Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

Moutan Cortex (MC), a well-known medicinal herb and distributed in multiple regions of China, has been found to be benefical for improving diabetic nephropathy (DN). The geoherb one worked particularly well with specific structural composition of components (SCC). However, whether its better efficacy of geoherb MC than others on DN was attributed to SCC was still unclear. Medicinal plants of MC were collected from ten regions of China, including Gansu, Chongqing, Shandong, Sichuan, Zhejiang, Hunan, Guizhou, Hebei, Henan and Anhui provinces, which were classified for three categories by cluster analysis. HPLC analysis demonstrated that the content and structural composition of 13 compounds were discrepancy. Principal component analysis (PCA) showed six compounds including oxypaeoniflorin, paeoniflorin, trigalloyl glucose, pentagalloyl glucose, benzoylpaeoniflorin and paeonol were the main contributors to its anti-DN activity via increasing superoxide dismutase (SOD) activity, decreasing malondialdehyde (MDA) content, reducing IL-6 and MCP-1 levels in vitro. The results of advanced glycation end products (AGEs)-induced HBZY-1 cells and STZ-induced DN rats showed a significant efficacy difference among MC from ten regions on catalase (CAT), glutathione peroxidase (GSH-Px) activities, intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-β1 (TGF-β1) and fibronectin (FN) protein expression in vivo and in vitro. The recombination of SCC in Hebei MC (worst efficacy) to Anhui MC (best efficacy) showed a similar efficacy. The results demonstrated that the efficacy of MC was closely related to its SCC. Our findings provide evidence for the importance of SCC in quality control about Chinese materia medica and also provide an novel insight into the better efficacy of geoherb one.


INTRODUCTION
The quality of Chinese materia medica (CMM) is a key factor to ensure the efficacy.The quality inconsistency of CMM from different regions has been found to be closely related to non-repeatability of clinical efficacy and safety [1].
However, the reason why the inconsistency in efficacy and safety of CMM from different regions is not only associated with contained compounds, but also with structural composition of components (SCC).The stable SCC acts as a crucial contributor to the quality consistency of CMM which is related to the better efficacy and monitored safety.
Geoherbs, the famous authentic herbs, which grew under the specific natural conditions and ecological environment, play an indispensable role in the prevention and treatment of diseases in clinic [2].Geoherbs had been considered as the best quality of CMM with better efficay than others [3].The best quality with better function is caused by the special and stable proportion of active components, namely SCC, not the unique compounds or higher content of some compounds in geoherbs.Therefore, the structural proportion of these components could not only reveal the constitutive characteristics of CMM, but also provide effective strategy for the quality control of CMM.
Moutan Cortex (MC), the dried root bark of dicotyledonous plants Paeonia suffruticosa Andr., has a significant efficacy on detoxification and blood circulation [4,5].MC, recorded in the "Shen Nong's Herbal Classic", was used for the treatment of cardiovascular disease, hepatoprotective effect, hypoglycemic, hypolipidemic, hypotensive and the central nervous system [6][7][8].Additionally, our previous research has confirmed the attenuation of MC on oxidative stress for renal injury in AGEs-induced mesangial cells dysfunction and streptozotocin-induced DN rats [9][10][11].MC, distributed in multiple regions of China, has a good protective effect on DN, especially the geoherb one.There were great differences between SCC and efficacy in MC from different regions.To reveal the better efficacy of geoherb MC and provide further experimental support for quality control of CMM, the present study was designed to compare the difference on SCC and efficacy of MC between geoherb and nongeoherb and also clarified the SCC related to the active components in geoherbs.

Comparison of MC from different regions on CAT and GSH-Px activities in AGEs-induced HBZY-1 mesangial cells
As shown in Table 1, the CAT (2.29 ± 0.21 U/mL) and GSH-Px (204.17 ± 37.10 U/L) activities in HBZY-1 mesangial cells were decreased remarkably by 200 μg/mL AGEs, compared with blank control group (P < 0.001).The treatment of MC from different regions could improve AGEs-decreased CAT and GSH-Px activities significantly (P < 0.001, P < 0.01).Interestingly, Anhui MC, geoherb one, was considered to have the best activity on improving CAT (6.81 ± 0.42 U/mL) and GSH-Px (413.44 ± 8.45 U/L) activities.The results demonstrated that MC from different regions made different contributions to attenuate AGEsinduced oxidative stress.Especially, the geoherb MC (Anhui) held the better activity on improving the activities of CAT and GSH-Px than others.

