Abstract
OBJECTIVE:
To study the anti-inflammatory and anti-tussive effects of Qingfei Dayuan granules (清肺达原颗粒, QFDY), and to evaluate the acute and sub-chronic toxicity of QFDY.
METHODS:
Anti-inflammatory effects were evaluated by murine model of xylene induced ear edema in mice. Ear swelling degree was calculated and tumor necrosis factor-α, interleukin-1β and interleukin-6 were determined. Anti-tussive evaluations were carried out in the mouse cough model induced by ammonia liquor. Latent period cough and number of cough within 3 min were counted. In acute toxicity study, the rats were randomly divided into test group and solvent control group. Body weighs, food intakes and general clinical signs were monitored. In the sub-chronic toxicity study, QFDY was administered to rats at 0, 4, 8 and 16 g/kg per day for 28 and 30 d of post treatment was conducted. Mortalities, clinical signs, body weight changes, food intakes, ophthalmological examinations, hematological parameters, biochemical indicators, electrolyte indicators, urinalyses and histopathological examinations were monitored.
RESULTS:
QFDY significantly inhibited the development of ear edema in anti-inflammatory assay and decreased cough frequency caused by ammonia liquor. The results presented a dose-effect relationship. In acute toxicity study, no abnormality exhibited at dose of 24.0 g/kg per day during the 14-d observation period. In the sub-chronic toxicity study, higher reticulocyte count, lymphocyte and lower Cl-, blood urea nitrogen were analyzed compared with the solvent control group. But the differences were considered to be incidental and not clinically toxic. Obvious dose-effect relationship of urine color was observed, and the three test groups at the end of the experiments resulted in significant increase in urobilinogen, bilirubin, ketone body and urine leukocyte. However, all the positive indicators returned to normal in the recovery period. Therefore, no toxicological changes were found during the study period.
CONCLUSION:
QFDY showed significant anti-inflammatory and anti-tussive effects in mice. The lethal dose (LD50) of per oral QFDY in rats was estimated to be more than 24.0 g/kg per day and the no observed adverse effect level was over 16 g/kg per day, which suggested that QFDY is relatively safe for oral medication at the present dose on rats. Our experimental results provide a reference for the further development and research of QFDY.
Keywords: anti-inflammatory agents; anti-tussive; toxicity tests, acute; sub-chronic toxicity; Qingfei Dayuan granules
1. INTRODUCTION
The prevalence of coronavirus disease 2019 (COVID-19) and severe acute respiratory syndrome have a dramatic effect on health and socio-economics. Traditional Chinese Medicine products, such as Lianhua Qingwen Capsule (连花清瘟胶囊), have played an essential role in treating COVID-19.1,2 Qingfei Dayuan granules (清肺达原颗粒, QFDY), also be called as “Hubei Pneumonia No. 1”, is widely used for treating COVID-19 with the significant preventive effect, which had been proven by previous studies.3,4 The prescription was recommended by the Hubei Headquarters for the Prevention and Control of Novel Coronavirus Pneumonia Epidemic. It is composed of Chaihu (Radix Bupleuri Chinensis), Huangqin (Radix Scutellariae Baicalensis), Houpu (Cortex Magnoliae Officinalis), Binglang (Semen Arecae), Caoguo (Fructus Tsaoko), Dangshen (Radix Codonopsis), Chenpi (Pericarpium Citri Reticulatae), Gualou (Fructus et Semen Trichosanthis), Zhimu (Rhizoma Anemarrhenae), Banxia (Rhizoma Pinelliae), Chishao (Radix Paeoniae Rubra), Huzhanggen (Radix Polygoni Cuspidati) and Gancao (Radix Glycyrrhizae).5,6 According to the reported pharmacological research, many components in the prescription have anti-inflammatory and anti-tussive effects, such as saikosaponin-A, saikosaponin-C and saikosaponin-D,7,8 baicalin, baicalein and wogonin,9,⇓-11 magnolol and honokiol.12,13 Previous reports showed that QFDY can prevent the progress of mild/moderate symptom convert to severe course of COVID-19 disease, contribute to relieve cough symptoms, and the absorption of lung inflammation that can alleviates the clinical symptoms of patients.3,⇓-5 However, most of them only retrospective clinical reports without scientific experimental verification, and some adverse reactions were reported when taken inappropriately. In this paper, the anti-inflammatory and anti-tussive effects of QFDY were studies in mice for the first time, as well as the acute and sub-chronic toxicity of QFDY in rats were evaluated.
