Background
Although traditional medicine provides front-line pharmacotherapy for millions of Chinese, its application is often viewed with skepticism by the Western medicine establishment [
1]. There has been wide concern about the toxicity of herbal medicine, and several side effects (such as allergic reactions, hepatotoxicity, nephrotoxicity, and cardiac toxicity) of herbal medicines have been reported in recently years [
2].
Psoriasis is a chronic inflammatory skin disease affecting more than 125 million people worldwide [
3]. Currently, there is no cure for psoriasis. Patients with psoriasis in China often turn to alternative and complementary treatments, which are considered to be effective and safe [
4]. Jueyin granules (JYG), an effective formula consisting of eight Chinese herbs (
Haliotis diversicolor, Flos Lonicerae Japonicae, Radix Rehmanniae exsiccate
, cortex moutan,
Herba Hedyotisdiffusae, Folium isatidis, Smilax china L. and Radix Curcumae) were discovered in the 1950s by Han Xia (a well-known Chinese surgeon) and have been used to clinically treat psoriasis for over 50 years by Yueyang Hospital of Integrated Traditional Chinese and Western Medicine. Our previous study showed that JYG can reduce inflammation and proliferation of keratinocytes and prevent psoriasis in animal models [
5]. Moreover, the major ingredients, including
Haliotis diversicolor, Flos Lonicerae Japonicae, Herba Hedyotis diffusae, Folium Isatidis, Smilax china L., Radix Curcumae, have been demonstrated to have anti-inflammatory effects in vitro and in vivo models [
6‐
12]. The ingredient
Cortex Moutan has been reported to have an inhibitory effect on proliferation of HaCaT cells in vitro models [
13]. However, the toxicity of JYG has not been well studied. The objective of this study was to evaluate the safety of JYG in animal models.
Methods
Testing materials
Jueyin granules(manufactured by Tianyin Pharmaceutical Co. Ltd., Jiangsu Province, China; Certified Number of 20,120,103) were prepared using a water–alcohol extraction method and its quality control was performed using high-performance liquid chromatography (HPLC) by detecting chlorogenic acid and paeonol as shown in a previous publication [
5]. The suspension of the drug was prepared by purified water. Its composition is shown in Table
1.
Table 1
Ingredients of JYG used with English translations
Haliotis diversicolor
| Concha Haliotidis | Haliotis diversicolor Reeve | 15 g |
Flos Lonicerae japonicae
| Honeysuckle flower | Lonicera japonica Thunb | 12 g |
Radix Rehmanniae exsiccata
| Dried Rehmannia root | Rehmannia glutinosa(Gaertn.)Libosch | 15 g |
Cortex Moutan
| Tree peony bark | Paeonia suffruticosa Andr | 12 g |
Herba Hedyotisdiffusae
| Oldenlandia | Hedyotis diffusa Willd | 15 g |
Folium isatidis
| Dyer’s woad leaf | Isatis indigotica Fort | 15 g |
Smilax china L.
| Chinaroot greenbrier rhizome | Smilaz china L | 15 g |
Radix Curcumae
| Turmeric root tuber | Curcuma longa L | 9 g |
Animals
ICR mice weighing 17.2–19.8 g, purchased from Shanghai Super B & K Laboratory Animal Corp., Ltd., were used for the acute toxicity test. Six-to-seven-week-old SPF grade Wistar rats, purchased from Beijing WeiTongLiHua Experimental Animal Technology Co., Ltd., were used for the chronic toxicity test (Animal certificate no. 11400700011308). All animals were housed in groups of five rats per cage under a schedule of 12 h light/12 h dark and in a controlled temperature of 21 °C–24 °C. Animals had free access to standard laboratory animal feed and water. The experimental protocols were approved by the institutional Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (No. 14480 and 14,486).
Testing methods
Acute toxicity test
Forty ICR mice were randomly divided into two groups, each comprising 10 males and 10 females. For 18 h before the start of the experiment, the mice had access to water but no food. The mice were fed JYG oral solution, 40 ml/kg, by gavage twice a day for 14 days. The animals’ skin, mucous membrane, changes in fur color, eyes, circulation, central nervous system, respiration, and conscious behavior were observed daily. Body weights were also measured once a week. Mice were euthanasized with CO2 inhaltion on the 14th day.
Chronic toxicity test
A total of 160 Wistar rats were randomly divided into four groups, each group comprising 40 animals of 20 males and 20 females. All rats in the experimental groups were fed JYG oral solution once a day at graded doses of 7.5 (JYG-H), 3.75 (JYG-M), and 1.875 (JYG-L) g/kg for 3 months and 6 months, respectively, followed by a recovery period of 4 weeks. Those in the control group were administered distilled water at 20 ml/kg/d. The rats were observed daily for abnormal behavior and other adverse signs of toxicity. Consumption of food and water as well as body weight were recorded weekly. All animals were euthanasized with CO2 inhaltion at the end of testing, and blood samples were obtained for the biochemical assays. The liver, kidney, lung, heart, spleen, brain, ovaries, testes and adrenal gland were all collected, weighed, and homogenized. A portion of each organ was removed for histological studies.
