During hundreds of years of medication, it is believed that the steamed
During hundreds of years of medication, there was a description for
Despite differences in pharmacological effects of RPN and SPN, there is no clear standard for the steaming of SPN. Until now, the processed PN has never been recognized by any pharmacopeias of different countries or regions [
Moreover, different from the mechanism of chemical drugs, traditional Chinese medicine (TCM) has the characteristics of multicomponents, multitargeted, and multichannels, which emphasize integrity and systemicity [
Furthermore, prevailing evidence demonstrated that Box-Behnken design response surface methodology (BBD-RSM) is widely used in the researches of the extraction, preparation, and processing of TCM, which is a set of mathematical and statistical techniques used to establish models for evaluating multiple parameters and their interactions with quantitative data and statistically and effectively optimizing the complex extraction procedures [
SPN samples were obtained by steaming the RPN powder in an autoclave (Zhejiang, China) at different steaming conditions and then drying in an electric-thermostat-blast drying oven (Shanghai, China) at 40°C for 3 hours. The SPN samples were crushed and sieved through a 40-mesh sieve. Ginsenosides Re, Rd, Rh1, Rh2, Rk3, Rh4, 20(
Specific-pathogen-free BALB/c mice were purchased from Guangdong Medical Laboratory Animal Center, China (SCXK(Yue)2018-0002), and kept in standard environment in the lab animal room in Clinical College, Hubei University of Chinese Medicine. The anemic model of mice was established according to the methods described by previous studies [
Numerous studies have shown that saponins are the major active components of SPN, with pharmacologic effects such as blood enriching, antioxidation, antiplatelet, and anticoagulant [
To predict the hematopoietic active ingredients of SPN, we need to analyze the complex relationship between the constituents and targets associated with anemia. So, the “constituent-target-disease” network was constructed to help us study the blood-enriching mechanism. In the network, the nodes with different colors and shapes represented different compounds and targets, and the edges represented the interactions between the targets and compounds. Cytoscape 4.3 was used to visualize and analyze the network and calculate the topological features of each node in the network [
To verify the hematopoiesis activities of the active constituents, mice were randomly divided into 18 groups (
Trizol reagent (Invitrogen, USA) was used to extract total RNA from the serum of different groups in mice according to the manufacturer’s instruction. Quantitative real-time PCR (qRT-PCR) analyses were performed with SYBR® Premix Ex Taq™ (Takara, USA). Relative quantification was performed using the 2-
In order to make the SPN have better blood-enriching effect, the active ingredients are used as evaluation indexes. And variables including the steaming time (2-10 h), steaming temperature (90-130°C), and producing area (Baoshan, Dali, Honghe, Lijiang, Puer, Qujing, Tengchong, Wenshan, and Yuxi) were investigated by using the BBD-RSM. A 500.0 g PN sample powder from different producing areas was separately placed in an autoclave (Zhejiang, China) and steamed at a given temperature and time. Subsequently, a 5.0 g sample powder of SPN was extracted by 50 mL of 70% ethanol for 1.5 h at 85°C in a water bath (Bang Xi Instrument Technology Co., Ltd. Shanghai, China). Repeat the extraction process three times, and the extract solution was combined, centrifuged, filtered, and concentrated to obtain the saponins of SPN which are related to hematopoietic effect.
The sample solutions were prepared by dissolving the crude saponins of SPN of 2.4.1 into a 20 mL volumetric flask and dissolving with pure water, shaken, filtered, and the subsequent filtrate reserved. The mixed standard solution was prepared by adding standard substances including Rd, Rh1, Rh4, Rk3, and 20(
HPLC chromatograms of reference solution of (a) PN and (b) SPN samples (taken from the sample of SPN steamed for 5 h at 120°C as an example). Peaks 1, 2, 3, 4, and 5 correspond to ginsenosides Rh1, Rd, Rk3, Rh4, and 20(
According to the published articles [
The BBD-RSM was adopted to determine the optimal steaming conditions of SPN. And based on the results of the single-factor experiment analysis, we finally designed three-factor, three-level experiments involving steaming time (4-8 h), steaming temperature (110-130°C), and producing area (Honghe, Qujing, and Wenshan). The dependent variable (the content of saponins) was considered as the response. In this design, the test variable was transformed to the range of -1 to 1 for the evaluation of factors (Tables
Experimental domain of BBD-RSM.
