key: cord-0921403-b1luvx38
authors: Varnasseri, Mehran; Siahpoush, Amir; Nejad, Khojasteh Hoseiny; Amini, Fatemeh; Karamian, Masoumeh; Yad, Mohammad Jafar Yad; Cheraghian, Bahman; Khosravi, Azar Dokht
title: The Effects of Add-on Therapy of Phyllanthus Emblica (Amla) on Laboratory Confirmed COVID-19 Cases: A Randomized, Double-blind, Controlled Trial
date: 2022-01-29
journal: Complement Ther Med
DOI: 10.1016/j.ctim.2022.102808
sha: f98fcc13bf5a7e9346761af6fc908d594b63ebee
doc_id: 921403
cord_uid: b1luvx38

OBJECTIVE: This randomized, double-blind, controlled trial (RCT) aimed to evaluate the effect of Phyllanthus Emblica (Amla) as an add-on therapy on COVID-19_ related biomarkers and clinical outcomes in COVID-19 patients. METHODS: In this RCT, sixty-one patients were randomly assigned into two arms [the intervention (n=31) and control arms (n=30)]. The effect of Amla on diagnostic Reverse-transcription Polymerase Chain Reaction (RT-PCR) test results between the first and the last days of the study, the length of stay (LOS) in hospital, the percentage of lung involvement on CT scans, changes in the clinical symptoms, and the laboratory markers were assessed. RESULTS: The two study groups had similar baseline demographics and characteristics in terms of medical history. The mean of LOS in the intervention arm (4.44 days) was significantly shorter than in the control arm (7.18 days, P<0.001); RT-PCR results were not significantly different between the two arms (P=0.07). All clinical variables decreased over time in the two groups (P<0.001). However, the difference between the two groups in terms of fever (P=0.004), severity of cough (P=0.001), shortness of breath (P=0.004), and myalgia (P=0.005) were significant, but this intergroup comparison was not significant with regard to respiratory rate (P=0.29), severity of chills (P=0.06), sore throat (P=0.22), and weakness (P=0.12). Out of the eight evaluated para-clinical variables, three variables showed significant improvement in the intervention arm, including the mean increase in oxygen saturation (SpO2) level (P<0.001), the reduction in the mean percentage of lung involvement on CT (P<0.001), and the improvement in C-reactive protein test results (P<0.001). CONCLUSION: Organic herbal Amla tea cannot significantly affect the RT-PCR results and or degree of lung involvement. Nevertheless, it showed an ameliorative effect on the severity of clinical signs and CRP levels. Also, Amla tea may shorten the recovery times of symptoms and LOS in COVID-19 patients.

The progression of the coronavirus (COVID-19) pandemic caused numerous difficulties for people worldwide. Although some eligible treatment options identified against COVID-19 such J o u r n a l P r e -p r o o f as immunotherapies [], there is still a strong desire to utilize herbal medicines, especially in the Middle East. Therefore, scientific evaluation of the efficacy of herbal medicines and their possible side effects can provide a more precise strategy for the administration of this class of drugs. Although some of the therapeutic options have shown partially good effects on COVID-19 patients' recovery, there are a number of side effects related to these treatments, such as bradycardia in critical patients following Lopinavir-Ritonavir (2) , and breathing problems following extended Remdesivir consumption (3) .

Prevention and treatment are well-known strategies in both traditional Persian medicine and Chinese medicine. In addition to their therapeutic effect, the issue of cost-effectiveness is also a potential benefit of these disease management practices (4) . One of these medicinal plants in traditional medicine is called Emblica Officinalis Gaertn or Phyllanthus Emblica Linn.

(Euphorbeaceae), popularly referred to as Indian gooseberry or Amla. Due to the special medicinal and pharmaceutical properties, each part of this plant has fruitful anti-inflammatory, nootropic, antioxidant, anticancer, adaptogenic, anti-diabetic, antimicrobial, antiviral, as well as immunomodulatory effects besides preserving the vitality of the human cells (5) (6) (7) (8) .