Difference of MC on ICAM-1, TGF-β1 and FN expressions in AGEs-induced HBZY-1 mesangial cells
Western blotting was used to evaluate the protein expressions of ICAM-1, TGF-β1 and FN after HBZY-1 mesangial cells exposed to AGEs in the presence of MC from different regions.As shown in Figure 1A, the stimulation of AGEs could increase significantly these protein expressions.The treatment of MC from different regions could significantly decrease the levels of inflammatory factors in AGEs-induced mesangial cells.The pharmacological order of MC from different regions on decreasing ICAM-1 expression from high to low was Anhui, Guizhou, Zhejiang, Henan, Shandong, Hunan, Hebei, Sichuan, Gansu and Chongqing (Figure 1B).Similarly, the order of MC to decrease the TGF-β1 expression from high to low was Anhui, Sichuan, Shandong, Gansu, Chongqing, Hunan, Henan, Zhejiang, Guizhou and Hebei (Figure 1C).The results shown in Figure 1D indicated that the pharmacological order of MC decreasing FN expression from high to low were Shandong, Anhui, Sichuan, Guizhou, Zhejiang, Gansu, Hunan and Hebei.Generally, Anhui MC, geoherb one, was considered as the best to decrease ICAM-1, TGF-β1 and FN expressions.

Effects of MC from different regions on CAT and GSH-Px activities in DN rats
After the treatment of MC from different regions, CAT and GSH-Px activities in serum of DN rats were shown in Table 2. CAT and GSH-Px activities of control group were 5.42 ± 0.410 U/mL and 458.01 ± 39.112 U/L, respectively.However, these activities in model group were reduced significantly (CAT: P < 0.01, GSH-Px: P < 0.001).Compared with the model group, their activities were increased in MC-treated groups and positive drug (AG) group.The ability to improve CAT activity from high to low was Anhui, Sichuan, Hunan, Guizhou, Chongqing, Gansu, Zhejiang, Shandong, Henan and Hebei; while the ability to increase GSH-Px activity from high to low was as follow: Anhui, Shandong, Henan, Zhejiang, Chongqing, Sichuan, Hebei, Hunan, Guizhou and Gansu.The results also showed that Anhui MC had the best efficacy on increasing CAT and GSH-Px activities in vivo.

Reduction of MC on ICAM-1 and TGF-β1 expressions in vivo
As shown in Figure 2, there were no obvious pathological changes in kidney tissues of control group.Namely, ICAM-1 and TGF-β1 expressions were observed normal under light microscope.In DN model group, ICAM-1 and TGF-β1 expressions were significantly increased compared with control group.When DN rats were treated with MC from 10 regions and AG, the relative expression levels of these inflammation cytokines ICAM-1 and TGF-β1 in DN rats was relived significantly.Figure 2A showed that MC from Anhui, Sichuan and Shandong declined ICAM-1 expression more obviously, while Hunan MC decreased TGF-β1 expression more obviously than others (Figure 2B).

Pharmacodynamic cluster analysis of MC from ten regions
The classification results were given directly by cluster analysis.By the comparative analysis of physiological, biochemical indexes after administration of MC from 10 regions, which was proved that the efficacy  was different between MC from different regions.Cluster analysis method could effectively classify different efficacy of MC.As shown in Figure 3A, MC from Guizhou, Zhejiang, Henan and Anhui were classified as a category due to the high efficacy.MC from Hunan, Hebei, Chongqing, Gansu, Sichuan and Shandong were classified as another class for the poor efficacy.It was indicated that there were no obvious differences of efficacy between MC from Anhui, Guizhou, Zhejiang and Henan.However, it also indicated the essential differences in quality of MC from different regions.