Independent variables | Unit | Symbol | Coded levels | ||
---|---|---|---|---|---|
-1 | 0 | +1 | |||
Steaming temperature | °C | 110 | 120 | 130 | |
Steaming time | h | 4 | 6 | 8 | |
Producing area | Honghe | Wenshan | Qujing |
The BBD matrix and the experimental data for the responses.
Treatment No. | Steaming temperature (°C) | Steaming time (h) | Producing area | Content of saponins (%) |
---|---|---|---|---|
1 | -1 | 0 | 1 | 3.037 |
2 | 0 | -1 | -1 | 3.889 |
3 | 1 | 1 | 0 | 3.214 |
4 | 0 | 1 | -1 | 3.643 |
5 | 1 | -1 | 0 | 3.635 |
6 | -1 | -1 | 0 | 2.914 |
7 | 0 | 0 | 0 | 3.896 |
8 | -1 | 1 | 0 | 3.015 |
9 | -1 | 0 | -1 | 3.025 |
10 | 0 | 1 | 1 | 3.658 |
11 | 0 | -1 | 1 | 3.895 |
12 | 0 | 0 | 0 | 3.992 |
13 | 0 | 0 | 0 | 3.979 |
14 | 0 | 0 | 0 | 3.812 |
15 | 1 | 0 | 1 | 3.482 |
16 | 0 | 0 | 0 | 3.967 |
17 | 1 | 0 | -1 | 3.846 |
To determine the hematopoiesis activity of the optimal SPN, mice were randomly divided into six groups (
Results were obtained as the
In our study, the twenty-four kinds of saponins of SPN which have been reported in the literature were investigated [
The “constituent-target-disease” pharmacology network of SPN saponins. The red triangles represented the saponins of SPN, the blue dots represented the indirect targets of those saponins, the yellow dots represented the targets of anemia, the purple dots represented the interactional proteins of the anemia targets and saponins of SPN, and the yellow squares represented the common targets of SPN saponins and anemia.
To predict the target proteins of SPN, the pharmacological network of “constituent-target-disease” was constructed. As shown in Figure
The active constituents and the corresponding common target proteins predicted by network pharmacology analyses.
Constituent | Structure | Target proteins |
---|---|---|
Ginsenoside Rd | P16442 | |
Ginsenoside Re | P16442 | |
Ginsenoside Rh2 | P16224 | |
Ginsenoside Rh4 | P16442 | |
Ginsenoside Rh1 | P16442 | |
Ginsenoside Rk3 | P22830 | |
Ginsenoside 20( | P22830 | |
Ginsenoside 20( | P16442 |
Remark: P16442: histo-blood group ABO system transferase; P06702: protein S100-A9; Q06124: tyrosine-protein phosphatase nonreceptor type 11; P30163: pyruvate kinase PKLR; P22830: FECH, mitochondrial.
To further verify the hematopoiesis activities of ginsenosides Rk3, Rh4, Rh1, 20(
Effects of ginsenosides Rk3, Rh4, Rh1, Rh2, Re, Rd, 20(
Effects of the active constituents responsible for SPN hematopoietic function on visceral index in mice (
Grouping | Liver index (mg/g) | Kidney index (mg/g) |
---|---|---|
Control | ||
Model | ||
Rk3-L | ||
Rk3-M | ||
Rk3-H | ||
Rh4-L | ||
Rh4-M | ||
Rh4-H | ||
Rh1-L | ||
Rh1-M | ||
Rh1-H | ||
20( | ||
20( | ||
20( | ||
Rd-L | ||
Rd-M | ||
Rd-H |
Note: model groups compared with the control groups,
Meanwhile, the expressions of Kitlg, Csf1,Csf3, IL-6, IL-11, and Lif in different groups were determined. Kitlg, also known as stem cell factor, is a factor that induces the growth of the earliest hematopoietic stem cells. It mainly stimulates the proliferation of hematopoietic stem/progenitor cells (HSC/HPC) and promotes the formation of hematopoietic colonies in coordination with various Csfs [
Effects of ginsenosides Rk3, Rh4, Rh1, Rh2, Re, Rd, 20(
Steaming temperature is one of the most important factors that could affect the content of saponins. The contents of saponins under different steaming temperature were shown in Figure
The results of single-factor experimental analysis.