Because of the pandemic spread of the SARS-CoV-2 infection and the morbidity and mortality associated with COVID-19 and its threat to human health and economies worldwide, finding an effective and affordable treatment is crucial (9) . Therefore, we aimed to evaluate the effects of Phyllanthus Emblica (Amla) as an add-on therapy on laboratory-confirmed admitted COVID-19 patients using a randomized, double-blind, controlled trial (RCT).

The present study was conducted to determine the effects of Amla on diagnostic RT-PCR test results in patients with COVID-19 between the first and last days of the study (day 10), the length of stay (LOS) in hospital, the symptoms and signs of patients with COVID-19, including, chills, cough, shortness of breath, weakness, sore throat, respiratory rate, myalgia and fever, the laboratory results of patients with COVID-19 between the first and last days of the study including the mean number of lymphocytes, the mean hemoglobin (Hb) level, the polymorphonuclear (PMN) count, the mean number of platelets (PLT), the mean erythrocyte sedimentation rate (ESR) level, the C reactive protein (CRP) level and final RT-PCR result, J o u r n a l P r e -p r o o f oxygen saturation (SpO2), as well as the mean percentage of lung involvement on computed tomography (CT) scans of patients with COVID-19 between the first and last days of the study.

The three hypotheses of this study included observing significant differences in the results of RT-PCR, daily recorded signs and symptoms and paraclinical results in the intervention arm compared to the control arm. The null hypothesis included the rejection of the aforementioned hypotheses.

This clinical trial was conducted from May 1 st , 2020 to June 1 st , 2020 at Razi and Sina Hospitals, affiliated with Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran. In this RCT, 61 patients were randomly assigned into two arms, i.e., the intervention (n=31) and the control arms (n=30). The effects of Amla on the clinical and para-clinical symptoms were assessed, including the diagnostic Reverse-transcription Polymerase Chain Reaction (RT-PCR) test results on the first and last days of the study (day 10), the length of hospital stay (LOS), the percentage of lung involvement on CT , as well as the laboratory markers.

Study inclusion criteria were age over 18 years, a positive RT-PCR test for COVID-19, pulmonary involvement on chest imaging, hospitalization with the following criteria: fever (≥38°C based on oral or axillary measurements) or respiratory rate above 24 per minute and cough within 8 days of disease onset. The exclusion criteria were disapproval by physicians or any condition that did not allow the protocol to be followed safely, severe liver disease, advanced kidney disease, an allergic reaction to the Amla, pregnancy or breastfeeding, transfer to another non-targeted hospital within the next 72 hours, being administered any experimental treatment for COVID-19 in the 30 day-period prior to evaluation and a history of taking angiotensin-converting enzyme inhibitors; also, patients with a WHO severity score > 6 were excluded from the study [10].

J o u r n a l P r e -p r o o f

All eligible patients enrolled in this study had voluntarily signed the written informed consent forms. None of the participants were deprived of their routine treatments for COVID-19 during the trial. Patients had the choice to leave the study freely at any time without disruption of their routine treatment.

After diagnosing COVID-19, an infectious disease specialist prescribed 200 mg hydroxychloroquine sulfate tablets along with Lopinavir/ritonavir (Kaletra) tablets (every 12 hours, two tablets after meals for 7 to 14 days) as the first-line therapy for all patients. In the intervention arm, patients received 2 grams of the sachet powder of Amla or 100 cc Amla tea per day for 10 days in addition to routine COVID-19 treatments. In the control arm, patients received a placebo along with routine treatment for COVID-19. The placebo was 2 g of sachet powder of Starch for oral solution. At the beginning of the intervention, precise instructions and "dos and don'ts" were explained to all patients in detail.