MS analysis and comparison on the content of active components of MC from different regions
MCs from 10 different regions were collected in our research (Figure 4A).The results in Table 3 showed the extraction rate of MC from different    origins was different.As shown in Figure 4B and Table 4, it showed no significant difference in the categories of components but SCC with respect to the active components was different among MC from 10 regions.Thus, the cause of pharmacodynamic differences of MC from different regions was the stability of these efficacy components.

Cluster analysis for MC from different regions
The peak areas of 13 chemical compositions in MC from different regions were set as quantitative traits to conduct cluster analysis by SPSS16.0.Combined with the cluster analysis of peak area, MC from Zhejiang, Henan and Anhui were considered as a class for the same constituted composition.It was indicated that geographical advantages would affect the quality of the CMM.MC from Zhejiang, Henan and Anhui were grouped as another class due to the similar constitution.As shown in Figure 3B, Hebei MC was divided into a separate class due to the significant difference in the ratio of active components compared with Anhui MC.

Principal component analysis for active components in MC
A few indicators could be filtered by principal component analysis to reflect the overall message.To select representative compounds among 13, the peak areas of 13 compounds (mudanoside B, paeoniflorin sulfonate, oxypaeoniflorin, paeoniflorin, trigalloyl glucose, tetragalloyl glucose, tetragalloyl glucose, pentagalloyl glucose, hexagalloyl glucose, galloylpaeoniflorin, benzoylpaeoniflorin, mudanoside A, paeonol) from 10 regions were analyzed by principal component analysis using SPSS 16.0.The 13 compounds were set to x1~x13 as variables to establish the data file of 10 regions.As shown in Table 6 and Table 7, they revealed that the cumulative contribution rate of the first six principal components main up to 95.08%, which indicated that the six representative compounds could be used as the main components due to their high content.The six main components were known as oxypaeoniflorin, paeoniflorin, benzoylpaeoniflorin, paeonol, trigalloyl glucose and pentagalloyl glucose by correlation matrix analysis.These six components were preferred as the representative active components contributing to the efficacy.

Effects of active components on SOD activity and MDA content in AGEs-induced HBZY-1 mesangial cells
We measured SOD activity and MDA content to evaluate the activity of these components.As shown in Figures 5A and 6B, SOD activity was significantly decreased whereas MDA content was increased remarkably by AGEs, compared with the control group (P < 0.05).After the treatment of these active components, SOD activity was increased while MDA content was decreased, which means that oxypaeoniflorin, paeoniflorin, benzoylpaeoniflorin, paeonol, trigalloyl glucose and pentagalloyl glucose can reduce the AGEsinduced oxidative stress.

Effects of active components on IL-6 and MCP-1 levels in AGEs-induced HBZY-1 mesangial cells
The contents of IL-6 and MCP-1 in supernatants of HBZY-1 mesangial cells were measured by ELISA kits according to the manufacturer's protocols.As shown in Figures 5C and 6D, the contents of IL-6 and MCP -1 could be increased by AGEs significantly (P < 0.001).After the treatment of these active components, the contents of IL-6 and MCP-1 were reduced significantly (P < 0.05, P < 0.01, P < 0.001).The results showed that the screened active components (oxypaeoniflorin, paeoniflorin, benzoylpaeoniflorin, paeonol, trigalloyl glucose and pentagalloyl glucose) could reduce IL-6 and MCP-1 contents in AGEsinduced mesangial cells.

Reduction of active components on FN expression in AGEs-induced HBZY-1 cells
As shown in Figure 6, FN protein expression was reduced by the active components (P < 0.01, P < 0.001) when compared with the AGEs-induced cells.Statistical analysis was performed based on the value of scanned grayscale.Results showed that these six components could inhibit FN secreting.
To clarify the pharmacological effects of oxypaeoniflorin, paeoniflorin, trigalloyl glucose, pentagalloyl glucose, benzoylpaeoniflorin and paeonol, their effects on pathological phenomena of AGEs-induced HBZY-1 mesangial cells, such as oxidative stress, inflammation, thickening of the basement membrane were observed and detected.The results showed that the six preferred active components could increase SOD activity, decrease MDA content, reduce the content of IL-6, MCP-1 and FN protein.

