Traditional Chinese herbal medicine has provided clinical benefits to patients infected with coronavirus 2019 (COVID-19) in China. Jinhua Qinggan granule (JHQGG) is a Chinese multi-herbal formula previously developed for the treatment of H1N1 influenza and has been encouraged for use in patients with clinically suspected COVID-19 infection. However, the immunopharmacological mechanism for the efficacy of JHQGG has not yet been confirmed. To obtain insight into this issue, the present study examined the acute effects of JHQGG ingestion on hematological and immunological parameters using uninfected individuals as subjects. For this purpose, 18 healthy volunteers were enrolled, all of whom tested negative for prior and current severe acute respiratory syndrome coronavirus 2 infection. Peripheral blood samples were collected 1 h after a single oral JHQGG administration and subjected to hematological, biochemical and cytokine tests. JHQGG rapidly induced a significant decrease in the plasma level of interleukin (IL)-6 (P=0.00309) and an increase in the plasma level of interferon (IFN)-γ (P=0.0268). A decrease in IL-6 and an increase in IFN-γ levels were observed in 14 (77.8%) and 13 (72.2%) subjects, respectively. Notably, JHQGG significantly decreased the proportion of neutrophils (P=0.00561) and increased that of lymphocytes (P=0.00485); accordingly, the neutrophil/lymphocyte ratio (NLR) was significantly reduced by JHQGG (P=0.00649). These findings suggest that the clinical benefits of the use of JHQGG against COVID-19 are, at least in part, associated with its rapid modulatory effects on IL-6, IFN-γ and NLR. Considering that IL-6 and NLR are critical biomarkers for severe COVID-19 infection, JHQGG may thus be suitable not only for suppressing disease onset in suspected and asymptomatic cases, but also for preventing disease progression in patients with mild to severe infection. The present open-label, single-arm study has been prospectively registered on the University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) under the trial no. UMIN000040407 on May 15, 2020.
Traditional herbal medicine has provided clinical benefits to patients with coronavirus 2019 (COVID-19) in China (
On the basis of its therapeutic efficacy for influenza, JHQGG has been recommended for use in patients clinically suspected of COVID-19 infection to prevent disease onset (
Although network pharmacological studies have provided
Since JHQGG has been recommended for use in individuals clinically suspected of COVID-19 infection under medical observation to prevent disease onset, the present study employed uninfected individuals as subjects in a single-arm trial. Participants were recruited through the University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) website, the authors' clinical website (
JHQGG was kindly provided by Dr Hugh Wang, Juxiechang (Beijing) Pharmaceutical Co., Ltd. The subjects were instructed to take a packet (5 g) orally 40 min their lunchtime meal in accordance with the administration protocol of the Chinese official guideline (
To examine the acute hematological and immunological effects of JHQGG, peripheral blood samples were obtained from each subject immediately prior to and at 1 h following the administration of JHQGG. Hematological and blood biochemical tests were outsourced to SRL, Inc. Plasma cytokine levels were quantified using the V-PLEX Proinflammatory Panel 1 Human kit (K15049D-1; Meso Scale Diagnostics) and the Human IL-18 ELISA kit (ab215539; Abcam). The primary outcome measure was changes in the plasma levels of inflammatory-related cytokines [interleukin (IL)-6, IL-1β, IL-18, IL-12, IL-2, IL-8, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α] at 1 h after the JHQGG ingestion compared with the baseline levels. The secondary outcome measure was changes in hematological parameters (as listed in
No outliers were taken into account, and all collected data from all patients (n=18) were subjected to statistical analysis. The normality of the data was tested using the Shapiro-Wilk test. On the basis of the results from the normality test, the two-tailed Wilcoxon signed-rank test was employed at the significance level (α) of 0.05 for subsequent statistical analysis of the data. All statistical analyses were performed using EZR version 1.53 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) (
A total of 18 healthy volunteers were screened for eligibility, found to be eligible and enrolled in the trial in the present study (
It was found that hematocrit levels (Z=2.06, P=0.0394, r=0.486) and mean corpuscular volume (Z=2.30, P=0.0216, r=0.542) were slightly altered within the reference ranges, although there were no significant differences in the other measurements of complete blood count and blood biochemistry between pre- and post-JHQGG ingestion (
In the blood cytokine analysis, the plasma levels of IL-6 and IFN-γ were significantly decreased and increased, respectively, compared with those in the pre-JHQGG ingestion (IL-6: Z=2.96, P=0.00309, r=0.697; IFN-γ: Z=2.20, P=0.0268, r=0.518) (
Post hoc two-tailed power analysis was performed (significance level, α=0.05; sample size, n=18) and statistical powers were obtained (1 - β) of 0.720 (neutrophils), 0.735 (lymphocytes), 0.706 (NLR), 0.775 (IL-6), 0.524 (IFN-γ) and 0.445 (IL-18) following the completion of the trial.
