Optimization of Processing Technology Based on Chemical Composition Difference and Anti-Fibrotic Activity

Background Cornus ocinalis, a kind of edible herbal medicine, has been widely used in liver and kidney protection due to its anti-inammatory, anti-tumor, and anti-oxidant activities, which can be enhanced with wine-steamed (WS) processing. Methods Based on the activations of HSC-T6 and HK-2, our study used single-factor plus orthogonal design to investigate the anti-brosis of C. ocinalis processed with steamed (S), high-pressure steamed (HPS), WS, high-pressure wine-steamed (HPWS), wine-dipped (WD), and wine-fried (WF). The chemical constituents in processed C. ocinalis with higher anti-brotic activities were detected by UPLC-Q-TOF-MS/MS. Results Results showed that C. ocinalis with HPWS signicantly inhibited the activations of HSC-T6 and HK-2. Moreover, compounds in C. ocinalis with HPWS were obtained via UHPLC-Q-TOF-MS/MS, indicating that 27 components were changed compared with raw C. ocinalis. These results demonstrated that HPWS is the optimal processing technology for anti-brosis of C. ocinalis. shown that its anti-brosis activity can change with processing. Based on this information, our experiment used enhanced anti-brosis effects as the indicator to optimize the processing technology of C. ocinalis through single-factor and orthogonal tests. Finally, we identied the processing that can produce optimal anti-brosis activity, namely, C. ocinalis with HPWS, whose chemical composition was identied by UHPLC-Q-TOF-MS/MS analysis. The experimental result is a further step. The results of this study also provided material basis for further exploring the role of C. ocinalis in liver and kidney protection.


Introduction
Cornus o cinalis, commonly used in traditional Chinese medicine (TCM), is the dry mature fruit of C. o cinalis Sieb. et Zucc (Cornaceae) and is rede ned as a class of herb and edible plant [1]. C. o cinalis, with a mild warm nature, belongs to the meridians of the liver and kidney according to TCM theory; thus, it is commonly used in the prevention and treatment of liver and kidney diseases [2]. Moreover, it can be found in foodstuff, such as medicinal dishes and healthcare products and drinks due to its various pharmacological activities, including anti-in ammatory, antioxidant and anti-apoptotic [3]. To date, about 305 components have been isolated and identi ed from C. o cinalis, including iridoids, alkaloids, polysaccharides, avones, organic acid, essential oils and terpenoids [1]. Among these compounds, loganin and morroniside-active ingredients isolated from C. o cinalis-have been shown to alleviate osteoarthritis in mice by inhibiting pyroptosis and NF-kappaB activity [4][5]. Furthermore, morroniside could ameliorate neuropathic pain through the regulation of glucagon-like peptide-1 (GLP-1) receptors. 5-hydroxymethylfurfural (5-HMF), which are mainly isolated from processed C. o cinalis, could prevent human umbilical vein endothelial cells (HUVECs) from oxidative stress induced by glucose [6]. However, the compounds in C. o cinalis and its pharmacological activity may change with processing. In traditional crafts of China, C. o cinalis is often processed using fried, steamed, wined, fated, salted processing techniques, among others, to achieve different clinical effects. For example, wined C. o cinalis is most commonly used in clinical preparations, such as the Liuwei Dihuang pills in China, which has better effects in nourishing the liver and kidney compared to raw C. o cinalis. However, there is no uni ed requirement for steaming and braising times and wine amount, all of which could change in the compositions and activities of C. o cinalis. Therefore, the present study aimed to optimize the processing technology of C. o cinalis through the single-factor method and an orthogonal experiment design based on chemical composition difference and the anti-brotic activity of C. o cinalis.
Tissue brosis, de ned as the excessive deposition of extracellular matrix (ECM), is the outcome of chronic tissue damage, leading to the formation of scar tissue and even organ dysfunction and failure [7].
ECM is mainly derived from proliferative and brogenic myo broblasts, which are broblast-like cells that possess contractile properties [8,9]. Activated hepatic stellate cells (HSC-T6) are the main source of myo broblasts, which serve as a key driver of hepatic brosis in liver injuries [9]. In normal liver, HSC-T6 are in quiescent state and are mainly used to store vitamin A [10]. In comparison, during liver injury, quiescent HSC-T6 are activated and indicated by the high expression of alpha smooth muscle actin (α-SMA) and excessive deposition of ECM [11]. Similar to HSC-T6, human proximal tubular epithelial cell (HK-2) cells play a key role in tubulointerstitial brosis (TIF), which is the nal result of chronic kidney disease and is closely related to the degeneration of renal functions [12]. Moreover, epithelial mesenchymal transition (EMT) is the main pathogenesis of renal brosis and can transform differentiated epithelial cells into myo broblasts that are also characterized by the high expression of α-SMA [13,14]. During brogenesis, EMT may be driven by various pro-brotic growth factors, among which transforming growth factor β (TGF-β) is the most important factor [15]. Apart from these, TGF-β is regarded as a common vital switch for brosis in tissue or organs in response to chronic injuries [16].
In summary, we will evaluate the effects of C. o cinalis on anti-brosis via the expression of α-SMA in activated HSC-T6 and differentiated HK-2 cells induced by TGF-β, with the goal of exploring the protective effect of C. o cinalis on the liver and kidney, and nally selecting the processing technology with higher anti-brotic activity. Moreover, changes of chemical constituents in processed C. o cinalis will be detected by ultra-high performance liquid chromatography coupled with hybrid triple quadrupole time-ofight mass spectrometry (UHPLC-Q-TOF-MS/MS).