Another significant factor that would affect the content of saponins is the steaming time. Thus, in the present study, the effect of steaming time on the content of saponins was evaluated. The steaming temperature and the producing area were set as 120°C and Wenshan, respectively. The steaming time was then sequentially set as 2 h, 4 h, 6 h, 8 h, and 10 h. As shown in Figure
PN, a traditional and precious medicinal material unique to China, has special requirements for the environment. Its distribution is limited to the mid-high altitude area near 23°30
As shown in Tables
The
The analysis of variance (ANOVA) was used to estimate the statistical significance of factors and their interaction [
The ANOVA results for the response surface quadratic models for the content of saponins of SPN.
Source.(content of saponins) | Sum of squares | DF | Mean square | ||
---|---|---|---|---|---|
Model | 2.36 | 9 | 0.26 | 31.47 | <0.0001 |
0.6 | 1 | 0.6 | 71.64 | <0.0001 | |
0.081 | 1 | 0.081 | 9.67 | 0.0171 | |
0.014 | 1 | 0.014 | 1.64 | 0.2408 | |
0.068 | 1 | 0.068 | 8.17 | 0.0244 | |
0.035 | 1 | 0.035 | 4.24 | 0.0785 | |
1 | 0.9621 | ||||
1.41 | 1 | 1.41 | 169.42 | <0.0001 | |
0.1 | 1 | 0.1 | 12.21 | 0.0101 | |
1 | 0.9572 | ||||
Residual | 0.058 | 7 | |||
Lack of fit | 0.036 | 3 | 0.012 | 2.1 | 0.2434 |
Pure error | 0.023 | 4 | |||
Cor total | 2.42 | 16 | |||
0.9759 | |||||
0.9449 | |||||
CV | 2.55 | ||||
AP | 14.822 |
The comparison between the predicted and measured values of the content of the saponins from the SPN.
In this model, as shown in Table
Figure
Response (a) surface and (b) contour plots showing the significant (
In order to make the SPN have a better blood-enriching effect, the steaming conditions of PN need to be optimized to obtain the maximum saponin content which is associated with blood-enriching effects. So, the quadratic models were established, and the predicted maximum value (3.99%) of the test variables was found under the following conditions: steaming time 5 h, steaming temperature 120°C, and place of origin Wenshan. Experiments were performed under the recommended optimum steaming conditions; the actual values of the content of saponins were
As shown in Figures
The hematopoiesis effects of SPN steamed at 120°C for 5 h evaluated by peripheral blood parameter measurement. Data were expressed as
The effects of SPN steamed at 120°C for 5 h on hematopoietic factor expressions. Data were expressed as
In the research, to obtain a kind of SPN which has better blood-enriching effect, the bioactive constituents of SPN were unveiled and verified by network pharmacology coupled with pharmacology experiments. Results showed that Rd, Rh1, Rh4, Rk3, and 20(
Adequate precision
Box-Behnken design response surface methodology
Hemoglobin
Red blood cell specific volume
Platelet
Prediction error sum of squares
Regression coefficient
Red blood cell
Ribavirin
Raw PN
Steamed
Traditional Chinese medicine
White blood cell
Kit ligand
Colony-stimulating factor
Interleukin-6
Interleukin-11
Leukemia inhibitory factor
Hematopoietic stem/progenitor cells.
All data used to support the findings of this study are available from the corresponding author upon request.
The funder had no role in the study design, data collection and analysis, interpretation of data, or writing of the manuscript.
The authors declare that they have no competing interests.
Shuhan Zhou, Nan Jiang, and Min Zhang contributed equally to this work.
This research was supported by grants of Inheritance and Innovation in Traditional Chinese Medicine from Hubei University of Chinese Medicine (grant number 5428/1011010301) and the Hubei Provincial Department of Education (grant number Q20101805).