The standard treatment received by all subject were vitamin C, vitamin D, and paracetamol if needed. It treatment based on COVID-19 management guidelines issued by the Indonesian Ministry of Health. Subjects were followed up for 4 weeks. The primary endpoints were neutrophil and lymphocyte percentages as well as the neutrophil/lymphocyte ratio (NLR) on day 7 of the treatment. The secondary endpoints were the high-sensitivity c-reactive protein (hs-CRP) level on day 7, the PCR-based conversion time, and recovery rate on weeks 2 and 4. Recovery was defined if the subject's two consecutive RT-PCR-swab tests over 24 h were both negative. Compliance was determined by counting the left-over study capsules. Any adverse events according to the patient's report were noted.

The measurement of leucocyte and differential blood count
A total of 3 ml of blood sample was taken from each subject. The measurement of leucocyte and differential blood count (neutrophil, lymphocyte, eosinophil, monocyte, and basophil) were measured by Sysmex 800i. Whereas, the hs-CRP level was assessed by immunoturbidimetry (Architect c8000) at baseline and after 7 days of treatment.

Statistical analysis
Statistical analysis with SPSS 25 version was performed using a computer program. Continuous variable values are expressed by means and standard deviations (SD) if normally distributed, and median (min-max) if unevenly distributed. Statistical comparisons between pre-and post-therapy were made using paired t-test, while between-group comparisons used an independent t-test or Mann-Whitney test. The Chi-square test was used for analyzing the proportion of the recovery. Statistical significance was defined at a p-value < 0.05.

Results
The results showed that there were no differences in the initial characteristics of the experimental group receiving P. guajava and the control group (Table 1). A total of 90 subjects (45 in each group) were enrolled, but there were 40 subjects in the P. guajava group and 41 subjects in the control group who finally completed this research, had post-treatment values, and were included in the analysis. The flow of subject disposition is described in a flowchart (Fig. 1).
There was a significant decrease in the percentage of neutrophils and the NLR and a significant increase in the lymphocyte in the experimental group on the seventh day of administration of P. guajava extract; however, no significant changes were made in the parameters of the number of leucocytes, monocytes, and hs-CRP. In the control group, no significant differences were obtained on the seventh day on all parameters assessed, both the number of leucocytes, neutrophils, lymphocytes, monocytes, NLR, and hs-CRP (Table 2).
There were significant differences in the length of conversion based on PCR examination between the treatment group and the control group. The treatment group had a shorter recovery duration than the control group. Likewise with the proportion of recovery after 2 and 4 weeks, there was a greater proportion of recovery in the P. guajava group (Table 3). During the implementation of the study, no side effects were found in the administration of P. guajava in all subjects who got it.