2. MATERIALS AND METHODS
2.1. Chemicals and reagents
Aspirin and pentosyverine were purchased from Bayer Medical Care Co., Ltd. (Wuhan, China), enzyme linked immunosorbent assay (ELISA) were obtained from Solarbio (Beijing, China). QFDY (Batch number, Z202006269) was produced by Jing Brand Chizhengtang Pharmaceutical Co., Ltd. (Huangshi, China). The samples were analyzed with 91 fractions by ultra-high performance liquid chromatography coupled with time-of-flight mass spectrometry and the average chemical contents of baicalin, paeoniflorin and hesperidin were 18.05, 2.69 and 3.30 mg/g, respectively (supplementary Figure 1).
Figure 1. Effects of QFDY on swelling degree and inflammatory cytokines in the xylene-induced ear edema in mice model.
A: swelling degree; B: TNF-α; C: IL-1β; D: IL-6. SC groups: treated with distilled water; AC groups: treated with 520 mg/kg aspirin; LQ, MQ, and HQ groups were given 3.90, 5.46 and 7.80 g/kg QFDY per day, respectively, each group was administered once a day for 7 d. SC: solvent control group; AC: aspirin control group; LQ: low-dose group; MQ: medium-dose group; HQ: high-dose groups; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; IL-6: interleukin-6. aP < 0.01, bP < 0.05, significantly different compared with SC group; cP < 0.05, significantly different compared with AC group.
2.2. Experimental animals and housing conditions
Specific-pathogen-free Sprague-Dawley (SD) Kunming mice and rats were obtained from Hubei Experimental Animal Research Center (Wuhan, China). The animals were maintained at constant room temperature [(25 ± 2) ℃] on a 12-h light/dark cycle with free access to food and water for 24 h or more before experiments. All the experimental procedures were approved by the Animal Management and Use Committee of Hubei Food and Drug Safety Evaluation Center (approval No. 201602014) and were in line with good laboratory practice laboratory requirements (No.16002082), which had been detailed in prior study.14
2.3. Anti-inflammation and anti-tussive experiments
The eligible male mice weighing 25-30 g were chosen as described before15 and randomly divided into 5 groups of 10 mice each, Low-dose (LQ), medium-dose (MQ), and high-dose (HQ) groups were given 3.90, 5.46, 7.80 g/kg per day QFDY, respectively, while solvent control (SC) group was given distilled water. Positive control group was given 520 mg/kg per day aspirin (AC) for anti-inflammation experiments and 30 mg/kg per day pentosyverine (PC) for anti-tussive experiments.
The anti-inflammation experiments were carried out as described previously.16 Each group was administered once a day for 7 d of 20 mL/kg. An hour after the last administration, 0.05 mL xylene was applied to the anterior and posterior surfaces of the left ear evenly. After 0.5 h, mice were sacrificed and a punch was excised from both ears. Anti-inflammatory was assessed using ear swelling degree. Finally, the left ears were lyophilized in liquid nitrogen, shattered, dissolved in 1 mL tissue protein extract, sonicated for 10 min and filtered. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), were detected by ELISA according to the instructions.
Anti-tussive effects were investigated by using a classical mouse cough model induced by ammonia liquor.17 Half an hour after intragastric administration, the mice were placed in a 1000 mL beaker and sprayed with 0.2 mL ammonia liquor. The mice were taken out from the beaker a minute later. Latent period cough and number of cough within 3 min were recorded. Inhibition and cough activity were calculated.
2.4. Acute oral toxicity study
Forty healthy SD rats of both genders with the body weight ranging from 180 to 220 g were randomly divided into test group and solvent control group. The test group was given 24.0 g/kg per day QFDY for two times within 24 h, while the solvent control group was given the same volume of distilled water. Weight of the experimental animals was recorded on days 1, 2, 3, 7, 14, and 15 (day of planned anatomy). Food intakes were calculated on days -1 (day before administration), 1, and 8. Mortality and general clinical signs were monitored every day. At the end of the observation period, all animals were anesthetized and autopsied after bloodletting to observe gross necropsy.
