A Novel Strategy For Screening Active Components in Cistanche Tubulosa Based On Spectrum-Effect Relationship Analysis And Network Pharmacology Ⅱ

3. Results and Discussion

3.1. HPLC Fingerprints

3.1.1. Method Validation

The validation for the HPLC method showed that the relative standard deviation (RSD) for method precision, reproducibility, and stability was less than 2.85% for the relative peak area (n = 11) and 0.77% for the relative retention time (n = 11). The precision of the same sample solution appeared within the range of 0.05–0.77% for relative time and 0.28–2.70% for the relative area of the common peaks. The reproducibility of the experiment was within the range of 0.03–0.20% for the relative time and 0.23–2.59% for the relative area of the common peaks. The sample stability was 0.09–0.24% for relative retention time and 0.75–2.85% for the relative area of the common peaks. These results indicated that the established fingerprint was satisfied. The linear relationships for geniposidic acid, echinacoside, acteoside, tubuloside A, and isoacteoside are shown in Table S4. The value of R square was 1.0000, indicating good linearity. The results of sample recovery showed that the average recoveries of geniposidic acid, echinacoside, acteoside, tubuloside A, and isoacteoside were 100.37%, 103.59%, 98.46%, 100.81%, and 101.19%, and the RSD for sample recoveries was less than 2.68%.

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3.1.2. Peak Area (PA) and Relative Retention Time (RRT)

The reference fingerprints and fingerprints of HMs, WEs, and HRs from 11 batches of C. tubulosa are presented in Figure 2. Eleven peaks, which exhibited good separation and resolution, were identified as common peaks among HMs, WEs, and HRs. The five standard compounds were identified as geniposidic acid (A2), echinacoside (A8), acteoside (A9), tubuloside A (A10), and isoacteoside (A11). The standard compound, echinacoside, which was present in all chromatograms (average retention time 12.86 min) with a suitable peak area and good stability, was selected as the reference peak and used to calculate the relative retention times (RRTs) of the other ten common peaks. The RRTs of these different forms are in the 0.16–1.51 range. The PA and coefficient of variance (CV%) of these common peaks are listed in Tables S5–S7. From the data, the CV% values for PA in various forms are 25.78%–142.02%, 23.36%–150.38%, and 28.91%–112.78% for HMs, WEs, and HRs, respectively. These results reveal significant differences in the concentration of each Cistanche tubulosa compound among the different forms. The fingerprints of HMs, WEs, and HRs are shown in Figure 3.

Figure 2 HPLC fingerprints of standard samples

3.1.3. Contents of HMs, WEs, and HRs

Five standard constituents of C. tubulosa were measured. The contents of the main components are shown in Table 1. The comparison between HMs, WEs, and HRs is shown in Table 2 and Figure 4. PhGs in C. tubulosa are biologically active but thermosensitive. Heat-sensitive components dissolving in water can be efficiently extracted using a reasonable method. Generally, Cistanche herba is extracted with water and then evaporated into a concentrated solution for the following chemical analysis [26, 27, 30]. After extraction and concentration, spray-drying technology was used and the procedure was modified from the previous article. Water was quickly removed from the liquid steam, and then dry extracts of raw materials from plants were obtained. In this step, adding maltodextrin is considered a common carrier to enhance the dispersion and extend the storage time. Through a series of manufacturing processes, herbal plants were then pressed into formula granules with additives. This step of adding excipients was not included in the experiment. Generally speaking, our production process includes extraction, concentration, and spray drying, as described in Section 2.2, in parallel with a formula granule production process. In order to produce these semifinished products, the above three steps must be followed. The procedure of forming WEs involves concentration and spray-drying, which easily cause the loss of thermosensitive components, but HRs are obtained after extraction and drying of HMs. We wonder whether it is possible that active components remain in HRs. According to our results, the content of verbascoside reduced significantly from HMs to WEs and HRs ( and , respectively). The thermal stability of verbascoside is investigated by monitoring the changes in the peak area through HPLC during the heating process. After heating for 4 h, 41.6% of verbascoside is left. It indicates that verbascoside is thermosensitive [31]. Isoacteoside, tubuloside A, and echinacoside in WEs remained stable after complex processing procedures. During the long-term drying process, the accumulation of PhGs showed a significant decrease, which might be attributed to the thermal degradation of these thermosensitive components [32]. In terms of the other target components, HRs and WEs did not differ significantly except for verbascoside. Our understanding of this difference will enable us to develop better quality standards for herbal dregs in the future and advance them into products.

Table 1 Contents of 11 batches of C. tubulosa

Table 2 Comparison of contents of main components (n = 11).

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Figure 4 Content determination of five components from different forms (n = 11). , ns: not significant.