Discussion
The administration of Psidium guajava leaf extract resulted in significant changes in the number of leucocytes after therapy in the treatment group compared to the control group. At the beginning of the study, there were no significant differences between the P. guajava group and the control group, including the number of leucocytes and their differential count, NLR, and hs-CRP. Although the total number of leukocytes on day 0 and day 7 was within the normal limit, the differential count after day 7 changed, such as the percentage of neutrophils and lymphocytes. Neutrophils increased while lymphocytes decreased so the ratio of lymphocyte neutrophils decreased. It is known that the ratio of neutrophils and lymphocytes is one of the inflammatory markers. The decrease in NLR indicates an improvement in inflammatory conditions in COVID-19 patients in this study.
P. guajava has been proven to have potential benefits as an antiinflammatory product (Sriwilaijaroen et al., 2012) Sudira et al. (2019) found that P. guajava leaf extract contains several active substances that act as an anti-inflammatory, such as flavonoids, phenols, tannins, and terpenoids. In this study, P. guajava extract,  which contains total flavonoids ! 0.2%, was administered at a daily dose of P. guajava 1000 mg/8h (Jusuf, 2020). Study by Savov et al. using 2 Â 20 mg/kg BW quercetin showed a decrease in lipid peroxidation (LPO) by 25%. The LPO is an oxidant that plays a role in the incidence of inflammation (Savov et al., 2006). Study by Weni et al. in rats obtained an anti-inflammatory effect of P. guajava leaf extract at a dose of 0.125 g/kg BW/5h. The maximum antiinflammatory effect was obtained at a dose of 0.25 g/kg BW/5h. The anti-inflammatory effect at this dose was better than indometacin 10 mg/kg BW. There were no side effects found in experimental animals at higher doses (Weni et al.. 2011). In this study, there was a significant decrease in the neutrophil count after treatment. The SARS-Cov-2 virus infection causes a massive increase in neutrophils that cannot be fully explained. However, there is evidence that neutrophils play a role in increasing the body's antiviral defenses through interactions with other immune cells, viral internalization, viral destruction, cytokine release, oxidation bursts, and neutrophil extracellular traps (Cavalcante-Silva et al. 2021). Research shows that P. guajava leaf extract can reduce neutrophils by decreasing pro-oxidants (Savoy et al., 2006). Flavonoids in P. guajava can reduce inflammatory markers of IL-6, TNF-a, neutrophils, and interferon-ɣ at doses of 0.2-1.2 g/day. Flavonoid supplementation reduced the incidence Protein P53 is known to increase the secretion of proinflammatory IL-1b and IL-8. The IL-8 is known as a cytokine that plays a role in increasing the neutrophil count (Khalil et al., 2017).
This study also found an increase in the lymphocyte count by administration of P. guajava leaf extract. Methanol extract of P. guajava leaves in small doses (100 mg/kg BW), moderate doses (150 mg/kg BW), and high doses (200 mg/kg BW) can increase the number of peripheral lymphocytes in rats. Administration of P. guajava leaf extract showed an immunomodulatory effect and can prevent mortality after myelosuppressive administration at moderate and high doses (Shabbir et al., 2016). Research in mice showed guajava leaf extract has the potential to reduce the expression of proinflammatory mediators lipopolysaccharide-inducible nitric oxide synthase and cyclooxygenase-2 proteins levels through down-regulation of nuclear factor-transcription activity jB (NF-jB). The P. guajava leaves can also be used as an immunity stimulant because they modulate the lymphocyte proliferation response (Diaz-de-Cerio et al., 2017). In COVID-19 patients, there is a decrease in lymphocyte count, which is predicted to be caused by the inadequate formation of interferon I and III and increased regulation of cytokines and chemokines. Chemokines CXCL-9 and CXCL-10 are leukocyte chemoattractants that decrease peripheral lymphocytes. It is not yet known the effect of P. guajava leaf extract, flavonoids, or the active substance quercetin directly against the chemokine inhibition of CXCL-9 and CXCL-10, but decreased cytokines play a role in the induction of T helper 1 cells (Cavalcante-Silva et al., 2021). The silico studies show that flavonoids can bind to the spike protein, helicase, and protease at the ACE2 receptor with high affinity for cells getting infected with the SARS-Cov-2 virus (Ngwa et al., 2020).
Research shows flavonoids have antioxidant, antiinflammatory, antimutagenic, and anti-carcinogenic properties (Panche et al., 2016). Flavonoids also have an inhibitory effect against the enzymes xanthine oxidase, cyclo-oxygenase (COX), lipoxygenase, and phosphoinositide-3-kinase. The molecular docking technique shows that flavonoid derivatives bind to the influenza virus neuraminidase hemagglutinin 1 neuraminidase (H1N1) and can inhibit the viral activity (Lu et al., 2012). Research shows the antiviral effect of flavonoids with various degrees of antiviral activity, both against RNA (RSV, Pf-3, polio) and DNA (HSV-1) (Kaul et al., 1985). Quercetin has an antibacterial activity effect (Cushnie and Lamb, 2011). Various in silico analyzes have been carried out to determine the benefits of flavonoids. Da  examined the effect of flavonoids on SARS-CoV-2 non-structural proteins, namely 3-chymotrypsin-like cysteine protease (3Clpro) and RNA-dependent RNA polymerase (RdRp). The research found flavonoid glycosides and their metabolites have a role as inhibitors of these two proteins. Herbacetin flavonols, quercetin, and isobavachalcone show the effect of antiviruses on SARS-CoV-2 and other coronaviruses (Solnier and Fladere, 2020). 28 The administration of P. guajava extract in this study also had benefits for accelerating the conversion of PCR-swab test for SARS-COV-2 from positive to negative and increasing the 2-week and 4-week recovery rates. The anti-inflammatory effect of P. guajava is predicted to play a role in shortening the conversion time, in addition to its stimulating effect on the immune system. 21 Further, the addition of P. guajava extract to the standard of care was safe in subjects with mild and asymptomatic COVID-19. This study has the limitation of a small sample size.

Conclusions
In conclusion, the favorable outcomes of this study indicated that P. guajava extract at the daily dose of 1000 mg/8h for 7 days has an effect on inflammation markers and conversion of RT-PCR results in mild and asymptomatic patients. P. guajava extract as a supplement might improve the inflammatory condition of patients with mild and asymptomatic COVID-19 infection by reducing neutrophil count and the NLR. The PCR-based conversion time was shorter and the recovery rate at 2 and 4 weeks was higher in subjects receiving P. guajava extract. The P. guajava extract along with the standard of care was safe for patients with mild and asymptomatic COVID-19. Further research with larger sample size, higher degree of disease, and using various inflammatory markers is needed to confirm the promising results of the study.

Data availability
The data used to support the findings of this study are available from the corresponding author upon request.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.