The fruits of the dried Amla plant had been purchased from a valid pharmaceutical plant were also warned to avoid taking the medicine with food. Both types of sachet powder were identical in terms of cover and taste and were prepared by a person who was not involved in the clinical trial.

The sachet powders were distributed among the nurses in charge for each case, and they were asked to administer medication for 10 days. Furthermore, all of them were asked to contact the investigation team in case of any side effects or drug poisoning. Also, we tracked them by phone on a daily basis to inquire about any probable problems. We explained to the nurses that the participants who had left at least 20% of the sachet powders after 10 days would be considered as non-compliant and excluded from the study; however, COVID-19 treatment would be continued for them.

The primary outcome was a change in the COVID-19 diagnostic test results between the first and last days of the study using RT-PCR. Secondary outcomes included changes in the LOS, clinical symptoms [e.g., daily body temperature, respiratory rate, chills, cough, sore throat, myalgia, weakness and shortness of breath], and laboratory results between the first and last days of the study, including peripheral blood lymphocytes, CRP levels, blood hemoglobin (Hb) levels, mean PMN cell counts, PLT counts, ESR levels, SpO2; also, the pulmonary imaging results between the first and last day of the study, i.e., chest radiographs and CT scans, were assessed as secondary outcomes.

Due to the lack of data on possible effects of this new treatment, and having no hypothesis regarding it, this study was an explorative and or pilot study consisting of a minimum number of 30 COVID-19 cases in each group.

The allocation of patients to each treatment arm was done by the block randomization method. The randomization unit was the individual; the randomization sequence was created J o u r n a l P r e -p r o o f using WinPEPI program (version 11.43), and was stratified by center with a 1:1 allocation ratio using a random block size of 6, and allocation concealment was done by assigning unicode.

In this parallel-group, double-blind, placebo-controlled RCT, the drugs for both groups were in the same aluminum containers, and the Amla plant did not have a specific odor and its color and taste were similar to the placebo. The sachet powders were placed into a matte envelope and an unique code assigned by www.sealedenvelope.com was pasted on every envelope. The list of numbers was given to a statistical consultant for subsequent data analysis.

All patients, physicians and investigators were blind to the kind of drugs and nurses were responsible for prescribing medication to patients.

In this study, the continuous variables were reported as mean with standard deviation and the comparisons between groups and within groups were performed by the independent sample t-test and paired sample t-test, respectively. Categorical variables were presented as numbers and percentages and the comparisons between groups were performed by chi-square and/or Fisher exact test. The comparisons for continuous clinical variables and ordinal variables of intragroup and intergroup during time (intervention and control group) were performed using the repeated measures ANOVA test and Generalized Estimating Equations (GEE), respectively. Plots were created by Graphpad prism 8. The data were analyzed using the statistical package for social science (SPSS Inc., Chicago, version 26) and p-value < 0.05 was considered statistically significant. 

Sixty-one patients with laboratory confirmed COVID-19 were assigned to this RCT and were randomly divided to two arms, i.e. an intervention arm (n= 31) and a control arm (n=30).

One patient left the intervention arm due to a feeling of stiffness in the throat. Finally, 30

subjects were analyzed in each study arm. Figure 1 shows the CONSORT flow diagram associated with subjects. The patients' mean (SD) age in the intervention and control groups were 47.87±14.31 and 44.27±11.20 years, respectively, without any statistically significant difference (P:0.28). Moreover, there was no significant difference between the groups in terms of height, weight, body mass index (BMI), gender, marital status, education level, occupation, living setting, history of pulmonary infectious disease, treatment history, history of type of treatment and comorbidities (P>0.05), as shown in Table 1 . Therefore, the two arms of the study had similar baseline characteristics.

At the beginning of the study, all participants had positive RT-PCR results for COVID-19, and at the end, there was no significant difference between the two arms (P=0.07).