3.1.4. Fingerprint Similarity Analysis

The similarities among the three C. tubulosa groups were evaluated. The herbal material-water extract, herbal material-herbal residue, and water extract-herbal residue similarity values were in the ranges 0.943–0.994, 0.847–0.995, and 0.938–1.000, respectively (Table 3).

Table 3 Similarities of HM-WE, HM-HR, and WE-HR for 11 batches of C. tubulosa.

3.2. Antioxidant Activity Test Results

The antioxidant activities of the various forms of C. tubulosa were determined using the DPPH, , and  scavenging capacity assays, and the relevant results are presented in Figure 5. In Table S8, the ranges for the DPPH,  and  scavenging capacity assay results were 0.04–37.80, 0.98–843.90, and 0.32–27.65 mg/mL for the three different forms among the 11 batches of C. tubulosa. In three antioxidant activity tests, HMs and WEs exhibited close inhibition activity, whereas HRs showed the weakest inhibition.

The spray-dried WEs were found to exhibit significant activities even at low concentrations. A previous report indicated that a spray-dried Vernonia amygdalina WE achieved 50% scavenging inhibition at 0.17 mg/mL [33]. The application of long extraction times and high temperatures is a double-edged sword. On the one hand, increasing the extraction time and spray drying inlet temperature improves the yield and efficiency. Moreover, the extracts achieve strong antioxidant activity and higher concentrations of biological components than those plants [34]. On the other hand, excessively hot inlet air degrades the bioactive compounds. Such elevated air inlet temperatures led to losses of antioxidant Bidens pilosa extract activity and were attributed to decreases in phenolic compounds [35]. Present results are consistent with the aforementioned report. For instance, the WE in S6 exhibited weaker radical inhibitory abilities than both HM and HR. Furthermore, HR in S5 exhibited stronger DPPH and superoxide anion scavenging abilities than HM and WE. The structure of PhGs consists of glycosidic bonds and acetyl groups that are hydrolyzed easily under enzymatic action or decomposed at high temperatures. These reactions may account for decreases in some main components during large-scale production. However, the hydrolysis or isomerization of certain components might accelerate the synthesis of other components. Such transformations are common when processing Cistanches herbs [36–38]. PhGs being water-soluble implies that most biological components can be utilized via water extraction. The contention that the majority of the active components remain in WEs has persisted for decades, so it seems reasonable to assume that the wet residual materials can be discarded after extraction. However, it is incorrect to regard HRs of C. tubulosa as waste. Researchers point out that PhGs are unstable, and they are susceptible to enzymatic or hydrolytic degradation [39]. Hydrolysis or isomerization reactions that contribute to decreases in biological ingredients within phytomedicines during processing might at the same time present new opportunities for exploiting HRs. By converting traditional extraction methods, medicinal residues can be developed and utilized more effectively. Enzymatic hydrolysis was performed to convert the Panax ginseng residue into mono sugars. Yields of polysaccharides and ginsenosides increased, such as sugar, succinic acid, ginseng polysaccharides, and ginsenosides [40]. Sophora flavescens residues are reextracted by ultrasonic waves with ethyl acetate [41]. The updated technologies for utilizing herbal residues are summarized by Huang et al. [42].

Based on the PLSR and BCA results, the top five peaks of different forms were screened using the DPPH, superoxide anion, and hydroxyl radical scavenging assays to identify the most important peaks. The results are illustrated in the Venn chart (Figure 7). A2, A6, A8, and A10 are the common peaks that are shared by HM, WE, and HR (Figures 7(a) and 7(c)) in the superoxide anion and hydroxyl radical scavenging assays, whereas HM, WE, and HR share no DPPH assay peaks. Meanwhile, the BCA models show that A1, A2, A3, and A6 are the common peaks shared by HM, WE, and HR (Figures 7(d)–7(f)). Notably, the overlaps in the Venn diagram indicate that the BCA model appears more suitable than the PLSR model, the former exhibiting more repetition. The BCA model coefficients and antioxidant ability IC50 values were analyzed via RDA. As the RDA shown in Figure 8, A1, A3, and A6 from HM and HR are related positively to the antioxidant indexes, except that A3 is related negatively to the hydroxyl radical scavenging capacity. A1 and A6 from WE have strong correlations with DPPH and the superoxide anion. The A6 peaks noted from the various forms exhibit the strongest connection to the DPPH, superoxide anion, and hydroxyl radicals. A1 and A3 also exhibit a similar connection.

Figure 7

Venn diagrams of PLSR and BCA model: (a) DPPH assay. (b)  scavenging assay. (c)  scavenging assay were analyzed by the PLSR model. (d) DPPH assay. (e)  scavenging assay. (f)  scavenging assay were analyzed by the BCA model. The overlapping section was the common peaks shard by HM, WE, and HR.