Patients with severe COVID-19 infection present with neutrophilia, lymphocytopenia and a resulting elevated NLR, which are closely associated with poor clinical outcomes (
JHQGG contains various active pharmaceutical ingredients, mainly rutin, luteolin, wogonin, myricetin, quercetin, ursolic acid, chrysoeriol and glabridin (
There is a large body of evidence to indicate that patients with COVID-19 have aberrantly increased blood levels of pro-inflammatory cytokines and chemokines (
INF-γ is produced predominantly by type 1 helper T (Th1) and natural killer (NK) cells and stimulates innate immunity and inflammation, particularly through the activation of macrophages and dendritic cells. In the present study, JHQGG ingestion induced a slight, yet significant increase in the blood level of INF-γ. This pro-inflammatory activity appears to be contradictory to its clinical benefits against COVID-19. Notably, IFN-γ is known to serve as a central mediator of a broad spectrum of antiviral immunity by interfering with viral replication directly, potentiating the effects of IFN-α/β, activating Th1-dependent immune responses, and promoting the activation of the MHC class I pathway (
Patients with severe COVID-19 infection have significantly lower numbers of CD4+ T-, CD8+ T- and NK cells, with a decreased capacity to produce IFN-γ, which results in a decrease in IFN-γ production by CD4+ T-cells (
The main limitations of the present study are the small number of participants and the selection of uninfected individuals as the study subjects. Since JHQGG has been recommended for use in individuals clinically suspected of COVID-19 infection under medical observation or asymptomatic patients to prevent disease onset, uninfected individuals were employed as the study subjects. The present study demonstrated that JHQGG significantly up- and downregulated the plasma levels of IFN-γ and IL-6, respectively. Further studies with larger cohorts of patients with moderate to severe infection are thus essential to confirm the conclusion in patients and determine generalizability. Randomized controlled trials in patients with moderate to severe infection are also essential to confirm whether cytokine responses to JHQGG are sustained and whether they are associated with significant clinical improvement. Further validation studies are also required to determine whether the blood levels of JHQGG-derived compounds increase at 1 h following oral administration. The present study tried to measure the blood levels of representative active ingredients of JHQGG before and after oral administration; however, since some difficulties were encountered in finding optimal Liquid chromatography-mass spectrometry conditions, such data could not be obtained. In addition, since JHQGG contains 12 herbal components, the quality control or batch-to-batch quality consistency poses a great challenge in clinical practice.