SCC in Anhui geoherb MC and Heibei nongeoherb MC
As shown in Figure 3B, the result of cluster analysis showed that MC from Zhejiang, Henan, Anhui were considered as a class for the same constituted compositions, while Hebei MC was divided into a separate class for the significant difference in active components compared with the Anhui MC.Contents of six active components of geoherb (Anhui) and non-geoherb (Hebei) MC were measured.The test solutions of Anhui MC and Hebei MC were a mixture of oxypaeoniflorin, paeoniflorin, trigalloyl glucose, pentagalloyl glucose, benzoylpaeoniflorin and paeonol in the ratio 1:7.5:0.3:0.4:0.7:10.7,respectively.The concentrations of each component were oxypaeoniflorin (1.94 μg/mL), paeoniflorin (14.56 μg/mL), trigalloyl glucose (0.58 μg/mL), pentagalloyl glucose (0.58 μg/ mL), benzoylpaeoniflorin (2.04 μg/mL) and paeonol (20.78 μg/mL) in cell experiment.Ratio of the six active components in geoherb MC was 1:7.5:0.3:0.4:0.7:10.7, the result was consistent with ratio of peak area in Table 5.As shown in Table 8, the ratio of Hebei non-geoherb MC was 1:1.2:0.03:0.03:0.1:1.3,similar with the result of comparison of the peak area.The results demonstrated that the prevention and treatment of geoherb MC on DN was due to SCC instead of the categories of compounds.

Effects of SCC transformation of non-geoherb MC on SOD activity, and MDA, IL-6, MCP-1 levels
The results of Table 9 demonstrated that, after recombination, the efficacy was superior to the geoherb one.The SCC of oxypaeoniflorin, paeoniflorin, trigalloyl glucose, pentagalloyl glucose, benzoylpaeoniflorin and paeonol in Hehei was adjusted from 1:1.2:0.03:0.03:0.1:1.3 to 1:7.5:0.3:0.4:0.7:10.7 as Anhui MC's.SOD activity of Anhui was 7.71 ± 0.98 U/mL, while Hebei recombination one was 7.17 ± 0.53 U/mL.MDA content of Anhui was 0.46 ± 0.05 nM while Hebei recombination one was 0.43 ± 0.06 nM.Meanwhile, IL-6 and MCP-1 contents of Anhui MC were 3.20 ± 0.1 ng/L and 13.67 ± 0.12 ng/L, respectively.These two contents in   Hebei recombination were 3.62 ± 0.10 ng/L and 3.85 ± 0.16 ng/L.There was no significant difference in efficacy between Hebei recombination and Anhui MC.This further validated that SCC acted as a crucial contributor to the quality consistency of MC which was related to the better efficacy.