2.5. Sub-chronic oral toxicity study
2.5.1. Animal assignment and treatment
The sub-chronic toxicity test was performed in 120 healthy SD rats of both genders. Animal with body weight ranging from 180 to 220 g were randomly divided into 4 groups of 30 rats each (15 males and 15 females), SC, LQ, MQ and HQ groups were given 0, 4, 8 and 16 g/kg QFDY per day, respectively. After 28 d administration, 10 male and 10 female rats were collected from each group. Blood collection and necropsy were performed to detect planned indicators. The remaining 5 animals of each group were continued to observe for 4 weeks after drug withdrawal, reversibility, persistence and delayed effects of toxicity were detected on the 57th day.
2.5.2. General clinical symptom observations
Abnormalities and mortality were recorded daily for all rats. Body weight and food intakes were calculated once before administration (day-1) and once a week throughout the experiments. Ophthalmic examinations including cornea, conjunctiva, iris and lens were performed on all surviving animals monthly.
2.5.3. Clinical testing
The following hematological parameters were determined using an automatic blood analyzer (Sysmex XT-2000iv, Kobe, Japan): red blood cell (RBC), hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular-hemoglobin concentration (MCHC), platelet (PLT), reticulocyte count (Retic), white blood cell count (WBC), differential count (DC), neutrophils (NEUT), Eosinophils (EOS), basophilic granulocyte (BASO), monocytes (MONO), and lymphocyte (LYMP). And prothrombin time (PT) and activated partial thromboplastin time (APTT) were determined using a coagulation function analyzer (Sysmex CA-510, Kōbe, Japan).
The following blood biochemical indicators and electrolyte were analyzed by an automatic biochemical analyzer (Backman AU-680, San Francisco, CA, USA): alkaline phosphatase (ALP), glucose (GLU), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), alanine aminotransferase (ALT), blood urea nitrogen (BUN), γ-glutamyltransferase (γ-GT), creatinine (CREA), total cholesterol (TCHO), total bilirubin (TBIL), creatine phosphokinase (CK), and triglyceride (TG), concentration of K+, Na+, and Cl-.
Overnight urine was collected at fourth week and eighth week. The following urine detection indexes were recorded with a urine analyzer (H-800, Wuhan, China): urine color and clarity, proportion, pH, urobilinogen (UBG), urine leukocyte (LEU), urine glucose (UGLU), bilirubin (BIL), ketone body (KET), occult blood (BLD), and protein (PRO).
2.5.4. Pathology examinations
All the surviving rats were anesthetized and performed a complete necropsy. The selected organs including brain, heart, livers, spleen, kidneys, thymus, adrenal gland, testicle (male), epididymides (male), uterus (female) and ovaries (female) were isolated and weighted. The organ coefficient (organ/body) and the relative ratio of organ weight to brain weight (organ/brain) were calculated according to the final body weight and the brain weight. All organs and tissues from the SC and HQ groups, macroscopic lesions and masses in each group were embedded in paraffin and sectioned. After being stained with hematoxylin-eosin, the slides were examined with a microscope (Thermo CX41F, San Francisco, CA, USA) to detect lesions.
2.6. Statistical analysis
The data, was analyzed using SPSS GraphPad Prism software version 8.0 (GraphPad Software, Inc. La Jolla, CA, USA), and expressed as mean ± standard deviation ($\bar{x}$ ± s). Statistical significance was defined as P < 0.05, P < 0.01 or P < 0.001.
3. RESULTS
3.1. Anti-inflammation activity
The swelling degree of mice in LQ, MQ and HQ groups was all lower than that in SC group, with extremely significant difference in the high dose group (P < 0.01), and presented a dose-effect relationship (Figure 1A). Compared with SC group, cytokines decreased significantly. TNF-α and IL-6 decreased more significantly in the low dose groups (Figure 1B-1D). These results suggested that QFDY can be used to inhibit the development of ear edema and decrease inflammatory cytokines to help preventing or controlling diseases, such as influenza and pneumonia. Anti-tussive activity.
The anti-tussive activity of QFDY was demonstrated by prolonging the incubation time, and reducing coughing times in 3 min. Compared with the SC group, a significant and dose-dependent decrease in cough responses was observed in mice treated with QFDY (MQ and HQ groups). HQ groups showed good capability of reducing cough times with the inhibition of 65.8% and cough activity of 92.8%, which close to PC group (Table 1).