Biochemical assay
Hematological assessments
Hematological parameters, such as total white blood cell count, red blood cell count, packed cell volume, hemoglobin, and platelets (PLT), were determined using a fully automated hematology analyzer (Simens, Bayer ADVIA120, Germany).
Liver, renal function and serum electrolytes tests
A Hitachi 7020 Automatic Biochemical Analyzer was used to detect aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, blood urea nitrogen, total protein, albumin, blood glucose, total bilirubin, creatinine, total cholesterol, triglycerides, creatine kinase, sodium ion concentration, potassium ion concentration, and chloride ion concentration.
Histopathology
The brain, liver, spleen, adrenal gland, epididymis, uterus, heart, kidney, testis, ovary, lung, and thymus were all weighed to calculate organ coefficients. The thyroid, stomach, pancreas, testis, prostate, aorta, bladder and bone marrow were preserved in 10% Faure Marin solution, fixed for 36–48 h, and subjected to conventional histological processes for histopathological examination. The tissue sections were examined under a microscope with a 40× objective to check cell morphology and quantity.
Statistical analysis
The experimental data were analyzed using SPSS 21 statistical software. All data were expressed as the mean ± the standard error of the mean (SEM).Significant differences among the groups were determined by a one-way analysis of variance and post hoc testing was performed for inter-group comparisons for least significant differences (LSDs) using a statistical analysis program for social science (SPSS).
Discussion
Psoriasis, first defined by Ferdinand von Hebra as a distinct entity in 1841, is a common chronic inflammatory skin disorder. It is characterized by abnormal hyperproliferation of epidermal keratinocytes and infiltration of immunocytes along with angiogenesis [
14]. The prevalence of psoriasis has been reported to range from 0.91% to 8.5% in adults and from 0 to 2.1% in children [
15]. Despite the advances in what is known about the pathogenesis of psoriasis, modern therapies have had limited effects. People are looking for novel drugs to treat psoriasis, but many patients cannot afford the new therapies because of their high cost [
16]. Moreover, patients suffering from psoriasis are subjected to considerable physical and psychological disorders,which can aggravate the severity of psoriasis. This situation impairs the quality of life of people with psoriasis [
17‐
19].
Recently, more people with psoriasis have turned to complementary and alternative medicine (CAM) because of its low cost and minimal adverse effects [
8]. Traditional Chinese medicine is part of such therapies. Deng et al. [
20] reported that herbal formulations could significantly improve the modified psoriasis area severity index score. In addition, these herbs and/or their constituents have anti-inflammatory, anti-angiogenic, anti-proliferative, and tissue repair actions. However, some herbal remedies or herbal formulae can produce a wide range of adverse reactions, even death [
21‐
24]. Therefore, in recent years scientific investigation of CAM has been focused mainly on safety and toxicological evaluations [
25].
In the present study, we investigated the acute and chronic toxicity of JYG, which was created in the 1950s by a well-known Chinese surgeon named Han Xia. Although clinical experience and animal studies have demonstrated the effectiveness of JYG in treating psoriasis, some patients suffering from weight loss, gastrointestinal symptom and abnormal liver function were reported in a recent publication [
26]. Thus an evaluation on the safety of JYG is necessary.
The acute toxicity test results show that JYG cause no abnormalities or mortality with a maximum dose of 21.5 g/kg, equivalent to 143 times the clinical dose (0.15 g/kg) for a person weighing 60 kg. Therefore JYG could be regarded as a partially nontoxic compound.
In the chronic toxicity test, food consumption and body weight showed a tendency to decrease in the JYG-treated group, especially in male rats. All rats’ weight was recovered at 14 days after the withdrawl of the treatment. Although reduced food intake and weight loss were consistent with clinical observations, there was no significant dose response observed among animals in any of the three groups with different doses. In addition, organ weight and histopathological examinations also remained close to or within the normal range suggesting JYG showed no toxic effects on digestive system in JYG-treated rats. Therefore, we speculate that the reason for the differences is that animals could not adapt to the solution administered. In addition, we recommend that caution still should be taken in determining the dosage of JYG for children.
The serum enzyme levels, organ weight and histopathological examinations remained close to the control values indicating chronic administration of the drug JYG neither impaired the physiology of the liver nor the cellular structures of the liver, which is inconsistent with the clinical observation. However, we speculated the abnormal liver function in the patient may be caused by other reasons such as alcohol consumption rather than JYG administration, since the patient had an alcohol drinking history [
26]. No hematological or other biochemistry alterations, or delayed toxic reactions were found in JYG-treated rats. These results were consistent with the recent clinical study showing that patients treated with JYG formulation twice a day for a continuous four-week period had no adverse effects in hematology or hepatorenal functions [
26].
Conclusion
Jueyin granules at the given doses did not produce acute and chronic toxicity in animal models. There were no statistically significant alterations found in behavior, biochemistry, hematological parameters, organ weight, or histopathology. But children, the elderly and those with abnormal digestive function should be used with caution.
Acknowledgements
We thank SIFDC for their contribution, especially director Lian Ning, whose work helped us to overcome technical problems.
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