DISCUSSION
CMM is the material basis of TCM to prevent or treat disease.Its quality directly affects the clinical efficacy.Geoherbs are the representative with particular area, excellent quality, and good efficacy making them become the proverbial superiors of TCM [1].The material basis of geoherbs efficacy is their internal components.Its SCC contributes to the level of efficacy and the pros and cons of quality [12].The scientific connotation of geoherbs quality and the relationship between the quality and efficacy of geoherbs are the emphases and difficulty in the study of geoherbs.MC is one of the commonly used TCM with significant activity against DN [13].The prevention of MC on DN included series pharmacological effects, including its improvement on the basement membrane thickening, oxidative stress, inflammation, and so on.In our present study, body weight, the content of blood glucose, urinary albumin and serum creatinine of DN rats were determined to reflect the treatment of MC from 10 regions.Among them, Anhui MC hold more significant reduction on the blood glucose, 24 h urine protein and serum creatinine.Therefore, Anhui MC, geoherb one, had a better anti-DN efficacy than other nongeoherb MCs (Figure 2).To further confirm the superior effect of Anhui MC on the treatment of DN, CAT, GSH-Px activity, the protein expression of ICAM-1, TGF-β1 and FN were detected to reflect the efficacy of MC from different regions.Finally, these samples were classified by cluster analysis, results indicated that geoherb MC from Anhui Tongling held the best efficacy in the treatment of DN.MC from Guizhou, Zhejiang and Henan were classified as a category due to their similar efficacy.MC from Hunan, Hebei, Chongqing, Gansu, Sichuan and Shandong were classified as another classification based on their poor efficacy .The material basis of geoherbs is composed of various components with a stable compositional characteristic, related to the stable SCC but not new compounds.Structural inconsistencies directly result in pharmacodynamic differences.Based on the overall concept of Chinese medicine and "component structure theory" [3], we compared the difference of efficacy and components between geoherb and non-geoherb MC.The efficacy of MC from different regions was not same.Unique stable SCC in geoherb MC contributed to a better efficacy on prevention and treatment of DN.Our study confirmed the different traits and extraction rate of MC from different regions, but there was no significant difference in the types of active components.The cluster analysis of efficacy component was introduced combined with peak areas of MC from 10 regions.After analyzing the belonging of the 13 components, we found that composition of Zhejiang was the same as Henan and Anhui MC.The composition of Hebei MC was quite different with Anhui.According to PCA, SCC of six components (oxypaeoniflorin, paeoniflorin, trigalloyl glucose, pentagalloyl glucose, benzoylpaeoniflorin and paeonol) have been recombinated for further study.These six active components also have been reported to have a good therapeutic effect on kidney disease and diabetes [14,15].In the present study, both SOD activity and MDA content of AGEs-induced mesangial cells were increased or decreased by active components screened from MC.The results indicated that the screened active components could inhibit oxidative stress of DN.IL-6 expression in mesangial cells was enhanced by the screened six active components of MC.The screened active components of MC could significantly down-regulate AGEs-induced MCP-1.The selected components of MC also reduced FN secretion, down-regulated FN protein expression, which revealed that MC could inhibit the synthesis of FN to prevent the thickening of basement membrane.
In conclusion, the SCC of oxypaeoniflorin, paeoniflorin, trigalloyl glucose, pentagalloyl glucose, benzoylpaeoniflorin and paeonol in Heibei was 1:1.2:0.03:0.03:0.1:1.3, while the ratio with distinct pharmacodynamic effect in Anhui geoherb MC was 1:7.5:0.3:0.4:0.7:10.7.Hebei MC had worse efficacy and different SCC from Anhui MC.The results showed that the stable and orderly structural integrity of active components in MC from Anhui, geoherb one, acts as an important contributor to the prevention and treatment of DN.Adjusting SCC of Hebei MC (with worst efficacy) to geoherb MC from Anhui (with best efficacy) showed a similar activity in AGEs-induced HBZY-1 cells.These demonstrated that the efficacy of MC was related closely to its stable SCC.
This paper revealed the SCC of geoherb MC, initially provided the material basis for a better prevention and treatment of geoherb MC on DN.The results demonstrated that the quality of MC materia medica was related closely to its stable SCC.Our findings provide evidence for the importance of SCC in CMM efficacy, and also provide a new insight into quality control of TCM.After being taken and weighted, kidney tissues were fixed with 10 % formalin.The blood was centrifuged at 3000 rpm for 10 min.The supernatant was collected and stored at -80 o C.

Instrumentation and analysis conditions
MC powder of 0.2 mg was dissolved in 1 mL methanol with an ultrasound for 15 min.Then the samples were centrifuged at 11000 rpm for 10 min.The supernatant was obtained for HPLC analysis.
Varian 310 triple quadrupole mass spectrometer equipped with ESI ion source (Varian, USA) was used for MS analysis in our study.Negative ion detection mode was conducted in this analysis.Nitrogen (N 2 ) was set at a flow rate of 0.45 L/min as atomization gas, 0.2 L/min as the curtain gas.The depolarization potential (DP) was -20 V while focusing potential was -80 V.The mass data was recorded within the range of 50-1200 Da.

Assays for SOD, CAT, GSH-Px activity and MDA content
Cell suspension or serum was centrifuged with 3000 rpm for 10 min and the supernatant was collected for the measurement.The levels of CAT, GSH-Px, SOD and MDA were determined according to manufacturer's protocols.Optical density (OD) value of samples was determined with microplate reader (Thermo Scientific, MA, USA) at wavelength of 405 nm (CAT), 412 nm (GSH-Px), 550 nm (SOD) and 532 nm (MDA) respectively.