Plant materials and processing technology
The samples of C. o cinalis were selected from the PanAn City of Zhejiang Province and identi ed by Professor Jianwei Chen of the Department of Chinese Medicine Identi cation, School of Pharmacy, Nanjing University of Chinese Medicine. According to single-factor experiments, C. o cinalis samples were steamed at different times (1, 2, 4, 6, and 8 h) at different temperatures (100°C, 105°C, 110°C, 115°C, 120°C and 125°C) and then dried at 60°C, respectively. Next, the samples were braised at different times (0.5, 1, 2, and 4 h) with different dosages of rice wine (w/w) (15%, 20%, 25%, 30%, and 40%), respectively. These samples were then steamed again for 1 h at 105°C and then nally dried at 60°C. The orthogonal experimental design was based on the results of single-factor experiments, as follows: C. o cinalis samples were processed with three factors (steamed times, steamed temperatures, braised times) and at different dosages of rice wine in line with L 9 (3 4 ) design orthogonal table shown in S1, S3, and S5, respectively.

Extraction preparation
Both raw and processed C. o cinalis were crushed and sifted through 16-mesh screen. The powder (5 g) was re uxed with 90% ethanol (50 mL) at a water bath (100°C) twice and ltered with four layers of gauze. The ltrates were collected and combined. Next, the ltrates were transformed into freeze-dried powder through vacuum concentration and lyophilization.

UHPLC-Q-TOF-MS/MS analysis
The LC-MS/MS analysis were performed using an UHPLC (Shimadzu LC-30AD, Japan) coupled with a Triple TOF 5600 Plus System (AB Sciex, USA). The following parameters were followed: column temperature was 30°C, mobile phase was acetonitrile (B) and 0.1% formic acid in water (A), ow rate was 0.3 mL/min, and the injection volume was 3 µL. The gradient elution procedure was as follows: 0-3. and negative modes were set as follows: the ion source temperature was 550°C, IonSpray Voltage was 5500-5500 V, auxiliary spray gas was nitrogen, Ion Source Gas1 (Gas1) was 55 psi, Ion Source Gas2 (Gas2) was 55 psi, curtain gas (CUR) was 35 psi, declustering potential (DP) was 60 V, and collision energy was 30V. The scanned ranges of TOF-MS and TOF-MS/MS were 100-2000 and 50-1000 Da, respectively.
When establishing liver brosis in vitro, the HSC-T6 and HK-2 cells were cultured in six-well plates and treated with TGF-β (PeproTech, USA), respectively. To detect the effects of C. o cinalis on anti-brosis, cells were treated with extracts derived from raw and processed C. o cinalis.