The baseline and final para-clinical results (laboratory and radiologic findings) are summarized in Table 2 . Para-clinical variables refer to laboratory and radiological parameters. The mean SpO2 level had no significant difference between the two arms before the intervention (p=0.11); but, at the end of the study, the intervention arm showed a significant increase compared to the control arm (P<0.001). Also, the mean SpO2 level had a significant increase at the end of the study in both arms (p<0.001) compared with their corresponding baseline values. At the end of the study, both groups showed a significant decrease in the mean percentage of lung involvement on CT scans (p<0.001), without a significant difference between the groups (p>0.05). The mean number of lymphocytes was higher in the intervention arm than the control arm at the beginning and end of the study, yet, there was neither a significant difference between the two arms at the beginning (p=0.15) nor at the end of the study (p=0.08). However, at the end of the study, a significant decrease in the mean number of lymphocytes was noted in the intervention arm The mean ± SD of LOS in the intervention arm (4.44 days) was significantly shorter than in the control arm (7.18 days; P<0.001).

No adverse effects occurred in either the intervention group or the control group.

At the initiation of this RCT, both groups of patients had similar baseline demographics and characteristics regarding medical history. There was no significant difference in RT-PCR results between the groups. All evaluated COVID-19 symptoms decreased significantly in both arms during the study period. However, out of 8 signs, 4 signs had a significant reduction in the intervention arm compared with the control arm, including fever, the severity of cough, shortness of breath and myalgia. Also, at the end of this RCT, out of 8 para-clinical variables, 3 variables had a significant improvement in the intervention arm compared with the control arm, including the mean increase in SpO2 level, the reduction in the mean percentage of lung involvement on CT scans and the improvement in CRP levels. Also, the LOS in patients who had consumed organic herbal Amla tea was significantly shorter than that of the control group.

In a recent study published by Ul Qamar et al. concerning SARS-CoV-2, out of 32,297 Chinese medicinal compounds, Amla along with eight others have been proposed as novel non-toxic, druggable natural compounds that bind to the enzyme 3-chymotrypsin-like protease (3CLpro)

receptor binding site and the catalytic dyad (11) . This enzyme plays a pivotal role in the viral replication process. It breaks down the gene-derived 800 kDa polypeptide of beta-coronaviruses J o u r n a l P r e -p r o o f in 11 specific sites and produces a variety of non-structural viral proteins (12, 13) . In an in-vitro study, the antiviral effect of Amla on the herpes simplex virus has been discovered (14) . In another in-vitro study, the antiviral effects of Amla along with six other Thai medical plants have been investigated on the porcine reproductive and respiratory syndrome virus (PRRSV). Amla can inhibit PRRSV infection at a low concentration of 78 µg/mL. It was also ranked second among plants in terms of antioxidant activity against free radicals (15) . A review study has reported that the therapeutic potential of Amla is due to its spasmolytic, expectorant and antitussive effects (5) . Furthermore, the dose-dependent cough suppressive activity of Amla has been described in an animal study (16) . Although the severity of cough showed a significant decrease compared to corresponding baseline values in both arms at the end of the study, this reduction was significantly more in the intervention arm compared to the control arm. Moreover, other animal studies have reported the antipyretic and analgesic activities of Amla (17, 18) . In this regard, although fever and myalgia at the end point of the trial showed a significant decrease compared to their corresponding baseline values in both groups, this reduction was significantly more in the intervention arm compared to the control arm.

The present study has several limitations, including 1) failure to assess adherence and certain clinical findings, e.g., the patients' smoking status and disease complications; 2) prolonged follow-up outcomes after cessation of treatment were not documented; 3) the sample size of this clinical trial was relatively small. This study has the following strengths: firstly, more comprehensive data have been presented in this RCT study compared with other studies.

Secondly, this was a double-blind RCT, which is among the most reliable study designs regarding COVID-19. However, outcomes, follow-up findings and laboratory or immunological investigations with repeated chest imaging for all participants, should be evaluated for at least a few months following treatment cessation to give more reliable information about the medicinal effects of this plant on COVID-19. Also, we would suggest the assessment of all these factors in different age subgroups. Finally, although this study was conducted in a single province, other nationwide multicenter studies are recommended to assess additional unknown factors, such as race, geographic location, and climate, which may affect the safety and efficacy of Amla.