ELISA for inflammatory cytokines IL-6 and MCP-1
Cell supernatant was taken for the determination of inflammatory cytokines.IL-6 and MCP-1 ELISA kits were used to determine cytokines levels according to manufacturer's protocols.Wavelength at 450 nm was chosen to read the OD value of samples with microplate reader (Thermo Scientific, MA, USA).The contents of inflammatory cytokines were calculated according to the standard curve.

Western blotting analysis for TGF-β1, ICAM-1 and FN
HBZY-1 cells were washed with ice-cold PBS and cell lysate was performed on the ice for 30 min.The lysates were centrifuged at 1500 rpm for 3 min, then supernatant was taken for measuring.An equal amount of total protein was separated by 10 % SDS-PAGE and then transferred from the SDS-PAGE gel to PVDF membrane.After being blocked with 1 % BSA in tris-buffered saline Tween-20 (TBST), membrane was incubated with the primary antibodies ICAM-1(1:400), TGF-β1 (1:400), FN (1:400) overnight.Then membrane was washed thrice and incubated with the peroxidase-conjugated secondary antibody (1:1000).Finally, the bands in the membrane incubated with chemiluminescence color and photographed by an ECL minicamera.β-actin was used as loading control.The quantification of proteins was analyzed by Image pro plus (IPP 6.0, Media Cybernetics, MD, USA) software.

Immunohistochemistry for TGF-β1and ICAM-1 expressions
Kidney tissues were dehydrated by serial gradient concentrations of alcohol, and then embedded in paraffin blocks for 3 h.After the paraffin embedded, the tissues were cut into serial sections at the thickness of 5 μm.Sections were heated and washed with 10 mM sodium citrate buffer.After quenching endogenous peroxidase and blocking with normal goat serum, the sections were incubated overnight at 4 ˚C with rabbit anti-rat antibodies specific against TGF-β1 (1:200) and ICAM-1(1:200), respectively.After washing with PBS, the sections were incubated at 37 ˚C for 2 h with the secondary antibodies, and then stained by DAB.Finally, the results were observed under IX83 microscopic (Olympus, Japan).

SOD activity, MDA, IL-6, MCP-1 content after component compatibility
AGEs-induced HBZY-1 cell model was established as in the section "Cell culture and extract treatment", cells were divided into six groups.200 μg/mL AGEs was used to induce mesangial injury.Aminoguanidine (AG) of 10 μM was used as the positive control while BSA of 200 μg/mL as blank control.Drug groups were given extractions of MC (200 μg/mL) from Anhui, Hebei and Hebei transformations which the intrinsic ratio of oxypaeoniflorin, paeoniflorin, trigalloyl glucose, pentagalloyl glucose, benzoylpaeoniflorin and paeonol was adjusted from 1:1.2:0.03:0.03:0.1:1.3 to 1:7.5:0.3:0.4:0.7:10.7,respectively.Medium was added to 2 mL before culture plates were incubated at 37 ˚C, 5% CO 2 for 24 h.And then, SOD activity, MDA content and inflammatory cytokines like IL-6, MCP-1 were determined according to manufacturer's protocols.

Figure 2 :
Figure 2: Effect of Moutan Cortex from different regions on ICAM-1 and TGF-β1 protein expression levels in kidney of DN rats.After being treated with STZ and/or MC extract of 5g/kg or , immunohistochemistry was conducted to evaluate the expression levels of ICAM-1 (A) and TGF-β1 (B) of renal tissues."a" represents normal control; "b" represents model group (DN rats); "c" represents Positive control 0.1 g/kg AG; "d-m" represent Gansu, Chongqing, Shangdong, Sichuan, Zhejiang, Anhui, Hunan, Guizhou, Hebei, Henan.

Figure 3 :
Figure 3: Cluster analysis of active ingredients (A) and efficacy dendrogram (B) of Moutan Cortex from different regions.SPSS16.0 sofeware was performed for Cluster analysis.

Figure 4 :
Figure 4: Pieces characteristics, powder characteristics and composition differences analysis.(A) Characteristics comparison of Moutan Cortex and their powder from different regions.(B) HPLC chromatogram of Moutan Cortex from different regions.The order of absorption peaks that from front to back was from A to J. (C) The chromatogram of active ingredients in Moutan Cortex.

Table 6 : Characteristic value and the variance contribution rate
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