Table 1.
Effects of QFDY on the ammonia liquor induced cough in mice ($\bar{x}$ ± s)
| Group | n | Dose | Latent period cough (s) | No. of coughs | Inhibition (%) | Cough Activity (%) |
|---|---|---|---|---|---|---|
| SC | 10 | 0 g/kg | 7.6±1.3 | 41.2±12.7 | - | - |
| PC | 10 | 25 mg/kg | 16.5±6.8a | 12.0±3.6b | 70.9 | - |
| LQ | 10 | 3.90 g/kg | 12.4±4.9 | 39.1±12.4 | 5.1 | 7.2 |
| MQ | 10 | 5.46 g/kg | 13.1±2.8b | 25.4±6.3a | 38.3 | 54.1 |
| HQ | 10 | 7.80 g/kg | 18.9±3.7b | 14.1±3.5b | 65.8 | 92.8 |
Notes: SC groups: treated with distilled water; PC groups: treated with 30 mg/kg pentosyverine per day; LQ, MQ, and HQ groups were given 3.90, 5.46 and 7.80 g/kg QFDY per day, respectively, each group was administered once a day for 7 d. SC: solvent control group; PC: pentosyverine control group; LQ: low-dose group; MQ: medium-dose group; HQ: high-dose groups. aP < 0.05, bP < 0.01, significantly different compared to the SC group.
3.2. Acute oral toxicity of QFDY
There were no experimental rats died during the experiments. As shown in supplementary Figure 2, the daily food intake of female rats in the test group was reduced 7.2% in the first two day with statistically significant (P < 0.05). But it returned to normal from the eighth day of the second week after treatment, indicating that the test substance caused a temporary and slight decrease in the food intake of female rats. There were no significant differences in the body weight of male and female rats in the test group. And clinical observations and gross necropsies of animals showed no abnormality during the 14-d observation period.
Figure 2. Effects of sub-chronic toxicity experiment of QFDY on mean body weight and food intake.
A: body weight in male; B: body weight in female; C: food intake in male; D: food intake in female. SC groups: treated with distilled water; LQ, MQ, and HQ groups were given 4, 8 and 16 g/kg QFDY per day, respectively, each group (n = 30, 15 males and 15 females) was administered once a day for 28 and 30 d of post treatment was conducted. SC: solvent control group, LQ: low-dose group, MQ: medium-dose group, HQ: high-dose groups. aP < 0.05, bP < 0.001, significantly different compared to the SC group.
3.3. Sub-chronic 30-day oral toxicity study in rats
3.3.1. General clinical signs
No accidental death occurred in the experiments, no obvious abnormalities were observed in the clinical observation of animals in both the SC group and the three test groups, and no abnormalities were found in ophthalmic examinations.
3.3.2. Body weights and food intakes
There were no significant differences in body weight between the rats in all groups during the whole experiments (Figure 2A, 2B). The food intake of HQ group was lower than that in the SC group (Figure 2C, 2D). However, it returned to normal after stopping administration. Therefore, we speculated that decreasing in food intake may be due to large daily doses of medication but not because of the side effects of the drug.
3.3.3. Hematological parameters
As shown in supplementary Table 1, the RET of female in the HQ group (end of administration) and LYMP in the LQ group (recovery stage) were higher than that in the SC group with statistically significant differences (P < 0.05). Other hematological parameters were normal during the experiments. Our results showed that no toxicological changes of hematologic markers were observed.
3.3.4. Blood biochemical and electrolyte indicators
The Cl- in the male rats of the HQ group and the BUN in female rats of the LQ and HQ groups was lower than that in the SC group (P < 0.05 or P < 0.01), both still within the range of normal values. Other indicators were normal during the experiments (supplementary Table 2). Thus, no changes of serum biochemical indexes with toxicological significance were observed in the present study.
Table 2.