In conclusion, the findings of the present study suggest that the clinical benefits of JHQGG against COVID-19 are, at least in part, associated with its rapid immunomodulatory effects; JHQGG can rapidly decrease the blood levels of neutrophils and IL-6, two critical exacerbating factors of COVID-19, and increase the blood levels of lymphocytes and IFN-γ that are essential for coordinated antiviral immune responses. JHQGG has been recommended for use in suspected and asymptomatic cases of COVID-19 to suppress disease onset, as per the Chinese official clinical guideline (
The authors would like to thank Dr Hugh Wang [Juxiechang (Beijing) Pharmaceutical Co., Ltd.] for generously providing Jinhua Qinggan granule and for his helpful advice for clinical trial planning.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YK and TE were involved in the conceptualization and methodology of the study, as well as in performing the experiments and data collection, obtaining resources, data curation, reviewing and editing of the manuscript, and project administration. KA, KK and MM were involved in performing the experiments and data collection, obtaining resources, and in the reviewing and editing of the manuscript. TA was involved in the conceptualization of the study, and in the reviewing and editing of the manuscript. TN was involved in the conceptualization and methodology of the study, as well as in formal analysis, and in the writing of the original draft, as well as in the preparation and creation of the published figures and tables. YK, TA and TN were also involved in study supervision. YK and TN confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
The present study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). All procedures were reviewed and approved by the Ethics Committees of Takanawa Clinic (approval no. 2020-2). A signed informed consent form was obtained from each participant prior to inclusion in this study.
Not applicable.
YK, KA, KK, MM and TE are employees of Takanawa Clinic. TA and TN serve as research advisers to Takanawa Clinic and receive advisory fees. The authors declare that there is no conflict of interest between the study group and Juxiechang (Beijing) Pharmaceutical Co., Ltd., the Chinese pharmaceutical company that provided Jinhua Qinggan granule for the study.
CONSORT flow diagram of the study trial.
JHQGG modulates the proportions of neutrophils and lymphocytes, neutrophil-to-lymphocyte ratio, and the plasma levels of IL-6 and IFN-γ. Data (n=18 subjects) were analyzed using the two-tailed Wilcoxon signed-rank test at the significance level (α) of 0.05. The single asterisk (*) indicates one subject with undetectable IL-6 before and following oral JHQGG administration and double asterisks (**) indicate two subjects with undetectable IFN-γ following oral JHQGG administration. The values of 0.5 x lower limit of detection (IL-6, 0.03 pg/ml; IFN-γ, 0.185 pg/ml) were used to correct the undetectable IL-6 and IFN-γ data. Pre, pre-ingestion (baseline); Post, post-ingestion; JHQGG, Jinhua Qinggan granule.
Alterations in hematological parameters and cytokine levels in subjects administered JHQGG.