Western blot analysis
Cells were lysed using RIPA buffer supplemented with 1% phenylmethanesulfonyl uoride (PMSF) (Solarbio, Beijing, China) and then quanti ed by BCA protein assay kit (Beyotime, Shanghai, China). Equivalent protein samples were separate by sodium dodecyl sulfate-polyacrylamid gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene uoride (PVDF) membranes (GE, Freiburg, Germany). The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with antialpha smooth muscle actin (α-SMA) antibody (Abcam, UK) overnight at 4°C. The membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature and visualized using ECL Reagent (Beyotime, Shanghai, China). Finally, the membranes were stripped and probed with GAPDH that a loading control. The intensities of bands were quanti ed by Quantity One software (Bio-Rad, Hercules, CA, USA).

Statistical analysis
The data in the experiments were present as mean ± SD. The comparison of the results was evaluated by GraphPad Prism program (Graphpad Software, Inc., San Diego, CA, USA) with one-way analysis of variance (ANOVA) and Tukey's multiple comparison tests. Statistical signi cance between groups was considered with a p-value less than 0.05. Difference analyses were detected by MarkerView TM and T Test.
The original data were imported into MarkerView TM and statistical analysis via T Test. The t-test was employed to identify signi cant differences among processed products. Here, P < 0.05 was considered a signi cant difference, and a t-value greater than 0 indicated an increase in ingredient contents.

Evaluation of raw C. o cinalis on anti-brosis
The cytotoxicity of C. o cinalis on HSC-T6 and HK-2 cells were evaluation by CCK8-a widely used method in the detection of cytotoxicity and drug sensitivity. To establish a preferable brosis model in vitro, HSC-T6 and HK-2 were treated with different concentrations of TGF-β at different times. The results showed that TGF-β (1, 5, 10, and 20 ng/mL) activated HSC-T6 and HK-2 cells at different times (24,48, and 72 h), which manifested by the higher expressions of α-SMA compared to the normal group ( Figures 1D and E, respectively). Upon comparing the results, TGF-β (10 ng/mL) at 48 h was used to activate the cells. Moreover, results in Figure 1D showed that C. o cinalis at different doses (0.25, 0.5, and 1 mg/mL) inhibited the expression of α-SMA in HSC-T6 and HK-2 cells induced by TGF-β, with the best effect identi ed at a dose of 1 mg/mL ( Figure   1F).  (Figures 2A and B, respectively). Furthermore, braised C. o cinalis for 0.5, 1, 2 and 4 h inhibited TGF-β-induced over-expression of α-SMA in HSC-T6 and HK-2 cells, especially at 1 h in HSC-T6 and 4 h in HK cells ( Figure 2C). C. o cinalis processed with different dosages of rice wine (w/w) also showed different degrees of anti-brosis activity, especially 15% in HSC-T6 and 25% in HK-2 cells ( Figure 2D).