Effects of QFDY on urine indexes in end of treatment period (rat numbers)
| Parameter | Level | Males | Females | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| SC | LQ | MQ | HQ | SC | LQ | MQ | HQ | |||
| UBG | - | 10 | 10 | 10 | 7 | 10 | 9 | 10 | 6 | |
| ± | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| + | 0 | 0 | 0 | 3 | 0 | 1 | 0 | 4a | ||
| BIL | - | 10 | 10 | 8 | 2 | 10 | 8 | 9 | 3 | |
| ± | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| + | 0 | 0 | 2 | 8b | 0 | 2 | 1 | 7b | ||
| KET | - | 5 | 0 | 0 | 0 | 10 | 8 | 7 | 2 | |
| ± | 5 | 9 | 7 | 4 | 0 | 2 | 3 | 8b | ||
| + | 0 | 1a | 3a | 6b | 0 | 0 | 0 | 0 | ||
| LEU | - | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | |
| ± | 1 | 1 | 0 | 0 | 4 | 0 | 1 | 0 | ||
| + | 8 | 9 | 9 | 5 | 6 | 9 | 7 | 5 | ||
| ++ | 1 | 0 | 1 | 4 | 0 | 1 | 1 | 5c | ||
| +++ | 0 | 0 | 0 | 1a | 0 | 0 | 0 | 0 | ||
Notes: SC groups: treated with distilled water; LQ, MQ, and HQ groups were given 4, 8 and 16 g/kg QFDY per day, respectively, each group (n = 20, 10 males and 10 females) was administered once a day for 28 d. SC: solvent control group; LQ: low-dose group; MQ: medium-dose group; HQ: high-dose groups; UBG: urobilinogen; BIL: bilirubin; KET: ketone body; LEU: urine leukocyte. aP < 0.05, bP < 0.001, cP < 0.01, significantly different from the SC group.
3.3.5. Urinalysis
The urine colour of male and female rats in SC, LQ, MQ and HQ groups were normal, light red, medium red, and high red, respectively, showing an obvious dose-effect relationship.
At the end of the experiments, the positive rates of UBG, BIL, KET, and LEU in test groups were higher than those in the SC group with statistically significant differences (Table 2). However, all the positive indicators returned to normal in the recovery period (Table 3).
Table 3.
Effects of QFDY granules on urine indexes in end of recovery period (rat numbers)
| Parameter | Level | Males | Females | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| SC | LQ | MQ | HQ | SC | LQ | MQ | HQ | |||
| UBG | - | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
| ± | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| + | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| BIL | - | 4 | 5 | 4 | 5 | 5 | 5 | 5 | 5 | |
| ± | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| + | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | ||
| KET | - | 3 | 2 | 0 | 0 | 5 | 5 | 5 | 5 | |
| ± | 1 | 3 | 4 | 4 | 0 | 0 | 0 | 0 | ||
| + | 1 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | ||
| LEU | - | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |
| ± | 0 | 2 | 1 | 0 | 2 | 3 | 2 | 5 | ||
| + | 5 | 3 | 4 | 5 | 1 | 2 | 3 | 0 | ||
| ++ | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||
| +++ | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||
Notes: SC groups: treated with distilled water; LQ, MQ, and HQ groups were given 4, 8 and 16 g/kg QFDY per day, respectively, each group (n = 10, 5 males and 5 females) was administered once a day for 28 and 30 d of post treatment was conducted. SC: solvent control group; LQ: low-dose group; MQ: medium-dose group; HQ: high-dose groups; UBG: urobilinogen; BIL: bilirubin; KET: ketone body; LEU: urine leukocyte.
To analyze urine color and index abnormalities, the tested QFDY were mixed into different concentration solutions (143, 0.071, 0.036, 0.018 and 0.009 g/mL) for color observation and routine urine detection in an additional experiment. It’s showed that the color of solution becomes darker with the increase of concentration, and the colour of the tested substances had an influence on the determination results of UBG, BIL, KET, and LEU (supplementary Table 3).
3.3.6. Pathology
The kidney/brain of female rats in the LQ (end of administration) and HQ (recovery stage) groups were lower than those in the SC group (P < 0.05), both were no dose-response relationship and no toxicological significance. The statistical differences were considered to be incidental and of no clinical significance. There were no other significant differences in the organ weight, organ/body and organ/brain in the treatment groups of female and male rats (supplementary Tables 4 and 5). No toxicological changes in the rat organs were observed in the present study.