J o u r n a l P r e -p r o o f

This study revealed no significant difference in final RT-PCR results and or degree of lung involvement on CT scans. But, significant reductions in fever, the severity of cough, shortness of breath, myalgia, LOS, and a significant improvement in SpO2 and CRP levels were noted in the group treated with Amla. Moreover, no adverse effects were observed. Nevertheless, further studies are needed to examine Amla supplements in light of evolving standards of care as well as its mechanism of action.

All authors worked equally and contributed to all parts of the study.

None Assessed for eligibility (n=98)

Not meeting inclusion criteria (n=26)

Declined to participate (n=11) Fever and respiratory rate were continuous variables and the rest of variables were ordinal as: no, mild, moderate and severe Based on repeated measure ANOVA, the difference between two groups for fever was significant (P=0.004) but for respiratory rate was not (P=0.297)

Based on Generalized Estimating Equations (GEE), the differences between two groups in terms of severity of cough, shortness of breath and myalgia were significant (P=0.001, P=0.004 and 0.005) but for severity of chills, sore throat and weakness were not (P=0.059, P=0.220 and P= 0.121)

The levels of all variables decreased over time in both groups (P<0.001).

Variables into the first and second graphs were continues while others were ordinal variables. So, the ordinal variables made histogram graphs.

 Organic herbal Amla (Phyllanthus Emblica) tea cannot significantly affect the RT-PCR results.

 Amla tea did not significantly reduce the severity of the lung involvement.

 Amla tea showed an ameliorative effect on the severity of clinical signs and CRP levels.

 Amla tea can shorten the recovery times of symptoms and LOS in COVID-19 patients. Continuous variables reported using mean±SD and categorical variables reported using n(%). P values were calculated based on the t-test and or Chi-squared test. 

Advances in immunotherapy for COVID-19: A comprehensive review

Lopinavir-Ritonavir Treatment for COVID-19 Infection in Intensive Care Unit: Risk of Bradycardia

Remdesivir for 5 or 10 days in patients with severe Covid-19

Becoming a Faithful Defender: Traditional Chinese Medicine against Coronavirus Disease 2019 (COVID-19)

Therapeutic potential of Phyllanthus emblica (amla): the ayurvedic wonder

Emblica officinalis (Amla): A review for its phytochemistry, ethnomedicinal uses and medicinal potentials with respect to molecular mechanisms

In vitro anti-herpes simplex virus activity of 1,2,4

O-galloyl-β-D-glucose from Phyllanthus emblica L. (Euphorbiaceae)

Emblica officinalis (amla) on cultured human primary dental pulp fibroblasts

Clinical management of COVID-19: interim guidance, 2020. World Health Organization

Structural basis of SARS-CoV

3CLpro and anti-COVID-19 drug discovery from medicinal plants

Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs

Structures of the Middle East respiratory syndrome coronavirus 3C-like protease reveal insights into substrate specificity

In vitro anti-herpes simplex virus activity of 1, 2, 4, 6-tetra-O-galloyl-β-D-glucose from Phyllanthus emblica L. (Euphorbiaceae)

In vitro screening antiviral activity of Thai medicinal plants against porcine reproductive and respiratory syndrome virus

Antitussive activity of the fruit extract of Emblica officinalis Gaertn. (Euphorbiaceae)

Evaluation of anti-pyretic and analgesic activity of Emblica officinalis Gaertn

Evaluation of analgesic activity of Emblica officinalis in albino rats

The authors would like to thank the Vice-Chancellor for Research Affairs of Ahvaz Jundishapur University of Medical Sciences for financial support. We also thank Tahmineh Khalilian and Roshanak Roshanfar for technical support.