Pre-JHQGG administration | Post-JHQGG administration | ||||||
---|---|---|---|---|---|---|---|
Measurements | Median | (IQR) | Median | (IQR) | Z value | P-value | r value |
Complete blood count | |||||||
Red blood cell count (x104/µl) | 454 | (419-468) | 457 | (416-469) | 1.26 | 0.206 | 0.298 |
Hemoglobin (g/dl) | 13.5 | (12.3-14.4) | 13.6 | (12.2-14.0) | 1.60 | 0.111 | 0.376 |
Hematocrit (%) | 41.0 | (36.5-43.7) | 40.6 | (36.2-42.8) | |||
MCV (fl) | 90.7 | (88.2-94.7) | 90.3 | (87.8-95.4) | |||
MCH (pg) | 30.1 | (29.4-31.5) | 30.3 | (29.5-31.7) | 0.166 | 0.868 | 0.0392 |
MCHC (%) | 33.2 | (32.6-33.7) | 33.4 | (32.9-33.7) | 1.58 | 0.115 | 0.372 |
White blood cell count (/µl) | 6200 | (5730-6680) | 6450 | (6030-6780) | 1.11 | 0.265 | 0.263 |
Platelet count (x104/µl) | 24.4 | (22.1-28.7) | 24.7 | (22.7-28.3) | 0.214 | 0.831 | 0.0504 |
White blood cell differential | |||||||
Neutrophils (%) | 62.3 | (59.6-68.5) | 61.7 | (58.9-65.6) | |||
Eosinophils (%) | 1.10 | (0.850-2.08) | 1.05 | (0.725-2.38) | 0.0713 | 0.943 | 0.0168 |
Basophils (%) | 0.450 | (0.300-0.575) | 0.500 | (0.325-0.600) | 0.431 | 0.666 | 0.102 |
Monocytes (%) | 5.25 | (4.40-6.38) | 5.65 | (4.73-6.48) | 1.06 | 0.288 | 0.250 |
Lymphocytes (%) | 29.1 | (23.6-32.9) | 29.9 | (25.3-33.0) | |||
Neutrophil/lymphocyte ratio | 2.24 | (1.82-2.97) | 2.08 | (1.79-2.52) | |||
Blood biochemistry | |||||||
AST (U/l) | 16.5 | (15.0-19.5) | 16.0 | (14.3-18.8) | 0.00 | 1.00 | 0.00 |
ALT (U/l) | 15.0 | (10.3-18.5) | 15.0 | (10.3-18.5) | 0.632 | 0.527 | 0.149 |
γ-GT (U/l) | 14.0 | (12.3-21.0) | 14.0 | (13.0-21.0) | 1.51 | 0.132 | 0.355 |
LDH (U/l) | 143 | (134-170) | 145 | (131-166) | 1.07 | 0.285 | 0.252 |
Albumin (g/dl) | 4.70 | (4.50-4.88) | 4.65 | (4.53-4.90) | 0.206 | 0.837 | 0.0486 |
Urea nitrogen (mg/dl) | 12.2 | (11.8-14.2) | 12.4 | (11.4-14.4) | 0.0237 | 0.981 | 0.00559 |
HDL cholesterol (mg/dl) | 69.0 | (61.8-77.8) | 68.5 | (61.3-77.0) | 1.02 | 0.306 | 0.241 |
LDL cholesterol (mg/dl) | 109 | (96.5-124) | 109 | (91.8-123) | 1.30 | 0.193 | 0.307 |
Triglycerides (mg/dl) | 62.0 | (41.3-108) | 66.0 | (42.0-111) | 0.687 | 0.492 | 0.162 |
CRP (mg/dl) | 0.0500 | (0.0225-0.0875) | 0.0550 | (0.0225-0.0875) | 1.61 | 0.107 | 0.380 |
Cytokines | |||||||
IFN-γ (pg/ml) | 3.33 | (1.80-4.72) | 3.85 | (2.99-5.00) | |||
IL-6 (pg/ml) | 1.40 | (0.731-3.31) | 0.988 | (0.690-1.59) | |||
TNF-α (pg/ml) | 2.49 | (2.01-3.55) | 2.63 | (2.29-3.39) | 0.893 | 0.393 | 0.210 |
IL-1β (pg/ml) | 0.504 | (0.336-1.45) | 0.377 | (0.224-2.36) | 0.806 | 0.442 | 0.190 |
IL-18 (pg/ml) | 145 | (99.6-232) | 109 | (90.1-304) | |||
IL-12 (pg/ml) | 0.262 | (0.106-0.452) | 0.330 | (0.185-0.472) | 1.02 | 0.325 | 0.241 |
IL-2 (pg/ml) | 0.280 | (0.0955-0.655) | 0.389 | (0.157-0.891) | 1.94 | 0.0539 | 0.457 |
IL-8 (pg/ml) | 448 | (105-1220) | 415 | (115-766) | 0.719 | 0.495 | 0.169 |
IL-10 (pg/ml) | 0.259 | (0.184-0.500) | 0.268 | (0.213-0.556) | 0.327 | 0.766 | 0.0770 |
Statistically significant results are presented in bold font
a(P<0.05 and
bP<0.01). The undetectable IL-6 data in one subject and the undetectable IFN-γ data in two subjects were corrected with the values of 0.5 x lower limit of detection (IL-6, 0.03 pg/ml; IFN-γ, 0.185 pg/ml) (