The results of histopathological examinations showed that some animals showed inflammatory cell infiltration in livers and renal interstitium, proliferation and aggregation of mononuclear macrophages in lungs, calcium deposit in renal tubules, dilation of renal tubules, degeneration of epithelial cells, dilation of uterine cavity and other pathological changes during administration period and recovery period. The above pathological changes occurred simultaneously in the SC group, the animals of this strain were common spontaneous lesions, and the number of lesions was very small. These pathological changes should be unrelated to the test substance and have no toxicological significance. The details are showed in Figure 3, supplementary Tables 6 and 7.
Figure 3. Histological findings of SC group and HQ group in the recovery period.
A: histological findings of SC group in lungs (10 × 40); B: histological findings of HQ group in lungs (10 × 40); C: histological findings of SC group in livers (10 × 20); D: histological findings of HQ group in livers (10 × 20); E: histological findings of SC group in kidneys (10 × 20); F: histological findings of HQ group in kidneys (10 × 10). SC groups: treated with distilled water, HQ groups: given 16 g/kg QFDY per day, each group (n = 30) was administered once a day for 28 and 30 d of post treatment was conducted. All stained with hematoxylin-eosin. SC: solvent control group; HQ: high-dose groups.
4. DISCUSSION
It is well known that TCM have been utilized in various illnesses treatment in China. However, the characteristics of multi-ingredient, multi-target, multiple-pathway and overall regulation make some safety and effectiveness issue.18 The present study firstly investigated the anti-inflammatory and anti-tussive effects of QFDY, a traditional Chinese prescription used to treat COVID-19 by improving symptoms of fever, cough and inflammation, etc. Moreover, the acute and sub-chronic toxicity in rats were evaluated.
Coughing and inflammatory processes have been involved in the pathogenesis of pneumonia.16 In vivo activity analysis, QFDY showed a significant anti-inflammatory effects in the xylene-induced ear edema in mice model. The swelling degree of mice in treatment groups were lower than that in control group, and presented a dose-effect relationship, while TNF-α and IL-6 decreased more significantly in the low dose groups. QFDY significantly prolonged the incubation time and reduced coughing times in the mouse cough model induced by ammonia liquor. The high-dose group showed good capability of reducing cough times with the inhibition of 65.8% and cough activity of 92.8%, which close to pentosyverine control group.
There are a variety of Traditional Chinese Medicines in this prescription, and some of them have been reported to have side effects, such as Houpu (Cortex Magnoliae Officinalis), Binglang (Semen Arecae), etc.13,19 In the acute toxicity test, the maximum allowable dose was calculated to be 24.0 g/kg per day, which was about 48 times of the clinical intended dose.
In the sub-chronic toxicity test, the high-dose group was set to be 16 g/kg per day, which was 66.67% of the maximum allowable dose and 32 times of the clinical intended dose. During the study period, higher RET, LYMP and lower Cl－, BUN were analyzed. But the differences were considered to be incidental and not clinically toxic. Obvious dose-effect relationship of urine color was observed, and the three test groups at the end of the experiments resulted in significant increase in UBG, BIL, KET and LEU. However, all the positive indicators returned to normal in the recovery period. The darker color of urine due to the fact that the drug itself is reddish brown. After long-term high dose administration in rats, the metabolites were discharged from the urine, resulting in the color of urine. The positive rates of UBG, BIL, KET, and LEU in urinalysis may be due to the fact that these indicators were measured by color reaction,20 and the urine color itself interfered with the test results. Therefore, no toxicological changes were considered on clinical observations, body weights, food intakes, ophthalmological examinations, hematological parameters, biochemical indicators, electrolyte indicators, urinalyses and histopathological examinations in the present study.
In conclusion, QFDY showed significant anti-inflammatory and anti-tussive effects, and presented a dose-effect relationship. The LD50 of QFDY in SD rats was more than 24.0 g/kg per day in the acute toxicity test, equivalent to crude drug 124.0 g/kg. And the no observed adverse effect level of QFDY was more than 16 g/kg per day in oral sub-chronic toxicity test, equivalent to crude drug 82.7 g/kg. The above two doses are much higher than the clinical dosage. Therefore, QFDY is relatively safe for oral medication at the present dose on rats. Our experimental results will provide a reference for the further development and research of QFDY.
5. SUPPORTING INFORMATION
Supporting data to this article can be found online at http://journaltcm.com.
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