Optimization of the processing technology with orthogonal test
In accordance with the terms of the single-factor experiments, the processing parameters were optimized with an orthogonal L 9 (3 4 ) test design. Our results showed that C. o cinalis processed at three levels of two factors (steaming times and steaming temperatures), as shown in Table S1, inhibited the expressions of α-SMA in HSC-T6 and HK-2 induced by TGF-β ( Figure 3A). The composite score of the anti-brosis effects was calculated with non-negative analysis, and the results indicated that the maximum was 1.92 (Table S1). Furthermore, in selecting the better processing term, the values of K and R are shown in Table   S1, and the variance analysis results are shown in Table S2. These results showed that C. o cinalis processed for 1 h at 125°C (known as high-pressure steaming (HPS) showed optimum anti-brosis effect. The craft conditions of WS C. o cinalis were also optimized with an orthogonal design (Table S3), and its anti-brosis effects on both HSC-T6 and HK-2 were evaluated via the over-expression of α-SMA induced by TGF-β ( Figure 3B). The results indicated that WS C. o cinalis with different inhibited the activation of myoblasts, which was mainly manifested by the decrease of α-SMA expressions ( Figure  3B). Combined with the results of variance analysis (Table S4) and the values of K and R in Table S3, the optimal processing term with anti-brosis effects were as follows: steaming time of 2 h, braising time of 0.5 h, and rice wine dosage (w/w) of 30%. Similarly, the optimal parameters of high-pressure wine steamed (HPWS) C. o cinalis were identi ed (steaming for 1 h at 115°C, braised time of 1 h, and rice wine dosage (w/w) at 25%), all of which were optimized with orthogonal design (Table S5), variance analysis (Table S6), and the expression of α-SMA ( Figure 3C).

Validation of processed C. o cinalis in terms of antibrosis effects
The optimal processing technologies of processed C. o cinalis were determined by single-factor and orthogonal experiments. According to the terms of HPWS C. o cinalis and the stipulation in Chp, winedipped (WD) and wine-fried (WF) C. o cinalis samples were prepared. The expressions of α-SMA were detected by Western blot to verify the anti-brosis activity of all processed C. o cinalis. The results showed that both raw and processed C. o cinalis inhibited the expressions of α-SMA induced by TGF-β in both HSC-T6 and HK-2, especially C. o cinalis processed with HPWS ( Figure 4A). Then, immuno uorescence assay for α-SMA in HSC-T6 was conducted to con rm the anti-brosis effect enhanced by C. o cinalis processed with HPWS. The results also showed that C. o cinalis inhibited the positive expression of α-SMA (in green) induced by TGF-β, especially the sample processed with HPWS ( Figure 4B).

Ingredient identi cation of C. o cinalis processed with HPWS
The analysis of the compounds in both raw and HPWS samples were identi ed by ESI positive and negative modes ( Figure 5). The chemical name, molecular mass, molecular formula, and molecular structure of components in C. o cinalis were retrieved and download from the database, after which the accurate mass-to-charge ratio of plasma morphology were calculated in both ESI positive and negative modes. The raw data were imported into the PeakView TM software. All the chemical components were encoded and a new session was established under the XIC Manager template. Then, rst-level data matching was conducted with reference standards, standard mass spectrometric database, and literature according to m/z. The chromatographic peak with the retention time error within 0.2 min and the m/z error within 10 ppm was identi ed as a uni ed compound. Further, the identi cation validation and chromatographic peak attribution were based on molecular structures and secondary fragments of compounds.
As shown in Table 1, 27 components in C. o cinalis were changed in the HPWS condition, including avonoids, iridoid glycosides, and organic acids. Moreover, 5-HMF was a typical emerging compound after C. o cinalis was processed with HPWS. We take quercetin as an example to illustrate the identi cation procedure: [M -H] − of peak 11 was 301.0354, formula was calculated as C 15 H 10 O 7 with Mass (Da) 302.0427, the main secondary fragment was 121.0305, and neutral loss was 180.0019. After consulting the literature [17] and comparing the results with the standard, we con rmed that the compound is quercetin.