3.4. Network Pharmacology-Based Analysis

3.4.1. Construction of C-T Network

A total of 4359 targets related to the antioxidant activity were obtained from the GeneCards database and the OMIM database. At the same time, active components were screened from the TCMSP database and the SwissTargetPrediction database. Then, 198 targets were collected and standardized through the UniPort database. There were 159 target genes shared by active components and antioxidant-related diseases (see Figure S1). The C-T network was constructed to illustrate the correlation between the compounds and the key gene targets (Figure 9).

Figure 9 C-T network. The network showed the correlation between active components and the key gene targets.

3.4.2. Construction of the PPI Network and Screening of Key Targets

PPI was visualized using the STRING database (Figure 10). The network included 159 nodes and 2528 edges. In the entire interaction network, the connecting components or the nodes with more target points may be the key component or target gene that plays an antioxidant role in C. tubulosa. The results were downloaded and introduced into Cytoscape for visualization. The higher the DC value, the darker the color, and the larger the combined score value, the thicker the edge. We found that RAC-alpha serine/threonine-protein kinase (AKT1), interlukin-6 (IL6), tumor necrosis factor (TNF), and vascular endothelial growth factor A (VEGFA) were centrally located (Figure 11), indicating that they were key targets when active components exerted an antioxidant effect. It is reported that echinacoside reduces mitochondrial dysfunction via regulation of mitogen-activated protein kinases (MAPK) and AKT and their phosphorylated forms [43]. Researchers speculated that the antidiabetic effect of glycosides of C. tubulosa might be due to the antioxidant activity of PhGs by downregulating proinflammatory cytokines, such as IL-6 and TNF-α [44]. In addition, echinacoside could impair ovarian cancer cell growth by downregulating the expression of VEGFA to inhibit angiogenesis [45], which is closely correlated to the ROS system for ROS induces the expression of VEGF signaling [46].

Figure 10 PPI network.

3.4.3. Enrichment Analysis and C-T-P Network Establishment

The potential antioxidant compounds acted on numerous biological functions, including BP, CC, and MF. In Figure 12(a), the top 10 pathways are shown. The predicted targets from the PPI network mainly responded to many biological processes, such as organic cyclic compounds, xenobiotic stimulus, inorganic substances, oxygen levels, and positive regulation of the cellular component movement. The cellular component analysis showed that the genes were mainly related to the membrane raft, extracellular matrix, secretory granule lumen, transcription regulator complex, and apical part of the cell. These targets are also involved in many molecular functions, including DNA-binding transcription factor binding, protein homodimerization activity, protein domain-specific binding, and cytokine receptor binding.

Figure 12 Enrichment analysis: (a) GO enrichment analysis. (b) KEGG enrichment analysis.

To investigate the biological functions of these major hubs, a pathway enrichment analysis was conducted. From KEGG enrichment results, a bubble diagram was drawn to show top 20 pathways. The larger the spot was, the more genes were included in the pathway. As shown in Figure 12(b), the key pathways of C. tubulosa were related to pathways in cancer, lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, chemical carcinogenesis—receptor activation, and MAPK signaling pathway. Effects of C. tubulosa on apoptosis and cellular redox homeostasis were investigated. The data suggest that C. tubulosa can be a promising candidate for anti-colon-cancer therapy [47]. C. deserticola extract is found in aged people [48].

Figure 13 illustrates the correlation between the pathways and their related targets and the relationship between the overlapping target genes and biologically active components of C. tubulosa. A global view of the C-T-P network was generated, which consisted of 12 ingredients, 159 targets, and 20 pathways. Most of the targets were shared by the candidate active compounds. These candidate active ingredients with high interconnection degrees were responsible for the high interconnectedness of the C-T-P network, especially quercetin (degree = 131). The majority of the targets, such as AKT1, IL6, TNF, and VEGFA, were mapped to KEGG pathways associated with pathways in cancer.

Figure 13 C-T-P network.

4. Conclusions

In this study, we primarily probed complex situations when considering the spectrum-effect relationships among HM, WE, and HR of C. tubulosa. The HPLC fingerprints and antioxidant assays were used to identify the differences between Hs, WEs, and HRs of C. tubulosa. According to the HPLC fingerprints, 11 peaks were common among the 11 batches of Hs, WEs, and HRs. Geniposidic acid, echinacoside, verbascoside, tubuloside A, and isoacteoside were identified among these peaks. The contents of these five components were determined. In addition, the antioxidant effects of the C. tubulosa Hs, WEs, and HRs varied due to the alterations in the chemical compositions caused by complex manufacturing conditions. Based on diversified statistical models, the spectrum-effect relationship study indicated that peak A6 might be the most decisive component among the three forms of C. tubulosa. The study was based on network pharmacology to explore potential mechanisms of C. tubulosa on antioxidation through screening of compounds, prediction of key targets, construction of networks, and conduction of enrichment analysis. Our results provide a theoretical basis for recycling the herbal residues and the potential of C. tubulosa in the treatment of antioxidant-related diseases.