Discussion
In TCM, C. o cinalis is often used in replenishing the liver and kidney due to its tonic effect [1]. Furthermore, modern pharmacology indicates that C. o cinalis shows low toxicity on cells only at high concentrations [18], as con rmed by the results of CCK8 in the present study. The present study also provides a theoretical basis for further research on liver-and kidney-related diseases. In liver and kidney brosis, persistent or dysregulated brogenic reactions may hamper regeneration and promote dysfunction [19], which could ultimately raise susceptibility to organ failure and death [20]. During these processes, TGF-β, which is a key cellular factor of brotic reactions, can contribute to a brogenic phenotype by activating broblasts cells [19], including HSC-T6 and HK-2 cells. Taking all these elements into account, the present study aimed to develop a therapeutic implementation of anti-TGF-β approaches. The results of this study showed that C. o cinalis downregulated the TGF-β-induced expression of α-SMA by inhibiting the activation of broblast cells, thus providing a direction for us to optimize the processing technology of C. o cinalis with higher anti-brotic activity.
Wined C. o cinalis is a traditional and common processing method that is applied until now. However, different indexes of evaluation are accompanied by varying wine processing techniques. Thus, the current research aimed to explore the suitable wined technology of C. o cinalis based on the anti-brosis synergistic effects produced by each technique. The results of univariate elements lay the foundation for subsequent orthogonal experiments. However, considering the time bene t, the term of orthogonal experiment was nally determined as follows: steaming times were 1, 2, and 3 h; steaming temperatures were 105°C, 115°C, and 125°C; braising times were 0.5, 1, and 1.5 h; and rice wine dosages (w/w) were 25%, 30%, and 35%. Due to the fact that steaming time, steaming temperature, braising time, and rice wine dosage are vital criteria in the processing of C. o cinalis [21], processed samples using different technologies have varying antidiabetic effects [22]. Aside from this, the results of our study showed that the samples had different inhibitory effects on activated brosis cells. Therefore, optimization of processing criteria is the essential step in screening the technology with the optimal anti-brosis effect. Studies have shown that the orthogonal test design is a common method for the optimization of experimental conditions [23][24]. Thus, the current study used the orthogonal L 9 (3 4 ) test design to detect the anti-brosis of C. o cinalis processed with different factors for the single-factor experiments and obtained several kinds of samples via S, WS, and HPWS processing technologies.
Based on the single-factor and orthogonal tests, variance analysis, and the evaluation of anti-brosis activity, C. o cinalis processed with HPWS showed better anti-brosis activity than other processed products. The pharmacological activities of C. o cinalis are mainly due to various active components found in the C. o cinalis [25], which are transformed during processing, thereby leading to differences in the anti-brosis activity of C. o cinalis in liver. Therefore, exploring the composition changes in C. o cinalis processed with HPWS can provide material basis for further clarifying the mechanism of C. o cinalis with HPWS-enhanced anti-hepatic brosis.
To further excavate the differences in C. o cinalis between raw and HPWS products, the data were standardized with SIMCA 14.1 (version, country) and analyzed via PLS-DA under supervised recognition mode. The results in Figure 5 present the PLS-DA score plot of C. o cinalis before and after processing. As can be seen, the raw and HPWS processed C. o cinalis products were obviously clustered into two categories, thereby indicating that the processing has changed the chemical composition of the sample.
The current results also showed that, after processing via HPWS, the chemical composition of C. o cinalis changed qualitatively, including 5-HMF, linoelaidic acid, and quercetin. 5-HMF prevents L02 hepatocytes from injury induced by GalN/TNF-α [26] and attenuates liver brosis by inhibiting oxidative stress in mice [27]. Furthermore, quercetin can protect the liver and kidney, as reported in another study [28]. Thus, the above research could reveal a chemical basis for the enhanced anti-brosis activity of C. o cinalis processed by HPWS.

Conclusion
As a kind of medicine and edible herbal, C. o cinalis has been shown to suppress liver and kidney brosis by inhibiting the activation of HSC-T6 and HK-2 cells. Studies have also shown that its antibrosis activity can change with processing. Based on this information, our experiment used enhanced anti-brosis effects as the indicator to optimize the processing technology of C. o cinalis through singlefactor and orthogonal tests. Finally, we identi ed the processing that can produce optimal anti-brosis activity, namely, C. o cinalis with HPWS, whose chemical composition was identi ed by UHPLC-Q-TOF-MS/MS analysis. The experimental result is a further step. The results of this study also provided material basis for further exploring the role of C. o cinalis in liver and kidney protection.